The interaction of magnetic nanoparticles with biological entities has been recently explored by various parties yielding great interest within the scientific community. The manipulation of individual and small groups of cells has long been of interest and magnetic nanoparticles interesting properties within both dynamic and static magnetic fields have seen recent exploration. Here we show results from experiments where magnetite (Fe3O4) nanoparticles (< 50nm) are used in conjunction with microelectromagnetic devices to both position biological cells (erythrocytes) non- destructively and to perform a surgery on the cells so that a controlled haemolysis is performed on collections of cells within the magnetic trap, without harming cells spatially located a few microns nearby. This ability to both position cells non-destructively and to perform a surgery on them without damaging nearby tissues has been of interest to the medical community with the recent emergence of magnetic hyperthermia for the treatments of cancers. The interaction between the magnetic traps and the nanoparticles allows for a surgery to be performed on the individual groups of cells, after being non-destructively positioned, in this case resulting in cell haemolysis, most likely through a localized thermal process. The entire process takes place in over a short period of time allowing for real time video analysis to be used to help analyze samples during the cellular surgery which typically takes place in less than 5 seconds. The results lay the groundwork for explorations for spatially controlled magnetic hyperthermia experiments as well as offering a technique beyond that of optical tweezers to spatially manipulate and control cells non-destructively with a resolution of just a few microns. Also, the results provide an interesting exploration into the interaction of magnetic nanoparticles with living cells, which is another avenue of interesting research.

Gold/nickel barcode nanowires of various lengths and surface chemistries will be presented for use as cell-imaging contrast agents, for control and separation of osteosarcoma cells (OSCA-8), and for therapeutic applications employing localized surface plasmon resonance and rotational magnetic fields. Identifying and separating cell populations from a mixture of analytes in small volumes has both clinical and commercial significance, especially in health and security. Existing technologies such as immunomagnetic isolation and flow cytometry have significant limitations in specificity. Multi-segment nanowires provide a unique combination of optical and magnetic readout signals for each cell/analyte based on the multilayers of the nanowires, much like how a unique barcode is associated with a product at a convenient store. Here, fabrication of the nanowires was done using sequential electrodeposition in anodic aluminum oxide (AAO) pores. Dark-field optical microscopy images confirmed uptake of these nanowires by osteosarcoma (OSCA) cells and their use as contrast agents. SEM/TEM images suggested that the nanowires were enclosed inside cell vesicles. Initial toxicity results indicated that the barcode nanowires were non-cytotoxic. The effects of incubating nanowires with osteosarcoma cells under different conditions were studied. Initially, increasing the concentration of nanowires in the cell culture led to a decreased cell separation yield due to aggregation. This aggregation was overcome by functionalizing the nanowires using hetero-bifunctional polyethylene glycol (PEG). Kinetics such as sedimentation and diffusion were observed, and the effect of various surface chemistries on aggregation and cellular uptake was studied using UV/VIS spectroscopy and Fourier Transform Infrared (FTIR) spectroscopy. Cell imaging was enhanced by nanowire surface plasmons, and cellular motion (including separation) was easily controlled using external magnetic fields. In short, barcode nanowires hold much promise for multiplexed diagnosis and therapy.

The use of nanoparticles for tissue-specific imaging and targeted therapy has shown great potential for detection and treatment of disease. A quintessential key in nanotechnology is to self-assemble multifunctional nanoparticles with well-defined properties, i.e. morphology, size, charge, surface ligands. To facilitate high precision self-assembly we turned toward a bio-inspired approach, specifically plant viral nanoparticles (VNPs). Production of VNPs is highly scalable and economic using molecular farming in plants. Genetic and chemical engineering can be applied to introduce targeting ligands, therapeutics and imaging moieties. High multivalency of VNPs render them an attractive candidate material for applications in medicine; thousands of imaging moieties and/or drugs (chemotherapies or photosensitizers) can be displayed on the interior or exterior surface of these nanomaterials. In this presentation, I will discuss the application of genetically and chemically engineered VNPs, such as icosahedral cowpea mosaic virus, rod-shaped tobacco mosaic virus, or filamentous potato virus X for tissue-specific imaging and treatment of cancer and cardiovascular disease. Acknowledgements: This work was supported by NIH/NIBIB grants R00 EB009105 and P30 EB011317, a Case Western Reserve University Interdisciplinary Alliance Investment Grant, and a Case Comprehensive Cancer Center grant P30 CA043703 (NIH/NCI).

Two-photon fluorescence materials have attracted much recent attention for their many promising applications, especially in the growing field of biomedical imaging. Among the best performing two-photon fluorescence materials are semiconductor quantum dots such as CdSe and related core-shell nanoparticles. At the same time, however, heavy metals as the essential elements in available high-performance semiconductor quantum dots have prompted serious health and environmental concerns. Therefore, the search for benign alternatives has become increasingly important. Here we show that photoluminescent silica and metal oxide nanoparticles can be prepared in the absence of any fluorophores or metal doping via a novel wet-chemical methodology of surface-functionalization. This approach overcomes laborious multi-step procedures of nanoparticle functionalization. The fluorescent particles display good water solubility, good dispersibility, low cytotoxicity, strong photoluminescence and photostability under two-photon imaging conditions. The cytotoxicity of the functionalized particles were tested in dendritic cells (DCs) that have received broad scientific and clinical interest due to their key role in anti-cancer host responses and potential use as biological adjuvants in tumor vaccines. Similarly, bioinert and photoluminescent zirconium, hafnium and tantalum oxide nanoparticles were prepared as multifunctional nanoprobes for two-photon and CT-imaging. For example, uniform and hollow tantalum oxide nanoparticles were prepared from core-shell Fe-Ta2O5 nanoparticles with a sacrificial Fe core. The particles were modified by silanization. The silanization of the surface permitted the attachment of functional units (e.g. polyethylene glycol (PEG)). The particles could be loaded with low molecular drugs, that were released over a period of up to 24 hours. PEG was introduced to endow the nanoparticles with biocompatibility and antifouling activity, whereas the luminescent Ta2O5 core enabled simultaneous fluorescence imaging as well as CT-imaging. The ease and versatility of the synthesis, the aqueous stability and dispersibility, the intrinsic fluorescence emitting capacity, the easiness of any biomolecule conjugation using a simple step and the extreme robustness of the photophysical properties make the photoluminescent oxide particles excellent candidates for biomedical applications.

We will describe the utilization of single molecule spectroscopic methods and unique semiconductor-based reagents to superresolution imaging and to the study of molecular machines in-vitro, in live cells and in small organisms

Among the clinically relevant non-invasive imaging techniques, magnetic resonance imaging (MRI) is perhaps the most widely used. In the majority of cases, image contrast is generated from the physiological variance in the nuclear magnetic resonance characteristics of water protons. Practically, MRI contrast is normally enhanced by the presence of contrast agents. Gadolinium, due to its exceptionally large paramagnetic moment and unique relaxation properties, has long been the center of the clinically used T1 contrast agents[1]. In coupling an MRI contrast agent to a biomolecule, one may be able to effectively marry the advantages afforded by increased molecular bulk with those engendered by natural biological targeting[1-3]. In the present work, the synthesis of stable, MR contrast enhancing and biologically vectoring mesoporous silica nanoparticles (MSNs) is reported. One of the principal aims was to generate T1 contrast particles not only with relaxivity characteristics exceeding those of any prior report (r1=33mM-1s-1), but also tuneable through surface chemistry and functional protein conjugation. Significantly, controlled biomodification could be achieved with retention of high relaxivity and luminescence characteristics which facilitate subcellular analysis of uptake and distribution. Moreover, a reversible relaxivity was developed through competitive protein displacement. This level of control over surface chemistry and biomodification additionally lays the ground for both enabling and directly imaging protein delivery in vivo. [1] W. Y. Huang, J. J. Davis, Dalton T 2011, 40, 6087-6103. [2] K. M. L. Taylor, J. S. Kim, W. J. Rieter, H. An, W. L. Lin, W. B. Lin, Journal of the American Chemical Society 2008, 130, 2154-+. [3] W.Y.Huang, J.J.Davis, Biovectoring and T1 Contrast Gating with Mesoporous Silica Nanoparticles, manuscript in preparation

We have developed arginine-glycine-aspartic acid (RGD)-attached gold (Au) half-shell nanoparticles containing methotrexate (MTX) for the treatment of rheumatoid arthritis (RA), where MTX is the most widely used disease modifying anti-rheumatic drug (DMARD) for the treatment of RA, and RGD peptide is a targeting moiety for inflammation. Upon near-infrared (NIR) irradiation, heat is locally generated due to Au half shells, and the drug release rate is enhanced, delivering heat and drug to the inflamed joints simultaneously. RA is a chronic inflammatory disease characterized by synovial inflammation in multiple joints within the penetration depth of NIR light. When combined with NIR irradiation, these nanoparticles containing a much smaller dosage of MTX (1/930 of MTX solution) showed greater therapeutic effects than that of a conventional treatment with MTX solution in collagen-induced arthritic (CIA) mice. This novel drug delivery system is a good way to maximize therapeutic efficacy and minimize dosage-related MTX side effects in the treatment of RA. Furthermore, these multifunctional nanoparticles could be applied to other DMARDs for RA or other inflammatory diseases.

Engineered cerium oxide (CeO₂) nanostructures exhibit enhanced physical and chemical properties compared to their bulk counterparts. CeO₂ has established uses in catalysis, fuel cells, glass polishing and semiconductor manufacturing. Beyond these industrial applications, CeO₂ shows antioxidant activity at physiological pH, opening a new route for its application in biomedicine. Cellular targeting with CeO₂ nanoparticles (CNP) is therefore exploited in protecting cells against radiation damage, inflammation and oxidative stress. We present our research on superoxide dismutase (SOD) and catalase mimetic activities of CNPs, which in healthy cells would normally occur to reduce the oxidative stress. Having high surface to volume ratio, tunable Ce⁴⁺/Ce³⁺ ratio and low coordinated surface cerium (Ce) sites which can interact with reactive oxygen species (ROS) without involving oxygen vacancies (Vo), CNPs are able to behave as an auto-regenerative free radical scavenger. We investigate the interaction between ROS and low coordinated surface Ce sites suggesting the underlying atomistic mechanism for adsorption/desorption of ROS, which becomes crucial when developing highly optimized, sized and shaped CNPs for cellular targeting and free radical scavenging. Using first principle calculations we find that the presence of Ce³⁺ is not only correlated to the presence of Vo as is generally believed, but also to the presence of surface hydroxyl groups. Furthermore the heat of reduction is strongly affected by the surface composition; for example the presence of water molecules promotes further surface reduction. To understand the effect of low coordinated surface Ce sites on the adsorption of ROS, we use the faceted CeO₂{111} surface. The faceting resembles the CNP edges and surfaces yielding low coordinated surface Ce sites with local charge that can be easily tuned by adsorbing ROS. Adsorption of hydroxyl radicals and desorption of less dangerous ROS such as water and hydrogen peroxide is found to be more favorable on reduced hydroxylated faceted surfaces than on the oxidized hydroxylated faceted surfaces. This behavior is however strongly site dependent with edge adsorption sites yielding more stable adsorption than corner sites.

Magnetic nanoparticles (NPs) hold great promise for biomedical applications. However, the development and control of safe in vivo applications for NPs call for the study of cell-NPs interactions and cell viability. Up until now poly(ethylene glycol) (PEG) has been used to increase NPs solubility in water and to prevent unspecific interactions with other molecules. We report the use of monosaccharides such as glucose to this end, as they do not increase the global hydrodynamic size of the NP, they are easy to work with, and they are inexpensive. Monosaccharides could be used as passivation molecules but may also serve as targeting molecules, as carbohydrates present in the organism play a major role in many biological processes. Furthermore, carbohydrate-functionalized NPs could have another potential role, namely overcoming biological barriers such as the plasma membrane or the blood-brain barrier. Therefore, the functionalization of a NP with a single carbohydrate moiety could confer this structure three capacities (blocking protein unspecific adsorption, active cell targeting and cell internalization) when multifunctionalization is usually required to achieve this goal. Given the huge potential of NPs and in order to further characterize these challenging materials, it is important to study their cellular uptake as well as their potential toxicity. Here we performed systematic studies to evaluate the cellular internalization of 6 nm magnetic NPs coated with several types and various densities of biomolecules, such as glucose, galactose and PEG, as well as an extensive set of cytotoxic assays. We found that the density of the grafted molecule was crucial to prevent unspecific uptake of NPs by Vero cells. Moreover, we describe that the cellular uptake of NPs is largely dependent on the type and density of carbohydrate attached to their surfaces. Surprisingly, the glucose coated NPs described here showed cellular uptake as a result of lipid raft instead of clathrin-mediated cellular internalization. Our studies demonstrate that carbohydrate-modified magnetic NPs are non-toxic and represent an advance in the field, in which few examples can be found.

In recent years, RNA interference (RNAi) has become a major area of interest for directing stem cell fate. RNAi involves using small interfering RNA (siRNA) to selectively silence genes or pathways to control cellular behavior. However, a major challenge in this area is developing an efficient methodology to deliver siRNA into stem cells. The most common methods include solution-based delivery using viruses, non-viral cationic lipids, nanoparticles or polyplexes. However, when treated with exogenous materials, stem cells are more prone to undergo cell death or undesired differentiation patterns. In contrast, methods based on substrate-mediated delivery, wherein cells uptake siRNA from their microenvironments, are extremely advantageous as they provide a way to improve the efficiency of siRNA delivery by simply changing the cellular microenvironments. However, the fact that nanotopographical features of the extracellular microenvironment can be used to deliver siRNA into stem cells remains to be explored. To this end, we have developed a nanotopography-mediated reverse uptake platform (NanoRU) to demonstrate a simple technique to deliver siRNA into neural stem cells (NSCs). NanoRU consists of a self-assembled nanoparticle film coated with a mixture of extracellular matrix (ECM) proteins and the desired siRNA of interest. We show that siRNA delivery to NSCs is dependent on the size of the nanoparticles and that only the siRNA molecules, and not the nanoparticles, are taken up by the NSCs. Furthermore, we use this technique to enhance the neuronal differentiation of NSCs by suppressing the expression of specific proteins and transcription factors. This platform was also used to demonstrate efficient siRNA delivery into various other mammalian cell lines, while maintaining superior cellular viability compared to commercial transfection agents. Our results show that NanoRU relies on the ability of the cells to sense the nanotopographical features and take up only the siRNA from its microenvironment, and it does not require the use of exogenous agents that can perturb the cells. We believe this is valuable delivery platform which can truly complement conventional genetic manipulation tools in cellular biology.

Chlorambucil (CHL) is a recognized lipophilic anticancer agent that has been used clinically against some types of cancer. However, the use of CHL is restricted because of its undesirable side effects. A promising approach to overcome this problem and to improve therapeutic efficacy is to encapsulate the CHL with a suitable drug delivery system (DDS). Special interest has been focused in the use of nanoparticles (NP) prepared from poly(DL-lactide-co-glycolide) (PLGA), due to their biocompatibility and biodegradability. Drugs and/or other compounds can be encapsulated into NP or may be chemically attached or physically absorbed in the surface of them in order to enhance the solubility and reduce toxicity. In this work, PLGA-NP encapsulating CHL were prepared by the solvent evaporation technique. These nanoparticles were evaluated in relation to size, zeta potential, drug encapsulation efficiency, release profile, and external morphology and spectroscopic properties. Toxicity of breast cancer cell was evaluated using a cell viability measurement (MTT). The NPs dried product (white powder appearance) resulted a yield among 49-62%. CHL-PLGA-NP and PLGA-NP showed hydrodynamic diameters of 525±20 nm and 456±43 nm, respectively. Negative data with Laser Doppler Electrophoresis (LDE) were acquired for zeta potential (-24±0.8 to CHL-PLGA-NP and -14±0.6 to PLGA-NP) showing low aggregation index. The encapsulation efficiency was determined by direct method employing spectroscopic measurements and the value obtained was between 49-53%. The reduction on MCF-7 cell viability after CHL-PLGA-NPs treatment (89-50%) was more pronounced when compared with free CHL (92-72%) at similar concentrations. Treatment in fibroblasts presented toxic as well but with lower intensity (100-89% CHL and CHL-NP 100-84%). The MTT assays demonstrated that the conjugate decreased de number of viable cells in MFC-7 and could be more potent antiproliferative agent than free CHL due cytotoxicity was concentration-dependent in both treatment, but it was more pronounced at shorter times in CHL-PLGA-NP than CHL free. The analyzes of the produced NPs showed that this is a reproducible procedure that results in nanoparticles with therapeutic applicability. It can be argued that the encapsulated resulted in increased efficiency of the drug reaching the target than to its application in free form. Financial Support: CNPq, CAPES, FAPDF, FINATEC

The objective of the present in vitro study was to investigate cardiomyocyte functions, specifically adhesion and proliferation, on injectable pHEMA (poly (2-hydroxyethyl methacrylate)), RNT (rosette nanotube) and CNF (carbon nanofiber) composites to determine their potential for myocardial tissue engineering applications. RNTs are novel biocompatible nanomaterials assembled from synthetic analogs of DNA bases guanine and cytosine that self-assemble within minutes when placed in aqueous solutions at body temperatures. Thus, RNTs were used in this study as a material that could potentially improve cardiomyocyte functions and solidification time of pHEMA and CNF composites. Since heart tissue is conductive, CNFs were added to pHEMA/RNT composites to increase composite conductivity. pHEMA was chosen here since it has been one of the most widely tested injectable biocompatible hydrogels for tissue repair applications. Contact angle, conductivity and scanning electron microscopy experiments characterized the surface energy, conductivity and structure of the composites. Results showed that cardiomyocyte density increased after 4 h , 1day and 3 days with greater amounts of CNFs and greater amounts of RNTs in pHEMA composites (up to 10mg/ml CNFs and 0.05mg/ml RNTs). With greater amounts of CNFs and RNTs, factors that may have increased cardiomyocyte functions include greater wettability , conductivity and increased nanometer surface features mimicking the natural nanometer features of heart tissue itself . Specifically, contact angles measured on the surface of the composites decreased while the conductivity and surface roughness in the composites increased as CNFs and RNTs content increased. In summary, the properties of these injectable composites make them promising candidates for myocardial tissue engineering applications and thus, should be further studied.

Self-assembled monolayers (SAMs) provide an efficient means to control surface functionalities on substrates. We have developed a versatile photoprotected alkane thiol monomer to functionalize gold substrates. This single monomer can generate two unique functional groups depending on the deprotection conditions utilized. When the monomer was photoirradiated in a nitrogen rich environment, a cyclooctyne moiety was formed, which can then be used for copper free “Click” chemistry. However, when the monomer was deprotected in the presence of oxygen, a diketone was formed, which can then be used in stabilized Schiff base reactions. This monomer provides us a handle to precisely control the distribution of cyclooctyne and diketones on a gold substrate based on the conditions of the photodeprotection. By utilizing our commercially available direct-write photolithography system, we have been able to generate both punctate and gradient patterns of cyclooctyne and diketone functional groups on gold substrates. Strained cyclooctynes react with azide moieties to form triazole rings. The diketones react with hydrazines and aminooxy groups to form hydrazones and oximes, respectively. As a first proof of principle, gold nanoparticles were functionalized with a thiol azide monomer, which could then be “Clicked” to the patterned cyclooctyne regions of the substrate. Proteins labeled with an azide functionality were also coupled to gold substrates. Moreover, proteins labeled with hydrazine functionalities could be coupled down to the diketone regions of the substrate. These patterns were characterized by a variety of methods including, surface plasma resonance imaging, scanning probe microscopy, and fluorescent microscopy.

Silk fibroin (SF) has attracted great attention in the field of tissue engineering due to its intrinsic characteristics such as biocompatibility, bioactivity, and flexibility. Moreover, SF can be used to create three-dimensional nano- fibrous scaffolds since it can readily be fabricated by electro-spinning to imitate the innate structure of the extracellular matrix (ECM). However, SF does not have desirable mechanical strength which is essential for bone tissue repair. Hydroxyapatite, a hard and brittle ceramic material, comes in handy to improve the stiffness and osteoconductivity of SF scaffold. Here we suggest electro-spinning of SF solution combined with nano-hydroxyapatite (nHAp) at different nHAp/SF ratios to examine the change of mechanical strength to determine the optimal content of nHAp for ideal SF scaffolds with tunable modulus and flexibility. A SF solution was prepared with the following procedure: Bombyx mori cocoons were boiled in 0.02 M of Na2CO3 aqueous solution until sericin was completely removed. Dehydrated SF was then dissolved in 9.3 M of LiBr aqueous solution at 60 °C, followed by dialysis against distilled water. The final SF aqueous solution was diluted to 9 % (w/v). Then, 11% of nHAp slurry was added to the SF solution by giving a variation in the percentage: nHAp:SF (w/w) of 0:100, 5:100, 10:100, 20:100, 30:100, and 40:100. A syringe was filled with this mixed SF-nHAp solution and was connected to the metal tip of a 17G needle. The solution-filled syringe was then placed 21cm apart from the grounded cylindrical collector and was pushed by the syringe pump with a constant rate of 1.2 mL/h. The applied voltage was kept at 12kV. The electro-spun fibers had a diameter of 300 nm on average. SEM and TEM image analysis confirmed that nHAp was uniformly distributed within the SF nano fibers. The external morphology of the SF fibers appeared rougher when nHAp content increased in each sample groups compared to SF fibers without any nHAp. The aggregation of nHAp was only seen in limited locations. Manual tensile tests have also shown that tensile strength of SF scaffold increased as the amount of nHAp increased. Above the nHAp-SF ratio of 30:100 the scaffold became too brittle to act as a scaffold. Further characterization of the SF/nHAp hybrid scaffolds will be performed to find the quantitative relationship between the ratio of nHAp:SF and mechanical characteristics such as tensile, compressive, and torsional modulus. Biocompatibility and osteoconductivity will also be tested in rat calvairal defect models.

Recently, gold nanoparticles (AuNPs) protected by a mixed surface monolayer composed of linear alkanethiol ligands were observed to spontaneously penetrate cell membranes even when endocytosis was inhibited. Penetration seems to depend on the structure of the ligand monolayer; particle uptake was greatest when the surface self-assembled into a “striped” morphology consisting of alternating ribbon-like domains of hydrophilic and hydrophobic ligands. Here, we show that striped AuNPs prefer to embed within simpler single-component lipid bilayers and vesicles, supporting the observation of non-specific penetration in full cellular membranes. Stable embedding is surprising given the large thermodynamic penalty associated with exposing charges to the hydrophobic core of the bilayer, which would be seemingly necessary because the width of the striped domains is smaller than the thickness of the bilayer. However, the flexibility of the ligands allows the surface monolayer to adopt configurations more thermodynamically favorable than would be expected when in the presence of the bilayer, permitting stable embedding. In this work, we provide a thermodynamic analysis of monolayer-protected AuNP-bilayer complexation supported by implicit bilayer simulations. We calculate the free energy change associated with embedding particles of mixed, striped, and random morphologies in a bilayer as a function of the particle size, salt concentration, and fraction of hydrophilic ligands in the monolayer. Our results show that stable embedding critically depends on the deformation of surface ligands to avoid exposing charged moieties to the bilayer core, mimicking the “snorkeling” behavior exhibited by some transmembrane proteins. We demonstrate that the free energy change is strongly dependent on particle size, but does not appear to depend on the morphology of the monolayer. We reconcile this deviation from experimental results by showing the effect that added branching points have on the free volume in the monolayer. Our results improve our understanding of particle-bilayer interactions and will enable the design of nanoparticles for drug delivery and biosensing applications.

Hybrid plasmonic-magnetic nanoparticles possess properties that are attractive in bioimaging, targeted drug delivery, in vivo diagnosis and therapy. The stability and toxicity, however, of such nanoparticles challenge their safe use today. Here, biocompatible, SiO₂-coated, Janus-like Ag/Fe₂O₃ nanoparticles are prepared by one-step, scalable flame aerosol technology. A nanothin SiO₂ shell around these multifunctional nanoparticles leaves intact their morphology, magnetic and plasmonic properties but minimizes the release of toxic Ag⁺ ions from the nanosilver surface and its direct contact with live cells. Furthermore, this silica shell hinders flocculation and allows for easy dispersion of such nanoparticles in aqueous and biological buffer (PBS) solutions without any extra functionalization step. As a result, these hybrid particles exhibited no cytotoxicity during bioimaging and remained stable in suspension with no signs of agglomeration and sedimentation or settling. Their performance as bioimaging agents was explored by selectively binding them with live tagged Raji and HeLa cells enabling their detection under dark-field illumination. Therefore, these SiO₂-coated Ag/Fe₂O₃ nanoparticles do not exhibit the limiting physical properties of each individual component but retain their desired functionalities facilitating thus, the safe use of such hybrid nanoparticles in bio-applications. Furthermore, both the plasmonic and superparamagnetic components can be facilitated for photothermal ablation or hyperthermal treatment of cancer cells, as seen from their heat dissipitation under alternating magnetic field, as well as their temperature increase under laser irradiation. [1] Sotiriou GA, Pratsinis SE. Engineering Nanosilver as an Antibacterial, Biosensor and Bioimaging Material. Curr Opin Chem Eng 2011, 1: 3-10. [2] Sotiriou GA, Sannomiya T, Teleki A, Krumeich F, Vörös J, Pratsinis SE. Non-Toxic Dry-Coated Nanosilver for Plasmonic Biosensors. Adv Funct Mater 2010, 20: 4250-4257. [3] Sotiriou GA, Hirt AM, Lozach PY, Teleki A, Krumeich F, Pratsinis SE. Hybrid, Silica-Coated, Janus-Like Plasmonic-Magnetic Nanoparticles. Chem Mater 2011, 23: 1985-1992. [4] Sotiriou GA, Teleki A, Camenzind A, Krumeich F, Meyer A, Panke S, Pratsinis SE. Nanosilver on Nanostructured Silica: Antibacterial Activity and Ag Surface Area. Chem Eng J 2011, 170: 547-554. [5] Sotiriou GA, Pratsinis SE. Antibacterial Activity of Nanosilver Ions and Particles. Environ Sci Technol 2010, 44: 5649-5654. [6] Sotiriou GA, Franco D, Poulikakos D, Ferrari A. Optically Stable Biocompatible Flame-Made SiO₂-Coated Y₂O₃:Tb³⁺ Nanophosphors for Cell Imaging. ACS Nano 2012, 6: 3888-3897.

Phosphate is an essential building block for all creatures and universally used in genetic material (DNA), proteins, and energy carriers (ATP). Phosphate exchange and translocation in living cells is very fast and can lead to a fierce competition for phosphate between different species and typically results in very low free phosphate availability in the environment. The lack of phosphate often limits biomass growth, e.g. in open ocean water. In contrast, abundant phosphate (e.g. from overfertilisation) promotes eutrophication with excessive algae growth. Generally, by withdrawing any essential nutrient from a life cycle, a population can be limited. Here, lanthanum oxide nanoparticles are employed to remove essential phosphate from nutrient solutions and thus to limit and even expunge algae, fungi, and bacteria populations. Lanthanum, a transition metal in the group of the rare earths elements, is known for its high affinity to phosphate. Here, we show that the extreme competition and scavenging of phosphate by the species of the rare earth metal lanthanum additionally results in the death of microbes. The effect of lanthanum oxide nanoparticles in phosphate-containing bacteria, fungi and algae cultures is examined. While in phosphate-rich solutions no toxic effect was observed, below a certain phosphate to lanthanum oxide ratio all organisms were killed by the particles. As long as free phosphate and unsaturated lanthanum was available, the phosphate was bound by the lanthanum. Only when very little free phosphate was present, the growth was inhibited and the organisms were directly attacked and ultimately killed by the lanthanum oxide. Besides the antibacterial properties against E. coli (a Gram-negative bacteria) we similarly investigated S. carnosus (a Gram-positive bacteria), P. roqueforti (a fungus), and Chlorella vulgaris (a single-celled green alga). This toxicity of lanthanum oxide can be controlled by the addition of phosphate (a completely non-toxic and biocompatible agent) and render the antimicrobial material non-active. Since the undesired growth of microbes in industrial or house-hold scenarios (e.g. water purification) occurs in media with very low phosphate concentrations (sea water, rain, industrial cooling waters, drinking waters), the use of the phosphate starvation strategy may be a generally applicable technical alternative to nano-silver, copper-paints, or antibiotics. The ability to switch the toxicity off by a second factor (phosphate) additionally promises external control of the antimicrobial activity. Compared to most work on toxicity of metal oxide nanoparticles presenting either increased toxicity or no observable effect to a given organism, we show that nanoparticle originated toxicity can be technically utilized if the antimicrobial action is well understood and targetable towards given organisms under specific environmental conditions. Literature: L.C. Gerber, et al., Chem. Commun., 48, 3869-3871 (2012).

The biological and medical applications of layered double hydroxide (LDH) nanoparticles have attracted wide interest. LDHs consist of brucite-like layers and the exchangeable anions-containing gallery space. The present abstract reported that an anti-restenotic drug, low molecular weight heparin (LMWH), was intercalated into LDH interlayers which improved the biological and therapeutical effects of LMWH in vitro and in vivo. Results from powder X-ray diffraction and transmission electron microscopy demonstrated successful intercalation of LMWH into LDH interlayers by observation of enlarged LDH interlayers. LMWH-LDH presented a hexagonal plate-like shape with ~90 nm in size. Under physiological conditions, the intercalated LMWH was released from LDH in a sustained manner, resulting from the diffusion of LMWH from LDH and the dissolution of LDH layers. The biological examination on cultured rat smooth muscle cells (SMCs) demonstrated the low cytotoxicity of LDH nanocarriers. Comparison of the effects of unconjugated LMWH and LMWH-LDH conjugates showed the enhanced inhibitive effects of LMWH on SMC proliferation and migration. The cellular uptake of LMWH by SMCs was also increased (more than 10 times) by conjugation to LDH nanoparticles. After internalization by SMCs, LMWH-LDH was found to undergo the endocytic pathway, and (unlike unconjugated LMWH) escape from endosomal compartment, thus avoiding biodegradation of LMWH. Compared with unconjugated LMWH, LMWH-LDH enhanced suppression of mitogen-activated protein kinas signal transduction, probably due to the sustained release and improved cellular uptake of LMWH-LDH; the enhanced suppression of MAPK signal transduction is associated with enhanced inhibition of SMC proliferation and migration. To target deliver LMWH-LDHs to the site of arterial injury, LMWH-LDH was conjugated with a targeting moiety (an antibody to XLF) and examined in a rat model. Our preliminary results showed that targeted delivery of LMWH-LDH with anti-XLF effectively limited restenosis and thrombus formation, which suggested the potential of this technique for clinical application.

Regulation of gene expression using oligonucleotides, such as antisense oligodeoxynucleotides (AS-ODNs) and small interfering RNAs, is one of attractive methods for gene therapy. It is important to deliver ONs into cells effectively, and to inhibit expression of target gene selectively. In many cases, cationic liposomes (CLs) are used for effective delivery of them because stable and small complexes of CL and ON, called “lipoplex”, are formed by electrostatic interaction. Although lipoplex could be introduced into cell effectively, the efficiency and selectivity of gene suppression are insufficient. Previously, we reported that high hydrostatic pressurization (HHP) treatment for the complex of CL and plasmid DNA could improve the transfection efficiency. In this study, we investigated ONs delivery using lipoplex treated with HHP to inhibit a target gene effectively and selectively. Further, physicochemical properties of the HHP-treated lipoplex were examined in order to elucidate relationship between the structure and the transfection efficiency of the HHP treated lipoplex. AS-ODNs were designed to inhibit expression of firefly luciferase. Cationic liposome consisting of DOPE and DOTMA was prepared according to the extrusion method. Lipofectamine2000 was also used. The complexes of ONs and CLs at various ratios of C/A ratios were prepared, and then pressurized hydrostatically at various atmospheres (~10,000 atm) for 10 min. After removal of pressure, the pressurized lipoplexes were used. The complexes of these AS-ODNs and lipofectamine2000 or DOPE/DOTMA with/without pressurization were transferred into HEK293 cells expressing luciferase stably. Although the levels of gene suppression were different between ONs used, the efficiency of gene silencing was enhanced by the HHP-treated lipoplex compared to the non-pressurized lipoplex. In order to clarify the different transfection efficiency of lipoplex with/without HHP treatment, the properties of them was investigated by TEM, AFM and fluorescent spectral analysis. For TEM observation, the lamellar structure of the non-treated lipoplexes was observed, whereas the amorphous structure including small and regular structure was observed for the HHP-treated lipoplexes. For AFM observation, spherical shape of the non-treated lipoplexes with the diameter of 100-200nm was observed, whereas bell-shaped form of the HHP-lipoplexes was observed. The hydrophobic microenvironment of lipoplex was investigated by using fluorescent hydrophobic probes, such as Laurdan and pyrene. The fluorescent spectrum and intensity of these probes differed between lipoplexes with/without pressurization. These results suggest that the structure of lipoplex was changed by the HHP treatment. We believe that this change of lipoplex structure by the HHP treatment affected the efficiency of gene suppression. This HHP treatment for lipoplex appears to be a promising contribution to gene and ON delivery.

Recent medical technologies have primarily paid attention to the development of new diagnostic material with intrinsic biocompatibility and specific selectivity and strong intensity. Several bio-inspired hybrid materials containing fluorescent organics or inorganics have been intensively considered for the design of new diagnostic tool kits. Moreover, these materials for the diagnostic imaging system require high signal intensity as contrasted with the surroundings. To achieve the issues, a variety of candidates have been developed1. Here, we presented a synthetic light-emitting proto-cell resembling a natural luminescence bacterium. It simply composed of three subdivisions including outer membrane, interfacial polymeric matrix and core fluorescence dye-DNA complex. Interestingly, compared with conventional chemical fluorophores or luminescent organisms, its physicochemical properties were varied depending on the precursor compositions and preparation methods. It demonstrated the intrinsic biocompatibility, strong serum stability, outstanding targeting and higher sensitivity, which might be attractive in a modern diagnostics. Moreover, it was possible to construct a multi-colored labeling platform for multiplexing. It is speculated that combined with generic and genetic DNA nanotechnology developed previously by the author2, it may have many potential at the field of several biotechnologies and even system biology. 1. Rudin, M. and Weissleder, R. Molecular imaging in drug discovery and development, Nature Drug Discovery, 2, 123-131 (2003); Breaker, R. R. Natural and engineered nucleic acids as tools to explore biology, Nature, 432, 838-845 (2004); Resch-Genger, U. Quantum dots versus organic dyes as fluorescent labels. Nature Methods 5(9), 763-775 (2008). 2. Um, S. H. et al. A cell-free protein-producing gel. Nature Materials 8(5), 432-437, (2009); Um, S. H. et al. Enzyme-catalyzed assembly of DNA hydrogels. Nature Materials 5(10), 797-801 (2006); Um, S. H. et al. Dendrimer-like DNA-based fluorescence nanobarcodes. Nature Protocols 1(2), 995-1000 (2006).

Silver and gold nanoparticles are in the focus of modern materials science studies due to their distinct chemical, physical and biological properties. There is a wide range of applications in biological sciences and nanomedicine. However, the preparation of such noble metal nanoparticles is often performed in organic solvents where the absolute yield is low and the transferability to biological media is limited. Here we present a facile synthesis of small silver, gold and alloyed silver-gold nanoparticles and a thorough investigation of their comparative effects on hMSCs. To assure good transferability and stability in biological media, the noble metal nanoparticles were synthesized by a modified Turkevich method. Silver (diameter 15-25 nm), gold (diameter 5-6 nm) and alloyed 50:50 (n:n) silver-gold nanoparticles (diameters 6-7 nm, 10-12 nm, 16-20 nm, depending on the synthesis conditions) were obtained by reducing tetrachloroauric acid or silver nitrate, respectively, with a mixture of tannine and citrate , followed by stabilization with either tris(3-sulfonatophenyl)phosphine (TPPTS) or poly(N-vinylpyrrolidone) (PVP). Human mesenchymal stem cells (hMSC) were cultured in the presence of all three types of nanoparticles. The cell viability of the treated cells was analyzed by fluorescence microscopy (green fluorescent calcein assay). Furthermore, the release of the cytokines IL 6 and IL 8 was studied as an indicator of cell activation by nanoparticles. To examine a size-dependent effect, the silver-gold nanoparticles were prepared and analyzed in three different sizes. The toxicity of silver nanoparticles strongly increased with concentration and incubation time due to the release of silver ions. Gold nanoparticles showed only a weak effect on the cell viability at the highest concentration. After prolonged incubation, the small gold nanoparticles induced a concentration-dependent cytotoxicity, which was much lower than the effect of silver nanoparticles. Interestingly, no significant influence on the viability of hMSC was observed after incubation with the larger (10-12 nm and 16-20 nm) alloyed silver-gold nanoparticles. The smaller 6-7 nm alloyed nanoparticles showed a concentration dependent toxicity between that of Ag- and Au-nanoparticles. The IL-6 release was reduced by the silver, gold and small alloyed nanoparticles, whereas the larger alloyed nanoparticles had no significant impact. Silver and silver-gold nanoparticles induced the release of IL-8, whereas the gold nanoparticles reduced the interleukin release. It is notable that there was no observable difference between a coating with TPPTS and with PVP in any case.

The density and conformation of poly (ethylene glycol) on nanoparticles plays an essential role in mitigating protein binding and macrophage uptake, while extending circulation time in vivo. Additionally, long-circulating nanoparticles are necessary for effective tumor accumulation by means of the EPR effect. In this work, PEG density effects are observed by in vitro and in vivo studies. A series of calibration-quality 80 nm x 320 nm hydrogel nanoparticles were fabricated using the soft lithographic technique known as PRINT (Particle Replication in Non-wetting Templates). Standard fluorescence analysis of fluorescein-labeled PEG grafts was used to calculate the PEG surface coverage. PEG surface density was varied resulting in two particle sets with distinct conformations - brush (0.083 ± 0.006 PEG/nm2) and mushroom (0.028 ± 0.002 PEG/nm2). In protein binding studies via isothermal titration calorimetry, the PEG-brush particle exhibited a four- and three-fold decrease in protein binding as compared to the non-PEGylated and PEG-mushroom formulations, respectively. Macrophage association studies (fluorescence-activated cell sorting) with mouse alveolar macrophage cells displayed decreased association of at least five-fold upon PEGylation. Brush and mushroom PEG conformations led to increased circulation times as measured by intravital microscopy, with both PEGylated particles behaving similarly. PEGylation resulted in at least a 17-fold increase in circulation half-life, a 136-fold decrease in clearance, and an 86-fold increase in AUC over non-PEGylated NPs. Overall, we have shown significant improvement in particle behavior in vitro and in vivo with lower PEG densities than previously reported. In future work, these long-circulating PRINT particles will be conjugated to acid-labile chemotherapeutic pro-drugs for delivery to the tumor bed via EPR effect. Finally, active targeting will be utilized for enhanced cellular uptake and efficacy.

The objective of the present study was to evaluate the in vitro photodynamic effects of poly (lactic-co-glycolic acid) (PLGA) nanoparticles loaded with the photosensitizing agent methylene blue (MB) on Enterococcus faecalis (ATCC 29212), a gram-positive commensal bacterium inhabiting the human gastrointestinal tract, a leading cause of nosocomial infections, and a microorganism associated with failures of endodontic treatment. Sensitization of E. faecalis species in planktonic phase with MB-loaded nanoparticles for 10 minutes followed by exposure to red light of 665 nm led to approximately 2 log10 bacterial killing. The uptake and distribution in E. faecalis was investigated by transmission electron microscopy using PLGA conjugated with colloidal gold particles (10-15 nm). Nanoparticles were found to be concentrated mainly on the cell walls of microorganisms. In biofilms, the synergism of nanoparticles and red light eliminated approximately 1 log10 of biofilm species in experimentally infected root canals of extracted teeth. E. faecalis species in experimentally infected root canals, in both planktonic and biofilm phase, showed susceptibility to photodynamic therapy. In conclusion, PLGA nanoparticles as carriers of photoactive drugs may be a promising adjunctive method to established antimicrobial regimens.

Over the past decade there has been growing interest in producing dynamic, stimuli-responsive materials through self-assembly. Recent work in the Scherman group has demonstrated the formation of supramolecular diblock co-polymers using cucurbit[8]uril (CB[8]) as a “molecular handcuff” to bring two polymeric species together in water with extremely high equilibrium association constants (Keq up to 10^(14) M^(-2). This system has recently been applied to the preparation of multi-stimuli responsive micelles for the controlled delivery of cancer therapeutics. We have extended this initial system through development of multivalent copolymers which can serve as a platform for the preparation of supramolecular crosslinked materials. It is clear that the ability to finely tune the solution viscosity of an aqueous system is critical in many applications ranging from large-scale fluid-based industrial processes, to free-standing hydrogels important in regenerative medicine, controlled drug delivery, as well as “green” self-healing materials. The present work focuses on the preparation of reversible, stimuli-responsive supramolecular hydrogels driven by strong host-guest interactions with CB[8]. We demonstrate that a solution of multivalent copolymers, bearing good guest moieties, can be immediately transformed into a supramolecular hydrogel with precise control over the mechanical properties. Moreover, the non-covalent crosslinks are dynamic and stimuli-responsive and the material properties can be modulated by a variety of external stimuli. Hydrogels with extremely high water content (up to 99.75% water by weight) have also been prepared by utilizing renewable cellulose derivatives. Their rapid formation and shear-induced flow properties make these materials perfectly suited for use as “injectable” hydrogels for delivery of therapeutics. Indeed, model proteins can be easily encapsulated and their extremely sustained release is observed over the course of 6 months. This sustained release far surpasses the current state of the art for protein release from a hydrogel, highlighting these materials as important potential candidates for sustained therapeutic applications. These hydrogels are easily processed and the simplicity of their preparation, their availability from inexpensive renewable resources, and the tunability of their mechanical properties are distinguishing for many important applications. The fundamental knowledge gained from the study of these dynamic materials has facilitated progress in the field of drug delivery, smart, self-healing materials, self-assembled hydrogels, and controlled solution viscosity.

We have developed the first example of nano-sized polyion complex (PIC) vesicles (Nano-PICsomes) with a unilamellar PIC membrane obtained by the simple mixing of water-soluble and oppositely charged block copolymers composed of biocompatible poly(ethylene glycol) (PEG) and poly(amino acid)s in an aqueous medium. Compared with conventional vesicles, many advantages of Nano-PICsomes have been revealed so far, such as no need for an organic solvent, easy encapsulation of water-soluble macromolecules, semipermeability of the vesicle wall, protease resistance, tunable size ranging from 100-400 nm and so on. Another important aspect of Nano-PICsomes is facile cross-linking of PIC layers using an amide coupling reagent. In fact, stability of Nano-PICsomes under physiological conditions was greatly improved after cross-linking, and much prolonged blood circulation was demonstrated irrespective of their sizes. Furthermore, excellent tumor accumulation was confirmed in mice upon using PICsomes with the diameter of 100 nm. Thus, Nano-PICsomes are expected to be a versatile platform to deliver a wide variety of macromolecules and nano-materials to tumor tissues. However, the drawback of this system is difficulty in loading highly charged objects into Nano-PICsomes, mainly because charged objects often disturbs the PICsome formation. In this report, we established a new method that allows for loading nano-sized materials with various properties, such as highly charged QDs and enzymes, magnetic resonance imaging (MRI) contrast agents with the size of ~ 50 nm, etc..., utilizing characteristic properties of supramolecular system. Indeed, we found that MRI contrast agents loaded into Nano-PICsomes showed a better contrast in tumor tissues compared to the use of MRI contrast agents alone. Also, we confirmed potential utility of enzyme-loaded Nano-PICsomes as a nano-reactor that can be delivered to tumor tissues. This new method can broaden the utility of PICsomes for both in vivo imaging and therapy.

The development of drug resistance, low bioavailability and poor cellular uptake are major problems with chemotherapy agents. It is much importance to develop novel drug and gene delivery systems to overcome these multiple extra- and intracellular obstacles as well as a deep understanding of the mechanism of uptake and intracellular trafficking. MiR-21, as a therapeutic target for glioma, could efficiently enhance the chemotherapeutic effect in cancer therapy when it was downregulated. Here we reported a novel amphiphilic star-branched cationic micelle that was prepared from PDMAEMA-PLA-PDMAEMA copolymers and applied for the delivery of miR-21 inhibitor and DOX into glioma cells. The super-branched star copolymers with symmetric A3 (B3)3 structures were obtained by the “assembly process” as firstly conjugation of PLA onto the 1, 3, 5-benzenetricarboxylic acid chloride followed by the click reaction between the azide end-caps of PLA and the acetylene end-groups of PDMAEMA. The copolymers exhibited amphiphilic behavior in the aqueous solution, nano-sized micelles, positive surface charges and sustained drug release profile. The micelles revealed a low cytotoxicity compared with PEI and had a high capacity for DOX encapsulation, with a DOX loading level of up to 17%. Gel retardation assay showed that the micelles loaded with DOX could effectively complex with miR-21inhibitor at N/P ratios of 4/1 and achiveved high gene transfection. MTT and flow cytometry assay indicated that co-delivery of DOX and miR-21 inhibitor enhanced the chemo-sensitivity of human glioma cells to DOX by a maximum inhibition. More importantly, confocal laser scanning microscope and flow cytometry studies on FITC-miR-21 inhibitor complexed with DOX-loaded micelle revealed that the polyplexes were delivered into the same cells LN229 through clathrin-mediated endocytosis pathway and effectively escaped from lysosomal degradation. The results of this study demonstrated the promise of novel amphiphilic star-branched SPLA-b-PDMAEMA copolymers for efficient drug and gene delivery in a timely and spatially controlled manner.

Bone repair and regeneration of defects with bioresorbable implant materials are in great demand in reconstructive surgery. Many investigated biomaterials consist of calcium phosphates which excel in their biocompatibility, bioactivity and osteoconductivity. However, current commercially available products are limited for clinical applications because of their brittleness, incompressibility and difficulty to shape. Amorphous, flame spray-derived calcium phosphate (a-CaP) nanoparticles with advantages in terms of size, accessible composition and reactivity [1] offer a platform for new biomaterial products. The high temperature-derived nanoparticles find application either as a direct biomedical product or as part of a nanocomposite implant material. On one hand a-CaP can be used to render mechanically improved high-density polyethylene bioactive [2]. On the other hand it can also be introduced into a biodegradable polymer, poly(lactide-co-glycolide) (PLGA) to produce a bioactive bone fixation device [3]. Preparation of a highly porous fibre network by electrospinning using the a-CaP-PLGA composite system resulted in a flexible, cotton wool-like material suitable for the filling of bone defects [4]. The formation of nano-sized hydroxyapatite confirmed the high bioactivity of the compressible material suggesting applications for non-load bearing bone defects. Biocompatibility of the nanocomposite was assessed in two different animal models [5] [6]. By adding collagen to the a-CaP/PLGA system an easy to handle anisotropic bilayer was fabricated. The double membrane presented compositional and structural similarities to natural bone tissue on one side and pure PLGA fibres on the other side [7]. [1] Loher, S et al., Chem Mater (2005) [2] Hild, N et al., Biomed Mater accepted (2012) [3] Mohn, D et al., Polym Eng Sci (2010) [4] Schneider, OD et al., J Biomed Mater Res B (2008) [5] Schneider, OD et al., Acta Biomater (2008) [6] Schneider, OD et al., The Open Orthopaedics Journal (2011) [7] Hild, N et al., Nanoscale (2011)

Direct and fast (10s of seconds) deposition of flame-made, high surface-area aerosol films on polymers and polymeric microfluidic devices is demonstrated. Uniform TiO₂ nanoparticle films were deposited on cooled Poly(methyl methacrylate) (PMMA) substrates by combustion of titanium(IV) isopropoxide (TTIP) - xylene solution sprays. Films were mechanically stabilized by in-situ annealing with a xylene spray flame. Plasma-etched microfluidic chromatography columns, comprising parallel microchannels were also coated with such nanoparticle films without any microchannel deformation. These microcolumns were successfully used in metal-oxide affinity chromatography (MOAC) to selectively trap phosphopeptides on these high surface-area nanostructured films. The chips had a high capacity retaining 1.2 mu;g of standard phosphopeptide. A new extremely fast method is developed for MOAC microchip stationary phase fabrication with applications in proteomics.

Recently, there has been observed growing interest in nanomaterials, especially nanoparticles, which have been widely incorporated to the biomedical field due to their unique properties associated with nanometric sizes. Superparamagnetic iron oxide nanoparticles (SPION) are especially used as Magnetic Resonance Imaging (MRI) contrast agents [1,2]. As every diagnostic technique MRI has some advantages and disadvantages. The most important benefit is practically unlimited tissue penetrating depth. On the other hand, there is worse spatial resolution. Whereas, fluorescent microscopy is a complementary technique to MRI with rather limited depth of imaging, but high spatial resolution. Moreover, when there are two or more fluorescent probes with different spectra, simultaneous visualization of these modalities is possible [3,4]. Thus, the combination of these two methods in one nanoparticle gives more complete information about an imaged object and has been developed nowadays [5]. In this report we present the synthesis and physicochemical properties of novel SPION coated alternately with cationically and anionically modified chitosan that surface was modified by fluorescamine serving as a fluorescent probe. The obtained materials in the future could be used as multimodal imaging agents for biomedical applications. [1] A.K. Gupta, M. Gupta, Synthesis amd surface engineering of iron oxide nanoparticles for biomedical applications, Biomaterials 26 (2005), 3995-4021 [2] S. Laurent, D. Forge, M. Port, A. Roch, C. Robic, L. Vander Elst, R.N. Muller, Magnetic Iron Oxide Nanoparticles: Synthesis, Stabilization, Vectorization, Physicochemical Characterizations and Biological Applications, Chem. Rev. 108 (2008), 2064-2110 [3] Z. Liu, F. Kiessling, J. Gatjens, Advanced Nanomaterials in Multimodal Imaging: Design, Functionalization, and Biomedical Applications, J Nanomater 2010, art. no. 894303 [4] N. Chekina, D. Horak, P. Jendelova, M. Trchova, M.J. Benes, M. Hruby, V. Herynek, K. Turnovcova, E. Sykova, Fluorescent magnetic nanoparticles for biomedical applications, J. Mater. Chem. 21 (2011), 7630-7639 [5] L.E. Jennings, N.J. Long, ‘Two is better than one&’ - probes for dual-modality molecular imaging, Chem. Commun. 24 (2009), 3511-3524 Acknowledgements: This work was supported by the European Union from the resources of the European Regional Development Fund under the Innovative Economy Programme (grant coordinated by JCET-UJ, No POIG.01.01.02-00-069/09).

Carbon black nanoparticles (CBNPs) with adsorbed poly aromatic hydrocarbons (PAHs) are known carcinogens and thus pose severe health risks to humans. We adopt an engineering strategy to mitigate the risks posed by CBNPs by utilizing atomic layer deposited (ALD) Al₂O₃ thin films to coat the CBNPs. It is expected that these ‘nano-cloaks&’ of Al₂O₃ would act as a diffusion barrier layer and prevent interaction of PAHs with the immediate biological environment. ALD on CBNPs of diameter 8-10nm were made in a home-build rotary ALD system using alternate exposures of Al(CH₃)₃ (trimethylaluminum (TMA)) and H₂O at 150 °C. A wide set of characterization tests were then conducted on sets of CBNPs with and without ALD Al₂O₃. Raman spectroscopy on uncoated CBNPs showed an intense ‘D&’ band (1325 cm^-1), indicative of a high concentration of surface defects on CBNPs. Fortunately, these defects act as easy nucleation sites for the initiation of ALD deposition. The ‘D&’ band is quickly quenched with increasing ALD Al₂O₃ cycles on CBNPs and for a 30 cycle ALD Al₂O₃ film, no evidence of the ‘D&’ band remains. Likewise, TEM images revealed 3-4nm conformal films of Al₂O₃ around CBNPs and SEM contrast images showed evidence of uniform charging due to Al₂O₃ coating on otherwise highly conducting CBNPs. Evidence of particle agglomeration to sizes of 100-300nm after ALD Al₂O₃ is confirmed using SEM and DLS data. Thermogravimetric analysis (TGA) showed the Al₂O₃ films were thermally stable till around 350 °C at which point the stresses due to thermal mismatch between the CBNPs and Al₂O₃ is high enough to cause rupture of the films and loss of mass due to release of adsorbates. Leaching experiments on ALD Al₂O₃ coated, PAH-loaded CBNPs indicate the effectiveness of Al₂O₃ as a barrier layer to prevent release of carcinogenic agents.

A novel hydrogel-based sensor, developed using a biocompatible, hollow cylindrical ferromagnetic contrast agent, enabling a magnetic resonance imaging (MRI) sensing capability, is proposed in this paper. Unlike the existing bottom-up, chemically synthesized agents, these hollow cylindrical agents, fabricated using top-down MEMS technology, are geometrically distinct, yielding a local magnetic field modification which results in distinct spectral resonance properties. The ability to sense the local in-vivo microenvironment based on environmentally responsive hydrogels could enable true functional imaging capability using MRI. Compared to previous works on Ni-based cylindrical-nanoshells and Fe-based double-disk particles, biocompatibility issues were strongly considered in this development of a simplified MEMS fabrication process incorporating an environmentally responsive hydrogel. COMSOL Multiphysics Solver was used to simulate the magnetic field distribution within and surrounding two hollow magnetic cylinders. The simulation demonstrates a homogeneous magnetic field distribution within the cylinders, which is distinctive from the surrounding field. The fabrication methodology is as follows: (a) Photolithography on the photoresist coated wafer to form a micropost array pattern; (b) iron oxide thin film deposition by RF reactive sputtering; (c) ion milling to remove the up-facing iron oxide layer and redeposition of iron oxide on the post sidewall; (d) oxygen plasma ashing to remove all photoresist. (e) polymerization of the coated hydrogel. Utilizing this process, biocompatible cylinder arrays with 200nm in thickness, 5um in diameter, 7um in height were successfully fabricated. The specific hydrogel incorporated into the fabrication process may be responsive to a variety of environmental stimuli, such as temperature, pH, or the presence of specific enzymes, among others. In the case of protease sensing hydrogels, these may be tuned to respond to the presence of a specific enzyme based on the peptide sequence which is incorporated into the hydrogel. The peptide sequence chosen is derived from an active segment of the natural substrate of the enzyme of interest, the presence of which results in the degradation of the hydrogel. The protease responsive hydrogels will serve to eliminate the diffusion of water through the internal volumes of the cylinders. In the presence of the targeted enzyme, the hydrogel will eventually degrade and allow for diffusion of water, thereby turning the MRI signal inducing properties of the particles “on”, enabling an in vivo sensing capacity for the presence and possibly concentration of the target enzyme. The fabricated MRI sensor presented, to the best of our knowledge, is the first mcirofabricated, biocompatible hollow cylindrical agent enabling a sensing capability. The further development of this new class of MRI sensors will offer the potential for multiplexing as well as functional imaging capacity.

The purpose of this study is to synthesize mesoporous biobased calcium silicate (CaSiO3) through ecofriendly technique and to evaluate its possible applications as fillers for polymers and as biomaterials. Calcium silicate was synthesized from calcium carbonate (obtained from egg shell) and anhydrous silica gel in a solid state reaction in a high energy ball mill. The nanostructure of the synthesized powder was characterized by FESEM/EDS and TEM analysis. XRD analyses were carried out to study the crystal structure. These results revealed that nano-sized polycrystalline calcium silicate particles were formed during 10 hours of ball milling with particle sizes ranging from 20-100nm.Thermal and mechanical properties of thin films and electro-spun fibers made from composites of PMMA reinforced with Calcium silicate (1%, 2% and 5%) were tested. These results showed an improvement in tensile and thermal properties as compared to the neat PMMA films and fibers. Human osteoblast cell interaction studies with the bio based calcium silicate showed that this material is biocompatible and promotes bone cell proliferation.

Microfluidic technology is appealing because of its ability to handle small fluidic volumes making it ideal for high-throughput applications. Flow focusing devices have the ability to form droplets with well defined size distribution while utilizing suitable microfabrication techniques. Controlling the wetting of the two immiscible phases with the microchannels surface is important for the control of the generated particles. Here we use silk fibroin as an appealing biopolymer to fabricate microparticles because of its mechanical integrity, optical clarity, and biocompatiblity which can maintain the biological activity of embedded antibodies and enzymes. Using flow focusing devices to fabricate micro and submicron sized particles in silk can find use for advanced materials, high technology, biomedical devices, and biological sensors. The high-throughput and high yield of these devices make it a promising method to fabricate monodisperese spheres for multiple biological and chemical applications. The flow focusing devices were fabricated on a silicon wafer using photolithography with 30mu;m thick SU-8 resist which determines the channel height. The silicon wafer was used to pattern liquid polydimethylsiloxane (PDMS) which was cured creating a negative mold. The PDMS negative was oxidized in a plasma chamber and bonded to a glass surface. The hydrophobic nature of PDMS is lost through oxygen plasma treatment during the bonding process making the surface hydrophilic. Surface modification such as silanization of the PDMS makes the surface hydrophobic and changes the performance of the microfluidic device. Polyvinyl alcohol (PVA) (5%) was the continuous phase with varying concentrations of aqueous silk solution as the discrete phase. Scanning electron microscope (SEM) images and dynamic light scattering analysis (DLS) were used to measure the size of micro and nanoparticles. Different contact angles of PDMS were explored to offer better control of microsphere size and dispersity. Experimental evidence suggests that hydrophilic PDMS generates smaller particles (100s of nanometers) which are more polydispersed while hydrophobic PDMS makes large particles that are monodispersed (approximately 1-5mu;m). Different separation techniques can be used to provide a monodisperse selection of different diameter nano and microspheres made of silk fibroin. We have examined the wettability of PDMS flow focusing devices to control the size and dispersity of silk fibroin particles. Due to silk&’s ability to act as a biologically favorable carrier that enables bio-dopants to maintain their functionalities, this device can be used to produce functionalized, controllable monodisperse nano and microparticles for biological applications. By varying the wettability of PDMS, reproducible, high yield silk spheres can be fabricated using extremely small fluidic volumes.

Silk is now finding application as a useful biocompatible material, as an environmental friendly polymer and in photonic devices. As a biomaterial silk is being used for tissue engineering and body implantation because of a confluence of favorable properties such as biocompatibility, biodegradation, non-toxicity. Micro and nanospheres made with silk provided new options for drug delivery due to their biocompatibility, biodegradability and their tunable drug loading and release properties. We present improved results on nanoparticles&’ synthesis and modification and new results in their use for nano optics and nano medicine. All the chemical processes are inspired by green chemistry guidelines, typical of the water-based processing that silk biomaterials are known for. Using a microfluidic device we were able to achieve a better control on the nanoparticles&’ size, in term of size dispersion, and we were able to synthesize smaller particles respect to the actual published methods. This can be very useful whenever the control over the particles&’ degradation kinetic is necessary and also for application in optics, since many optical properties are size dependent. To extend the field of applicability we introduced new functions in the silk by chemical modification of the nanoparticles. We were able to introduce carboxyl, ammine and thiol functions. Those functions can be used as a dangling bond on which other molecules can be grafted, increasing the versatility of the nanoparticles as a carrier or as a probe. In our study we bound small gold nanoparticles on the silk nanoparticles&’ surface. These small gold particles represents the first step for the fabrication of a gold coating on top of a silk core. We studied the syntesis of both gold nanoparticles-coated silk nanospheres and the gold-silk nanoshells. Gold decorated silk nanoparticles can be useful to exploit the combined properties of gold nanoparticles with a silk drug carrier. We demonstrate also the possibility of tuning the optical properties of the gold decorated silk nanospheres by controlling the growth of the gold nanoparticles. These gold decorated nanospheres constitute the first step towards manufacturing of Au-silk nanoshells. Nanoshells are well studied nano objects known for their use for both therapy and diagnostic. Until now nanoshells were synthesized using a inert silica core. Since silk is a proven material for drug delivery, with the capability of a controllable drug elution time and protection of the drug activity, nanoshells and gold decorated silk nanoparticles made with an active core can couple the properties of gold nanoparticles and nanoshells with the potential of an active, biocompatible, biodegradable and bio active core extending the utility of these nanoscale devices. In our work we will present the latest results on the synthesis of gold-coated bioactive silk nanoparticles and their potential as optically activated drug delivery platforms.

Remote activation of ion channels in neurons using radio-frequency electromagnetic-field heating of magnetic nanoparticles (NPs) has a potential as a minimally invasive tool for analyzing complex networks and treating neurological disorders in vivo. To achieve neuronal signaling at biologically relevant time scales requires materials that can heat the cell membrane at a rate of ΔT>5°C/s. Recently, core-shell NPs composed of magnetically harder and softer oxides were demonstrated to achieve heating rates nearly 5 times greater than conventional single-material NPs. To synthesize exchange-coupled tertiary ferrite core-shell NPs (shell@core: YFe2O4@XFe2O4, X,Y=Mn,Co,Ni), we have developed a robust metal-oleate chemistry. The decomposition of metal-oleate precursors at high temperatures yielded monodisperse NPs that can be synthesized on a large scale. Using these NPs as seeds, we have synthesized uniform core-shell NPs with a standard size deviation < 5%. Energy-dispersive X-ray spectroscopy mapping on a Scanning Transmission Electron Microscope confirmed the NP&’s core-shell geometry, with a 2 nm shell consisting of ZFe2O4 (Z=Co or Ni) coating a 10 nm MnFe2O4 core or a MnFe2O4 shell coating a ZFe2O4 core. Magnetic exchange interaction between ZFe2O4 and MnFe2O4 of the core-shell NPs was investigated using specific loss power (SLP) and SQUID hysteresis curve measurements. We found that our core-shell NPs possess an SLPs nearly two times greater than NPs without an additional coating. To target these NPs to the neuronal membranes, we have directly conjugated them to streptavidin. Proteins of interest on the cell membrane can then be biotinylated and directly linked to the streptavidin functionalized NP. The cellular response to the remote actuation of ion channels through radiofrequency heating of the core-shell NPs is currently being characterized.

Fluorescent nano-sized carbon dots (Cdots) are an emerging bioimaging agent with excellent chemical inertness and marginal cytotoxicity in comparison to widely used semiconductor quantum dots. In this work, we report the application of Cdots to real time bioimaging for target specific delivery of hyaluronic acid (HA) derivatives. Polyethylene glycol (PEG) diamine capped Cdots were synthesized by the pyrolysis of citric acid in a hot solvent. The synthesized Cdots showed strong fluorescence under UV excitation with emission properties dependending on the excitation wavelength. HA-Cdot conjugates were synthesized by amide bond formation between amine groups of Cdot and carboxylic groups of HA. After confirmation of the negligible cytotoxicity of Cdots and HA-Cdot conjugates, in vitro bioimaging was carried out for target specific intracellular delivery of HA-Cdot conjugates by HA receptor mediated endocytosis. Furthermore, in vivo real time bioimaging of Cdots and HA-Cdot conjugates exhibited the target specific delivery of HA-Cdot conjugates to the liver with abundent HA receptors. Taken together, we could confirm the feasibility of HA derivatives as a target specific drug delivery carrier and Cdots as a promising bioimaging agent.

Gold nanomaterials (GNMs) are of special interest due to the distinctive advantages of tailorable surface plasmonics and photon-to-heat conversion as well as enlarged surface area. GNMs also enable abundant surface chemistry through surface modification by thiolized organic/inorganic/biological molecules. Recently, GNMs with different morphologies and surface chemistry have been used in a number of research areas from fundamental researches to biological applications. In this study, porous GNMs were prepared by templated electrochemical routes using porous alumina membranes. Alternative deposition for gold and gold-silver alloy was repeatedly performed in a pulsed chronopotentiometric mode, and subsequent silver etching of the resulting barcode-type nanomaterials in nitric acid simply enables enough amounts of porous gold nanomaterials. Depending on electrochemical deposition time for gold-silver alloy segment, we could easily obtain porous gold nanodiscs, nanoparticles, and nanorods, i.e., different aspect ratios of porous GNMs, after the silver component etching completely. The resulting porous GNM particles were examined for controlled drug release under light illumination. Hot electrons excited by light give local heating at the nanopores of the GNMs, which increases degree of freedom of encapsulated molecules therein. Hence encapsulated drug molecules within nanopores could be selectively released when light was illuminated. Here we compared controlled release behavior of the GNMs for doxorubicin with respect to different GNM aspect ratios and surface chemistry. Since a doxorubicin molecule has five hydroxyl functional groups, several surface coupling agents were used to understand releasing behavior depending on the number of hydroxyl groups of the coupling agents. The results indicate that the unique porous GNMs with optimized drug releasing property would be useful as carriers for drug delivery applications.

Biological functionalization of inorganic nanoparticles is of great interest for biomedical applications. One current challenge in nanomedicine is the lack of a method for the directional attachment of biological molecules to nanoparticle surfaces without damaging structural and functional properties of the biological molecule. We have engineered a novel mechanism for conjugation of peptides and proteins to iron oxide nanoparticle surfaces using red and green fluorescent proteins as a model system. This novel conjugation mechanism provides advances over current technologies in two ways. First, proteins can be specifically and directionally attached to the surfaces of nanoparticles, allowing for improved medical functionalization. Second, nanoparticle attachment can be performed under physiological conditions, minimizing structural damage to the conjugated proteins and, thus, enhancing biological functionality. In the present study, we present the expression and purification of several proteins functionalized for nanoparticle attachment and show the specificity of nanoparticle attachment provided by our technology. Future studies will apply this conjugation method to expand the use of inorganic nanoparticles as a platform for biomedical applications.

Methotrexate (MTX), is a potent immunomodulating drug, is widely used in the treatment of malignancies including childhood acute lymphocytic leukemia, osteosarcoma, non-Hodgkin&’s lymphoma, Hodgkin&’s disease, head and neck cancer, lung cancer, breast cancer, psoriasis, choriocarcinoma and related trophoblastic tumors1. Despite its efficacy, the use of MTX is greatly limited due to its toxicity, have been reported such as toxicity to normal cells, drug resistance, nephrotoxicity, bone marrow suppression, acute and chronic hepatotoxicity, interstitial pneumonitis and chronic interstitial obstructive pulmonary disease2. To solve these problems, various delivery vehicles for MTX for antitumor therapy were developed. In this study, we prepared MTX-incorporated in Tetraethyl orthosilicate (TEOS) nanoparticles using the sol-gel process3, which has been considered as one approach to overcome these limitations. These particles were evaluated in relation to size using dynamic light scattering (DLS), zeta potential, drug encapsulation efficiency, release profile, and external morphology using scanning electron (SEM) and spectroscopic properties. Cell toxicity was evaluated using a cell viability measurement (MTT). The MTX was loaded into TEOS nanoparticles, with an entrapment efficiency of 72 ± 4%. SEM and DLS revealed that the particles are spherical in shape, have a diameter between 180 and 260 nm, and show low tendency to aggregate, confirmed by zeta potential of 16.7 ± 3.5 mV. The release of the complexes from nanoparticles shows that MTX-NP showed a biphasic release pattern, which was characterized by an initial complex burst during the first 24 h, followed by a slower release phase complex profile, due to a few pores observed in surface of nanoparticles Cell toxicity showed that TEOS nanoparticles without MTX are non toxic for cells in study. These results suggests that TEOS nanoparticles can be used as a potential drug delivery to MTX in cancer treatment. References: 1Seo, D-H. Colloid Surface B. 69 (2009) 157-163 2Singka, G.S.L. Eur. J. Pharm Biopharm. 76 (2010) 275-281 3Tang, W. Photochem. Photobiol. 2005, 81, 242. Acknowledgements: This work was supported by CNPq, CAPES, FAPDF and FINATEC

Metal nanomaterials (NMs) have been used in a wide range of commercially available consumer products ranging from cosmetics to household detergents. This is especially true for silver (Ag) NPs, which is known to have antimicrobial properties. Besides that, Ag NMs with different morphology have distinctive electronic and optical properties, making them ideal candidates for biological, medical and defense applications. Therefore, it is of great importance to evaluate the potential biological impact of Ag NMs with different shapes before employing them in applications. This study investigates Ag NMs with different morphologies on biological responses in the human skin fibroblast cells to model potential exposure to Ag NMs. Ag nanocube and nanorod was synthesized and then characterized using scanning electron microscope, dynamic light scattering, X-ray Diffraction and UV-Vis spectroscopy. Furthermore, the cellular responses were evaluated by cell viability, reactive oxygen species (ROS) generation, alternation in cell cycle. The cell uptake and translocation of Ag NMs was further confirmed by microscope and scanning electron microscope. In addition, we demonstrate that morphology appears to play a significant role in mediating the cellular response to Ag NMs.

The stability of the binding of various, complex ligands on gold nanoparticles is typically studied by first, preparing gold nanoparticles by a standard synthesis such as the reduction with sodium citrate and subsequently, introducing the desired ligands in an exchange reaction. This procedure has the disadvantage that some of the original ligands are still present on the nanoparticle surface and hence, influence the stability and other properties of the final system. In a novel approach, gold nanoparticles are formed by hydrolytic decomposition of a novel molecular precursor [NMe4][Au(CF3)2] without any stabilizing ligand [1]. In this case, the particles are simply stabilized by their surface charge. We use this approach for an in situ functionalization of gold nanoparticles with mono-, di-, and trivalent thiol ligands with a polyethylene glycol (PEG) end group. For this purpose, the literature syntheses of the mono- and divalent thiol ligands were improved [2] and a novel trivalent ligand was synthesized The gold nanoparticles synthesis is reproducible and rapidly done in the microwave. The obtained nearly monodisperse particles are compared with gold nanoparticles prepared by the standard citrate synthesis and subsequently, stabilized with the same three thiol ligands in a ligand exchange reaction. First, we demonstrate, by UV and TEM measurements that the gold nanoparticles functionalized by the in situ synthesis with the PEG thiol ligands are more stable under high salt concentrations than the classical cap exchanged gold nanoparticles. Further, we studied the influence the multivalency of the capping ligands. The nanoparticles stabilized by the di- and trivalent ligands were stable in high salt concentrations, up to 5 M in contrast to those with the monovalent thiol ligands. The digestion of the particles by NaCN was investigated as a function of the multivalency of the ligand, the particle size, and the synthesis process of the functionalized gold nanoparticles. In general, a higher number of anchoring groups per ligand leads to a stronger colloidal stability. Also in this case the stability of the in situ functionalized nanoparticles is significantly higher than that of the cap-exchanged ones. [1] D. Zopes, S. Kremer, H. Scherer, L. Belkoura, I. Pantenburg, W. Tyrra and S. Mathur Eur. J. Inorg. Chem. 2011, 273-280. [2] B.C. Mei, E. Oh, K. Susumu, D. Farrell, T. J. Mountziaris and H. Mattoussi Langmuir 2009, 25 (18), 10604-10611.

We have utilized bio-inspired Moth eye nanostructures and further improved this bio-mimetic structure to enhance the medical imaging scintillator materials external quantum efficiency significantly. With the increasing interest in reducing the front surface reflection, intensive effort has been focusing on the development of surface structures and process methodologies to achieve broadband antireflection. Nano-structure comprising an array of circular protuberances (corneal nipples) on the facet lenses in Moth eye has been widely utilized to achieve this purpose. This bio-inspired nano structure is only one of many fascinating surface structures in nature. Instead of broadband antireflection properties, this structure can also be utilized to enhance the light extraction of the scintillator materials. Inorganic scintillators are widely used in modern medical imaging modalities as converter for the x-rays and γ-radiation that are used to obtain information about the interior of the body, like x-ray, CT and PET scan. Most of the high-density scintillators have a high refractive index, so when the light travels from inside the crystal to the air, the total reflection critical angle is small, most of light is in total reflection within the crystal and trapped in the crystal, only 10-30% of the light from the scintillator can enter into the photodetector, the majority of light couldn&’t be effectively extracted, which seriously affected the detection system's efficiency and detection sensitivity. Due to the results of our fabrication technique, we have further improved the bio inspired Moth eye structure for the scintillator materials light output enhancement. As a proof of concept, we have demonstrated very high light output efficiency enhancement for Lu2SiO5:Ce3+ (LSO:Ce) film in large area, the X-ray mammographic instrument was employed to demonstrate the light output enhancement of the Lu2SiO5:Ce thin film with bio-inspired Moth eye-like nano structures. Our work could be extended to other thin film scintillator materials and is promising to achieve lower patient dose, higher resolution image of human organs and even smaller scale medical imaging.

Engineered nanomaterials have shown a tremendous potential in various biomedical applications because of their unique size-dependent properties. Nanoconstructs based on gold nanoparticles are particularly attractive because of their biocompatibility, size dependent plasmonic properties, and ease of surface modifications. The high Z number for gold (Z=79) makes them an ideal candidate to enhance the therapeutic efficacy of the radiation therapy. The use of small size nanoparticles is critical for clearance and final distribution of the nanoparticles in vivo. The relative ease of surface modifications with biocompatible materials like polyethylene glycol imparts long circulation and higher degree of stability to the ultrasmall gold nanoparticles. Also, the feasibility of covalently linking fluorescent molecules to the gold nanoparticles enables optical tracking of the nanoparticles in in vitro and in vivo environments. Here we present the synthesis and characterization of fluorescent PEGylated gold nanoparticles of sub-10nm size as efficient radiosensitizing agents which can be tracked using inherent photoluminescence from the conjugated fluorophore. We have synthesized ultrasmall gold nanoparticles (UGNPs) with a core diameter of 2-4nm. These nanoparticles were further conjugated with different ratios of methoxy and amine PEG for imparting long circulation properties and functional groups on the nanoparticle surface. The amine groups on the nanoparticles surface were further conjugated to the fluorescent molecule, Alexa fluor 647 (AF647). The UGNP were characterized using dynamic light scattering, transmission electron microscopy, zeta potential measurements and photoluminescence studies. Long term stability studies carried out with the PEGylated nanoparticles showed no aggregation or further growth of the nanoparticles for more than 3 months. The cellular uptake was studied in prostate cancer cell line PC3 by fluorescence confocal microscopy and the results indicated a robust uptake of the nanoparticles in the cells. The quantitative estimation of the nanoparticles&’ uptake was performed with BCA assay. For biocompatibility studies, cell survival assay (MTS assay) carried out with UGNPs showed no toxicity over a range of 0-2.5mg/ml of nanoparticle concentrations. The radiation damage enhancement of these nanoparticles was confirmed using PC3 cells. The cells treated with UGNPs were irradiated with kilovoltage X-rays. γH2AX and MTS assays were used for quantitation of the DNA damage with and without nanoparticles as controls. The results indicated increased DNA damage in cells treated with gold nanoparticles. The results obtained from these studies will lay a foundation for the in vivo application of these nanoparticles in enhancing the radiation therapy along with a real time assessment of the therapeutic response and disease progression by optical imaging. Work supported by NSF-DGE- 0965843 and HHS/1U54CA151881 CORE1.

We describe efforts towards developing nanoparticle based platforms for localized delivery of therapeutics in conjunction with radiation therapy in prostate cancer. This new approach involves the fabrication of inert spacers doped with nanoparticles encapsulating anti cancer drugs and fluorophores. The use of nanoparticles in fabricating brachytherapy spacers provides a sustained release depot of drug delivering chemotherapeutic drugs at the target site locally, resulting in local radio-sensitization of the prostate with the use of lower radiation doses and thereby leading to less rectal toxicity. We have synthesized mesoporous silica based nanoparticles encapsulating radiosensitizing anti-cancer drug, Docetaxel (DTX) and conjugated with a near infra-red (NIR) fluorophore Cy 7.5 (for optical tracking of the nanoparticles) via the micro-emulsion method. Silica nanoparticles were characterized by dynamic light scattering (DLS), transmission electron microscopy (TEM), UV-Visible absorbance and photoluminescence studies. The nanoparticles encapsulating DTX showed a sustained release of the drug in buffer. In vitro cytotoxicity studies with PC3 cells showed the biocompatibility of the blank silica nanoparticles whereas an efficient therapeutic response with DTX encapsulated nanoparticles. Further, these nanoparticles were incorporated into a polymer matrix of PLGA (poly (lactide-co-glycolide)) and formulated into small cylinders of standard brachytherapy spacer&’s length. These polymeric spacers were characterized by SEM for the nanoparticles distribution within the polymeric matrix. The release kinetics studies were performed to estimate a sustained release of the drug from the nanoparticles entrapped in the polymer matrix. In vitro cytotoxicity assays with PC3 cell line showed an efficient therapeutic response with the drug released from the spacers. This approach provides localized in-situ delivery of the sensitizer to the tumor and avoids the toxicity associated with current systemic delivery of radiosensitizers. This combinatorial approach is expected to enhance the therapeutic ratio of radiation therapy without introducing additional patient interventions over current brachytherapy procedures and also aids in real time monitoring and assessment of the disease progression by optical imaging. Work supported by NSF-DGE- 0965843, HHS/1U54CA151881 CORE1 and ARMY/ W81XWH-12-1-0154.

The macroscopic properties of ligands such as the net charge, size and hydrophobicity have been considered as major factors determining the biological behavior of nanoparticles. We show that the microscopic arrangement of surface charges also strongly influences biocompatibility, clearance behaviors, and effective permeability. Newly synthesized betaine containing polyimidazole ligands (BPILs) allows us to prepare stable, bright, and compact zwitterionic QDs, and to analyze the behavior of ionic QDs versus non-ionic QDs both in vitro and in vivo. Surprisingly, even for neutral QDs, the level of nonspecific binding to cells and serum proteins ranges from ‘minimal&’ to ‘significant&’ depending on the level of exposed amines. Also, despite similar size and zeta potential, ionic QDs display significantly faster clearance and lower effective permeability than nonionic QDs.

In this study, various materials (such as polymers and paper towels) were coated with selenium nanoparticles using a novel quick precipitation method to introduce antibacterial properties. Selenium nanoparticles were synthesized through a simple reaction between glutathione and sodium selenite (4:1 molar mixture) and were precipitated on the surface of different substrates for 30s. SEM (Scanning Electron Microscope, HITACHI 2700) images of the substrate surfaces were taken to determine the distribution and coverage of selenium nanoparticles. An AFM (Atomic Force Microscope, MFP3D, Asylum Research, Sharp tipped cantilever, K = 0.06N/M, Contact Mode) was used to demonstrate that the coated selenium nanoparticles increased the surface selenium exposure and surface roughness. Bacterial assays with Staphylococcus aureus was implemented to test the effectiveness of these selenium coated materials at preventing biofilm formation. A bacteria solution was prepared at a concentration of 106 bacteria/ml. The sterilized samples were transferred to a 24-well plate, treated with the prepared bacterial solutions (106 bacteria/ml) and cultured for either 24, 48 or 72 hours in an incubator (37°, humidified, 5% CO2). After the treatment, the samples were rinsed with a PBS (phosphate buffered saline) solution twice and placed into 1.5ml microfuge tubes with 1ml of PBS. These tubes were shaken at 3000 rpm for 15 minutes on a vortex mixer to release the bacteria attached on the surface into solution. Solutions with bacteria were spread on agar plates and bacteria colonies were counted after 18 hours of incubation. The selenium coated samples showed a high effectiveness at killing bacteria and preventing bacteria from attaching. For example, on the surface of paper towel samples, bacteria colonization decreased significantly when the paper towels were treated with selenium. The selenium coatings inhibited the growth of bacteria (S. aureus) on the surfaces after 24, 48 and 72 hours by about 89% compared to the uncoated samples. Importantly, this was accomplished without using any antibiotics and establishes a paradigm shift in treating medical device infections without drugs that bacteria are developing a resistance to anyway.

Highly fluorescent two-photon active polymer nanoparticles were synthesized for high contrast in vivo fluorescent imaging of cancer cells. Strong two-photon-fluorescence was observed from these luminescent nanoparticles when excited using femtosecond laser pulses at 800 nm. The measured two-photon action cross-sections of the polymer nanoparticles are comparable to those of quantum dots reported. Interestingly, unlike the quantum dots, these nanoparticles were found to be non-toxic in cell viability studies. The dose and time dependent cytotoxicity effects were investigated. Two-photon microscopic imaging confirmed cellular uptake by Human acute monocytic leukemia cells. The deep penetration of the near-infrared laser pulses together with large two-photon absorption cross-sections demonstrated here renders the fluorescent polymer nanoparticles as ideal candidates for high contrast in vivo fluorescent imaging.

Considering their unique structure and properties compared with bulk material, nanoparticles exhibit different, and potentially hazardous, chemical and electrochemical properties when exposed to bioenvironment. While efforts in colloid and polymer science are mainly focused on phase diagrams and mechanisms of nanoparticle stability and synthesis, little fundamental work appears at the interface between soft-matter (colloid/polymer) and toxicology. Thus, little is known about molecular mechanisms of health hazards and cytotoxicity of nanoparticles. This presents a problem when new nanomaterials are introduced for biomedical applications. Since the nanomaterials exposed to bioenvironments may participate and even interfere with biochemical reactions; oxidation and oxidative stress are among the risks posed to the cellular and sub-cellular environment. These redox properties may not be directly measurable within a bioenvironment, but can be quantified through well-defined electrochemical reactions. In this work we have developed electrochemical measurement methods to measure and quantify redox properties of nanoparticle-ligand complexes. Electrochemical methods, when combined with complementary techniques, such as optical spectroscopy, offer unique approaches to investigate and understand structure-property-toxicity relationships. The traditional approach to study the redox chemistry of nanoparticles is to treat them as dispersed analyte, yet this method would lead to solution degradation due to the presence of electric field and disturbance of functional groups on nanoparticles. In order to avoid these drawbacks we have adopted controlled self-assembly technique to fabricate well-defined, solid-state thin-films of nanoparticle-ligand complexes. We have examined the changes in redox properties of a wide range of nanoparticles as a function of size, shape, material and ligands; it was observed that the redox properties of nanoparticle-ligand complexes strongly depend on physical and chemical properties of nanoparticles and ligands. We also investigated and quantified participation of ligands in redox reactions via spectroscopic-potentiostatic hybrid measurements.

The last decade has seen a surge in nanoparticle-based imaging agents due to their advantageous spectral properties, multivalent targeting, and opportunities for multimodal imaging and/or therapy. Despite these advantages, there is much concern regarding the toxicity profile of nanoparticles, which most often contain inorganic materials and are too large to be cleared through the renal system. Here we present a versatile targeted imaging and therapeutic delivery strategy that employs self-assembled fluorous nanoparticles. The fluorous nanoparticles are synthesized by emulsion of a solution containing 7 wt% perfluorodecalin, 3 wt% perfluorotripropylamine, 3 wt% of an 8.5 kDa polyoxyethylene-polyoxypropylene block copolymer, and 0.4 wt% soybean phospholipids in an aqueous buffer. The fluorous nanoparticles are ~450 nm in diameter and stable at room temperature over multiple weeks. Fluorophores and therapeutic agents can be sequestered to the center of the nanoparticles by tagging them with a perfluoroalkyl chain. Ligands, antigens, or peptides can be anchored to the outside of the fluorous nanoparticles for active targeting by covalently linking the small-molecule targeting agent to the hydrophilic end of a fluorophobic/hydrophilic chain. The simplicity of this self-assembly strategy should allow for facile access to differentially functionalized nanoparticles. Importantly, these particles are fluid and can be easily excreted as their small-molecule components, mitigating the toxicity concerns that surround most nanoparticles. Perfluorocarbon emulsions have previously been FDA approved for use as blood substitutes, providing additional support for the biocompatibility of the fluorous nanoparticles.

Hyperthermia is a cancer treatment modality in which the tumors are exposed to higher temperatures to kill cancer cells. Optimizing tumor&’s obliteration efficiency and regulating the ablation of adjacent healthy tissues is challenging. Rf induced ablation is non-selective and stimulates thermal necrosis in both healthy and malignant cells. These rf procedures can be cell-selective when combined with directly or systemically injected functionalized nanoparticles (NPs) as rf absorption enhancers. Specific absorption rate (SAR) is used as a dosimetric measure to quantify loss enhancement from NPs. However, the caloric contribution of the NPs&’ dispersion medium at MHz frequencies can&’t be ignored for precise SAR calculations. In this work, we have analyzed the rf losses due to interaction of the NPs in aqueous media with both electric (E_rf) and magnetic (H_rf) fields. Our hyperthermia setup comprises a high quality factor inductance-capacitance-resistance (LCR) resonator designed to generate E_rf and H_rf fields up to 100 kV/m and 50 kA/m, in 12-50 MHz frequency range at low input power. The capacitor is built with two copper plates isolated by a single crystal sapphire and the solenoid is made of six turns of copper tube wound into a coil with the sapphire placed inside. Sapphires are water-cooled to maintain a sink temperature of 20°C. The samples to be characterized are placed in a quartz tube mounted on sapphire. Measurements of temperature change vs. time were carried out for gold, silica and superparamagnetic iron oxide (SPIO) NP suspensions. Controls for the NPs samples were prepared by ultracentrifuging the NP suspensions with subsequent purification of supernatants using desalting columns. All components of rf losses in the samples including conservative and induced fields were analyzed. E_rf and H_rf originated SARs exclusively for NPs with controls taken into account were determined. Magnetic SAR for SPIO was calculated as 4 kW/kg and was normalized to field and frequency to get 3x10^-12 Wsm²/kgA². Electric SARs for SPIO, gold and silica NPs were calculated as 27 kW/kg,~10³ kW/kg and 2 kW/kg respectively. Our results showed that in MHz range of frequencies SAR numbers for SPIO and gold NPs are overestimated. Electric SAR of gold NPs is three orders smaller than reported in literature. The relaxation mechanisms for magnetic NPs heating in H_rf have been well established; however the E_rf loss can be explained by Ohmic/dielectric loss of SPIO&’s polymer matrix. Charge on any NPs forms ionic double-layers around them enhancing the E_rf loss from the surface polarization. Additionally, to analyze the effect of physiologically relevant environments, bovine serum albumin (BSA) with concentrations of 0.5% to 4% w/v were adsorbed on silica NPs. A synergistic heat enhancement was observed for BSA adsorbed silica NPs with respect to bare silica NPs and was calorimetrically analyzed to study the SAR enhancement of bare silica NPs.

Integration of nanoparticles in biotechnology is receiving a great deal of interest owing to the unique properties and vast applications that these offer superior to its bulk counterpart. Superparamagnetic/luminescent capped core/shell heterostructured nanoplatforms have been recently advised for cancer treatment; however the release of Cd2+ ions which is acutely cytotoxic and environmentally unfriendly hampers their clinical applications. In this work, non-toxic Fe3O4/ZnS:Mn nanoparticles are wisely proposed in order to surmount this hurdle and promote both in vivo and in vitro biomedical treatments and focus principally on photodynamic therapy. We herein used a simple aqueous chemical route to synthesize these nanoparticles and employed chitosan to make them biocompatible. This preliminary synthetic-part work has been successfully tested through electron microscopy, PL, UV-Vis Raman and EDS spectrometry, diffractometry and magnetometry. Moreover, the photochemical properties are also being studied as well as the generation of reactive singlet oxygen species and will be presented.

The potential of superparamagnetic iron oxide nanoparticles (SPIONs) in various biomedical applications, including magnetic resonance imaging (MRI), sensing, and drug delivery, requires that their surface be derivatized to be hydrophilic and biocompatible.¹ We report here the design and synthesis of a compact and water-soluble zwitterionic dopamine sulfonate (ZDS) ligand with strong binding affinity to SPIONs.² After ligand exchange, the ZDS coated SPIONs exhibit small hydrodynamic diameters (HD), and stability with respect to time, pH, and salinity. Furthermore, small ZDS coated SPIONs were found to have a low non-specific affinity towards serum proteins; streptavidin/dye functionalized SPIONs were bioactive and thus specifically targeted biotin receptors. References: [1] Insin, N.; Tracy, J. B.; Lee, H.; Zimmer, J. P.; Westervelt, R. M.; Bawendi, M. G. ACS Nano 2008, vol 2, page 197, [2] Wei, H.; Insin, N.; Lee, J.; Han, H. S.; Cordero, J. M.; Liu, W. H.; Bawendi, M. G. Nano Letters 2012, vol 12, page 22

Nanobiocomposites are believed to predict a bright future through the use of them in the orthopedic domain. In human bone structure, the interaction of hard hydroxy appatite crystals (HA) and elastic collagen fibers in the nanoscale structure of bone contribute to its unique mechanical properties. In this work, an attempt will be made to introduce a novel biodegradable nanobiocomposite by employing carbon nanotubes(CNT), HA nano particles and biodegradable polyurethane (PU) to mimic the structure of natural bone. This research suggests that the better osteoconductivity would be achieved by incorporation of HA nano particles. On the other side, high mechanical properties of CNT make it possible to fabricate biodegradable biocomposite with adequate mechanical strength. A typical example of bone tissue engineering strategy is the development of a biodegradable 3-dimensional (3-D) porous scaffold and seeding of osteogenic cells into it with appropriate in vitro culture. In this scenario, characteristics for the scaffold, cultured cells, and osteoconductive growth factors are critical to optimize bone regeneration. Experimental works herein have three steps: First, Synthesis of PU with suitable biodegradation rate. Second step would be functionalizing the surface of CNTs, and third step is developing and characterizing of the novel nanocomposite. The novel nanobiocomposite evaluated to characterize its morphological, mechanical, chemical and biological properties by using following methods: attenuated total reflection Fourier transformed infrared (ATR-FTIR) spectroscopy, NMR and scanning electron microscopy (SEM), wateruptake test, hydrophilicity test, and mechanical properties, Diffractional Scannig Calorimetric (DSC).

We have investigated the influence of ZnO and TiO2 nanoparticles (NPs) on the infectivity of J774.A1 macrophages by Leishmania tropica (L. tropica). We found that compared to control, the number of macrophages infected was 50.0% , 15.0%, and 35.0% higher after exposure to 0.01 mg/ml of ZnO, TiO2 anatase, or TiO2 rutile NPs 24 hours prior to infection, respectively. TEM cross sectional microscopy indicated that the Leishmania parasites and nanoparticles were sequestered in the same parasitophorous vacuole. Exposure to UV light reduced the number of ingested Leishmania by 8.0% in the control group, but by more than 35.0% in the experiments with exposure to nanoparticles. This enhancement, we believe, is due to the proximity, which makes the nanoparticle toxicity to Leishmania more effective. Hence, even though infectivity is increased under the influence of nanoparticles, the toxicity of nanoparticles to the parasite is greater, especially under the influence of UV light. Consequently, these nanoparticles may provide an inexpensive alternative treatment for leishmaniasis that mainly affects humans residing in developing countries.

Effective cellular uptake, controllable release, and safe excretion from the biological system after drug functioning are critical features of nanoparticle (NP) based nanomedicine. In particular, the controllable release and the safe excretion from biological system are critical for drug&’s potential clinical translation. In the present study, we demonstrate simultaneously achieving controlled drug release and carrier decomposition in SiO2-drug composite NPs. Escape of the drug molecules from the nanoparticles triggers the SiO2 carrier decomposition, which starts from the NP center and eventually leads to its complete fragmentation. The small size of the final carrier fragments is critical in enabling their easy excretion from renal systems. As SiO2 itself has long been considered as one of the promising carrier systems (chemical stability in physiological environment and natural hydrophilic surface for efficient cellular uptake), the simultaneously achieved controllable drug releasibility and self decomposability make it an ideal nanocarrier system for various diagnostic and therapeutic applications. This worked is supported by a grant from GRF of HKSAR (project No. 414710).

Polyvalent interactions are characterized by the simultaneous binding of multiple ligands on one biological entity to multiple receptors on another. Comparing to corresponding monovalent interactions, polyvalent interactions often have cooperative effect, resulting in a significant enhancement of binding affinity and specificity. Better performance of polyvalent interactions strongly relies on precise control of relative spatial position of multiple ligands. In the past decades, DNA has been demonstrated as an ideal material to fabricate nearly arbitrary one-, two- to three-dimensional nanostructures. Besides, the well-established modification and accurate addressability of DNA make these nanostructures ideal scaffolds to hold ligands in position and show polyvalent effects. More recently, successes on DNA nanomachines and dynamic DNA nanotechnology provide a promising method to study dynamic behavior at nanometer scale. Here we report a DNA machine which can tune the spatial distance between two functional domains, and reversibly regulate their target binding affinity. This strategy provides a nanoscale platform for studying polyvalent systems and will also benefit the understanding of the mechanism of the polyvalent interactions.

MPA capped CdTe Quantum dots (QDs) were synthesized by Organometallic route in aqueous solution. The QDs thus obtained were encapsulated with glycolipid by the EDC (1-ethyl-3(3-dimethylaminopropyl) carbodiimide hydrochloride ) sulfo-NHS (N-Hydroxysulfo-succinimide) coupling reaction. We used microbially synthesized lipid (Sophrolipid, SL), which itself has various properties like anticancerous and antimicrobial for the encapsulation of CdTe in order to make them biocompatible. Various characterization techniques such as UV-visible-NIR, Photoluminescence (PL), X-Ray Diffraction (XRD), Zeta potential, Dynamic light Scattering (DLS), Fourier Transform Infrared Spectrocopy (FTIR), Confocal microscope, High Resolution Transmission electron microscope (HRTEM), Scanning electron Microscope (SEM), Energy dispersive X-Ray spectroscope (EDAX) analysis were employed to validate the various properties of the material. FTIR shows that encapsulation of CdTe by sophorolipid is based on bond formation between carboxylic group of thiol capped QDs with Carboxylic group of sophorolipid via EDA linker. Photoluminescence of CdTe Quantum dots and SL coated QDs are similar but with a small blue shift. EDAX analysis confirms the presence of lipid on the surface of Quantum dots. Cytotoxicity testing was done by MTT assay to check the biocompatibility of the particles on two different cell lines- NIH3T3 (mouse fibroblast cell line) and MCF7 (Breast cancer cell line). The particles were studied under fluorescence microscope to image Mycobacterium smegmatis cells as fluoroscent nanoparticle dye. Cell imaging was also done to study the fate of the particles inside the cells. Zeta Potential of the SL-coated QDs shows good dispersibility and stability which is required for the biological applications. Confocal microscopy shows the internalization of lipid coated QDs in the cell line suggesting that the particles have good potential in bioimaging and disease diagnosis applications. Cytotoxicity testing of SL coated QD with NIH3T3 and MCF 7 show a far better viability as compared to the cases of a) the QDs and b) a physical mixture of QDs and SL without the EDA linker. These results will be presented and discussed.

The immobilisation of vesicles and liposomes via recognition units such as complementary DNA strands, electrostatic interactions and protein-ligand pairs has attracted increasing attention in recent years. By using these recognition units, it was possible to attach liposomes and vesicles to a variety of substrates, to prepare microarrays of liposomes, to construct sensing platforms and to investigate reactions in immobilized liposomes, including single molecules reactions. Coiled-coil motifs are abundant in proteins where they exhibit an array of functions like gene regulation, cell signalling, transport of small molecules and membrane fusion. Native SNARE proteins control fusion processes between and within cells (e.g. exocytosis). The common feature of all these coiled-coils is that at least two α-helical peptide strands bind, thereby acting as molecular Velcro. The specific molecular recognition between helices has enabled scientists to develop self-assembled, highly structured materials based on the coiled-coil motif. Here we describe a completely new function for the coiled-coil peptide binding units - their application in material science and surface modification. In this communication we report that an α-helical coiled-coil pair shown to exclusively form parallel heterodimers, denoted “peptide E” (EIAALEK)3 and “peptide K” (KIAALKE)3, act as selective recognition units through which liposomes and vesicles can be selectively immobilized in surface patterns obtained using microcontact printing. In summary we described a novel system for the immobilization of liposomes and the formation of supported bilayers by using a coiled-coil binding motif which was printed using microcontact printing-induced click chemistry. It was possible to obtain well defined structures of liposomes and supported bilayers. This technique shows great potential to study fluidity and recognition processes of supported lipid bilayers. Further experiments will be conducted in the future to investigate orthogonality of the coiled-coil binding motifs as well as the production of lipid arrays consisting of different lipid compositions

We introduce self-assembled, anti-fouling polyelectrolyte complexes, or polyplexes, based on reducible dextran-siRNA conjugates for in vivo tumor-targeted siRNA delivery. Dextran has been widely used as anti-thrombotic agents, blood plasma substituents, and drug adjuvants because of its biocompatibility. In this work, dextran is used to prevent non-specific protein adsorption and stabilize colloidal polyplexes, contributing to the stabilization and intracellular delivery of siRNA. The dextran-siRNA conjugates are synthesized by grafting thiol-terminated siRNA to thiolated dextran via disulfide bonding and electrostatically complexed with linear polyethylenimine to produce stable polyplexes. The polyplexes show excellent colloidal stability in the presence of serum proteins due to the dextran exposed to the complex surface. The enzymatic degradation of siRNA is also effectively suppressed within the polyplexes. The intracellular translocation and targeted gene silencing of the dextran-siRNA conjugate polyplexes are very efficient compared to those of the naked siRNA polyplexes especially in serum-containing media. Folates are introduced as an active tumor-targeting moiety for the polyplexes by conjugating them to the hydroxyl groups of dextran. The folate-decorated polyplexes are very efficiently targeted to cancer cells in vivo as demonstrated using an ovarian cancer xenograft mouse model. These results suggest that the dextran-siRNA conjugate polyplexes are a promising nanocarrier to stabilize and deliver therapeutic siRNA to cancer cells in clinical applications.

The highly specific functions of DNA can be employed for designing novel functional materials. However, aqueous solubility and biochemical instability of DNA impede its direct utilization as a functional component. Herein, preparation of a hybrid material encapsulating the DNA molecules (double-stranded, salmon sperm, 50-5000 base pairs) in robust host,sol-gel derived silica, has been described. The encapsulation was carried out in two-steps: hydrolysis of an acidic tetraethylorthosilicate [Si(OC2H5)4] sol, was followed by condensation near physiological pH upon addition of alkaline DNA-containing solutions. The gelation behavior and structural properties of the DNA-silica hybrids were investigated by 29Si NMR and by nitrogen adsorption. The selective adsorption of an DNA-interactive reagent molecule (ethidium bromide) in their diluted aqueous solutions on DNA-silica hybrids confirmed that the DNA molecules remained entrapped within the silica host without any deterioration. DNA encapsulation mechanism correlating the silica microstructure and DNA holding efficiency has been proposed.

Carbon nanotubes (CNT), with an annual world production reaching several hundreds of tons, represent a special category of nanomaterials with exceptional characteristics. In particular, they are currently investigated for different biomedical applications, ranging from medical imaging (MRI, Raman) to targeted drug delivery and tissue engineering. Double-walled CNT [1], at the frontier between SWNT and MWNT, are very promising for applications in the biomedical field. Although the toxicity and the environmental impact of CNT have now both been investigated by many different groups (although the latter never focused much attention until very recently), there is yet a controversy about the results and still no answer to the simple question: "are CNT toxic?" The fact is that the large range of kinds of CNT and methods to produce and then (in most cases) process them make any comparison almost impossible. The results presented here were obtained with the same batch of CCVD-produced DWNT [1] investigated for biomedical applications, and concern both the investigation of their potential impact on human health (in vitro / in vivo models) [2, 3] and the environment (in vivo models) [4]. They lead to the conclusion that all the experimental parameters (dealing both with CNT and biological models used) play a very important role and can easily explain the large differences between the results obtained by the different researchers. References: [1] E. Flahaut et al., Chem. Comm. (2003) 1442. [2] E. Flahaut, M.C. Durrieu, *, Ch. Baquey, Carbon, 44, (6), (2006), 1093-1099 [3] J. C. Debouzy, D. Crouzier, E. Flahaut, Env. Toxicol. Pharmacology, 30, (2), (2010), 147-152 [4] E. Flahaut, Nanomedicine, 5, (6), (2010), 949-590

We have investigated the effects of electrical fields on the growth of primary hippocampal cells and human embryonic stem cells, and measured their activities using carbon nanotube electrode array. The cell growth was nearly not affected by applying ac electric fields, whereas the cell viability was significantly reduced with dc electric fields. However, when both ac and dc electric fields were applied, the cell viability was not reduced and the neural network was elongated along the dc electrical fields. Using MEA, the propagation of the extracellular field potentials were measured parallel and perpendicular to the direction of applied dc field. Different time dependence was observed and its possible origins are discussed.

Alginate hydrogels are excellent candidates for constructing in vitro models of muscle contractility because they have close structural and mechanical similarity to the extracellular matrix (ECM) microenvironment. However, they are generally not amenable to protein adhesion and patterning. Hence, we sought to (i) develop strategies to couple ECM proteins to soft alginate hydrogel surfaces to make them suitable for cell culture and (ii) create chemical and structural heterogeneities for anisotropic tissue formation. Alginate films were prepared on aminosilane-activated glass coverslips by applying a CaCl₂ loaded agar stamp on top of an aqueous alginate droplet. Streptavidin was then covalently attached to alginate using carbodiimide chemistry. Biotinylated ECM proteins, fibronectin and laminin, could then be directly attached to these surfaces to make them suitable for cardiac and vascular smooth muscle cell culture respectively. Two schemes were employed to engineer anisotropy in subsequent tissues: Microcontact printing of extracellular matrix proteins to guide cellular processes with chemical cues, and Micromolding of alginate surface to guide cellular processes with topographical cues. Neonatal rat ventricular myocytes as well as human umbilical artery vascular smooth muscle cells successfully attach to both these micropatterned substrates leading to subsequent formation of anisotropic cardiac and vascular smooth muscle tissues. Furthermore, muscular thin film cantilevers cut from micropatterned cardiac substrates exhibited synchronous and anisotropic contraction and are being employed for functional characterization of engineered cardiac tissue. Both tissue alignment and contractile stresses were quantitatively evaluated and were found to be similar to cardiac muscular thin films constructed on PDMS substrate. Efforts are underway to collect functional readout from vascular smooth muscle assays constructed on alginate surface. The novel protocols of micropatterning native non-fouling alginate hydrogels and subsequent anisotropic tissue formation will enable in vitro assays for cardiovascular contractility to be constructed on biomimetic scaffolds.

Bone cancer is among one of the most deadly diseases where the patients are often required to receive treatment with bone substitute materials for natural bones that had to be removed. These bone substitute materials and implants need to meet the criteria of biocompatibility for cell adhesion and cell growth, and ideally biodegradability so that the natural tissue can gradually replace it. Additionally, a hierarchical structure and mechanical properties are also important for the artificial implants as they provide critical structural, mechanical and chemical cues. Of the many natural and synthetic polymeric materials that are suitable and currently used for the synthesis of bone tissue scaffolds, the polysaccharide chitin in its nanofibrillar form has in recent years attracted increasing research attention due to its outstanding biocompatibility and mechanical properties. In this contribution, we describe the manufacture of novel, highly porous tissue scaffolds from chitin nanofibers by a versatile cold processing technique called freeze-casting, which is the directional solidification of water-base solutions and slurries. Chitin nanofibers of 20-30 nm in diameter were prepared from crab and lobster shells by a series of chemical treatments involving HCl, NaOH, and ethanol to remove minerals, proteins, and pigments. The dimension of the nanofibers and the fiber morphology were found to depend on particular chemical processing parameters. FTIR analysis showed that the surface of chitin nanofibers are deacetylated (< 10%) into chitosan, which is of benefit as it aids dispersion of the chitin nanofibers in water-based solutions and allows the surface of the nanofibers to be chemically functionalized for drug release, for example. For freeze casting, the chitin nanofibers were dispersed in water, followed by a directionally frozen process, and finally freeze dried to remove the ice crystals that template the architecture of final scaffolds. The resulting material is a honeycomb-like scaffold with highly aligned and interconnected pores, whose pore size, cell wall porosity and surfaces properties can be carefully controlled and tailored for a given application. Mechanical testing in compression revealed at 99% porosity, mechanical properties are appropriate for low to medium load bearing applications. The scaffolds&’ modulus and strength were found not only to be affected by the amount of chitin in the solution, but also the nanofiber&’s degree of deacetylation, and diameter and length of the chitin nanofibers.

Using sacrificial microfiber networks formed from shellac, a material with pH-dependant solubility, we have formed 3D microfluidic networks throughout thick slabs of a gelatin hydrogel. While fabrication of 2D microfluidic structures in structural materials such as polydimethylsiloxane is straightforward, it is currently extremely challenging to form microfluidic structures in biomimetic materials such as hydrogels. Moreover, the 2D patterning techniques (e.g. photolithography) typically used do not readily scale to form complex 3D structures. There are, however, many applications for which the accuracy of photolithography can be forfeited in order to obtain scalability and compatibility with a wider range of materials. In particular, 3D microfluidic networks that mimic natural vascular systems have many promising applications, ranging from providing cells with nutrients in thick artificial tissue to supplying healing agents in self-healing polymer systems. The functionality of these networks will depend less on the exact placement of each channel, and more on the size, density, and interconnectivity of the channels. We have therefore used sacrificial microfiber networks produced using a common cotton candy machine to form complex microfluidic networks in 5% gelatin hydrogels crosslinked with microbial transglutaminase. These networks have large extent (on the order of cm) in all 3 dimensions. By using a sacrificial material with pH-dependent solubility, we are able to maintain an entirely aqueous process, wherein the dissolution of the sacrificial material is triggered by changing the pH of the surrounding environment. Larger structures of shellac deposited with a hot glue gun onto the microfiber network yield macrochannels that allow interfacing between the microfluidic network and external tubing. Fluorescent spheres were introduced into the 3D microchannel network, and multiphoton microscopy revealed the complex 3D channel architecture. The resulting 3D microscopy data was analyzed quantitatively using plugins in the FIJI ImageJ package to obtain a distribution of channel diameters and a distribution of interchannel spacing. These distributions (as analyzed for 5×10^9 total cubic voxels, each with edge length of 1.24µm) indicate that over 70% of the hydrogel volume is within 100µm of a channel wall, and that the average channel diameter is 17µm. These microfluidic materials allow soluble compounds to enter the material volume both by diffusion and advection, and the simple, inexpensive fabrication process used to form them is highly scalable.

Most tissues in organisms are composed of repeating cellular structures (i.e., functional units), such as the lobule in the liver and kidney, islets in the pancreas. In vivo, the cells in these functional units are imbedded in a three-dimensional (3D) microenvironment composed of extracellular matrix (ECM) and neighboring cells with defined spatial distribution. Tissue engineering approaches therefore attempt to recreate the native 3D architecture in vitro. Recently, the convergence of nano and microscale technologies and hydrogels has resulted in the emergence of bottom-up methods where cell-laden microgels can be used as building blocks for tissue engineering and regenerative medicine. Although various microgel fabrication and assembly methods have been developed based on modifying interfaces and using microfluidics, so far, two main challenges remain: (1) to fabricate microgels composed of multiple cell types spatially confined in 3D as functional units, and (2) to assemble microgels into large complex 3D constructs rapidly in an efficient way. Recently, we developed several technologies based on magnetics, acoustics and bioprinting to create 3D complex tissue constructs. We have shown a simple, multilayer photolithography method to fabricate co-culture microgels as tissue units (co-culture units) in a high throughput manner, with control over composition of cell types and their microscale spatial organization. We demonstrate the utility of this method by fabricating four different types of co-culture units, where the quality of the units was optimized by the photomask design. Also, we developed a magnetic assembler that utilizes nanoparticles and microscale hydrogels as building blocks to create 3D complex multi-layer constructs via external magnetic fields using different concentrations of magnetic nanoparticles. These methods enable a biologically relevant in vitro platform to investigate cell-cell interactions in a 3D microenvironment, holding great potential in various areas, spanning tissue engineering, regenerative medicine, pharmacological studies and high throughput applications.

The ongoing debate over the use of hydrogels based on naturally derived versus synthetic polymers in the context of tissue regeneration continues. One of the numerous claimed merits of the latter approach is the argument that many synthetic polymers act as protein-resistant blank-slate templates upon which instructive biological functionalities present within natural matrices (e.g., adhesion peptides, protease-sensitive cross-links, etc.) can be grafted. Since the interactions between cells and these materials can be more explicitly controlled through the judicious grafting of such cues, these functionalized synthetic hydrogels are often touted as ideal in vitro model systems in which to dissect the role of the extracellular matrix (ECM) on cell fate on 3D. One area in particular where we have attempted to exploit the features of synthetic hydrogels is in our efforts to dissect the impact of ECM mechanics on vascular morphogenesis in 3D. Unlike native biopolymer systems, in which substrate mechanical properties, adhesion ligand density, and proteolytic sensitivity are intimately linked, the mechanical properties of synthetic hydrogels can theoretically be tuned independently. However, in our experience, this is a classic case of where theory and practice disagree. Firstly, altering cross-link density to change mechanical properties in synthetic hydrogels typically restricts cell spreading and macromolecular diffusive transport. Secondly, synthetic hydrogels are typically amorphous, and thus lack the fibrillar architecture found in native ECM; these nano- and microscale architectural cues may also be instructive to cells. Thirdly, the mechanical properties of many systems may change significantly with time, due either to passive hydrolysis, cell-mediated proteolysis, or both. Finally, at least in our hands, the ability of synthetic materials to support complex, multicellular, morphogenetic processes (like angiogenesis) in 3D in vitro models is significantly inferior to that of naturally derived materials, despite some promise in vivo. Thus, interpreting “controlled” mechanics experiments in synthetic hydrogels may be no simpler than those in naturally derived materials. Nevertheless, given the relative strengths and weaknesses of both synthetic and naturally derived materials, our approach has been to embrace and carefully measure the complexity within naturally derived hydrogels in order to engineer the simplicity of synthetic ones. The development of methods and tools to quantify microscale mechanics and cell-generated deformations in naturally derived hydrogels in 3D, along with new insights provided by these methods and their impact on synthetic material design, will be discussed as examples of our approach.

The important practical target of regenerative medicine is the development of the non-destructive detection methods to understand the fate of cells and constructs during the fabrication, implantation and the repair process. Multi-photon microscopy (MPM), which is a non-linear optical imaging method can accomplish that with high contrast in 3D while having resolution comparable to the confocal imaging methods. MPM combines backscattered second harmonic generation (SHG) and two-photon fluorescence (TPF) signals suitable for obtaining structural and functional information at depth in thick living tissues. The fluorescent contrasts afford further opportunities to understand remodeling within biomaterials. We induced the mammalian embryonic G-Olig2 stem cells utilizing Olig2 to become neural lineage cells and followed their early differentiation and migration within 3D fibrillar-collagen systems non-destructively with the SHG and TPF signals. The fibrillar collagen is one of the widely used biomaterials for the nerve guide fabrication and our finding for an encapsulated as opposed to a topographic model will be described. For the encapsulated 3D model, the cells were embedded and differentiated within hydrogels prepared from two different initial concentrations: 2 g/l and 4 g/l. For the topographic model, the embryonic stem cells were differentiated on the hydrogels cross-linked with 0.1 M EDC, NHS mixed with EDC (1 part of 0.1 M NHS mixed with 4 parts of 0.1 M EDC) or genipin and compared to the cells differentiated on the uncross-linked counterparts. The rate of cellular differentiation was controlled by the micro-structure of the collagen materials, not simply by the chemical induction with a retinoic acid. For example, in the embedded models, embryonic stem cells differentiated faster in the 2 mg/ml collagen materials than in the 4 mg/ml materials. We additionally observed high heterogeneity in the final collagen alignment around the differentiated cells implying that the mechanical properties will be very heterogeneous as well. Our data and methods provide an outline to monitor non-destructively in situ the extent of differentiation for the stem cells while employing collagen-based substrates in tissue engineering applications.

Changing lifestyles and stress caused by urbanization are further contributing to the increased prevalence of heart failures. Given to the patient morbidity associated with current procedures such as open chest surgery and transplantation, it is desirable to develop minimally invasive technologies such as injectable therapeutics. Despite advantages such as safety and improved functioning, the dynamic myocardial microenvironment prevents peripherally or locally administered cells from homing of therapeutic cells owing to its highly sensitized nanoscale cues that it exerts. Lack of precise cytokine regulation and microenvironment support in scarred myocardium might be a plausible reason for colossal cell loss and ineffective homing of injected cells. Inspired by the natural myocardial microenvironment, we designed a cell recruiting, chemotactic nanofiber based solution by unique combination of two constituents that currently accepted at clinical levels: core-shell nanofiber composites of poly (L-lactic acid)-co -poly (epsilon-caprolactone)/collagen (PLACL/Col) containing chemokine SDF-1 in its core of magnitude less than 600nm. Guided by nanoscale mechanical cues provided by the under relying random nanofibrous scaffold, the tissue constructs displayed anisotropic re-arrangement of cells, characteristic of the native cardiac tissue. The cell morphology, growth and the expression of an interactive healthy cardiac cell population were exquisitely sensitive to differences in the composition of porous nanofiber biocomposite that features importance of the myocardial ECM. The hydrophilic core shell nanofibers fostered extensive cell-biomaterial interaction, active cellular communication (P<0.5), increased migration by 200% compared to control and differentiation of human adipose derived stem cells in myocardial milieu. Our investigations demonstrate the feasibly of chemokines application in form of chemotactic nanofiber based system along with therapeutic cells into infarcted hearts that may serve as an alternative to heart transplantation and aid myocardial regeneration.

Directing stem cell lineage requires exposure to various environmental cues and soluble mediators. We have managed to dictate stem cell lineage choice by altering polymer substrate chemistry and mechanics. Using the mouse C3H10T1/2-derivative, C9, that expresses hBMP-2 under control of the doxycycline (Dox)-repressible promoter, Tet-Off, we previously showed that growth on polybutadiene (PB) promoted the expression of chondrogenic markers (aggrecan and alcian blue staining) while growth on sulfonated polystyrene (SPS) promoted the expression of osteogenic markers (BSP and alizarin red staining). Here we explore the influence of mixed cues, such as those which occur when the substrate is a polymer blend of PB and SPS at ratios of 1:3, 1:1, and 3:1 spun cast on HFX-Si wafers and annealed for 24-hours at 170oC under a vacuum of 10-4 Torr. The non-transduced C3H10T1/2 cells were used as controls. AFM examination of the substrates showed that they had phase segregated into micron scale domains of PB and SPS, where the PB regions had a significantly lower modulus than those of the SPS. The cells were plated and incubated on replicate surfaces and grown in DMEM with 10% fetal bovine serum, 0.2 mM L-ascorbic acid 2-phosphate, 2 mM glutamine, and 10 mM b-glycerol phosphate in the presence and absence of 1 mg/ml doxycycline (Dox) which represses hBMP-2 expression in C9. Samples were examined at weekly intervals up to 21 days using confocal microscopy of cells stained with Alexafluor 488-linked phalloidin and propidium iodide, and by scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDAX). At all ratios of polymer blends, C9 and to a lesser extent C3H10T1/2 were found to biomineralize with and without DOX on the rigid SPS domains but not on the PB domains. Chondrogenic and osteogenic markers were evaluated by qRT-PCR as a function of surface on the different surfaces. The influence of the patterns, both chemical and mechanical, and the role of length scale and morphology on determining the differentiation pathway will be presented.

Stimuli responsive polymeric systems with tunable hydrophobicity properties are of interest for many biomedical and industrial applications. The goal of this project is to develop a 3-dimensional (3D) polymeric system that utilizes a hydrophobic photolabile protecting group (1-(2-nitrophenyl)ethyl (NPE)) to facilitate a 3D hydrophobic to hydrophilic transition upon exposure to UV light. This hydrophobic to hydrophilic transition at the molecular level translates into a dramatic increase in the wettability of the material in 3D. Three dimensional non-woven polymeric meshes were created using an electrospinning technique yielding 150-300 nm fibers with 3-7 µm beads layered into 300 µm thick meshes (determined using SEM). The apparent contact angles (ACA) of these meshes was measured by applying a 4 µL drop of deionized water onto the surface of the meshes and the rate of wetting was determined by recording the water droplet penetration into the mesh. These meshes had an apparent contact angle (ACA) of 135° before UV irradiation and an ACA of 0° after 60 minutes of UV exposure. The rate of wetting increased with increasing UV exposure time up until 60 minutes of UV exposure where the maximum wetting rates were observed due to complete NPE deprotection (confirmed by NMR). The size and the morphology of the nano-fibers and micro-beads in the meshes provided a dual scale roughness known to exaggerate the hydrophobicity or hydrophilicity of a material where the contact angle of smooth cast films of the polymer before UV irradiation was 113° and after 60 minutes of UV exposure was 108° (vs. 135° and 0° respectively when electrospun). This indicates that the observed ACAs of this material and the dynamic wetting properties are due to the roughness inherent in the electrospun meshes. Control over the 3D wettability of the UV-activated meshes was obtained by using a combination of UV exposure time to control the depth of water penetration and a photomask to control the shape of the wettable region within the mesh. A 1.59 mm in diameter circular photomask was used to selectively expose a small region of the meshes to UV light. The water penetration was measured by applying a 4 µL drop of 80 mgI/mL Visipaque CT contrast agent to the center of the UV exposed region and using a µCT imaging system to visualize the penetration of the liquid into the mesh. We found a linear relationship between UV exposure time and depth of water penetration. However, the hydrophobic to hydrophilic boundary was not strong enough to prevent water penetration out of the pre-defined channel leading to anisotropic wetting properties. Future work will focus on strengthening this boundary region in order to make these meshes useful for drug delivery, microfluidic device, and 3D cell culture applications.

Cellular constructs closely emulating in vivo tissue complexity are needed to improve predictions of clinical outcomes from in vitro models for applications ranging from drug development to disease pathogenesis. Microfluidic devices are better able to mimic the in vivo cellular microenvironment than conventional cell culture techniques by reproducing features such as tissue microarchitecture and flow-induced shear stress. However, microfluidic cell cultures are often conducted with cell lines, which are convenient and readily available, while the most authentic response is provided by well-differentiated primary cells obtained from the tissue of interest. Our aim is to develop a biomimetic co-culture of primary lung cells that emulates the morphology and physiology of the human conducting airways. A key component of the culture is the use of primary human bronchial epithelial cells. When cultured at the air-liquid interface, these cells become highly differentiated and turn into a pseudostratified, barrier-forming epithelium with authentic physiological functions such as beating cilia and mucus secretion. Microfluidic culture of sensitive primary cells presents challenges related to device material selection and perfusion requirements. Cell differentiation is highly dependent on the membrane support material and optimal for nanoporous polytetrafluoroethylene (PTFE) membranes (Millicel®). To achieve bonding of PTFE, we have developed a novel approach based on surface functionalization of polydimethylsiloxane (PDMS) elastomere. PDMS microfluidic devices with two independent flow compartments separated by a PTFE membrane were fabricated and adequate bonding strength for perfusion operation was demonstrated. Using selected tubing and constriction components, we have developed a microfluidic perfusion system optimized for long term culture (> 3 weeks) as required for bronchial epithelial cell differentiation (three to four weeks). The gravity-based perfusion system achieves a constant flow rate as low as 0.5 microliter/min and defined by a constant hydrostatic head pressure. This perfusion system requires no external power and enables autonomous operation over 60 hours as well as parallelization of the device cultures. Viability of lung epithelial cell cultures in the microfluidic device cultures is characterized by live/dead staining and compared to static controls. The devices are designed to include, in addition to the lung epithelial cells, a model of the interstitium, represented by collagen embedded fibroblasts, as well as endothelial cells representing the vasculature. This co-culture closely emulates the air-blood interface in the conducting airways providing a powerful research tool to more accurately predict the human response to respiratory challenges and therapeutics.

Fifteen million people are suffering from visual impairment due to corneal dysfunctions. Transplantation with donor corneas is the only possible treatment for a majority of cases. Unfortunately, there is a severe shortage of good quality donor tissue, such that the development of a corneal equivalent is urgently required. For the first time, a composite of gelatin fibres and alginate gel has been fabricated to mimic the microstructure and composition of corneal stroma, key for the transparency and toughness of natural cornea. Gelatin (hydrolysed collagen) was used to produce nano-fibres by an electrospinning process. These electrospun gelatin fibre mats were then infiltrated with alginate gel, resulting in a transparent material with a tensile modulus ranging from 0.5 - 65 MPa depending on the water content. This new material hence possesses similar mechanical and optical characteristics to native corneal stroma. Furthermore, it is biocompatible with cells since the production process does not involve any toxic chemicals. Therefore, this material could potentially be used as a scaffold to support corneal cells during the regeneration process, and could be used to restore vision when a donor cornea is unavailable.

Cells sense the mechanical stiffness of their environment to control cell shape, differentiation, survival, proliferation, and migration. How cells sense the Young&’s modulus of an elastic environment to make these vital decisions is not clear. One possible mechanism for this sensing is a “motor-clutch” mechanism, where myosin motors transmit forces to F-actin filaments, which in turn are linked to the substrate via plasma membrane-spanning cell adhesion molecules, which act as molecular “clutches.” When the molecular clutches are engaged, F-actin retrograde flow, driven by myosin-II motors, is resisted and force is effectively transmitted. Conversely, when clutches disengage the F-actin will flow at rates approaching the unloaded velocity of myosin-II and force transmission will be poor. We recently demonstrated that this simple model, which models the clutches as reversible bonds that fail exponentially in the force transmitted through the bond, exhibits stiffness sensitivity (Chan and Odde). In particular, the retrograde flow rate exhibits a biphasic response to substrate Young&’s modulus, an effect that we confirmed using embryonic chick forebrain neurons. We now further explore the behavior of the motor-clutch model, and assess which model parameters control the stiffness at which sensing is optimal. Simulations were performed as previously described (Chan and Odde, 2008) with minor modifications to ensure elastic equilibrium and limit each iteration to a single event via the Gillespie Stochastic Simulation Algorithm. Our simulations revealed that no single parameter in the motor-clutch model can strongly control the set-point for optimal stiffness sensing. Rather, parameters need to be changed coordinately to effectively change the set-point. In particular, coordinate increases of both motor and clutch numbers effectively increases the set-point stiffness. In conclusion, we found that the motor-clutch model can explain stiffness sensing across a broad range of Young&’s moduli.

Peptides were designed to intramolecularly fold into β-hairpins once they are exposed to physiological conditions and then consequently self-assemble into a rigid hydrogel with a network structure of branched and entangled, 3nm-wide fibrils. These physical hydrogels can be injected as preformed solids, because they can shear-thin and consequently flow under an appropriate shear stress but immediately recover back into solids on removal of the stress with gel stiffness restoring over time. In this work, mechanisms of gel shear-thinning and immediate recovery were elucidated by investigating gel behavior during and after flow via mechanical and structural characterizations. Importantly, hydrogel flow behavior was studied in a capillary geometry that mimicked the actual situation of syringe injection. Hydrogel flow profiles were obtained via fluorescent particle tracking and the profile shape was found dependent on flow rate and gel stiffness. Due to injectable-solid behavior of the hydrogels, one can co-inject multiple gels in order to produce multicompartment, solid hydrogels for biomedical applications. Hydrogel nanostructure was probed with small angle neutron and x-ray scattering and electron microscopy. The results demonstrate that these hydrogels can be excellent candidates for tissue regeneration substrates and injectable therapeutic delivery vehicles.

Human glioblastoma multiforme (hGBM) is the most common, aggressive, and deadly form of brain cancer. While in vivo tumor models are the gold standard, in vitro assays offer potential for rapid quantitative studies with potential for patient-specific insights. A significant problem is that the majority of studies depend on dish culture (flat, stiff, plastic); while designed for easy analysis, this environment is known to alter proliferation, gene expression, and malignancy profiles relative to the native tumor microenvironment. Three-dimensional biomaterials that flexibly present defined sequences of matrix and cell cues to mimic the complex native hGBM microenvironment are critically needed. A very exciting idea is that microfluidic technology can be used to fabricate optically translucent biomaterials containing heterotypic cell and matrix environments; such a platform could unlock a new generation of studies into the role the biophysical microenvironment plays on GBM invasion and malignancy. We have developed a library of synthetic hydrogel precursors to control the local mechanics, hydrophilicity, and biomimetic presentation of artificial ligands and cadherins surrounding individual a common GBM cell line (U87MG). This platform as allowed us to examine the synergistic influences of matrix composition, mechanical properties, microstructure, and biotransport on key genomic and functional markers of GBM malignancy and invasive potential. In parallel we have demonstrated a microfluidic mixing approach to create single small-volume hydrogels termed microgels containing counter-gradients of matrix chemistry and cell content for an application investigating niche regulation of hematopoietic stem cell fate. We are modifying this microfluidic technology to fabricate microgel tumor chips that recapitulate cell-matrix (chemistry, stiffness) and cell-cell (GBM cell-glial cell) interactions associated with GBM malignancy and invasive potential. This biomimetic approach will provide a more physiologically relevant brain tumor model to quantify the influence microenvironmental parameters on tumor progression and invasion.

Single cell-based microfluidics offers excellent properties for biological assays. The compartmentalization of cells into droplets provides an independently controllable environment which leads to a reduction of sample volume and consequently a reduction of costs and time accompanied by a significant increase in assay sensitivity. Additionally, the general features of microfluidics, most predominantly high-throughput operation and reduced sample sizes and volumes render single cell-based microfluidics a powerful tool for biological sample treatment. However, many applications, e.g., long term cell culture, cannot be performed in aqueous droplets due to the need for medium exchange or washing steps. While the encapsulation of cells in aqueous droplets provides an excellent microenvironment for single cells in emulsion, a transfer into aqueous media will directly lead to a dissolution of the drops and hence, the disintegration of the cell&’s microenvironment. To make droplet-based microfluidics more suitable for biological assays we work on the incorporation of single cells into hydrogel beads. Due to the 3-dimentional network structure of the hydrogel, an easy and convenient manipulation of the microenvironments of the encapsulated cells can be achieved. The hydrogel offers a superb scaffold for cell encapsulation which can be manipulated with micropipets or microsyringes and can also be used to build 3-dimentional structures, e.g., tissue design and engineering. Currently, we are focusing on the application of alginate microgels that offer high biocompatibility paired with the possibility of chemical modification and functionalization of the network-forming polymer. These features render alginate an enormous versatile candidate for cell culture applications. Using microfluidic flow focusing, cell containing droplets of liquid alginate solution were generated. After droplet formation, crosslinking was induced via internal gelation using acidic dissolution of suspended calcium carbonate nanoparticles. In a second approach, a coflow device was used to create a laminar flow of three well-separated layers of alginate, water and calcium chloride. Gelation of the alginate was induced via mixing of the three layers during droplet formation using a crossjunction drop maker which resulted in monodisperse alginate beads. The preparation of hydrogels with adjustable sizes in a range of 20-80 µm was successfully achieved. After transferring the gels into an aqueous environment the encapsulated cells can be cultured for several days without losing the integrity of the microenvironment provided by the surrounding hydrogel scaffold. Besides the general applicability of hydrogels for on-chip biological assays, the microfluidic fabrication of microgels with different geometries were investigated.

Cell migration in three-dimensional (3D) tissue depends on a physicochemical balance between cell body and extracellular matrix (ECM) constraints. Collagen degradation through MT1-MMP/MMP-14 promotes interstitial migration of mesenchymal cells but not of amoeboid-moving leukocytes, however it is unresolved how tissue space, cell deformation, and ECM remodeling jointly control migration efficacy in vitro and in vivo. Using in vitro-reconstituted 3D collagen lattices of different density and/or pore size, MMP-independent migration is a linear function of pore size, with subtotal and complete inhibition at respective 10 and 7µm² pore cross-sections for tumor cells, and 4 and 2µm² for T-cells and neutrophils. Decreasing pore size imposed different types of deformation of the nucleus towards minimal cross-section of 10%, followed by migration delay and, ultimately, arrest. Conversely, with MMPs engaged migration was rescued by proteolytic widening of narrow pores, preventing exhaustion of nuclear deformability. To validate the interdependence of scaffold porosity and nuclear deformation for cell migration in vivo, we used intravital infrared-excited multiphoton second and third harmonic generation microscopy for the visualization of invasion routes of the collagen-rich stroma of the mouse dermis. Invasion in vivo resulted predominantly from non-destructive contact-guidance type migration exploiting preformed tracks of multi-interface topography, including 1D, 2D and 3D dimensionalities of pore diameters from 2 to 20µm, whereas more confined regions were avoided. The data suggest 3D cell migration as space-guided process into and along enabling 3D tissue tracks maintained by shape change and, with space restricted, MMP-mediated ECM degradation or pathfinding for alternative routes.

Investigating the basic behavior of carbon nanotubes (CNTs) in organs is important to understand applications of these nano-vehicles in drug delivery for cancer therapy. However, recent studies on CNT organ delivery are designed for different purposes and there are large differences among these protocols. Moreover, these studies are commonly conducted on animal organs, which could be inherently difficult for studies of CNT permeation profiles. In addition, in-vivo real-time whole animal imaging after nanotube delivery has insufficient resolution to trace both local heterogeneities of the organs and nanotube transport. In this research, we utilized a 3D model tissue cultured in inverted colloidal crystal (ICC) scaffolds to compare the diffusivity of CNTs with relatively small molecules. We also investigated basic aspects of CNTs penetration through model tissue and compared the diffusion with or without the targeted factor TGF-beta1. Through high-resolution fluorescent imaging and data processing, it was found that nanotubes have exceptionally fast diffusion rate in model tissues compared to the low molecular weight molecules of the similar charge, and the diffusion rate is significantly higher with targeted factors. This result further proved the advantages of CNTs as nanoscale drug carriers and helps us to understand design new drug carriers for both treatment and diagnostics.

Spheroids are three-dimensional(3d),multicell aggregates of cells which serve as an effective in-vitro model to study tumorous growth of cancerous cell lines. Such 3D cell aggregates enhance in-vitro tests for basic biological research as well as for therapeutics development. Studies have shown that the stiffness of various metastatic cancer cells is as little as 30% of normal healthy cells, but the stiffness of cells in spheroids is still relatively unexplored despite their clinical relevance.We report results using AFM to explore the nanomechanical properties of cells in spheroids, derived from Lewis Lung Carcinoma (LLC) cell lines. Arrays of nano-indentations can reveal differences in cell stiffness when cultured as a spheroid, versus isolated or in a monolayer as is commonly studied by AFM. Cell stiffness is also mapped and analyzed as a function of depth, providing contrast from sub-membrane cytoskeletal structures.Such studies are important for understanding links between the growth and distribution of cytoskeletal structures in both individual and spheroid (tumor) based cells, their potential connection to metastatic behavior, and/or their response to therapeutic agents.

Introduction: Articular cartilage repair therapies based on scaffolds and human mesenchymal stem cells (hMSCs) are complicated by incomplete graft integration and uncontrollable hMSC differentiation. In the present work, composites based on three-dimensionally woven poly(ε-caprolactone) (PCL) scaffolds with cartilage-mimetic mechanical properties, hMSCs, and bone explants were studied with respect to construct integration potential and hMSC differentiation in vivo. Materials & Methods: Four groups of specimens were implanted ectopically (i.e., subcutaneously) in nude rats, and harvested after eight weeks. Specimen groups were: 1) pre-cultured constructs, i.e., hMSCs seeded onto PCL and differentiated three weeks in chondrogenic medium then sutured onto vital bone; 2) hMSCs freshly seeded onto PCL then sutured onto vital bone; 3) PCL sutured onto vital bone; 4) PCL alone. All hMSCs were expanded in monolayer and seeded onto PCL after one passage. Vital bone was obtained from fresh bovine calf femurs and cut into 2mm thick by 8mm diameter sections. PCL scaffolds were made by weaving 156 µm diameter multifilament PCL strands (EMS-Griltech, Dormat, Switzerland) in the x, y, and z directions on a custom built miniature loom. Finished scaffolds were approximately 0.7mm thick and 8mm in diameter. Assessments included histology (von Kossa and toluidine blue stains) and, after removal of the bone explant, molecular analyses (qPCR) for cartilage and bone markers. Other assessments (adhesive strength of construct to bone, construct compressive modulus, immunostaining, and construct GAG, collagen, and calcium contents) are pending. Results: At the time of harvest, pre-cultured constructs were firmly adhered to the bone explant, in contrast to either freshly seeded or unseeded scaffolds. Histological cross-sections of specimens in the pre-cultured group revealed a smooth transition zone between construct and bone explant. Pre-cultured constructs appeared ossified (strong positive von Kossa stain; minimal metachromasia by toluidine blue stain). Molecular (qPCR) analyses of constructs in the pre-cultured group revealed expression of chondrogenic (SOX9, COL2A1, ACAN, COMP) and osteogenic (CBFA1/RUNX, COL10A1, BSP, OC, OSX/SP7) transcripts. In contrast, specimens based on PCL with freshly seeded hMSCs and unseeded scaffolds showed poor integration with bone explants, minimal construct osteogenesis (by von Kossa stain and qPCR), and minimal construct chondrogenesis (by toluidine blue stain and qPCR). Conclusion: Three dimensionally woven PCL scaffolds seeded with hMSCs and pre-cultured in chondrogenic differentiation medium integrated very well with bone explants and underwent a process resembling endochondral ossification over eight weeks of ectopic implantation. Funding: NIH R42-AR055414 Copyright copy; 2012 by The Charles Stark Draper Laboratory, Inc MIT and Case Western Reserve University all rights reserved

Tissue engineering aims to save lives by producing synthetic organs and bone. This study is attempting to determine what effects a polycaprolactone (PCL) scaffold will have on the blood flow of Rattus norvegicus, as measured by the amount of platelets. Prior to experimentation, it was hypothesized that the polycaprolactone scaffold would maintain and/or increase the amount of platelets when compared to the control group. This was developed based on prior research that showed polylactic acid (PLA), a polymer being used currently, and polycaprolactone had similar characteristics like boiling point, melting point, and glass transition temperature. To test this hypothesis, the PCL, created from an existing protocol, was used to mold a scaffold in vitro. Three groups of rats were identified, then further split into an "A" and "B" subdivision with 5 members in each. All "A" subdivision members received the scaffold, while the "B" factions lacked it. Each rat underwent surgery to remove 1mm of the right ventricle, which was replaced by the PCL scaffold in the experimental group. The control group did not have the scaffold replacement. Without this piece of the right ventricle, prior research conducted at the University of Virginia in 2006 suggests that the rats would die within one week. However, in the experimental group of rats, the missing piece of the ventricle was replaced with the scaffold, so if it were accepted then the rats would survive beyond 1week. All rats in the experimental group died exactly 1 week after the control group as predicted before experimentation. After all of the rats had a 1-week acclimation period, a 1mm^2 slice of the heart was extracted and then the amount of platelets was counted using a phase contrast microscope. The heart extraction was prepared in a petri dish and then placed into a hemocytometer, splitting the dish into smaller sections making it possible to count. Also, at the conclusion of the blood flow work, a biopsy was done every two days to see how much of the PCL scaffold was remaining. These results were compared to prior PLA data from a Duke University study in 2008. The data supports the hypothesis whereby an average 12% increase in the amount of platelets in the rats with the PCL scaffold versus the group without it was seen. This increase in platelet count reflects an increase in blood flow. A statistical t-test was conducted on each trial (n=5 per group, n=10 total per trial) comparing experimental versus control group to calculate a p-value. The p-values were 0.034, 0.045, and 0.022, respectively which indicates statistical significance since the value is less than 0.05. After all experimentation, the benefits of using PCL in tissue engineering were examined. For example, PCL costs $80 less to produce per kilogram than polylactic acid. Moreover, the biopsy results show that PCL exits the body 2 weeks before PLA. This study suggests that PCL would be a viable candidate for tissue engineering in humans.

Severe high energy and penetrating traumas can result in acute shock and death. Emergent care at the scene is essential for optimal outcome prior to surgical care, as uncontrolled bleeding can result in death. An ideal hemostatic agent for field use that is effective, easy to use, safe, practical and provide consistent hemostasis for several hours is needed. We report, herein, the design of a reversible hydrogel based on a nanoscale dendritic approach, as a portable system for emergent hemostasis of abdominal and extremity wounds. Our approach introduces the novel capability of controlled dissolution in the surgical theater setting to allow gradual wound re-exposure during definitive surgical care. The hydrogel formation, its degree of swelling and reversible nature as well as its rheological characterization will be discussed.

The success of tissue engineering applications in regenerative medicine requires advances in low-temperature preservation. Cryopreservation of cell-containing constructs is in high demand in tissue-engineering applications to produce the tissue engineered products ‘‘off-the-shelf&’&’. Dimethyl sulfoxide (DMSO) has been used for several decades as the most efficient cryoprotective agent for many types of cells and tissues. Currently, 10% DMSO solution the most efficient cryopreservation technique is commonly used for cell preservation in cell banks around the world, in spite of its cytotoxicity and its effect on cell differentiation. However, cryopreservation of regenerated tissues including cell sheets and cell constructions is not easy compared to cell suspensions because of the weakness of cell-cell interaction and their inhomogeneous structures. Recently we showed that carboxylated poly-L-lysine, which is classified as a polyampholyte, has a cryoprotective effect on cells in solution without any other cryoprotectant [1, 2]. Cells are killed because of the damage caused by the intracellular crystallization of water during freezing. Therefore, a membrane-permeable chemical such as DMSO is usually added in order to cryopreserve the cells. However, the cryoprotective effect of polymers such as polyampholytes that do not penetrate the membrane cannot be explained by the same mechanism of DMSO. The recent study suggests that extra cellular environment might affect the cell viability after cryopreservation. In this study, we attempted to make cell scaffolds using polyampholytes for the development of the novel cryoprotective cell scaffolds. Here we report that polyamholytes with an appropriate ratio of amino and carboxyl groups show higher cryopreservation efficiency and lower cytotoxicity than DMSO. A novel polyampholyte based on naturally available polymer Dextran, in which we introduced both amino and carboxyl groups shows an excellent post thaw-survival efficiency of more than 90% of murine L929 cells. It can serve as the sole high molecular weight cryoprotecting agent (CPA) without animal derived materials. This polyampholyte can be converted to a hydrogel by slight modifications, which can be used as a cell scaffold for cryopreservation of tissue-engineered constructs and in the drug or protein delivery systems platform. [1] Matsumura K, Hyon SH. Biomaterials 30: 4842-4849 (2009). [2] Matsumura K, Bae JY, Hyon SH. Cell Transplant.19; 691-699 (2010).

Cryoprotective agents (CPAs) such as dimethyl sulfoxide (DMSO), glycerol, ethylene glycol, and propylene glycol have been used for the cryopreservation of cells and tissues. DMSO is the most effective CPA but shows high cytotoxicity and can effect differentiation. The cryoprotective properties of glycerol are relatively weak, and DMSO shows high cytotoxicity and affects the differentiation of neuron-like cells, cardiac myocytes, and granulocytes, and needs to be eliminated rapidly after thawing. Human bone marrow-derived mesenchymal stem cells (hBMSCs) are known for their potential to undergo mesodermal differentiation into many cell types, including osteocytes, adipocytes, and chondrocytes. Therefore, hBMSCs could be used for a variety of regenerative medicine therapies. In fact, hBMSC-derived osteocytes have already been used in bone reconstruction. This study discusses the viability and the differentiation properties of hBMSCs that have been cryopreserved in the absence of proteins or DMSO, by using a novel CPA. This CPA is based on carboxylated poly-L-lysine (COOH-PLL) and was prepared by a reaction between ε-poly-L-lysine and succinic anhydride. hBMSCs can be cryopreserved for 24 months at minus;80°C by using a 7.5% (w/w) cryopreserving solution of COOH-PLL which introduces carboxyl groups that result in over 90% cell viability after thawing. Furthermore, the cryopreserved hBMSCs fully retained both their proliferative capacity as well as their potential for osteogenic, adipogenic, and chondrogenic differentiation. Confocal laser-scanning microscopy findings showed that the polyampholyte CPA did not penetrate the cell membrane; rather, it attached to the membrane during cryopreservation. These results indicate that the cryoprotective mechanisms of COOH-PLL might differ from those of currently used small-molecule CPAs. These results also suggest that using COOH-PLL as a cryoprotectant for hBMSC preservation can eliminate the use of proteins and DMSO, which would be safer if these cells were used for cell transplantation or regenerative medicine. And also we will discuss the mechanisms of the cryoprotection of these polyampholytes from the viewpoint of the interaction between polymer chains or polymer chain and water and salts.

Due to their self-renewal capacity and differentiation potential, embryonic stem (ES) cells have great potential in the fields of drug discovery, tissue regeneration and cell therapy. To achieve the full potential of these cells, strategies must be implemented to maintain them in a pluripotent state during expansion. Cell attachment is essential for maintaining ES cells in an undifferentiated state and preventing them from forming unwanted embryoid bodies. Fully defined synthetic substrates are ideal candidates for the development of simple, broadly applicable, uniform culture systems for pluripotent stem cells. The use of three-dimensional (3D) polymeric materials as scaffolds has been well documented and offers several advantages for supporting ES cell maintenance. However, these 3D polymeric substrates have not been chemically optimized for stem cell maintenance and expansion. Using a rapid high-throughput polymerization and screening platform, a chemically defined substrate was identified from a comprehensive library of 66 monomer-grafting poly (ether sulfone) (PES) membrane surfaces. This novel substrate supports optimal cell attachment, expansion and long-term maintenance of mouse ES cells. The optimal chemistry has been further incorporated into PES nanofibrous scaffolds for 3D ES cell culture. Briefly, we have electrospun PES at 2.5, 5.0 and 7.5% to form nanofibers of various diameters and modified them with our optimal surface chemistry using photo-induced graft polymerization. The morphology and diameter of the nanofibers were visualized by environmental scanning electron microscopy (ESEM). The grafting of the optimal chemistry to nanofibers was confirmed by X-ray photoelectron spectroscopy (XPS) analysis. Mouse ES cells were seeded onto these chemically modified 3D nanofibrous substrates and cell morphology examined under ESEM. Cell expansion was then quantified by the MTT assay and scanning electron microscopy. Maintenance of pluripotency of ES cells grown on these modified fibers was confirmed by expression of pluripotency markers, Oct4, SSEA-1, and alkaline phosphatase. Our data demonstrates that chemically modified 3D nanofibers (from 7.5% PES) support excellent ES expansion with the maintenance of stem cell pluripotency. Altogether, this monomer-grafted, chemically defined, scalable, sustainable and controllable, 3D polymeric substrate provides a new opportunity for stem cell expansion and differentiation.

The avascular structure of load-bearing soft tissues such as cartilage, disc and meniscus is the main inhibition of self-derived healing process in these tissues. Hence, several methods form materials science and tissue engineering have been hired to generate artificial constructs for regeneration of new tissues. Load-bearing soft tissues are specialized area in need of artificial grafts mimicking native tissues both anatomically and physicochemically. In this study, highly porous cross-linked gelatin (G)/chitosan (Cs) scaffolds were chosen because of their outstanding logistic features such as material processability and supply, and comparable mechanical property with native soft tissues. Different concentrations of G and Cs were used for the preparation of primary samples and then they were cross-linked through two different methods (mixing cross-linking and scaffold cross-linking) with four different categories of variant parameters such as polymers and cross-linker agent concentration, cross-linking time and temperature. The underlying cross-linking parameters and their effects on the scaffold characteristics such as pore morphology, porosity and mechanical properties were fully investigated. Furthermore, human articular cartilage and meniscus-derived chondrocytes were seeded on the samples and evaluated in terms of proliferation, morphology and MTT assay. The cross-linked scaffolds possessed different porous structures and effectively adjusted mechanical property depending on the ratio of components and cross-linking methods. The state-of-the-art in this research could effectively obtain controllable microstructure, biodegradation profile and mechanical maturation in G/Cs scaffolds. The mechanical, biodegradation and biological activity results also indicated that the composite scaffold G60/Cs40 cross-linked by 1% genipin concentration at room temperature for 24 h through scaffold cross-linking method might be considered as a specific candidate for defect treatment of load-bearing soft tissues.

Recent biomimetic engineering has been driving toward a multifunctional cellular mimic system, which is an agglomeration for all living metabolites: for instance, there is a mimicry of a nuclear pore transporter for selective mass transfer at the interface of nucleus and cytosol1,2, a virus-like assembly for effective drug delivery3, an artificial dendritic cell for tunable immune system4. Inspired by these achievements, we sought to design a gene-networked hydrogel (G-net-gel), resembling an organized chromosome in the natural cell nuclear territories. It was spherodically manufactured using both advanced lithography and DNA nanotechnology5. Stable transgene cross-networks have been optimized in various parameters to accomplish enhanced in vitro protein expression efficiency. Furthermore, once with lipid capping, it completely reproduced the natural nucleus system, demonstrating the improved level of messenger RNAs relative to solution phase vectors. It is speculated that the proto-nuclear bio-inspiration would be a substitute to the conventional gene therapy, by which it overcome several barriers such as impaired transgene incorporations, and be a ground-breaking stratagem for gene-associated molecular therapeutics. 1. Jovanovic-Talisman, T. et al., Artificial nanopores that mimic that transport selectivity of the nuclear pore complex. Nature 457, 1023-1027 (2009). 2. Kowalczyk, S. W. et al., Single-molecule transport across an individual biomimetic nuclear pore complex. Nature Nanotechnology 6, 433-438 (2011). 3. Grigoryan, G. et al, Computational design of virus-like protein assembles on carbon nanotube surfaces. Science 332, 1071 (2011). 4. Cohen, I. R. et al., Real and artificial immune systems: computing the state of the body. Nature Immunology, 7, 569-574 (2007). 5. Um, S. H. et al. A cell-free protein-producing gel. Nature Materials 8(5), 432-437, (2009); Um, S. H. et al. Enzyme-catalyzed assembly of DNA hydrogels. Nature Materials 5(10), 797-801 (2006); Um, S. H. et al. Dendrimer-like DNA-based fluorescence nanobarcodes. Nature Protocols 1(2), 995-1000 (2006).

The results of experimental studies presented in the report concern the biocompatibility of a 3D porous matrix (tissue-cellular scaffolds) made of bioresorbable polymers (polycarbonate, polyamide, polyetherketone), including with the biocompatible oxide ceramics additive (TiO2, Al2O3, ZrO2 and hydroxyapatite), for implantology and cellular engineering. The test porous samples were prepared by the selective laser sintering (SLS) method. We compared the surface roughness as a pure porous polymer matrix, as a ceramic-polymer porous matrix on the cell morphology, proliferation and adhesion. The culture of multipotent mesenchymal stem cells (MMSC) and laser sintered titanium sample were as the reference groups. The surface microstructure and roughness were studied by the dynamical optical microscopy. It was shown that the materials carried out obviously have biocompatible properties comparable with Ti based scaffolds [1]. The experimentally estimated cell duplication speeds per day were highest for the pure polycarbonate group (0.279 doubling/day) and the PEEK + Al2O3 = 3:1 group (0.30 doubling/day) against 0.387 doubling/day for the pure steam cell group and 0.270 doubling/day for pure titanium group. [1] I.V. Shishkovsky, S.E. Volchkov, O.V. Tumina, MRS Online Proceedings Library, 1417, mrsf11-1417-kk03-15, (2012). Keywords: Selective laser sintering (SLS), porous scaffolds, multipotential mesenchymal stromal stem cells (MMSSC).

Tissue-culture polystyrene (TCPS) is the standard growth substrate in cell culture laboratories. TCPS, with an elastic modulus (E) of 3 GPa, is several orders of magnitude stiffer than physiological values. Many cell types, including stem cells, are highly sensitive to substrate stiffness. Human mesenchymal stem cells (hMSCs), in particular, are subject to in vitro aging, which is hallmarked by telomere shortening, slowed proliferation, and decreased differentiation capacity. It has also been shown that hMSCs stiffen over many population doublings, possibly as a result of stiff culture substrates. Research has indicated that cell culture conditions are contributing to the in vitro aging effects of cell stiffening and decreased differentiation capacity. Therefore, long-term cell maintenance solutions are needed to realize the potential of hMSCs in clinical applications. Thermoplastic polyurethane (TPU) is a biocompatible polymer with an E of ~12.5 MPa. It does not biodegrade, so it is suitable for long-term stem cell maintenance. It can be injection molded (IM), allowing for easily reproducible cell culture substrates, including petri dishes. Therefore, it may be an appropriate material to prevent the in vitro aging of hMSCs. hMSCs were cultivated on TCPS and IM TPU to investigate the effect of substrate elasticity on in vitro aging. At each passage, differentiations were carried out. To induce osteogenesis, hMSCs were incubated in osteoinductive media for 4 weeks. To induce myogenesis, azacytidine was added to media once a week for 4 weeks. Local cell E was measured at every passage using atomic force microscopy indentation. Gene and protein expression was examined using qRT-PCR and immunocytochemistry, respectively, for osteogenic and myogenic markers. Actin stress fiber diameter was quantified using ImageJ. On TCPS, the cell E and stress fiber diameter increased from population doubling (PD) 7 to PD 24. The modulus dropped dramatically at PD 26, and was accompanied by limited proliferation. In addition, the differentiation capacity of the hMSCs was dramatically affected by long-term maintenance on TCPS. For myogenic hMSCs, muscle markers peak at the specific population doubling that corresponds to the average E of muscle cells (~15 kPa). For osteogenic hMSCs, bone markers peak at earlier PDs and decline thereafter. However, on TPU, the E and stress fiber diameter are retained throughout the experimental timeframe. Osteogenic marker expression in osteogenic hMSCs did not decrease until a very late PD when cells are grown on TPU. In myogenic hMSCs on TPU, there was higher expression of myogenic markers overall, suggesting that TPU maybe be more appropriate than TCPS for myogenic differentiation. This study gives insight into how the substrate elasticity affects in vitro aging of hMSCs and suggests that IM TPU may be substituted for TCPS as a suitable long-term cell culture material for hMSC maintenance and differentiation.

Mechanical properties of the extracellular environment play an important role in regulating many cellular processes, including migration, proliferation, and differentiation. Cell response to material elasticity seems to be cell-type specific and correlates to elasticity of a cell&’s native tissue. While previous studies have illustrated the importance of elasticity in regulating stem cell differentiation, the role of substrate mechanics in directing neuronal differentiation is not clear. Differentiated neurons (dorsal root ganglia and cortical neurons) have been shown to develop longer neurite extensions on softer materials than stiffer ones, but previous studies do not address the ability of neural stem cells to undergo differentiation as a result of material elasticity. In this study, we investigate neuronal differentiation of C17.2 neural stem cells due to growth on polyacrylamide gels of variable elastic moduli. Neurite growth, synapse formation, and mode of division (asymmetric vs. symmetric) were all assessed to characterize differentiation. C17.2 neural stem cells were engineered to express fluorescently labeled utrophin (an actin binding protein) and doublecortin (a tubulin binding protein) to be used as a tool for the study of cell division during differentiation. Using these cells, we were able to visualize the actin filaments and microtubules during division on various substrates, and therefore, study the role of the cytoskeleton in regulation of division plane in response to substrate stiffness. C17.2 neural stem cells were seeded onto polyacrylamide gels coated with Type I collagen. The cells were then serum starved over a 14 day period, fixed, and analyzed for biochemical markers of differentiation. During differentiation, neuronal precursors will undergo asymmetric or symmetric cell division, in which contents of the parent cell are divided equally or unequally between the two daughter cells. The type of division plays a role in determining the final fate of the cells, and the engineered cells provide a way to study the cytoskeleton during cell division and differentiation. For division studies, time-lapse imaging of both native and fluorescently tagged cells on various substrates was performed over extended periods of time using the Nikon Biostation. Division events were analyzed using ImageJ to quantify sizes of resulting daughter cells and distribution of fluorescent proteins. Data indicates that C17.2 cell differentiation (as dictated by number and type of division events) is dependent upon substrate stiffness, with softer polyacrylamide surfaces (140 Pa) leading to increased populations of neurons and increased neurite length. In addition, early data indicates that the ability of neural stem cells to express synaptic proteins and develop synapses is dependent upon material elasticity.

Recent work has shown that actomyosin-mediated contraction plays an important role in determining the morphology of 3D microtissues during directed self assembly in self-constrained nonadhesive molds. Here, we have developed a bio-chemo-mechanical model to investigate the morphological evolution of microtissues by explicitly considering the interplay of actomyosin-mediated force and tissue geometric shape. The key features that we have incorporated in our model include: 1) Hill relation for single cell response whereby the speed of shortening decreases with increasing load and the isometric force/maximum velocity is related to myosin recruitment; 2) feedback mechanism whereby the equivalent stress due to mechanical constrain leads to anisotropicmyosin recruitment and themyosin recruitment permitted by biochemistry can be saturated; 3) necking instabilities in active material whereby a section of the tissue with reduced area can rapidly elongate if the contraction speed in narrowed regions is slower than other regions. Using quasi-1D and 3D finite element implementation of our model, we have qualitatively analyzed in situ data of microtissue morphology, including thinning and narrowing rates, shape evolution, and the formation and the disappearance of gaps between the microtissue and the constraining pegs in our moulds during different phases stress generation and failure. In addition to these mechanochemical coupling parameters, our analysis provides a quantitative estimate of cell density or mechanical properties by measuring the contraction and failure of self constrained microtissues.

Tissue engineering provides a promising strategy for tissue regeneration, which combines the utilization of scaffolds, cells and growth factors. An ideal scaffold for bone tissue engineering should possess interconnected porous structures, adequate mechanical support, controlled degradation rate, and appropriate surface chemistry. Surface topography and nanostructures of biomaterials can significantly influence cell growth and viability. To get a high-performance porous bone tissue engineering scaffold, a 2-level-3-dimentional (2-L-3-D) collagen- hydroxyapatite (HAp) micro/nano porous composite scaffold was constructed by using porcine acellular porcine dermal matrix (PADM) as the micro-porous organic framework, and coated with a nano-structured hydroxyapatite layer on the channels of PADM by biomineralization in simulated body fluid. In vitro assessment proved that the HAp nano-structure on surfaces of PADM channels can enhance the mechanical property, decrease the bio-degradation rate, and be benefit to the proliferation and differentiation of the pre-osteoblasts. The experimental results illustrated that biomineralization time can tune mechanical properties of the composite scaffolds and also the biodegradation rate by controlling the thickness of the HAp nanostructure on the surface of PADM channels. Cell cultivation with MC3T3-E1 revealed that this nano-structured HAp can support cell proliferation and increase the expression of the osteogenetic marker proteins. In vivo comparative assessments of the scaffolds demonstrated that the 2-L-3-D PADM-HAp composite scaffolds can successfully repair the rat mandibular 4mm defects very rapidly. Micro-CT observation and histological examination of bone defects in rat mandible were carried after scaffold implantation up to15 weeks. Results revealed that no closure and no repair of the critical bone defects were observed in the blank group (without scaffold), and little in the control group (scaffolds without HAp), while the composite scaffolds promoted the repair of critical bone defect and the defects were completely regenerated after 15 weeks in vivo. In conclusion, the incorporation of nano-structured hydroxyapatite layer on the surfaces of micro-porous PADM channels not only increases the bio-stability of the collagen scaffold, but also benefits cellular activity in vitro and promotes the repair of critical bone defect of rat mandible in vivo.

Functional vascular networks are fundamental for the fabrication of 3-dimensional thick tissues and organs for regenerative medicine. In this study, we combined crosslinkable hydrogel and electrochemical detachment of cells for rapidly engineering vascular-like structures. The crosslinkable hydrogel was composed of hydrazide-modified gelatin (gelatin-ADH) and aldehyde-modified hyaluronic acid (HA-CHO). The gelation occurred in less than 30 s after mixing these two components. The hydrogel was characterized for its stiffness, pore size, cytotoxicity, and cell adhesion. In addition, we demonstrated that cells adhering to a gold surface covered with an oligopeptide layer could be transferred to the hydrogel by applying a negative electrical potential, which desorbs the oligopeptide layer from the gold surface. To apply this approach in the fabrication of vascular-like structures, we used a gold-coated cylindrical rod. On applying an electrical potential and extracting the surface layer of the rod, endothelial cells were transferred from the rod to the internal surface of the microchannel in the hydrogel. This technique can be a promising approach for fabricating vascularized 3-dimensional tissues. References [1] Y. Seto, R. Inaba, T. Okuyama, F. Sassa, H. Suzuki, J. Fukuda, “Engineering of capillary-like structures in tissue constructs by electrochemical detachment of cells,” Biomaterials, 31(8), pp. 2209-15, 2010.

Hydroxyapatite (HA) [Ca10(PO4)6(OH)2] is the main mineral constituent in human bone. It crystallizes in hexagonal and monoclinic phase, which are very similar in structure and properties. A critical measure for healthy bones is the Ca/P ratio which in turn affects the dielectric constant of the mineral constituent. The dielectric constant of HA varies between 5 and 20 depending on the Ca/P ratio in the sample [J. Mater. Sci.: Mater. Med. 21, 399]. We suggest exploiting this large span in the dielectric constant in a non-destructive method to measure the Ca/P ratio in bone by optical spectroscopy. Using density functional theory we calculate the long-range corrected phonon dispersion. We find that only modes around 330 cm-1 are strongly affected by the dielectric constant. The shifts in frequency can be up to 20 cm-1 as you span the range of the dielectric constant. Thus, by measuring the optical shift and comparing with calibrated samples it is possible to draw conclusions on the Ca/P ratio in the mineral. Importantly, we find the same modes in both the monoclinic and hexagonal phases to be sensitive to changes in the dielectric constant.

Recently, anisometric structures of composite materials have attracted intense research interests since they are considered to open up novel capabilities and related applications. Herein, anisometric particles of biocompatible hydrogels were prepared and their responsive properties were explored. Janus particles of chitosan and alginate hydrogels were also prepared by ionic gelation for the development of biocompatible embolic particles. They showed reversible self-aggregation behavior through the short range ionic interactions, which was dependent on various external stimuli such as pH and salt concentrations. The reversible self-aggregation behavior of these particles promoted the efficiency of transcatheteral delivery and artery embolization in the in vivo experiment of rabbit kidney arterial emblization. Anisometric hydrogels showed a slower swelling rate and a lower equilibrium swelling ratio than their control symmetric hydrogels. As a result, controlling the morphology and structure of anisometric hydrogels can make a wide range of mechanical and swelling properties available for novel applications. These particles could provide intelligent functionality to the current embolization technology and other particle applications.

Over the past decades, it has been reported that both AC and DC electrical stimulations accelerate regeneration of different tissues. Electrical fields have been shown to provide a neural cell activity enhancing the regeneration of both peripheral and central nervous systems. Direct current electrical stimulation is also known to enhance and direct neurite outgrowth. It has been shown as well that bone cells respond to electrical stimulation leading to a faster bone growth. Piezoelectrics are an interesting group of materials because they produce an electrical response when mechanically stressed. As a result, an electrical stimulus may be created without direct connection to a voltage source. The direct piezoelectric effect has been exploited in many different practical applications, including voltage and power sources, sensors and actuators, piezoelectric motors, reduction of vibrations and noise, among others for industrial, manufacturing and medical instrumentation applications. However the exploitation of the phenomenon for tissue growth and regeneration is almost unknown. Within our current studies on the exploitation of piezoelectrics for tissue regeneration, this study aims to assess the influence of the polarization of a piezoelectric, biodegradable and biocompatible polymer Poly (L-lactic) acid (PLLA), approved by the FDA, on the viability and adhesion of cells from neural tissue. PLLA samples were prepared with different techniques: PLLA dense films were obtained by solvent casting and PLLA nanofibers meshes by electrospinning. Some samples were submitted to posterior heat treatment to vary the crystallinity and some were electrically poled using a corona discharge (10 kV, 10 µA for 30 min at 100 degree C and during posterior cooling). This large set of PLLA samples: dense films, nanofibers meshes, as processed and crystallized, non poled, negatively and positively poled were evaluated for their biocompatibility. Specifically, neuroblastoma SH-SY5Y cells were cultured on the different PPLA substrates and the latter were evaluated for their cytocompatibility adhesion, and effect on cell morphology. Results are discussed in terms of the effects of the different PLLA samples on characteristics such as topography and crystallinity on cells behavior. The effect of polarization and how negatively poled PLLA surfaces generally enhance cellular bioactivity is also discussed.

Embryonic stem (ES) cells have the capacity to form any adult cell type and therefore possess extensive therapeutic potential. ES cell behaviour is regulated by a combination of extracellular cues, including growth factor signalling, cell -cell contact and cell -extracellular matrix (ECM) binding. Current two dimensional culture techniques do not fully exploit these interactions and as a consequence the effective propagation and differentiation of ES cells is limited. Self-assembling peptide hydrogels have recently been the focus of significant attention as they offer the potential to design functional ECM mimics. Their defined, fully synthetic nature and ease of functionalisation makes then very attractive for the design of 3D cell culture environments. β-sheets forming peptides in particular allow the design of very stable hydrogels that have been used for the 3D culture of a number of cell lines. We have recently focused on developing and optimising a reproducible and transferable protocol for the encapsulation of stem cells into peptidic hydrogels. Mouse ES cells were seeded as single cells within FEFEFKFK hydrogels and were shown to proliferate into aggregates retaining their pluripotent nature (pluripotent markers: Nanog, SSEA-1 and E-cadherin). Proliferation within the gels was found to be dependent on both cell seeding density and peptide concentration. ES cells could also be retrieved and directly passaged into fresh gels, with cells sequentially cultured for 17 days remaining positive for pluripotent markers Rex1, Klf4, Nanog and Oct4 and retaining the capacity to differentiate into cells from all three germ layers. The hydrogels were also successfully functionalised with a heparin binding peptide resulting in an increase in stability and retention of heparin. The nanofibrillar environment of the peptidic hydrogels represents a highly versatile culture system which has significant therapeutic potential, replicating the architecture of the ECM and providing a truly 3D culture environment for stem cells.

Triply-periodic minimal surfaces are shown to be a more versatile source of biomorphic scaffold designs than currently reported in the tissue engineering literature. A scaffold architecture with sheetlike morphology based on minimal surfaces is discussed [1], with significant structural and mechanical advantages over conventional designs. These sheet solids are porous solids obtained by inflation of cubic minimal surfaces to sheets of finite thickness, as opposed to the conventional network solids where the minimal surface forms the solid/void interface. Using a finite-element approach, the mechanical stiffness of sheet solids is shown to exceed that of conventional network solids for a wide range of volume fractions and material parameters. We further discuss structure-property relationships for mechanical properties useful for custom-designed fabrication by rapid prototyping. Transport properties of the scaffolds are analyzed using Lattice-Boltzmann computations of the fluid permeability. The large number of different minimal surfaces, each of which can be realized as sheet or network solids and at different volume fractions, provides design flexibility essential for the optimization of competing design targets. [1] S. Kapfer et al, "Minimal surface scaffold designs for tissue engineering", Biomaterials 32, 6875 (2011)

Neural stem cells (NSCs) are multipotent and differentiate into neurons and glial cells, which can provide essential sources of engraftable neural cells for devastating neural diseases and injuries. One of the major challenges involved in the differentiation of NSCs is to identify and optimize factors which result in an increased proportion of NSCs differentiating into neurons as opposed to glial cells. To this end, soluble cues such as brain-derived neurotrophic factor, sonic hedgehog, and retinoic acid, have been shown to significantly increase neuronal differentiation of NSCs in vitro. However, the research toward studying the function of the two microenvironmental cues - cell-cell interactions and insoluble cues - during the neuro-differentiation of NSCs is limited, mainly due to the lack of availability of methods for the investigation. Herein, we demonstrate the effect of extracellular matrix (ECM) protein patterns and nanotopography, as insoluble cues, on the differentiation of NSCs. Bio-surface chemistry combined with soft lithography was used to generate patterns with varying geometries and dimensions of the ECM protein, laminin, to study the influence of surface patterns and cell-cell interactions on the differentiation of NSCs. In addition, we used varying nanotopographies, generated by films of different sizes of nanoparticles coated with laminin, to deliver genetic material to enhance the neuronal differentiation of NSCs. Our results confirmed that ECM protein patterns with variant geometries and dimensions guided cell-cell communications and cell-ECM interactions in a controlled manner, which ultimately led to a pattern geometry-dependent and dimension-dependent neuronal differentiation. In addition, by controlling gene expression levels using nanotopography-based delivery, we observed a remarkably higher number of NSCs undergoing neuronal differentiation. Our results could be significant for neural tissue engineering, wherein NSCs can be transplanted into the damaged regions of the brain or spinal cord with scaffolds containing specific geometries or nanotopographies for delivery of genetic material into stem cells. Scaffolds having nanotopographies and patterns promoting cell-cell interactions in a controlled manner could potentially lead to increased neuronal differentiation in vivo.

Adipose-derived stem cells (AdSCs) have shown great potential for tissue engineering applications due to their relative abundance, ease of harvest and ability to differentiate into multiple lineages such as adipocytes, osteocytes and chondrocytes. As such, the incorporation of AdSCs into scaffolds is a promising approach for the replacement or repair of damaged tissues and organs. Ideally, a scaffold should be three-dimensional (3D) with high porosity and interconnected pores that facilitate nutrient and waste exchange as well as provide a suitable microenvironment to support or direct cell proliferation or even differentiation. Unlike traditional 2D cultures, these 3D cultures serve as a better representation of the actual in vivo situation where cells grow in a complex 3D environment. Moreover, cells grown in 3D cultures were found to have different morphology, differentiation, cell-cell and cell-matrix interactions as compared to 2D cultures. Thus 3D cultures serve to bring in vitro analysis to a whole new level that is more capable of mimicking the actual in vivo microenvironment for studying cellular behaviors. Although the use of scaffolds for tissue engineering applications has become increasing common, limited research had been carried out on developing scaffolds with tunable properties after fabrication. Such development in scaffold design will enable in situ modification and precise tailoring of scaffold properties at the point of cellular implantation both in vitro and in vivo according to the different needs of various applications. The properties of the scaffold matrix e.g stiffness or porosity have direct implications on cellular behavior as they sense the mechanical cues from their immediate 3D microenvironment. The effect of various soluble factors on cell behavior has been widely studied, but little is known on how the 3D microenvironment affects cell proliferation or differentiation. Therefore, this study aims to investigate the ability of AdSCs to proliferate and differentiate in tunable 3D scaffolds. Alginate was used as the scaffolding material due to its mild gelling, superior biocompatibility and biodegradability. 3D alginate scaffolds were fabricated using different concentrations of cross-linker in order to alter the final properties of these scaffolds. Adipogenic and osteogenic differentiation of AdSCs in the scaffolds were observed to occur in the absence of differentiation supplements. In addition, a reciprocal relationship was shown between adipogenesis and osteogenesis. Thus by combining detailed bulk and surface material characterization studies with cellular and molecular studies, the role of different material properties on cellular behavior in a 3D microenvironment could be investigated. This study serves to provide new insights on the role of 3D tunable scaffolds in providing environmental cues that directs cellular behavior and function without the need for biological supplements.

Dental pulp stem cells (DPSCs) are known to be stimulated along odontogenesis by the presence of dexamethasone. The purpose of this study was to investigate the in vitro differentiation of DPSCs into odontoblasts in the absence of dexamethasone, when seeded onto enzymatically cross-linked gelatin hydrogels of different stiffness (8KPa to 100Pa). DPSCs were cultured on hard and soft substrates till 35 days with and without dexamethasone (dex). SMFM indicated that the cell modulus responded to the stiffness of the substrate but was independent of Dexamethasone. SEM and EDX analysis showed an increase in the hydroxyapatite mineralization from day1 to day 35of incubation. RT-PCR analysis showed an upregulation of dentin sialophosphoprotein (DSPP) expression indicating a dex independent odontogenesis that was long term on the hard gels. Results from this study indicated that odontogenic differentiation of DPSCs can be achieved without dex on the cross-linked gelatin scaffolds as early as day 1. These scaffolds are also capable of self-biomineralization thus proving to be a potential biodegradable scaffold for dentin regeneration.

Interventional therapies for renewal of mammalian tissues that suffer from degenerative conditions such as diseases or trauma require an in-depth understanding and control over cellular processes and microenvironment including spatial and temporal variability in soluble signals, signal cascades and matrix mechanics. Synthetic mimics of extra cellular matrix (ECM) that can replicate the organization and physical attributes of the physical and biological signaling environment of the cell are therefore highly desirable. Synthetic environments derived from hydrogels can mimic many of the physiochemical characteristics of living systems. As a result hydrogels derived from water-soluble synthetic and biopolymers have been extensively explored as carriers for cell and in vivo engineering of tissue. Several challenges remain to be overcome in realizing a well-defined synthetic ECM mimic, such that it is clinically translational. These include replication of stiffness and topographical attributes of the ECM, and rendering of a biologically well-defined environment, all in an injectable system. Polysaccharides (PS) have gained a favorable reputation in regenerative therapies due to their cell/tissue compatibility, relative ease of use, and more importantly translation into humans. Among the various mechanisms by which PS can be gelled, helical-helical interactions represent a highly evolved structural interaction that is highly specific. In fact many marine polysaccharides exhibit helical-helical interactions, and undergo thermo reversible gelation as a consequence. We have studied extensively the effect of backbone modification on the association helices in PS, and shown that the association behavior of helices can be impacted significantly so as to impart new physicochemical properties to the gel. We have validated this premise, by conducting molecular dynamic simulations on agarose as a model system, using structural data obtained from X-ray diffraction. Key findings from the simulation have been confirmed using rheology, atomic force microscopy and light scattering. Exploiting this backbone modification strategy, gels with shear modulus that span the range from muscle to neural tissue (G&’ range 10*E4 Pascal) can be achieved independent of polymer variables. These novel biomaterials upon further modification with cell-ECM binding peptide motifs and have been shown to support the growth and differentiation of a diverse set of primary human cells including mesenchymal stem cells, articular chondrocytes and umbilical vein endothelial cells. An important aspect of the peptide modified gels is that they do not induce any inflammatory responses as assessed by Chorioallantoic Membrane (CAM) assay and when taken in sum with their ability to support primary cells, make them a promising candidate as a translational synthetic ECM environment for regenerative medicine.

The goal of this study was to blend conducting polymer with a hydrogel in a manner that could provide guidance cues for axonal regeneration. It has been suggested that presentation of topographical, electrical, or chemical cues can enhance and direct axon growth. To provide these guidance cues, our platform was designed with conducting polymer to enable electrical stimulation and electrically controlled release of growth factors, aligned nanofibers to provide a directional topography, and a hydrogel to provide softer mechanical properties preferred by neural tissue. We fabricated conductive hydrogel films and nanofibers by photopolymerization of poly(ethylene glycol) diacrylate and poly(ethylenedioxythiophene)-poly(styrene sulfonate) (PEDOT:PSS). Concentrations of PEDOT:PSS were varied from 0% to 25% of total polymer weight content. Films were first cast and cross-linked to serve as the foundation for the scaffold. Aligned conductive hydrogel nanofibers were then electrospun over these films to provide the desired topography. While conductive nanofibers have often been fabricated by a hard templating method involving electrodepositing conducting polymer around an electrospun template fiber, the method used here is a single-step fabrication as the conducting polymer is directly electrospun into the nanofiber. The sheet resistance and impedance were measured as a function of PEDOT:PSS concentration. Impedance results revealed that higher percentages of PEDOT:PSS resulted in higher conductivity of samples. Swelling ratios were measured to characterize the gel properties of the materials. We used energy dispersive spectroscopy to confirm the presence of PEDOT:PSS in the nanofibers, and examined the nanofiber morphology by scanning electron microscope. We also examined degradation of conductive hydrogel samples by measuring the mass loss over four weeks to ensure its suitability for future cell culture experiments. Recently, we have incorporated nerve growth factor into the conductive hydrogel samples. We will quantify the release of nerve growth factor by actuation of the conductive hydrogel through electrical stimulation. Stimulated release of growth factor will be compared to its passive release. The platform we have developed is a promising candidate for future use in axonal regeneration applications.

The synthesis of new carbohydrate-based polymers that are chiral and retain the cyclic pyranose ring found in natural polysaccharides is an important challenge for polymer chemists. Replacing the ether linkage found in natural polysaccharides with an amide linkage is a strategy that allows the molecular-level control available with organic synthesis while maintaining the unique properties of natural polysaccharides. To this end, we have synthesized chiral, controlled molecular weight poly-amido-saccharides (PASs) via the anionic ring opening polymerization of β-lactam sugar monomers. Characterization of these new polymers using NMR, IR, DLS, CD, and microscopy will be discussed and related to the molecular structure. The ability to change PAS solubility by changing monomer stereochemistry, co-monomer ratios, and by adding charged groups will be described. Finally, we will address the potential biomaterials applications of PASs by studying their interaction with natural carbohydrate receptors.

The “London tragedy” - a clinical trial in which severe toxic shock symptoms occurred in human volunteers - dramatically demonstrated the risks of in vivo drug testing. Organotypic tissue cultures establish a highly promising approach for performing in vivo type of studies in vitro, viz. within the preclinical phase. Currently, however, very limited survival times of only a few days for adult tissue due to substrate issues often severely limit their application. We propose a novel biotechnological concept, which allows for unprecedented long culture times even in absence of biochemical growth factors, as demonstrated for adult retinal and nervous tissue. As tissue distortion is commonly preceded by migration of individual cells from the tissue to the substrate, tailoring the substrate properties unfavorable for adhesion or migration of individual cells while ensuring good adhesion of the overall tissue constitutes the key for successful long-term organotypic tissue culture. Employing TiO₂ nanotube array substrates, whose interaction properties with individual cells and the overall tissue can be tuned by the tube parameters, we verify this concept for different adult neuronal explants, which are successfully cultured for the first time longer than 14 days with no indications of degeneration. It turned out that adequate nanotube diameter, wall thickness and surface roughness vary for successful long-term culture of adult mammalian retinae and brain slices. Additionally, we found that the intrinsic super-hydrophilicity of the substrates allows for a continuous supply of fresh medium without exposing the explants to the bulk liquid. These findings additionally pave the way for in vitro drug testing, as well as retina and brain tissue regeneration. [1] Valentina Dallacasagrande, Mareike Zink, Steven Huth, Alexander Jakob, Marcus Müller, Andreas Reichenbach, Josef A. Käs and S. G. Mayr, Adv. Mat. 24, 2399 (2012)

Statement of Purpose: Poly(vinyl alcohol) (PVA) and poly(vinyl pyrrolidone) (PVP) hydrogels have been investigated for nucleus replacement, but have not been a successful candidate for injectable nucleus replacement. The addition of a chemical crosslinking agent to the PVA/PVP hydrogel eliminates the need to freeze/thaw the hydrogel and enables the gel to form a cohesive implant in situ. Poly(ethylene glycol) diglycidyl ether (PEG-DGE), a diepoxide, was used to chemically crosslink PVA/PVP hydrogels to form an injectable nucleus replacement. The purpose of this study is to determine the effect of adding PEG-DGE versus PEG, varying PEG-DGE and varying PVA concentration on compressive modulus and swelling ratio (V/Vo) as well as to the cytotoxic effects of the PVA hydrogel crosslinked with PEG-DGE. Methods: PVA was chemically stabilized with PVP in a ratio of 99:1 to form a solution in deionized water, barium sulfatewas added. PEG (non-crosslinked gels) or PEG-DGE (crosslinked gels) with 9.4M NaOH were stirred into the PVA solution to form hydrogels. Swelling Studies: Hydrogels were immersed in a solution of PEG in PBS to examine the changes of the properties over time in an osmotic environment (0.2 MPa). V/Vo was calculated from the masses of the samples before and after swelling (n=3). Mechanical Testing: Hydrogels (n=5) were swollen in 0.2 MPa osmotic solution before testing. Unconfined, uni-axial compression was applied. The samples were compressed to a total strain of 30%; a chord modulus was measured between 10 and 20% strain. Cytotoxicity: Leachables were extracted from purified hydrogels in PBS for 72 h at 37oC. L929 mouse fibroblast cell suspension was added to 96-well tissue culture plates and the cells were incubated for 6 h at 37oC. PBS with extracted leachables was added to the cultured cells, which were further incubated for 48 h. CellTiter-Glo® luminescent cell viability assay was used to measure the ATP of living cells. Results: Swelling Studies: At a constant 30% PEG or PEG-DGE, PVA concentration was increased from 15.1 to 20%. As PVA concentration increased V/Vo increased from 0.95 to 1.10 for crosslinked gels and 0.98 to 1.10 for non-crosslinked gels. As PEG or PEG-DGE is increased from 9 to 29%, at a constant 14.3% PVA, V/Vo increased from 0.72 to 0.93 for crosslinked gels and 0.91 to 0.94 for non-crosslinked gels, except for 9% PEG and PEG-DGE which did not form gels. Mechanical Testing: At a constant 30% PEG or PEG-DGE, as PVA content was increased from 15.1 to 20% modulus increased from 0.51 to 1.54 MPa for crosslinked gels and 0.34 to 0.76 MPa for non-crosslinked gels. At a constant 14.3% PVA, as PEG or PEG-DGE content increased from 19 to 29% the modulus increased 0.05 to 0.37 MPa for crosslinked gels and 0.01 to 0.09 MPa for non-crosslinked gels. Cytotoxicity: Purified hydrogel extractions in PBS showed no greater than a 4.3% cytotoxicity, when ATP of cells with extractions is normalized to tissue culture plastic.

The objective of this work is to examine potential uses for electrically conductive hydrogel composites as a 3D scaffold for tissue growth with clinically relevant cell lines. The composite is comprised of carbon nanobrushes embedded in a biocompatible poloxamer gel. This work assesses the ability of such composite gels to support the growth of fibroblasts and myocytes and eventually serve as a matrix to stimulate wound closure. In such a model, fibroblasts and myocytes are seeded on the hydrogel and bathed in culture medium. The experimental model assesses the ability of fibroblasts and myocytes to grow into and adhere to the gel. The work demonstrates that carbon nanobrushes can be dispersed within poloxamer gels, and that fibroblasts and myocytes can proliferate within homogenously dispersed carbon nanobrush-containing poloxamer gels. Future work will examine the effects of design parameters such as carbon nanobrush content and matrix structure on wound healing, as well as the growth of tendons and other cell lines within the hydrogel composites. This work has relevance for tissue engineering and tissue regeneration in clinical medicine.

In tissue engineering, biomaterials that play an active role in the tissue regeneration process are greatly needed. Although not as common as polymeric materials, bioceramics play a critical role in tissue engineering. Research in the use of bioceramics for tissue engineering has often involved the implementation of sacrificial phases for forming a replica of a natural construct (e.g. marine sponge). Utilizing a direct foaming method, we have processed bioactive ceramics with potential for orthopedic tissue engineering applications. Direct foaming gives us the ability to control overall porosity and pore dimensions of these ceramics, a great improvement over other methods for fabricating porous scaffolds. Before moving into tissue engineering applications, it is first important to thoroughly assess the biocompatibility of a material system. We will present biocompatibility studies of ceria, known for its free radical scavenging ability, and barium titanate, a piezoelectric material. These active material properties can play an active role in the development and proliferation of cells for tissue engineering. We have demonstrated the ability for bone specific cell types to attach and proliferate on these materials with no significant toxic effects. We will present these findings as well as preliminary inflammatory response measurements using THP-1, a human monocytic leukemia cell line, which has the ability to mimic the foreign body response of peripheral blood monocytes. Our work shows that these bioactive ceramics have great potential for more actively supporting tissue development in the field of regenerative medicine.

The development of hydrogel carriers with remote controlled capabilities is an emerging strategy in drug delivery. The release of drug molecules entrapped in the hydrogel matrix can be triggered remotely by external stimuli, providing a flexible control of the release rate and profile, according to the specific needs of the patient [1]. Near-infrared (NIR) light is harmless and deeply penetrates in the living tissues, hence being an attractive stimulus source for remotely controlled bioapplications. Carbon nanotubes (CNTs) absorb NIR light and convert the absorbed energy into heat very effectively and can therefore act as light activated nanoheaters to induce phase transitions of thermosensitive hydrogels and to remotely control the release of molecules. In this work we report the development of temperature and NIR light responsive hydrogel composites comprising a thermosensitive polymer matrix (κ-carrageenan) and multi-wall CNTs (MWCNTs) as dispersed phase. The effects of the MWCNT concentration and surface functionalization on the microstructure, strength, swelling and release properties of the resultant composites were investigated. MWCNTs acted as multifunctional fillers by enhancing the mechanical properties of the hydrogels, the effect being mostly CNTs load dependent and conferring light responsive characteristics to the composites. Surface functionalization of nanotubes had a major influence on the swelling of the composite hydrogels. The release enhancement of a drug model in in vitro conditions, from κ-carrageenan hydrogel composites by NIR photothermal conversion of MWCNTs was demonstrated. In face of these promising results we believe that these hydrogel composites have potential in the development of remotely controlled light activated drug delivery systems. [1] Timko BP, Dvir T, Kohane DS. Adv. Mater. 2010, 22, 4925 Acknowledgements: The authors acknowledge FCT (Pest-C/CTM/LA0011/2011), FSE and POPH for funding.

Statement of Purpose: Biologically and mechanically well-characterized hydrogels have generated broad interest in tissue engineering and regenerative medicine. Hydrogels are widely used to seed cells in two dimension (2D) or encapsulate cells in three dimensions (3D) to form biomimetic constructs. Hyaluronic acid (HA) and gelatin (hydrolyzed collagen) are natural polymers and major components of the extracellular matrix (ECM) in most of the tissues (e.g. cardiovascular, cartilage, neural). When used as a hybrid material, HA-Gelatin hydrogels may enable mimicking the ECM of native tissues. Although HA-gelatin hydrogels are promising biomimetic substrates, their material properties have not yet been characterized in the literature. Herein, we generated hybrid hydrogels using different ratios of HA and gelatin, determined their material properties and assessed the cellular response in both 2D and 3D. Methods: We synthesized hyaluronic acid methacrylate (HAMA) and gelatin methacylate (GelMA) as reported before. Biomimetic hybrid hydrogels were subsequently generated by mixing different ratios of HAMA (0, 1, or 2% (w/v)) and GelMA (0, 3, 5 or 10% (w/v)). The hydrogel constructs were fabricated by a photocrosslinking strategy in the presence of 0.1% (w/v) photoinitiator (Irgacure 2959) upon exposure to ultraviolet (UV) light. The swelling ratio, compressive modulus and degradation of these hydrogels were then determined. In addition, cell adhesion and spreading on and within HAMA-GelMA hydrogels were determined using human umbilical cord vein endothelial cells (HUVECs). Results: The swelling behavior of HAMA-GelMA hydrogels is found to be tunable by varying the mass percentage of the gel components. HAMA-GelMA hydrogels were also determined to be mechanically tunable, which could be useful for a variety of different tissue engineering applications (e.g. cardiac, cardiovascular, cartilage, skeletal muscle). The increase in the concentration of GelMA rseulted in slower gel degradation by 2.0 U/ml collagenase demonstrating the tunable degradation behavior of HAMA-GelMA hydrogels. Furthermore, these hydrogels were demonstrated tunable cell adhesion behavior when seeded on 2D, and tunable cell spreading when encapsulated in 3D within the hybrid hydrogels. Conclusions: Methacrylated HA and gelatin were successfully used to generate ECM-mimetic hydrogels with biologically relevant concentrations. The material properties of resulting hydrogels were characterized and found that they can be biologically and mechanically tuned to yield in a range of different cellular response for HUVECs. Due to their abundance in the native ECM, HA and collagen hybrids have great potential to be used in a wide range of drug delivery, cell transplantation, and tissue engineering applications.

Bone and cartilage defects, which are caused by a variety of reasons such as traumatic injuries, osteoarthritis, osteoporosis or bone cancers, represent common and severe clinical problems. Each year, over 6 million people visit hospitals in the U.S. for various knee, wrist, and ankle problems. Tissue engineering approaches for the repair of bone and cartilage defects is currently an area receiving focused attention. Since bone and cartilage are nanocomposite materials, the goal of the current work is to use nanomaterials and nanofabrication methods to create novel biologically inspired TE bone and cartilage scaffolds for facilitating human bone marrow mesenchymal stem cell (hMSC) osteogenesis and chondrogenesis. For this purpose, through electrospinning and particle leaching techniques, we designed a series of novel 3D biomimetic nanostructured scaffolds based on nanocrystalline hydroxyapatites (nHAs), biomimetic carbon nanotubes, DNA based rosette nanotubes and polymers. Specifically, a series of electrospun fibrous biocompatible poly(L-lactic acid) (PLLA) and polycaprolactone (PCL) scaffolds with controlled fiber dimension and surface nanoporosity were fabricated in this study. In vitro hMSC studies showed that stem cells prefer to attach in the scaffolds with smaller fiber diameter or suitable nanoporous structures. More importantly, our in vitro differentiation results demonstrated that incorporation of the biomimetic nHA, rosette nanotubes and carbon nanotubes can induce more osteogenic and chondrogenic differentiations of hMSCs than controls, which make them promising for bone and cartilage tissue engineering applications.

The wound healing process is an intricate integration of overlapping events involving a multitude of cells, cytokines, and extracellular matrix components. In healthy individuals, this process is generally carried out to completion, resulting in timely wound resolution. However, elderly individuals and those suffering from diseases such as obesity and diabetes display an increased risk of disruption in this process, resulting in non-healing chronic wounds. While growth factor therapy has demonstrated a limited ability to promote chronic wound healing, the current therapeutic modalities do not accurately recreate the natural wound healing process. Here, I will discuss the development new therapeutic dressings that more accurately recreate the dynamic signaling observed in vivo. By using layer-by-layer technology, I will show the ability to modulate the temporal delivery of multiple growth factors that have been implicated in wound healing. Furthermore, the impact of these dressings on an in vivo chronic wound healing model will be discussed.

The field of tissue engineering has gained remarkable importance in the recent years. Bone tissue engineering makes use of the principles of biology and engineering to produce biocompatible substitutes that restore and maintain the function of human bone tissue. It is performed by the in vivo implantation of the tissue engineered biomaterial with the purpose to stimulate and to direct bone tissue formation at the site of implantation. We prepared biocomposite materials for bone tissue engineering using foaming and cryogelation techniques. Foam and cryogel samples of polyvinyl alcohol-tetraethoxyorthosilicate (TEOS)-alginate-calcium oxide (PTAC) were taken and their degradation rate was monitored over a period of one month by incubating them in sterile and freshly prepared simulated body fluid (SBF) inside a water bath maintained at body temperature, i.e., 37 degrees C. The average percentage degradation over a period of one month for biocomposite cryogels was faster as compared to biocomposite foams while the mechanical properties of biocomposite foams were better as compared to biocomposite cryogels. Although, the mechanical study results also suggest that the biocomposite foams were quite brittle while the cryogels were ductile in nature. Further, in order to determine the microstructure and porosity of these biocomposite materials scanning electron microscopy (SEM) study was conducted and the results propose that although the pore size range was slightly higher in the case of biocomposite foams, yet the overall porosity and interconnectivity was better in the case of biocomposite cryogels. Also, the density measurement data for these biocomposites indicate that both the foams and cryogels have the densities almost lying in the range of trabecular bone although foams have higher density as compared to cryogels. Therefore, these results provide a comparative study of two different kinds of biocomposite materials, i.e., biocomposite cryogels and foams and the results implicate that the prepared biocomposite foams and cryogels fulfill many basic requirements for a bone tissue engineered biomaterial. Thus, these biomaterials have the potential to develop into a biomaterial for bone tissue engineering purposes.

Prophylaxis, diagnostics and therapy are three fundamental tools used by the contemporary medicine to address health issues of the modern societies. Diagnosis and therapy are in a particularly close causative, spatial and temporal association. Recently coined term “theranosis” suggestively reflects that interdependence. One of the basic tools used by modern diagnostics are biomarkers - substances (often proteins such as enzymes, or hormones, but also cells or genes) whose presence (and concentration) are indicators of the presence of a particular disease or physiological process(es), both advantageous and disadvantageous, taking place in the organism. We have synthesized and studied a new hybrid adsorbent for compounds bearing adenine moiety, nucleotides in particular. An important example is adenosine triphosphate (ATP), which is a biomarker for neutrophilic airway inflammation and ischemia and may have considerable potential in the management of ischemic heart disease and stroke. Submicrometer sized silica gel particles were coated with a polyanion and a polycation bearing thymine chromophores. The polymer coated particles were found to selectively adsorb adenine and ATP, as compared to other nucleobases and nucleotides, respectively. The adsorption was enhanced by the irradiation of the particles in the presence of adenine which resulted in the molecular imprinting of adenine and improved selectivity vs. other nucleobases and nucleotides. ATP adsorption was strongly pH-dependent. Acknowledgement: Financial support from National Science Centre, grant “Hybrid adsorbents and sensors of adenine-containing compounds” is gratefully acknowledged. Project financed in part within the Foundation for Polish Science Team Programme co-financed by the European Regional Development Fund, PolyMed, TEAM/2008-2/6.

Hydroxyapatite (HA) is a well known bioceramic and major choice of hard tissue replacement implants. Although is it highly biocompatible, it is non magnetic that limits its application for treating malignant bone tumors and providing hyperthermia therapy. In order to introduce magnetism, ferromagnetic magnetite (Fe3O4) is mechanically mixed with HA. In the present work, HA-xFe3O4 (x: 5 wt.%, 10 wt.%, 20 wt.% and 40 wt.%) powder compositions were sintered using uniquely designed spark plasma sintering conditions (three stage sintering with final holding temperature of 1050 oC). All sintered samples were ~ 98% dense, and X- ray diffraction (XRD) characterization revealed no dissociation of HA or Fe3O4 materials after sintering. A systematic increase of saturation magnetization as well coercive field strength with Fe3O4 addition was recorded using vibrating samples magnetometer (VSM) measurements. In order to assess the cytocompatibility of developed, human fetal osteoblastic cell line (hFOB) were seeded for 48 hrs under standard culture conditions. Fluorescence and scanning electron microscopy confirm the proliferation of hFOB cells on the HA-based magnetic biocomposites.

Orthopedic interfaces such as the tendon-bone junction (TBJ) present unique challenges for biomaterials development. This work describes the development and analysis of a multi-compartment collagen-GAG (CG) scaffold with discrete mineralized and non-mineralized regions joined by a continuous interface. We report the role of discrete compartment structural properties on the overall mechanical and functional parameters of the multi-compartment scaffold. Non-mineralized collagen-GAG (CG), mineralized CG (CGCaP), and multi-compartment (CG-CGCaP) scaffolds were fabricated via lyophilization allowing independent control over scaffold microstructure, chemistry, and mechanical properties. We applied structural (histology, SEM, µCT), compositional (EDX, XRD), and mechanical (compression) characterization techniques to assess differences in the performance of single scaffold compartments versus the multi-compartment scaffold as a whole. Mechanical and permeability analysis identified significant differences in scaffold performance as a function of microstructural organization and composition. Multi-compartment scaffolds displayed heterogeneous properties, with the CG compartment representing the limiting factor in mechanics. Towards developing a platform for driving specific multi-lineage stem cell differentiation, we examined the proliferation, metabolic activity and gene expression profiles of human mesenchymal stem cells (hMSCs) in each scaffold compartment. hMSC proliferation and metabolic activity increased for both CG and CGCaP scaffolds in growth media, but key markers of early hMSC osteogenic differentiation (BSP, OC, and RUNX2) were elevated in the CGCaP compartment, suggesting guided hMSC differentiation based on scaffold properties alone. Osteogenic media supplementation led to further increases in osteogenic differentiation as measured by a significant increase in scaffold mechanical properties with long term (4 week+) culture relative to growth media. To better investigate distinct compartment behaviors, we have employed ultrasound elastography techniques to monitor spatial and temporal changes in local strain fields and fluid mobility environment within the scaffold. This tool allows us to monitor compartment-specific strain profiles in CG-CGCaP scaffolds and investigate spatial and temporal changes that occur at different regions within the scaffold with hMSC remodeling during osteogenic differentiation.

Denture stomatitis is a disease commonly associated with wearing of prosthetic appliances. Although Candida albicans is the predominant oral fungal pathogen, other species may also play an integral role in pathogenesis, such Candida glabrata and S. mutans. Studies have showed that coatings containing hydrophilic or zwitterion monomers are effective to reduce protein adsorption and also promote a reduction in microbial adhesion. Thus, the use of these coatings could reduce the development of denture stomatitis. Therefore, the purpose of this study was to evaluate the ability of experimental photoactivated coatings containing hydrophilic or zwitterion monomers to reduce the multi species (C. albicans, C.glabrata and S. mutans) biofilm adhesion (90 minutes) and biofilm formation (48 hours) to a denture base acrylic resin. Methods: Two hundred-eighteen disc-shaped specimens (10 x 2 mm) of microwave acrylic resin (Vipi-Wave) were prepared with rough or smooth surfaces and were either left untreated (control) or coated with one of the following experimental coatings (n=9/group): 3-hydroxypropyl methacrylate - HP and sulfobetaine methacrylate - S. The concentrations of these constituent monomers were 35%. After treatment with coatings, all specimens were stored in water for 48 h at room temperature for release of uncured residual monomers. Thereafter, the specimens were sterilized with sterile distilled water and ultrasonicated for 20 minutes, following by ultraviolet light exposure in a laminar flow chamber for 20 minutes. Subsequently half of the specimens of each varnish were exposed for 30 minutes in saliva. All specimens were inoculated with C. albicans, C.glabrata and S. mutans (1x107 cell/mL) to biofilm adhesion (90 minutes) or biofilm formation (48 hours) at 37°C under agitation. The number of viable microorganisms in the accumulating biofilm layer was evaluated by XTT method. Results: The absorbance values ranged from 0,28 to 1,58 for the evaluated groups. There was no significant difference between rough or smooth surfaces and among with or without saliva exposition, for biofilm adeshion or biofilm formation. Moreover, the control groups obtained the highest values of absorbance compared to experimental groups in all conditions. Conclusion: The experimental photoactived coatings produced a reducing in biofilm multi-species adhesion to a Vipi-Wave denture base acrylic resin. Grants: Fapesp 2010/00545-9, 2011/23543-4 and 2012/01528-6.

Collagen-apatite (Col-Ap) scaffolds have been employed for bone tissue engineering but their applications are limited due to their poor mechanical strengths. A hierarchical biomimetic Col-Ap scaffold has attracted much attention as the increased level of organization offers higher active surface area and better mechanical strength than traditional Col-Ap scaffolds. In the current study, we have developed a new technique to prepare Col-Ap scaffolds mimicking the hierarchical structure of natural bone. First, mineralized collagen hydrogel was prepared by a one-step co-precipitation method which includes collagen self-assembly and simultaneous in situ apatite precipitation in a collagen-containing modified simulated body fluid (m-SBF). Second, the mineralized hydrogel with different fibrillar concentrations were formed into shape using a simple plastic compression process. Third, the compressed hydrogel was frozen unidirectionally under different freezing temperatures and then lyophilized. The resulting scaffold exhibits a novel hierarchical structure. At the primary level, the scaffold is comprised of basic building blocks of hierarchically organized collagen fibers and poorly crystalline apatite nanoparticles mimicking the microstructure of natural bone. At the secondary level, each lamellae consists of many aligned nanolayers . At the tertiary level, the scaffold exhibits unidirectional macro porous structure with pore sizes ranging from 40 to 150 mu;m extending through the entire scaffold. Furthermore, the lamellar spacing and wall thickness are tunable depending on fibrillar density and freezing kinetics. This novel hierarchical lamellar scaffold exhibits increased anisotropic mechanic strength compared to equiaxed conventional lamellar structured scaffolds. To the best of our knowledge, this is the first report that a three-dimensional Col-Ap hybrid scaffold with controllable pore size and pore orientation ranging from nanoscale to macroscale is fabricated. In vivo evaluation of these scaffolds was also performed by loading the scaffold with mouse calvarial osteoblast cells in a two-hole mouse calvarial defect model. It was found that the Col-Ap scaffold well supported osteoblastic activities. It successfully repaired the critical-size defect after four weeks of implantation.

The potential of electrospun nanofibers as tissue engineering scaffolds and their use in many areas of application such as drug delivery, implantable devices has led to an increase in their research. We report here the fabrication of electrospun nanofibers from Poly(methyl methacrylate) (PMMA) and Poly (D, L-lactide-co-glycolide) (PLGA), hybrane polymers and other biodegradable polymers functionalized with hydroxyapatite, calcium magnesium silicate nanoparticles and hydroxyapatite/silver nanoparticles using single and two different solvents systems (chlorofoam and dichloroformamide). The morphology, structure, mechanical and thermal properties of the PMMA, PMMA/HA, PLGA, PLGA/HA, PLGA/CaMgSiO6 composite nanofibers were characterized by scanning electron microscopy (SEM), energy dispersive spectrum (EDS) spectroscopy, X-ray diffraction (XRD), Fourier transform-infrared (FT-IR) spectroscopy and thermogravimetry (TGA) and differential scanning calorimetry. The ability of the composite electronspun nanofibers to sustain and support the growth of bone cells was investigated. The FESEM images revealed that the nanofibers are well-oriented and incorporated with the nanoparticles well distributed in on the nanofibers. The energy dispersive spectrum (EDS) confirmed the inclusion of the nanoparticles on the surface of the nanofibers.

Intracellular delivery of nucleic acids as gene regulation agents typically requires the use of cationic carriers. Yet, issues related to cellular toxicity or immune responses often hamper their attractiveness as therapeutic candidates. The discovery that spherical nucleic acids (SNAs), structures comprised of densely packed, highly oriented oligonucleotides covalently attached to the surfaces of nanoparticles, can effectively enter more than 50 different cell types presents a potential strategy for overcoming the limitations of conventional transfection agents. Here, we demonstrate that the rapid uptake kinetics and intracellular transport of SNAs stem from the arrangement of oligonucleotides into a three-dimensional architecture, which facilitates their endocytosis via the lipid-raft/caveolae pathway. Moreover, endocytosis of SNAs is regulated by heterotrimeric G proteins, dependent on microtubules, and mediated by Class A scavenger receptors. These results reinforce the notion that SNAs can serve as a therapeutic payload and targeting structure to engage biological pathways not readily accessible with linear oligonucleotides.

Nanoparticles are being developed as drug delivery systems because of their ability to increase drug solubility, improve otherwise unfavorable pharmacokinetics, protect encapsulated drugs from degradation, provide controlled and sustained release of a drug and, target specific sites within the body thereby improving the drug efficacy and minimizing the systemic side effects. We have developed polymeric pH-responsive expansile nanoparticles (eNPs) that encapsulate hydrophobic drugs, such as the chemotherapeutic agent paclitaxel. Paclitaxel (Pax) is maintained within eNPs at neutral pH (7.4); when eNPs are endocytosed by tumor cells, the local pH within the endosome / lysosome decreases to ~ pH 5. This acidification cleaves acetal-protecting groups on the polymer resulting in a compositional change from hydrophobic to hydrophilic and infiltration of water into the particle. Hydration of eNPs, and subsequent swelling, allows Pax to diffuse out leading to significant intracellular accumulation of the drug and, consequently, cell death. Currently, we are investigating the use of paclitaxel-loaded eNPs (Pax-eNPs) for treating mesothelioma. Mesothelioma affects the pleural, pericardial, and peritoneal linings of the thorax, and, though rare, is universally fatal. Previously published results demonstrate that eNPs administered to the intraperitoneal (IP) space localize to IP mesothelial tumor without the need for targeting ligands. We hypothesized that this localization would provide increased accumulation of Pax, administered as Pax-eNP, thereby improving its efficacy compared to the equivalent, standard-of-care formulation—Pax in Cremophor/Ethanol (Pax-C/E). To test this hypothesis, we utilized a previously developed model of established i.p. mesothelioma in mice. Animals (n = 6) bearing established tumor burden were treated with four weekly doses of either saline, unloaded-eNPs, Pax-C/E, or Pax-eNPs. Animals receiving Pax-eNP treatment showed a significant improvement in overall survival compared to all other treatment and control groups. Additionally, four of six Pax-eNP treated animals demonstrated a complete clinical response. These results suggest that Pax-eNPs may offer a novel means for effectively delivering Pax to IP mesothelioma tumors thereby improving overall treatment outcomes.

One of the advantages of RNA Nanotechnology is the feasibility to construct therapeutic particles carrying multiple therapeutics with defined structure and stoichiometry. Herein, we report the construction of thermodynamically and chemically stable X-shaped and tri-star shaped RNA nanoparticles to carry one, two three or four siRNA, miRNA, RNA aptamer, or miRNA using reengineered RNA fragments derived from the central domain of the motor pRNA of bacteriophage phi29 DNA packaging motor. The nanoparticles self-assemble very efficiently in the absence of divalent salts, resistant to denaturation by 8 Molar urea, and remains intact at ultra-low concentrations. The delta G of the nanoparticles is extremely low and the slope of the temperature melting curve is close to 900. We proved that each arm of the helices in the X-motif or tripods can harbor one siRNA, ribozyme, or aptamer without affecting the folding of the central X or 3WJ core, and each daughter RNA molecule within the nanoparticle fold into respective authentic structure and retain their biological and structural function independently. Gene silencing effects were progressively enhanced as the number of the siRNA in each pRNA-X nanoparticles gradually increased from one to two, three, and four. More importantly, systemic injection for bio distribution assay of the ligand-containing nanoparticles into the tail-vein of mice revealed that the RNA nanoparticles remained intact without showing any signs of dissociation or degradation; and strongly bound to cancers without entering liver, lung or any other organs or tissues. Pharmacokinetic analysis revealed that its half-life was extended 100-fold compared to the siRNA counterpart. Particles tested in vivo revealed that the they did not induce cytokines, interferon-I, antibody, and toxicity while retaining favorable pharmacokinetics profiles. References: (1) Guo P. 2010. The Emerging Field of RNA Nanotechnolog. Nature Nanotechnology, 5: 833-842. (2) Shu D, Shu Y, Haque F, Abdelmawla S and Guo P. 2011. Thermodynamically stable RNA three-way junction for constructing multifunctional nanoparticles for delivery of therapeutics. Nature Nanotechnology. 6:658-67.

Creation of effective cancer therapeutics requires understanding of microenvironment gradients in tumors. Metabolic heterogeneity and diffusion resistance both significantly reduce the efficacy of most standard cancer therapies. Fortunately, therapeutically resistant microenvironments are unique to tumors and are distinguishing characteristics that enable targeting. Because of this dichotomy, tumor heterogeneity is both a curse and a blessing. My group and I have developed multiple tools to quantify tumor drug response, including in vitro tissue models and mathematical models. Our microfluidic tumor-on-a-chip device is capable of quantifying the transport properties of experimental therapeutics on tumor tissue for prolonged exposure times. With the microfluidic device we have demonstrated the importance of cell binding and particle release for drug efficacy. The mathematical tool we developed characterizes transport properties for individual patients based on dynamic contrast enhanced magnetic resonance imaging. We have shown that this mathematical model predicts drug efficacy and the likelihood that a patient will respond to neoadjuvant chemotherapeutic therapy. In addition, we have shown with these tools that 1) an optimum molecular weight exists for which drugs are more effective and 2) positively charged gold nanoparticles are more effective at delivering therapeutic payload to cells surrounding blood vessels. Bacteria have unique capabilities that make them perfect anticancer agents. Current treatments, including chemotherapy and radiation, are toxic to normal tissue and do not completely destroy all cancer cells. Three major causes of these problems are incomplete tumor targeting, inadequate tissue penetration and limited toxicity. Bacteria can overcome these limitations because they specifically target tumors and can be engineered to perform a wide array of functions. We have engineered Salmonella that 1) produce toxic compounds, 2) have genetic circuits that amplify drug production, 3) have gene promoters that can be induced by chemical and radiation triggers, 4) can sense the local tumor microenvironment, and 5) specifically chemotax to intratumoral regions that are unresponsive to standard therapies. With these engineered organisms we have shown that responsiveness to external signals enables precise control of the location and timing of cytotoxicity, and promotes tumor shrinkage and increased survival in mice. We envision that these results will lead to improved tools and more effective, personalized therapeutics.

RNA interference (RNAi), which can selectively knockdown target genes, has shown great potential in the treatment of various diseases. Thus far, numerous nanoparticle (NP) platforms such as lipoplex and polyplex have been developed to facilitate the safe and effective delivery of small interfering RNA (siRNA), which represents a major hurdle for the clinical applications of RNAi. Nevertheless, these NP systems lack the sustained siRNA release property, and thus can only induce transient gene silencing due to the short lifetime of siRNA. Therefore, the delivery of siRNA using controlled-release NPs would be necessary for achieving sustained gene silencing. Herein, we will present a novel NP platform for safe and effective siRNA delivery in a sustained manner, which can be developed through self-assembly of biodegradable and biocompatible polymers and lipids. The lipid-polymer hybrid NPs show excellent in vitro knockdown efficacy at low doses of siRNA, and promising in vivo results for delivering siRNA to xenograft tumors. More importantly, these NPs can control the temporal release of siRNA, with the half-release time of ~ 9 days, for sustained silencing of target gene expression. Results demonstrate that the luciferase expression is less than 15% at day 6 when the lucifrease-expressed HeLa cells were transfected with the NPs containing 40 pmol anti-luciferase siRNA for 1 day, and is about 30% at day 10. As a comparison, the luciferase expression is largely recovered (~ 85%) at day 6, after 1 day transfection with lipo2000-siRNA complexes (containing 40 pmol siRNA). Furthermore, this NP platform has been applied to co-deliver siRNA (e.g., anti-Bcl2) and chemotherapeutic drugs (e.g., taxanes) for the effective treatment of drug resistant cancers. We believe that the lipid-polymer hybrid NP platform with controlled release of siRNA could hold potential in both fundamental studies and clinical applications.

Ovarian cancer ranks as the sixth most common cancer worldwide and the seventh leading cause of cancer-related deaths in women mainly due to late stage diagnosis. Current treatments include laparotomy and vigorous chemotherapy; however, disease relapse occurs in 70% of cancer patients due to chemoresistance. Alternative treatment methods for ovarian cancer are necessary and nucleic acid-based cancer therapies have been helmed as a potential solution. The development of non-toxic and efficient gene and siRNA delivery systems is therefore crucial for the advancement of this cause. Some of the factors to consider in the design of nucleic-acid carriers include low toxicity, ability to protect the nucleic acids from enzymatic degradation and the ability to deliver cargo into the cell and unload it in the cytosol. Polyethylenimine (PEI) 25kDa is currently the gold standard as a polymer-based gene and siRNA carrier. However, its usefulness in the clinics is hindered by high toxicity levels. In light of this, low molecular weight PEI 2kDa has gained much interest due to its non toxic properties. However, it is inefficient as a nucleic acid carrier. To address the problems of low efficiency, our lab has developed a facile and efficient method to substitute primary amine in PEI with various hydrophobic functional groups in a one-pot reaction step. Remarkably, hydrophobic modifications greatly enhanced transfection efficiency PEI 2 kDa as evidenced by luciferase transfection results. The modification did not affect PEI&’s buffering capacity significantly compared to their unmodified counterpart and this is important for endosomal escape after cellular uptake. In addition, our studies showed that only a single group modification was needed to increase transfection efficiency by at least 20 fold on both liver carcinoma cell line, HepG2, and ovarian cancer cell line, SKOV-3. Moreover, the modified PEIs were able to bind and pack genes into nanoparticles (<200 nm) with small polydispersity indexes (PDI), which is important for in vivo applications. The ease and flexibility of the modifications also allows for the mix-and-match of different side groups to form multi-functionalized PEI equipped with targeting moieties such as sugars, like mannose, which increases specificity to mannose receptor-rich dendritic cells. This opens the possibility for potential use for the modified PEI in immunotherapy for cancer.

Autonomously moving micro-objects, or micromotors, have attracted the attention of the scientific community over the last decade, but the incompatibility of phoretic motors with solutions of high ionic strength and the use of toxic fuels have limited their applications in biologically relevant media. We demonstrate that ultrasonic standing waves in the MHz frequency range (commonly used by medical ultrasound devices) can levitate, propel, rotate, align and assemble metallic micro-rods (2 µm long and 330 nm diameter) in water as well as in PBS buffer solution. Fast axial motion of metallic micro-rods at ~200 mu;m/s was observed at the resonant frequency, corresponding to a driving force of ~1 pN. Segmented metal rods (AuRu or AuPt) were propelled unidirectionally with one end (Ru or Pt, respectively) consistently forward. A self-acoustophoresis mechanism based on the shape asymmetry of the metallic rods is proposed to explain this axial propulsion. The ability to control the speed, steer the direction, shift the location and spin along the axis of the ultrasonically driven micromotors open the possibility of potential biomedical applications.

Increasing numbers of advanced strategies for in vivo tissue imaging and drug delivery are being applied to current biomedical research. We describe here our work on embedding diverse chemical and biological handles into apoferritin protein nanoparticles with the aim of modifying the apoferritin with drugs, imaging agents and tailoring a variety of hybrid protein scaffolds. The hollow core of these functionalised protein nanoparticles can be utilised to encapsulate a PbS nanocrystal (quantum dot) or drug molecules. Previously, we have reported that PbS quantum dots (QD) which emit in the near infrared ‘optical window&’ (700-1100 nm) can be entrapped in the cavity of an apoferritin shell [1], and hence offer minimal tissue absorption in vivo. Our current interest is focused on imparting human apoferritin with four different types of functional groups either by genetic manipulation or enzymatic modification: (1) we have expressed genetically modified human ferritins in bacteria to generate recombinant proteins bearing the non-canonical amino acid L-azidohomoalanine at target sites; (2) we have designed fusion genes to localise a luciferase on the outer surface of apoferritin; (3) apoferritin mutants with cysteine residues have been site-specifically introduced into protein scaffolds; (4) the use of E.coli biotin ligase enables the AvitagTM sequence of apoferritin to be conjugated with an azide-bearing biotin analogue. Using bioorthogonal “click chemistry” and thiol-conjugation, a range of targeting molecules and chemical probes can be covalently conjugated to apoferritin scaffolds. Moreover, by combining a variety of functionalised apoferritin forming hybrid protein cages, a series of versatile multifunctional nanoparticles for deep-tissue imaging and drug delivery can be produced. [1] B. Hennequin, L. Turyanska, T. Ben, A.M. Beltrán, S.I. Molina, M. Li, S. Mann, A. Patanè and N.R. Thomas, Adv. Mater. 20, 3592 (2008).

Gold nanoparticles (AuNPs) have been extensively used in recent years for biomedical applications1 due to their unique properties such as the presence of a strong absorption band (SPR) in the visible region and its relatively ease of the functionalization by the high affinity of the bond formed between Au and S atoms2. Besides, small interfering-RNAs (siRNA) show significant potential to down-regulate specific abnormal gene expression in cancerous or viral-infected cells. In our case, the target selected has been the proto-oncogene c-Myc. To overcome the problem of the low rate of transfection by itself the dsRNA have been successfully conjugated to AuNPs previously optimized with other molecules that promote the efficient targeting and transfection (TAT, RGD, carbohydrates) and detection (fluorophores). The AuNPs used have a core of 14-nm synthesized by the method of Turkevich and Frens, followed by its stabilization with thiolated chains of polyethylenglycol that end in carboxylic and azide groups used for the binding of the molecules previously described. The attachment of the siRNA has been achieved by two different approaches, either through electrostatic interactions or direct binding of the Au atoms of the core with thiol-modified oligonucleotides3. For this purpose, two different models of AuNPs have been completely developed. In the first approach, the surface has been fully saturated with PEG chains followed by a further functionalization with a highly charged molecule while in the second case the saturation has been decreased so as to leaving free spaces for the siRNA to get close to the core and attach to it. In both cases stable and multifunctionalized AuNPs have been obtained and controlled. The in vitro and in vivo studies carried out with a cancer cell line, Hydra and C57BL/6j mice have confirmed a high efficiency for the specific silencing of c-Myc expression in these three biological systems. 1. El-Sayed I. H.; Huang X.; El-Sayed M. A.; Cancer Letters, 2006, 239, 129-135. 2. Sperling R. A.; Parak W. J.; Phil. Trans. R. Soc. A., 2010, 368, 1333-1383. 3. Alivisatos A. P.; Johnsson K. P.; Peng X.; Nature, 1996, 382, 609-611.

This study aims to provide understanding on the relationships between stress relaxation mechanisms and the macroscopic viscoelastic properties of collagen matrices. The relaxation behavior of collagen matrices are studied with the effects of hydration, crosslinking and initial stress. Hydrated collagen gel and dehydrated collagen thin film were exploited as two different hydration levels of collagen matrices. Genipin solution (0.03%, 0.1%, and 0.25%) was used to induce different levels of crosslinking in collagen matrices. Biaxial stress relaxation tests were performed with different initial stress level to characterize the viscoelastic behavior of collagen matrices. The rate of stress relaxation of both hydrated and dehydrated collagen matrices shows a linear initial stress level dependency. Furthermore, increased crosslinking reduces viscosity in collagen gel, but the effect is negligible for thin film, which is due to the tight hierarchical structures of the collagen thin film forming from dehydration preventing fiber and fibril sliding. Relaxation time distribution spectrum was obtained from the stress relaxation data by inverse Laplace transform. For most of the collagen matrices, three peaks at the short (0.3s ~ 1s), medium (3s ~ 90s), and long relaxation time (>200s) were observed in the continuous spectrum, which corresponds with relaxation mechanisms involve fiber, inter-fibril, and fibril sliding. The intensity of the long-term peaks increases with higher initial stress levels indicates the engagement of collagen fibrils at higher levels of tissue strain. Splitting of the middle peak was observed at higher initial stress levels suggesting increased structural heterogeneity at the fibril level with mechanical loading. Finally, a viscoelastic constitutive model combining hyperelastic and generalized Maxwell model was established with viscous material parameters obtained directly from analysis of the relaxation time spectrum. The simulations fit the experimental results well. The coupled experiment-modeling method is promising in connecting the macro-level viscoelastic behavior of collagen matrices with micro-level structure changes.

Femtosecond laser micromachining and microfabrication have been shown to be powerful methods to produce complex microstructures for various applications, from optical devices to biology. The nonlinear nature of the light matter-interaction achieved with fs-pulses, confines the interaction to the focal volume of the laser beam, allowing the fabrication of microstructures by moving the laser focus throughout the sample. In this work, we describe the use of fs-laser microfabrication to produce micro-environments specially designed for biological applications. We have explored the possibilities of doping the fabricated microstructures with active components, such as antibiotics and sensitizers, in order to investigate aspects related to photodynamic therapy and drug action in the developed micro-environments. The laser microfabrication was carried out with 100-fs pulses from Ti:sapphire lasers, either amplifiers or oscillators, operating at 800 nm. The sample was positioned in the axial z-direction using a motorized stage, and the laser beam was scanned in the resin x-y-direction with a set of galvanic mirrors. After fabrication, the microstructures were characterized by scanning electron micrographs. Such microstructures were then used as platforms to investigate the growth of bacteria (E. coli) and cells, which was performed by optical transmission, fluorescent and confocal microscopy. The approach employed here is a promising alternative for the fabrication of tailor-made scaffolds for biological applications, opening new venues for advanced drug delivery systems.

Adult bone marrow derived mesenchymal stem cells (MSCs) represent an important source of cells for tissue regeneration. Control of MSC migration and homing is still unclear. The goal of this study was to identify potent chemoattractants for MSCs and characterize MSC chemotaxis using a microfluidic device as a model system and assay platform to observe cells in a stable linear chemokine gradient. The three chemokines compared in this study were Stromal Derived Factor-1 (SDF-1), Thymus Chemokine-1 (TCK-1), and AMD 3100. Microfluidic devices made of polydimethysiloxane (PDMS) were fabricated by soft lithography techniques and successfully coated with (3-aminopropyl)-trimethoxysilane for optimal cell attachment. Cell movement in response to a linear chemokine gradient was captured by timelapse photos and tracked over 20 hours. Chemokine potency was measured via several chemotaxis parameters including: velocity in the direction of interest (V), center of mass (Mend), forward migration indice (YFMI), and directionality (D). Cell movement throughout the 20 hour time period was mapped onto a cell tracking plot to compare the migratory paths of the cells. The following results were measured in the direction of interest (towards higher concentrations of chemokine): For velocity, only cells in the SDF-1 condition had a statistically significant (p=.0028) average velocity (V=.301 ± .270 um/min) when compared to the control condition (V=.016 ± .127 um/min). For the center of mass, where the displacement of cells from their starting positions were compared, again only SDF-1 (Mend= 99.417 ± 70.913 um) stimulated statistically significant (p = .00066) displacement of cells compared to the control condition (Mend = 4.421 ± 26.397 um). For the forward migration index, the efficiency of cell movement was measured. Indices in the SDF-1 (YFIM = .355 ± .229) and AMD 3100 (YFIM = .347 ± .237) conditions were statistically significant (p = .0002 for SDF-1 and p = .0005 for AMD 3100) when compared with the control index (YFIM = .036 ± .134). For the directionality index, which describes the directness of cell movement, all three chemokines, SDF-1 (D = .459 ± .173), AMD 3100 (D = .445 ± .169), and TCK-1 (D = .403 ± .208) induced statistically significant (p= .0019, p= .0025, and p= .01 respectively) directness in cell movement when compared with the control result (D = .240 ± .184 ). This study demonstrated that a microfluidic device can be a viable platform for chemotaxis studies. A stable linear chemokine gradient was maintained over an extended time scale to obtain biologically relevant cell migration results. SDF-1 was statistically determined to be the most potent chemoattractant in this study; these chemoattractive properties promote its use in the future development of biomaterials to recruit MSCs to an injury site and enhance bone regeneration.

Cells sense and respond to chemical and physical cues in their environment, which in vivo is provided by other cells and the extracellular matrix (ECM). The ECM is a 3-D fibrillar network with tightly regulated fiber diameter, composition and orientation that gives structural support and serves as an insoluble mechano-sensitive signalling network. It has proved challenging to mimic the ECM and achieve similar control over fibers within tissue engineered constructs. Traditional fabrication techniques such as phase separation and electrospinning enable the formation of highly porous networks of fibers with diameters as small as tens of nanometers but are limited in their control of size distribution, orientation, shape, and composition. We hypothesized that our bio-inspired, surface-initiated assembly approach would enable fabrication of protein nanofibers and nanostructures with enhanced control by mimicking cell-mediated matrix assembly. Here we report surface-initiated assembly of monodisperse fibronectin nanofibers ~50 nm thick with length to width aspect ratios of 2.5:1 and 10:1. Prior to release, nanofibers had initial widths of 20.21 + 0.32 mu;m and 20.32 + 0.41 with lengths of 50.14 + 0.37 mu;m and 199.95 + 0.65 mu;m, respectively. Upon thermally triggered release from the poly(N-isopropylacrylamide) surface, the nanofibers detached from the surface and were observed to contract as the constituent ECM molecules refolded. Released nanofibers had final widths of 3.85 + 0.26 mu;m and 3.97 + 0.31 mu;m and lengths of 35.23 + 2.18 mu;m and 162.57 + 3.63 mu;m, respectively. This resulted in fibronectin nanofibers with final aspect ratios of ~10:1 and ~40:1, with standard deviation at <10% of the width or length. We also show the ability to form nanostructures with shapes more complex than simple nanofibers such as star and cross patterns, which cannot be created using typical fiber fabrication techniques. In summary, these results demonstrate our ability to engineer fibronectin nanofibers that are monodisperse, and similar results are achievable using other ECM proteins such as laminin, fibrinogen and collagen type IV. In the future, we hope to leverage surface-initiated assembly to engineer ECM nanofibers with specific chemical composition, size distribution and orientation for use in cardiac tissue engineering applications.

Strokes are the leading cause of adult disability. However, current treatments are focused on rest and rehabilitation and lack the ability to promote wound healing and tissue regeneration of the stroke cavity. Hydrogel scaffolds are currently being explored as therapeutics to aid in regeneration and healing of the stroke cavity. We have been investigating the design of cell-instructive hydrogels that can deliver bioactive signals to promote angiogenesis and repair and also be used to deliver neural progenitor cells to the stroke cavity. We found that Hyaluronic acid (HA) hydrogels crosslinked with matrix metalloproteinase degradable peptides did not elicit an inflammatory reaction in normal and stroke brain, indicating that it is an ideal material to design cell instructive scaffolds for stroke treatment. The mechanics of the hydrogels, however, did impacts their inflammatory reaction, with stiffer hydrogels resulting in increase immune cells at the injection site. To induce angiogenesis into the stroke site, vascular endothelial growth factor (VEGF) delivery is being engineered as well as integrin adhesion into the material. In vitro, the presentation of VEGF within biomaterials (slowly released, covalently bound or clustered) was found to impact endothelial cell activation (VEGF receptor-2 phosphorylation) and the resulting morphology of blood vessels formed. Stem cell survival is a critical for the success of transplantation strategies. To improve transplanted cell viability, we investigated the effect that each component of the transplantation process has on cell survival. We found that the needle gauge, infusion speed and the use of a hydrogel as the delivery medium impact viability with larger gauges, slower infusion speed and the presence of the hydrogel resulting in enhanced cell survival.

Living cell encapsulation currently attracts much interest owing to the new applications offered by this technology such as bioreactors, biocatalysis, biosensors or cell therapy. In the medical field, this technology is particularly promising to overcome the shortage of organ donors. In fact, the progress made in this specific domain could improve the compatibility between organisms and current encapsulating materials. For instance, in cell therapy, biocompatibility encompasses three major criteria: (1) the use of materials that are compatible with both the encapsulated cells and the human body (to target a graft for an artificial organ), (2) the development of synthesis methods that permit the in-situ construction of a matrix without damaging the cellular integrity and finally (3) the control of pore size in the host material, allowing nutrients and metabolites to permeate throughout the support. The encapsulation of animal cells is a challenging task. In particular, immuno-isolation is a key factor to successfully develop cell therapy technologies where cells are protected against rejection by the immune system whilst allowing nutrients and metabolites to be evacuated. This protection can only be conferred by a biocompatible and semi-permeable membrane. Moreover, the materials should be sufficiently resistant with time to ensure long-term implantation of the graft. This work investigates the in vivo biocompatibility of mechanically resistant hybrid beads, a model of artificial organ, synthesised via the encapsulation of living cells (HepG2) within alginate-silica composites. Two types of beads (alginate-silica hybrid (AS) or alginate/silica hybrid subsequently covered by an external layer of pure alginate (ASA)), with or without HepG2 cells, were implanted into several female Wistar rats. After four weeks, the potential inflammatory local response that might be due to the presence of materials was studied by optical microscopy. The results showed that the performance of ASA beads was better compared to AS beads where less abnormal rat behaviour and less inflammatory cells in histological sections were observed in the case of ASA beads. This suggests that a direct contact of silica gel with living tissues has to be avoided. For that reason, alginate-silica composite materials coated with an extra-alginate shell offer much promise in the development of robust implantation devices and artificial organs. The present work opens a new avenue to develop biocompatible biomedical devices for Diabetic diseases.

Saccharomyces cerevisiae yeast cells have been encapsulated with lightly cross-linked pH-responsive layer-by-layer (LbL) hydrogel nanoshells made from amine-functionalized polymethacrylic acid. Enhanced cell viability rate (~90%) demonstrated high biocompatibility of such synthetic hydrogels in comparison to electrostatically assembled LbL shells. By keeping encapsulated cells above the isoelectric point, the cell proliferation process was suppressed and delayed in comparison to the cells held at pH below the pKa of methacrylic acid (pKa=5.0). At the same time, expression of green fluorescent protein was not affected by pH variation and showed stable green fluorescence at pH 7. Progressive ionization and charge accumulation within synthetic shells evoked a structural change in the outer shell which affected the membrane transport. We suggest that the variation in surface charges caused by deprotonation/protonation of unbound carboxylic groups in the nanoshells controlled cell growth and cell function, which can be utilized for external chemical control of cell-based biosensors. The ability to manipulate perceptible response from the encapsulated cells by keeping them in “dormant” conditions (constrained replication) for the extended time can be rewarding for biosensing applications when the early onset of cell growth can compromise the long-term performance.

Electrospun fibrous membranes have been investigated extensively in vascular tissue engineering because of their similar structure to the natural extracellular matrix as well as suitable mechanical properties to natural vessels. In the reconstruction process of vascular vessels, vascular endothelial cells (VECs) proliferated firstly on the inner lumen of the scaffold, and then the growth of vascular smooth muscle cells (VSMCs) on the outer layer could make the regeneration tissue more stable. The designed different release devices to control the rapid delivery of vascular endothelial growth factor (VEGF) and prolong release of platelet derived growth factor-bb (PDGF) were required. The goal of this study was to develop a bioactive composite electrospun scaffold loaded with dual VEGF and PDGF to promote the cells adhesion, proliferation, and vascular regeneration. For preparation of the composite fibrous scaffolds, the hydrogel/coaxial electrospun fibrous membrane loaded with VEGF was used as the inner layer, and the emulsion/coaxial electrospun fibrous membrane loaded with PDGF was selected as the outer layer. The release behaviors of VEGF and PDGF were evaluated, while the adhesion and proliferation of VECs and VSMCs on the membranes were examined by both scanning electron microscope and laser confocal scanning microscope in vitro. Results showed that the cumulative release of VEGF was about 66% at 6 days, and PDGF exhibited sustained release with only 38% at 6 days, which reached about 90% at 28 days. The cell culture results showed that the VECs rapidly adhered and proliferated in the first 6 days, while the VSMCs had a fast growth rate after the 6th day. The cell growth behaviors were consistent with the release behaviors. The tubular scaffold with 2 mm diameter was implanted into rabbit carotid artery in vivo to evaluate the potential applications as small-diameter vascular grafts. After implantation for 1 month, the tubular scaffolds permitted VECs adhesion on the lumen, and VSMCs grew on the outer layer without thrombosis. This study suggests that the electrospun scaffold combined with dual VEGF and PDGF is potentially to be feasible substitutes for vascular reconstruction. Acknowledgements Fengxuan Han, Hong Zhang and Xiaoling Jia contributed equally to this work. Financial support of this work was provided by Natural Science Foundation of Tianjin, China via grant No. 09JCZDJC18600. Corresponding authors: Yubo Fan, Tel. & Fax: 86-10-82339428, E-mail: [email protected]; Xiaoyan Yuan, Tel. & Fax: 86-22-87401870, E-mail: [email protected]

The formation of a stable vascular network in a scaffold is one of the most challenging tasks in tissue engineering and regenerative medicine. Despite the common use of porous scaffolds in these applications, little is known about the effect of pore size on the neovascularization in these scaffolds. Here we fabricated poly(D, L-lactide-co-glycolide) (PLGA) inverse opal scaffolds with uniform pore sizes of 79, 147, 224, and 312 mu;m in diameter and then used them to systematically study neovascularization in vivo. Histology analyses revealed that scaffolds with small pores (<200 mu;m) favored the formation of vascular networks with small vessels at high densities and poor penetration depth. By contrast, scaffolds with large pores (>200 mu;m) favored the formation of vascular networks with large blood vessels at low densities and deep penetration depth. Accordingly, we proposed a model to describe neovascularization in a three-dimensional porous scaffold based on collective effects of the surface pore size, the pore size and the window size of the scaffold. In addition, we have developed a novel technique using multiscale photoacoustic microscopy (PAM) to non-invasively monitor neovascularization in an inverse opal scaffold in vivo up to six weeks. By combining with optical coherence tomography (OCT) based on optical scattering contrast, we were able to simultaneously image and analyze the vasculature and the scaffold. By quantifying the PAM volumetric data, we could further examine the effect of pore size (200 µm versus 80 µm) of a scaffold on neovascularization. The data collected from PAM were consistent with those obtained from traditional invasive, labor-intensive histologic analyses. We expect our finding about the effect of pore size on neovascularization to be highly useful in serving as a guideline for future design of porous scaffolds by providing a new insight into the mechanisms that control the ingrowth of blood vessels into porous scaffolds. Meanwhile, we believe that our non-invasive imaging technique will find wide applications in the investigation of neovascularization in tissue-engineered 3D scaffolds.

Nanoparticle-based cancer therapeutics promises to improve safety and efficacy of cancer therapy. However, fabrication of consistent theranostic nanoparticles with high and controllable therapeutic loading remains a challenge, primarily due to the cumbersome, multi-step synthesis processes conventionally applied. We present a simple and highly controllable method for assembly of theranostic nanoparticles, which may greatly reduce batch-to-batch variation. The nanoparticle system is comprised of superparamagnetic iron oxide nanoparticle (SPION) core. For the surface coating of nanoparticle, a series of multicomponent and biodegradable poly (beta-amino ester) (PBAE) and poly (beta-amino amide) (PBAA) copolymers have been developed. The nanoparticles are loaded with either chemotherapeutic agent doxorubicin (DOX) or therapeutic radionuclides. Polymers with also contain biotin as targeting moieties. Here the polymer pre-loaded with drug or radionuclides is directly assembled to the surface of SPIONs forming a drug-loaded nanoparticle. The loading efficiency, colloidal stability and magnetic properties of nanoparticles were examined. The drug release analysis showed fast DOX release at endosomal pH and virtually no release at physiological pH, demonstrating pH-sensitive drug release kinetics. In vitro evaluation of DOX-loaded nanoparticles on drug-resistant C6 glioma cells demonstrated a 300% increase in cellular internalization at 24 h post-treatment and 65% reduction of IC50 at 72 h post-treatment when compared to free DOX. The in vivo evaluation of nanoparticles will employ pretargeted strategy in xenograph mouse model. A fusion protein containing streptavidin and antibody units will be injected first, followed by biotinylated nanoparticles. Blood half-life, biodistribution and anti-tumor activity of both drug-loaded and radionuclide-loaded nanoparticles will be determined. These nanoparticles could serve as a foundation for building smart theranostic formulations for sensitive detection through MRI or gamma imaging and effective treatment of cancer by overcoming drug/radiation resistance.

This work investigates the rheological properties of nanocomposite hyaluronic acid (HA)-Clay, gelatin-clay and HA-gelatin-clay hydrogels physically crosslinked by varying the clay concentration. Resulting hydrogels were subjected to oscialltory shear rheometry which allows the evaluation and comparison of the shear storage moduli (G'), an index of the stiffness of the hydrogels. The stress and frequency sweep measured G' and G" as a function of clay concentration and the results suggested the formation of a stable, three dimensional network. The stiffness of the hydrogel increased with the increase in clay concentration. SMFM and Phase contrast microscopy done on these hydrogels with dermal fibroblasts indicated that cells responded to the stiffness of the hydrogel and could be cultured on these surfaces efficiently. To enhance the attachment of cells, gelatin was used to coat the surface of HA- Clay hydrogels producing a more biocompatible hydrogel. Effect of glucose as a physiological additive indicated a drastic decrease in the hydrogel stiffness at the diabetic glucose concentration. Finally, the controlled release of Salicylic acid indicated that HA-clay hydrogels can be used as efficient drug delivery system. Our results emphasize the preparation of a biocompatible hydrogel with no chemical additives that can aid in controlled drug delivery and biomedical applications, including rigid cell scaffold structures.

We performed a systematic study of the bactericide and bacterial anti-adhesive properties of nanocrystalline diamond (NCD) and microcrystalline diamond (MCD) in comparison to other important industrial materials, such as copper, silver, polyethylene (Poly), and stainless steel (SS). The data show that NCD has better bactericide and bacterial anti-adhesive activity than Ag, but not as good as Cu. MCD, on the other hand, does not appear to have significant anti-bacterial activity. The superlative properties of NCD, such as mechanical hardness, resistance to oxidation and corrosion, and biological compatibility with blood and tissue, enable its use as antibacterial coating for medical implants. This is an application that cannot be achieved by Cu, which is known to be a highly effective antibacterial material but is not biocompatible. We also discuss possible underlying mechanisms to help understand the bactericide and bacterial anti-adhesive properties of the NCD surface. Diamond & Related Materials 22 (2012) 77-81. E-mail address: [email protected] (O. Medina).

Macro self-assemblies of lipids into different structures depend on parameters like temperature and water content. In bulk, phases are formed and stable only as long as water is not removed from the system. Thus, transfer and handling of these lipid phases is difficult. Carbon nanotubes are compatible with amphiphilic molecules like lipids due to their hydrophobic nature. Grown in the form of aligned arrays, they demonstrate the ability to act as 3D scaffolds for the assembly and possible function of biomolecules. In this work we have investigated self-assembly of lipid molecules in the presence of aligned arrays of carbon nanotubes using Monoolein, Dioleyolphosphatidylcholin and Dioleyolphosphatidylethanolamine. We found surface adsorption of the lipids within the arrays in a precisely aligned way with respect to the CNT growth axis in the narrow channels between the nanotubes. We have observed various lyotropic nanostructures that are found for corresponding lipids in the bulk under dry and hydrated conditions. Most interestingly, we were able to generate a cubic phase assembly within the CNT arrays, forming a solid, transferable 3D ordered network of lipid with incorporated water channels. This makes clear, that we are not focussed on stabilising the CNTs in aqueous suspensions rather than creating and investigating macro assemblies of different lipids around the CNT arrays in a 3D structure. We suggest that these lipid/CNT scaffolds would act as templates for bigger molecules, potentially creating a crystallisation cell for proteins. Most recent progress reveals that the lipid/CNT arrays assemblies act as docking structures for protein incorporation, which very strictly depends on the lipid phase formed. This is manifested in a widening of the lipid structure by a value related to the incorporated protein. The structural studies were performed using small and wide angle X-ray scattering techniques. Different phases of lipids formed can be identified by their specific d-spacing, which also gives conclusions about the widening of the structure. By varying the sample detector distance, different angle ranges can be measured and thus different information of distance within the sample obtained. Moreover, a two dimensional x-ray detector allows us to conclude on the alignment of the structures with respect to the CNT orientation in the arrays.

Calcium carbonate is the most abundant biomineral and is biogenically formed with a vast array of nano and microscale features. Among the less stable polymorphs present in mineralised organisms, the most soluble, amorphous calcium carbonate (ACC), formed in chitin exoskeletons of crustacea, is of particular interest since the aqueous stability of isolated ACC is limited to a few hours in the absence of polyanions or magnesium [1]. Amorphous compounds are also thought to play a significant role as precursors to crystalline phases in both invertebrate [2] and vertebrate hard tissues [3]. Here we investigated the influence of a selection of biopolymers and demineralized lobster shell on the formation and stability of calcium carbonate, and then went on to investigate the ability of this phase to act as a precursor for the synthesis of a bone-like collagen scaffold. Using a gas diffusion technique, calcium carbonate mineralization was achieved in all biopolymers tested, but was particularly abundant in collagen hydrogels, in which a significant proportion (~18%) was found to be amorphous, as determined by FTIR and DSC measurements. In dense collagen gels, this amorphous fraction did not crystallize for up to six weeks in deionised water at room temperature. Attempts were also made to remineralize demineralised lobster shell using the same technique; although amorphous-like deposits were observed forming on the chitin matrix using SEM, ACC could not be determined using spectroscopic or calorimetric techniques. Our results strongly suggest that fibre diameter, fibre spacing, and the amphoteric nature of collagen fibres were critical factors in stabilising ACC. Upon immersion in phosphate containing solutions, the calcium carbonate present within the collagen hydrogels was readily converted to carbonated hydroxyapatite, enabling the formation of a stiff bone-like composite containing 78 wt% mineral, essentially equivalent to cortical bone. Since the initial calcium carbonate carbonate containing gels remained flexible and then stiffened on conversion to hydroxyapatite, this demonstrates the potential of this approach for the formation of readily mineralizable hard tissue scaffold materials that could be applied through minimally invasive techniques. Our work also points to the similarity in nanostructure, particularly the curvature of nanofibres present in collagen and chitin being a key factor in their ability to stabilise amorphous calcium minerals. References 1) L. Glazer, et al. J. Biol. Chem. 2010, 285, 12831. 2) H. A. Lowenstam, S. Weiner, Science 1985, 227, 51. 3) a) J. Mahamid, et al. Proc. Natl. Acad. Sci. USA 2008, 105, 12748; b) N. J. Crane, et al. Bone 2006, 39, 434; c) R. M. Biltz, E. D. Pellegrino, Clin. Orthop. Rel. Res. 1977, 279.

Biogenic silica nanostructures, derived from diatoms, possess highly ordered porous hierarchical nanostructures. They afford flexibility in design, due to the availability of a great variety of shapes, sizes, and symmetries from the over 100,000 known species. The complex 3D arrays of silica pores have very unique properties, making it the powerful nanofluidic device to select and capture molecules and colloids. This advantage can be exploited to study fluidic transport phenomena of ions and molecules with the goal of developing selective filters ultrasensitive biosensors, and for manipulating cellular behavior. In this research, we used Coscinodiscus wailesii, because it is a rather large (about 200 micrometer in diameter), has a well ordered pore structure and exhibits a narrow size distribution of nanopores on the order of 40 nm diameter. This size range is challenging to accomplish using top-down fabrication methods, which makes a bottom-up approach using these particular diatom shells very compelling. Our hybrid silicon MEMS/biogenic silica platform offers the user the ability not only to detect nano-particles or bio-molecules permeating through the nanopores, but eventually be employed to study the cellular response to these treatments.. In this study, we will demonstrate that the biogenic silica nanostructure diatoms integrate into microsystems and devices. Micromachined silicon acts as the substrate hosting the biogenic structures. Positioning of the diatoms on top of the micro-pore on the silicon chips is accomplished using linker chemistry and photoresist lift-off. This platform will be employed to address and interrogate certain populations of cells in a selective manner. Different cancer cells will be cultured directly on one side of MEMS/biogenic silica platform, while different molecules, (e.g. dyes, drugs), and nanoparticles (e.g. quantum dots, gold nanorods) will be released on the opposite side of the diatom membrane. Transport through the nanopores in the diatoms will be monitored by optical microscopy. Only cells cultured directly above the diatom surface will be exposed to small-molecule or nanoparticle treatments. This will allow us to interrogate cellular response to drugs and imaging agents with a focus on how selective manipulation of certain cells influences the fate of surrounging cells (e.g. bystander effect). These studies will also allow us to leverage the understanding of the fluidic transport phenomena through well-characterized hierarchical nanopore structures towards designing hybrid devices for applications in separation, sensor and biomedical device technologies.

Poly(lactic acid) (PLA) is a biodegradable aliphatic polyester currently used for tissue engineering applications. This FDA approved polymer material is biocompatible, easy to form, and consequently, widely used in medical procedures. The absence of neo-endothelium coverage on the intimal surface of small-diameter PET vascular substitutes is known to be one cause of failure upon implantation of these prostheses in humans. As coating with proteins does not improve the endothelialization capability of textile prostheses, it was sought to replace this traditional protein (collagen or gelatin) coating with a PLA nanofiber mesh scaffold obtained by an innovative air spinning system. The ultimate goal of this fine-tuned interface between blood and textile threads is to provide an adequate scaffold for endothelial cells to proliferate as monolayer. Thanks to the inherent biodegradability of PLA, it is expected that this synthetic scaffold will be gradually replaced by the natural extracellular matrix. In this context, biodegradation mechanisms of PLA must be understood to evaluate, and eventually modulate parameters which control the PLA degradation reaction. It is therefore the objective of this study to identify and quantify of stereochemical and structural parameters that would eventually allow modulating the PLA degradation rate. Two commercial PLAs have been air-spun and allowed to degrade in air, water, or PBS during 90 days. On one hand, quantitative measurement of the number of polymer end-chains, which allows determining PLA molecular weight, was performed using classical 1H-NMR spectroscopy every 15 days. On the other hand, the utilization of homonuclear decoupled 1H-NMR spectroscopy allowed studying the stereosequences evolution during the degradation process. In addition, the polymer crystallinity evolution was highlighted by classical and modulated differential scanning calorimetry (DSC and TMDSC) and X-ray diffraction (XRD). Finally, polymer chain assembly behavior was studied with vibrational spectroscopy technique (FTIR). The NMR data have shown that the two investigated PLAs displayed different degradation rates, despite having very similar average initial molecular weights. Using homonuclear decoupled NMR, it was evidenced that PLA hydrolysis is also related to this polymer L/D stereosequence ratio. Further DSC experiments will focus on assessing the role of PLA crystallinity on the degradation mechanism. Finally, FTIR spectroscopy will be used to understand the PLA interchain macromolecular interaction and the conformational influence in the cristallinity. These experimental analyses allow a global understanding of PLA degradation behavior from the monomer assembly to the polymer chain assembly. This model will therefore constitute the first step toward the understanding of in vivo PLA degradation and will serve to make PLA nanofibrous scaffold with tailored degradation behavior.

Biofilms are a major source of hospital-acquired infections, due to their persistent growth on medical devices and surfaces. Therefore, there is a clinical need to create surfaces that resist biofilm formation. Recent studies have shown a great potential to create antibacterial surfaces via engineering surface nanoroughness. In addition, some specific metabolites, such as fructose, increase the efficiency of antibiotics and eradicate biofilms. Here, we combined the antibacterial effect of nanorough topographies with fructose metabolites to further decrease bacterial growth and biofilm formation on medical device surfaces without using antibiotics. Nanorough (NanoR) topographies on polyvinyl chloride (PVC) surfaces were created by soaking conventional PVC in a 0.1% solution of Rhizopus arrhizus lipase dissolved in 1M potassium phosphate buffer (PBS). To coat surfaces with metabolites, sterilized samples were soaked in 10 mM and 100mM fructose solutions and incubated overnight. For bacteria assays, 100 µL of a 10^3 Staphylococcus aureus (ATCC #25923) solution was seeded onto a single well of 96-well plate containing either NanoR or an untreated PVC sample soaked in a 10 mM or 100 mM fructose solution. Unsoaked samples were used as controls. After 24 hours, samples were removed. Optical density was measured at 562 nm to analyze the planktonic bacteria growth. Vortexing method was used to remove sessile bacteria and colony forming units were counted to quantify biofilm formation. All experiments were conducted in triplicate and repeated at least three times. We showed for the first time that nanorough surfaces are more resistant to bacterial growth than PVC without using antibiotics. Planktonic bacteria growth decreased more on NanoR and control PVC surfaces soaked into 10 mM and 100 mM fructose solutions after 24 hours. Bacteria growth reduction was also dependent on fructose concentration. Fructose soaking also decreased biofilm formation on both PVC and NanoR surfaces. A 38% decrease in growth was observed on PVC surfaces soaked in a 10 mM fructose solution compared to PVC surfaces. In addition, biofilm formation decreased (45%) on NanoR surfaces compared to the PVC surfaces (p < 0.1). Also, a 60% decrease was observed on NanoR surfaces soaked in a 10 mM fructose solution compared to conventional PVC surfaces (p < 0.05). We present for the first time that the presence of fructose on nanorough PVC surfaces decreases planktonic bacteria growth and biofilm formation. We envision that this method has potential to impact the engineering of PVC surfaces to possess novel nanostructured features leading to more successful clinical outcomes in terms of longer medical device lifetimes, minimized bacteria infections and decreased antibiotic usage that can support decreased antibiotic resistance. Acknowledgements: The authors thank the Hermann Foundation for funding.

Chronic neural electrode arrays capture the electrical activity of neurons from a number of discretely sampled volumes of the brain. This translates to a push towards smaller electrodes that are more biologically transparent and biocompatible with a high density of recording sites that remain functional for long period of time. As electrode dimensions approach the microscale, initial electrode impedance increases, therefore, sensitivity and signal quality decreases. Thus, there is a tradeoff between the size (special selectivity) and the quality of signal recordings (sensitivity). Moreover, producing biocompatible microelectrodes with long term reliable signals remains a challenge. Here we report a novel method for fabrication of conducting polymer micro and nanospherical cups (CPMNSCs) for improvement of electrical properties of neural electrodes and triggered release of drugs at electrode tissue interface. The fabrication process involves electrostatic spraying of biodegradable poly (lactic-co-glycolic acid) (PLGA) on the gold sites, followed by electrochemical polymerization of conductive polymers poly (3,4-ethylenedioxythiophene) (PEDOT) by applied current density 0.5 mA/cm2 on the gold sites and around the micro/nanospheres to create CPMNSCs. We were able to modulate the diameter of the PLGA spheres by controlling the electrospraying parameters such as polymer concentration, flow rate, and voltage. The diameters of the PLGA spheres were ranged from 500nm to 3mu;m, and wall thickness of the PEDOT spherical cups varied from 50 nm to 100 nm. By changing the polymerization time, we could reproducibly control the opening size of the CPMNSCs. We have characterized the electrical properties of modified microelectrodes using impedance spectroscopy and cyclic voltammetry. Preliminary results indicated the impedance of microelectrodes decreased about two orders of magnitude and capacity of charge transfer increased about three orders of magnitude. We also compared the electrical properties of PEDOT MNSCs and PEDOT film. PEDOT MNSCs exhibited lower impedance and higher capacity of charge transfer than PEDOT film. This can be explained by the increasing effective surface area during CPMSCs formation. These results demonstrate the superiority of CPMNSCs for neural recordings and stimulations in which the low-impedance and high capacity of charge injection electrode tissue interface is essential. We have recently incorporated an anticancer agent BCNU into the CPMNSCs. We will examine the triggered release of BCNU using electrical actuation of CPMNSCs.

Ventilator associated pneumonia (VAP) is a serious, costly complication of mechanical ventilation. Endotracheal tubes (ETTs) present a special concern to clinicians because they are often colonized by oropharyngeal bacteria during long-term mechanical ventilation. Cost effective ETTs that are resistant to bacterial infection would be essential tools in the prevention of VAP. The objective of this study was two fold, first to develop strategies to decrease bacterial adhesion on ETTs using nanotechnology and secondly to develop better methods to assess in vitro bacterial adhesion and biofilm formation on ETTs using a bench top experimental model and an associated finite element model (FEM). Nanoroughened ETTs were created by exposing polyvinyl chloride (PVC) ETTs (Sheridan^®) to a 0.1% mass solution of R. arrhizus (Sigma Aldrich) lipase dissolved in a potassium phosphate buffer for 48 hours at 37° C. Nanomodified ETTs were tested in a custom made bench top model airway. ETTs were connected to an Infant Star 950 ventilator (Puritan Bennett, Covidien) and bacterial media consisting of 480 mL of trypticase soy broth media (TSB) inoculated with 10³ colony forming units/ml (CFU/mL) Staphylococcus aureus (ATCC #25923) or Pseudomonas aeruginosa (ATCC #25668) was introduced into the system over the duration of the 24 hour test. At the end of each 24 hr trial, ETTs were cut into ten 1.5 cm pieces, added to 2 mL of media, and vortexed at 3000 rpm for 1 min. This media was then serially diluted and plated overnight to determine CFU/mL on each section. Dynamic flow conditions influenced the location of bacterial adhesion and biofilm formation in both the unmodified and nanomodifed ETTs. Areas of tube curvature, such as at the entrance to the mouth and the connection between the oropharynx and the larynx, were correlated with larger amounts of biofilm. Notably, the dynamic values for biofilm density along many areas of the tube were substantially less than previously reported static values, with a 1.5 log reduction in CFU/mL of S. aureus on the nanomodified ETTs compared to the untreated tubes. In dynamic tests with P. aeruginosa, a 2.7 log reduction in CFU/mL was seen on the nanomodified ETTs when compared to the untreated tubes. While the overall pattern of bacterial growth around areas of curvature remained consistent, the location of bacterial adhesion and biofilm formation varied between S. aureus and P. aeruginosa, with P. aeruginosa biofilms forming closer to the proximal end of the ETT. Dynamic studies showed that lipase etching can create nano-rough surface features on PVC ETTs suppressing both S. aureus and P. aeruginosa growth. The results of both the static and dynamic models suggest that combining improved nanomodified materials with changes in clinical procedure (patient positioning, tube curvature) could provide clinicians with an effective and inexpensive tool to combat VAP, and should be studied in greater depth.

Semiconducting single-walled carbon nanotubes (SWNTs) fluoresce in the near-infrared and are promising as optical sensors when suitably functionalized to enable selective analyte recognition. Their detection limit has been extended down to single molecules, as the SWNT emission can stochastically fluctuate in a quantized manner in response to single-molecule adsorption and desorption events. SWNT sensors with enhanced emissions have been hypothesized to have greater analyte sensitivities, and reductive brightening reagents, such as dithiothreitol (DTT) and Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), have been shown to enhance nanotube brightness via the passivation of defect sites. In this work, we examine the effect of the brightening agents, Trolox and DTT, on the sensitivity of a d(AT)15 DNA oligonucleotide wrapped-SWNT sensor selective for nitric oxide (NO). While Trolox and DTT nearly double the mean brightness of the d(AT)15-SWNT construct, the resulting sensor sensitivity to NO is correspondingly reduced. The NO adsorption rate constant, a measure of NO sensitivity, decreases from 0.0007+/-0.00003 s^(-1) µM^(-1) to 0.0003+/-.00003 s^(-1) µM^(-1) and 0.0004+/-.0001 s^(-1) µM^(-1) upon pre-treatment of 1 mM Trolox and 1 mM DTT, respectively. The results are consistent with a model where Trolox and DTT behave as competitive binding agents with the analyte, NO, occupying one of a finite number of available SWNT binding sites and altering the strength of NO binding to the surface. Hence, in the use of brightening agents, such as Trolox and DTT, to enhance molecular detection, a trade-off is predicted between signal intensity and analyte sensitivity.

Chitosan and gelatin have been used as suitable matrices for cell support due to their bioactivity. However, hydorgels fabricated from these polymers have commonly low mechanical strength. Several approaches were attempted to improve the mechanical strength of natural hydrogels such as using different types of crosslinking agents and the preparation of composite polymers. In this study, we investigated the effect of addition of a nano-fibres for improving the mechanical strength of natural hydrogels. The nano-fibres were prepared from cellulose, which is the most abundant and renewable biopolymer and proved to be biocompatible. The cellulose fibres consist of highly ordered microcrystalline structures and a less ordered structures known as ‘‘amorphous regions&’&’. These fibres are used as bio-based nano-reinforcement owing to their superior mechanical properties. In this study nano whiskers of cellulose were produced by hydrolysis of eucalyptus pulp followed by neutralisation and used as a source of nano-fibre. The solution contained nanowhiskers were used for dissolving the polymers. Genipin and photocrosslinking were also used as non-toxic approach for the preparation of hydrogel. The results of this study demonstrate it is feasible to produce a uniform distribution of cellulose nanowhiskers (CNW) in crosslinked structure of hydrogels.The swelling properties of hydrgels were increased at least two fold by addition of CNWs. The mechanical strength was also enhanced more than 3 fold. These results demonstrate that cellulose nanowhisker are promising material for improving the mechanical strength of hydrogels and promote their biological activities.

Titanium is the one of materials which are widely used for medical implants. It provides good bio compatibility but it doesn&’t bond to surrounding bones directly because of its inertness. Thus, the method of making the surface of an implant functionalized has been studied. For better osseointegration, nanostructured surface and cell interaction have been investigated extensively. In our study, we measure the degrees of functionalization by osteoblast attachment and the amount of alkaline phosphatase(ALP) using a substrate structured with titania(TiO2) nanowires. TiO2 nanowires are coated with hydroxyapatite or RGD(Argine-Glycine-Aspartic acid) peptide which are known to help the acceleration of osseointegration. The degree of distribution and the attachment of the coating materials are evaluated by cell/substrate interaction. The cell/substrate interaction is measured by the number of cells, ALP activities that indicate the cell differentiation, and the morphology of cells. TiO2 nanowires are grown on Ti alloy substrates by oxidation process. Firstly, titanium plate samples (1.5mm thick, 99.7%, Sigma Aldrich) are immersed in a mixture of 2ml 48wt% HF and 3ml 70% HNO3 for 5min to remove native oxide layers. The nanowire layers are then prepared by oxidation in oxygen environment at 700-900 oC for 7-9 hours. After annealing, the samples are cooled down to a room temperature by air quenching. For functionalization, samples are immersed in the solutions of coating materials for half an hour, 1, 2, and 3 hours. Hydroxyapatite and RGD(Argine-Glycine-Aspartic acid) are then applied as coating materials. After the functionalization of the TiO2-nanowires, human osteoblast cells are cultured on the substrate in SBF at 37oC in an atmosphere. Then, the number of cells, ALP activity, and the morphology of cells are measured. After the cell culturing, the dependence of functionalization on nanowire parameters such as diameter, length, and others are evaluated. Depending on the conditions of TiO2 nanowires, the attachments to the coating materials vary. Generally, nanowires enhance more attachment and proliferation of cells. For more functionalized surface, nanowire surface containing nanocomposites with antimicrobial agents or bio-signal agents will further be investigated.

For biomedical applications, a nano carrier is required to have multiple functionalities, such as drug delivery, fluorescence imaging, and cell targeting. However, nano materials may be limited by their intrinsic behaviors in terms of structure, size, property, and biocompatibility. To address these issues, a versatile nano-assembly is designed and developed based on the concept of "plug and play." The system is engineered on a nanoscaled graphene nano-sheet (GNS) platform via host-guest chemistry between the hybrid and functional payloads. Cyclodextrin (CD), an oligosaccharide consisting of six, seven or eight glucose units (α, β, or γ-CD, respectively), is utilized as the “host” molecule. This cone-shaped cavity of CDs can serve as hosts for a great variety of functional or biological “guest” molecules by taking advantage of its geometric compatibility and hydrophobic interactions between the CDs and the guest molecules. The nanohybird GNS/β-CD is fully capable of selectively accommodating/releasing various biological and functional agents, in a controlled fashion, including antivirus drug amantadine, fluorescent dye (5(6)-Carboxyfluorescein), and the RGD targeting ligands. The loading capability of 5(6)-carboxyfluorescein reaches as high as 110 % with a drug concentration of 0.45 mg/mL. The cyclic RGD is found to exhibit remarkable targeting effect for the HeLa cells.

Significance of this work is to develop three-type disinfectants to inactivate bacteria and advancement of healthcare materials. A green chemistry method using natural product as reducing agents was employed to produce nanoscaled metal silver (Ag) and Ag-modified titania (TiO2). This approach allows for improving the environmental impact of hazardous waste. The second approach, a hydro-solvothermal, to prepare MOFs as disinfectants provides a new paradigm to mitigate bacterial contamination from food and water, which otherwise would pose a severe threat to the public health and disease control. The recent outbreak of bacteria leading to human fatalities is a motivational force for us to develop anti-bacterial agents with high potency and long-term stability. The first generation of disinfectant is focused on Ag material derived from green chemistry using coffee and tea as reducing agents. Although silver as a bac-tericide has been extensively investigated since antiquity, its mode of action is not as well-known. Our study indicated that the Ag catalyzed the microbe membrane peroxidation, causing the cell death. In addi-tion the Ag development, Ag-modified TiO2 was produced using the same green colloidal chemistry. This Ag-TiO2 was found to be highly effective at inactivating bacteria under visible light condition due to the decrease in band gap. This activity overcomes to the public concern of applications of Ag-TiO2 system in as photocatalyst, optical application, and antibacterial treatment, which requires untraviolet energy. Both gen-eration of disinfectants displayed high potency and long-term stability, however the nanomaterials required prolonged time period to inactivate bacteria. Therefore, a novel cobalt (Co) based metal-organic framework (MOF) was prepared and tested to eliminate the above problem. Importantly, the MOFs were usually used in the hydrogen storage and carbon dioxide capture. Our study of using MOFs in the biological field will attract a lot of attention from the inorganic chemists. Gram-negative Escherichia coli (E. Coli) and Gram-positive bacteria, Staphylococcus aureus (S. aureus) were selected to determine the antibacterial activities of these three types of anti-bacterial materials. The results indicated that high potency of the Co-TDM disinfectant was evaluated using minimal bac-tericidal concentration (MBC) benchmark and was determined to be 10-15 ppm, within a short incubation time period (< 60 min). Compared with Ag nanoparticles and Ag-TiO2 nano-composites for the same time period, the MBC and effectiveness of Co-TDM are superior. Electron microscopic techniques were used to characterize the morphology of Ag, Ag-TiO2 and Co-MOFs. The Ag and Co active sites rapidly cat-alyzed the lipid peroxidation, causing rapture of the bacteria membrane followed by inactivation, with 100 % recycling and high persistence (> 4 weeks).

We use the dissipative particle dynamics to design a biomimetic synthetic vesicle that can selectively capture and encapsulate specific solutes such as polymer macromolecules and nanoparticles. The “smart” vesicle consists of a lipid membrane that encloses a stimuli-sensitive microgel particle. When the responsive microgel particle swells due to an external signal, its size increases causing membrane rapture and the formation of transmembrane pores. These pores are reversible and close when the signal is turned off leading to microgel deswelling. In our simulations, we formulate the criteria for the controlled pore opening and closing, and probe how this responsive synthetic system can be harnessed to “ingest” highly-specific macromolecules dispersed in solvent surrounding the vesicle. In particular, we use target macromolecules that can preferably bind to the gel particle. Thus, when the gel swells, the macromolecules diffuse through pores and attach to the gel surface. After gel deswelling, the target macromolecules remain in the vesicle interior protected by the lipid membrane. Our findings will be useful for developing artificial phagocytic agents and creating new active sampling systems for biomedical applications.

Graphene is a promising material, due that it has a large theoretical specific surface area, high intrinsic mobility, high Young's modulus, thermal conductivity, and good electrical conductivity that merit attention for many applications. As a robust yet flexible membrane, graphene provides essentially infinite possibilities for the modification or functionalization of its carbon backbone. A study of the bacterial growth curves of Pseudomonas aeruginosa in presence of graphene and graphene oxide (GO) particulates in suspension were done, the last one shows a high accelerated rate of bacterial population growth respect to the standard and graphene curves. SEM images of graphene and GO films show a strong interaction between GO surface and the bacteria, and a less density of colonies distribution on its surface compare with the graphene. These results open new gates for another research fields no yet explored, and no recommend the use as coating in tools, implants and medical device in biomedical industry. The microbial activity of GO was examined by observing the growth curve using 640nm wavelength. The graphene oxide hat we did was produced by mean of Hummers-Offerman reaction method. The GO was characterized using Scanning Electron Microscopy (SEM), Transmission Electron Microscopy (TEM), Fourier Transform Infra-red Spectroscopy and Raman Spectroscopy.

Poly(ethylene Oxide)(PEO)s are well-known as anti-fouling polymers that are non-toxic, water soluble, chemically stable in physiological condition. PEOs reduce protein adsorption, macrophage attack, platelet adhesion and decrease immune response because of increasing hydrophilicity on the surfaces they are modified. To improve anti-fouling efficiency, dendritic PEOs were synthesized by living anionic ring opening polymerization of ethylene oxide. Anionic polymerization is the most powerful method to obtain well defined structure like star shaped or dendritic PEOs. Anionic polymerization is carried out in a moisture free vacuum line that leads to molecular weight control and narrow molecular weight distribution. Dendritic PEOs were synthesized by divergent method and each generation was carried out in THF using diphenylmethyl potassium (DPMK) as a strong base catalyst at 40 oC for three days. To introduce branch point, allyl bromide was reacted with polymer chain ends and dihydroxylation was carried out. Allylation and dihydroxylation results are analyzed by 1H NMR spectroscopy and molecular weight and molecular weight distribution were characterized by GPC. Allylic double bonds were observed at 6.03-5.79 (CH=CH2) and 5.33-5.11(CH=CH2) ppm and completely disappeared after dihydroxylation according to analyzing by 1H NMR spectroscopy. Dendritic PEOs of third generation were obtained with a very low Mw/Mn values below 1.03 and excellent control of molecular mass by GPC. Focal point and end groups were functionalized for surface modification on biomedical application. Focal point was converted to N-hydroxyl succinimide (NHS) group by activating carboxylic group that could be reacted with amine groups on the surfaces. End groups of dendritic PEOs were functionalized various type of functional groups for additional surface modification or binding other molecules and drug. Anti-fouling effect of dendritic PEOs showed excellent compared to linear PEOs. Living cells were modified with dendritic PEOs and coverage effect and viability test were performed. The results showed high coverage effect and low toxicity. Cell function was not changed and cells were lived for long time than cells that modified linear PEOs. Dendritic PEOs have fewer binding site to the surface, but higher coverage effect than linear PEOs. They also have high efficiency to prevent adsorption of proteins because they have bulky structures.

Despite the lot of research done to understand the correlation between surface properties, such as roughness and surface energy, and the biological response of the materials, there is very few information about the effect of the atomic ordering. In this work, we deposited amorphous and crystalline thin films to study the effect of the atomic ordering on the surface properties and the bacterial adhesion. The films were deposited from a metallic target using a magnetron sputtering method and a reactive Ar/O2 atmosphere (80:20). The amorphous films were deposited at room temperature, and the crystalline films at a substrate temperature of 250oC. The films were deposited on pure Titanium substrates, but having different roughness. The films were characterized by X-ray diffraction, X-ray photoelectron spectroscopy, water contact angle, atomic force microscopy and secondary electron microscopy. Bacterial adhesion experiments were done using E. Coli and S. Aureus, two pathogen strains. The number of attached bacteria was obtained after 24 hours of incubation by the method of counting the number of colony forming units, which measures the number of viable cells that were initially attached to the surface. The results suggested that the number of bacteria was larger for the crystalline films. The correlation between this result and the surface properties is discussed.

Magnesium and its alloys have attracted great attention as biodegradable metallic materials for orthopedic applications due to their degradability and similar mechanical properties to cortical bone. The degradation of magnesium alloy is accompanied with the sustained release of magnesium ion (Mg2+) at the site of implantation. Previous studies suggested that Mg2+ stimulated bone formation and promoted fracture healing, however, the precise mechanism is unclear. In this study, the corrosion behavior of commercially available pure magnesium (p-Mg) in simulated body fluid (SBF) was evaluated. The results demonstrated that accompanying with the hydrogen release the pH values increased rapidly, which was due to the formation of Mg(OH)2. The pH value gradually got stable and reached maximum of 7.8 in next 3 days, and then the corrosion became steady. The surface morphology and corresponding chemical composition were characterized by scanning electron microscope (SEM) equipped with energy-dispersive X-ray spectrometer (EDS) and X-ray diffraction (XRD). A thin amorphous apatite layer, which was rich in Mg, Ca, P and O, formed on the surface of p-Mg after 24h immersion. The corrosion rate decreased and the formation of pitting corrosion was significantly delayed due to precipitation of Mg-containing apatite. The biocompatibility of p-Mg was evaluated in vitro and the intracellular signaling pathways activated by Mg2+ in osteoblasts were further investigated. The degradation of p-Mg promoted the attachment and accelerated the proliferation of preosteoblast MC3T3-E1, which might at least in part be due to the alkaline microenvironment around the p-Mg. The expression levels of some key proteins related to mitogen-activated protein kinases (MAPK) signaling pathway was evaluated by western blot. We found that Mg2+ induced the phosphorylation of extracellular regulated protein kinases (ERK), enhanced the c-fos level, and then stimulated osteogenic gene expression. In summary, the above results indicated that the degradation of p-Mg builds an alkaline microenvironment around the implant and subsequently enhances osteoblastic activity. Furthermore, the intracellular MAPK/ERK signaling pathway is involved in the Mg2+ for stimulating bone formation.

A methodology to study the cellular contraction forces using Polydimethylsiloxane (PDMS) micropillar arrays in the frequency domain with improved accuracy is reported herein. The cellular contraction data was expressed as a Fourier series, and the complex modulus of PDMS was measured using a dynamic nanoindentation technique (DNT). An improved method for measurement of complex modulus was developed with the use of a flat punch indenter. The cellular contraction force was calculated by finite element analysis (FEA). In the contraction of cardiac myocytes, oscillatory motion is often involved. In this work, the contraction deformation history was expanded into Fourier series for analysis in the frequency domain. Two representative states of cell were selected, corresponding to 3 min (stimulated) and 7 min (desensitized) after the isoproterenol (ISO) perfusion. The discrete Fourier transform (DFT) technique was used to fit the experimental data to the Fourier series and good agreement was achieved. The results were also used to identify the cell beating frequencies under difference states. The complex modulus of PDMS was measured by DNT. Compared with the conventional dynamic mechanical analysis (DMA), DNT features minimal sample preparation and high spatial resolution. The DNT tests were conducted on an Agilent G200 Nano Indenter system using a 2.01 mm diameter sapphire flat punch indenter. The frequencies of the tests were in the range of 1~45 Hz. In this work, we developed an improved model to extract the complex modulus from the dynamic nanoindentation. The advantage of our method is that there is no linear constitutive model used in derivation, therefore the frequency-dependent viscoelastic behavior is precisely captured without assumptions. The cellular contraction force was calculated using finite element analysis by ABAQUS. The geometry of micropillar was modeled in three dimensions with the consideration of enlarged root and notched sidewall. The cellular contraction in Fourier series was applied to the boundary condition at the free-end, and the complex modulus of PDMS was incorporated for the viscoelastic modeling. The direct-solution steady-state dynamic analysis was conducted, and the magnitude of simulated forces for 3 min and 7 min states were 15.9 nN and 10.8 nN, respectively. It indicates that the effects of viscoelastic properties and appropriate beam model are very important in the calculation of cellular contraction forces using PDMS micropillar sensor arrays. The dynamic nanoindentation technique is extremely useful for soft polymeric materials for which the dynamic mechanical properties are a challenge to characterize at small scales. It is beneficial for the design of other cantilever-based soft polymer bio-transducers. The methodology provides a general guideline for complex biological analysis in the frequency domain.

A carbonated form of hydroxyapatite (HA) [Ca10(PO4)6(OH)2] is one of the most abundant materials in mammal bone. It could prove invaluable understanding the material&’s properties in sight of possible medical applications to not only repair but also preventively detect bone degradation. Using density functional theory, we analyze the vibrational and electronic properties, surface energy and reactivity of HA for different orientations and terminations. We identify the chemically stable low energy surface configurations as function of the OH, PO4 and Ca concentration. In the experimentally relevant OH-rich regime we find only two surfaces competing for the lowest energy. The surface most stable over almost the entire OH-rich regime is OH-terminated. We study the adhesion between water and this surface, and when substituting Na, Mg, Al, Si, S and Cl on appropriate nuclear sites as a first step towards studying its bioactivity and how it can be modulated.

Osteoarthritis, a degenerative disease marked by breakdown of cartilage and subchondral bone in joints, is a growing public health concern throughout the world. It is associated with increasing age and body mass and, with a prevalence of approximately 27 million cases in the United States alone, its specific mechanical and biochemical causes are unknown and there is no cure. Symptoms resulting from the friction between degrading cartilaginous surfaces include subchondral cysts, sclerosis, and osteophytes, leading to pain and stiffness in the joint. A promising material that can mitigate these symptoms is one that interacts with the damaged tissue in a specific way, is long-lasting, and exhibits minimal or no off-target toxicity. We report, herein, the preparation of heterofunctionalized dendrimers from biocompatible synthons for the treatment of osteoarthritis. These biomaterials boast three different modalities: i) multiple positive charges for adherence to native elements in joint cartilage, ii) therapeutic moieties for disease treatment, and iii) a computed tomography contrast agent for attenuation of x-rays during imaging. Specifically, a polycationic dendron scaffold derived from L-lysine, L-tyrosine, and L-dihydroxyphenylalanine monomers has been synthesized with an iodinated contrast agent at the point of convergence through facile carbodiimide-mediated couplings and copper-catalyzed alkyne-azide heterocyclizations. The resulting branched macromers were characterized by conventional techniques including nuclear magnetic resonance and infrared spectroscopy and size exclusion chromatography. Computed tomography imaging studies are ongoing to analyze the interaction between the dendrimer and ex vivo bovine cartilage to facilitate dendrimer optimization. With this branched scaffold in place, attachment of therapeutic molecules may allow for targeted treatment of the disease state. Dendritic molecules featuring functionalities with both therapeutic and diagnostic character may serve as efficient biomaterials for treating and monitoring osteoarthritis.

The ability to generate and control neuronal growth in vitro from stem cell precursors is a growing area of interest in biomedicine. Neuronal development occurs in various stages from immature precursor cells to fully integrated and functionally mature neurons. These developmental steps are classified into two categories: activity independent and activity dependent. Independent landmarks are thought to be genetically determined and include neuronal differentiation, migration and axon guidance. Activity dependent stages of neuronal growth are heavily regulated by secreted molecules such as hormones and neurotransmitters. The overall effects of the secreted molecules in vivo are well researched; however, the effects on in vitro differentiation are not fully understood. Researchers are unable to identify and measure the small molecules in vitro, as the secreted hormones are absorbed by neighboring cells. Therefore, a substantial opportunity exists in neuronal interface research to develop a material platform that allows for both the proliferation and differentiation of stem cells into neurons and the ability to quantify the secretome of neuronal cells. Anodic aluminum oxide (AAO) membranes are biocompatible and composed of highly-ordered nanopores that penetrate the entire material. The inert properties of AAO membranes support the growth of neuronal cells and the nanopores allow for selective concentration of secreted molecules. Nanopore sizing, surface functionalization and morphologies are controllable based upon experimental parameters and allow for precise segregation and selection of secreted molecules. The AAO membranes were fabricated in a mild two-step anodization procedure. The voltage was varied during both anodization steps to control the pore size and morphology of the AAO membranes. Pore sizes ranged from 34 nm to 95 nm. After the growth of the membranes, the remaining barrier on the back of the membranes was removed by an etching process to create pores that penetrated the entire material. Characterization of the pore structure was performed by scanning electron microscopy (SEM). ImageJ software was used to determine average pore size. To assess the potential of the AAO membranes as a neuronal differentiation platform, C17.2 neural stem cells (NSCs), an immortalized and multipotent cell line, were used. The NSCs were forced to differentiate via serum-withdrawal. Cellular growth was characterized by Alamar Blue, immunocytochemistry (ICC) and SEM. Differences in overall cell numbers and phenotypes were observed through Alamar Blue and ICC, respectively. ImageJ software was used to obtain phenotypic cell counts. Differences in cell morphology were studied through ICC and SEM experiments. Results indicate a highly tunable correlation between AAO nanopore sizes and cell phenotype populations. By selecting AAO membranes with specific nanopore size ranges, control of neuronal network density and neurite outgrowth length was achieved.

A library of mono and diacid acrylamide derivatives have been synthesized using lower critical solution temperature (LCST) monomers N-isopropyl acrylamide(NIPAM) and N,N&’diethyl acrylamide (DEAA) as models for design. The synthesized polymers include poly(N-acryloyl alanine), poly(N-acryloyl β alanine), poly(N-acrlyoyl N&’-methyl alanine), poly(N-acrlyoyl N&’-methyl β alanine), and (N,N&’-iminodiproprionic acid acrylamide) which act as amino acid analogs in solution and have been tested for their various LCST properties, due to their structural similarity to poly(NIPAM) and poly(DEAA). A demonstrated LCST transition creates the possibility of similar applications as poly(NIPAM) currently enjoys, but with reduced toxicity due to their biocompatibility. Polymerizations were performed by using activators generated by electron transfer (AGET) atom transfer radical polymerization (ATRP). Polymerizations used CuBr2 or FeCl3 as the metal catalyst, sodium ascorbate as the activating agent, water as the solvent, and the monomer as the complexing ligand. No group has yet reported synthesis of a compound via ATRP without using a separate ligand. Furthermore, there are very limited reports of a carboxylic acid acting as a ligand for a copper ATRP center. Additionally, there are only sparse reports of the polymerization of monomers containing free carboxylic acid moieties due to the sensitivity of general ATRP to acidic media. These polymerizations conditions are also in line with the principles of “green” chemistry, as iron is low in toxicity, and a green solvent is used. Polymers were then characterized using NMR and GPC, as well as DSC in water to quantify their solution transitions.

Electrochemical methods based sensors for detecting D-glucose is widely adopted due to its simplicity and relative ease in calibration. However, the response time of these sensors is extremely slow and due to their inherent large size they cannot be used for in-vivo measurements. Applications such as monitoring patient&’s glucose level in intensive care as well as continuous monitoring of glucose in patients demand for a miniature glucose sensor with faster response time. Presented in this work is the development of a simple, micron scale, highly sensitive, conductance based D-glucose biosensor in which highly aligned single walled carbon nanotubes (SWNTs) with immobilized glucose oxidase (GOD) act as the sensing element. These developed D-glucose sensors have a large detection range (0~150 mg/dl) with a lower limit of detection (0.1 mg/dl). Template guided fluidic assembly and non-covalent functionalization processes are employed to fabricate these sensors while immobilization of the GOD onto SWNTs is effected through a succinimidyl ester. The active area of the sensor is <10micron2 paving the way for in-vivo mode applications. Two-electrode configuration is implemented to allow continuous resistance response monitoring for the D-glucose detection. The electronic properties of the SWNTs had a significant impact on the performance of these sensors. Use of a different immobilized enzyme instead of GOD can potentially lead to sensors for detection of other targeted biomolecules.

Given their ability to encapsulate small-molecule, liposomes have been extensively investigated for the delivery of cytotoxic drugs to cancer cells. However, liposomes are often unstable in biological environments, leading to low shelf-life and short circulation time. Moreover, the lack of functionality in lipid components and the need to preserve the lipid structure make it difficult to modify liposome to incorporate functions for selective targeting and release. To overcome these limitations, polymer-caged nanobins (PCNs) have been developed by inserting a cholesterol-terminated poly(acrylic acid) into the lipid bilayer of liposome templates to create a protective polymer cage around the liposome core. This greatly stabilizes the liposomal structure and preserves the encapsulated drugs under physiological conditions. In addition, the polymer cage allows for facile pH-triggered drug release in the acidic environment of tumor interstitium and cellular endosomes as well as provides a multifunctional environment for functionalizing the PCNs with targeting groups, such as Herceptin cancer-targeting antibodies, and imaging entities, such as gadolinium(III) MRI contrast agents. This presentation will focus on the successful fabrication of a Herceptin- and gadolinium(III)-conjugated PCN (Her-GdIII-PCNDXR) platform that encapsulate doxorubicin chemotherapeutic agent inside its core. The versatility of PCN as a cancer-targeting and -theranostic platform will also be discussed as a function of its excellent cellular uptake, enhanced MR contrast, pH-triggered drug-release, and good stability. Given these outstanding properties, the PCN platform may be a promising candidate for cancer-targeting theranostic applications where it can deliver therapeutic agents to target tissues or organs while simultaneously provide important timely feedbacks regarding the effectiveness of the treatment.

Carbon is one of materials of highest biocompatibility and often used as medical devices. Especially, hydrogenated amorphous carbon (a-C:H) film coating has emerged as a potential technique for improvement of biomaterial. There are many reports suggested available applications of a-C:H films for medical devices. a-C:H film including diamond-like carbon has low frictional coefficient, high wear and corrosion resistance, chemical inertness, high electrical resistivity, infrared-transparency, high refractive index, and excellent smoothness, etc. Recent trials on a-C:H film coatings for tissue engineering have indicated promise for hasten the cell growth applications. Moreover, cellular response of a-C:H films is wide range from cell adhesion level without cytotoxicity. In our previous works, we have developed a-C:H film deposition technique for cell growth and deposited on micro segmented polyurethane (SPU) scaffold fibers. We investigated structural and compositional effects of the a-C:H film on cell growth and cell behaviour at each fiber surface. It was observed that the a-C:H film coating enhanced cell growth and cell behaviour. Especially, nitrogen-doped a-C:H (a-C:H:N) film coating had high level cell growth. In this study, in order to investigate biocompatibility of a-C:H:N film coating SPU scaffold fiber (a-C:H:N/scaffold) sheet in in-vivo test, the scaffold sheet was implanted at partial aorta descendens of a goat for 27 days. The surface composition, roughness, potential, and structures of the a-C:H:N/scaffold sheet was estimated by X-ray photoelectron spectrometer (XPS), atomic force microscopy (AFM), and surface energy measurement. Moreover, in in-vitro test, mouse fibroblasts (NIH 3T3) cells were grown on the a-C:H:N/scaffold sheet. The cell behaviour was monitored by video camera and analysed. As a result of the in-vivo test, it was observed that the a-C:H:N/scaffold surface was uniformly covered by endosporium. The a-C:H:N/scaffold surface had no thrombus formation as an inflammatory reaction and it was shown that the a-C:H:N film coating had a good blood compatibility. Additionally, in in-vitro test, it was observed that amount of nitrogen content of the a-C:H:N film was possible to control cells behaviour on the scaffold sheet and induced cell growth. These results suggest that a-C:H:N/scaffold has good blood compatibility and cell compatibility and it is a promising approach for improvement of biocompatibility of synthetic vascular graft surfaces.

Many advances in nanomaterials synthesis have been recorded during the last 30 years. Biological ways of production acquired the major importance as green ways. Inorganic and organic materials such as magnetite particles, metallic nanoparticles (silver, goldhellip;) and quantum dots (ZnS, CdS..) have been produced by microorganisms such as bacteria, molds, algae and yeasts. In addition, bacterial cellulose (BC) produced by bacteria belonging to the genera Acetobacter, Rhizobium, Agrobacterium, and Sarcina [1] is one of many eco-friendly materials with great potential in biomedical field. BC consists of a network of nanofibers; the most efficient producer is Acetobacter xylinum [1]. The shape of BC is sensitive to the container shape and incubation conditions (agitation, carbon source, rate of oxygenation, electromagnetic radiation, temperature, pH..) [2, 3]. The challenge is to control the dimensions of BC nanofibers by the optimization of culture conditions. In addition, the production of 3D structures based on BC is important for many industrial and biomedical applications such as paper and textile industries, biological implants, wound and burn dressing material, and scaffolds for tissue regeneration [2]. Acetobacter xylinum is wide-spread in nature where the fermentation of sugars takes place, it could be found in fruits, fermented food, homemade vinegar,.. In our work, some wild strains Acetobacter spp. were isolated from fermented fruits, fruit juices, and vinegar then purified in order to be used in the cellulose production. The purified strains were then used to inoculate a volume of 100 ml of different media containing essentially glucose at different concentrations, yeast extract, and some mineral sources in 1 L Erlenmeyer flask to produce BC. Our study presents a new approach in understanding the behavior of the used bacteria during the biosynthesis in order to control the physical parameters of the produced material. Two ways of BC biofabrication were investigated. The first culture was performed under agitation (stirring and/or shaking) in order to produce spherical and cylindrical 3D BC and the second culture consisted on static growth of bacteria in order to synthesize 2D or monolayer BC films. The viscoelastic properties of the culture media during the biofabrication are monitored in order to evaluate the influence of the environment on the bacterial activity. The resulting BC nanofibers are characterized using different methods. The dimensions and texture of the nanofibers were studies using scanning electron microscope. X-ray diffraction was used to investigate the crystallinity of the materials. FTIR is used to determine the chemical nature of the fibers. Mechanical properties of the resulting material are also determined. [1] Klemm D. et al., 2001. Prog. Ploym. Sci., 26:1561-1603. [2] Paul Gatenholm et al. 2010. MRS Bulletin, 35:208-213. [3] Surma-Slusarska B. et al., 2008. FIBRES & TEXTILES in Eastern Europe, 16:108-111.

Virus capsids have provided ideal building blocks for nanotechnology with their advantages of biocompatible and biodegradable properties over other inorganic and organic substances. Due to their well-defined structure and self-assembling system, a large number of virus capsids have been employed as drug/gene delivery vehicles. Cowpea Chlorotic Mottle Virus (CCMV) is an icosahedral plant virus with a diameter of 25 nm that specifically infects the cowpea plant, or black-eyed pea. One of the most profound characteristics of CCMV is that the viral capsid can undergo reversible pH and metal-ion structural transition (swelling), without any loss of viral function or structural damage. Here we report the surface modification of CCMV capsids by copper catalyzed azide-alkyne cycloaddition (CuAAC) reaction, the best example of “click” chemistry. The bioconjugation of chemoselective oligo-ethylene glycol (OEG) and RGD-containing peptide addressed onto the exterior of CCMV capsids were performed via carbodiimide activation and CuAAC reaction. The intact viral particles were confirmed by mass Spectrometry (MS), transmission electron microscopy (TEM), fast protein liquid chromatography (FPLC), and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) with fluorescence visualization analysis. Cell attachment of the modified CCMV with NIH-3T3 fibroblast cells were further studied using unmodified CCMV as control. The results illustrate the potential application of virus based nanomaterials in drug delivery for future.

Formation of biomimetic coatings capable of retaining high cell survivability and selective permeability to analytes is a primary objective for cell-based sensor applications. The layer-by-layer (LbL) approach is an attractive method for encapsulation of individual cells with the possibility to control metabolic properties by regulating transport through artificial shell membranes. We have explored the formation of soft hydrogel network shells on the basis of hydrogen-bonded interactions between natural polyphenols and neutral polymers, as well as by using lightly crosslinked amine-functionalized polymethacrylic acid. Increased viability (85-95%) of yeast cells without loss of cell function makes these polymer-based shells attractive candidates for many cell-based applications. All-natural, biocompatible, highly stable conformal shells based on unmodified silk as well as silk functionalized with polyamino acids provided additional advantages for sustained long-term performance of cell-based sensors. We explored the formation of silk-based shells with different chemistries of interactions, such as electrostatic factors between poly-Glu and poly-Lys side chains embedded in the backbone of functionalized silk, and intrarmolecular hydrogen bonding between β-sheets of unmodified silk. Viability and cell function greatly depend on the cell type and the chemistry of interactions used for encapsulation.

Silk, the natural protein extracted from the Bombyx mori caterpillar, is an attractive material for applications in biophotonics due to its biocompatibility and unique mechanical and optical characteristics. A series of achievements for micro- and nano-fabrication using silk have proven that the silk structure can be a useful platform for biophotonic application. In addition, bio-dopants such as enzymes and drugs can be incorporated into a silk film under an all aqueous and mild process. Photonic crystal (PhC) and surface plasmonic resonance (SPR) structures have independently enabled micro/nano photonic devices with novel functionality. Here we report a silk-based hybrid photonic-plasmonic structure (HPPC) and its application as a biocompatible refractive index (RI) sensor. The HPPC composed of a silk inverse opal (SIO) and thin silver (Ag) film on the structured surface of the SIO was fabricated using a PMMA opal template. We used RI matching fluids as analytes. In transmission measurements, we observed simnultaneously the presence of extraordinary transmission (EOT) at the 470-nm wavelength and a band-gap shift around the 650-nm wavelength. Each mode supported by this structure is theoretically verified. Along with these two effects the transmission spectra revealed the complex response as a result of the interplay between the 2D plasmonic crystal and the 3D pseudo-PBG material, making the structure suited to multispectral RI sensing. From the transmission spectra, we estimate the sensitivity to be of 200,000 nm Δ%T/RIU (refractive index unit) which is an order magnitude higher than reported values from tquasi-3D plasmonic crystals and nano-slit arrays. The strong response observed in this experiment is caused by the interplay between the SIO and the plasmonic crystal, This sensitivity can be further improved by broadening the measured wavelength-region to cover more than the EOT resonances and the pseudo-PBGs.

Self-assembling peptide amphiphile molecules form nanofibers through electrostatic interactions among the peptides, where the nanofibers organize into three-dimensional networks similar to the extracellular matrix. Peptide amphiphiles have various application areas, including tissue engineering and drug delivery. One tissue engineering application is to use biologically active peptide amphiphiles to induce angiogenesis. Heparin needed for growth factor activity can be eliminated by using heparin-mimetic peptide nanofibers decorated with bioactive chemical groups (sulfonate, carboxylic acid, hydroxyl). PA molecules with differing bioactive groups revealed no significant differences in mechanical and structural properties analyzed by circular dichroism (CD), rheology, atomic force microscopy (AFM), scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The in vitro angiogenesis assays revealed that sulfonate group itself is not sufficient for an optimal angiogenic outcome. By using the other bioactive groups along with the sulfonate group, in vitro vessel-like structure formation by mouse and human endothelial cells was induced on Heparin-mimetic peptide scaffold without further addition of growth factors and other angiogenic supplements. Endogenous angiogenic growth factors that bind to the bioactive PA nanofibers were sufficient for vessel formation. Heparin-mimetic PA is shown by quantitative RT-PCR studies to actively trigger endothelial cells to enter into angiogenic route. Heparin-mimetic PA nanofibers present the critical functional groups of heparin in a special conformation to the cells and induce more sustained growth factor signaling.

Cancer is a complex and deadly disease that is a large burden on public health world-wide. The disease has been linked with multiple causes (both environmental and genetic) in addition to multiple factors influencing clinical outcome and is increasingly recognized as being of an highly heterogeneous nature. In recent years the role of small sub-populations of cells within tumors such as cancer stem cells and cells undergoing an epithelial-mesenchymal transition have been of increasing interest in research as to their role in cancer but also in the clinical context as potential targets for treatments such as immunotherapy. A wide range of materials including nanomaterials (specifically nanoparticles) and biomimetic substrates have been and are being developed for use in a diagnostic or therapeutic role in disease treatment. However a comprehensive understanding of the responses of cells (including those isolated from tissue of a cancerous nature) to different materials is currently lacking. Cancer research in vitro would benefit from a greater understanding of how cells interact with materials having varying physical and chemical properties. This contribution highlights a multidisciplinary experimental approach to increase our understanding of the response of different tumor derived cell lines (such as melanoma and prostatic adenocarcinoma) to inorganic materials. Cell behavior in the presence of silica based materials that vary in terms of surface functionality and the surface topology is presented. Experimental data presented includes physicochemical characterization of the materials, cell proliferation and adhesion assays and analysis of the influence of the surfaces on the cytoskeleton. The direct relationship of cellular responses to material interactions in the local environment are probed as well as the effect of the presence of cells on the materials themselves. With an improved understanding of cellular responses to different materials, the design of materials which exert a greater control over cancer cells will be better informed and the resulting materials more specifically tailored. These materials in turn can be used to address specific issues in the cancer field, including the development of materials as tools for biomedical research into cancer cell sub-populations.

Elastin is essential to accommodate physiological deformation and provide elastic support for blood vessels. As a long-lived extracellular matrix protein, elastin can suffer from cumulative effects of exposure to chemical damages, which can compromise its biomechanical properties. The mechanical properties of elastin are closely related to its microstructure and the external chemical environments. The purpose of this study is to investigate the changes in the macroscopic elastic properties of purified porcine aortic elastin under the effect of glycation. Glucose was used for the glycation of elastin. Elastin samples were incubated in 2M glucose solution and were allowed to equilibrate at room temperature for 2, 4, 7, and 10 days. Control experiments were performed in 1× phosphate buffered saline (PBS) solution. After treatment with glucose, the side lengths of the elastin sample decrease, however the thickness increases after 2 days and then decreases for longer glucose treatments. Biaxial tensile experiments were performed to characterize the mechanical behavior of elastin. Tangent stiffness was calculated from the stress-strain curves to better compare the mechanical properties of elastin with and without glucose treatments. Experimental results reveal that the elastic behavior of elastin changes with glucose effect. However, the changes are anisotropic in the longitude and circumferential directions. After glucose treatment of 2 days and 4 days, elastin exhibits an increase in stiffness in the longitude, but remains similar mechanical response in the circumferential direction. For 7 and 10 days, glucose treated elastin also becomes stiffer in the circumferential direction. Further studies are needed to understand the structural and anisotropic biomechanical changes in elastin with glucose effect.

Biologically-inspired synthetic polymers prepared from carbohydrate building blocks are promising materials for both basic studies and applications. Robust synthetic methods are needed to allow the structural complexity present in natural polysaccharides to be incorporated into chemically synthesized polymers. Building on our recent success in preparing chiral, controlled molecular weight poly-amido-saccharides via the anionic ring-opening polymerization of a β-lactam sugar monomer, we report the synthesis and characterization of alkyl ether derivatives. We will describe the design, synthesis, and characterization of the ethyl ether monomer, a previously unreported lactam. The formation of homopolymers and co-polymers, and their characterization will be discussed. In addition, the properties of alkylated poly-amido-saccharides will be compared to previously reported alkylated polysaccharides. A notable advantage of our strategy is that different substitution patterns can be achieved with greater levels of control. This control results from the alkyl groups being installed on the monomer before polymerization, in comparison to polysaccharides that are alkylated as macromolecules. Alkyl ether derivatives of natural polysaccharides such as cellulose have been utilized in a variety of biomedical applications. Alkylation of hydroxyl groups is a way to increase the hydrophobicity of the hydrophilic parent poly-amido-saccharides. The modulation of hydrophobicity is important for biomaterials applications such as drug delivery and tissue engineering.

Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) can be used for bone scaffolds or muscular tissue scaffold [1]. PHBV has many attractive properties, such as degradability, biocompatibility, and its natural origin of bacterial fermentation [2]. In scaffold aided regeneration of muscular tissue, composites and blends are utilized as a temporary substrate to stimulate the tissue growth. Electrical stimulation has been shown to enhance the tissue regeneration process [3]. Consequently, conducting polymers are very attractive for the construction of scaffolds. Polyaniline (PANI) has attracted considerable attention in consequence of its good environmental stability, good electrical conductivity, good redox reversibility, ease of synthesis and low costs. Recently studies showed some evidence of ability of PANI to supporting cell growth and nervous tissue [3, 4]. In this study the PANI/PHBV blends were prepared, and thermal properties, crystallization behavior, microstructure of the blends were investigated. The PANI/PHBV blends were prepared by dissolution of PANI (esmeraldine base doped with dodecylbenzenesulphonic acid, DBSA) and PHBV in chloroform and films were obtained by casting. PANI amount in the blend was varied from 0.1 to 1% wt. PANI/PHBV blends were characterized by FTIR spectroscopy, wide angle x-ray (WAXS) and the thermal behavior were analyzed by differential scanning calorimeter (DSC). FTIR spectra of the pure PHBV and PANI/PHBV blend had similar peaks. However, blends spectra shows an enlargement of bands, due interaction of the chain PANI with PHBV matrix. The crystallization behaviors were investigated using DSC, with at a scanning rate of 10oCmin-1.. Curve of pure PHBV showed two melting peaks (159.1oC and 172.3oC). With the increase of PANI amount in the PHBV matrix, both of the melting peaks became wider and shifted to lower temperatures. The decrease trend of first and second melting points with increase of PANI amount, suggests a reduction in the crystallinity of the blends. This behavior was confirmed by WAXS. Keywords: Conducting polymer, scalffold, PHBV, PANI. Work supported by Fapesp. [1] A. Borrietto, V. Guarino, L. Schiavo, M. A. Alvarez-Perez, L. Ambrosio, J. Mater. Sci: Mater. Med. 22, 1053 (2011). [2] Y. D. Zheng, Z. H. Chen, J. Biomed. Mater. Res. B 1, 236, 2007. [3] L. G. Mobarakeh, M. P. Prabhakaran, M. Morshed, M. H. Nasr-Esfahani, H. Baharvand, S. Kiani, S. Al-Deyab, S. Ramakrishna, J. Tissue Eng Regen Med. 5, e17 (2011).

Hydroxyapatite (HAP) is the main component of the biological hard tissues, such as dentine, enamel, and bone and it is considered, together with the collagen matrix, the main building block of the bone. It represents the hard part of the bone; it has a critical role in determining the stiffness and the strength, since it carries most of the tensile load, due to the higher mechanical properties compared to the collagen matrix. The combination of two rather distinct materials, with a staggered arrangement of HAP brittle platelets embedded in a soft collagen matrix, is believed to lead to materials with superior mechanical properties. Geometric confinement can control the mechanical behavior of many biological materials at the nanoscale, and such effects have been suggested to play a crucial role in enhancing the strength and toughness of bone. In this presentation, we will focus on determining the influence of a defect of a particular size on the HAP mechanical properties. In particular, we are interested in seeing how mechanical properties, such as the strength and the fracture toughness, the stress field, and the crack propagation, vary with the presence of different initial crack sizes. Here we study the effect of geometric confinement on the fracture mechanism of hydroxyapatite (HAP) crystals that form the mineralization phase in bone. We perform molecular simulations of HAP crystals with an edge crack, where the crack is situated on the (001) plane. The size effect is taken into account by varying the sample height whilst keeping the width constant. We find that by decreasing the sample height, the stress concentration disappears, in contrast with what is generally described by the classical fracture mechanics, and the material show a different failure mode: by decreasing the sample size, a clear crack path can be recognized in larger samples, whilst the failure of the smaller samples is due to the formation and the interaction of small defects over the whole sample volume. Moreover, the area of high stress concentration appears to be more pronounced, as the sample size increases. The results from this study will help in understanding the high toughness of bone and bone-like materials, although they are mainly made of a brittle material. The methodology developed here can be further extended to include collagen to study the defect tolerance of bone-like materials at the nanoscale.

The goal of this project is to create a biologically relevant graft material with dual functionality. The electrospun mesh features two distinct components, one hydrophobic and the other hydrophilic; meant to influence biological events. The hydrophobic side could function as a barrier to maintain separation of biological fluids whereas the hydrophilic side would act as a scaffold for future cell ingrowth. Controlled deposition allows fabrication of both a bilayer mesh as well as a Janus-type mesh. Three polymers were synthesized from a Poly(glycerol-co-ε-caprolactone) (PGC) starting material. Two polymers were functionalized with a hydroxyl group (PGC-OH) and an acid (PGC-COOH), respectively to give hydrophilic properties whereas the third polymer was functionalized with an octadecyl alkyl chain (PGC-C18) to instill it with hydrophobicity. Polycaprolactone was electropsun with doping of any of the PGC-based polymers. Electrospinning parameters such as polymer concentration, collection distance, voltage and ejection rate were tuned to obtain bead-free fibers. Insulating materials such as electrical tape and glass were utilized to control deposition for Janus-type deposition. Resulting meshes were characterized via scanning electron microscopy (SEM) and contact angle. SEM was used to determine fiber morphology and size. All meshes yielded bead free micron-sized fibers. Contact angles for PGC-OH, PGC-COOH and PGC-C18 doped meshes were 48.3±8.0, 51.3±10.9 and 142.3±3.3 degrees, respectively. Contact angles were taken at 30 seconds after initial mesh-droplet contact, otherwise PGC-OH and PGC-COOH demonstrated complete wetting. Three polymers were synthesized and successfully electrospun into bead-free fibrous meshes that were bilayer or Janus-type. The components have the desired hydrophobic and hydrophilic characteristics. Future work is under way to examine side specific cell interactions and protein binding.

Self-assembled nanostructured materials attracted much attention due to well-defined porous structures. Various types of mesostructured materials have been synthesized via self-assembly method employing surfactants and block copolymers. Here, we present synthesis of self-assembled nanostructured materials such as mesoporous carbons and silicates and their applications to biosensors. Large cellular porous silica materials were synthesized by the self-assembly of commercially available block copolymers, P123. One-pot multi-catalyst system, so called “nanofactory”, was developed entrapping magnetic nanoparticles and oxidases in large cellular mesoporous silica with high loadings of simultaneously above 40 wt% MNPs and 20 wt% enzymes. Our approach provided highly loaded MNP system and any highly loaded enzymes with superior activity, stability, and reusability, thereby making further applications as versatile sensors for detecting DNA, protein, and cell highly promising. A conductive nanocomposite consisting of Fe3O4 magnetic nanoparticles (MNPs) and oxidative enzyme co-entrapped in the pores of mesoporous carbon is fabricated for highly efficient and robust electrochemical biosensing platform. Pt/Fe3O4/mesocellular carbon foam was prepared and the constructed nanocomposite yielded up to 50 times higher catalytic efficiency than that of free MNPs, thereby enabling ultrafast colorimetric immunoassays to detect clinically important target molecules such as human epidermal growth factor receptor 2 (HER2) or diarrhea-causing rotavirus in only 3 min with excellent linearity in a wide range of target concentrations at RT.

Pseudomonas aeruginosa is a gram-negative bacterium potentially pathogenic for humans. The biofilms formed by these bacteria cause high resistance to antibiotics and disinfectants. There is high interest in developing antibacterial coatings to prevent biofilm growth. Hydrogenated amorphous carbon (a-C: H) deposited at near room temperature is a potential candidate for protective and antibacterial coatings of surgery tools, medical implants and devices. The relation between the bacterial colonization factor, and the material&’s composition, structure, topography, surface energy and mass-density was examined. a-C: H were grown by distributed electron cyclotron resonance plasma from C2H2. A wide variety of films, from low-mass density (1.2 g/cm3) polymer like carbon to high density (2.4 g/cm3) tetrahedral a-C: H was considered. The films composition, structure, topography and physical properties were characterized by Raman spectroscopy, electron energy loss spectroscopy, electron diffraction; laser induced surface acoustic waves, Fourier transform infrared spectroscopy, spectroscopic ellipsometry, atomic force microscopy and contact angle measurements.

In the US alone, the forest products industry is as big as the automotive and plastics industries. Sustainability and other environmental concerns have motivated the creation of a wide array of new wood adhesives and engineered wood products. A key necessity in this field is to characterize and understand the effects of chemical modifications on mechanical properties of the amorphous and semi-crystalline polymers that make up the cell walls in wood. Toward that goal we have developed techniques that transform nanoindentation into a local probe for mechanical spectroscopy. In mechanical spectroscopy viscoelastic and viscoplastic properties are assessed across broad spectra of time scale, deformation rate, temperature, and moisture content. Broadband nanoindentation creep (BNC) measures viscoplastic properties across 4-6 decades of strain rate, and broadband nanoindentation viscoelasticity (BNV) measures viscoelastic properties across greater than 8 decades of time scale. Variable-temperature BNC and BNV are also performed. All of these measurements can be made on volumes of material that are less than one micrometer across, and there is no fundamental limit against much faster measurements or much smaller volumes. In validation studies we have applied BNC and BNV on poly methyl methacrylate (PMMA), polycarbonate (PC), polystyrene (PS), low density polyethylene (LDPE), and polyether ether ketone (PEEK). For all of these materials the viscoelastic moduli and loss tangent obtained with BNV agrees with conventional dynamic mechanical analysis (DMA) measurements. Likewise, the flow stress-strain rate characteristics obtained with BNC agree with measurements made in conventional experiments. Both BNV and BNC are sensitive to small changes in structure brought about, for instance, by differential cooling rates in the cross-sections of extruded polymer sheets, polymer plasticization, and chemical modification of polymers in wood cell walls.

Electrocatalytic detection of biological species, such as cardiolipin is complicated by the adsorption of products of electrode reactions or matrix components that cause electrode failure. To address this problem electrochemically assisted deposition of sol-gel films (50-200nm thickness), from tertaalkyl orthosilicate and with ca. 10-15nm pores normal to the electrode surface can be achieved by attaching generation-4 poly(amidoamine) (PAMAM) dendrimer onto the electrode prior to film formation [1]. By end-capping negatively charged silanol sites of the film as a post-treament, perturbation of the voltammetry of ions is eliminated [2]. An alternative is to use organically modified precursors to preclude the need for the end-capping procedure. Such films may find an application in designs of batteries, supercapacitors, fuel cells and biosensors, especially in conjunction with immobilization of electron-transfer catalysts. In the present study, extending the range of size of the pores was investigated. Electrode surfaces were modified with aminopropyl trimethoxysilane (APTES) to obtain positive sites. Next, polystyrene (PS) spheres in the range 50-75 nm were electrostatically attached. After removal of PS, the surfaces were characterized by scanning electron microscopy. Of particular interest was whether the array of the resulting pores could be varied in terms of density by changing the surface coverage by APTES. Attempts to vary the size of the PS and, hence, the pore diameter was also investigated. Dirhodium-substituted polyoxometalates were employed as catalysts for the oxidation of biological compounds including cysteine, cystine, methionine, and the phospholipids, phosphatidylcholine and cardiolipin. Various methods such as electrostatic binding within the pores and adsorption to metal nanoparticles on the electrode surface were studied as means of immobilizing the catalysts. Optimized electrodes were evaluated for the voltammetric determination of selected test species in matrices that include known fouling agents such as bovine serum albumin. They were also used for electrochemical detection after HPLC separation. References: [1] M. Wandstrat, J.A. Cox, W. U. Spendel, G. E. Pacey, Electroanalysis 19, 139 (2007). [2] J.A. Cox, B. L. Mehdi, K. M. Wiaderek, B. P. Gudorf, D. Ranganathan, S. Zamponi, M. Berrettoni, J. Solid State Electrochemistry, 15, 2409 (2011).

Biomimetic cilia flow sensors and manipulators were developed for use with existing and emerging microfluidic platforms. The development of low cost, portable, small sample detection systems is a highly energized area of medical research. Applications of such systems include early disease detection point-of-care for rural clinics, retirement communities, and developing countries. The systems also have applications in complete blood counts(CBC) for cancer patients who have CBC&’s done multiple times per week during treatments. Many of the unique and brilliant detection systems published have great potential, but they often rely on a large set of external pumps, flow detectors and complicated circuitry to enable their output. In order to address this cilia, Nature&’s microflow sensors and manipulators, were mimicked through the use of magnetic nanowires fabricated by electrodeposition in an oxide template. These cilia were either placed directly on Giant Magnetoresistance(GMR) sensors or suspended above GMR sensors within a polymer microflow channel. The wires were released from their oxide templates so that they could bend with an applied flow. The shape anisotropy of the wires under zero flow conditions produced a field that was perpendicular to the GMR&’s plane, yielding a base voltage. When the wires were bent due to the action of a flowing fluid, the component of the field parallel to the surface of the sensor increased, yielding a voltage change in the sensor which was easily sensed. This design is an improvement over other sensors on the market in a variety of ways. First, our design is easily integrated into sensor systems, and it takes up less than a 100µm2 area. The sensor uses no external amplification, produces a SNR of 700 and only uses 140µW of power. This combination of low power consumption and the inherent temperature stability makes this type of sensor well suited for portable applications. Similar to cilia, the nanowires can mimic flagella through the application of a rotatingexternal magnetic field. Therefore, the sensor can also be used for manipulation and flow agitation to promote mixing and flow. This dual function of the sensor sets it apart from many in the literature which only have a single capability. Therefore, our flow sensor has unique potential to be integrated into a variety of systems that need flow characterization manipulation, or mixing in order to make analyte detection systems capable of meeting the quality and precision control that the FDA demands.

Natural extracellular matrix (ECM) proteins, e.g. collagen, gelatin, and elastin, are used to create hydrogels for different biomedical applications, such as drug delivery and tissue engineering. ECM proteins benefit from their superior biological properties; however, poor mechanical performance often limits their broad application. The conjugation of these proteins with a synthetic polymer to generate hybrid structures that possesses a superior mechanical strength may address this issue. The aim of this study was to develop a copolymer with good mechanical properties that is actively conjugated to protein based polymers. The copolymer consisted of thermo responsive N-isopropylacrylamide (NIPAAm), an active N-acryloxysuccinimide (NAS) group for polymer functionalization, 2-hydroxyethyl methacrylate-poly(lactide) (HEMAPLA) to enhance mechanical strength, and hydrophilic oligo(ethylene glycol) monomethyl ether methacrylate (OEGMA) blocks. NMR results showed that it was feasible to synthesize and make tailored combinations of these copolymers. The composition of monomers had a significant impact on the lower critical solution temperature (LCST). The results of FTIR analyses demonstrate that protein conjugated efficiently (>90%) to the co-polymer. Of the panel of constructs, a co-polymer composition was selected on the basis of having a combination of gelation time <10 min, a gelation temperature between 25°C and 37°C and desirable mechanical properties suitable for soft muscular-skeletal applications. The conjugated polymer produced hydrogel and possessed a linear elastic modulus of 195 ± 11 kPa and initial compression modulus of 70 ± 11 kPa. The result of this study demonstrates the potential of using the conjugated products for a broad range of proof-of-concept biomedical engineering applications, including load bearing tissue engineering.

We present in-vitro biocompatibility studies of biomolecule-wrapped CoMoCAT (6,5) single-walled nanotubes. Non-covalent functionalisation of single-walled nanotubes by blood protein (bovine serum albumin, BSA) and, separately, the oligonucleotide (AC)15 was achieved. Absorption and photoluminescence spectra of the resulting functionalised nanotube in an aqueous solution were measured and used to calculate the mean surface binding density. This was found to be ca. 40 BSA molecules and ca. 100 (AC)15 per nanotube. Mouse macrophage (RAW264.7) was used as a model system to investigate the dynamics of phagocytosis, and the effect of carbon nanotube uptake on cellular viability. Through monitoring the Raman spectra and the cell growth in a culture solution containing 8mg/mL of functionalized CNTs, uptake by the macrophages was found to be ca. 70mg/mL for confluent coverage. The growth of macrophage was suppressed after an incubation period of 2 days, which implies possibly the onset of negative response.

Polymer nano- and microparticles are intensely investigated as drug carriers for biomedical applications. Targeted delivery, controlled cell uptake, and controlled release of cargo molecules are demanding functions that have to be implemented into the carriers. In nature this is mastered to perfection by viruses. They recognize cells, attach to their cell membrane and release their cargo (DNA) into the cell interior. The current strategy for polymeric carriers is similar, involving cell recognition and membrane attachment, but further requiring carrier internalization, e.g. by endocytosis, to deliver the cargo into the cell interior. We show by using covalently attached fluorescent dyes and quantum dots, that block copolymer micelles and vesicles (polymersomes) deliver hydrophobic drugs into cells without internalization. Examples include the delivery of hydrophobic dyes and drugs for cancer phototherapy. As the polymeric carrier does not have to be internalized, this considerably reduces the number of functions that have to be implemented into the carrier together with significantly reducing its cell toxicity.

The cell membrane is a natural barrier that prohibits foreign polar molecules from entering the cell interior. Although it is an excellent defense system against harmful substance in the extracellular environment, it also prevents therapeutic agents especially macromolecules to access their intracellular targets. Over the years, numerous researchers have devoted themselves to develop drug delivery approaches to overcome this natural barrier. Various strategies have been invented, including the utilization of liposomes, cell-penetrating peptides, virus capsids, and other vehicles. With these vehicles mediate the cellular uptake of therapeutic macromolecules primarily via an endocytotic pathway. This process, however, is commonly associated with limited endosomal release, and efficiency depended on cell type. Although nature presents us excellent delivery strategies to bypass endocytosis, e.g. paramyxovirus, the non-endocytotic pathway is rarely explored. Recently, we developed a novel strategy to deliver protein and genes across the plasma membrane via a non-endocytotic pathway. By encapsulating proteins in a cationic polymer shell, we could intracellularly deliver the proteins with high efficiency. The delivered protein is uniformly distributed in cytosol after cellular uptake. Moreover, similar levels of cellular uptake were achieved even in the presence of inhibitions of low pH-dependent endocytosis. Similar results can be observed in the case of DNA cassette delivery and in various cell types. We believe that delivery through non-endocytotic pathways represent a breakthrough novel approach in the drug delivery field.

The rapid growth of the field of nanomedicine has seen the concomitant increase in the demand of multifunctional tools for rapid disease detection both in vitro and in vivo and highly effective therapy. Noble metal nanoparticles (NPs) are especially useful in this context because they can be easily synthesized in different sizes and shapes, can be functionalized with multiple moieties, and most importantly, can support surface plasmons that can induce extremely high electromagnetic field enhancements, especially useful in techniques such as surface enhanced Raman spectroscopy (SERS) and photothermal therapy. In this contribution I will present our results in the synthesis and application of multifunctional gold NP dimers capable of specific cell targeting, optimized uptake, and highly resolved imaging. The systems presented here are based on spherical gold NPs functionalized to target U87 glioblastoma cells, whose synthesis was optimized with the aid of simulations developed in our group. Dithiolated linkers have been used to lock the NPs in a dimer conformation and also act as SERS reporters owing to their strategic positioning within the intermetallic junction, also known as hot spot. In our experiments, heterobifunctional polyethylene glycol (HS-PEG-NH2) was used to stabilize the NP systems, avoiding coalescence and improving biocompatibility, while exposing to solution a pendant amino group that renders the dimers amenable of further functionalization. Red emitting rhodamine molecules were bound to the surface of the NP dimers to confirm our results via the more traditional fluorescence confocal microscopy, and to test the feasibility of dual mode targeting platforms. The use of cyclic RGD peptides to target αvβ3 integrins overexpressed on the surface of cancerous cells ensured an efficient targeting and uptake of the SERS tags. Combined analysis carried out via confocal and Raman microscopy demonstrated the efficient tagging and imaging of U87 cells via SERS-based Au NP dimers-based tags at only three hours of incubation, compared to the 18 hours necessary with fluorescence. In addition, and for the first time, we observed via TEM that the tags retained their dimeric conformation upon endocytosis, hence demonstrating their stability against enzymatic cleavage. Finally, preliminary results demonstrate the applicability of our tags in photothermal therapy, thus opening the way to a novel SERS-based theranostic platform. Current studies are now targeting melanoma cells, to demonstrate the versatility of our system and set the ground for new SERS-based theranostic tools.

Successful development of a non-invasive cancer detection method to replace tissue biopsy for screening high risk patients and assessing treatment response and surveillance of patients could be highly valuable. Non-invasive biomarker detection strategies relying on circulating tumor cells (CTCs) and microvesicles (MVs) are likely to be of biological relevance to tumorigenesis. We have recently developed a novel and simple ‘tethered ImmunoLipoplex Nanoparticle (tILN) microarray&’ method whereby intracellular molecular probes, such as molecular beacons (MBs) and/or peptide aptamers, and cell surface ligands including antibodies, are encapsulated and inserted respectively in liposomal nanoparticles tethered on a solid surface. After CTC capture, cells will internalize tILNs and the encapsulated MBs can detect specific microRNAs and/or mRNAs as intra-cellular biomarkers. The same tILN is also able to capture and detect microRNAs and/or mRNAs biomarkers contained in MVs such as exosomes and free microRNAs in serum from patient blood samples. We have developed a simple method for preparing the tILN microarray by micro-contact printing using a micropillar array stamp and an ink mixture of lipidic anchor molecule and a biotin-conjugated thiol molecule onto a gold coated glass substrate. To prevent nonspecific cell binding, we introduced a mixture of polyethylenegylcol (PEG) thiol molecules with 2,000 and 20,000 Da to protect the 80% non-disk area. To detect intracellular biomarkers in captured living cells and MVs, we can encapsulate MBs in tILNs. We used the biotin-avidin linkage method to incorporate antibody ligands onto tethered lipoplex nanoparticles at room temperature. Utilizing this concept, uptake and internalization of MB-containing tILNs allows direct detection of intracellular biomarkers in the captured living cells. Similarly, fusion of MVs onto the same MB-containing tILNs would allow direct detection of intracellular biomarkers in those circulating MVs. As an example, our tILN microarray has successfully detected miR-21 and TTF-1 mRNA in captured lung cancer cell line, A549 cells and normal epithelial cells after 4 hr by encapsulating detection probes in the tethered liposomal nanoparticles. A549 cells showed higher fluorescence intensities from the over expressed miR-21 and TTF-1 fluorescence signals comparing with normal epithelial cells, which is consistent with qRT-PCR results. Using blood samples from lung cancer patients, our preliminary data showed that the same tILN can also capture CTCs from PBMC and exosomal cancer microRNA and mRNA from serum using anti-EpCAM as the cell surface marker and miR-21 and TTF-1 as intra-cellular and serum markers. This facile method has great potential for non-invasive early cancer detection and treatment surveillance. The captured living CTCs can be used for further functional assessment, not achievable by any existing methods

We developed nanoconjugation technique to allow controlled formulations of sub-100 nm, mono-modal polymeric nanoconjugates with definable drug loading, quantitative drug loading efficiency and controlled release profiles. Nanoconjugates were prepared through drug-initiated ring-opening polymerization followed by nanoprecipitation. Hydroxyl-containing therapeutic agents were used as initiators to initiate living polymerization of cyclic ester monomers (e.g., lactide), and resulted in polyester-drug conjugates. The precipitation of the polyester-drug conjugates gave rise to the desired polyester-drug nanoconjugates. Using paclitaxel as a model drug, we formulated paclitaxel-polylactide nanoconjugates with 100% drug incorporation efficiency and up to 37% drug loading. This new type of nanoparticles showed excellent cancer targeting capability when cancer-specific aptamer ligands were conjugated to the surface of nanoconjugates. I will also discuss our recent research progress on developing size controlled silica nanoconjugates and assessing these silica-drug nanoconjugates for tumor penetration, biodistribution and anticancer efficacy.

The allure of single walled carbon nanotubes (SWCNTs) in biomedicine is their ability to translocate into various mammalian cells and accumulate in several compartments. Majority of reported findings point to the cytoplasm as the final destination for internalized carbon nanotubes. Recent Reports show that there is an optimal nanoparticle size at which the speed of endocytosis is fastest. This size also controls the intracellular fate of nanoparticles and their eventual release. In this work, we show the efficient internalization of two different sizes of Ultra Short single walled carbon nanotubes by Human Umbilical Vein Endothelial Cells (HUVEC), HeLa and Liver cells. Whiles both HeLa and Liver cells transported the Ultra short nanotubes to their nucleus and efficiently excreted them, nuclear accumulation of the Ultra short nanotubes in HUVECs was minimal with prolonged intracellular persistence. Taken together, these results show that the internalization and intracellular fate of SWCNTs in mammalian cells can be controlled. Also, nuclear accumulation and efficient excretion is both length and cell dependent.

Although the answer to the question of the potential toxicity of carbon nanotubes is not very clear yet, some promising biomedical applications are already in development. These applications mainly include 2 categories, related to different fields of applications. In the first case, the nanotubes are intended to be localized in a specific area of the body, such as inside an implant where they would be used as a substrate for cell growth (tissue engineering) [1]. In the second one, they are intended to be used for systemic distribution, and used as cargoes for drug-delivery, or contrast agent for imaging [2]. These 2 situations represent very different scenarios in terms of potential toxicity. In this work, the DWNTs are synthesized by a Catalytic Chemical Vapour Deposition process developed at the CIRIMAT [3]. We are presenting different potential applications of double-walled carbon nanotubes (DWNTs) currently in development in the two categories defined above, as substrate for the guided growth of neurons, MRI contrast agents or cargoes for drug-delivery (transfection). Our latest results in terms of interaction between neuron cells and DWNTs will be presented, showing that they enhance significantly the development of neurites and limit the differentiation into scar tissue, which is very attractive for in vivo applications (implants, or monitoring electrodes). We will also illustrate how carbon-encapsulated carbon nanoparticles, a typical by-product of the CCVD synthesis of carbon nanotubes, may also be used for combined MRI imaging and hyperthermia [4]. References: [1] A. Beduer, L. Vaysse, E. Flahaut, F. Seichepine, I. Loubinoux, Ch. Vieu, Microelec. Eng., 88, (2011), 1668-1671; [2] V. Sanz, C. Tilmacîu, B. Soula, E. Flahaut, H.M. Coley, S.R.P. Silva, J. McFadden, Carbon, 49, (15), (2011), 5348-5358; [3] E. Flahaut, R. Bacsa, A. Peigney, Ch. Laurent, Chem. Commun., (2003), 1442-1443; [4] P. Lukanov, V. K. Anuganti, Y. Krupskaya, A-M. Galibert, B. Soula, C. Tilmaciu, A.H. Velders, R. Klingeler, B. Büchner, E. Flahaut, Adv. Funct. Mater., 21, (2011), 3583-3588;

A new era of polymer nanoparticles is currently being developed for use in photothermal ablation therapies in the treatment of bacterial soft tissue infections as well as cancer. We have used nanotubes (NT) composed of the electrically active polymer poly (3,4-ethylenedioxythiophene) (PEDOT) for photothermal ablation. PEDOT NT have a broad absorption band from 700 to 1400nm. It has been proposed that infrared absorption of PEDOT is dominated by bipolarons strongly coupled to phonons; we hypothesize that non-radiative decay of these states leads to heat generation. The optimal window for body tissue transparency to infrared light is between 700-1100nm. Therefore, since PEDOT NT generate heat upon absorption within this range, we have chosen to use them for photothermal ablation. We first evaluated the use of PEDOT nanotubes for photothermal ablation of both gram-positive (Staphylococcus aureus) and gram-negative (Escherichia coli) bacteria. Complete eradication of bacterial colonies incubated with 100ug/ml of PEDOT NT occurred with a single exposure to 1064nm light (3.8W/cm2) for 60s. PEDOT NT were also shown to elicit a mild antibacterial response upon incubation with bacteria and no infrared exposure. PEDOT NT have the same capacity for photothermal ablation of bacteria as compared to single-wall carbon nanotubes; therefore, they represent an exciting new class of polymer based nanoparticles for medically-relevant photothermal therapies. We have also evaluated the use of PEDOT NT for photothermal ablation of colorectal cancer cells in vitro. PEDOT NT do not elicit a cytotoxic response up to 1mg/ml concentrations. Upon exposure of 808nm light at 0.6W for five minutes, two different colorectal cancer cell lines, RKO and HCT 116 showed an 80% reduction in cell viability. This work shows that PEDOT NT are safe for medical photothermal ablation procedures; however the broad infrared absorption is not optimal. Thus, we have developed low band gap donor-acceptor (D-A) electrically conducting polymer (ECP) nanoparticles that can be tuned to absorb near infrared light and in turn generate hyperthermia. An aqueous dispersion of spherical nanoparticles composed of cyclopentadithiophene with 2,1,3-benzothiadiazole (PCPDTBT) and 2,1,3-benzoselenadiazole (PCPDTBSe) were synthesized. These polymers absorb around 800 nm and have average diameters between 50-500nm. Incubation of the nanoparticles with colorectal cancer cells, in the absence of infrared light, showed that the particles are not cytotoxic up to 1mg/ml. Upon exposure of 808nm light at 0.6W for five minutes, RKO and HCT 116 cells showed an 80% reduction in cell viability, which was similar to our results using PEDOT NT. These results demonstrate that there is a new subset of nanoparticles, composed of electrically conductive polymers, that are inherently non-toxic, and very useful for photothermal ablation techniques that could be used clinically.

Near-infrared (NIR) photothermal therapy using nanomaterials, such as carbon or metal nanoparticles, has garnered much attention as a minimally invasive and efficient treatment for cancer. Under NIR stimulation, excitation of vibrational resonances in the nanomaterial allow for heat generation, which destroys surrounding cancer cells. We have recently developed donor-acceptor (D-A) electrically conducting polymer (ECP) based nanoparticles that can be used for NIR photothermal therapy. Low band-gap D-A ECPs are polymers that incorporate both an electron donating monomer and an electron accepting monomer into their backbone. Through judicious choice of co-monomers used, D-A ECPs can be tuned to have excellent absorption properties in the NIR window from 700-900 nm, where body tissue is most transparent. We have utilized low band gap D-A ECPNs along with NIR light to photothermally destroy cancer cells in-vitro. The D-A ECPs chosen for this study are based on cyclopentadithiophene with 2,1,3-benzothiadiazole (PCPDTBT) and 2,1,3-benzoselenadiazole (PCPDTBSe) because both of the copolymers absorb around 800 nm (E_g = 1.46 and 1.37 eV), which is near the wavelength that we have identified for laser treatment (808 nm) of cancer cells. We have employed a facile technique to disperse the D-A ECPs in aqueous media to form D-A electrically conducting polymer nanoparticles (ECPNs). Dynamic light scattering (DLS) experiments showed that nano-PCPDTBSe had an average diameter of 154.3 nm, while nano-PCPDTBT had an average diameter of 298.4 nm. The heating efficacy of the D-A ECPNs was tested and compared to a nanomaterial known to heat very well under NIR stimulation (oxidized multi-walled carbon nanotubes). It took ~23 mu;g of nano-PCPDTBT and ~45 mu;g nano-PCPDTBSe to change the temperature of cell medium solution by 20^oC after five minutes. The NIR heating of the D-A ECPNs was shown to be reproducible over 5 heating/cooling cycles. Cytotoxicity studies were undertaken and the results confirmed that nano-PCPDTBT and nano-PCPDTBSe showed no significant toxicity towards either HCT116 or RKO colorectal cancer cells from 10-1000 mu;g/mL in the absence of NIR light. Thermal ablation studies were performed and demonstrated that for concentrations above 100 µg/mL (125 and 250 mu;g/mL), cell survival for HCT116 and RKO was less than 20% for both nano-PCPDTBT and nano-PCPDTBSe. This study confirms that a new nanoparticle, based on a donor-acceptor polymeric structure, can be used to photothermally ablate colorectal cancer.

Magnetic iron oxide nanoparticles are increasingly being utilized as contrast enhancing agents in magnetic resonance imaging (MRI), magnetically targeted drug delivery agent and in magnetic hyperthermia. Recent developments have shown that size and size distribution of nanoparticles play a very critical role in the biomedical application of these magnetic nanoparticles. We have previously demonstrated the synthesis of monodisperse iron oxide nanoparticles using the high-temperature thermal decomposition of organometallic iron precursors. Nanoparticles synthesized by this method are uniform in size with very narrow size distribution and the size tunable by changing the experimental conditions. In this work, we have carried out a systematic study on the effect of particle size of iron oxide nanoparticles on its MRI contrast and magnetic hyperthermia. In the first step, nanoparticles of various sizes from 4 nm to 20 nm were synthesized by the thermal decomposition method in organic solvents and then coated with phospholipids containing PEG. The use of PEGylated phospholipid enables water solubility, imparts better dispersity and long circulation in blood stream. This results in a core-shell like morphology with iron oxide nanoparticle forming the core and phospholipid PEG forming the shell. The nanoparticles were characterized for their size and morphology using dynamic light scattering (DLS) and transmission electron microscopy (TEM). The hydrophobic core can also act as a depot for hydrophobic chemotherapeutic drugs. In the second step, hydrophobic drug docetaxel (DTX) was loaded with the core of the different size iron oxide nanoparticles. We further studied the effect of external magnetic field on its ability to trigger the release of drugs. The use of external oscillating magnetic field ensures the localized release of drugs only at the site of accumulation thus minimizing damage to adjacent areas. In vitro studies were carried out to study the efficacy of DTX in both HeLa cervical cancer and PC3 prostate cancer cell lines. Results from the size dependent MRI contrast from phantoms and cancer cells incubated with nanoparticles and the effect of magnetic field on triggered release of drugs will be presented. This work was supported by IGERT Nanomedicine Science and Technology Program (NSF 0965843), HHS/1U54CA151881 CORE 1 and Northeastern University.

Gold nanorods (GNRs) have emerged as promising nanomaterials for diagnostics, imaging, photothermal treatment and therapeutic delivery in cancer diseases. We report that gold nanorods, synthesized using chemical methods, demonstrate rapid aggregation in biologically relevant media, which results in poor photothermal properties. As a consequence, we generated poly(amino ether)-functionalized gold nanorods, or PAE-GNRs, using a layer-by-layer deposition approach; polymers from a poly(amino ether) library recently synthesized in our laboratory were employed to generate PAE-GNR assemblies. PAE-GNRs demonstrate long-term stability, which, in turn translates to reproducible photothermal response. Polymer-modified gold nanorods demonstrated hyperthermic ablation of prostate cancer cells, when irradiated with near infra-red light. In addition, sub-toxic concentrations of PAE-GNRs generated using polymers from our laboratory demonstrated higher transgene expression and exhibited lower cytotoxicities when compared to similar assemblies generated using 25 kDa poly(ethylene imine) (PEI25k-GNRs), a current standard for polymer-mediated gene delivery. Our results indicate that surface engineering using biocompatible polymers leads to multifunctional gold nanorod-based assemblies that combine high stability and low cytotoxicity with photothermal ablation, transgene delivery, and optical imaging capabilities on a single theranostic platform.

Biomotors in nanotechnology are as important as macroscopic motors in society. The bacterial virus phi29 DNA packaging biomimetic motor is one of the strongest motors assembled to date using chemically synthesized or purified recombinant components. The ingenious motor design with an elegant and elaborate channel has inspired its application in nanotechnology and nanomedicine. The motor is geared by a hexameric RNA ring and driven by an ATPase to translocate dsDNA through a dodecameric channel which forms a ring with a central 3.6 nm channel acting as a path for dsDNA to enter during packaging and exit during infection. The observed single direction dsDNA transportation provides a novel system with a natural valve to control dsDNA loading and gene delivery in bioreactors, liposomes, or as a high throughput DNA sequencing apparatus. Insertion of the motor into the membrane of the liposome has created a bioreactor with pores that can easily be turned on and off. We elucidated the sequential action of the motor ATPase and additional motor components in motor action. The contact of ATPase to ATP resulted in its conformational change to a higher binding affinity towards dsDNA. It was found that ATP hydrolysis led to the departure of dsDNA from the ATPase/dsDNA complex, an action to push dsDNA to pass the connector channel. DsDNA packaging went through a combined effort of both the gp16 ATPase for pushing and the channel as a one-way valve to control the dsDNA translocation direction. Combination of the two distinct roles of gp16 and connector renews the perception of previous dsDNA packaging energy calculations and provides insight into the discrepancy of the number of base-pairs translocated per ATP. Relevant Publications: 1. Schwartz C, Fang H, Huang L & Guo P (2012). Sequential action of ATPase, ATP, ADP, Pi and dsDNA in procapsid-free system to enlighten mechanism in viral dsDNA packaging. Nucleic Acids Res 40:2577. 2. Guo P (2010). The emerging field of RNA nanotechnology. Nature Nanotechnology 5:833. 3. Wendell D, Jing P, Geng J, Subramaniam V, Lee T, Montemagno C, & Guo P (2009). Translocation of double stranded DNA through membrane adapted phi29 motor protein nanopore. Nature Nanotechnology 4:765.

Hydrogels are a class of biomaterials that are useful for tissue regenerative therapies and drug delivery. Controlling the degradation of the hydrogel can aid in the integration of the newly formed tissue. For numerous applications, the rate of hydrogel degradation should approximate the rate of new tissue formation. Furthermore, upon injury to tissue, the surrounding cells secrete extracellular proteases that hydrolyze the amide-containing components of the extracellular matrix. If the hydrogel is peptide-based, secreted proteases can provide a means to degrade the hydrogel scaffold with temporal resolution. The degradation profile of five self-assembling β-hairpin peptide hydrogels by proteolysis with matrix metalloproteinase-13 (MMP-13) was assessed by oscillatory rheology, analytical HPLC and mass spectrometry. Each hydrogel peptide was designed to vary in its susceptibility to MMP-13. Results showed that degradation of peptide based scaffolds can be controlled by varying the peptide&’s amino acid sequence from 5% to 70% after exposure to MMP-13 for 14 days. Furthermore, degradable peptide hydrogels are non-cytotoxic and allow for migration of SW1353 cells.

Mucins - large heavily glycosylated proteins populating membranes of epithelial cells - have a dual biological purpose: to protect the epithelial surface by creating a physical barrier and to serve as mediators of cellular communication and signaling. Despite their importance, very little is known about the molecular mechanisms of mucin function. This is due mainly to the structural complexity and heterogeneity of mucins and the fact that their glycans are metabolic products and, thus, outside of direct genetic control. Inspired by mucins, we have developed synthetic materials that emulate the architecture and function of native mucins but afford exquisite control over glycan structure, valency and presentation. Using the RAFT polymerization technique, we have now generated a number of mucin mimetics endowed with useful functionalities for their integration with synthetic materials (supported lipid bilayers, silicon wafers, or nanoparticles), as well as living cells. In my presentation, I will show that mucin mimetic microarrays can be used to evaluate in a high-throughput manner how parameters, such as the valency of mucins and their spatial separation, may determine the modes of their interactions with receptors. I will also show that the mucin mimetics can be introduced into the Glycocalyx of living cells to perturb the biophysical properties of their surface and, consequently, their interactions with the surrounding matrix.

Mesoporous materials have attracted a great deal of research interest both industrially and academically as adsorbents, catalysts, sensors and fuel cells owing to their well-ordered structure, organized porosity, high specific surface area and pore volume. Among them, mesoporous silica materials having Fm3m symmetry and well built 3D porous architecture such as FDU-12 are considered to be more advantageous as applicants in the field of adsorption, encapsulation and separation of proteins wherein large biomolecules are involved. Despite numerous methods available in the open literature to synthesis FDU-12, the focus have recently been shifted towards microwave assisted irradiation technique, which facilitates the design of mesoporous materials quite rapidly saving reaction time, energy and cost by manifolds and without compromising its structural and textural qualities. In addition, the fabrication of large pore FDU-12 is also critical to encapsulate huge amount of biomolecules with a large size. In the present work, novel 3D mesoporous FDU-12 silica materials with ultra-large nano-cages tuneable porous structure have been synthesized for the first time using non-ionic surfactant via rapid high temperature microwave-assisted process. We demonstrate that the pore diameter of the FDU-12 materials can be finely tuned from 5 to 22 nm with a simple adjustment of the reaction temperature of the microwave process from 100 to 250 °C. The physico-chemical traits of the prepared materials are thoroughly explored employing high-end techniques such as powder X-ray diffraction, N2 adsorption studies, high resolution scanning electron microscopy (HRSEM), high resolution transmission electron microscopy (HRTEM) and FT-IR spectroscopy. All the samples exhibit XRD patterns that are characteristic of three-dimensional mesoporous FDU-12 silica materials with inherent Fm3m symmetry and ultra-large nano-cages. N2 adsorption results reveal that all the FDU-12 materials produced possess excellent textural characters including large pore volume and large pore diameter. Finally, the immobilization of the biomoelcules over FDU-12 with tuneable pore diameters is studied. It is found that the pore diameter of the FDU-12 plays a critical role in controlling the amount of immobilized protein inside the mesochannels. The material with the largest pore diameter offers a quick and a high adsorption of the large biomolecules such as lysozyme, myoglobin and cytochrome c. The large adsorption capacity of the FDU-12 with the largest pore size could be due to the easy diffusion of the biomolecules that can freely access the adsorption sites that are available even in the interior part of the pores without any steric or diffusion constraints. The effect of pH and textural parameters of the adsorbents affecting the immobilization of biomolecules is also investigated and the results will be presented during the presentation.

Lack of technology for chronic high-throughput neural recordings and simultaneous stimulation is a major obstacle in the development of neuroprosthetics, and in understanding of the electrophysiological signatures of the diseased or damaged nervous system. Capitalizing on fabrication methods developed by optical fiber industry, we create a new generation of electrode arrays and optoelectronic neural scaffolds, which aim to minimize tissue damage, produce reliable neural recordings and potentially facilitate repair of damaged neural tissues. Taking advantage of the recently developed method of “optogenetics”, which uses light to stimulate neurons, we have previously developed a miniaturized device optetrode that combines optical stimulation with simultaneous electronic recordings from tens of individual neurons in freely moving animals (Anikeeva et al., Nat. Neurosci. 2011). Our streamlined design, which centered around an optical fiber, produced minimal tissue damage and maintained high quality recordings over the course of several months. We now extend the concept of fiber-electrode composites to integrated hybrid polymer-metal flexible arrays incorporating tens to hundreds of electrodes (2-3 µm in diameter). In addition, our fabrication approach allows for incorporation of drug delivery channels and optical waveguides. Finally, we employ fiber-electrode composites as scaffolds for individual neurons. We demonstrate the potential of our devices as a platform for testing of neuronal viability and electrical activity as well as a tool for investigation of neuronal growth under controlled conditions.

Contact guidance reactions are highly conserved, active cellular responses to structural components in the extracellular matrix. Cell-topography responses are likely to govern decision-making processes regarding interactions with micro- and nano-particles such as opsonizaiton and phagocytosis versus frustrated phagocytosis. Contact guidance has also been implicated in fundamental processes in development, cell migration, and neural regeneration. Here I will present recent advancements in from our laboratory in the rationale design of programmable substrate topography to elucidate the mechanism of contact guidance in mammalian cells. A facile processing strategy for the fabrication of these surfaces will be presented. This new class of materials will be used as a critical tool in combination with high-resolution live-cell imaging to measure filopodia dynamics of IC-21 macrophages. Filopodia dynamics will be measured in response to the instantaneous presentation and removal of topographic grating cues with feature sizes of approximately 1 micron in wavelength and 300 nm in amplitude. Empirical observations from these experiments are used to inform a new hypothesis for contact guidance that is based on passive filopodia diffusion. Specifically, these data will be used to support a working model that is based on biased filopodia migration dynamics in a potential field via transition state theory. Possible implications for this model of contact guidance will be discussed in the context of designing micro/nanoparticles and biomaterials interfaces.

Quantification of certain neurochemicals could be a useful diagnostic tool for the early detection of neurological disorders. In particular, monitoring of changes in extracellular glucose concentration in brain may improve diagnosis and therapy for diabetes and brain tumors. Electrochemical biosensors produce a electronic signal, which is proportional to the concentration of a specific analyte. High performance biosensors require sensor specificity within acceptable sensitivity and limits of detection for each analyte. We have developed a novel method for fabrication of highly sensitive amperometric glucose biosensor by utilizing poly(ethylene oxide) (PEO) hydrogel and doped conducting polymer poly(3,4-ethylenedioxythiophene) : poly(styrenesulfonate) (PEDOT:PSS). The fabrication process includes electrospinning of PEO-PEDOT:PSS composite nanofibers on the surface of platinum electrodes. The glucose oxidase (GOx) enzyme was incorporated into PEO-PEDOT:PSS composite nanofibers during electrospinning. To prepare hydrogel nanofiber, PEO-PEDOT:PSS nanofibers were crosslinked using UV polymerization in the presence of a photoinitiator . immobilized GOx in the PEO-PEDOT:PSS hydrogel nanofibers was immediately detected glucose molecules in phosphate buffered saline solution at low potential. We characterized the surface morphology of the conductive hydrogel nanofibers using scanning electron microcopy (SEM) SEM images showed that 4%w/w PEO hydrogel nanofibers with immobilized 200U/mL GOx had diameters ranged from 100 to 300 nm. The amperometric current response was measured to a successive addition of glucose at a working potential of 300mV (vs. Ag/AgCl). The current was linearly dependent on glucose concentration up to 5 mM of glucose. The sensor had a sensitivity of 0.054µA×cm-2×mM-1 and low limit of detection (0.1mM). The future goal of this work is to improve the longevity and stability our biosensor developed for continuous in-vivo monitoring of glucose.

The merger of living microorganisms with classic material sciences results in responsive systems that exhibit desired functions on site. In order to build so called Living Materials, microorganisms are suspended in artificial habitats sandwiched between a supporting base layer made of acrylic lacquer and a porous cover layer that permit the exchange of gases, nutrients, and secondary metabolites and allow the organisms to grow in their confined spaces but are not enabled to leave it. [1] To demonstrate the activity and test the self-cleaning effect of living materials, the fugus P. roqueforti was incorporated into a living material. A sugar solution applied to the surfaces in order to simulate an artificial food spill was consumed within a few days while the hyphae density inside the living material increased continuously. Importantly for application-oriented materials, the stability and cyclic behavior of the living materials was proven. Fungi are able to form spores, which survive long periods of dryness, temperature extremes and lack of nutrients. [1] The use of microorganisms in technical applications and production units is commonly referred to as biotechnology. Today, many molecules used in medicine or building block chemicals are produced biotechnologically, and the utilization of transgenic organisms further increases the product diversity and production capacity. The combination of biotechnology and surface science now leads to living materials that are able to produce and release products. Here, we show a self-sterilizing, antibacterial material that is capable to produce and release penicillin on site due to the incorporation of the penicillin-producing mold P. chrysogenum. [2] This novel concept eliminates any preloading steps, refilling, or replacement as needed in classic and smart materials, extends the material&’s functionality and shelf life, and the released amount of agent can be minimized due to the biological responsive behavior. The incorporation of other organisms, e.g. other fungi, algae, or bacteria into living materials uncovers aplenty of novel smart materials. Besides fanciful applications such as photosynthetic, self-cleaning high-rise buildings, living materials can be used as production units. By continuous nourishment of living materials, steadily formed secondary metabolites are delivered via a membrane into the environment. This provides a powerful production strategy, a convenient in-situ separation of product, and promises many application scenarios in material science and biotechnological production. Literature: [1] L.C. Gerber, F.M. Koehler, R.N. Grass, W.J. Stark, Incorporating microorganisms into polymer layers provides bio-inspired functional living materials, Proc. Natl. Acad. Sci. USA, 109(1), 90-94 (2012). [2] L.C. Gerber, F.M. Koehler, R.N. Grass, W.J. Stark, Penicillin producing living materials provide chemically reactive and self-sterilizing surfaces, (under review).

Novel techniques such as computerized tomography (CT) and positron emission tomography (PET) have been developed in medical imaging for cancer diagnosis. Bismuth germanate (BGO) has been the focus of several studies due to its applications as biomaterial. It has been employed as detectors in scientific research, industry and medicine. However, to the best of our knowledge, this is the first work that studies the properties of BGO nanoparticles for biomedical applications. BGO powders were synthesized by sol-gel and subsequent annealing. The annealed powders were analyzed by X-ray diffraction (XRD), transmission electron microscopy (TEM), photoluminescence (PL) and radioluminescence (RL.) The PL properties of BGO nanoparticles were analyzed in body fluids at different pH concentrations. Oxidative stress in lipids and proteins produced by BGO in different tissues of zebra fish were analyzed. Finally, BGO was functionalized with W6/32 monoclonal antibody which recognized the human antigen HLA-I in the membrane of human acute monocytic leukemia cell line THP-1. The BGO nanoparticles yielded a blueish-white luminescence with a maximum emission peak at 485 nm, associated to the electron transition 3P1→1S0 of Bi3+ ions, when they were excited with lambda;ex= 280 nm. The physical characterization proved that BGO nanoparticles have scintillator properties similar to BGO single crystals. BGO nanoparticles presented high optical stability in organic components. Additionally, they showed luminescence properties under different pH concentrations, which make them extremely useful as biomarker. The BGO nanoparticles can be functionalized with W6/32 antibodies that recognized human tumor cells antigens.

Fluorescence imaging is one of the most versatile and widely used visualization methods in biomedical research. However, tissue autofluorescence is a major obstacle confounding interpretation of in vivo fluorescence images. Here, we report that the unusually long emission lifetime (5-13 µs) of porous silicon nanoparticles can allow time-gated imaging of tissues in vivo, completely eliminating shorter-lived (< 10 ns) emission signals from organic chromophores or tissue autofluorescence. Using a commercial animal imaging system (eXplore Optix) that is not optimized for such long-lived excited states, we demonstrate substantially improved signal to background contrast ratio when imaging porous silicon nanoparticles. Time-gated imaging of porous silicon nanoparticles accumulated in a human ovarian cancer xenograft following intraveinous injection is demonstrated in a mouse model.

Besides glucose, lipids are the major fuel in the blood for energy consumption and tissue proliferation. Lipoprotein lipase (LPL) is the master regulator of vascular lipid metabolism. LPL mediates the release of fatty acids from triacylglycerol that are transported by lipoproteins in the blood. It acts thereby as a gatekeeper for fatty acid uptake comparable to the role glucose transporters for glucose uptake. Here, we present an in vivo sensor for LPL activity based on superparamagnetic iron oxide (SPIO) and quantum dot (QD) nanocrystals. A recombinant lipoprotein model named nanosomes which carries different species of nanocrystals was recently established (Bruns et al. Nature Nanotechnology 2009, Bartelt et al. Nature Medicine 2011). Given the high flexibility and exceptional signal properties, nanosomes are the ideal platform for LPL sensing. Nanosomes allow to sense LPL activity in vivo by non-invasive real-time MRI imaging. In mouse models the upregulation of LPL activity in brown adipose tissue (BAT) after cold exposure could be detected by MRI and high-speed intravital confocal imaging. Inhibition and removal of LPL from tissue could be detected by in vivo imaging. These results were confirmed with quantitative measurements using radiolabelled nanocrystals. Furthermore MRI in combination with online interventions allowed to image vascular/endothelial lipoprotein processing in BAT in real-time in vivo. Sensing LPL activity by fluorescence microscopy and non-invasive MR imaging will allow measuring the biological importance of LPL function and dysfunction in target organs online in vivo. A sensor will thereby allow detecting changes in disease-associated LPL modulation in tissues in a non-invasive manner. Therefore, in future LPL activity in disease-affected tissues might be used to monitor effects of a therapeutic intervention from early on. References: Bartelt A, Bruns OT, Reimer R, Hohenberg H, Ittrich H, Peldschus K, Kaul MG, Tromsdorf UI, Weller H, Waurisch C, Eychmüller A, Gordts PLSM, Rinninger F, Bruegelmann K, Freund B, Nielsen P, Merkel M and Heeren J, Brown adipose tissue activity controls triglyceride clearance. Nature Medicine, 2011 Feb;17(2):200-5. Bruns OT, Ittrich H, Peldschus K, Kaul MG, Tromsdorf UI, Lauterwasser J, Nikolic MS, Mollwitz B, Merkel M, Bigall NC, Sapra S, Reimer R, Hohenberg H, Weller H, Eychmüller A, Adam G, Beisiegel U, Heeren J, Real-time magnetic resonance imaging and quantification of lipoprotein metabolism in vivo using nanocrystals. Nature Nanotechnology, 2009 Mar;4(3):193-201.

In recent years magnetic nanoparticles (MNPs) in the size range 1-100 nm have been found very useful in several biomedical applications [1]. Reproducible synthetic protocols and several chemical routes allow modification of their surface for ensuring their colloidal stability in liquids and functionalising with biological molecules. Furthermore, MNPs like iron oxides are biocompatible in nature and are able to circulate in the blood stream, cross biological membranes and interact closely with biomolecules because of their superparamagnetic properties. Their unique properties and the local magnetic field they create can be used for manipulating them in vitro or in vivo or to disturb the relaxation times of surrounding protons for imaging purposes. Moreover, MNPs exhibit different kinds of losses under an ac magnetic field, such as hysteresis, Brownian and Néel loss, which can be used for generating heat, enabling thermal treatment of cancer cells or controlled and targeted drug release. In this talk I will review some recent work in this field, with an emphasis on iron oxides [2, 3] but also including more complex oxides such functionalized manganese ferrite [4] and manganese perovskites [5] MNPs. [1] A. G. Roca et al., J. Phys. D: Appl. Phys. 42, 224002 (2009) [2] K. Kaaki et al., Langmuir 28, 1496 (2012) [3] I. Milosevic-Markovic, Ph. D. Thesis, University of Orléans (November 2009) [4] U. I. Tromsdorf et al., Nano Lett. 7, 2422 (2007) [5] R. Epherre et al., J. Mater. Chem. 21, 4393; 14990 (2011)

The use of semiconductor quantum dots for bio-imaging requires the development of a surface chemistry such that i) they can be transferred into water, ii) their non specific adsorption is minimal, iii) they can be functionalized with macromolecules, iv) they retain their fluorescence, v) their size is kept as small as possible (although in some cases, this last requirement may not be necessary). I will present recent developments we have performed in order to get a surface chemistry for QDs that fulfills all these requirements. Two approaches will be discussed. One is based on the ligands exchange with a hydrophilic polymer based on the radical random copolymerization of two methacrylamides: one containing the dithiol function, the other including the sulfobetaine group. Such a multiplication of attachment points and hydrophilicity-promoting groups in the same structure covering the quantum dot surface resulted in extremely stable QDs even in they are strongly diluted. We will show that QDs coated with this ligand are ideal for long-term live-cell imaging. The second approach is based on the encapsulation of QDs into a polymer cage that confers them excellent stability.

We have developed a novel concept for nanoabtibiotic particles based on a combination of nanoimprining technology and “key-lock” colloid interactions. These nanoantibiotic materials were fabricated via several preparation steps which involve producing “negative” inorganic replica of the pathogen particles in the form of nanoshells that match closely the pathogen shape and size. Further, these inorganic nanoshells were subjected to partial fragmentation which allowed the templated cells to be removed from the shells interior by a bleaching process while retaining their ability to “recognise” the shape and size of the pathogen particles upon binding. The nanoshell-target interaction is enhanced by several orders of magnitude by the increased contact area between their surfaces when such “recognition” event takes place. The binding of these partially fragmented inorganic nanoshells to the original target cells can be further enhanced by additional surface functionalisation with suitable bio-specific coating which promotes the cell-shell adhesion. Once in contact with the target microbe (or virus), the nanoantibiotic particle can bind specifically to its surface and can deactivate them by impairing its biological activity. Nanoantibiotics designed for microbial pathogens can bind on a large area of the cell wall and stop its further division and reproduction. This technology is also expected to work for development of nanoantiviral agents which can bind specifically to viruses matching their shape and size thus preventing the attachment of the viral capsid to the host cell and the subsequent release of the viral DNA. In a proof of principle study we demonstrate that such "tailor made" inorganic nanoshells can bind to cells with specific shape and size. In our work we used silica and other inorganic materials to template several different types of cells and produced shape specific nanoshells. We coated the targeted cells and the fragmented silica nanoshells with polyelectrolytes to explore the effect of surface adhesion on the binding efficiency depending on the shape recognition. Our statistical analysis of the binding events showed high degree of nanoshell-target orientation which leads to favourable binding. Here we present experimental results illustrating the nanoantibiotics shape-specific binding and analyse the effect of the surface coating on the binding efficiency. It is anticipated that this novel class of nanoantibiotic particles could be designed to bind and potentially deactivate highly antibiotic resistant lines of bacteria (like MRSA, E-coli) and many others where most conventional antibiotics are powerless. Nanoantibiotic particles can also find applications as non-toxic antibacterial agents, for example to prevent harmful bacteria like E-coli from growing on food and personal care formulations.

Introduction: The emergence of methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of hospital-acquired infections (HAI). These events affect approximately 1.7 million patients each year in the U.S., resulting in up to 100,000 excess deaths and leading to an estimated cost of more than $35 billion per year. Hence, there is an urgent need to develop new therapies to reduce infection, without resorting to the use of antibiotics for which bacteria are developing a resistance towards. In this study, we developed superparamagnetic iron-oxide nanoparticles (SPION) to treat MRSA biofilms and showed that their efficacy increases with antibacterial metal conjugation in the presence of fructose metabolites. Materials & Methods: SPION were synthesized using a high temperature synthesis method in triethylene glycol (TREG) and capped with 4 mmol dimercaptosuccinic acid (DMSA). Then, SPION solution was conjugated with 1 mM or 4 mM salts of AgNO3, ZnCl2, and FeCl3. For bacteria experiments, Mu50 strain of MRSA (ATCC #700699) was used. 10^8 cells/mL were incubated for 24 hours in a 96-well plate for biofilm formation. Biofilms were treated with 1 mg/mL of unconjugated (UC) and metal conjugated SPION. Different concentrations of a fructose solution (1 and 10 mM) were added to investigate their effect on SPION efficacy. After 24 hours of treatment, biofilms were analyzed by crystal violet (CV) staining and viable cell counts (CFU/mL). 1 mg/mL of vancomycin, oxacillin and gentamicin were tested using the same experimental design. Results & Discussion: A 6.69±0.88 log reduction in viability was observed when biofilms were treated with UC (p<0.001). In addition, conjugating with 1 mM and 4 mM Fe3+ (Fe1, Fe4), Zn+2 (Zn1, Zn4), and Ag+1 (Ag1) further decreased viable MRSA within the biofilm (p<0.001). Moreover, all of the conjugated SPION were significantly more effective than vancomycin, the antibiotic of last resort (p<0.05). Most importantly, for the first time, it was observed that the anti-biofilm efficacy of UC as well as Fe1 (p<0.05) and Zn1 (p<0.01) significantly increased in the presence of fructose. On the other hand, gentamicin increased biofilm mass (p<0.01). Further analysis showed that fructose increased the uptake of SPION within the biofilm, thus, enhanced SPION efficacy. Conclusions: Our results have important implications. First, we have shown that SPION are more effective than currently used antibiotics for treating resistant biofilms. SPION can be conjugated with different metals to increase their anti-biofilm properties. Moreover, for the first time, the presence of fructose metabolites was shown to enhance the efficacy of the SPION against MRSA biofilms. It is envisioned that this simple and inexpensive approach could lead to successful clinical outcomes in terms of minimized infections and decreased antibiotic usage. All of these events can support decreased antibiotic resistance in the clinical settings.

Many species of bacteria form surface-attached communities known as biofilms. Surrounded in secreted polymers, these bacterial aggregates are difficult to both prevent and eradicate, posing problems for medicine and industry. We show that nature generated a solution to prevent biofilms in the form of mucus, the hydrogel that coats the wet surfaces of animals. We found that mucin biopolymers, the main functional constituents of mucus, act as natural dispersants by promoting the segregation of planktonic bacteria, and preventing their adhesion to underlying surfaces. We furthermore show that the loss of motility and production of an alginate matrix allow Pseudomonas aeruginosa to overcome the inhibitory effects of mucins and form suspended, antibiotic-resistant flocs. We conclude that mucins may offer new strategies that target bacterial virulence, and moreover, have the potential to solve some vexing engineering challenges, such as the design of antimicrobial coatings of implants.

Silver nanoparticles (nanosilver) are broadly used today in textiles, food packaging, household devices and in bioapplications prompting for a better understanding of their toxicity and biological interactions. However, the cytotoxicity mechanism of nanosilver is still not clear as limitations regarding the controlled synthesis of particles with different sizes and coatings hinder such investigations. In this work, nanosilver with well-defined sizes (5.7 to 20.4 nm) immobilized on nanostructured silica was produced by flame synthesis and its cytotoxicity was assessed in vitro. The nanosilver dispersibility in aqueous solutions was facilitated by the presence of silica, and therefore, no surface coatings which could otherwise inhibit the Ag⁺ ion release were required. Detailed physicochemical characterization of these nanoparticles with X-ray diffraction, N₂ adsorption and electron microscopy revealed that nanosilver size was adequately controlled. Smaller nanosilver released larger fractions of its mass as Ag⁺ ions upon its dispersion in water. This strongly influenced the cytotoxicity of such nanosilver upon their incubation with murine macrophages. The nanosilver size dictated its mode of cytotoxic activity. The toxicity of small nanosilver that releases high fractions of Ag⁺ ions was mostly mediated by the released ions. In the case of large nanosilver, which released substantially less Ag⁺ ions, the toxicity was attributed to both the released ions but also to the direct nanoparticle-cell interactions. [1] Sotiriou GA, Pratsinis SE. Engineering Nanosilver as an Antibacterial, Biosensor and Bioimaging Material. Curr Opin Chem Eng 2011, 1: 3-10. [2] Sotiriou GA, Sannomiya T, Teleki A, Krumeich F, Vörös J, Pratsinis SE. Non-Toxic Dry-Coated Nanosilver for Plasmonic Biosensors. Adv Funct Mater 2010, 20: 4250-4257. [3] Sotiriou GA, Hirt AM, Lozach PY, Teleki A, Krumeich F, Pratsinis SE. Hybrid, Silica-Coated, Janus-Like Plasmonic-Magnetic Nanoparticles. Chem Mater 2011, 23: 1985-1992. [4] Sotiriou GA, Pratsinis SE. Antibacterial Activity of Nanosilver Ions and Particles. Environ Sci Technol 2010, 44: 5649-5654.

Contamination of surfaces, fomites, and medical instruments, such as endoscopic and dialysis devices, by opportunistic pathogens is the main culprit for hospital-acquired infections. The costs in human lives, extended hospital stay and antibiotic therapies add considerable burden on healthcare. The problem extends to implantable materials, where infection can jeopardize the success of prosthetic devices. For instance in orthopedics, infection is responsible for the septic failure of almost 5% of implants, requiring their replacement. While rigorous cleaning, disinfection and sterilization protocols are there to remain, the search is still on for materials that will inherently discourage the spread of microorganisms. To address this challenge, there has been significant effort to engender materials capable of conjugating direct physicochemical cueing that limits bacterial adhesion with the capacity to release antibiotics in a controlled manner. In this context, we investigate the effects of mesoporous surfaces generated by simple oxidative nanopatterning on the adherence of two common bacteria responsible of implant-associated infections and one yeast strain found in hospital settings. The adherence of E. coli, S. aureus and C. albicans to such mesoporous titanium discs was compared to ones with polished surfaces. In order to reduce experimental variability, E. coli were also grown on discs with side-by-side treated and control surfaces. Qualitative and quantitative results from this study indicate that the bacteria adhesion is significantly hindered by the mesoporous surfaces as compared to smooth titanium. The side-by-side surfaces demonstrated that E. coli bacteria show a visually evident preference to colonize the smooth. In addition, we provide evidence indicating that the chemically generated mesoporous surfaces also have an impact on the structural integrity of C. albicans. Mesoporous titanium surfaces created by oxidative nanopatterning are also very attractive for their capacity to act as metallic platforms for controlled drug release directly at the implantation site. In this context, we have loaded treated surfaces with Vancomycin, a commonly used antibiotic, and studied the elution profile engendered by the 3-dimensional network of nanosized pits. Experimental data show that mesoporous titanium surfaces can store the antibacterial agent and provide sustained elution. Achieving such a capacity is expected to further strengthen the innate ability of mesoporous surfaces to fight bacterial colonization. To conclude, these results demonstrate the efficiency and great potential of mesoporous titanium surfaces produced by oxidative nanopatterning in controlling bacterial adhesion, and open the door to their broad application in both industry and medicine to control bacterial colonization.

Bacteria primarily exist in robust, surface-associated communities known as biofilms, ubiquitous in both natural and anthropogenic environments. Mature biofilms resist a wide range of antimicrobial treatments and pose persistent pathogenic threats. Treatment of adherent biofilm is difficult, costly, and, in medical systems such as catheters or implants, frequently impossible. At the same time, materials strategies for biofilm prevention based on surface chemistry treatments have been found to only transiently affect initial attachment. Here we report that Slippery Liquid Infused Porous Surfaces (SLIPS) prevent 99.6% of Pseudomonas aeruginosa biofilm attachment over a 7-day period, as well as Staphylococcus aureus (97.2%) and Escherichia coli (96%), under both static and physiologically realistic flow conditions. In contrast, both polytetrafluoroethylene and a range of nanostructured superhydrophobic surfaces accumulate biofilm within hours. As quantified by crystal violet staining and fluorescence microscopy, SLIPS shows ~35x reduction of attached biofilm versus best-case-scenario, state-of-the-art PEGylated materials—and over a far longer timeframe. We screen for and exclude as a factor cytotoxicity of the SLIPS liquid, a fluorinated oil immobilized on a PTFE substrate. The inability of biofilm to firmly attach to the surface and its effective removal under mild flow conditions (~1 cm/s) result from the unique, non-adhesive, “slippery” character of the smooth liquid interface, which does not degrade over the experiment timeframe. SLIPS-based anti-biofilm materials are low-cost, passive, simple to manufacture, stable in extreme pH, salinity, and UV environments, and can be incorporated into arbitrary surface geometries. We anticipate that our findings will enable a tremendous range of anti-biofilm solutions in the clinical, industrial, and consumer spaces.