Unfortunately there has not been significant innovation to provide effective technologies for sealing tissues, especially internal tissues that are wet. While sutures and staples have been used for decades, they exhibit considerable limitations (they can induce significant tissue damage and their placement is often slow and challenging during minimally invasive procedures). This talk will explore multiple Next-Gen bio-inspired adhesive technologies to address several unmet medical needs. Through inspiration from the gecko, nano- and micro-topography has been harnessed to achieve biocompatible and controlled tissue adhesion. Towards development of a tissue adhesive glue to seal septal defects in children, we have harnessed inspiration from insect footpads, slugs and sandcastle worm viscous secretions that can create stable underwater adhesive bonds. We have been inspired by porcupine quills to develop mechanically interlocking adhesives that could replace staples with strong adhesion yet less tissue damage. We have also been inspired by the spiny-headed worm to develop an array of microneedles with biomimetic swellable tips that can achieve strong bonds to tissue through a mechanically interlocking mechanism and can be used to securely affix skin grafts to prevent seromas. This design provides universal soft tissue adhesion with minimal damage and reduced risk of infection compared to staples, and provides opportunity for delivery of bioactive therapeutics.

Bone is a composite structure that is made up of collagen (organic), calcium phosphates and trace elements (inorganic). Together with strict mechanical requirements, many scaffold-based tissue engineering strategies are now focused on developing composite systems that provide sufficient mechanical stability and biological stimulation to dictate osteogenic events in vivo. On this note, processing techniques for bioresorbable composites for bone tissue engineering have traditionally involved solvents and heat, which may not be ideal due to toxicity and material degradation, respectively. As such, alternative processing methods are actively pursued and evaluated. To this end, cryo-nanoprocessing has recently surfaced as a potential alternative processing technique for composites. By subjecting composite materials to temperatures sufficiently below glass transition, effective attrition into fine powders (sub-micron and nano) may be achieved. In addition, this attrition process facilitates distribution of the filler, resulting in a homogeneous composite powder. This homogeneous distribution may be retained in further post-processing techniques. A mechanistic explanation for this phenomenon may be that of solid-state diffusion, where effective interfacial interactions are created, resulting in the retention of their homogeneous distributions. The implications of this phenomenon such as mechanical and biological responses are important for bone tissue engineering strategies centered on scaffold technology.

We present the synthesis of an upright nanotubular coating with discrete, exposed nanotubes on top of superelastic Nitinol via anodization and the characterization of the surface elemental composition and nickel release rates of these substrates. We demonstrate, for the first time, that this coating could improve re-endothelialization by increasing the cell spreading and migration of primary human aortic endothelial cells on Nitinol. The coating also has the potential for reducing neointimal hyperplasia by decreasing the proliferation and expression of collagen I and MMP-2 in primary human aortic smooth muscle cells (HASMC). Furthermore, we did not observe the nanotubular surface to induce inflammation through ICAM-1 expression in HASMC as compared to the flat control. This coating could be used to improve Nitinol stents by reducing restenosis rates and, given the extensive use of Nitinol in other implantable devices, act as a generalized coating strategy for other medical devices.

Electrospun nanofibers can be routinely prepared from a wide variety of polymers, incuding those biocompatible and biodegradable polymers that have been approved by the FDA for clinical applications. Owning to their small diameters, controllable structures and alignments, electrospun nanofibers represent an ideal class of substitutes for the extracellular matrix and have found widespread use in regenerative medicine. In this talk, I will focus on their use in the repair or regeneration of tendons, tendon-to-bone insertions, dural tissues, and peripheral nerves. I will specifically discuss the issues that still need to be addressed before this class of nanomaterials can eventually find use in clinical applications.

Alginate hydrogels are well-characterized, biologically inert materials that are attractive for many biomedical applications including the encapsulation of small molecules, proteins, and cells. Unfortunately, canonical covalent alginate hydrogels are limited in their usage for cell or protein encapsulation and subsequent delivery, due to crosslinking and functionalization strategies that can be biologically harmful. In this work we modified alginate polymers with tetrazine or norbornene groups and subsequently formed click alginate hydrogels with a wide range of mechanical properties. The highly specific, bio-orthogonal, and rapid click reaction is irreversible and allows for easy incorporation of cells with high postencapsulation viability. Additionally, preformed click alginate hydrogels can be easily modified with cell adhesive ligands to create a cytocompatible substrate with tunable ligand densities for 2D cell culture. Furthermore, click alginate hydrogels are minimally inflammatory, maintain structural integrity over several months, and reject cell infiltration when injected subcutaneously in mice. Click alginate hydrogels combine the numerous benefits of alginate hydrogels with powerful bio-orthogonal click chemistry to create new materials for a variety of tissue engineering applications.
Funding Sources: Wyss Institute, NIH 2 R01 HL069957, ARO W911NF-13-1-0242

Understanding transport of nanoscale carriers within organs/tissues is crucial for their adequate use in biomedical imaging and drug delivery.Animal studies of such processes encounter formidable challenges associated with spatial resolution of in-vivo real-time imaging, inherent variability of animals, and high costs.Conversely, Petri dish cultures of immortalized cell are too simplistic to account for cellular sophistication of organs.Three-dimensional (3D) cell cultures approach the complexity of the actual organs and may enable high temporal and spatial resolution of permeation dynamics. Here, we investigated transport behavior of CNTs with and without targeting ligand -- transforming growth factor β1 (TGFβ1) -- in highly uniform spheroids of hepatocellular carcinoma (HCC) obtained in inverted colloidal crystals (ICC) scaffold that closely replicate human liver tumor tissue. Surprisingly, apparent diffusion rate of CNTs in tissue replica is higher than can be predicted theoretically and comparable with diffusion rates of similarly charged small molecule. Moreover, efficiency of CNT transport in tissues is enhanced after functionalization by TGFβ1 despite the considerable increase of volume and molecular mass. Such unexpected diffusion is attributed to surface diffusion of nanotubes on cellular membrane. These findings demonstrate that tissue spheroids allow for accurate optical tracking and theoretical description of CNT transport. They also indicate that nanotubes and similar nanostructures can serve as efficient drug delivery vehicles deep into the tissue that was often difficult to realize with other drug delivery carriers.

Peripheral nerve injury incidences are quite high mainly due to the unprotected nature of peripheral nervous system when compared to the central nervous system. Although regenerative capacity of peripheral nerves is much higher than those of the central nervous system, therapy is not much successful when the gap between proximal and distal stumps is long and because of poor functional outcomes. Autografts are applied in such cases although this method holds donor site morbidity (sense loss) and neuroma formation risks leading to intense pain experience by patients. Moreover unsatisfactory operational recovery after nerve injury is still a significant clinical challenge. Polymeric nerve guides are developed to be used in such cases to provide peripheral nerve regeneration by isolating nerve stumps in a hollow tube. However, although these nerve guides improve regeneration up to some level, clinical success ratio is not much high. There are some reports indicating patients undergone recession surgery due to intense pain at the injury site and neuroma formation and without regeneration after nerve guide transplantation. Considering the high cost of these nerve guides along with being not as effective as desired, it is clear that further modifications are required to improve their therapeutic effect. Commercially available polymeric nerve guides are inert materials not possessing any regenerative signal; they are just hollow tubes in which nerve stumps are being isolated from the surrounding tissue. In this study, it is aimed to improve effectiveness of nerve guides used in peripheral nerve injury therapy. For this purpose, peptide nanofiber gels are made up of neuroinductive peptide molecules one of which is derived from laminin (IKVAV-PA) while the other mimics growth factor binding glycosaminoglycans (GAG-PA). It is recently published that neural cells cultured on PA gels formed from these two molecules can extend quite long neurites. Nerve guides were filled with various form of peptide gels or sucrose that were applied to 10 mm gaps formed by full transaction in rat sciatic nerve. Functional recovery of sciatic nerve in nerve guide/PA and autograft was remarkably faster in comparison of other groups. Behavioral tests, electrophysiological measurements and histological analysis revealed that these laminin and glycosaminoglycan mimetic peptide nanofibers induce neuritogenesis with a great efficacy compared with autologous nerve grafts. Our results demonstrated that these two bioactive peptide amphiphile are fundumental for the optimal regeneration in peripheral nerve injuries and also they will be investigated further for their clinical potentials.

Despite the tremendous progress in the past decade in translating nanomaterial&’s into cell contacting applications, there is still a huge knowledge gap to be filled with regards to deciphering the interplay between soluble signals and nanoscale physical cues present in the cellular microenvironment, on cell fate decisions. De novo bone regeneration is still challenging due to the complexity of the bone environment. Bone formation during development from cartilaginous tissue via endochondral ossification (EO) provides a unique framework to implement a developmental engineering paradigm in restoration of large volumes of bone. Recently, human nasal septum chondrocytes (hNCs) have been successfully used in nasal and knee cartilage reconstruction. However, the main challenge in using chondrocytes (Ch) in bone repair lies in invoking hypertrophy in phenotypically stable Ch.
We hypothesized that the stiff mineral phase juxtaposed with the softer collagen matrix, provides a biophysically heterogeneous environment that could play an important role in driving hypertrophy in Ch. With the aim of unraveling the role of physical cues offered by the bone environment, we have developed a platform that brings together concepts of self-assembly with control over protein conformation, to realize bone-like biomimetic hydroxyapatite (BBHAp) on synthetic substrates. Using polyelectrolyte-mediated induction of conformation changes in proteins, a nanoscale coating of biomineralization promoting proteins derived from avian and mammalian sources was realized on a fibrous polymer mesh, which when exposed to source of calcium and phosphate yielded BBHAp. That BBHAp coating in addition to being uniform, had similarity to the HAp phase found in bone in both the orientation of crystals along c-axis (002) and crystal dimensions (average BBHAp dimensions L: 58 nm; W: 30 nm; T: 3.90 nm versus HAp phase in bone: L: ~50 nm; W: 25 nm; T: 4 nm).
The ability of the BBHAp environment to upregulate EO related events was studied using hNCs and human articular chondrocytes (hACs)in vitroand in vivo in nude mouse model. In vitro, RT-PCR analysis of hNCs and hACs cultured on BBHAp revealed 12-fold down-regulation in COL II, and respectively 4- and 8-fold up-regulation in COL X in comparison to non-HAp controls. This drastic change in COL X/Col II ratio is one of the hallmarks for initiation of hypertrophy in Ch, which was also confirmed by immunohistochemistry (IHC). On going in vivo studies indicate the onset of EO-like events within the provisional cartilaginous matrix in an ectopic environment, including the ingress of blood vessels into the remodelling matrix, which was confirmed by the presence of CD31⁺ cells and concomitant positive staining for bone sialoprotein. These finings in sum implicate an important role for BBHAp in triggering EO related cellular events.

In the current presentation, development of three-dimensional and macroporous electrospun biodegradable scaffolds with the moldability and increased mechanical properties is discussed. The presented electrospun scaffolds were developed to overcome the limitations of the conventional electrospinning technique including i) restricted shapes (two-dimensional and sheet-like shapes) and ii) limited cellular infiltration into the scaffolds. In our previous study, by the core/sheath electrospinning technique with the polystyrene (PS) and the poly (ε-caprolactone) (PCL) followed by selective leaching procedure, three-dimensional, macroporous and highly moldable nanofibrous structure was fabricated. The scaffolds could be shaped into a variety of shapes with various shaping methods including molding, rolling and manual shaping without compromise of nanofibrous structures on the outer surface and inside of the scaffolds. The scaffolds, however, have relatively low mechanical strength which possibly limits the applications. To resolve the issue, various additives were added to PCL. With additives such as carbon nanotubes (CNTs) and polydopamines, the ultimate tensile strength and Young&’s modulus of the scaffolds were increased to 2- or 3-fold without compromising their flexibility, moldability and macroporosity. In in vitro characterization with the cells, the cells in the scaffolds showed the homogeneous cellular infiltration into the scaffolds and increased cell viability. Furthermore, the characterization with the viral vectors adsorbed on the surface of the scaffolds showed significantly enhanced the intensity of transgene expression. In vivo experiments showed that cells from host tissues infiltrated into the moldable scaffolds, whilst the cells were located only on the surface of the conventional electrospun scaffolds. In conclusion, nanofibrous and biodegradable scaffolds with macroporosity with high moldability were fabricated and with the inorganic and organic additives, the mechanical strength could be significantly enhanced. The presented system is a facile and versatile technology which has substantial potential to contribute to tissue engineering and regenerative medicine.

Alumina ceramics is one of the most important materials for bone repairing. Although alumina has been used as prostheses for many years due to its chemical stability, mechanical strength and fracture toughness, it has no direct bonding ability to bone. To obtain long-term fixation of the materials to bone, osteoconductive materials have been paid much attention because it can bond to bone through a bone-like apatite layer. Thus, surface modification technique to change alumina from bioinert to octeoconductive has been awaited for a long time. We previously reported that calcium-modified alumina prepared by hydrothermal treatment with CaCl₂ aqueous solution formed bone-like apatite on its surface after soaking in a simulate body fluid. This result suggested that the calcium-modified alumina might be an osteoconductive material. In this study, we performed in vitro biological analyses using rat bone marrow stroma cells to evaluate the osteoconductivity of the calcium-modified alumina.
Commercial alumina disks were immersed in distilled water or 50 mmol/L of CaCl₂ (aq) in Teflon vessel with stainless steel jacket. Then, it was kept 125°C for 7 days for hydrothermal treatment. After rinsed with distilled water, one of the disks was analyzed by X-ray photoelectron spectroscopy (XPS) to confirm the Ca binding onto the surface. Bone marrow stroma cells harvested from rat tibias were cultured in various periods on the surface of the sterilized alumina disks, and then analyzed in terms of initial cell attachment, proliferation, and bone-like nodule formation. The alumina disks without hydrothermal treatment were denoted as NT, and the alumina disks after hydrothermal treatment in distilled water or 50 mmol/L of CaCl₂ (aq) were denoted as HT0 or HT50.
From the XPS results, Ca binding was detected only on the surface of HT50 whereas no Ca binding was detected on the other alumina disks. The initial cell attachment rate after 7 hours of culture on the surface of hydrothermally treated alumina disks was the same as sintered hydroxyapatite. The cell number on the surface of Ca0 and Ca50 was significantly higher than that on NT. In the scanning electron microscopic observation after 24 hours, well spreading cells on the surface of Ca50 whereas cells were spherical shape on the surface of NT. Cells on all alumina disks proliferated greatly over 7 days of culture. After 3 days, the cell number on HT50 was significantly higher than other samples. Bone-like nodule formation was observed only on HT50 surface after 9 days culture. In contrast, there were no differences regardless of the type of the disks after 15 days of culture. These results showed that biological properties of alumina to bone marrow stroma cells could be significantly improved by the surface modification with Ca. Therefore, it is concluded that calcium-modified alumina prepared by the hydrothermal treatment with calcium chloride solution has high possibility to be osteoconductive material.

To improve current problems facing anti-adhesion products, we propose to use elctrospinning to fabricate porous nanofibrous membranes as anti-adhesion barriers. Nanofibrous membranes have microporous pores to allow diffusion of nutrients, while block penetration of cells at surgical sites to prevent adhesion formation. The membranes also have good mechanical properties and flexibility for application at surgical sites by surgeons after surgery. Anti-adhesive molecules, platelet-rich plasma (PRP) or silver nanoparticles could be incorporated into nanofibers to develop a multi-functional anti-bacterial membrane to prevent peritendinous adhesion and to promote tendon healing. We propose to combine FDA-approved biomaterials with anti-adhesion properties, polycaprolactone (PCL), chitosan (CS), polyethylene glycol (PEG), hyaluronic acid (HA), silver nanoparticles (Ag) and platelet-rich-plasma (PRP), to produce anti-adhesion barrier nanofibrous membranes for translational medicine study. The electrospun membranes contain single, mixed polymers or core-shell nanofibers. Five kinds of membranes be studied including (1) CS-grafted PCL nanofibrous membranes, (2) HA-grafted PCL nanofibrous membranes, (3) PCL+PEG blended nanofibrous membranes, (4) HA/PCL+Ag core-shell nanofibrous membranes, and (5) HA+PRP/PCL core-shell nanofibrous membranes. Grafted CS or HA on nanofibers can effectively reduce cell adhesion, thereby reducing adhesion formation. Blended PEG and PCL nanofibrous membrane can increase the efficiency of penetration of nutrients, and it is also can reduce the formation of adhesions. The HA releasing core-shell nanofibrous membranes contain a HA core region and a PCL shell layer for extended release of HA to reduce peritendinous adhesions. With the release of growth factors from incorporated PRP to promote tendon healing and the anti-bacterial activity from incorporated silver nanoparticles, multi-functional anti-adhesion barrier membranes could be obtained. Silver nanoparticles were added to the outer PCL shell layer during electrospinning to endow the nanofibrous membrane with anti-bacterial properties. The long-term growth factors releasing core-shell nanofibrous membranes contain a PRP/HA core region and a PCL shell layer for extended release of growth factors in PRP to promote long-term tendon healing. We characterize the physico-chemical properties of the fabricated membranes, and carry out in vitro and in vivo tests of the safety and efficacy of those membranes.

Bone and other natural material exhibit a combination of strength and toughness that far exceeds that of synthetic structural materials. Bone&’s toughness is a result of numerous extrinsic and intrinsic toughening mechanisms that operates synergistically at multiple length scales to produce a tough material. At the system level however no explanation or organizational principle exists to explain how so many individual toughening mechanisms can work together. In this paper we utilize the concept of phonon localization to explain, at the system level, the role of hierarchy, material heterogeneity, and the nanoscale dimensions of biological materials in producing tough composites. We show that phonon localization and attenuation, using a simple energy balance, dynamically arrests crack growth, prevents the co-operative growth of cracks, and allow for multiple toughening mechanism to work simultaneously in heterogeneous materials. In turn the heterogeneous, hierarchal and multi-scale structure of bone (which is generic to biological materials such as bone and nacre) can be rationalized because of the unique ability of such a structure to localize phonons of all wavelengths.

There is immense potential for biomaterials in translational medicine, when quantitative measures of biomaterial properties coupled with quantitative measures of biological responses lead to predictive, mechanistic models. We will present our recent studies [1,2] of an implantable electrospun polyamide nanofibrillar scaffold that has demonstrated promising wound healing properties for the central nervous system including in vivo mitigation of astrocytic glial scarring. The biomaterial properties of the nanofibrillar scaffolds and three controls were characterized by novel AFM and contact angle measurements. When four properties: nanoscopic elasticity, work of adhesion and surface roughness, and surface polarity were compared statistically, it became clear that the four environments presented significantly different sets of properties to astrocytes (ANOVA followed by post hoc comparisons with Tukey&’s test, P<0.05). Properties of the nanofibrillar scaffolds uniquely regulated multiple astrocyte features that were consistent with reduced reactivity. Reactive dBcAMP-treated and untreated astrocytes cultured on nanofiber scaffolds demonstrated minimal morphologic features changes, including process length, cell spreading and cell shape index. The relative levels of RhoGTPase expressions did not change between treated and untreated astrocytes cultured on nanofibrillar scaffolds, unlike all other cultures surfaces. Pairwise comparisons of untreated and treated astrocytes on the nanofibrillar scaffolds also indicated a statistically significant decrease in expression of inhibitory proteoglycans (CSPGs) with no accompanying decrease in cell density. A separate study demonstrated FGF-2 growth factor up-regulation by astrocytes on the nanofibrillar scaffolds with increased neurite outgrowth by co-cultured neurons. In sharp contrast, the responses to the properties of one control exhibited features reminiscent of glial scar formation. Confocal, super-resolution and AFM imaging revealed details of chain-like clustering with interwoven processes.
The emerging disciplinary field of translational medicine has identified “prevention of basic research findings being tested in a clinical setting” as a major roadblock. Often the block is at the first translation, when the correspondence between in vitro laboratory findings and in vivo animal model results is weak. Recent work, including ours, indicates that biomaterial properties can be tailored to reproduce in vivo responses in the laboratory with far greater accuracy and further suggests that precise control holds potential for inducing preferential cell differentiation in both in vitro and in vivo environments.
[1] Tiryaki, VM, Ayres, VM, Ahmed, I, Shreiber, DI. Nanomedicine, Epub ahead of print 2014 (DOI: 10.2217/NNM.14.33)
[2] Tiryaki, VM, Ayres, VM, Khan, AA, Ahmed, I, Shreiber, DI, Meiners, S. Int. J. Nanomedicine. 2012 7:3891-3905 (DOI: 10.2147/IJN.S32681)

It has been found that the nanopillars on cicada wings are inherently antibacterial, irrespective of surface chemistry (Ivanova et al., Small, 2012). Thus, fabrication of devices presenting such nanostructures would obviate the requirement for any special surface chemical modification. Nano- and microstructured antibacterial surfaces have been previously proposed, including the Sharklet microstructured film (Chung et al., 2007), black silicon (Ivanova et al., 2013) and multi-scale wrinkled polymer films (Freschauf et al., 2012); none of these approaches can be used on ordinary polymer surfaces or easily scaled up. Thus, we applied industrial nanostructring techniques to generate biomimetic antibacterial nanostructures at the surfaces of the commonly used polymer poly(methylmethacrylate) (PMMA). Compared to flat films, replicated cicada wings 1) exhibited reduced surface adhesion of live E. coli determined by a standard fluorescence based viability assay, and 2) killed these bacteria, as evidenced by a decrease in colony forming units in suspension over time (up to 24 hours). To make the nanopatterning technique more industrially viable and generate a larger patterned area, we next employed nanoimprint lithography. We utilized several nano-lined and nano-holed molds (Lightsmyth) as well as a commercially available antireflective stamp (Holotools, Germany) with a nanopillared pattern very similar to that of the cicada&’s wing, to imprint large, flat, nanostructured polymer thin films. We determined that, compared to flat films, the pillars generated by the antireflective stamp were more antibacterial, as assessed by live/dead staining. Recent efforts have focused on using polymer coatings with self-assembled nanostructures to decease the feature size of the nanopatterns, and to allow for the application of nanopillars to curved surfaces. Our surfaces could be used for a wide variety of environmental and medical applications, including surgical trays / instruments and door handles (which function in air), and for implantable medical devices or catheter tubes (which function in aqueous environments).

Nanocarriers have been studied to prolong the circulation time, bioavailability or stability of a certain biomolecules and to direct its delivery to a specific tissues or cells. In the whole scenario, mesoporous silica nanoparticles (MSNs) have several advantages; they are a very tunable platform for size and shape. Due to the flexibility of this platform and the vast possibilities for its further functionalizations MSNs represent a suitable targeted delivery system. Further, surface tailoring allows circumventing unwanted biological interactions, facilitating bioavailability and cellular uptake. In the present study we exploit this feature in order to study a molecular mechanism that leads to Human immunodeficiency virus-1 (HIV) neurotoxicity. HIV promotes neuronal injury. This process contributes to HIV-associated neurocognitive disorders, affecting more than 50% of HIV-infected individuals, even in the presence of combined antiretroviral therapy. However, the mechanisms of HIV-associated neurotoxicity remain unclear, thus precluding effective treatment of the neurological complications. Here, we report that the HIV envelop protein gp120 binds with high affinity to neuronal specific b-tubulin isotype III (TUBB3), by a twenty amino acid domain of the α-helix region that we synthetized (Helix-A), but not to tubulin isoforms expressed by other cell types. Using in vitro studies we have found that gp120 binds to recombinant TUBB3, tubulin dimers, and assembled MTs. The aim of the present study was to evaluate the neuronal protective effect of Helix-A from gp120 toxicity. Unfortunately, Helix-A peptide did not penetrate inside cells cytoplasm. Thus, we bound Helix-A to MSNs (Helix-A nano) to allow for its penetration into primary rat cortical neurons. We characterized the Helix-A nano delivery system for its stability and biological activity. Helix-A nano prevented gp120-mediated decrease in mitochondrial function (MTT) as well as alteration in mitochondrial morphology. In addition, Helix-A nano prevented gp120-mediated neuronal loss (Hoechst/PI). Since Helix-A did not bind to gp120 the proposed mechanism of neuroprotection of Helix-A involves its competitive binding to TUBB3 and MTs. Thus, neuroprotection conferred by Helix-A strongly suggests that the direct interaction of gp120 with tubulin is one of the main central mechanisms by which gp120 promotes axonal degeneration. Hence, our study identifies a novel mechanism of HIV neurotoxicity and provides a potential novel therapeutic approach to reduce HIV-mediated neurodegeneration.

The fast dissolving property of PVP makes it very suitable for transdermal delivery. But high water absorption and low mechanical strength limits its microneedles to penetrate into skin. In this work, we increase the mechanical strength of PVP by simply compositing cyclodextrin (CD) derivatives. PVP/CD film and microneedles are prepared by mixing and casting composite solution to PDMS mould, respectively. The water absorption, mechanical and thermal properties are tested by saturated salt method, microindentation and DSC, respectively. Comparing to pure PVP, The water absorption of PVP was reduced by 36%-40% at different RHs as the PVP/CD inclusion complexes formed, even after 10 or 20 days. The Young&’s modulus and hardness of the PVP/CD could be greatly improved, especially for low molecular weight PVP. Besides these, the thermal properties, the glass transition temperature (T_g) of the composite increased by up to 39 0C. With these improved properties, the composite microneedles can easily penetrate into pig skin with fewer cracks than pure PVP microneedles. This kind of microneedles is more suitable in transdermal delivery than pure PVP.

Regenerated Bombyx mori silk fibroin (SF) has excellent biological and mechanical properties, including biocompatibility, programmable biodegradability, and remarkable strength and toughness. Diverse and adaptable properties of Sf are possible by varying the structural form using different processing conditions. One of the important physical forms for biomaterials is the formation of hydrogels, which has been extensively studied for a variety of polymers. The sol-gel transition depended on the concentration of the protein, temperature, and pH. In the SF hydrogel, random coil to β-sheet(physical cross-linking) structural transitions were noted during the process of hydrogelation. Due to the β-sheet formation, SF exhibits relatively slow degradation in vitro and in vivo, compared to collagen and many other biopolymers.
Tissue engineering has potential to address this need through the combination of biomaterials, growth factors, and cells. Highly porous scaffolds are generally used as the substrate for anchorage dependent cells and to facilitate nutrient and metabolite distribution to guide cell growth leading to new bone tissue formation. For bone tissue engineering, biodegradable synthetic polymers such as poly(glycolic acid) (PGA), poly(lactic acid) (PLA), and copolymers of poly(DL-lactic-glycolic acid) (PLGA), and biodegradable naturally derived polymers such collagen and fibrin.
Hydroxyapatite (HAP) has been investigated for bone replacement since this material mimics natural bone mineral features. HAP has been studied extensively in cell culture and possesses osteoconductivity.
In this study, the synthesis and characterization of bone-like mineral HAP into highly porous biodegradable silk fibroin scaffold with via chemical cross-linking reaction of SF by gamma-ray (γ-ray) were investigated. These 3-D SF scaffolds had different secondary structures, elasticity and nanostructure compared with β-sheet induced hydrogels.The effect of degradation rate, elasticity and mineralization on osteogenic responses of osteoblast was assessed with respect to bone tissue engineering

Stimuli-responsive drug-delivery systems (DDSs) are playing an increasingly crucial role in a variety of applications for cancer treatment. We herein report an ATP-responsive nanogel consisting of three distinct functional components: an ATP-responsive DNA scaffold with doxorubicin (Dox), protamine and a hyaluronic acid (HA)-crosslinked shell. The DNA scaffold was composed of the ATP aptamer and its complementary single-stranded DNA (cDNA),which also provided faithful loading sites for Dox. When exposed to ATP, the dissociation of the DNA duplex resulting from the formation of the ATP/ATP aptamer complex caused the intercalated Dox to be released from the duplex. Cationic protamine was utilized to complex the DNA motif, which had enhanced cell penetration, endosomal escape and nuclear targeting effects. Anionic HA was coated on the complex core followed by UV irradiation-mediated photocrosslinking to form a protective crosslinked gel shell, and also serves as ligands to bind the CD44 receptors that are overexpressed on the cell surface of many cancers. This ATP-responsive nanocarrier showed an enhancement in the chemotherapeutic inhibition of tumor growth using xenograft breast tumor-bearing mice. In addition, a liposome-based co-delivery system, composed of a fusogenic liposome encapsulating the Dox-loaded ATP-responsive DNA scaffold and an ATP-loaded liposome, was further developed for ATP-mediated drug release triggered by liposomal fusion. Directly delivery of extrinsic liposomal ATP facilitated the Dox release from the fusogenic liposome in the acidic intracellular compartments by a pH-sensitive membrane fusion and therapeutic efficacy was enhanced both in vitro and in vivo.

Actin exists in almost all of the eukaryotic cells (except nematode sperm) and participates in many important cellular processes, including cell motility, morphogenesis and endocytosis. Specifically, the force generated by polymerization and depolymerization of actin filaments is commonly believed to play a key role in how these processes take place. Substantial progress has been achieved in the past decade in understanding how actin networks assemble themselves and evolve. In particular, essential proteins crucial for actin based motility have been identified allowing researchers to reconstruct actin-propelled motions of artificial cargos (microspheres or lipid vesicles coated with proper proteins) in vitro. Based on this approach, fundamental issues like the temporal and spatial distribution of polymerization-induced stress and the interplay between load surface geometry, as well as elasticity, and actin assembly can all be probed.
My present study is focusing on:
(1) Investigating the rotational movements of artificial cargos (microspheres) propelled by actin polymerization.
Many experiments have proved that Listeria monocytogenes will rotate along its long axis and form right-handed helical trajectories during actin-based motility. But whether artificial cargos propelled by actin polymerization have rotational movements has not been verified. To look into this subject, polystyrene beads bound with small fluorescent beads and coated by VCA were used to generate motility in assays reconstituted from purified proteins. The spatial positions of polystyrene beads and fluorescent beads were recorded by confocal microscope during the motion process. Whether rotational movements exist can then be confirmed by tracking their relative positions.
(2) Modeling actin polymerization induced shape changes of artificial lipid vesicles based on energetic considerations.
Lipid vesicles have been applied to investigate actin-propelled motions in vitro. It&’s been discovered that lipid vesicles can induce the structural deformation of actin filaments from branched networks to bundles. Bundled actin filaments exert forces on the surfaces of vesicles which will then generate filopodium-like protrusions. Built on the experimental results and the principle of minimum energy, we come up with a model attempting to explain the shape changes of artificial lipid vesicles induced by actin polymerization.
Experimental findings, theoretical frameworks and some preliminary results will be presented.

Biomaterials and biotechnology have made significant advances in mRNA therapeutics for a variety of biomedical applications in the past decade. One major challenge to the broad application of nucleic acid therapeutics in humans is their safe, selective and efficient delivery to target tissues. We will describe the development of new nanomaterials for delivery of messenger RNA for transient protein expression. These results offer promise towards the future treatment of genetic disorders that require complex gene regulation.

Cationic polymers capable of encapsulating chemotherapeutic drugs and electrostatically binding small interfering RNA (siRNA) are a promising platform for the treatment of drug resistant cancer. Specifically, pH responsive cationic polymers that are in a collapsed state in the blood and swollen at the slightly acidic pH of the endosomes allow for enhanced therapeutic effect.
In this work, block copolymers of dimethylamino(ethyl methacrylate), diethylamino(ethyl methacrylate), or diisopropylamino(ethyl methacrylate) and poly(ethylene) glycol were synthesized by activators regenerated by electron transfer atom transfer radical polymerization (ARGET ATRP). The hydrophobic pH responsive monomer as well as the ratio of hydrophobic to hydrophilic block lengths were varied to modulate pH responsive behavior. NMR was used to verify incorporation of monomers and critical micelle concentration was determined by pyrene fluorescence. Particles were typically 70-100nm at pH 7 as determined by dynamic light scattering and carried a positive zeta potential. The ratio of amine containing methacrylate monomers to poly(ethylene) glycol allowed for the tunable modulation of the pKa 5.5 to 7.0. Cytotoxicity of the cationic polymers was assessed in human adenocarcinoma cells, and nanoparticle uptake in murine macrophages was observed using confocal microscopy. Chemotherapeutic delivery potential was studied by imbibition of doxorubicin at low pH followed by release in vitro at pH 7.4 for 2 hours and then pH 5.2 for 6 hours.
Block copolymers synthesized by ARGET ATRP demonstrated tunable pH responsive properties appropriate for co-delivery via endosomes. These block copolymer were cyto-compatible with model cells lines and demonstrated the ability to effectively load and release a model chemotherapeutic.
This material is based upon work supported by the National Science Foundation Graduate Research Fellowship Program (DGE-1110007).

High throughput, combinatorial approaches have revolutionized small molecule drug discovery. Here we describe our work on high throughput methods for developing and characterizing RNA delivery and gene editing systems. Libraries of degradable polymers and lipid-like materials have been synthesized, formulated and screened for their ability to delivery RNA, both in vitro and in vivo. A number of delivery formulations have been developed with in vivo efficacy, and show potential therapeutic application for the treatment of genetic disease, viral infection, and cancer.

Stem cell-based tissue engineering holds great potential for the regeneration and/or replacement of damaged tissue due to their capability of differentiation to several lineages. Controlling Mesenchymal stem cells (MSCs) differentiation into osteogenic lineage provides an important strategy for the restoration the function of bones after osteoporosis, osteoarthritis or orthopedic surgeries. To control the osteogenic differentiation, microRNAs (miRNAs) are found to be highly potent tools due to the delicate control of gene expression through RNA interference (RNAi) mechanism. Several promising miRNAs have been identified as key regulators of multiple growth factors simultaneously. Despite their great potential, miRNAs are negatively charged macromolecules with limited stability, thus hampering the biological functions. Therefore, a key challenge for controlling MSCs differentiation by miRNAs is the development of an effective delivery system. In addition, an ideal platform for tissue regeneration usually involves a three-dimensional (3D) macroporous scaffold to provide mechanical support for cell seeding and organization.
To address the delivery issue, we synthesized a lipid-like-material, so-called “lipidoid” by conjugating alkyl-epoxides to spermine. Thereafter, the lipid nanoparticles (LNP) self-assembled together with cholesterol and DSPE-PEG by the ethanol-injection method. We evaluated the delivery efficiency by silencing the expression of GFP in GFP expressing MSCs and the demonstrated a significant knockdown effect, higher than that of Lipofectamine 2000, with no toxicity detected.
To generate a proper mechanical support for MSCs, a 3D porous scaffold was fabricated by lyophilization of a silk fibroin (SF) solution. SF is a naturally occurring protein with great biocompatibility, high strength and elasticity. Our water insoluble 3D SF scaffold with macroporous structure was obtained after crystallization by ethanol treatment. The functionalization of scaffold with siRNA/miRNA containing LNP was achieved by a simple soaking and freeze-drying process. By tuning the feed concentration of particles, various loading amounts could be achieved. 3D culture of MSCs in siGFP functionalized scaffold resulting in gene silencing and the efficacy is proportional to the loading amount.
Furthermore, miRNAs including anti-miR-138 and anti-miR-34c could also be loaded into the 3D scaffold. Under osteogenic culture, significantly enhanced alkaline phosphatase (APL) activity was observed in MSCs after 14 days and increased calcium content was achieved after 28 days, indicating the promoting of osteogenesis.
In summary, we have engineered a novel platform based on 3D silk fibroin scaffold functionalized with miRNAs loaded LNP to promote osteogenesis of MSCs. The facile fabrication and the versatility of our delivery platform to load different miRNA payloads, favor its broad regenerative applications and targeted personalized therapy.

Calcium phosphate nanoparticles consist of the same material as human hard tissue (bone and teeth) and therefore have a high biocompatibility. They can be loaded with all kinds of (bio-)molecules which are protected from enzymatic degradation below a nanometer-thin shell of silica. On their surface, targeting molecules like antibodies or peptides can be covalently attached. The incorporation of fluorescing molecules permits to track their pathway into cells, in the case of near-infrared dyes (NIR) also in-vivo after intraveneous injection.
They are entering a cell by endocytosis or macropinocytosis, as shown by fluorescence microscopy in the presence of endocytosis inhibitors. Inside the cell, they first occur inside an endosome that later turns into a lysosome where the low pH leads to the dissolution of calcium phosphate, an increase of osmotic pressure and a rapid endosomal escape to release the cargo into the cytoplasm.
Different applications are shown, including in-vivo results: The delivery of DNA leads to an efficient transfection to turn on the production of proteins. The delivery of siRNA leads to an efficient gene silencing. The delivery of toll-like receptor ligands together with an antigen stimulates dendritic cells and specifically turns on the immune system against viruses. This is demonstrated both for prophylactic and for therapeutic vaccination. In additional experiments, it is shown that all kinds of molecules, both synthetic as well as biological (like proteins) can be carried across the cell membrane with the help of calcium phosphate nanoparticles.

RNA interference (RNAi), which can selectively knockdown target genes, has shown great potential in the treatment of various diseases including cancer. Thus far, numerous nanoparticle (NP) platforms, such as lipoplexes and polyplexes, 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, it is expected that the development of controlled-release siRNA NPs will lead to sustained gene silencing and more effective cancer treatment.
Herein, we present a robust NP platform for safe and effective siRNA delivery in a sustained manner, which can be developed through the self-assembly of biodegradable and biocompatible polymers and lipids. The lipid-polymer hybrid NPs show excellent knockdown efficacy at low doses of siRNA. 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. For example, results demonstrate that the expression of PHB can be effectively inhibited for over two weeks after short-term transfection with the NPs. The NP-mediated sustained silencing of PHB in turn generates more effective tumor cell growth inhibition in vitro and in vivo than the lipofectamine-siRNA complexes. Furthermore, this NP platform has been applied to deliver siRNAs against drug resistance and chemotherapeutic drugs for synergistic cancer treatment. We expect that the lipid-polymer hybrid NP platform with the property of sustained siRNA release could be of interest in both fundamental biological studies and clinical applications.

An artificial “closed-loop” system able to mimic pancreas activity and release insulin in response to glucose level changes has the potential to improve patient compliance and health. Herein, a new glucose-responsive formulation for self-regulated insulin delivery was constructed by packing insulin, glucose-specific enzymes into pH-sensitive polymersome-based nanovesicles assembled by a diblock copolymer. Glucose can passively transport across the bilayer membrane of the nanovesicle and be oxidized into gluconic acid by glucose oxidase, thereby causing a decrease in local pH. The acidic microenvironment causes the hydrolysis of the pH sensitive nanovesicle that in turn triggers the release of insulin in a glucose responsive fashion. In vitro studies validated that the release of insulin from nanovesicle was effectively correlated with the external glucose concentration. In vivo experiments, in which diabetic mice were subcutaneously administered with the nanovesicles, demonstrate that a single injection of the developed nanovesicle facilitated reduction of the blood glucose levels in for over one week.

Bioorthogonal chemistries enable new avenues for materials design when encapsulation of therapeutic proteins without loss of function is required. Recently, inverse electron demand Diels-Alder (IEDDA) chemistries have garnered interest for hydrogel synthesis applications in which bioorthogonality is paramount. To date, a thorough evaluation of protein stability after exposure to IEDDA functionalities has been missing. In this work, we have characterized two therapeutic antibodies—a monoclonal antibody (mAb) and an antibody fragment (Fab)—by size exclusion chromatography (SEC), ion exchange chromatography (IEC), capillary electrophoresis (CE-SDS), antigen-binding surface plasmon resonance (SPR) and mass spectroscopy (MS) after exposure to several IEDDA functionalities. As an exemplary application, we produced hyaluronate hydrogels with in situ antibody encapsulation to demonstrate the applicability of this chemistry to the design of a protein delivery system. Tethering of the therapeutic antibody to the hydrogel matrix via a transient covalent or non-covalent linker enables the development of a system for the sustained delivery of proteins. This work sheds new light on the applicability of IEDDA chemistries to biomaterial design and synthesis and demonstrates their applicability in a translational protein delivery system.

Inflammation or disease severity in inflammatory/autoimmune diseases is presented in highly fluctuated state. One such example is, immune response during post-transplantation of solids organs and vascularized composite allografts (VCA). Currently, systemic immunosuppression is used in vascularized composite allotransplantation (VCA). This treatment has considerable side effects and reduces the quality of life of VCA recipients. We have developed a self-assembled hydrogel in which immunosuppressive drug tacrolimus has been encapsulated. Hydrogel releases the drug in response to proteolytic enzymes that are overexpressed during inflammation (disease severity). This hydrogel showed robust hydrolytic stability to avoid burst release of drug, which is a hallmark of current delivery vehicles, and released the drug in response to the inflammation in a dose-dependent manner. We validated this hydrogel in limb transplantation model in rats. A one-time local injection of the tacrolimus-laden hydrogel significantly pro-longed graft survival in a Brown Norway-to-Lewis rat hindlimb transplantation model, leading to a median graft survival of >100 days compared to 33.5 days in tacrolimus only-treated recipients. Control groups with no treatment or hydrogel only showed a graft survival of 11 days. In conclusion, a single-dose local injection of an enzyme-responsive tacrolimus- hydrogel is capable of preventing VCA rejection for >100 days in a rat model and may offer a new approach for immunosuppression in VCA.

Hydrogen peroxide (H2O2) is an important reactive oxygen species generated in cells via oxygen metabolism and is involved actively in cell signaling and cell growth. However its uncontrolled over-production can cause detrimental oxidation of biomolecules and lead to aging, cancer and other diseases. In this talk, I will present our new method of developing dumbbell (Au-Fe3O4 and PdPt-Fe3O4) and core/shell (Au/MnO) nanoparticles as sensitive probe for H2O2 detection. The dumbbell nanoaprticles were prepared by controlled nucleation and growth of Fe3O4 on the pre-synthesized noble nanoparticles, while the core/shell Au/MnO nanoparticles were made by controlled oxidation of AuMn alloy nanoparticles. Both dumbbell and core/shell nanoparticles are active for electrochemical reduction of H2O2 with the detection limit reaching as low as 5 nM. The highly sensitive electrochemical sensor has been used to monitor H2O2 concentration levels released from living cells, from which tumorigenic cells were detected to have higher level of H2O2 than the non-tumorigenic ones. The composite nanoparticle probe can be used for sensitive cancer detection and may also help to increase efficacy for cancer therapy.

One of the most important issues in nanoparticle-carrier drugs is the release of the drug molecules from the carrier. In particular, sustained release is a critical feature that is desired in modern drug design. It aims to prolong therapeutic effect and avoid systemic toxic effect caused by burst release. In the present work, we show that the sustained release can be achieved by employing a self-decomposable SiO₂ nanoparticle system with specific loading schemes. We are able to manipulate the drug release profiles and achieved sustained drug release with long duration. In vivo results showed that under the same dose, the nanoparticle drug demonstrated prolonged and stable presence in plasma for 3 days, while the free drug itself was cleared out of the biological system in a couple of hours. In addition, the self-decomposable feature of the nanoparticles enables the carriers&’ easy renal clearance after the drug release, significantly reduced their undesired accumulation in livers and spleens. The present study shows a promising system with highly desired therapeutic features. It will have great impact on improving the management of chronic diseases which require long-term medication.

Near-infrared (NIR)-responsive drug delivery system have been studied for a decade from inherent characteristics of NIR such as good biocompatibility or deep penetration depth against human tissue. Herein, we prepared a NIR-responsive drug delivery carrier by the facile fabrication methods including mixing and extraction procedure. Nano-assembly of mesoporous silica coated gold nanorods (AuNR@mSiO₂) and a drug loaded phase change material (PCM) was fabricated and physicochemical properties were analyzed. The gold nanorods at the core of AuNR@mSiO₂ showed outstanding NIR sensitivity and the mesoporous silica shell was utilized for reservoir of loaded drug and PCM. The PCM played a pivotal role for drug loading as both a thermosensitive gatekeeper and a medium for a hydrophobic anticancer drug. An enhanced cytotoxicity of this system was observed against various cell lines in comparison with that of free DOX. It can be explained as a result of enhanced intracellular drug releasing through the rapid melting process of PCM, which was easily monitored by cellular imaging. Since any complicated synthetic methods are required for fabrication and PCM is easily capable of medium for various hydrophobic drugs, this system has an enormous potential for broad application in the biomedical field as a delivery system for hydrophobic drugs with NIR-responsive behavior.

Current methodologies towards developing selective drugs and nanoparticle-based therapies rely on non-specific drugs or metal nanoparticle, and a selective drug-delivery system (colloids and vesicles coated with desired antibodies or proteins) to deliver therapeutics to desired cell sites. Typically these non-specific drugs, once spatially delivered, kill the targeted cells (e.g. tumor or pathogen) and even healthy cells in close proximity. Here we present a novel nanoparticle-based therapy where the nanoparticles selectively target specific cell phenotypes and biological processes, based on their biochemical potentials. Using a specific light induced targeting mechanism, these Light-Activated Reactive Species (LARS) have the capability to target and kill specific-pathogens upon illumination with visible or infrared light, without affecting the growth of other cells. Furthermore, the growth of selected cells and the targeted pathogens is unaffected in dark, and we can trigger these designed LARS species externally with light to demonstrate bactericidal properties. To demonstrate the efficacy of the specific design of these LARS species, we will also show both proliferation and cell death using two different LARS in the same targeted cell phenotypes. Using Escherichia coli cells and HEK293 mammalian cells as model systems, we will demonstrate selective LARS which either specifically kill or proliferate E. coli growth, while leaving HEK293 cells unaffected in individual cultures as well as a co-culture of E. coli and HEK293 cells. These unprecedented results demonstrate the ability to tune the biochemical potential using LARS nanoparticles, allow targeting selective cell phenotypes over a range of organisms and enable a new class of antimicrobials and targeted organism-specific therapeutics.

The issues encountered with current cancer therapies stem from a vast array of different diseases that fall under the heading of cancer. In order to address this issue in the past, treatments have been developed that leave much to be desired with respect to efficacy and deleterious side effects. This is especially apparent with the systemic delivery of chemotherapeutics. To this end, a wide range of nanocarriers have been developed over the years to take advantage of the enhanced permeability and retention effect that leads to the localization of large particulates in the cancerous tissue. One subset of nanocarriers that has been extensively studied is thermoresponsive hydrogels combined with an external stimuli responsive nanoparticle. The actuation of the temperature responsive hydrogel by the heating caused the external stimuli responsive nanoparticle leads to a release of a drug payload. In the past, these systems have been based around nanogels of N-isopropyl acrylamide and its copolymers. The issue that arises with these systems is that, due to the high surface area to volume ratio, and lack of consideration for drug polymer interactions, these systems result in inefficient loading and premature release of the drug payload.
To this end, a new core/shell system has been developed to enhance drug-polymer interactions while maintaining the responsive nature of N-alkyl substituted acrylamides. These systems utilize a core of temperature responsive N-isopropylmethacrylamide, to elicit a temperature response above 37°C, coated with a variety of drug compatible polymers. In order to identify the perameters that greatly improve drug-polymer interactions bulk hydrogels of the various polymer coatings are tested for partition coefficient and permeabilies of model chemotherapeutics. The results of these studies are then compared to the physical properties of the gels, including swelling ratio and relative hydrophobicity determined by pyrene fluorescence. The core shell systems are then synthesized and loading and release experiments are performed to determine the improvement of loading efficiency and the eventual release allowed by the different systems.
The bulk polymer films studied include tert-butyl methacrylate (TBMA), phenyl ehtyleneglycol ether acrylate (PEEA), and phenyl methacrylate (PMA). These systems are chosen due to hydrophobicity and structural properties that lend themselves to the interaction with model chemotherapeutics. The partition coefficients demonstrated that TBMA and PMA have a much higher drug affinity than PEEA, likely due to greater hydrophobicity. However, the overall release under gentle heating is inefficient, releasing a small percent of the loaded drug. This can be overcome via the extreme heating capable with the inclusion of magnetic nanoparticles, utilizing a burst-like release often attained by sharp temperature changes in N-alkyl substituted nanogels.

Magnetic microbubbles, microbubbles coated with magnetic-nanoparticles, offer strong potential for targeted drug delivery due to their combined acoustic and magnetic properties. So far drug delivery based on magnetic microbubbles (MMBs) has been relying on their disruption/collapse upon applying strong ultrasonic forcing. During bubble collapse not only most of the carriers are released but also large shear stresses and microjets are generated. Yet the rapid collapse of bubbles may be harmful for the surrounding tissues and the synchronous burst release of drugs from many bubbles could exceed toxic limits. In summary, there is little control on the release of drugs using the bubble collapse.
In this talk, we will introduce on a new property of MMBs, in which MMBs continuously eject nanoparticles from the surface without collapsing under an acoustic filed. We drive the bubbles at their resonance frequency at a moderate amplitude inducing volume and surface modes. Particle release has been observed for both MMBs and functionalized MMBs (i.e. coated with a mixture of magnetic and drug containing particles) with medical relevant sized between 450 nm to 200 mu;m. We are able to predict the transport of the drug with a simple model force balance model. This technology posses interesting opportunities for targeted drug delivery through/into tissue barriers, which are hard to diffuse or penetrate without causing damage.
As a proof-of-concept, we deliver Doxorubicin-containing poly(lactic-co-glycolic acid) (PLGA) particles across the hydrogel barrier to target the cancer cells that are hard to reach for free Doxorubicin or Doxorubicin-PLGA particles.

Staphylococcus aureus (S. aureus) is a significant human pathogen responsible for a wide range of diseases including skin and wound infections, toxic shock syndrome, septic arthritis, endocarditis, and osteomyelitis. Increasingly, these infections are caused by bacterial strains resistant to standard antimicrobial treatments. We have demonstrated an effective alternative based on nanoparticle-mediated photothermal approach for elimination of S. aureus. In this work, we develop an integrated approach involving targeted delivery of antibiotics and photothermal effect to treat S. aureus. A smart polymer is developed to coat the surface of the nanoparticles for delivery of the antibiotics. The polymer responses to nanoparticle-mediated photothermal effect and expand its network to release controllable amount of antibiotics to eradicate the bacterial cells. Conjugation of a specific antibody to the nanoparticle platform increases the therapeutic effect to the S. aureus. This approach may lead to new treatment for implant-associated infections.

Although nanotechnology promises to revolutionize the treatment of infectious disease, existing state-of-the-art nanoparticle delivery vehicles, including many liposomal and polymeric nanoparticle formulations, suffer from limited capacities, uncontrollable release profiles, and complex, specialized synthesis procedures that must be re-adapted for each new cargo molecule, leading to drug- and disease-specific ‘one-off&’ approaches. To address these limitations, we have developed mesoporous silica nanoparticle-supported lipid bilayers (‘protocells&’ - see Nature Materials (2011), 10: 389-397) for high capacity, cell-specific delivery of various therapeutic molecules, including antibiotics. Protocells are composed of a mesoporous silica nanoparticle (MSNP) core encased within a supported lipid bilayer (SLB). MSNPs have high surface areas and can, therefore, be loaded with 20-55 wt% of acidic, basic, and hydrophobic antibiotics, capacities that are 100 to 1000-fold higher than similarly-sized liposomes and polymeric nanoparticles. Furthermore, by controlling the degree of silica condensation in the MSNP core, release rates can be precisely tailored from 100% release within 12 hours to 2% release for nearly two months. Fusion of liposomes to antibiotic-loaded MSNPs creates a coherent SLB that enhances the colloidal stability of protocells in blood and helps retain encapsulated drugs within the MSNP core until protocells reach their target organ or cell. Furthermore, the SLB provides a biocompatible interface for display of targeting and endosomolytic moieties, which we have shown trigger efficient, cell-specific uptake of protocells, followed by cytosolic dispersion of encapsulated antibiotics. Specifically, protocells loaded with the antibiotic, levofloxacin, and targeted to cells infected by Francisella tularensis SCHU S4 are internalized by target cells 10,000-times more efficiently than by non-target cells and kill intracellular SCHU S4 more effectively than free levofloxacin and levofloxacin-loaded liposomes at just 2 wt% loading (one-twentieth the protocell&’s maximum loading capacity). In summary, protocells combine high cargo capacities, long-term stability in blood, high targeting specificity, and controllable release kinetics and are, therefore, a promising nanoparticle-based delivery platform. We are currently adapting protocells for inhalational administration and testing the pharmacokinetics, efficacy, and safety of levofloxacin-loaded protocells in rodents and non-human primates infected with aerosolized Francisella tularensis. Furthermore, to facilitate clinical translation, we are addressing reproducibility, purity, scalability, cost, and shelf-life in the absence of cold chain.

The aim of this study was to evaluate the potential use of the antioxidant activity of surface -modified cerium oxide in rescuing human dermal fibroblast cells in the presence of hydrogen peroxide (H₂O₂) or hydroquinone (HYQ). It is widely known that various stressors like UV, heavy metals, drugs and other environmental agents constantly challenge cells. If unchecked, these stressors lead to diseases including inflammation, premature aging, neurodegeneration, various other disorders, and cancer. Oxidative stress is responsible for generating various types of reactive oxygen species (ROS), such as the superoxide anion (O₂^-), the hydroxyl radical (OH) and H₂O₂. ROS scavengers have become an important area to focus on nano-technology and apply to nano-medicine. With recent reports on cerium oxide nanoparticles being neuroprotective, radioprotective and anti-inflammatory, cerium oxide nanoparticles may be potential free radical scavengers which allow us to use it as a therapeutic agent to fight against cancer and other diseases due to a reduction in ROS. Considering these facts, cerium oxide nanoparticles may promote cell survival under oxidative stress. Ceria nanoparticles were synthesized from 1 mL aqueous solutions of 1 M cerium nitrate (Sigma Aldrich, St Louis, MO) and 2 mL of 0.1 M dextran T-10 (Pharmacosmos, Holback, Denmark) and these solutions were added drop wise to 6 mL of a 30% ammonium hydroxide (Sigma Aldrich, St. Louis, MO) solution while stirring for 24 hours at 25 ^omicron;C. Synthesized nanoparticles were characterized in terms of their size and chemistry via Transmission Electron Microscopy (TEM) and X-ray photoelectron spectroscopy (XPS). TEM results showed that the particles were around 3 nm. XPS data indicated the presence of cerium atoms in dried powders. Cytotoxicity (MTS) assays were also carried out with human dermal fibroblast (ATCC^® PCS-201-012^trade;) cells for 1 day in culture using DMEM (ATCC^® 30-2003^trade;), 10% FBS (ATCC^® SCRR-30-2020^trade;) and a 1% penicillin-streptomycin solution (ATCC^® 30-2300^trade;). Cells were seeded at a density 5,000 cells/well, allowed to adhere for 24 hours and the following day, the culture was treated with cytotoxic agents like H₂O₂ or HYQ with the concentration range from 100 to 800µM. After 24 hours of incubation, the MTS reagent was added and was determined. A dose of 500µM for H₂O₂ and 150µM HYQ cytotoxic agents was found effective in killing 50% of the cells. In order to determine the cyto-protective function of ceria nanoparticles, some of these cells were preincubated with ceria at a 500µg/mL concentration for 24 hours followed by the addition of cytotoxic agents. The culture was incubated for 24 hours before MTS assays. Results were compared with only 500µM of H₂O₂ and 150µM HYQ, but not ceria treated cells. The results showed that ceria treated cells were able to recover from the oxidative damage /cytotoxicity exerted by the drugs which suggests that ceria nanoparticles may act as antioxidants within the body.

Hydrogen peroxide (H₂O₂) is one of reactive oxygen species (ROS) and plays a key role as a secondary messenger in normal cellular signaling. However, its overexpression leads to oxidative damages to tissues and organs and has been implicated in inflammatory responses and various life-threatening diseases. Therefore, H₂O₂ has great potential as a diagnostic and therapeutic biomarker of various inflammatory responses and there is increasing interest in the development of strategies to exploit ROS for diagnostics and therapy. We have developed copolyoxalate (PVAX) as an antioxidant polymeric prodrug that is able to rapidly scavenge H₂O₂ and exert antioxidant, anti-inflammatory and anti-apoptotic activities. PVAX was designed to incorporate H₂O₂-responsive peroxalate ester linkages and antioxidant vanillyl alcohol (VA) in its backbone. PVAX nanoparticles readily undergo H₂O₂-triggered hydrolytic degradation and release VA, which is able to reduce the generation of ROS and exert anti-inflammatory and anti-apoptotic activity. The main culprit in the pathogenesis of ischemia/reperfusion (I/R) injury is the overproduction of H₂O₂ which is one of the most abundant forms of ROS and causes inflammation, apoptosis and subsequent tissue damages. We therefore evaluated the therapeutic potential of PVAX nanoparticles using a mouse model of hepatic and myocardial I/R injuries. During the hepatic and myocardial I/R injuries, PVAX nanoparticles specifically reacted with overproduced H₂O₂ and exerted highly potent anti-inflammatory and anti-apoptotic activities, leading to the reduced cellular damages. We anticipate that PVAX nanoparticles have great potential as a novel therapeutic agent for the treatment of oxidative stress associated diseases.

The outermost layer of skin, the stratum corneum (SC), protects the body from harmful environmental conditions such as ultraviolet (UV) exposure by serving as a selective barrier. We explore an emerging class of UV-blocking nanomaterials, inorganic zinc oxide (ZnO) and titanium dioxide (TiO₂) nanoparticles (as opposed to conventional micron-sized particles). These are applied to the SC to protect against the harmful effects of solar UV exposure and induce regenerative repair after injury. They diffuse and partition into the SC intercellular boundaries, and the resulting construct is highly effective in preventing erythema from solar UV radiation. However, it remains unclear if these treatments can prevent degeneration in the biomechanical barrier of the SC. We have previously shown that solar UV radiation poses a double threat to the SC by increasing the driving force for cracking while simultaneously decreasing the SC&’s resistance to cracking by significantly reducing cellular cohesion, largely dominated by the intercellular lipids and corneodesmosomes, thereby impairing the critical barrier function of the skin.
We explored the interaction of zinc oxide and titanium dioxide nanoparticles with SC in the presence of UV radiation. We quantitatively determined the drying stresses, and hence the driving force for damage, that occur with UV exposure and the ability of inorganic nanoparticles to mitigate this damage. We also explored the ability of the nanoparticles to protect the innate SC resistance to corneocyte separation, which has been shown to significantly decrease under UV exposure. We found that the inorganic UV inhibitors protected the SC&’s mechanical properties remarkably well under relatively large doses of UV radiation. We also compared efficacy of the inorganic particles to chemical (UV absorbing) sunscreens. Clinical implications of this work include prevention and treatment of sunburn and long term skin damage such as photo-aging.

The natural defenses of biological systems for exogenous oligonucleotides, such as synthetic antisense DNA and siRNA, present many challenges for the delivery of nucleic acids in an efficient, non-toxic, and non-immunogenic fashion. Indeed, because nucleic acids are negatively charged and prone to enzymatic degradation, researchers have historically relied on transfection agents such as cationic polymers, liposomes, and modified viruses to facilitate cellular entry and protect delivered biomolecules from degradation. However, each of these platforms is subject to several drawbacks, which include toxicity at high concentrations, the requirement of specialty nucleic acids to enhance stability, and severe immunogenicity.
Spherical nucleic acid (SNA) nanoparticle conjugates pose one possible solution for circumventing these problems in the context of both immunomodulatory and RNAi pathways. SNAs are typically synthesized from inorganic nanoparticle templates that are functionalized with a spherical shell of densely organized, highly oriented nucleic acids. Remarkably, these highly negatively charged SNA structures do not require cationic transfection agents or additional particle surface modifications and naturally enter all cell lines tested to date (over 50, including primary cells). This talk describes novel liposomal SNAs representing a new class of single entity gene regulation agents made exclusively from components that are biocompatible and components of FDA-approved therapeutics. These novel therapeutics are constructed of 30 nm liposomal cores stabilized with a dense shell of oligonucleotide with a hydrophobic tail that can intercalate between the phospholipids that define the liposome structure. First, we have discovered that oligonucleotide shells can stabilize small (sub-50 nm) liposomal particles and in the process create a potentially very useful class of metal-free SNA. As with conventional SNAs based upon gold cores, these novel liposomal structures rapidly enter multiple cell lines without the need for ancillary transfection agents and can be used to effectively knockdown gene expression via antisense pathways. Moreover, they are indefinitely stable in serum containing media and can be easily made at scale from readily available starting materials. The fact that they do not require the polymeric, viral, or lipoplex co-carriers employed by most researchers is critical since such materials pose significant obstacles in terms of toxicity and immunogenicity.

Self-folded redox/acid dual-responsive nanocarriers (RAD-NCs) with a well-defined core-shell structure were developed for physiologically triggered delivery of anticancer drug. Monodispersed RAD-NCs with a diameter of around 200 nm were self-assembled from a graft copolymer mainly comprised of polyethylene glycol (PEG) and polyserine, which are highly biocompatible. The redox-sensitive disulfide bonds were incorporated into the PEG backbone, while pH-sensitive hydrophobic ketal groups were introduced to the polyserine side chanis. Broad-spectrum anticancer drug doxorubicin (DOX) was non-covalently encapsulated in the hydrophobic core during a self-folding process attributed to the amphiphilic nature of the graft copolymer. Additionally, the mild acid-responsive behavior of ketal groups can induce confirmation change of the folded graft copolymer upon pH trigger, leading to the pH-responsive release of the encapsulated DOX. In our design, disulfide bond was directly incorporated into the polymeric backbone via a condensation polymerization. Importantly, these disulfide bonds not only served as a redox-sensitive moiety, but also provided potential for further modification of the RAD-NCs surface such as conjugation of tumor-targeting ligands. Chosen as a model targeting moiety, folic acid was decorated onto the surface of RAD-NCs for an enhanced anticancer efficacy. The drug release profile studied via dialysis suggested a redox/acid responsive two-phase pattern where an initial fast release phase was followed by a slow release phase. The intracellular trafficking of DOX loaded NCs were observed and confirmed by confocal laser scanning microscopy (CLSM). Evidenced by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, DOX loaded folic acid conjugated RAD-NCs (DOX/FA-RAD-NCs) displayed more potent cytotoxicity compared with both free DOX solution and DOX/RAD-NCs. The evidenced redox/acid responsiveness, cell internalization, and active tumor-targeting capability of RAD-NCs suggest their potential as a promising formulation for tumor-targeted chemotherapy.

Hospital-acquired infections (HAIs) have become a worldwide burden for the healthcare industry, costing pound;1 billion each year in the United Kingdom alone. Hospitals are under great pressure to control and prevent infections caused by, amongst others, methicillin-resistant Staphylococcus aureus (S. aureus), Clostridium difficile and Escherichia coli (E. coli). Around 80% of all nosocomial infections are transmitted by touch, as a significant proportion of patients, visitors and staff in healthcare environments rarely practice the high standards of personal hygiene that are expected. Frequently touched surfaces that bacteria colonise include door handles, computer keyboards, telephones, railings, walls and surgical/food trays, which then act as reservoirs of transmission. Thus, staff can be contaminated either by direct contact with patients or by contact with colonised surfaces, facilitating the spread of HAIs.
Photosensitisers (PS) are capable of inducing the destruction of a wide range of pathogens including bacteria and viruses, in addition to cancerous cells. Light activation of the PS leads to the generation of reactive oxygen species (ROS) resulting in the destruction of the target cell. These ROS attack multiple sites within the target cell such that the development of resistance to this form of treatment is highly unlikely, which is a key advantage in treating bacterial infections.
A novel strategy to incorporate photosensitiser dyes into medical grade polymers has resulted in the development of effective light-activated antimicrobial materials that induce the lethal photosensitisation of both Gram-positive and Gram-negative bacteria. Further enhancement of antimicrobial behaviour can be seen with the addition of metal oxide nanoparticles due to their unique physical, chemical and effective biological properties.
Crystal violet and ZnO nanoparticles (CVZnO) were incorporated into medical grade polyurethane polymer square samples by using a two-step “swell-encapsulation-shrink” method, developed by the Materials Chemistry Research Centre at University College London (UCL). Exposure of the polymer samples to a white light source induced the lethal photosensitisation of both S. aureus and E. coli.
In addition, this novel system demonstrated significant antibacterial activity under dark conditions against S. aureus within 2 hours, but more remarkably, a 99.9% reduction of E. coli within 4 hours in the dark. This is, to the best of our knowledge, the most potent “dark-kill” by a light activated antimicrobial agent ever reported. The results suggest that the encapsulation of ZnO nanoparticles into the polyurethane sample enhances the light-activated photobactericidal effects, and also the inherent antimicrobial properties of the crystal violet dye itself. The results of this study show great promise for this new strategy aimed at reducing the impact of HAIs in a clinical environment.

Gold nanorods (AuNRs) have demonstrated their applications in cancer therapy and diagnostics due to established protocols for surface functionalization, optical properties and biocompatibility. These novel characteristics make AuNRs attractive for utilization in simultaneous diagnostic and therapeutics in cancer treatment. The AuNRs were synthesized using the seed-mediated method and functionalized with 11-mercaptoundecanoic acid (MUA-AuNR) and (2-hydroxypropyl)-β-cyclodextrin (CD-AuNR) to afford loading of doxorubicin (DOX) at the hydrophobic core of the CD. UV-active dextran-phenyl-azo-benzoate (DexAzo) was used to coat the CD-AuNR while folic acid-terminated ligand (FA) was incorporated as cancer cell targeting moiety. The DOX-loaded AuNR complex was then incubated with HeLa cells to assess imaging and therapy applications. Imaging of HeLa cells incubated with AuNR was demonstrated using dark-field microscopy; while intracellular drug delivery after 5s UV treatment resulted in less than 30% cell viability after 24 h. Photothermal therapy (PTT) was demonstrated by IR treatment of HeLa cells with laser at 800 nm at 1.5 W/cm² resulting in cell death. Furthermore, the synergistic effects of UV and IR treatments of AuNR complex in cancer cells were exhibited using live/dead cell assay. The multifunctional AuNR complex demonstrated high drug loading capacity via CD functionalization while FA ensured intracellular delivery. The use of DexAzo as capping agent allowed UV-triggered drug release which in combination with IR-induced PTT resulted in higher therapeutic efficacy. Furthermore, the diagnostic application through dark-field imaging showed the AuNR complex as a promising new approach in cancer theranostics.

In this study europium- doped nanohydroxyapatite was synthetized using a simple aqueous precipitation method. Calcium nitrate tetrahydrate, Ammonium dihiydrogenphosphate and Europium nitrate, were used, respectively, as Ca, P and Eu precursors with a Ca:P ratio 1.67. The nanohydroxyapatite was doped with 3, 5, 10 and 20 wt% of europium. Powders obtained were studied after they were dried at 70°C and hydrothermal treated at 120°c by 2 hours. The samples were analyzed by (Transmission Electron Microscopy) TEM, (X-ray diffraction) XRD, (Fourier transform infrared spectroscopy) FT-IR, (Photoluminiscence) PL and (Dinamic Light Scattering) DLS. The Eu concentration toxicity effects of nano Eu:HAP were studied on human fibroblasts cells in vitro.
The sizes of the crystallites were about 10-70 nm with irregular morphology and present the (P63/m space group) phase corresponding to the JCPDS card 9-0432.The Eu-doped samples present photoluminescence lines at 590, 615, and 699 nm. The results of the toxicity experiments indicated that the powders were biocompatible and would not cause toxic reactions. This work provides an interesting view of the role of nanohydroxyapatite as ideal biomedical materials in future biomedical applications.

The use of gold nanoparticles in Surface-enhanced Raman Scattering (SERS) has provided a new means for imaging techniques, especially in cancer detection. In particular, varied gold nanoparticle morphologies could allow for different surface plasmon resonances, which make for easier identification of cancer markers. With this goal in mind, we have explored various synthesis methods for Star-like Gold Nanoparticles (SGNs) in order to optimize their properties for SERS with the expectation of increased enhancement as effective diameter decreases. This is accomplished through variation of reaction times, as well as the ratio of precursors, and the synthesis temperature. In addition, growth from gold seeds is also utilized as a means of modifying the resulting SGNs. The particles obtained using this method, which range from 30 nm to 140 nm, will be used in SERS in order to compare their relative enhancement factors to confirm that smaller particles provide greater enhancement, as predicted by Mie Scattering Theory. We expect that the presence of surface features on the nanoparticles will increase the overall surface area and effective diameter of the particles, therefore increasing the local surface plasmon resonance (LSPR). In order to prepare samples for comparison, the same mass of gold is used in each synthesis of SGNs, and the same concentration of particles is used during the SERS analysis.

Engineered nano- and microparticles that co-deliver antigen and immunostimulatory molecules are of interest as ‘smart&’ adjuvants, given their ability to mimic pathogens while avoiding toxicity and anti-vector immune responses. To demonstrate that mesoporous oxide nanoparticles warrant development as vaccine adjuvants, we co-loaded mesoporous silica nanoparticles (MSNPs) with a Francisella tularensis (Ft)-derived antigen (IglC) and an immunostimulatory RNA (isRNA) known to activate Toll-like receptor (TLR) 7 and TLR8 and then encapsulated cargo-loaded MSNPs in a supported lipid bilayer (SLB) that we further modified with various targeting and phagosomolytic moieties. We found that high-surface-area MSNPs are able to encapsulate 50-60 wt% of IglC or isRNA individually and simultaneously encapsulate ~30 wt% of both IglC and isRNA, capacities that exceed those of state-of the-art liposomes and polymerosomes by up to 100-fold. We, furthermore, found that conjugation of the SLB with a single-chain antibody fragment (scFv) against DEC-205 triggers efficient uptake of MSNPs by bone marrow-derived dendritic cells (BMDCs) and that the degree of silica condensation in the MSNP core can be controlled to tailor antigen release from burst (100% in 12 hours) to sustained (2-5% per day for several weeks) rates. We employed fluorescently-labeled IglC and isRNA to demonstrate that incorporating phagosomolytic lipids in the SLB enables phagosomal release of isRNA and cytosolic dispersion of IglC, which, in turn, trigger DC maturation and cross-presentation of IglC-derived peptides. Furthermore, DCs pulsed with DEC-205-targeted MSNPs induce vigorous in vitro proliferation of IglC-specific CD8⁺ T cells, whereas DCs pulsed with free IglC or IglC complexed with Imject Alum trigger weak T cell responses. Upon immunization of C57Bl/6 mice, MSNPs loaded with IglC and isRNA and targeted to DEC-205, induce high-titer (>10⁵), IglC-specific IgG responses that are 100- and 10,000-fold higher than the titers achieved using IglC complexed to Imject Alum and free IglC, respectively. Furthermore, these MSNPs also trigger IglC-specific CD8⁺ T cell responses, the magnitude of which is higher for MSNPs with sustained (vs. burst) release kinetics. Importantly, initial efficacy data demonstrate that two subcutaneous immunizations of MSNPs co-loaded with IglC and isRNA protect Fischer 344 rats against lethal aerosol challenge with Ft SCHU S4 without causing any gross or histopathological signs of toxicity or reactogenicity. We are currently extending our mesoporous nanoparticle-based adjuvants to additional Category A and B biothreats (e.g. Ebola virus, Dengue virus, Burkholderia pseudomallei) to demonstrate the universal nature of our platform. Taken together, our results indicate that mesoporous oxide nanoparticles induce potent immune responses without causing deleterious side effects and can easily be adapted for delivery of new antigens without requiring a complete re-design.

Magnetic microbubbles (MMBs) are microbubbles (MBs) coated with magnetic nanoparticles (NPs).¹ MMBs not only maintain the acoustic properties of the original MBs, but also serve as an important contrast agent for magnetic resonance imaging.² Such dual modality functionality makes the MMBs especially useful for a wide range of biomedical applications such as localized drug/gene delivery.^3-5 Currently, the exploration of MMBs relys on the disruption/collapse of MBs by applying a high energy ultrasound, which generates high shear flows and microjets for detaching drugs and facilitate the uptake of drugs by adjacent cells and tissues. However, this rapid collapse of bubbles (so called cavitation) might also damage surrounding tissues such as endothelium.⁶ In this study, we report the ability of MMBs to release its particle cargo on demand. When stimulated by ultrasound at resonance frequencies, MMBs of 450 nm to 200 µm oscillate in volume and surface modes. Above an oscillation threshold, NPs from the MMB shell are released and can travel hundreds of micrometers away from the surface of bubble. The migration of NPs from MMBs can be described with a force balance model. More interestingly, this phenomenon is observed not only from MMBs but other functional polymer- or silica-NP-MMBs as well. With this technology, we deliver Doxorubicin-containing poly(lactic-co-glycolic acid) (PLGA) particles across the hydrogel barrier to target the cancer cells that are hard to reach for free Doxorubicin or Doxorubicin-PLGA particles. The biostability of MMBs and its feasibility for controlled drug delivery in vivo were further tested in a mouse cancer model. The ability to remotely control the release of NPs from MMBs poses opportunities for targeted drug delivery through/into tissues which are hard to diffuse or penetrate, with minimal damage to health tissues.
References:
1. Zhao X, Quinto-Su P, Ohl C-D. Phys. Rev. Lett. 2009, 102(2).
2. Yang F, Li YX, Chen ZP, Zhang Y, Wu JR, Gu N. Biomaterials 2009, 30(23-24): 3882-3890.
3. Yang F, Zhang MA, He W, Chen P, Cai XW, Yang L, et al.Small 2011, 7(7): 902-910.
4. Vlaskou D, Mykhaylyk O, Krötz F, Hellwig N, Renner R, Schillinger U, et al.Adv. Funct. Mater. 2010, 20(22): 3881-3894.
5. Cai XW, Yang F, Gu N. Theranostics 2012, 2(1): 103-112.
6. Ay T, Havaux X, Van Camp G, Campanelli B, Gisellu G, Pasquet A, et al.Circulation 2001, 104(4): 461-466.

The significance of continuous monitoring of physiological parameters such as heart rate, pulse oximetry, glucose monitoring, blood pressure, and several others in the context of disease diagnosis and tracking disease progression is well established clinically. For physiological parameters, the clinical utility of measurements taken over time is quite significant compared to a single time point measurement. However, this kind of test is not practical for protein biomarkers due to the limitations of the current gold standard technology. The reasons are four-fold: low sensitivity, high cost, bulky instrumentation, and lengthy assay times. To solve these limits, we propose an architecture which consists of a (1) microfluidic mixer for mixing beads with serum (2) an input electrical impedance sensor (3) a capture region coated with antibodies followed by (4) an output electrical impedance sensor. The sample mixes with antibody coated beads, and we detect impedance changes due to the beads passing through the impedance cytometer to quantify the number of beads. Then the beads which have captured the target protein biomarkers get captured in the capture region, as a result, the number of peaks counted by the impedance sensor at the output will be less than the number of peaks counted by the impedance sensor at the input. We will present the design, fabrication, and characterization of our microfluidic electronic system based on glass and PDMS and it&’s capability for continuous detection at high temporal density. Because of the electronic nature of the detection, the platform can be fabricated on flexbile substrates and the readout electronics can be miniaturized resulting in a wearable platform.

Given the heterogeneity of diseases like cancer, multiplexed protein detection is necessary for biomarker discovery, validation and subsequently biomarker detection in the clinical setting. One of the most promising approaches for enabling biomarker based point of care disease diagnosis is by performing multiplexed protein detection electronically. In particular impedance based detection opens the potential for low cost, low powered, lightweight detection platforms. Currently, one of the most popular methods for multiplexed protein detection is the Luminex technology of optically barcoding beads with different ratios of fluorescent dyes. This is a very powerful technique enabling multiplexed detection of tens of biomarkers over multiple samples simultaneously, however requires very bulky and expensive optical instrumentation for performing the detection. In this presentation, we will present a novel electronic approach that use the electronically barcoded molecules. When the molecules pass the channel with microfabricated impedance sensors, the measured complex impedance will become modulated. A multifrequency measurement will be performed to distinguish the barcoded beads from each other electronically. More specifically, a metal-oxide-metal (MOM) structure covers half of the bead. Using this MOM coating, the measured capacitance of microparticle varies sharply with respect to the thickness of the oxide layer which is easily controlled using atomic layer deposition. Varying thicknesses will result in a change in the frequency at which the Claussiuss-Mossotti factor transitions from positive to negative, which translates to variance in the measured multi-frequency impedance measurement. These variances allow for electronically barcoding the bead, similar to the optical barcoding of beads used by Luminex, which is the basis of multiplexed protein detection.

The size-dependent optical properties of CdSe nanoparticles are desirable in bio-imaging and cell sorting applications because of their tunable photoluminescence in the visible range. Previous studies have already suggested that CdSe could be utilized for pathogen detection by using suitable capping agents to make it biocompatible; however, systematic works on the effect of crystallite size and composition of the nanocrystals are scarce. The present research will be focused on the effect of CdSe crystal size and composition (pure and doped systems) to systematically evaluate its applicability in detecting of pathogens, where Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) will be the detection target bacteria. Highly luminescent water-soluble CdSe QDs were firstly synthesized in aqueous phase in presence of thioglycolic acid (TGA) as capping agent. CdSe/TGA molar ratios, reaction temperature, time, and pH were evaluated in order to optimizer the QDs optical properties. XRD measurements confirmed the formation of the CdSe exhibiting hexagonal crystalline structure with an estimated averaged crystallite size in the 4-6 nm range. TEM analyses evidenced the formation of CdSe with particle sizes between 3-5 nm. UV-Vis measurements showed a strong exciton peak between 390-400 nm with an estimated band gap of 2.64 eV (bulk: 1.74 eV); additionally, a strong fluorescence peak was observed between 500-550 nm with an excitation wavelength of 400 nm. FT-IR analyses suggested the actual functionalization of the CdSe surface with TGA functional groups. Results of the CdSe/TGA coupling with the selected bacteria, (E. coli) and (S. aureus), will also be presented and discussed.

Medical research has demonstrated the importance of the utilization of stable, fluorescent nanoprobes. The present work addresses the applicability of biocompatible and fluorescent ZnO quantum dots as probes for detection of pathogens with the aim of achieving extremely low detection limits. For this purpose, ZnO surface must be functionalized for its subsequent interaction with bacterial cellular membrane (coupling), which will allow the corresponding detection and quantification. Herein we will discuss the aqueous synthesis of stable, water soluble and biologically compatible ZnO quantum dots (QDs) capped with L-glutathione (GSH). Is well known that glutathione protect red cell from oxidative damage in high quantities, have an antioxidant nature, can binds to toxins, heavy metals, pesticides and can transform them into a form that can be excrete by urine or bile. Due to this importance of glutathione, many studies have been recently performed to understand the reactivity of GSH with nanoparticles. ZnO QDs were synthesized in presence of GSH via a modified polyol route. The understanding of the interactions between GSH molecules and surface atoms in ZnO QDs became critical to foster the applicability of this nanomaterial in the biomedical and bioengineering fields. In this regard, the GSH/ZnO molar ratios, reaction temperature (40°C and 60°C), time (2hours-8 hours), and pH (6-9) became crucial factors to attain suitable tuning of the QDs properties. ZnO/GSH synthesized QDs were characterized by Transmission Electron Microscopy, X-Ray Diffraction, FT-IR, UV-Vis and photoluminescence (PL) spectroscopy. The QDs shape was spherical with a particle size between 5-10 nm. The UV-vis spectra revealed a strong exciton peak between 320-335 nm and a fluorescence peak between 330-350 nm using an excitation wavelength of 300 nm. The synthesis of ZnO/GSH under different experimental conditions and the corresponding coupling with E. Coli species, will be presented and discussed.

Theranostic agents present a promising clinical approach for cancer detection and treatment. We herein introduce a microbubble and liposome complex (MB-Lipo) developed for ultrasound (US) imaging and activation. The MB-Lipo particles have a hybrid structure consisting of a MB complexed with multiple Lipos. The MB components are used to generate high echo signals in US imaging, while the Lipos serve as a versatile carrier of therapeutic materials. MB-Lipo allows high contrast US imaging of tumor sites. More importantly, the application of high acoustic pressure bursts MBs, which releases therapeutic Lipos and further enhances their intracellular delivery through sonoporation effect. Both imaging and drug release could thus be achieved by a single US modality, enabling in situ treatment guided by real-time imaging. The MB-Lipo system was applied to specifically deliver anti-cancer drug and genes to tumor cells, which showed enhanced therapeutic effect. We also demonstrate the clinical potential of MB-Lipo by imaging and treating tumor in vivo.

Current guidance techniques for the surgical excision of breast tumors include implanted guide wires and injectable radioactive seeds; however, these methods provide significant disadvantages such as often requiring a second surgery and exposing the patient and staff to ionizing radiation. Biodegradable gas-filled hollow iron-doped silica nanoshells have been developed as alternative intrasurgical guide markers by performing as color Doppler ultrasound contrast agents. In vivo studies previously demonstrated that the nanoshells will remain stationary post-intratumoral injection and have an ultrasound longevity of 10 days. To determine the degradation and/or excretion profiles of Fe-doped and undoped nanoshells, in vivo experiments were performed in a murine model. We report on a novel method for trace silicon determination of Fe-doped calcined silica nanoshells in biological tissue using inductively coupled plasma optical emission spectrometry (ICP-OES). The procedure allows for the extraction of silicon by simultaneously dissolving the nanoshells and the nanoshell-bearing tissue and for the determination of nanoparticle concentration as a function of residence time in an animal. The procedure additionally provides a systematic method to calibrate each spectroscopic sample. The experiments were performed in healthy Swiss white mice in which either the Fe-doped nanoshells or their undoped counterparts were systemically administered into the tail vein, and the organs were harvested at designated time points over 10 weeks and analyzed using ICP-OES. 3.5% of the initial injected dose of the Fe-doped nanoshells and 2.3% of the initial injected dose of the undoped nanoshells were detected 10 weeks post-injection, consistent with excretion dominating biodegradability in systemic clearance. Fe-doped silica nanoshells may serve as a stationary marker for the guided excision of breast tumors and have demonstrated substantial systemic clearance in a mouse model. Although excretion is likely the dominant mechanism of in vivo clearance, biodegradation ensures that the small fraction retained by the liver will not bioaccumulate, allowing multiple doses.

Active targeting is predicted to better localize drugs in diseased sites and avoid collateral damage due to off-target effects. Ligand clustering can potentially result in a high local concentration of ligands for high avidity, synergistic interactions with cell receptors. Multivalent clustered ligands can also recruit and encourage receptor clustering to promote receptor-mediated endocytosis, thus enhancing the uptake of nanocarrier therapeutics into the targeted cells.
Here, we developed a monodisperse 15 nm micelle that has a long circulation half-life (29.5 hours) and favorable biodistribution. To present ligands on the micelle surface with control over multivalency, we designed the 3-helix micelle by mixing two independent coiled-coil peptide amphiphiles. The high recognition specificity of coiled-coils ensures the absence of cross-association between the peptides. The ligand presentation on the surface of a 15 nm nanocarrier can be modulated via the oligomeric state of the functionalized coiled-coil. The stability of the targeting micelle was maintained as confirmed by FRET. The uptake efficiency of micelles with ligands targeting VCAM-1 receptors on endothelial cells was evaluated and ~70% higher internalization was observed compared to non-targeted micelles. Ongoing efforts focus on the effects of ligand cluster size and linker length on targeted uptake.

Statement of Purpose: An on demand, non-invasive and portable insulin delivery method that can achieve pulsatile insulin release and effective regulation of blood glucose is highly desirable for type 1 and advanced type 2 diabetes management ^[1]. We report that integration of an injectable nano-network with a focused ultrasound system (FUS) can remotely regulate insulin release both in vitro and in vivo ^[2].
Methods: We prepared insulin-loaded nano-network by a double emulsion-based solvent evaporation method. Poly(lactic-co-glycolic acid) (PLGA) was selected as a matrix material due to its prominent biocompatibility and biodegradability. To acquire oppositely charged nanoparticles, two natural polysaccharides, chitosan (positively charged) and alginate (negatively charged) were respectively applied as surfactants during the emulsion procedure to coat PLGA cores. Further these two kinds of nanoparticles were mixed together to form cohesive gel like nano-network through electrostatic force. To demonstrate the pulsatile release profile triggered by Focused Ultrasound System (FUS), we performed multiple FUS treatment over time via optimized FUS condition.
Results: By serving as a synthetic insulin reservoir, the nano-network consisting of adhesive PLGA nanoparticles significantly promoted insulin release upon intermittent FUS triggers. Remarkably, a maximum of 80-fold increase in the insulin release rate was observed when the nano-network was exposed to the irradiation of ultrasound for 30 sec. In vivo studies validated that this method provided repeatable and spatiotemporal regulation of blood glucose levels in Type 1 diabetic mice.
Conclusions: We have developed a novel means of ultrasound-triggered controlled drug delivery based on the use of an injectable NN. The gel-like 3D scaffold of NN can be effectively triggered to release insulin upon FUS-mediated administration. This system provides an unprecedented useful tool for noninvasive, rapid and pulsatile regulation of BG levels for diabetes treatment. It also can be extended to deliver other drugs, therapeutic proteins, or peptides in an intermittent and spatiotemporal release fashion. Furthermore, this method can be integrated with an ultrasound imaging system for noninvasively monitoring degradation of the drug-contained formulation and facilitating the subsequent administration.
References:
[1] Ran Mo, Tianyue Jiang, Jin Di, Wanyi Tai, Zhen Gu*. Emerging Micro- Nanotechnologies Based Synthetic Approaches for Insulin Delivery. Chemical Society Reviews. 2014; DOI: 10.1039/C3CS60436E.
[2] Jin Di, Jennifer Price, Xiao Gu, Xiaoning Jiang, Yun Jing*, Zhen Gu*. Ultrasound-Triggered Regulation of Blood Glucose Levels Using Injectable Nano-Network. Advanced Healthcare Materials. 2013; DOI: 10.1002/adhm.201300490.

Polymeric micelle drug carriers were invented a quarter of century ago.¹ Today this technology has reached a clinical stage. Nearly a dozen of drug candidates based on polymeric micelles undergo clinical trials and one product, Genexol-PM, a polymeric micelle paclitaxel, was approved for cancer therapy in South Korea.² The value proposition of currently developed polymeric micelle drugs include increased drug solubility, increased extravasation and targeting to disease sites (e.g. tumors) as well as increased drug activity with respect to multidrug resistant cancers and cancer stem cells (CSC). One class of polymeric micelles is small aggregates (10 to 100 nm) formed by amphiphilic block copolymers. Hydrophobic drug molecules incorporate in polymeric micelles through cleavable covalent bonds or non-covalent interactions. Latest developments in this field include poly(2-oxazoline)-based polymeric micelles that can carry unprecedented high loading of hydrophobic drugs, such as paclitaxel, as well as blends of several insoluble drugs.³ Such formulations have much lower toxicity compared to conventional formulations, which use high amounts of unsafe excipients to dissolve poorly soluble drugs. Consequently, novel polymeric micelle formulations can be administered at much greater doses and are more efficient in killing cancer cells. Another class of polymeric micelles incorporates charged drug molecules and macromolecules by forming electrostatic complex with ionic block copolymers. In this format the incorporated molecules entrap into the polyion complex cores of micelles where they are protected from the biological environment by non-ionic water-soluble polymeric micelle shell. Upon reaching the target destination the micelles disintegrate and released their payload. This technology originally developed for antisense oligonucleotides,⁴ is now being used with chemotherapeutic agents, pDNA, siRNA and proteins. For example, extensive studies focus on the use of such systems for delivery of therapeutic enzymes (nanozymes) to the brain and other disease sites. In selected cases the nanozymes or are loaded into macrophages, which safely transport them, release at the sites of inflammation during disease.⁵ Moreover, the macrophages were shown to transduce the nanozyme particles as well as deliver genes into the host cells at the disease site.⁶ The proof of the principle has been obtained using animal models of stroke, hypertension, Parkinson&’s disease, eye inflammation, influenza virus infection, spinal cord injury, and other diseases. Recent work was supported by NIH (UO1 CA151806, R01CA184088, RO1 CA89225, RO1 NS051334, P20 RR021937), NC TraCS (4DR11404), DoD (W81XWH-09-1-0386, W81XWH-10-1-0806, W81XWH-11-1-0700), Rettsyndrome.org (HeART Award #3112) and Ministry of Education and Science of Russian Federation (11.G34.31.0004).
References:
¹ H. Bader et al. Angew. Macromol. Chem. 1984, 123/124:457; A. Kabanov et al. FEBS Lett. 1989, 258:343; M. Yokoyama et al. Cancer Res. 1990, 50:1693.
² M. Yokoyama et. al. J. Exp. Clin. Med. 2011, 3:8.
³ R. Luxenhofer et al. Biomaterials 2010, 31:4972; Y. Han et al. Mol. Pharmaceutics 2012, 9:2302; A. Schulz, et al. ACS Nano 2014, 8 (3), 2686-96.
⁴ A. Kabanov et al. Bioconj. Chem. 1995, 6: 639; A. Harada and K. Kataoka, Macromolecules 1995, 28: 5294.
⁵ A.M. Brynskikh et al., Nanomedicine 2010, 5:379-96; M.J. Haney, et al. Nanomedicine 2011, 6:1215.
⁶ M.J. Haney, et al., PLoS ONE 2013, 8(4): e61852; Y. Zhao, et al. PLoS One 2014, 9(9):e106867.

Innovation in Nanomedicine is driven by novel materials-based solutions to medical needs. Historically, the rate of translation of such concepts has been slow. The average nanomedicine product takes about 7-20 years to go through the pre-clinical phase, and another 5 years through clinical trials (Etheridge et. al., Nanomedicine, 9,pp1-14, 2013). Utilizing well-known materials and functionalizing such materials will help to accelerate the rate of translation, as oppsoed to devleoping entirely new nanomaterials. There are lessons to be learnt from the development of the first nanocarrier for cancer therapeutics, which took over 15 years to go from concept to FDA approval. In our own work, we have focussed on non-cancer therapeutics using nanomaterials. In particular, we have used the dual principle of localized and sustained delivery to enhance efficacy and aptient compliance. For this approach to succeed it is imperative that the problem of sustaining the delivery of therapeutics from nanocarriers be solved. Approved nanomedicine products currently have limited control over the release of drugs, due to the large surface area to volume ratio, as well as the short diffusion lengths. Using a prostaglandin derivative, latanoprost for lowering intra-ocular pressure, we will show how sustained efficacy of action can be achieved over several months with a single injection.
An area of nanomedicine where the size of the nanocarrier plays a role is in enhancing cellular uptake. Here again sustained action is a highly desirable feature which has not been satisfactorily addressed. We will describe our work on prevention of scarring following surgery in the eye, using sustained delivery of an siRNA molecule. The utility of polyelectrolyte layers in this regard will be highlighted.
The pre-clinical phase for the latanoprost nanomedicine was shortened to 4.5 years, while the siRNA delivery system will be ready for human trials in another year. Some of the factors behind this accelration will be discussed, as well as the options for expanding nanomedicinal approaches to treatment of other diseases.

Polymeric nanoparticle-based drug delivery systems are widely explored to improve the biological outcome of encapsulated drugs for therapeutic effects. However, nearly all synthetic materials, polymeric particles included, suffer from certain limitations in vivo. Poor vascular circulation, limited targeting and the inability to negotiate many biological barriers have prevented the overwhelming majority of polymeric particle drug delivery systems from entering the clinic. To further complicate the matter, the above requisites must be performed simultaneously while also limiting toxic effects to the patient. While attempts are being made to design such multifunctional carriers, the use of circulatory cells to augment the function of synthetic nanoparticles offers an exciting opportunity. I will discuss systems that make simultaneous use of natural systems (circulatory cells) and synthetic systems (polymer nanoparticles) to improve circulation and targeting.
Specific examples of such hybrid systems include nanoparticles adsorbed on the surface of red blood cells for prolonged circulation and lung targeting in vivo, and antibody-mediated attachment of particles to the surface of monocytes for specific targeting to inflamed tissues in vivo.
These hybrid systems offer a new design paradigm for nanomedicine.

Bio-responsive nanomaterials are of growing importance with potential applications including drug delivery, diagnostics and tissue engineering (1). A disagreeable side effect of longer life-spans is the failure of one#8232; part of the body - the knees, for example - before the body as a whole#8232; is ready to surrender. The search for replacement body parts has #8232;fuelled the highly interdisciplinary field of tissue engineering and regenerative medicine. This talk will describe our research on the design of new materials to direct stem cell#8232; differentiation for regenerative medicine (2). This talk will also provide an overview of our recent developments in the design of materials for ultrasensitive biosensing (3). We are applying these biosensing approaches both in high throughput drug screening and to diagnose diseases ranging from cancer to global health applications.
References
[1] Stevens MM, George JH, Exploring and engineering the cell surface interface., Science, 2005, Vol:310, 1135.
[2] Place ES, Evans ND, Stevens MM, Complexity in biomaterials for tissue engineering., Nature Materials, 2009, Vol:8, 457.
[3] Howes P, Chandrawati R, Stevens MM. Colloidal nanoparticles as advanced biological sensors.Science. 2014, Vol:346, 6205.

Molecular imprinting is the process by which molecules are imprinted into the matrix of a material through non-covalent bonding, including hydrogen bonding and van Der Waals interactions. In this study hydrogels were imprinted with glaucoma medication with the purpose of creating an ocular drug delivery device with reversible binding sites. The goal of this project is to create a reusable material with reversible binding sites for drug delivery applications. The material was synthesized and tested with UV-Vis Spectroscopy to determine the concentration profile of the drug release. Modifications were made to the material to ensure proper delivery over an adequate period of time, so that a sustained release was obtained.

Reactive oxygen species (ROS) are continuously generated as byproducts of the metabolism of oxygen in the body. Although the basal ROS level is essential in cell signaling, pathogen defense and homeostasis, overproduction of ROS are closely linked with initiation and progression of cancer, arthritis and other inflammatory diseases. Therefore, ROS are now considered as therapeutic targets in many diseases including cancer.
The use of antioxidants to scavenge ROS has emerged as a new strategy to treat oxidative stress-related diseases. For this purpose, low molecular weight antioxidants have been used to scavenge ROS in the body. In spite of their potential in antioxidant therapy, these molecules are generally susceptible to oxidation and readily lose their antioxidant activity under aerobic conditions resulting in low bioavailability and efficacy. In addition, the poorly controlled biodistribution and quick renal clearance of these small drugs limit their application.
This study aims to develop antioxidant polymeric micelles bearing catechol moieties, common structural units found in naturally occurring antioxidants. We synthesized diblock copolymers consisting of a hydrophilic poly(ethylene glycol) (PEG) block and a hydrophobic catechol-bearing block (PDA) by reversible addition-fragmentation chain transfer (RAFT) polymerization. The block copolymers self-assembled to form micelles with narrow size distribution. The micelles showed high oxidation stability under aerobic condition and efficiently scavenged ROS generated inside cells. Furthermore, we focused on the biological role of ROS in angiogenesis and explored the potential of the antioxidant micelles in anti-angiogenic therapy. In the endothelial cell tube formation assay and the chicken chorioallantoic membrane (CAM) assay, the micelles showed significant anti-angiogenic activity. These results suggest that the antioxidant micelles may be useful in antioxidant therapy.

Low-radiofrequency alternating magnetic fields (AMFs) penetrate deep into tissue with little attenuation, and hence serve as a convenient means to deliver signals into the bodyshy;shy; for minimally-invasive therapies. When coupled to AMFs, spinel ferrite magnetic nanoparticles (MNPs) undergo hysteresis to dissipate heat, which has been applied to cell-destructive therapy in cancer hyperthermia, to trigger action potential firing in neurons, and to turn on insulin expression in vivo. In order to decrease the MNP concentration required to achieve sufficient temperature gradients and reduce latency periods before onset of a cellular signal, significant improvement in the MNPs hysteretic heat dissipation efficiency (specific loss power, SLP) must be achieved. By tailoring the anisotropy energy of the MNP, which is dependent on its chemical composition, shape, and size to the AMF&’s field amplitude and frequency, we detail synthesis criteria to achieve optimal hyperthermic performance. We rely on the thermal decomposition of organometallic precursors to synthesize MNPs that exhibit some of the highest SLPs at clinically relevant AMF conditions by screening for MNPs with proper anisotropy energy varied over three orders of magnitude. We then passivate MNP surfaces with biocompatible coatings and demonstrate their application for magnetothermal neural stimulation.

Electrical biodetection is advantageous over optical detection in the sense that it is less expensive, lighter, label-free, and can be integrated into a portable device for analyzing a wide panel of markers. Traditionally, protein detection is performed using the sandwich ELISA (fluorescence based), which is bulky and lacks sensitivity. On the other hand, electronic detection at the single molecule level can give us real time measurements with high sensitivity, selectivity and reversibility, and also enable monitoring of binding kinetics. The current gold standard technique for single molecule electronic detection is nanopore technology. This involves measuring electrical current due to conduction of molecules through nanopores and is very sensitive to the size and shape of the nanopore. So, to achieve single molecule protein detection it is necessary to shrink the size of nanopore down to the same order of size as the target molecule, which is approximately 10-20 nm. Fabrication of devices at these dimensions is costly and low in throughput, and thus not suitable for mass production. We will present a novel micro-impedance sensor capable of detecting particles orders of magnitude smaller in dimension compared to the pore diameter. This multi-electrode architecture operates based on the principle that signal energy increases proportionally to the number of electrodes while noise stays constant. Here, we will present the microfabrication process for micron sized electrodes and micron sized pore, and it&’s ability to detect objects orders of magnitude smaller in size. We will present the theory of optimizing the design space for multi-electrode sensing, where N electrodes gives a SNR improvement of N compared to a single electrode pair. Using this sensing microstructure, peaks significantly below the noise floor can be extracted resulting in detection of nanometer sized particles.

Positron emission tomography (PET) is widely used by clinicians for disease diagnosis, staging, and patient management. Upon the administration of a small amount of radiotracer, PET imaging can provide a direct, highly sensitive and quantitative readout of its organ/tissue targeting efficiency and pharmacokinetics. Compared with radiolabeled antibodies, proteins, peptides, and other biologically relevant molecules, radiolabeled nanoparticles represent a new frontier in molecular imaging probe design by combining different imaging modalities and targeting ligands in a single vector and synergistically improving the imaging quality. The applications of radiolabeled nanoparticles are based on the premise that the radioisotopes are stably attached to the nanomaterials. Because of the fundamental differences in the various nanoparticles and radioisotopes, most radiolabeling methods are designed case-by-case. This talk will discuss some general rules about selecting appropriate isotopes for given types of nanoparticles, as well as adjusting the labeling reaction according to specific applications. Stability (colloidal and radiochemical) assessment of radiolabeled nanoparticles will be highlighted. Specific examples of PET imaging for evaluating biological fate of the radiolabeled nanoparticles and multimodal molecular imaging will be illustrated, emphasizing the importance of labeling strategies and caution in interpretation of PET data.

Orlistat, a chemically synthesized derivative of an endogenous lipstatin produced by Streptomyces toxytricini, is a selective inhibitor of pancreatic and gastric lipases, the principal enzymes responsible for the hydrolysis and subsequent absorption of dietary fat. At a therapeutic dose, orlistat can reduce the dietary fat intake by 30%. Meanwhile, excessive residual fat could lead to sickness in the digestive system. Moreover, because of low systemic absorption and first-pass metabolism, most of the drug is excreted unchanged. A self-tunable controlled release system can help improve the bioavailability and suppress side-effects. In this work, an amphipathic conjugated polymer (CP) was used to construct a fluorescent closed-loop drug delivery system, which contained functionality for imaging, real-time tracking, and smart controllable-releasing. To be specific, the CP worked as a near-infrared emissive carrier, and contained side chains of polyester (PCL), which can be cleaved by lipase. In aqueous solution, the PCL chain segments collapsed and encapsulated the drug, forming a hydrophobic and compact molecular fence. When the drug delivery system sensed lipase, the PCL fence would degrade and release orlistat. Working as a self-regulating controller, orlistat could adjust its releasing rate according to the concentration of lipase around. Once taken in vivo, the carriers broke down, resulting in the release of the weight loss drug and consequent weight loss of the mice. This procedure could be monitored by the near-infrared fluorescence from the CP, which indicated the release of the drug. It was found that that the carrier led to prolonged weight loss process while inhibited the side effects of the drug.

Near-infrared (NIR) quantum dots (QDs) have attracted much attention for bioimaging due to the lack of absorption by hemoglobin and water in the NIR range. As a result, NIR QDs can potentially image tissues deeper underneath the surface and at the same time minimize the interference by the tissue autofluorescence, which is mostly in the visible range. In this study, we examine a novel aqueous approach to synthesize SnS QDs with 2-aminoethanethiol (cysteamine) as initial capping molecule in glycerol followed by capping molecule lengthening by repeatedly attaching glycine to the capping molecule via peptide formation at 70^oC with a cysteamine:Sn:S molar ratio of 16:7:1. The suspension was further heat-treated at 200^oC for 4 hr. The obtained SnS QDs showed an NIR emission peak at 820-835 nm with an excitation wavelength at 690 nm. X-ray indicated that the SnS QDs contain mixtures of the orthorhombic and the zinc blend structures. The longer treatment time at 200^oC in water improved the crystallinity but reduced the colloidal stability of the QDs. Changing the solvent from water to ethylene glycol and glycerol improves colloidal stability while retaining the crystallinity. The crystallite sizes of the SnS QDs made in ethylene glycol and glycerol as estimated from the XRD peak widths using the Scherrer formula were 8.5 nm to 3.6 nm, respectively. In addition, due to the higher viscosity of glycerol, colloidal stability of SnS QDs made in glycerol was also enhanced. The as synthesized SnS QDs were found to have high positive zeta potential of ~ 30 mV and toxic to cells. By adding negatively charged molecules we neutralized the SnS QDs and minimized their cytotoxicity. The immunofluorescent staining of cancer cells was demonstrated by conjugating SnS QDs to antibodies. The antimicrobial effect of SnS QDs is also investigated by culturing the QDs with K12 E. coli cells in dark and fluorescent light exposure conditions.

Magnetic nanoparticles have shown great potential in biomedical applications, such as magnetic separation, drug delivery, MRI contrast enhancement and magnetic hyperthermia. Superparamagnetic nanoparticles have been widely investigated, because their zero-remanence and small particle size that can meet the required colloidal stability. However, superparamgnetism has relatively poor magnetic performance for some practice applications due to their reduced magnetization, requirement of a large field for saturation and low magnetic susceptibility. In order to enhance magnetic response, non-superparamagnetic nanoparticles have been explored for biomedical applications, particularly for hyperthermia and MRI. Exchange coupling has been used to enhance magnetic anisotropy energy, in order to achieve a large hysteresis loss. However, the use of other elements may cause toxicity and the relatively large remanence may lead in agglomeration. We have focused on magnetic nanoparticles with relatively large shape anisotropy based on iron oxide, such as disc and ring. Nanodisc and nanorings haveshown relatively good colloidal stability probably due to their vortex domain structure. These nanoparticles with relatively large shape anisotropy have shown excellent potential for biomedical applications, including MRI and hyperthermia.

Light-absorbing nanoparticles for localized heat generation in tissue have a variety of applications in diagnostic imaging, surgery, and therapies. Although numerous plasmonic and carbon-based nanoparticles with strong optical absorption have been developed, their potential cytotoxicity and long-term safety issues remain unresolved. Here, we show that melanoidins, “generally regarded as safe (GRAS)” and commonly used in food, offer a high light-to-heat conversion efficiency, biocompatibility, non-mutagenicity and efficient renal clearance with a low synthetic cost. Using the melanoidin nanoparticles, we demonstrated in vivo noninvasive photoacoustic mapping of sentinel lymph nodes via local delivery, photoacoustic tomography of gastro-intestinal tracts via oral delivery, and photothermal ablation cancer therapy. We could confirm the controlled biodegradation and renal clearance of melanoidins. Especially, melanoidin can be one of the best contrast agents for GI tract imaging as a component of foods. The versatile complex formation with metal ions like Fe³⁺ and functionalization with target moieties can make possible a variety of biomedical applications. Taken together, melanoidin can be a clinically feasible theranostic nanoparticle for photoacoustic and photothermal applications.

A quantum dot-based mRNA detection system that utilises toehold-mediated strand displacement has been developed which holds great potential for a novel diagnostic test for tuberculosis (TB).
Control of TB is hindered by a lack of effective diagnostic tools, particularly in regions of the world where HIV co-infection is highly prevalent, such as sub-Saharan Africa. There is an urgent need for a rapid, sensitive test for TB that can be performed in resource-limited settings. A recent study was successful in identifying gene signatures that can distinguish between patients with TB and latent TB infection (LTBI), and those with TB and other diseases [1]. These gene signatures, which can be detected in whole blood and are comprised of relatively few transcripts, represent promising biomarkers in the diagnosis of TB.
The gene signatures are composed of up- and downregulated transcripts which correspond to genes that are switched ‘on&’ and ‘off&’ according to whether an individual is infected with TB. Patients can be classified according to a disease risk score, where the summed intensity of the downregulated transcripts is subtracted from the summed intensity of the upregulated transcripts. This disease risk score method of patient classification is highly amenable to use with multiplexed quantum dots (QDs), where one QD species can be used to detect upregulated transcripts and another to detect downregulated transcripts.
In order to detect these transcripts, QDs were functionalised with probe DNA, which was able to hybridise to a reporter probe displaced by incoming target RNA. This toehold-mediated strand displacement reaction is capable of specifically detecting long RNA targets and is stable in purified RNA from whole blood, as well as human serum. We have investigated the use of rolling circle amplification as a means of creating an integrated system with a low limit of detection [2]. This quantum dot-based signalling mechanism represents a promising advance in the detection of mRNA molecules in complex mixtures of RNA.
References
[1] Kaforou, M., Wright, V. J., Oni, T., French, N., Anderson, S. T., Bangani, N., Banwell, C. M., Brent, A. J., Crampin, A. C., Dockrell, H. M., Eley, B., Heyderman, R. S., Hibberd, M. L., Kern, F., Langford, P. R., Ling, L., Mendelson, M., Ottenhoff, T. H., Zgambo, F., Wilkinson, R. J., Coin, L. J. and Levin, M. (2013) Detection of tuberculosis in HIVinfected and -uninfected African adults using whole blood RNA expression signatures: a case-control study. PLoS Med 10 (10): e1001538.
[2] Howes, P. D., Chandrawati, R. and Stevens, M. M. (2014) Colloidal nanoparticles as advanced biological sensors. Science 346 (6205): 1247390

The increasing application of gold nanoparticles (AuNPs) in the field of biomedicine stems, in part, from their potential for photothermal therapy and radiosensitizing effect in internal radiation therapy.¹ The remarkable x-ray attenuation of gold also makes these NPs a promising contrast agent for computed tomography (CT).² In addition, labeling with a paramagnetic element, such as manganese (Mn), will enable to track these NPs by magnetic resonance imaging (MRI), which has better soft-tissue contrast resolution than CT.³ Therefore, such multifunctional NPs would aid on the diagnostic, therapy and follow up of malignant tumours that are treated by internal radiation therapy, such as prostate cancer.
The AuNPs for biomedical applications are commonly prepared through well-established colloidal synthesis methods. However, the translation of those NPs to clinical trials has raised toxicity concerns related with the control of their size and surface properties as well as with the presence of chemical residues that are not completely eliminated through the purification processes.⁴ Hence, techniques that produce small and ligand-free NPs must be developed.
Pulsed laser ablation in liquids (PLAL) has recently emerged as an alternative technique for the synthesis of NPs, because it enables the production of small, ligand-free NPs, while avoiding the use of potentially toxic reducing agents.⁵ Consequently, toxicity concerns related with chemical traces could be avoided.
Therefore, in the present work, Au-Mn NPs were obtained by functionalizing AuNPs, synthesized by PLAL, with Mn²⁺ chelated by diethylenetriaminepentaacetic acid (DTPA). The ligand-free AuNPs were characterized by TEM, UV-visible absorption spectroscopy and XPS. The AuNPs have an average diameter of 8.0±2.6nm. Afterwards, these NPs were functionalized with polyethylene glycol and DTPA-Mn. The NPs were characterized at each step of the functionalization process by TEM, DLS, UV-visible absorption spectroscopy, XPS and FTIR. These analyses demonstrated that the functionalization procedure did not cause significant aggregation of the nanoparticles, that the different molecules were grafted on the surface of the nanoparticles, and that the final average hydrodynamic diameter is 40 nm. The final product was also analyzed by H¹ NMR and ICP-MS. The results of these techniques indicated that Mn²⁺ is chelated and that the final Au/Mn colloidal ratio achieved is appropriate for dual imaging by CT and MRI, as it was also confirmed by the imaging studies.
1.Dreaden, E. C. et al., Chem. Soc. Rev. 2012,41 (7), 2740-79
2.Ng, V. W. K. et al., J. Mater. Chem. B 2013,1 (1), 9-25
3.Alric, C. et al., J. Am. Chem. Soc. 2008,130 (18), 5908-5915
4.Albanese, A. et al., Annu. Rev. Biomed. Eng. 2012,14 (1), 1-16
5.Barcikowski, S.; Compagnini, G., Phys. Chem. Chem. Phys. 2013,15 (9), 3022-6

Magnetic resonance imaging contrast agents work by altering the relaxation times of water protons distributed around them. Around a paramagnetic ion, such as the Gd³⁺ ion, the relaxation rates of the bulk water are enhanced as a result of short-range interactions (inner-sphere relaxation from water molecules directly coordinated to the metal ions) and long-range interactions (outer-sphere relaxation from nearby H-bonded water). The lanthanide ion Gd³⁺ is by far the most frequently chosen MRI contrast agent because it couples a large magnetic moment with a long electron spin relaxation time. However the free Gd³⁺ ion has been shown to be toxic in both in vitro and in vivo studies. Therefore it must be sequestered by chelation or encapsulation for in vivo use. In this study, chelating amphiphiles of ethylene diamine tetraacetic acid (EDTA) and diethylene triamine pentaacetic acid (DTPA) complexed with gadolinium ions have been incorporated in phytantriol and monoolein cubosomes. Gd-loaded silicate particles with ordered lamellar, hexagonal and cubic, and disordered internal structure have also been prepared. For the soft organic and hard inorganic nanoparticle systems the effect of Gd-loading, particle size, internal particle water channel size and structure on relaxation rates has been investigated. Both soft organic particles and hard inorganic particles exhibit potential to be excellent MRI and multi-modal imaging contrast agents. Contrast agent performance of select nanoparticulate systems can be superior to existing commercialised MRI contrast agents.

Circulating tumor cells (CTCs) have become an emerging “biomarker” for monitoring cancer metastasis and prognosis. Although there are existing technologies available for isolating/counting CTCs, the most common of which using immunomagnetic beads, they are limited by their low capture efficiencies and low specificities. By introducing a three-dimensional (3D) nanostructured substrate - specifically, a silicon-nanowire (SiNW) array coated with anti-EpCAM - we can capture CTCs with much higher efficiency and specificity. The conventional methods of isolating CTCs depend on biomolecular recognitions, such as antigen-antibody interaction. Unlikely, we here proposed that nanoscaled local topographic interactions besides biomolecular recognitions inspired by natural immuno-recognizing system. This cooperative effect of physical and chemical issues between CTCs and substrate leads to increased binding of CTCs, which significantly enhance capture efficiency. Recently, we have also developed a 3D cell capture/release system triggered by aptamer enzyme, electrical potential and Temperature, which is effective and of “free damage" to capture and release cancer cells. The bio-inspired interfaces of cell capture and release open up a light to rare-cell based diagnostics, such as CTCs, fetal cells, stem cell and so on.
Financial support by the Chinese Academy of Sciences is gratefully acknowledged.
References
Liu X, Wang ST(2014) Chem. Soc. Rev. 43: 2385-2401
Liu H, Liu X, Wang ST, et al., (2013) Adv. Mater. 25: 922-928
Jin J, Wang ST, Liu DS, et al. (2013) Adv. Mater. 25: 4714-4717
Liu H, Li Y, Wang ST, et al. J. Am. Chem. Soc. 135: 7603-7609
Zhang PC, Chen L, Zhou J, Wang ST, et al. (2013) Adv. Mater. 25: 3566-3570
Wang ST, Liu K, Liu J, et al., (2011) Angew. Chem. Int. Ed. 50: 3084-3088

Lanthanide-doped nanoparticles have been attracting huge research interest in myriad fields because of their unique optical and magnetic properties. Particularly, the rich intermediate energy levels allow the sequential absorption low energy photons (usually near-infrared) and emission of high energy photons (usually visible or ultraviolet), which is often referred as “upconversion”. Because near-infrared light is considered bio-benign, lanthanide-doped upconversion nanoparticles have been considered as a class of powerful tool to introduce light into biomedical research. However, conventional upconversion nanoparticles are often excited by 980 nm laser and the overheating by laser is an inevitable problem. We rationally engineered the structure of upconversion nanoparticles by introducing various new dopants in the particle matrix so that they can be excited by 800 nm laser. This type of new upconversion nanoparticles absorb the 800 nm and convert them into visible light, subsequently excite the photosensitizers to generate singlet oxygen efficiently and enable photodynamic therapy. Compared to ultraviolet/visible-light-driven photodynamic therapy, this method relies on near-infrared laser, therefore is more precise and biocompatible, providing new opportunity for biomedical research.

We prepared cross-linked phenylboronic acid (PBA)-containing nanoparticles (NPs) using a simple surfactant-free dispersion polymerization method in phosphate buffer solution. The nanoparticle size could be controlled by simple adjustment of the feed ratio of monomers as well as by changing the pH of the polymerization solution. In this way nanoparticles could be obtained with hydrodynamic radius in the range 50-200 nm as determined by dynamic light scattering. Characterization of the nanoparticles by atomic force microscopy and field emission scanning electron microscopy revealed that the nanoparticles were composed of spheres resembling a framboidal structure. Since the size range of the nanoparticles is particularly suited for drug delivery application we investigated its chemical modification to introduce drug molecules. The presence of PBA groups on the NPs allow for further functionalization using the well-known Suzuki coupling reaction. Using a water-soluble phosphine ligand/palladium catalytic system we prepared nanoparticles carrying carboxylic acid, aldehyde and hydrazide groups. To get more insight in the nanoparticle structure we conjugated the hydrogen sulfide (H₂S) donor anetholedithiolthione with an amine to the carboxylic acid nanoparticles. When mixed with cell lysate from murine macrophages H₂S release was detected using a H₂S-specific fluorescent dye. Previously we showed that H₂S release from anetholedithiolthione is enzyme mediated. The observation that H₂S release is observed suggests a porous nature of the nanoparticles. The hydrazide nanoparticles were used to couple the drug doxorubicin (Dox) via the well-known acid-labile hydrazone linkage. By changing the chemical structure of the hydrazide we were able to control pH-induced Dox release. These Dox-nanoparticles all displayed a dose-dependent toxicity in the HT-29 human colon cancer cell line as well as RAW 264.7 murine macrophages.

Nanotechnology-based medicine (Nanomedicine) has received progressive interest for the treatment of intractable diseases, such as cancer, as well as for the non-invasive diagnosis through various imaging modalities. Engineered polymeric nanostructures with smart functions play a key role in nanomedicine, including drug carriers, gene vectors, and imaging probes. This presentation focuses present status and future trend of the development of self-assembled nanosystems from block copolymers for targeted therapy of intractable diseases.
Nanosystems with 10 to 100 nm in size can be prepared by programmed self-assembly of block copolymers in aqueous entity. Most typical example is polymeric micelles with distinctive core-shell architecture. Several micellar formulations of antitumor drugs have been intensively studied in preclinical and clinical trials, and their utility has been demonstrated^1,2. Compared with conventional formulations, such as liposomes, polymeric micelles have several advantages, including controlled drug release, tissue penetrating ability and reduced toxicity. Critical features of the polymeric micelles as drug carriers, including particle size, stability, and loading capacity and release kinetics of drugs, can be modulated by the structures and physicochemical properties of the constituent block copolymers.
The development of smart polymeric micelles that dynamically change their properties due to sensitivity to chemical or physical stimuli is the most promising trend toward nanomedicines, directing to the targeting therapy with high efficacy and ensured safety. Notable anti-tumor efficacy against intractable cancer, including pancreatic cancer and glioblastoma, of antitumor drug-incorporated polymeric micelles with pH-responding property was demonstrated to emphasize a promising utility of nanosystems for the cancer treatment. Furthermore, polymeric micelles loaded with platinum-based anti-cancer reagents successfully treated systemic metastasis even from the early stage of pre-angiogenic micrometastatic niches associated with inflammation.
Versatility in drug incorporation is another feasibility of polymeric micelles. Polymeric micelles loaded with oligonucleotides, including siRNA, have been successfully formulated with relevant properties as nanomedicines such as longevity in blood circulation to reveal significant gene silencing in xenografted as well as spontaneous mice tumor models by systemic injection³. These results demonstrates the promising features of polymeric micelles as platform nanosystems for tumor-selective delivery of a variety of molecular-targeted drugs with issues on pharmacokinetics and phramacodynamics.
1. K. Kataoka, A. Harada, Y. Nagasaki, Adv. Drug Deliv. Rev., 2012, 64, 246.
2. H. Cabral and K. Kataoka, J. Contrl. Rel., 2014, 190, 465.
3. K. Miyata, N. Nishiyama, K. Kataoka, Chem. Soc. Rev., 2012, 41, 2562.

Cancer cells, compared to normal cells, are under oxidative stress associated with the increased generation of reactive oxygen species (ROS) and are more vulnerable to oxidative stress induced by ROS-generating exogenous agents. Thus, manipulating the ROS levels provides a logical strategy to kill cancer cells preferentially without causing significant toxicity to normal cells. Fenton reaction is a chemical reaction between ferrous iron ions and with hydrogen peroxide (H₂O₂) to produce hydroxyl radical that is highly toxic and causes cell death. Cinnamaldehyde, a major component of cinnamon bark, has been well known to generate ROS and induce oxidative stress-mediated apoptotic cell death. Despite its ROS-mediated anticancer activities and excellent toxicity profiles, the clinical applications of cinnamaldehyde are limited by the low bioavailability and less therapeutic efficacy than commercial anticancer drugs. In order to overcome these limitations, we developed novel polymers (polyCAFe) consisting of H₂O₂-generating cinnamaldehyde and ferrocene, which can perform Fenton reaction in cells and induce cancer cell death. PolyCAFe self-assembled to form micelles that serve as NanoFenton reactors which generate hydroxyl radicals, leading to cell death. Cell culture studies demonstrated that polyCAFe micelles preferentially induce apoptotic cancer cell death through the generation of highly toxic hydroxyl radicals. A study of tumor xenograft mouse models showed that polyCAFe micelles accumulate in tumors and inhibit tumor growth. We therefore anticipate that the polyCAFe micelles as a NanoFenton reactor have promising potential as a novel anticancer therapeutic agent.

Stimuli-responsive nanocarriers capable of minimizing the systemic toxicity associated with most free anticancer drugs have attracted extensive attention in modern chemotherapy, among which pH responsive drug nanocarriers can utilize the pH gradient in tumor environment to achieved controlled drug release under complex pathological conditions for maximized therapeutic efficacy. We report here a DNA and DNAse based formulation for biocompatible and controlled drug delivery. This bio-inspired self-degradable drug delivery system consists of a DNA nanoclew weaved by long chain single stranded DNA and an acid degradable polymeric nanocapsule with DNAse I caged inside. The DNA nanoclew was synthesized by rolling circle amplification with its surface negatively charged and doxorubicin (DOX) was loaded into the DNA core by intercalating into the GC-base pair sequences (DOX/NCl). The polymeric nanogel containing positively charged monomers was cross-linked onto DNAse I surface by acid degradable cross-linkers, which made the DNAse I nanocapsule (NCa) positively charged as well as acid degradable. NCa was embedded into NCl via electrostatic interaction and formed a cocoon like structure with NCl as the "cocoon matrix" and NCa as "hibernating worms". The NCa was stable in physiological conditions while degraded in acid environment and exposed the encapsulated DNAse I to its substrate DOX/NCl, promoting the release of DOX. To further enhance the targetability of DOX/NCl/NCa, folic acid (FA) was conjugated to a DNA oligo complementary to NCl form DOX/FA-NCl/NCa. Rapid DOX/FA-NCl/NCa internalization and nucleus targeting in breast cancer cell line MCF 7 that overexpressed folate receptors was observed within 0.5 h. Embedding NCo into NCl reduced the half-maximal inhibitory concentration (IC50) of DOX from 2.3 mu;M in DOX/NCl to 1.2 mu;M in DOX/NCl/NCa, which was further reduced to 0.9 mu;M after the conjugation of folic acid.

Nanodiamonds are promising vehicles with uniquely faceted surface properties that have mediated marked improvements to therapeutic efficacy and safety. A recent example is the synthesis of NDX, a nanodiamond-doxorubicin agent that enhanced the pre-clinical treatment efficacy of multiple drug-resistant tumor models (e.g. liver, breast, and brain) with no apparent toxicity and the ability to improve drug tolerance [1,2]. In addition, magnetic resonance imaging contrast agents based on the conjugation of nanodiamonds to gadolinium(III) have also resulted in 12-fold enhancements in per-gadolinium relaxivity. This is among the highest ever reported values compared to clinical and nanoparticle agents . Recent advancements in the use of nanodiamond-anthracycline complexes to address hard to treat cancers as well as other applications in opthalmology and regenerative medicine will be highlighted [3]. In addition, comprehensive toxicity evaluation and the current translational roadmap for diamond-based nanomedicine which will also be discussed [4].
References
[1] EK Chow, et al. Science Translational Medicine, 73ra21, 2011.
[2] EK Chow and D Ho, Science Translational Medicine, 216rv4, 2013.
[3] LK Moore, et al. Advanced Materials, doi: 10.1002/adma.201300343, 2013.
[4]V Mochalin, et al. Nature Nanotechnology, 7, 2012.

In many cancers, including liver cancer, cancer stem cells (CSCs) are a key tumor-initiating subpopulation that is responsible for tumorigenesis, metastasis and recurrence following initial treatment. A common property of CSCs is the ability to escape traditional chemotherapy and a vast majority of cancer treatment failures can be attributed to intrinsic or acquired chemoresistance in cancer. Our lab and others have demonstrated that conventional chemotherapy is not useful in fully eradicating these CSCs and alternative methods of therapy are needed to target and eliminate these tumor-initiating cells. One such alternative method is through the use of nanomaterials to enhance drug delivery and retention in CSCs. Nanodiamonds (NDs), ~4nm carbon nanoparticles with a truncated octahedral structure, are a versatile drug delivery platform that can be functionalized with a broad array of molecules, including small molecules, proteins and genetic material. Because of their size, shape and stability, we and others have also demonstrated that NDs are biocompatabile and highly tolerated in vivo, lending them towards clinical applications. As such, we evaluated the use of NDs in cancer nanomedical applications, with a particular interest in their effectiveness against chemoresistant CSCs. Using multiple murine cancer models, including liver and breast cancer, we were able to demonstrate that NDs are capable of both increasing the efficicacy and safety of chemotherapeutics when delivered by NDs. Importantly, ND-drug delivery can overcome common mechanisms of chemoresistance in hepatic CSCs in vitro and in vivo and prevent subsequent tumor progression. We have also explored the mechanisms by which ND-drug delivery occurs at both the system and cellular level and identified several key mechanisms that contribute to enhanced drug retention, cancer cell specific drug release as well as lower systemic drug toxicity that can be applied to a wide range of other nanomedical applications for improved drug delivery. In addition to enhancing drug-delivery, unique chemical properties in NDs make them an ideal platform for enhanced cancer imaging, particular near infrared and magnetic resonance based imaging. This work provides a strong preclinical base for the application of nanodiamonds in cancer therapy and diagnostics.

Diagnostic ultrasound (US) is a prevalent medical imaging modality due to its low-cost, high resolution, and therapeutic capability when coupled with high intensity focused ultrasound (HIFU) systems. 500 nm rigid silica ultrathin nanoshells were synthesized as a chemically stable US tumor marking contrast agent with continuous in vivo US imaging lifetime. Iron (III) was included into the silica shell network to promote biodegradability from serum transferrin proteins. It was shown previously that the removal of iron from the silica shell network via transferrin fragments the nanoshells for efficient biodegradation. Folate was conjugated to the surface of the silica nanoshells via the 3-aminopropyltriethoxysilane (APTES) linker. Folate has been shown in the literature to bind to prostate specific membrane antigen (PSMA) with a high binding affinity due to folate hydrolase activity. Conjugating the silica nanoshell surface with folate targets the ultrathin silica nanoshells towards the LnCAP tumor where PSMA is significantly up-regulated. The surface modified ultrathin silica nanoshells were filled with liquid perfluorocarbon (PFC) which underwent acoustic droplet vaporization (ADV) during US insonation. The phase transition of PFC from liquid to vapor generated a large amount of PFC microbubbles that created high image contrast during US imaging. In vitro experiments with clinical diagnostic US have demonstrated that the ultrathin silica nanoshells can be imaged for at least 3 hours under color Doppler imaging, exhibiting a continuous US imaging lifetime. In vivo experiments have shown that non-folate conjugated silica nanoshells were able to accumulate in the LnCAP tumor via enhanced permeability and retention (EPR) effect for up to 3 days after initial tail vein intravenous (IV) injection and were observable via US imaging for 3 days. In contrast, folate conjugated silica nanoshells were able to accumulate and persist within the tumor region for up to 10 days post-injection, observable with US imaging. By synthesizing ultrathin silica nanoshells with a folate-conjugated surface, it is has been demonstrated that IV injected, folate-conjugated ultrathin rigid silica nanoshells can accumulate in the LnCAP tumor persistently for 10 days and was observable via diagnostic US imaging. Silica US contrast agents with long in vivo lifetime serve as effective injectable biomarkers for tumor localization useful for diagnostic and surgical planning.

Current oral hygiene treatments focus on antimicrobial strategies, which indiscriminately kill both pathogenic and commensal microorganisms present in the oral cavity. There is much controversy on how these broad-spectrum treatments affect commensal microflora and their subsequent negative effects on human health. We have developed antimicrobial-alternatives that interrupt cell-cell signaling and biofilm formation, with potential to be selective against pathogens while leaving commensal flora unperturbed. A drawback to such inhibitors is their limited efficacy when used in acute exposures (e.g., mouthwash or brushing). This motivated us to design and synthesize protein-based nanocapsules that are capable of loading inhibitory agents, adhering to the hydroxyapatite tooth surface, and sustainably releasing agents for prolonged time periods. Using a biomimetic design, we cloned and expressed a bi-functional protein that has a hydroxyapatite-binding region covalently linked to a vesicle-forming domain. The vesicle-forming domain is inspired by the corn-derived protein zein, which is designated as GRAS (generally recognized as safe) by the FDA. Nanocapsule synthesis, loading, and retention were studied using dynamic light scattering, fluorescence spectroscopy, atomic force microscopy (AFM), and confocal laser scanning microscopy (CLSM). Streptococcus mutans biofilm formation on nanocapsule-treated hydroxyapatite was also examined. Nanocapsules formed ~150-250 nm particles in a liquid-liquid dispersion synthesis procedure. Encapsulation efficiencies of small-molecules up to 60% were observed, depending on synthesis conditions. AFM and CLSM revealed that nanocapsules with hydroxyapatite-affinity tags displayed greater surface adsorption/retention than those formed with native zein. Nanocapsules loaded with biofilm inhibitors were shown to impact S. mutans biofilm on hydroxyapatite surfaces. This research demonstrates an innovative method to extend the time that dental plaque inhibitors are present at the tooth surface. This has the potential to delay recolonization of the tooth and reduce oral infection/disease. This platform technology has potential in other areas besides plaque inhibition, such as re-mineralization and restorative materials.

To date, polymeric and metallic nanoparticles have been utilized as DNA or drug delivery carriers, and they have shown different tumor targeting abilities depending on their size. Nanoparticles sized less than 20 nm can extravasate from both leaky blood vessels in tumorous regions as well as normal blood vessels, resulting in high side effect. In contrast, nanoparticles sized greater than 300 nm are easily eliminated by phagocytosis and hardly extravasate from even tumor blood vessels. Therefore, nanoparticles in the size range of 50-200 nm are highly recommended for tumor accumulation by the enhanced permeation and retention (EPR) effect. Another critical factor for drug efficacy is rapid responsiveness to the cellular environment and subsequent drug release after internalization into cells. After nanoparticles enter tumor cells, they should degrade to prevent cytotoxicity, followed by the release of their cargo, such as DNA or drugs. Therefore, clustered particles in which several small nanoparticles are assembled together for higher tumor accumulation and longer blood circulation should be disassembled into small nanoparticles after cellular uptake for low toxicity and efficient drug release.
Herein, we prepare a pH-responsive dynamic DNA nanocluster based on gold nanoparticles with highly packed nucleic acid assembly and evaluate its potential as a drug delivery vehicle with tumor-specific accumulation. Each gold nanoparticle was readily functionalized with various functional DNA sequences; in particular, we modified the surface of gold nanoparticles with bcl-2 antisense and i-motif binding sequences. Clustering of the gold nanoparticles induced by hybridization of each DNA sequence via i-motif DNA provided tumor targeting and drug loading capabilities. After cellular uptake, the drug was released by disassembly of the gold nanoparticle cluster into single gold nanoparticles in response to the pH decrease in the late endosome. Furthermore, the antiapoptotic Bcl-2 protein was down-regulated by the antisense-modified gold nanoparticles; thus, drug-mediated apoptosis was significantly accelerated by sensitizing the cancer cells to the drug. Our precisely designed nanostructure showed a synergistic effect for the antisense and anticancer drug and thus may overcome current impediments to using small nanoparticles in systematic administration.

Electrochemistry of self-assembled DNA monolayers represents an attractive strategy for understanding the intrinsic properties of DNA and for developing DNA-based biosensors. Thus, there is much interest in the discovery and characterization of new redox-active probes for application in DNA-based technologies. We have investigated the electrochemical properties of a perylene-3,4,9,10-tetracarboxylic diimide base surrogate when incorporated at various positions within a DNA monolayer. The redox chemistry of this perylenediimide probe is mediated by the DNA base pair stack, dependent on its location within the DNA monolayer, and activated thermally. The electrochemical features and general synthetic flexibility of the perylenediimide base surrogate suggest that it is well suited for bioanalytical assays. Our studies may therefore hold significance for the development of a new class of redox-active probes for electrochemical sensor applications.

Despite wide exploitation of corticosteroid drugs for the treatment of asthma, the poor therapeutic effect on a neutrophilic subtype of asthma prohibits the full recovery of asthma patients. In this work, we developed dexamethasone (Dexa) loaded Flt1 peptide - hyaluronic acid (HA) conjugate nanoparticles to overcome the limitation of corticosteroid resistance for the treatment of neutrophilic pulmonary inflammation. Hydrophobic Flt1 peptide was conjugated to HA by benzotriazol-1-yloxy-tris(dimethylamino)phosphonium hexafluorophosphate (BOP) chemistry and the resulting conjugates were self-assembled to nanoparticles due to hydrophobic interactions between the peptides. In vitro and ex vivo bioimaging revealed that Flt1 peptide - HA conjugate nanoparticles were effectively delivered and up-taken to the lung tissues by HA receptor mediated endocytosis with a prolonged residence time. vitro and in vivo cytotoxicity were negligible confirming the feasibility of Flt1 peptide - HA conjugate nanoparticles for further therapeutic applications. On the basis of bioimaging results for pulmonary drug delivery applications, we prepared Dexa-loaded Flt1 peptide - HA conjugate nanoparticles. Transmission electron microscopy (TEM), dynamic light scattering (DLS) and high performance liquid chromatography (HPLC) confirmed the formation of self-assembled nanoparticles encapsulating Dexa. The nanoparticles reduced cytokine levels of lipopolysaccharide (LPS)-stimulated cells more efficiently than free Dexa. Furthermore, according to the bronchoalveolar lavage (BAL) cellularity and histological analysis, Dexa loaded Flt1 peptide - HA conjugate nanoparticles showed remarkable therapeutic effects in both eosinophilic and neutrophilic asthma model mice.

Scientists have discovered that Chlorotoxin can target tumor cell in human brain with a great precision. The application of Chlorotoxin can assist future surgeons to avoid the risk of damaging healthy cells in the human brain. Chlorotoxin purified from scorpion venom is essentially a peptide containing 36 amino acids and demonstrates high affinity particularly to glioma and neuroectodermal tumor. Knowledge of the molecular structure and stability is immensely useful to understand transport of Chlorotoxin in a blood saturated environment. We employ equilibrium molecular dynamics simulations to examine the stability of Chlorotoxin at various temperature and ion concentrations of the neighboring solvent. We analyze the root mean square deviation, radial distribution function, and radius of gyration to predict the structure and stability of Chlorotoxin at different environments. The optimized temperature and ion concentration for Chlorotoxin diffusion in blood are determined based on the three properties of Chlorotoxin from the atomistic simulations.

In this paper, we suggest a new type of microfluidic systems that can generate shear stress gradient in one flow rate. The suggested system can be very high throughput by generating wide levels of shear stress, and can detect cell adhesions in real-time.
Most of the cells inside our body are adherent cells. They firstly adhere to the substrate or other tissue to be developed, and this adhesion plays an important role during the cell growth and tissue development. Many researcheres tried to measure the cell adhesion strength quantitatively, by suggesting various type of system that can generate hydrodynamic shear stress. However, some systems only generate one shear stress level in one flow rate, which can be very low throughput. Also, the other systems can be very high throughput by generating shear stress gradient in one flow rate, but the systems cannot detect cells in real-time due to the system setup.
The channel with the cross-section of an equilateral triangle have special features that the simple analytical solution was derived from the Navier-stokes equation. Also, the computational simulation was conducted by using commercially available software. The particle image velocimetry (PIV) analysis was used to measure the actual flow velocity in the suggested channel. Consequently, the flow characteristics were verified by comparing the analytical solution, computational simulation, and PIV analysis, which were well fitted.
Also, cell adhesion strength of NIH-3T3 cells were measured through the system by applying shear stress gradient. The cells were seeded in the microfluidic system, and incubated for 2 hours before adhesion assay. Then, the flow was generated by syringe pump. The cell adhesion can be found by screening the number of cell detached during 2 min. The critical adhesion strength was defined as the shear stress when the 50% of the cells detached for 2 min of flow.

Nanoparticle based drug delivery systems have attracted a great amount of interest due to their higher stability, higher capacity of carrying drugs and feasibility to incorporate both hydrophobic and hydrophilic substances. Due to its ability to switch from Ce⁺³ to Ce⁺⁴ reversibly at physiological pH values, cerium oxide nanoparticles have shown great potential as antioxidant and radioprotective agents for applications in cancer therapy. Additionally, polymer coatings on the surface of cerium oxide may improve their aqueous solubility to allow for surface functionalization of the nanoparticles. Considering these facts, here sub 5 nm dextran coated cerium oxide nanoparticles were synthesized to persist in bone tumors and selectively kill bone cancer cells over healthy bone cells. Ceria nanoparticles were synthesized from 1 mL aqueous solutions of 1 M cerium nitrate (Sigma Aldrich, St Louis, MO) and 2 mL of 0.1 M dextran T-10 (Pharmacosmos, Holback, Denmark); these solutions were added drop wise to 6 mL of a 30% ammonium hydroxide (Sigma Aldrich, St. Louis, MO) solution while stirring for 24 hours at 25 ^omicron;C. The synthesized nanoparticles were characterized in terms of their size and distribution via Transmission Electron Microscopy (TEM) and Dynamic Light Scattering (DLS). TEM and DLS results showed that the particles were around 3 nm and DLS results confirmed that there was no significant change in the size of the nanoparticles when they stored in +4 ^omicron;C over 2 months. The chemistry of the nanoparticles were analyzed via X-ray photoelectron spectroscopy (XPS). XPS data indicated the presence of cerium atoms in the dried powders. Cytotoxicity (MTS) assays were also carried out with osteosarcoma cells (MG-63; ATCC CRL-1427) for up to 5 days in culture using DMEM (ATCC^® 30-2003^trade;), 10% FBS (ATCC^® SCRR-30-2020^trade;) and a 1% penicillin-streptomycin solution (ATCC^® 30-2300^trade;). The cells were seeded at 5,000 cells/well, allowed to adhere for 24 hours and were treated with ceria nanoparticles at different pH values (9, 7.2 and 6.0) for up to 5 days. Different pH values were used to model various tumor environments, which are normally slightly acidic. Cell viability results indicated that dextran coated nano ceria particles had a drastic effect on killing osteosarcoma cells at concentrations between 250 and 1000 mu;g/mL, but at pH 6.0, ceria was much more active in killing cancer cells even at lower nanoparticle concentrations (100 mu;g/mL) compared to neutral and basic pH values. In summary, this study introduces a new chemistry for anticancer applications that can respond to the acidic tumor environment to effectively kill bone cancer cells.
Acknowledgements:
Authors would like to thank Prof. Katherine Ziemer and her team for their help in via X-ray photoelectron spectroscopy (XPS) analyses, William Fowle for Transmission Electron Microscopy (TEM) of the nanoparticles, and Northeastern University and Tubitak for the funding.

The ability to control cell adhesion on material surfaces is critical to the performance of implanted medical devices as adhesion is essential for biointegration of the implant. Of particular interest to our research is the development of an understanding of what role surface topography plays in cell adhesion, which could lead to simple and durable ways to engineer surfaces without having to chemically modify the surface of biomaterials used in implantable devices. Hu et al. showed that nanopillars of widely varying aspect ratios and surface energies had strong effects on cell morphology, discouraging cell spreading (Hu et al. 2010). Our group discovered that human embryonic stem cells grown on nanopillar structures have a significantly reduced number of focal adhesions per cell and concordantly exhibit increased cell motility on the nanopillars (Kong et al. 2013). Based on these findings, we hypothesized that the pillar nanostructures, especially nanopillars within the size range of 100-500 nm, would prevent cells from adhering. To show this, we first created a library of nanopillar structures, beginning with a biomimetic cicada wing replicate (cicada wing has a high density of nanopillars with dimensions that are ideal for our studies.) We molded a negative hPDMS stamp of the cicada wing and pressed the hPDMS stamp into polymethylmethacrylate films using a compression molding process. We also fabricated pillar arrays of different spacings from commercially available silicon (Lightsmyth) and nickel molds (Holotools, Germany) using nanoimprint lithography. Nanopillar structures were characterized with SEM and AFM (P1: diameter = 215 nm, center-to-center spacing = 595 nm, height ~300nm; P2: diameter = 267 nm, center-to-center spacing = 692 nm, height ~300 nm; P3: diameter = 86 nm, center-to-center spacing = 286 nm, height = 343 nm). To evaluate cell adhesion, we counted the number of fibroblasts adhering to flat polymer, nanolines and the nanopillars after 24-72 hours and examined cell morphology on the surfaces. We also determined the number of focal adhesion sites from immunostaining for vinculin, a major protein the focal adhesion complex. After 24 hours, we observed that the cells adopted different cell morphologies, suggesting changes in adhesion dynamics. Fibroblasts showed a spread-like morphology on the flat film while the cells on pillars were smaller and more equiaxed. Preliminary results show that there are noticeably fewer cells on PMMA pillars than on flat PMMA: there was a 48% decrease in cell number. Our study has shown that nanolines and nanopillars in the 100-500 nm range do affect cell adhesion dynamics. We found that structure dimensions modulate the adhesion of cells, which may provide researchers a useful means of controlling cell adhesion on surfaces of implants.

The hydroxyapatite/collagen (HAp/Col) has a similar nanostructure and chemical composition to bone and is incorporated into bone remodeling process, thus the HAp/Col might be good candidate for a bone paste material. The HAp/Col bone paste was fabricated by mixing with sodium alginate (Na-Alg) with addition of calcium citrate (Ca-Cit) as an anti-decay reagent; however,, pH of PBS decreased to 6.7 after anti-decay test, which may cause inflammation of surrounding tissues. To maintain pH of PBS, the HAp/Col paste was prepared by addition of both Ca-Cit and CaCO₃. Afer anti-decay property test of the paste, PBS maintained physiological pH, 7.4, in two mixing ratios of Ca-Cit and CaCO₃, and the paste maintained its injectability in all pastes. In this study, cytocompatibility of the HAp/Col paste prepared with Ca-Cit and CaCO₃ were investigated by using human osteoblast-like cells MG-63.
The HAp/Col at the hydroxyapatite and collagen mass ratio of 80:20 was synthesized by the simultaneous titration method, and the HAp/Col powder was prepared according to the previous report. Briefly, the HAp/Col synthesized was compacted to squeeze water, freeze-dried and crushed. Collagen molecules of HAp/Col powder were then dehydrothermal cross-linked and stirred in CaCl₂ solution to allow saturated absorption of Ca ions. Sodium alginate aqueous solution was used as a liquid phase of the paste. Mixing conditions were fixed to the HAp/Col powder and Na-Alg aqueous solution mass ratio of 0.6, and HAp/Col and Na-Alg mass ratio of 9:1. Mixing ratio of Ca-Cit - CaCO₃ were 8x-0x, 10x-0x, 12x-0x, 8x-2x, 10x-1x, 10x-2x and 12x-2x to the reaction equivalent Ca ion amount for Na-Alg gelation previously measured. The paste materials were mixed and formed to 5 mm in height and 7 mm in diameter. Human osteoblastic cell line, MG63, was used for cell culture test. On million of MG-63 cells were seeded in each well of 6-well plate and 1 day after seeding, the hardened paste specimens were placed in each well, the cells were harvested and counted by hemocytometer. Tissue culture polystyrene (TCPS) and HAp/Col dense body, soaked in the culture medium for 5 days, were used as controls.
All MG63 cells cultured with paste specimens showed good proliferation. The 8x-0x and 10x-0x showed lower proliferation tendency than the TCPS, but no significant difference was observed. The 12x-2x showed the lowest cell proliferation; however, cell proliferation itself seemed to be delayed in comparison others. In addition, the 12x-0x (acidic pH) and 8x-2x (alkaline pH) showed termination of proliferation at 3 days after specimen placed onto the well, even 10x-1x (alkaline pH) showed enough proliferation. These suggested that pH was not only the reason for cell proliferation, but large amounts of Ca and/or citrate releases also affect on it. Even though, these results could not be catastrophic because body fluid circulation would diminish or ignore completely the effects of those ions.

Genetically engineered polymers enable design of specific and tunable materials properties at the DNA level with control over function and structure unparalleled by synthetic polymers. The precise control afforded by these polymers, coupled with their biocompatibility, programmable assembly, and flexibility, transforms our ability to engineer materials for applications such as “smart skins,” and self-healing materials, and for the interfacial control with biological systems. We have created large (10^8) and diverse libraries of genetically encoded polymers and rapidly identified functional materials using a genetic technique akin to evolution. We will present our libraries, selection strategies and downselected polymers that induce mesenchymal stem cells to differentiate toward chondrocytes, without addition of chemicals nor extra cellular matrix.

It is widely recognized that stem cells can sense, react and adapt themselves to external physical stimuli. Indeed, numerous studies have highlighted the fact that physical cues in cellular microenvironment may elicit biochemical signals, which in turn may direct and mediate intricate cellular processes. In line with this, the present work is an illustration of how human mesenchymal stem cell (hMSC) differentiation can be achieved by stimulating the cell by directing electroactive nanoparticles (within and outside the cells) that are controlled by external electric fields. Using Layer-by-layer (LBL) assembly method, uniform layer of gold nanoparticles (GNPs) were electrostatically embedded onto the thin polyaniline (PANI) films. This not only improved the conductivity of the PANI substrate drastically, but also served as extracellular actuators for the adhered cells. Furthermore, hMSCs were internalized with monodipersed Poly-L-Lysine capped GNPs to trigger cellular uptake of these particles, thus rendering them to actuate from inside the cells in response to electric field. Hence, by the simultaneous application of electric field, it is inferred that a combination of nanoscale forces generated through internalized GNPs (intracellular actuation) and through substrate embedded GNPs (extracellular actuation) stimulate the adherent cell via cytoskeletally mediated traction forces.
In order to investigate whether varying the type of electric field stimuli can elicit different lineage commitment of stem cells, the study was bifurcated so as to understand the effect of steady direct current (D.C) stimuli (100 mV/cm) as well as that of pulsed D.C stimuli (1 Hz, 100 ms pulse width) on hMSC differentiation. From the altered cellular morphology, genotypic and phenotypic expression profile it was elucidated that steady D.C stimuli favored neural-like differentiation. In contrast, the pulsatile stimuli evoked cardiomyogenic differentiation of hMSCs. In particular, upon stimulation with steady D.C field, majority of hMSCs acquired longer outgrowth/filopodial extension and were positively stained for nestin and β III tubulin. Such observations were marked with higher mRNA expression level for nestin, Neurofilament, MAP2 and β III tubulin. Following pulsed stimuli, hMSCs exhibited stick-like morphology with higher expression of cardiomyogenic markers such as cardiac troponin T, desmin, GATA-4 and α-cardiac actinin as confirmed by semi-quantitative RT-PCR. Moreover, the study unfolded the role of molecular events such as transient ROS generation, G1 cell cycle arrest and intracellular Ca²⁺ levels in inducing differential response in hMSCs, as validated using a combination of FACS and time lapse fluorescence imaging techniques. Taken together, these comprehensive results support the concept of stem cell differentiation to neural/myogenic cells by inducing biophysical stresses by external means, in the absence of growth factors.

Droplet Digitaltrade; PCR (ddPCRtrade;) provides absolute quantification of target DNA or RNA molecules. ddPCRtrade; counts positive droplets and utilizes Possion calculations to accurately estimate the concentration of the target nucleic acid. Bio-Rad QX200trade; ddPCRtrade; system provides precise and sensitive solution for a wide variety of applications, which include cancer biomarker studies, copy number variation, pathogen detection, sample preparation for next generation sequencing, environmental monitoring and food testing. The benefits of the QX200trade; ddPCRtrade; system includes precise and sensitive measurement, flexibility of chemistry and assay set up, and its simple workflow. ddPCRtrade; has the potential to offer many advantages compared with traditional qPCR, such as no standard curve required in ddPCRtrade; for target nucleic acid quantification, in accessing the effectiveness of nanomaterials and nanomedicines used for therapy in diseases such as cancer, cardiovascular disease and HIV. ddPCRtrade; can be used as a high throughput and precise tool to evaluate the efficiency of nanomaterial treatment. For example, ddPCRtrade; can be used to quantify biomarkers with high precision and sensitivity, which are associated with diseases prognosis before and after treatment with nanomedicine.

We have constructed electronic devices in a wearable format that are capable of continuous, non-invasive, long-term health monitoring. The devices integrate electronic nanomaterials with elastomeric substrates and are thus soft, stretchable, and flexible. These mechanical properties are ideal for integration with the human body, as the softness enables mechanical matching to biological tissues, while the flexibility facilitates conformal contact between the devices and the curvilinear surfaces of the tissues. In this talk, I will discuss devices composed of nano-sized piezoelectric materials that are capable of converting changes in pressure to an electronic signal and vice-versa. We have used these devices to monitor transients in pressure associated with arterial blood flow in dogs and humans. I will also discuss preliminary results of using these piezoelectric nanomaterials for a number of other applications, such as measuring mechanical properties of skin and for performing acoustic-based imaging of near-surface subdermal structures, such as tumors and cysts.

Nanotubular structures offer exciting progress toward the design of multifunctional medical implants. To bring this to reality, the mechanical, physical, biocompatibility, and interfacial properties of such structures should be optimized. We have observed that the fabrication of TiO₂ nanotubes with elastic modulus close to actual bone promotes osteoblast growth. In addition TiO₂ nanotubes could be considered a suitable alternative route for the development of drug-eluting antimicrobial implants that can prevent unnecessary side effects caused by oral administration of drugs, increase drug efficiency, and prevent infection related implant complications and failures.
In the present work TiO₂ nanotubes with controllable size (AElig;60-130nm, L: 1-10mu;m) synthesized by a simple electrochemical anodization technique are used as drug-eluting nanostructures on the surface of rat mini implants. This ability to control the aspect ratio of TiO₂ nanotubes, gives an opportunity to manage their drug-eluting properties for optimal implant function. Sodium naproxene (anti-inflammatory drug) are loaded into TiO₂ nanotubes by using the mass-scalable method of self-sustained diffusion at room temperature and pressure.
In order to obtain a multifunctional implant, in addition to drug, silver nanopartilces were embedded within the nanotubes Transmission Electron microscope (TEM) analysis reveals the presence of Ag embedded inside and outside of the TiO₂ nanotubes. Energy-dispersive X-ray spectroscopy (EDS) analysis confirmed the presence of Ag on the TiO₂ nanotubes. This multifunctional surface is expected to perform superiorly mainly because Ag is embedded into the nanotubes resulting in more stable bonding between Ag and nanotubes, creating a slow, constant and controlled release of Ag ions to kill bacteria, and simultaneously create a minimum hazardous environment to the cells.

While oral drug administration is the preferred route due to its ease of use, low cost, and high patient compliance, it is accompanied by physiological barriers including mucosal and epithelial barriers and high intestinal fluid shear, limiting the oral uptake of most therapeutics. Recently, microfabrication techniques have been applied to create micron-scale oral drug delivery microdevices that significantly enhance drug uptake in vitro and in vivo. Microdevices are typically designed with a planar shape to provide an increased surface area and reduced shear force per mass, allowing the devices to bind to the intestinal epithelium for prolonged drug exposure. Herein, we present a novel nanofabrication approach to asymmetrically coat microdevices with polymeric nanowires to enhance drug-loading properties, bioadhesion, and permeation enhancement of microdevices. This nanowire coating provides high-throughput, low waste drug-loading via capillary action with loading efficiencies >90%. The nanowires also enhance bioadhesion with 91% of nanowire-coated devices and 17% of flat control devices remaining adhered to a Caco-2 epithelial cell monolayer upon exposure to fluid flow. Finally, the nanowire-coated microdevices increase the transport of FITC-BSA through the Caco-2 monolayer four-fold over a control bolus in a 4 hour period study. These nanowire-coated microdevices have the potential to increase the uptake of a wide range of small molecule drugs and biological therapeutics upon oral administration.

Insulin is the key hormone that regulates blood sugar levels. The inability of the human body to properly regulate glucose levels can have serious medical consequences, most notably diabetes, where the body exhibits insulin deficiency (type I) or insulin resistance (type II). A wide variety of schemes for detecting insulin have been studied over the past decade. There has been a significant amount of research on improving the performance of these sensors, in terms of increasing the sensitivity and selectivity, and lowering the limit of detection. However, most of these methods have so far been unsuccessful in achieving both the sensitivity and selectivity towards insulin in serum due to its low concentration in human blood.
Here, we present a nanoscale plasmonic interferometer in combination with magnetic beads and microfluidic channels for a highly selective and sensitive insulin sensor. Our capture and separation scheme shows the great selectivity towards insulin and the nanoscale interferometer enables us to detect insulin at physiological concentration in blood (10-300 pM). The detection technique takes less than 10 minutes, and the sample volume can be as low as 10² microliters.
The sensing scheme relies on a recently developed sensor device that uses waves generated by nano-scale scattering structures (e.g., a rectangular groove-slit pair) etched on the surface of gold or silver thin films. These waves, known as surface plasmon polaritons (SPPs), propagate along the metal / dielectric interface and are strongly confined to the surface of the metal. An SPP wave generated at the groove propagate through the material to be analyzed, then interferes with the original incident beam at the slit. The light intensity transmitted through the slit will be a function of the refractive index of the material on top of the metal film. One example of how this could be used is as a biochemical sensor - as a specific example, we were able to sense extremely low concentrations of glucose in the physiological range in human saliva.[1]
Microfluidic devices have also been integrated with a recently developed novel capture chemistry and separation technique for insulin using properly functionalized magnetic beads. The method can be used to enhance the selectivity of insulin in serum.
This method can be expanded for other biochemical sensors. In addition, due to the size of the plasmonic interferometer, we can detect the multiple analytes on a single chip. Overall, we have established a highly specific and sensitive biosensor in a compact lab-on-a-chip device using the nanostructured metal film.
[1] J. Feng , V. S. Siu , A. Roelke , V. Mehta , S. Y. Rhieu , G. T. R. Palmore , D. Pacifici, Nano Lett, 2012, 12 (2), 602-609

Cell tracking enables monitoring of the distribution and dynamics of biomarkers within live cells. Their successful implementation can be utilized for a broad range of applications such as: lineage tracing in developmental biology, signaling responses to chemical cues in stem cells and optimizing administration of cell therapeutics. The state-of-art typically relies on: 1)integrating reporter genes, 2)dyes (e.g. lipophilic dyes) and 3)nanoparticles (e.g. quantum dots) to distinguish them from unmodified cells. Despite progress made in a number of fields, each method is impeded by significant limitations. Gene modification can randomly introduce undesirable mutations, are limited to the engineering of robust cell types and require extended periods of culture for stable expression. On the other hand, particle and nanoparticle methods are culpable in the generation of false positive signals and fail to reveal cell status/function.
To address these limitations, a nanoparticle-based integration-free approach to confer sensor capability within individual cells is reported herein (nanosensor). This is made possible by the efficient packaging, intracellular delivery and sustained release of sensor molecules encapsulated within biodegradable polymers. It is demonstrated how sensors with poor intracellular access (i.e. oligonucleotides) or used as end-point assays (e.g. calcein acetomethoxy) can be adapted for monitoring status/function in live cells. Another benefit is the rapid labeling process, taking no more than 8 hours. Following intracellular sequestration, sensor molecules are released over a period of up to 30 days for detecting biomarkers of interest. A series of challenges using a range of different concentration and dynamic conditions showcase its real-time monitoring capability. This mode of sustained release presents sensor molecules below potentially toxic concentration levels, maintaining mesenchymal stem cell (MSC) functional and differentiation fidelity. Finally, platform versatility is demonstrated by developing formulations to examine: 1)cell viability, 2)nitric oxide, 3)mRNA transcripts.
This cell biosensor approach stands out in terms of speed and versatility by utilizing nanoparticle labeling. Yet, it improves discriminatory ability by detecting specific biomarkers instead. Its ease of application can supplant cell labeling techniques that are significantly more laborious for monitoring the distribution and dynamics of various biomarkers. We anticipate its impact on applications ranging from heterogeneity in cell population studies to ‘nano-diagnostics&’ for cell therapeutics.

Carbon nanotubes have three key attributes that make them of great interest for novel membrane applications 1) atomically flat graphite surface allows for ideal fluid slip boundary conditions and extremely fast flow rates 2) the cutting process to open CNTs inherently places functional chemistry at CNT core entrance for chemical selectivity and 3) CNT are electrically conductive allowing for electrochemical reactions and application of electric fields gradients at CNT tips. In general, the transport mechanisms through CNT membrane are a) ionic diffusion is near bulk expectation with no enhancement from CNT b) gas flow is enhanced by ~1-2 order of magnitude due to specular reflection off of flat graphitic surface c) and pressure driven flux of a variety of solvents (H2O, hexane, decane ethanol, methanol) are 4-5 orders of magnitude higher than conventional Newtonian flow [Nature2005, 438, 44] due to atomically flat graphite planes inducing nearly ideal slip conditions. Nearly all applications require chemical selectivity in what is allowed to pass across the membrane. However the act of placing selective functional chemistry at pore entrance or along the core of CNTs, dramatically/completely eliminates the enhanced flow effects by eliminating the near perfect slip boundary condition[ACS Nano 2011 5 3867]. Needed is a mechanism to pump chemicals through the pore where selective chemistry is. This is routinely achieved in protein channels where permeates are accelerated through regions of precise functionality. The CNT membrane, with tips functionalized with charged molecules, is a nearly ideal platform to induce electro-osmotic flow with high charge density at pore entrance and a nearly frictionless surface for the propagation of plug flow. Through diazonium electrochemical modification we have successfully bound anionic surface charge to CNT tips and along CNT cores. High electro-osmotic flows of 0.16 cm/s-V at are seen by the pumping of neutral caffeine molecules. Improvements in electroosmotic power efficiency of 25-112 fold are seen in CNTs compared to conventional nanoporous materials with atomically rough interfaces [RCS Nanoscale 2011 3(8) 3321-28]. In small diameter SWCNTs ion mobilities are seen to be ~6 fold enhanced due to induced electroosmotic flow. [Nat. Nanotech. 2012 7(2) 133-39]. Use of the electro-osmotic phenomenon for responsive/programmed transdermal drug delivery devices is discussed with the voltage gated delivery of clonidine and nicotine across CNT membrane at therapeutically useful fluxes [Proc. Nat. Acad. Sci.2010 107(26) 11698-702]. A working prototype coupling smart phone psychology based survey with blue tooth enabled wristwatch are also demonstrated.

One research area that has eluded investigators is rapid multiplexed profiling of proteins/receptors directly in cells without any labeling. Such devices if developed could have high significance and impact in isolation of cells for gene analysis. Many reports have been published on network nanotube devices on sensing proteins. While individual nanotubes and their networks are highly sensitive to their environment and many papers have been published in the literature on detection of pico-femto grams of proteins, sensors based on CNT networks as channels suffer from irregularity in their individual electrical and mechanical properties. This irregularity originates from diverse sources such as different synthesis methods, chirality distribution, gas adsorption and doping and variation of device contact resistance. Such irregularities of the individual CNT devices results in unpredictable and highly irregular behavior of CNT based sensors, impending their practical applications and commercialization. Our investigation has led to us to overcome effects such as doping in air, intertube attractions, functionalization protocol variability, and electrical parameters such as contact resistance in the development of nanotube based biosensors for cellular/protein sensing and enabled reproducibility of sensors to target specific proteins in cells. This led to identification of (EpCAM, cancer biomarkers) specific versus non-specific (IgG, MCF10A) interactions directly on the surface of the cells precisely. Extended testing of spiked cells in complex mediums such as buffy coats resulted in excellent reproducibility and statistically significant results. A statistical classifier was built to identify specific versus non-specific interactions in spiked buffy coats. The testing suggested ~100% sensitivity and 90% specificity for cells spiked in buffy coats for the trainer. A blind test was conducted to validate the classifier which enabled ~90.9% sensitivity and ~81.8% specificity. Such arrays with powerful statistical models can be useful in screening for cancer biomarkers from fine needle aspirates in few minutes in a clinical setting.

The failure of the implanted devices due to bacterial infection is the major concern in the field of biomedical. In this presentation, we will demonstrate the efficacy of a unique in vitro culture protocol involving vertical electric field stimulation of biological cells. In such protocol, any electrically conducting substrate can act as cell growth substrate and can be placed inside the culture medium, while another electrode can be placed outside the culture medium with a desired distance of separation according to the field strength to be applied during the in vitro study. The placement of the second electrode outside of the cell culture medium allows the investigation of cell response to electric field without the concurrent complexities of submerged electrodes such as potentially toxic electrode reactions, electro-kinetic flows and change transfer (electrical current) in the cell medium. During the presentation, the results obtained with the use of an amorphous macroporous carbon substrate as electrodes will be discussed to illustrate neural cell proliferation, differentiation and bactericidal property in electric field mediated culture conditions. The electric field was applied perpendicular to carbon substrate electrode that supported cultured mouse neuroblastoma (N2a) cells in vitro. The macroporous carbon electrodes are uniquely characterized by a higher specific charge storage capacity (0.2mC/cm²) and low impedance (3.3 k#8486; at 1kHz). The optimal window of electric field stimulation for better cell viability, neurite outgrowth and at the same time bacterial growth inactivation is established. When a uniform electric field was applied perpendicular to the amorphous carbon substrate, it was found that the N2a cell viability and neurite length were higher at low electric field strengths (le; 2.5 V/cm) compared to that measured without an applied field (0 V/cm). Interestingly, significant reduction in bacterial viability was recorded at the 2.5 V/cm electric field stimulation conditions. Also, the experimental results showed that the viability of bacterial cells was reduced with an increase in electric field duration in a cell type dependent manner (gram positive vs. gram negative). Overall, the results of the present study unambiguously establish the uniform/gradient vertical electric field based culture protocol to promote neurite outgrowth and to inhibit bacterial growth.

Usually, stabilization of central midface fractures (Le Fort I) is made by L-shaped titanium miniplates. Replacing of titanium by polyshy;meric materials like UHMWPE or PEEK requires adapted mechanical stability and improved biocompatibility of the polymeric materials. Therefore, beside suggestions for geometrical improvement, the polymeric materials were equipped with nanoporous surfaces.
UHMWPE (ultra high molecular weight polyethylene) and PEEK (polyetheretherketone) were laminated with HDPE (high density polyethylene) on both sides. Nanoporous surfaces were made by hot embossing with an alumina stamp having nanopores on top side. The nanopores were made by anodic oxidation of aluminum. With vacuum lamination, an almost perfect adhesion of HDPE on UHMWPE was achieved. For sticking HDPE on PEEK, additional medical grade glue was necessary. In both cases, peel tests were performed to prove adhesion. By using of two different stamps with mean pore sizes of 235 and 120 nm, nanostructured HDPE surfaces were produced. Polymer nanostructures were investigatedy by Scanning Electron Microscopy (SEM). Biocomshy;patibility was verified using cell culture of chondrocytes. Together with an optimization of mechanical properties by using finite element modelling (FEM), prototypes of polymeric implants having excellent biocompatibility were achieved. As next step, the implants will be used for an animal study.

Persistence in blood circulation is one of the main hurdles in the development of nanocarriers for cancer therapy. In this account, we have fabricated long circulating polymeric hydrogels of various sizes and shapes using the PRINT (Particle Replication In Non-wetting Templates) platform. Pharmacokinetics and biodistribution of these nanoparticles were explored in xenograft and allograft tumor models, in either orthotopic or heterotopic locations, and compared to naïve mice. While circulation profiles were constant across the various particle types, the biodistribution deviated notably. Interestingly, we found that tumor accumulation was not only dependent on particle size, but also on tumor model and disease site. Developing a deeper understanding how disease dictates particle sequestration at the target-site will help establish what types of cancer are most suited for nanoparticle-based drug delivery. In our future work, we will explore the potential of particle based therapeutics.

Purpose: This study presents a novel drug delivery platform aimed to optimize the local concentrations of systemic drugs through click chemistry and an implantable biomaterial. As a therapeutic proof of concept, we apply our discoveries to the construction of an antibiotic agent based on vancomycin.
Background: Click chemistry technology allows two reaction partners to interact exclusively with each other and ignore all other possible reagents present in nature. Our published studies have shown that the local concentration of a systemic radioprobe containing a click chemistry reagent can be increased by ten times to an area previously injected with an implantable biomaterial (alginate) modified with its click reaction partner (trans-cyclooctenes, TCO). Now we present a platform that does not only lead to localization but to subsequent release at the desired location.
Methods: We chemically synthesized the following molecules: an alginate modified with tetrazine (Tz-gel), as well as multiple agents modified with trans-cyclooctene (TCO): a releasable fluorophore (TCO-R-F), a non-releasable flourophore (TCO-NR-F) and a releasable vancomycin (TCO-R-Vanco).
Animal Model: After IACUC approval, in-vivo real-time biodistribution studies of fluorescence were carried out in nu/nu mice by injecting either nothing or a type of alginate (control vs Tz-Gel). Then subjects received a tail-vein injection of TCO-R-F or TCO-NR-F. The negative controls were: 1. No gels with TCO-R-F (Negative control); 2. control alginate with TCO-R-F (Gel control). The two experimental groups were Tz-gel and either TCO-R-F (Released protocol) or TCO-NR-F (Immobilization protocol). Fluorescence was measured with an IVIS Spectrum and reported in radiance.
Minimum Inhibitory Concentration (MIC) of Releasable Vancomycin: We mixed Vancomycin or TCO-R-Vanco with either a regular alginate gel or Tz-gel overnight. The following day luminescent methicillin sensitive Staph. aureus (MSSA, Xen 29) in broth were added to the mixture for 24 hours. Luminescence was measured with an IVIS Spectrum and reported in radiance.
Results
Animal Model
Released protocol showed the largest amount of radiance at 1hr that decreased over 5 hours. Immobilized protocol showed a large amount of radiance at 1 hr, 5 hr, 3 day and 1 month. Gel control showed a small amount of radiance at 1hr that decreased promptly. The Negative control did not show any focal radiance.
MIC of Antibiotics
The MIC for vanco was 0.5, for TCO-R-vanco with Tz-gel was 2.0, for TCO-NR-vanco with control alginate>4.0 (units: nmoles/mL)
Conclusions:
We present a system to chemically modify a biomaterial with small molecules after in-vivo implantation. This approach enables:
1. systemic small molecules to be immobilized at an implant and thenlocally released through a cascade reaction in a mouse model.
2. a modified antibiotic construct releases vancomycin and inhibits growth of luminescent bacteria (MSSA).