Symposium BM2 : Stimuli Responsive Organic and Inorganic Nanomaterials for Biomedical Applications and Biosafety

After a brief summary of the nanoparticles developed in the field of photodynamic therapy (PDT), the presentation will focus on the interest of using of multifunctional nanoparticles for the treatment of malignant gliomas [1-2]. PDT for brain tumors appears to be complementary to conventional treatments [2]. Number studies show the major role of the vascular effect in the tumor eradication by PDT [4]. For interstitial PDT of brain tumors guided by real-time imaging, multifunctional nanoparticles consisting of a surface-localized tumor vasculature targeting neuropilin-1 (NRP-1) peptide and encapsulated photosensitizer and MRI contrast agents, have been designed by our group. Nanoplatforms confer photosensitivity to cells and demonstrate a molecular affinity to NRP-1 [3]. Intravenous injection into rats bearing intracranial glioma exhibited a dynamic contrast-enhanced MRI for angiogenic endothelial cells lining the neovessels mainly located in the peripheral tumor. By using MRI completed by NRP-1 protein expression of the tumor and brain adjacent to tumor tissues, we checked the selectivity of the nanoparticles [5-6]. This is the first in vivo proof of concept of closed-head iPDT guided by real-time MRI using targeted ultrasmall nanoplatforms. Another approach will be also discussed: the combination of radiotherapy and PDT for the treatment of malignant cerebral gliomas [1-3], employing scintillating nanoparticles. With this novel therapeutic approach limited light penetration problem could be overcome and activation of the photosensitizer within tumors is performed using ionizing radiation [7-9].
1.Bechet et al., Trends Biotechnol., 26, 612-21, 2008.
2.Bechet et al., Cancer Treat. Rev., 40, 229-41, 2014.
3.Benachour et al., PLOSOne, 7,e48617, 2012.
4.Mriouah et al., Trends Biotechnol., PMID: 23021636, 2012.
5.Benachour et al., Theranostics, 2, 889-04, 2012.
6.Bechet et al., Nanomedicine NBM, 2014.
7.Bulin et al., J. Phys. Chem., 117, 21583-89, 2013.
8. Retif et al., Theranostics, 5(9):1030-1044, 2015.
9. Retif et al., Int. J. Nanomed, in press, 2016.

Atherosclerosis is a disease in which lipid containing plaques are built up on the inner walls of large and medium-sized arteries. This silent progress usually occurs for decades until the manifestation of severe clinical events such as myocardial infarction, myocardial ischemia and stroke. Therefore, it is critical to develop non-invasive, reliable and reproducible imaging techniques to diagnose atherosclerosis before the occurrence of the clinical events. Magnetic nanostructures (MNS) are promising contrast materials for high-resolution magnetic resonance imaging (MRI) to monitor plaque progression and regression. In this study, monodisperse MNS with Fe₃O₄ nanoparticles as core were synthesized and then functionalized with polyethylene glycol (PEG) by using NDOPA-PEG(600)-COOH. The resultant MNS-PEG had a pristine size of ~ 8 nm (by transmission electron microscopy – TEM) and a hydrodynamic size of ~ 91 nm (by dynamic light scattering – DLS). Human foam cells – lipid laden macrophages found in atherosclerosis plaques – were produced from THP-1 monocytes to investigate the toxicity and uptake of the nanoparticles. Our cell metabolism study showed that MNS-PEG concentrations up to 10 μg/mL were not toxic to foam cells while uptake was significantly dependant on time and concentration. The minimum MNS-PEG administered concentrations to result in positive Prussian blue staining in the foam cells were 50 and 5 μg/mL, respectively, for 2 and 24 hr exposure. We further examined the effects of surface charge on cellular uptake by coating MNS-PEG with different biopolymers, heparin (HEP) and protamine (PRM), using a layer by layer assembly technique. The zeta potentials of MNS-PEG, MNS-PEG-HEP and MNS-PEG-PRM were -43, -21 and +20 mV, respectively. Our results showed that foam cells only preferred to uptake negatively charged particles. Compared to foam cells, macrophages had double the capacity to uptake the MNS-PEG, while THP-1 monocytes did not show any uptake of these particles. As such, the amount of MNS-PEG uptaken by foam cells can be controlled by its surface charge, concentration and incubation time. Surface charge modified MNS have the potential to be further developed as a contrast agent for early detection of atherosclerotic plaques.

Stealth (PEGylated) liposomes have taken a central role in drug formulation and delivery combining efficient transport with low nonspecific interactions. Controlling rapid release at a certain location and time still remains a challenge dependent on environmental factors. Liposome-nanoparticle composite vesicles can overcome the dependence on the local environment by providing the possibility to directly manipulate the vesicle structure by external fields. Here we report a new method by combining solvent inversion and sonication to efficiently assemble and load superparamagnetic iron oxide nanoparticles (SPIONs) into liposomes. The new method demonstrates efficient loading of both SPION and a water-soluble model drug into small unilamellar liposomes (D ~ 50 nm) using a protocol that allows us to generally and independently control nanoparticle fraction and lipid composition (T_m) in the entire relevant range. Furthermore, we show how passive release is suppressed by detailed control over the stealth liposome assembly. Therefore, for the first time, the influence of lipid and SPION composition as well as structure on the magnetically triggered release kinetics could be investigated and optimized. Complete external control over the release kinetics from burst to pulsed gradual release is thereby demonstrated and the hypothesized mechanism for local magneto-thermal release investigated and verified.

In this paper, we report the development of two kinds of polymeric micellar nanoparticles (sphere and cylinder) prepared from amphiphilic tri-block copolymers containing Fe(III)-catecholate complexes as new T₁-weighted imaging agents with high relaxivity, low cytotoxicity, and long-term stability in biological fluids (serum and cell media). Relaxivity values of such nanoparticles are slightly higher than established gadolinium chelates and synthetic melanin-Fe(III) complex across a wide range of applied magnetic field strengths, which might be the result of long-lived second sphere water molecules hydrogen bonding with polar groups around metal ion centers inside the nanoparticles as suggested by ¹H NMRD. More interestingly, shape-dependent in vitro MRI performances of different micellar nanoparticles in Hela celles were also observed, suggesting new opportunities to optimize new parameter in the design of those self-assembled poly(Fe(III)-catecholate) nanoparticles for biodiagnosis that can not be achieved by synthetic melanins. We describe these materials in an initial proof-of-concept study and propose that this class of polycatechol-based shaped nanoparticle will be suitable for pre-clinical investigations where gadolinium-free, safe and effective T₁-weighted imaging by MRI contrast is highly desirable.

Finely regulated cell signaling is critical for controlling a number of cellular activities, such as differentiation, growth, and death. Magnetic nanoparticles have emerged as an important tool for the remote and noninvasive regulation of cell activities. In this study, we demonstrate a magnetic nano-switch, composed of magnetic nanoparticles conjugated with a targeting antibody, for activation of apoptosis signaling. Death receptor mediated extrinsic apoptosis signaling, one of the major targets for recent cancer therapy, is activated on cancer cells by magnetic nanoparticles under magnetic field. In addition, magnetic nano-switch can selectively induce cancer cell death in a single-cell level with the use of non-invasive external stimuli (e.g., magnetic field) which can give additional advantages of precision therapeutics with space and time selectivity for disease treatments with reduced off-target side effects. The efficacy of the newly developed magnetic nano-switch is comparable to that of the well-known death ligand, TRAIL. Moreover, it has demonstrated that it is possible to activate apoptosis signaling in vivo using magnetic nano-switch.

Magnetically induced heating of nanomaterials is being intensively studied for applications in hyperthermia therapy and drug delivery, yet the heating efficiency is usually characterized by macroscopic methods and little is known about the immediate nano environment temperature change. We are interested in quantifying this event on the nanoscale and comparing it with the observed macroscopic result. For this purpose, dual-core mesoporous silica nanoparticles were synthesized, where a lanthanide nanocrystal is embedded together with a magnetic nanocrystal. The magnetic nanocrystals generate heat in a high frequency oscillating magnetic field and the upconversion emission of the lanthanide nanocrystals shows a ratiometric response to the temperature variation as a result of two thermally coupled excited states. Thus, the silica nanoparticle interior temperature increase upon placing it in an oscillating magnetic field is quantified by the luminescence emission spectra. We found that the nanomaterial heating efficiency is related to the exposure time and the field strength of the magnetic field as well as the magnetic nanocrystal size. More importantly, the silica nanoparticles experience a much greater temperature increase than the surrounding media during the heating process. Our method measures local temperatures on the nanoscale and provides the information necessary to monitor and control the temperature for biomedical applications.

Novel applications of magnetic nanoparticles (MNPs) demonstrate their promise towards revolutionizing the field of biomedical research in various ways. MNPs,being a versatile imaging, detecting, targeting and therapeutic platform, enable new interdisciplinary approaches in experimental design in basic and translational research such asthe development and application of multimodal therapystrategies against cancer. We examined the effect of magneto-mechanical stress induced by endocytosed single-domain magnetic nanoparticles in an applied low frequency alternating magnetic field (AMF), on the proliferation rate of the human colon cancer cell line HT29. We used commercial ferrofluids (Chemicell GmbH) consisting of an aqueous dispersion of biocompatible starch-covered magnetic particles with a magnetite core, with an average hydrodynamic diameter of 100 and 200 nm. For the application of the magnetic fields, a fundamentally different from prior setups 3D printout of a polymer rotating holder was manufactured, with a DC rotating motor operating with variable voltage (3-12V) resulting in tunable rotation frequency and capable of generating magnetic fields of variousconfigurations and parameters (f=0-20 Hz, Β_o=0-200 mT). Seven characteristic three dimensional permanent magnet systems (AC, DC and rotating low frequency fields), whose component parts were NdFeB block magnets of several sizes and shapes, homogeneously magnetized in arbitrary direction, were employed. All magnet configurations used, were simulated, and the magnetic flux density was computationally calculated with COMSOL Multiphysics. Our findings suggest that the applied field frequency affects cell growth 72 hours post treatment. Interestingly, AC configurations induced cell growth, with a tendency to enhance cell proliferation by applying higher field frequencies, not only in MNPs-treated cells, but also in control, untreated cells exposed to the same field. Inhibition of cell proliferation was observed at the highest magnetic flux density values at all cases. The proposed novel setup represents a versatile and accurate systemfor studying,biological effects of magneto-mechanical stress in vitro.

Sound perception via mechano-sensation is a remarkably sensitive and fast transmission process, converting sound as a mechanical input to neural signals in a living organism. Although knowledge of auditory hair cell functions has advanced over the past decades, challenges remain in understanding their biomechanics, partly because of their biophysical complexity and the lack of appropriate probing tools. In this study, we present nanomagnetic force probes (nano-MFP) as a non-contact and ultrafast mechanical actuator to explore the audio frequency tonotopy of avian hair cells. The nano-MFP is designed to remotely deliver full avian audible frequency stimuli (50 ~ 5,000 Hz) to individual hair cell with spatial specificity in the tonotopic axis of the cochlea. The working principle of the nano-MFP is the use of a magnetic force generated from an electromagnetic probe tip, which is a non-contact stimulation enabling measurement of truly free-standing hair bundle motility. The switching rate of the magnetic field can be accurately controlled down to 9 μs, which corresponds to approximately 50 kHz, covering full-range audio frequency of avian, mammalian, and rodents. The hair bundle biomechanics in the full range of the avian cochlea which has different oscillatory dynamics were probed by using a nano-MFP. Two time constants are measured from the recovery motion of the avian hair bundle: one is related passively to the morphology of hair bundle, and the other is active and changing according to the applied stimulus and media condition, which shows the avian hair bundle possess biologically active components. Stimulating the full-range audio frequency of the individual hair bundles on the avian cochlea reveals that there exists tonotopic frequency specificity in hair bundles. The precise spatiotemporal controllability of our nano-MFP reported gives great promise to explore important questions in a variety of biomechanics research that have been difficult to answer with conventional probing techniques.

We present a high-sensitive DNA detection method using quantum dot (QD)-fullerene (C₆₀) based molecular beacons (MBs) assisted by magnetic nanoparticles. In this molecular beacon configuration, C₆₀ serves as an efficient quencher and CdSe/ZnS core-shell QD, the fluorophore. When C₆₀ and QD are located nanometer away from each other, C₆₀ efficiently uptakes photo-induced electron transferred from the QD and quenches QD emission. We observed about 60% of quenching efficiency in covalently incorporated QD-C₆₀ complex with 1:1 QD-to-C₆₀ ratio. Note that the quenching efficiency could be higher as the commercial QD employed in this experiment contains a 4-nm thick polymer coating which retards the electron transfer between QD and C₆₀. The inorganic QD-C₆₀ MB is developed to overcome the low signal-to-noise ratio and photobleaching issues in traditional organic dye based MBs. The MB probes were further attached on 200-nm sized magnetic particle to form a QD-C₆₀ MBs@MNP complex that facilitates manipulation of the probes in analytes using external magnetic field.
The DNA detection assay started with adding 5 ul QD-C₆₀ MBs@MNP solution in 5 ul analyte samples of various target DNA concentrations ranging from 10 fM to 1 uM. Hybridization takes 20 minutes. The solution was then loaded to a 500 um-thick flow cell for fluorescence analysis covered with silver coated reflector. The fluorescence imaging of hybridized molecular beacons was captured using monochromatic CMOS camera followed by measuring the photo-count of each sample. The QD-C60 MB provides highly sensitive detection with strong signal-to-noise ratio. No photo bleaching effect was observed. The detection limit of < 100 pM was observed. DNA detection based on QD-C₆₀ MBs@MNP is featured by its high sensitivity, stability, and low-volume assay. By applying external magnetic field to concentrate MBs, it is possible to amplify the fluorescence signal and therefore further reduce the detection limit. The sensitive detection method will find applications in the analysis of low-concentration target nucleic acids, such as cancer related circulating miRNA. In addition, the QD-based sensing configuration can be extended for multi-target detection by coding multiple MB probe sequences with different QD emission colors.

In the search for specific targeting of breast cancer cells, one of the mechanism in the use of functionalized nanoparticles is driven by nanoparticles-cells interactions. Significant studies done on nanoparticles-cell adhesion are yet to give insights at a nanoscale of ligands/antibody conjugated magnetite nanoparticles adhesion to cancer/normal breast cells. This paper presents the results of an experimental study of the adhesion forces between components of model conjugated magnetite nanoparticle systems/configurations for improved selectivity in the specific targeting of breast cancer. In this study, Atomic Force Microscope (AFM) was use to unravel and measure the adhesion interaction between biosynthesized magnetite nanoparticles (BMNPs)/conjugated BMNPs and breast cancer (MDA-MB-231) cells/normal breast (MCF 10A) cells. In each case, chemically synthesized magnetite nanoparticles (CMNPs) and conjugated CMNPs were used as a control. The BMNP constituents had adhesion forces (to breast cancer cells and normal breast cells) that were greater than that of CMNPs. The increased in adhesion interactions of BMNPs systems are attributed to Van der Waals interactions between conjugated nanoparticles and the over-expressed receptors (revealed by confocal images via immunofluorescence staining) on the surfaces of the breast cancer. In vivo studies also showed evidences of improve selectivity and specificity of conjugated BMNPs in detection of triple negative breast cancer via MRI imaging. The implication of the results suggest the potential use of BMNPs conjugates for rapid screening of potentials ligands/antibodies as well as the design of robust conjugated BMNPs as a contrast agent for enhancing magnetic resonance imaging (MRI) for specific targeting of triple negative breast cancer.

Stem cells have a number of useful properties, including their ability to proliferate, migrate and differentiate. For this reason, numerous clinical studies have utilized stem cell-based therapies for the treatment of various human diseases and disorders. An emerging area of interest lies in engineering cells to further enhance their innate abilities and confer them with new functionalities. Cellular-based gene therapies is one such example, wherein stem cells are genetically engineered to express therapeutic molecules. Such approaches have shown tremendous potential for cancer applications since stem cells have an innate ability to home to tumors. However, traditional stem cell-based gene therapies are hampered by the inability to control when the therapeutic genes are actually turned on, thereby resulting in detrimental side effects.

Herein, we report the novel application of magnetic core-shell nanoparticles for the dual purpose of delivering and activating a heat-inducible gene vector that encodes TNF-related apoptosis-inducing ligand (TRAIL) in mesenchymal stem cells (MSCs). By combining the tumor tropism of the MSCs with the spatiotemporal magnetic nanoparticle-based delivery and activation of TRAIL expression, this platform provides an attractive means with which to enhance our control over the activation of stem cell-based gene therapies. In particular, we found that these engineered cells retained their innate ability to proliferate, differentiate, and, most importantly, home to tumors, making them ideal cellular carriers. Moreover, exposure of the engineered cells to mild hyperthermia, by applying an alternating magnetic field, resulted in the selective expression of TRAIL from the engineered cells. As a result, significant cancer cell death was induced both in vitro and in vivo. Overall, stimuli-responsive stem cell-based gene therapy using multifunctional nanoparticles has immense potential for both cancer and other biomedical applications.

Inorganic nanoparticles (NPs) have been widely explored for the treatment, diagnosis, and detection of many diseases, because of their unique features as compared with their organic and polymeric counterparts. Among others, the response of superparamagnetic NPs such as iron oxide NPs (IONPs) to magnetic fields enables their contrast-enhanced magnetic resonance imaging (MRI), effective hypothermia therapy, and targeted delivery of therapeutic agents. The assembly of IONPs with polymers enhances their stability and biocompatibility in physiological environment, as the hydrophobic nature of these NPs synthesized by solvothermal synthesis approaches makes them unsuitable for direct use in biological conditions. Moreover, the clustering of MNPs within such as vesicular membranes can dramatically increase the MRI contrast and responsiveness to external magnetic field, because of their size-dependent transverse relaxivity and magnetic moment. In particular, vesicles embedded with IONPs in the membrane are ideal candidates of delivery vehicles, thanks to their high drug loading content, magnetically-triggered release, and high magnetization per vesicle. To date, a variety of IONPs-bearing vesicles (e.g., liposomes or polymersomes) have been developed for hyperthermia therapy and targeted chemotherapy. However, most of existing platforms suffer from insufficient magnetization or limited capability of encapsulating hydrophilic or hydrophobic drugs. To tackle the challenge, we have recently developed a versatile strategy for the design of drug-encapsulated magnetic nanovesicles with extremely high loading of IONPs (and gold NPs) in the polymer membrane for imaging-guided targeted delivery of therapeutics. In this talk, I will present the self-assembly of polymer-functionalized IONPs (and gold NPs) into vesicles with densely packed IONPs in the membrane and the application of the nanocarriers for imaging-guided chemotherapy of tumors and localized drug delivery for uveitis treatment.

Mesoporous silica nanoparticle-supported lipid bilayers (protocells) are an emerging class of nanocarriers that synergistically combine the advantages of mesoporous silica nanoparticles (MSNs), including tunable size, shape, and pore characteristics that facilitate high loading capacities, with the advantages of liposomes such as enhanced circulation, ease of synthesis, biocompatibility, flexible formulations, and capacity for targeting. Protocell assembly occurs upon fusion of the liposome to the surface of the MSN, resulting in encapsulation of the MSN core within a conformal supported lipid bilayer (SLB). Further conjugation of the SLB with polymers, such as PEG, or targeting ligands confer stability and enable specific binding and internalization. We have demonstrated this platform to be a plug-and-play nanocarrier, capable of housing disparate cargos ranging from small molecule drugs for cancer therapeutics to large biomolecules including large DNA constructs such as plasmids for use in gene therapy and applying recently developed CRISPR-Cas9 technology for precise gene editing and modulation. Furthermore, the flexibility of the system extends to the targeting potential viz surface chemistries that accommodate antibodies, affibodies, and other molecules to enhance cell-specific delivery. Zwitterionic protocells conjugated with an anti-EGFR antibody and loaded with the cytotoxic drug gemcitabine preferentially bound to and killed individual EGFR-positive REH leukemia cells ex ovo and avoided their EGFR-negative counterparts while maintaining properties that are generally viewed as necessary for successful therapeutic efficacy. These properties include size monodispersity, high colloidal stability over time, minimal nonspecific binding or uptake by the mononuclear phagocytic system, high capacity for and precise release of therapeutic cargos, and low cytotoxicity. Furthermore, cationic protocell formulations can package and deliver large plasmid constructs that encode for CRISPR-Cas9 components. In a HER-2 expressing cell line, protocells modified with an anti-HER-2 affibody successfully targeted and delivered plasmids resulting in GFP and RFP expression, demonstrating successful CRISPR delivery. Unmodified protocells were used for transfection of Cas9-mediated activation of TP53, resulting in increased p53 production. Overall we have demonstrated the potential of the protocell as a platform nanocarrier capable of targeting a range of cell-surface markers and delivering diverse cargos while maintaining advantageous properties that will be necessary for viable in vivo use.

The integration of multiple molecular functionalities into mesoporous silica nanoparticles (MSNs) enables the development of nanoagents for targeted drug delivery, as well as diverse imaging agents. We will provide an overview on our research regarding the synthesis of mesoporous nanoparticles, approaches towards targeting certain cell receptors, as well as controlled molecular release systems based on internal (such as pH and redox potential) and external (light, temperature, etc.) stimuli.

Folate and epidermal growth factor (EGF) has been used for successful cell targeting with MSNs. Considering triggered release in the endosome, a novel pH-responsive system has been created based on genetically modified carbonic anhydrase (CA) gatekeepers. This system relies on the spatial control of functionalization available for our previously developed mesoporous silica core-shell nanoparticles. A pH-dependent CA inhibitor was covalently attached to the surface of the MSN, resulting in the desired opening mechanism caused by the endosomal pH change.

Addressing endosomal escape, we have covalently attached a red-light sensitive phthalocyanine photosensitizer to the MSN, surrounded by a lipid bilayer. Photoactivation leads to endosomal membrane rupture in cells causing cargo release from the mesopores into the cytosol. Moreover, we have exploited the proton sponge effect during acidification of the endosome with polymer-functionalized MSN to achieve endosomal release. Selective extracellular release of bioactive molecules has also been achieved, taking advantage of overexpressed matrix metalloproteinases in cancer tissue. These enzymes were used to open an MSN release mechanism with high spatial selectivity.

It is of great interest to expand the scope of bioactive cargo molecules that can be released from MSN systems. We have developed organically functionalized large-pore and medium-sized pore MSNs with spatially controlled orthogonal functionalities that can reversibly adsorb oligonucleotides such as siRNA. Very high siRNA loading in the internal pore volume, efficient endosomal release and high biological activity was established. Moreover, cellular uptake of proteins such as small antibody fragments was successfully achieved using a novel release mechanism from MSNs. These antibodies were used to label cellular organelles with high specificity.

These and other examples demonstrate that mesoporous silica nanoparticles represent a promising and highly flexible platform for numerous biomedical applications.

A main challenge in nano-biomedicine is the engineering of nanostructures or nanomaterials that can efficiently encapsulate drugs at high load, cross cell membranes, and controllably release their cargo at target sites. One strategy to bypass the generic toxicity associated with the drug as often seen in cancer therapy is to entrap drugs into engineered mesoporous silica nanoparticles. Their pore sizes are tunable to fit different cargo sizes. Their framework is stable, their surface can be modified with different functionalities which permits a passive or active targeting of the tumor site. This enables an efficient drug payload into the malignant cell of tumors, while the non-malignant cells become minimally impacted.
TNF-α is one of the well-known anti-tumor factors. Due to its cytotoxic effect on tumor cells and its pro-inflammatory potential, TNF-α has the ability to slow down tumor growth. However, its pharmacological use is dramatically limited by its high systemic toxicity. Therefore, we devised a drug delivery system combining TNF-α loaded, dye functionalized dendritic mesoporous silica nanoparticles with a pH-sensitive PEI-PEG copolymer for pore coverage. The major objective of this study was the transport of TNF-α in a nontoxic carrier system followed by a tumor tissue specific release to induce anti-tumor activity without causing systemic toxicity.
We demonstrate the combination of mesoporous silica nanoparticles for transport and pH-sensitive PEI-PEG copolymer for pore coverage as a potent system allowing the transport of toxic anti-tumor biologicals. MTT assays were used to analyze the biological activity of encapsulated TNF-α. Solubility and functionalization of the mesoporous silica nanoparticles were monitored by zeta potential, dynamic light scattering and transmission electron microscopy measurements. Following the functionalization with anti-EGFR-antibody, which was controlled by FACS, mesoporous silica nanoparticles are tailored for use in humanized, tumor-bearing NOD/ScidtgHLA-A2⁺ mice.
Encapsulated TNF-α showed a 97% reduced toxicity compared to free TNF-α after 12h of treatment. TEM, DLS and zeta potential demonstrated stable and functional particles under normal culture conditions. FACS data demonstrated successful anti-EGFR conjugation to the particles PEI-PEG copolymer. Particle distribution in 3D tumor spheroids was studied by transducing C8161 cells with FUCCI (fluorescence ubiquitination cell cycle indicator) and growing as 3D tumor spheroids. Within 24h of treatment the silica particles were found throughout the spheroids. Whereas silica nanoparticles did not appear influence the cell cycle, TNF-α loaded particles induced G1 arrest before causing cell death. Our results allow further investigation using anti-EGFR functionalized DMSN to limit systemic toxic TNF-α effects in vivo.

Porous materials and capsules are interesting nano/micro system able to entrap desired molecules and act as delivery or imaging species. They can be created using soft species such as gels or polymers or inorganic precursor to obtain microporous and mesoporous silica based nanoparticles. In this talk I will focus on silica-based materials, and discuss how we can overcome the problem related to the fate of inorganic nanocontainers in in vitro and in vivo applications. Indeed the issue related to the use of materials for therapy and imaging in living organism, is their accumulation in vital organs that often prevent their use.
Recently, a new generation of breakable hybrid nanoparticles (NPs), able to response and degrade upon external stimuli (e.g. reductive agents, pH, etc.), have been developed in our group^1,2. The insertion of responsive linkers in the framework of these particles, results not only in the destruction and safe excretion of the nanoparticles from the cells, but also in a faster and better delivery of the payloads (see scheme 1). Moreover, to expand the breakability properties of this material for other purpose, the possibility to entrap proteins into a breakable silica shell has also been realized in our laboratory³. It has been shown that the activity of different proteins (Cytochrome C, EGFP, TRAIL Apo2 and onconase) remains intact after their delivery into cancer cells.

References.
1. Maggini.L et al, Nanoscale, 2016 , 7240-7247
2. Maggini.L et al, Chem. Eu. J., 2016, 22, 3697-3703
3. Prasetyanto, E.A. et al, Angew. Chem. Int. Ed. 2016, 3323–3327

Currently, cancer screening campaigns are widespread and allow early lesion detection. These small tumors with weak aggressive potential in the short term do not justify heavy treatment such as surgery or chemotherapy, and prompt the consideration of minimally invasive treatments. Recently, the development of nanosciences in the fight against cancer has been the object of tremendous efforts. In this context, the development of biodegradable and biocompatible nanotools could be of particular interest to achieve the combination of targeting, imaging, drug delivery and photodynamic therapy of cancers. Based on our recent results obtained through multidisciplinary and international collaborations we tried to develop a breakthrough technology in the field of nanomedicine. Membrane receptors overexpressed by cancer cells were identified and mesoporous silica nanoparticles carrying active molecules for imaging and therapy were developed and grafted with ligands specific of these receptors. This work at the interface between fundamental and applied research could lead to the design of effective and biocompatible prototypes for personalized medicine.

Mesoporous silica nanoparticles (MSNs) have attracted much attention in recent years due to their growing impact on various fields such as catalysis, storage and separation, and biomedical applications including bioimaging and biosensing, diagnostics and theranostics, bone and tissue engineering, and drug delivery. Their well-defined periodic pore system and their enormous flexibility regarding functionalization enable them to accommodate a variety of different guests. Moreover, using specific functionalization and design allows for controlled and targeted drug delivery from silica nanocarriers to specific target sites, such as cancer cells. However, there is still a great demand for spatial and temporal control of the release via external, non-invasive methods of actuation.

A particularly interesting concept of external actuation is the localized generation of heat at the intended target site that can trigger cargo release from suitably capped mesoporous silica nanoparticles. Examples of such a localized heating include high intensity focused ultrasound (HIFU) or superparamagnetic heating of iron oxide core – mesoporous silica shell nanoparticles in alternating magnetic fields (AMF). HIFU causes local heating due to focused sound waves, and superparamagnetic heating in AMFs causes a local increase in temperature of the nanoparticles that can be much higher than that in the bulk solution. Both modes of actuation feature good tissue penetration and are non-invasive and biocompatible. In this contribution, we will discuss how we can use these concepts of localized heating to design mesoporous silica nanoparticles that release their payload upon external actuation, thus giving spatio-temporal control over the triggered release process.

Enzyme-based biosensors have been of intense investigation and field-effect transistor (FET)-based sensors are one of the widely produced, miniaturized silicon-based semiconductor devices used to quantify ion concentrations in the analyte solution. Overall, current research has focused on enhancement of the sensitivity, detection limit, selectivity and the storage stability of these electrochemical biosensors. For this purpose, variety of modification methods on electrochemical biosensor surfaces is proposed. At present, use of new nanosized materials in biosensor design is a promising approach to improve analytical characteristics of the devices. Zeolites are perspective nanomaterials for biosensor modification. They are inorganic compounds, the structure of which is a crystal lattice consisting of alumina and silica tetrahedra bound by oxygen atoms. This framework forms a lot of pores and channels which considerably increases the zeolite surface. In the current study, a review on precise control over structural and chemical properties of zeolites and their consequent effect on electro-chemical biosensors will be given. All results suggest that the methodology of surface immobilization and zeolitic properties such as Si/Al ratio, surface roughness, particle size, hydrophilicity and the ability to create gold nanoparticles within the nanopores effect and ultimately enhance the biosensor sensitivity and stability. Considering the important biological role of urea as a diagnostic indicator of kidney failure and major uremic toxin, its determination was needed in medical diagnostics. It was shown that distinction of healthy people from people with renal dysfunction became easier by zeolite modified biosensors. Our results show that the performance of constructed ISFET-type biosensors strongly depends on Si/Al ratio of employed zeolite nanoparticles as well as the type of enzymatic reaction employed. All fabricated biosensors demonstrated high signal reproducibility and stability. The obtained results were used for the development and design for an experimental prototype of novel nanobiosensor located implant.

Effective detection and sensing of surfaces bacterial contamination is demonstrated. We have designed a multifunctional mesoporous silica-gated AuNCs@lys nanocarrier for sensing and killing bacteria. Our system offers a selective damage and sense of the bacterial walls; through the synergistic effect of lysozyme and kanamycin. The fluorescence of AuNCs decreases upon their interaction with bacterial cell wall, which allows us to monitor bacteria-nanoparticle interaction. The enhanced stability of AuNCs@lys (negatively charged) is obtained by immobilizing it on the positively charged silica via electrostatic interactions. The as prepared nanoparticles are embedded on Poly(ethylene oxide)/poly(butylene terephthalate) copolyether ester, (PEO/PBT copolymers) to fabricate the membrane. The absence of membrane fluorescence indicates bacterial contamination, while fluorinated membranes are enough indicators of membrane free bacteria.

Metal-Organic frameworks (MOFs), also known as coordination networks or porous coordination polymers, constitute an exciting new research area that has attracted a large amount of attention in recent years. These materials possess framework flexibility, large surface area, tailor-made framework functionalities, and tunable pore size.
In spite of their great advantages, MOFs generally have low tolerance to humidity. Recent reports have proved that MOFs could be stable in aqueous solutions and at high temperatures however, this requires the synthesis of a new class of MOFs with specific coordinating metals and ligands. Moreover, addition of certain functionalities such as surface charge, hydrophilicity/hydrophobicity etc. without deforming their excellent structure is not straightforward and demands intricate modifications. Thus, in our project we designed a novel method for coating nanoMOFs with polymeric shells by using one pot reversible addition fragmentation chain transfer (RAFT) polymerization. RAFT polymerization is one of the most facile and versatile techniques that tolerate all kinds of functionalities without losing control and livingness of polymerization. We envision that the designed system will have controlled and tailored stability depending on the nature of the polymeric shell used. Moreover, it will be used not only for CO₂ capture but also controlled gas release providing a versatile platform for CO₂ reuse.

One of the hallmarks of inflammatory arthritis (IA) is its variable disease activity with exacerbations (flares) of the chronic inflammatory joint process punctuated by periods of low disease activity. Treatment options are limited and often employ corticosteroids—agents with a plethora of toxic side effects. Use of a long acting intra-articular drug delivery method that titrates release to match disease activity would represent an attractive paradigm shift. We have developed an injectable inflammation responsive self-assembled hydrogel that can release the encapsulated drugs in response to enzymes, including matrix metalloproteinases (MMP-2 & MMP-9) and esterases, which are upregulated in inflammatory arthritis. Herein, we demonstrate the efficacy of this platform using triamcinolone acetonide (TA), a corticosteroid currently used in the clinic for the treatment of IA.

Release studies were performed in vitro to study on-demand delivery of TA in response to i) (MMP-2/ MMP-9/ esterase), ii) enzymes secreted from activated macrophages and iii) synovial fluid (SF) collected from arthritic patient's joint. TA loaded gels were evaluated in vitro for biocompatibility using chondrocytes and synoviocytes, and for anti-inflammatory efficacy using activated human macrophages. Therapeutic efficacy of TA loaded gels was evaluated in vivo using K/BxN serum model of IA developed in C57Bl/6 mice, and was compared to the therapeutic efficacy of Kenalog, the current clinically used formulation of TA.

Gels released TA in response to the enzymes that are expressed within arthritic joints (MMP-2, MMP-9 and esterases) as well as in conditioned medium of activated macrophages (Fig.1A and 1B). Incubation in PBS at 37^oC released moderate amount of drug during a 50-day incubation. To evaluate on-demand delivery, enzymes were added to the release medium on day 25, which triggered the release of TA (Fig.1C). In addition, gel released TA in response to SF collected from arthritic joints, but not when incubated with normal SF (Fig.1D).

TA loaded gels showed excellent biocompatibility with both chondrocytes and synoviocytes at 20 mg/ml concentration of TA, which implies that in vivo administration of these gels will not have any detrimental effects to the surrounding cells. TA as a gel formulation demonstrated improved anti-inflammatory activity in vitro in comparison to free TA, as evident by the reduced TNF-α and increased IL-10 secretions from activated human macrophages (Fig. 2). Finally, an in vivo efficacy study showed reduced clinical scores for animals treated with TA loaded gels (20 mg TA/ml), compared to the scores observed for untreated animals and animals treated with Kenalog (20 mg TA/ml) (Fig. 3).

Overall, our results suggest that an inflammation responsive hydrogel as self-titrating drug delivery system can offer improved therapeutic benefit in inflammatory arthritis.

Cancer remains one of the world’s most devastating diseases, with more than 10 million new cases every year¹. In traditional treatment methods, anti-cancer drug molecules circulate freely in the blood and do not exhibit targeted release and kill the healthy cells besides cancer cells. Because of these reasons and considering the emerging technology, the scientists from many disciplines study intensively for the development of new-generation nanocarrier systems which can do selective and controlled release^2-3. Nanocarriers have many advantages compared to traditional methods. First of all, the drug molecules are trapped into the nanocarriers and the toxicity of drug can be decreased in minimum levels. Thus uncontrolled delivery can be prevented and the drug dose can be adjusted to minimum but effective levels^4-5.
In this study, we have developed a novel ellipsoidal and pH/redox-sensitive hybrid nanocarrier system for triggered and targeted drug delivery applications. These multifunctional hybrid nanoparticles (Fe₃O₄@SiO₂@PLH-PEG/PEG-FA) composed of an ellipsoidal Fe₃O₄ core, a mesoporous silica shell and pH/redox-responsive poly(histidine)-co-poly(ethylene glycol) (PLH-co-PEG) polymer as gatekeeper and PEG-Folic acid (PEG-FA) polymer as the targeted agent to obtain an excellent platform for anticancer drug delivery. In particular, the PLH-co-PEG gatekeeper on the surface of the hybrid nanoparticle play a key role in accommodating anticancer drug molecules in the pore of the silica shell without premature release until crosslinked polymer shell gatekeepers are cleaved by glutathione (GSH). In addition to PEG-FA polymers provide to enhance the targeted cellular uptake of the nanoparticles by cancer cells. The experimental results showed that these smart nanoparticles exhibit fast DOX release profile in the presence of 10 mM GSH, due to the reductive cleavage of intermediate disulfide bonds of PLH-PEG polymer. It can be said that this multifunctional polymer shell is active for the controlling drug molecules in-and-out of silica channels. Moreover, the ellipsoidal smart nanoparticles allowed the perfect release profile under cellular pH environment.
As a result, this study will help the development of new generation nanocarrier systems and the obtained each result from every step will be very precious to reach excellent systems in this field and will provide very big contribute to the literature.

References
1. Stewart, B. W., Kleihues, P. 2003. Genova, 9-11.
2. Drbohlavova, J., Chomoucka, J., Adam V., Ryvolova, M., Eckschlager, T., Hubalek, J., Kizek, R. 2013. Current Drug Metabolism, 14, 547-564.
3. Duncan, R. 2006. Nat. Rev. Cancer, 6, 688–701
4. Mishra, B., Patel, B. B., Tiwari, S. 2010. Nanomed.-Nanotechnol. Biol. Med., 6, 9-24.
5. Peer, D., Karp, J. M., Hong, S., FaroKhzad, O. C., Margalit, R., Langer, R. 2007. Nat. Nanotechnol., 2, 751-760.
Acknowledgement
This work was supported by the TUBITAK Grant No. 115R280.

Microbial spoilage of food has been a constant nuisance and an unavoidable problem throughout history that affects food quality and food safety in a variety of ways. A simple and rapid test of foodborne fungi and bacteria in food and environmental samples is essential for proper management of food spilage and assurance of food quality and safety. A number of different techniques have been developed for detection and enumeration of foodborne pathogens including plate counting, enzyme-linked immunosorbent assay (ELISA), polymer chain reaction (PCR), nucleic acid sensor, electrical and microscopy methods. However, the significant drawbacks of these techniques are high demand of operation skills and the time and cost involved. In this report, we introduce a rapid method for detection of bacteria and fungi in food products using a specific interaction between a reducing agent (tris(2-carboxylethyl)phosphine (TCEP)) and the microbial surface proteins. The chemical reaction was transferred to a transduction system using gold nanoplates-enhanced chemiluminescence. We have optimized our nanoplates synthetic conditions, characterized the chemiluminescence parameters and optimized conditions for the microbial assay. The new detection method was applied for rapid detection of bacteria (E.coli sp. and Lactobacillus sp.) and fungi (Mucor sp.), with limit of detection as low as single digit cells per mL within 10 min using a portable luminometer. We expect our simple and rapid detection method to be a powerful alternative to the conventional plate counting and immunoassay methods for rapid screening of foodborne microorganisms.
Keywords: foodborne pathogens, gold nanoplates, chemiluminescence, reducing agents, luminol

Recent developments in cardiac tissue engineering have produced a range of synthetic or biological polymer based scaffold materials with proper mechanical and biological cues for cardiomyocytes. These scaffold materials promise advancements over traditional cardiac graft materials for treatment of heart damage. Despite these advances, such scaffolds primarily served as passive supports for cardiac tissue. Here we report a silicon nanowire based scaffold with the potential for localized optical stimulation of cellular components. As an optically responsive semiconductor, the silicon nanowires can function as a stimulation platform to direct synchronous beating of cells interfaced with the scaffold. Recently, our lab has demonstrated that silicon nanostructures can provide localized optical control of cellular membrane potential in excitable cells through photothermal or photoelectric stimulations. This functionality of silicon nanostructures was used to pace primary neonatal rat cardiomyocytes seeded on fibronectin coated silicon nanowire scaffolds. Immunofluorescent staining with connexin 43 and cardiac troponin I antibodies, verified that cells grown on the nanowire scaffold were viable, aligned, and were electrically connected to neighboring cells via gap junctions. These findings demonstrate a significant advancement towards a novel platform to optimize therapeutic strategies for patients who suffer from cardiac arrhythmias as a result of cardiac tissue damage.

Interfacing electronic devices with biological systems for clinical therapeutics has been of interest for many decades now. Traditionally, many of these devices are targeted at excitable cell systems. Here, we use silicon nanowire (SiNW) technology to study the role of membrane voltage in T cell activation. Previous work in the Tian lab has demonstrated that coaxial pin SiNWs can photoelectrically elicit action potentials in neurons. More specifically, we use coaxial pin SiNWs to photoelectrically depolarize Jurkat T cell membranes. This approach offers advantages over other optical approaches for the manipulation of cellular behavior such as optogenetics, which requires gene transfection of light-activatable ion channels into the target cell. In this study we demonstrate that upon green laser stimulation of the T cell-SiNW interface for various timepoints, we can depolarize T cell membranes. We also visualize the material-T cell interface using microscopy and show that T cell viability is unaffected by the presence of the SiNWs. Using chemical surface functionalization approaches for SiNWs with antibodies specific for T cell co-receptors, we improve the binding interface between the cells and SiNWs and assess the stimulation efficiency upon the creation of this enhanced binding interface. Our work will provide us with a platform to further study how T cell membrane depolarization can alter T cell signaling upon stimulation through the T cell receptor and more broadly will serve as an important foundation for improving treatments for patients with autoimmune diseases.

In this study, we have developed an efficient cancer-targeted nanoplatform based on Fe₃O₄/GQDs nanoparticles, which was functionalized with a redox sensitive cross-linker that activates in a reducing intracellular environment ensuring the drug can be released upon cellular uptake. To endow this nanoplatform with improved specificity, the composite surface was further modified with human transferrin protein, and doxorubicin hydrochloride (DOX) providing a dual-targeted capability to deliver the drug into the cells and stimulate the generation of reactive oxygen species therein. The cell viability was measured via MTS assay and flow cytometry, and the imaging and internalization through confocal microscopy. The singlet oxygen quantum yield was also quantified. Moreover, our findings indicate that the multifunctional nanocarrier shows an excellent drug loading ability with an enhanced efficiency for photodynamic therapy and improved specificity ascribed to the disulfide bond breakage upon cell entry. This piece of research represents a step forward to designing novel alternatives for treating cancer and describes an innovative protocol for theranostics.

Nanocomposites based on iron oxide (IO) and graphene (G) have recently emerged as novel materials for diverse applications, such as energy storage and biomedicine. Specifically, in biomedicine, the optimization of the loading amount of iron oxide nanoparticles on graphene that endows with the desired properties, has not fully investigated yet. Here, we report the optimization of loading ratio of iron oxide nanoparticles to graphene from IO:GO=1:3 to 4:3, in order to tune its magnetic as well as biological properties assessing its ability as novel MRI T₂-contrast agents. Our findings indicate that the T₂ relaxivity is increased by ~ 50% as the ratio of iron oxide nanoparticles is increased by 1, being ~183 mM^-1s^-1 the highest relaxivity recorded for IO:GO=4:3. This is attributed to the higher amount of IO content, which is responsible for producing higher susceptibility gradient in aqueous environment. The X-ray diffraction reveals that the peaks relevant to GO are attenuated as the concentration of IO increases in GO, evidencing the distortion of graphene oxide layers. The colloidal stability of this hybrid material is confirmed through dynamic light scattering (DLS) and zeta potential. The in vitro MTS assays show that cytotoxicity on HeLa, Human T lymphoblast, A549 and HepG2 cells increases for the sample of higher IO:GO ratio. The phantom imaging measurements and in vivo MRI tests will be also discussed.

Bridged silsesquioxane (BS) nanomaterials with chemical structures O_1.5Si-R-SiO_1.5 where R organic groups are emerging as the next generation of organosilica nanocomposites.¹ Physical and chemical properties of BS materials can be governed by the nature of homogenously distributed organic fragments within the siloxane network.² Nonetheless, due to the synthetic challenge to control the kinetic in sol-gel processes, most non-porous BS materials that have been extensively studied in the past two decades were macroscaled.³ Ideally, for biomedical purposes BS NPs should be non-aggregated sub-200 nm nanomaterials to benefit the enhanced permeation and retention (EPR) effect and, thus, accumulate in cancerous tissues and organs. Nature-inspired oxamide bridged silsesquioxane was used as a key component to endow nanoparticles with degradable feature. For all experiments sol-gel process was applied.
The designed nanomaterials were non-aggregated with biologically relevant sizes (sub-200 nm) for preferential accumulation in tumors. The unique constitution of the materials with a very high organic content (~50%) was found to be homogenously distributed within individual particle and confirmed by various techniques: FTIR, solid state NMR and STEM-EELS elemental mapping. According to nitrogen-sorption measurements with the BET (Brunauer-Emmett-Teller) theory obtained BS NPs are nonporous with 25 m²/g surface area. The biodegradation of NPs was demonstrated in the presence of the trypsin enzymes in simulated biological media. Furthermore, nonporous fluorescent BS NPs were obtained via incorporation of fluorescein isothiocyanate moieties inside the siloxane framework in order to image cancer cells. In-vivo studies show that concentration of silicon reaches maximum in mice urine on the second day after injection of BS NPs and goes to zero on the fifth day (according ICP-MS).
We describe the first example of enzymatically degradable bridged silsesquioxanes based on oxamide bridges. The oxamide functions were incorporated via a novel bridged alkoxysilane designed to mimic the enzymatic cleavage in the human metabolism. These novel hybrid organosilica NPs can find significant interest as future biomedical applications of inorganic silica NPs require higher biodegradability.

References

[1] Chen, Y.; Meng, Q.; Wu, M.; Wang, S.; Xu, P.; Chen, H.; Li, Y.; Zhang, L.; Wang, L.; Shi, J. J. Am. Chem. Soc. 2014, 136 (46), 16326-16334.
[2] Hu, L.-C.; Shea, K. J. Chem. Soc. Rev. 2011, 40 (2), 688-695.
[3] Creff, G.; Pichon, B. P.; Blanc, C.; Maurin, D.; Sauvajol, J. L.; Carcel, C.; Moreau, J. J.; Roy, P.; Bartlett, J. R.; Man, M. W.; Bantignies, J. L. Langmuir 2013, 29 (18), 5581-5588.

Antimicrobial photodynamic therapy (aPDT) is increasingly being explored for treatment of periodontitis. Here, we investigated the effect of aPDT on human dental plaque bacteria in suspensions and biofilms in vitro using methylene blue (MB)-loaded poly(lactic-co-glycolic) (PLGA) nanoparticles (MB-NP) and red light at 660 nm. The effect of MB-NP-based aPDT was also evaluated in a clinical pilot study with 10 adult human subjects with chronic periodontitis. Dental plaque samples from human subjects were exposed to aPDT - in planktonic and biofilm phase - with MB or MB-NP (25 µg/mL) at 20 J/cm² in vitro. Patients were treated either with ultrasonic scaling and scaling and root planing (SRP) or ultrasonic scaling + SRP + aPDT with MB-NP (25 µg/mL and 20 J/cm²) in a split-mouth design. In biofilms, MB-NP eliminated approximately 25% more bacteria than free MB. The clinical study demonstrated the safety of aPDT. Both groups showed similar improvements of clinical parameters 1 month following treatments. However, at 3 months ultrasonic SRP + aPDT showed a greater effect (28.82%) on gingival bleeding index (GBI) compared to ultrasonic SRP. The utilization of PLGA nanoparticles encapsulated with MB may be a promising adjunct in antimicrobial periodontal treatment.

Delivering microRNA is a conceivable new treatment for many diseases, however, in vivo extremely difficult. Therefore a delivery system, like microcapsules, is required. Microcapsules are containers, of less than 5μm in size, created by layer-by-layer formation. This process allows for precise control of the protective polymer shell and for the imbuing of targeting functionalities.

Many bioactive molecules can be functionally delivered intracellularly using microcapsules and have proven to be suitable vectors in vivo. This project aims to functionally deliver microRNA (miRNA) using magnetically-targeted microcapsules in vitro, which has not been previously demonstrated.

In many diseases, namely cancer, changes in miRNA levels results in detrimental changes in multiple downstream protein targets, this then leading to disease progression. Therefore, the rationale behind this therapeutic is to normalise these deficiencies by delivering mimics of the insufficient miRNA or miRNA antagonist to obstruct a surplus of miRNA.

This work focuses on miR-19a mimics and miR-19a antagonists being delivered using degradable magnetic microcapsules to colorectal cancer cells. miR-19a is responsible for mediators of cancer progressions. Interestingly, efficient delivery of miR-19a antagonists is challenging even in vitro with traditional techniques.

Encapsulation of miR-19a in magnetic microcapsules has been a success. Also Confocal Fluorescent Microscope images showing cell internalisation and consequent release of miRNA strongly prove that microcapsules are delivering miRNA. These images also confirmed effective targeting using a magnet. New data showing effectiveness of the miRNA-microcapsules in influencing downstream protein expression will be presented. Following successful manipulation of protein expression, miRNA-microcapsules will be tested in an in vivo model.

Multi-drug resistance (MDR) in stem-like cancer cells, also known as cancer stem cells, is emerging as a reason for failed treatment and cancer relapse. This work was conducted under the hypothesis that a one-pot synthesis could prepare targeted, biodegradable, dual-drug loaded nanoparticles, for intracellular co-delivery of a highly effective anticancer drug and a drug that inhibit the MDR-pathways. The commonly used and effective anti-cancer drug cisplatin (CDDP) and the Chinese herbal extract demethoxycurcumin (DMC) were loaded in biodegradable carboxymethyl-hexanoyl chitosan (CHC) nanoparticles targeted for CD133 antigen, which is often overexpressed on the membrane of stem-like cancer cells. The system was evaluated in vitro using stem-like A549-ON lung cancer cells with excellent results. Co-delivery of the drugs using the CHC nanoparticles achieved greatly enhanced efficacy at lowered drug concentration compared to free dual-drugs or individual drugs in nanoparticles, the targeting improved the efficacy even further. This significant result highlighted that CHC successfully synergized the effects of DMC and CDDP. The ease of preparation, biodegradation and biocompatibility of the CHC and the excellent in vitro results against stem-like cancer cells makes the system highly promising and realistic as a nanomedical approach to overcome MDR in cancer treatment. However, further demonstration of performance in vivo is needed and will be the next step towards clinical realization of the concept.

Controlled self-aseembly of anisotropic nanostructures has made great advances in nanomaterials and nanophotonics. An anisotropic inorganic-organic composite nanostructure with multi-compartments has different functionalities and physicochemical characteristics at each compartment, and their directional assembly formed via covalent- or noncovalent interactions induces unique optical properties, which can be applied to a variety of photonics-based biosensing applications. Especially, surface-enhanced Raman scattering (SERS) has been of great importance for biochemical detection because it has ultra-high sensitivity, narrow bandwidth as well as significant multiplexing capabilities. In this study, we report that anisotropic bimetal-polymer composite nanostructures were formed via oxidation-reduction, and were directionally clustered into forming superparticular structures composed of bimetal core and polymer shell via noncovalent interaction. The aniline monomers were surface-templated polymerized into poly(aniline) by reducing silver nitrate in the presence of a surfactant so that poly(aniline) and reduced silver were eccentrically deposited onto a variety of Au seeds with different physical dimensions, providing both bimetallic Au core - Ag shell compartment and poly(aniline) counter-compartment of the anisotropic composite nanostructures. Moreover, a variety of anistropic bimetal-polymer nanostructures were prepared by introducing various ligands or polymers onto metallic seeds, and they were directionally clustered via noncovalent interaction by selective surface modification of the bimetallic compartments, exhibiting significant enhancement of optical properties due to increased electromagnetic field in the interstices between them. As a proof of concept that these anisotropic bimetal-polymer composite nanostructures would be potential SERS nanoprobes for biosensing, we demonstrated the formation of sandwich-type immunocomplexes composed of SERS nanoprobes and magnetic beads in the presence of target proteins, and showed SERS-based quantitative analysis with a linear correlation between relative Raman intensity and concentration of the target proteins. Conclusively, these anisotropic composite nanostructures and directional clusters would be promising as advanced functional nanoprobes for SERS-based biosensing applications.

Silver nanoparticles are known to have antibacterial property. However, these nanoparticles are unstable and tend to form aggregates. To improve the stability of nanoparticles in aqueous medium and to apply the nanoplates as an anti-bacterial material, we developed triangular and plate-shaped silver nanoparticles (silver nanoplates) coated with gold atoms and then evaluated their dispersion stabilities and antibacterial activities on some pathogenic bacteria including Salmonella enterica serovar Typhimurium.
We evaluated antibacterial activities of silver nanoplates without gold coat (Ag NPLs) and those coated with one to four layers of gold atoms (Ag@Au1L NPLs, Ag@Au2L NPLs, Ag@Au4L NPLs) by colony-forming unit assay on LB agar plate. Ag NPLs and Ag@Au1L NPLs showed antibacterial activities. Surprisingly, addition of fetal bovine serum to LB medium enhanced the antibacterial activities. Furthermore, Ag@Au1L NPLs showed the strongest activity in this condition. We hypothesized that some components of the serum adsorbed on surface of the nanoplates and contributed to its stability in LB medium. Furthermore, concerning stability in LB medium, we examined changes in absorption spectrum, distribution of particle size and zeta potential of the NPLs in LB medium with or without serum. In the case of Ag@Au1L NPLs, there would be some defects on gold layer, and the silver atoms would be released as silver ions from them. Meanwhile, for Ag@Au2L NPLs and Ag@Au4L NPLs, silver ions would not release from inside because of thick gold layer.

Loaclized and recurring delivery of drugs is of great importance in many medical conditions such as in the treatment of solid tumors, localized infections and post-surgical wounds. Triggerable drug delivery platforms are often created from materials that undergo physical or chemical change in their structure when exposed to certain environmental stimuli such as light, temperature, pH, electric/magentic fields and enzymes etc. Among them, light stands out as potential candidate due to its non-invasiveness, high temporal resolution and ability to be controlled remotely. However, light-controlled systems have inherent problem: UV and Vis wavelength is required to initiate the photoreaction. Both of these lights are unable to penentrate beyond few millimeters in the body. An alternative is the use of near infrared (NIR) light since biological tissues are transparent to NIR wavelenghths. However, photoactive materials do not respond to this light. Lanthanide-doped upconverting nanoparticles (UCNPs) have emerged as excellent transducers for converting longer wavelength near-infrared (NIR) light to shorter wavelengths spanning the UV to the Vis regions of the spectrum via a multiphoton absorption process, known as upconversion. Here, we report the development of NIR to UV–Vis–NIR UCNPs consisting of LiYF4:Yb3+/Tm3+@SiO2 individually coated with a 10 ± 2 nm layer of chitosan (CH) hydrogel cross-linked with a photocleavable cross-linker (PhL). We encapsulated fluorescent-bovine serum albumin (FITC-BSA) inside the gel. Under 980 nm excitation, the upconverted UV emission cleaves the PhL cross-links and instantaneously liberates the FITC-BSA under 2 cm thick tissue. The release is immediately arrested if the excitation source is switched off. The upconverted NIR light allows for the tracking of particles under the tissue. Nucleus pulposus (NP) cells cultured with UCNPs are viable both in the presence and in the absence of laser irradiation. Controlled drug delivery of large biomolecules and deep tissue imaging make this system an excellent theranostic platform for tissue engineering, biomapping, and cellular imaging applications.

Three different size of cyclodextrin (α, β, and γCD) is used in the synthesis of cyclodextrin-graphite oxide-carbon nanotube (CD-GO-CNT) composites and compared for the electrochemical supramolecular recognition. All composites show very good electrochemical response to three biomolecules (dopamine, ascorbic acid, and thioridazine), and the response of the electrical current is linearly proportional to the concentration of the biomolecules. Bigger size of CD composite is more sensitive, resulting that αCD-, βCD-, and γCD-GO-CNT composites are in the order of the increasing sensitivity to the response. γCD-GO-CNT composite, therefore, gives the best electrochemical supramolecular recognition performance among the other composites because of the biggest cavity of γCD (0.95 nm of diameter) compared to αCD (0.57 nm of diameter) and βCD (0.78 nm of diameter), and detail results will be discussed.

Disinfection and implementation of new effective methods for sanitary practice are critical steps for health care. Metal oxides have been studied to overcome the limitations of current antimicrobial treatment like antibiotic resistance. In general, they have an antimicrobial activity based on the release of their ions and the induction of oxidative stresses via the generation of reactive oxygen species (ROS). Their antimicrobial activities are also associated with sorption potential to micro-organisms which may be related to the species-based sensitivity of metal oxides. In this study, we screened the metal oxides-based nanoparticles to have an enhanced antimicrobial effect on Escherichia coli (E. coli) and bacteriophage (phage). Zinc oxide-, magnesium oxide-, and copper oxide-based nanoparticles were used to evaluate their antimicrobial activities. First, their size, surface area and crystallography were monitored using a light scattering, BET analysis and x-ray diffraction. Morphology of metal oxide-based nanoparticles was determined using a scanning electron microscopy. For their antimicrobial activity tests, nanoparticles were added to culture medium and their dose-responses were characterized. Their combinations of nanoparticles were also tested, and measured the microbial activity against E.coli and phage. Nanoscale sized metal oxides with a high sorption capacity were confirmed. Zinc oxide-, magnesium oxide-, and copper oxide-based nanoparticles showed cytotoxicity on E. coli and phage in a dose-dependent manner. Combination of metal oxide-based nanoparticles also showed a strong antimicrobial effect. Our results demonstrate that metal oxide-based nanoparticles have a potential to be extensively used as antimicrobial agents. We anticipate that metal oxide-based nanoparticles can be applied for various disinfection steps in pharmaceutical industry and water purification systems.

Accelerating developments in the science and technologies afford modern civilization, however, simultaneously producing many kinds of material that cannot be degraded in natural environment. Development of evaluation methods and systems of the safety of nano-materials are urgently needed. However, it is extremely hard to confirm toxicity of the each nano-materials with different shapes and chemical structures.
We pay attention to primitive animal planarians, because they have tremendous vital power. For example, if they were cut down to small pieces, it can be regenerated from the all pieces. This distinguishing characteristic is based on pluripotent stem cell. In addition, planarian has primitive brain and regenerative capability. Therefore, they have very similar biological functions with higher animals including a human.
In order to discuss effects of toxicity of semiconductor materials to planarians, we produced foods that were combined organic semiconductor materials such as methyl ammonium lead- halide which used for solar cells. For preparing of the foods, the concentration ratio of the semiconductor material were changed. And then we gave the three different concentration foods to 20 planarians for one month, after that morphological observation was perform.
The results obtained from morphological observation, we found that the number of eyes of some individuals has changed. Generally, lead is known to affect the biological nervous system. However, whether the influence of the semiconductor material, it is necessary to further detailed examination.

Optode-based nanosensors have been widely implemented as tools for visualizing and monitoring of targeted ions and biomolecules. Here, we describe an improved optical ion sensor called calcium polymer-free nanosensor (CaPFN) for detection of free and elevated nanomolar calcium levels in living cells. The CaPFN is comprised of a plasticized matrix embedded with optode sensing chemistry and fluorescent dyes to selectively image calcium ion concentrations. Ratiometric fluorescent measurements show that CaPFNs are selective for calcium ions over magnesium by 4 orders of magnitude, exhibit a fully reversible response, and respond to a nanomolar dynamic sensing range both in solution and in the intracellular environment. For detection of intracellular calcium transients, nanosensors are delivered into the cell cytoplasm and their responsiveness to drug-evoked calcium store release are demonstrated by live-cell confocal microscopy. Overall, the newly optimized CaPFNs offer a new tool for optical determination of intracellular calcium, and demonstrate a new platform for detection of other target ions such as sodium and chloride, owing to its modular design and ease of preparation.

Endoscopes have been used to detect polys, bleeding and other abnormalities in the gastrointestinal tract. The endoscopes contain imaging tools and therapeutic devices together for effective cancer treatment. However, current endoscope system has a limitation in small cancers detection and long treatment time. Here, we developed a multifunctional endoscope system that consists of transparent bio-electronics and theranostic nanoparticles to overcome these problems. Bio-electronics can sense the change of temperature, pH, and impedance for detecting the colon cancer. Bio-electronics enables radio frequency ablation for treating the large-size cancer lesions. Theranostic nanoparticles can do fluorescence imaging and photo-triggered therapy. In-vitro, ex-vivo, and in-vivo experiments show that the endoscope system can be the closed-loop solution for colon cancer treatment.

Gloves made of materials such as latex, nitrile, and polyethylene are the most common types of protective equipment used to prevent cross-contamination and transmission of pathogenic bacteria in the food industry. In this study, we report a surface modification approach involving “fluorinated silica nanoparticles” (FSNs) to improve the protective ability against bacterial contamination of disposable glove surfaces. The bacterial antiadhesive (antifouling) properties of the modified gloves were evaluated with Salmonella Typhimurium LT2 and Staphylococcus aureus at bacterial concentrations of 8.6-9.0 log CFU/mL through the dip-inoculation approach. Bacterial attachment to glove surfaces were enumerated by the pour plating method as well as direct counting via scanning electron microscopy. The bacterial populations of S. Typhimurium LT2 and S. aureus on FSN-coated latex, nitrile and polyethylene gloves was reduced by 1-2 log units in comparison to bare gloves, which already reduce the bacterial attachment to some extent.

Nanoscale semiconductors are an important class of materials for use as biosensors, drug delivery carriers, and engineered tissue scaffolds, with silicon nanowires (SiNWs) being of particular interest because of their potential cytocompatibility, and their ability to be rationally designed, providing precise control over the devices structural and material properties. These unique characteristics make them an ideal material for designing a wide range of nanoscopic 'building blocks' which can be applied in a biological context. As an example of this, we recently demonstrated how kinked SiNWs can be used as both intra- and intercellular force probes for studying cell behavior. Despite these advances, there is still much to be understood about the bio-interface of these materials. For instance, a careful examination is still needed of how these large inorganic nanoconstructs can be internalized. Here we demonstrate that label free SiNWs can be internalized in multiple cell lines (~96% uptake rate), and that once internalized they undergo an active 'burst-like' transport process. Our results suggest that rather than through exogenous manipulation, SiNWs are internalized primarily through an endogenous phagocytosis pathway. To study this behavior we have developed a robust set of methodologies for quantitatively examining high-aspect ratio nanowire-cell interactions in a time-dependent manner, on both the single cell and ensemble levels. This approach represents one of the first dynamic studies of semiconductor nanowire internalization and offers valuable insight into designing devices for bio-molecule delivery, intracellular sensing and photoresponsive therapies.

We developed coaxial electrospun core-shell fibers of polycaproactone (PCL) in the inner core and PCL with zinc oxide (ZnO) nanoparticles (NPs) in the outer shell. Fibers were fabricated as an alternative antibacterial dressing to prevent infections during the treatment of dermal injuries. Antibacterial studies of inorganic NPs have become important due to the increased bacterial resistance against antibiotics. We used ZnO NPs (~25nm), which are chemically stable and possess an excellent photocatalytic property. ZnO NPs are low-cost material, listed as a “generally recognized as safe” (GRAS) by Food and Drug Administration (FDA). ZnO NPs have shown antibacterial activity (ABA) against a broad spectrum of bacterial strains attributed to two mechanisms: 1) Photocatalysis; i.e ZnO NPs in aqueous solution under UVA radiation produce reactive oxygen species (ROS) which penetrate the cell membrane; 2) Release of Zn⁺² ions, which in high concentration adhere to cell membranes. Both mechanisms causing bacterial death and occur on NPs surface. On the other hand, the application of inorganic NPs in medical treatment is limited because of the possible long-term side effects that might be caused by NPs release. In this study, ZnO NPs were dispersed into PCL electrospun fibers to prevent its release. The antibacterial effect of the ZnO NPs/PCL fibers have already been reported (1); however, the ZnO NPs were dispersed across the whole fibers and thus NPs in the center of the fiber did not participate in the ABA. In order to optimize the use of ZnO NPs concentration in PCL fibers, we developed core-shell coaxial electrospun fibers where the core of the fibers corresponded to PCL and the shell to mixture of PCL with ZnO NPs. In this way, ZnO NPs were only dispersed on the surface of the fibers increasing its use efficiency by means of superficial contact area. We evaluated the ABA against Escherichia coli at different concentrations of ZnO NPs embedded into uniaxial and coaxial PCL electrospun fibers under two different pre-illumination conditions: UVA light and sunlight. Preliminary results suggest that the ABA increases when ZnO NPs are only dispersed on the outer shell of coaxial fibers, indicating the potential to enhanced the ABA in coaxial fibers using the same amount of NPs than in uniaxial fibers. However, there were not significant differences in ABA between fibers under the UVA light and sunlight pre-illumination conditions. All fibers were characterized in their morphology, chemical composition, water wettability and crystalline structure by means of scanning electron microscopy, infrared spectroscopy, energy dispersive X-ray spectroscopy, X-ray photoelectron spectroscopy, X-ray diffraction and water contact angle. The coaxial electrospun fibers of ZnO NPs/PCL have a potential to be an antibacterial dressing.
References.
(1). R. Augustine, H. Nanda, D. K. Singhal, A. Mukherjee, D. Malakar, N. Kalarikkal, S. Thomas. J. Polym. Res, (2014), 21:347.

The specific targeting of diseased sites with therapeutic agents is considered as a key problem in medicine. A delivery ratio of only 0.01 to 0.1‰ of an injected drug molecule is estimated. The process of specific targeting can be separated into different stages, which demand different properties from the applied therapeutic system. Multi-stage delivery particles are promising candidates to fulfill these requirements. They consist of a micrometer sized carrier particle loaded with nanometer sized cargo. In this work, advanced multi-stage delivery particles are produced via scalable flame spray pyrolysis and spray drying. Heat responsive polymer microparticles are loaded with highly superparamagnetic Zn_0.4Fe_2.6O₄ nanoparticles and a therapeutic drug. During magnetic hyperthermia with the nano Zn-ferrites the temperature sensitive carriers can release the drug on demand. This effect was evaluated thoroughly and compared to literature. Furthermore, by optimizing shape and size of the system the targeting efficiency was evaluated ex vivo in a flow cell, showing the enormous potential of the produced particle system.

Availability of safe drinking water is of paramount importance. For this purpose, activated carbon (AC) bed is widely used as a water purification medium, which although removes most of the contaminants, however is incapable of killing pathogens. To resolve this issue, antibacterial property of copper nanoparticles (Cu-NPs) have been utilized. Therefore, the present work explores the potential of combining Cu and AC to demonstrate the application of Cu-NP impregnated AC for water disinfection in a continuous, flow-column set-up.

To this end, Cu-NPs with a mean diameter of 6.11 nm were synthesized successfully using a green synthesis method and it was successfully impregnated in plasma treated AC with 2.1 wt. % of Cu in the Cu-AC. In batch mode experiments, on using 8 mg/ml of Cu-AC, an initial E. coli cell concentration of 10⁴ CFU/ml was reduced to zero cells within 10 minutes. The experimental data fits well with an exponential decay curve of cell concentration, with a rate constant k = 0.757 min^-1. From this batch data, the maximum flow rate that could be maintained in a column (with 8 cm diameter and 25 cm height) for complete decontamination of water was calculated. A continuous flow-column (packed with only Cu-AC) showed zero cell count in the outlet water, when a contact time of 9 minutes (between E. coli and Cu-NP) was provided at a flow rate of 4.13 L/h, thereby successfully treating almost 694 L of contaminated water over an uninterrupted 7 day period. Simultaneously, in steady state continuous flow-column experiments, the maximum copper concentration in the outlet water was only about 83.4 µg/L, which is far below the upper recommended limit of 1000 µg/L (EPA).

Hence, water disinfection for potable quality water (zero E. coli count and < 1000 µg/L Cu) can be achieved in a continuous manner, with our packed Cu-AC column, with most of the Cu left intact (99.2 wt.% of total Cu loaded) in the Cu-AC hybrid for its continued, unabated performance over a long duration.

Gold nanorods are virus-sized objects that exhibiti brilliiant colors in the visible and near-infrared portions of the electromagnetic spectrum. The aspect ratio of the rods governs their maximum wavelengths of absorption and scattering. The surface chemistry of these nanorods greatly influences how biomolecules and even cells respond to these materials. In this talk I will describe recent results from our lab that involve molecular-level understanding of the nano-bio interface, as well as results that show that these nanorods can influence cell behavior.

Multifunctional nanoparticles have been widely investigated for biomedical applications, such as imaging, therapy, and drug delivery. Especially, photo-activated nanoparticles have received great attention as theranostic agents because of their heat-generating abilities after exposure to laser irradiation. However, photostability and safety issues have been the technical hurdles for further clinical applications. In this regard, carbon nandots (Cdots), a class of paracrystalline carbon nanostructures, have been the subject of extensive research because of their excellent biocompatibility, innocuousness, and photostability. They are known to have a structure that some sp² carbon clusters are embedded in a heterogeneous sp³ carbon matrix, presumably due to relatively “cold” reaction temperature, definitely insufficient to give a pure sp² carbon structure or carbon crystal. Here, we designed nitrogen (N)-doped Cdots (N-Cdots), which have strong absorption in the near-infrared region, high photostability, and excellent biodegradability. Optimized N-Cdots can be utilized not only as a new photoacoustic (PA) imaging agent but also as a superior photothermal therapy (PTT) agent in vivo because of their strong optical absorption at a specific wavelength. We used N-Cdots to perform in vivo/ex vivo noninvasive PA imaging of sentinel lymph nodes via local delivery and performed PTT for cancer ablation therapy. Finally, biodegradation and renal clearance were confirmed by performing whole-body PA monitoring and a degradation test.

Theranostics, which are nanoparticles (NPs) capable of cancer detection and treatment, have attracted much attention in recent years. In this investigation, a new type of theranostics based on multifunctional NPs was designed using a combination of different nanostructured materials and then fabricated. Their characteristics and performance were subsequently studied. Compared to their spherical counterparts, gold nanorods (AuNRs) are promising agent for photothermal therapy. Using the capping surfactant CTAB present on the AuNR surface as the template for mesoporous silica formation, a mesoporous silica coating can be made on AuNRs so as to improve the functionality and biocompatibility of AuNRs. Surface enhanced Raman scattering (SERS), which involves strong enhancement of Raman scattering from molecules adsorbed on nanostructured metal surface, can be used as a highly sensitive method for cancer detection. Therefore, based on our previous studies, we developed a facile method to fabricate Au capped AuNR core-mesoporous silica shell (AuNR@mSiO₂@Au) NPs, which are expected to not only provide sensitive SERS detection but also combine cancer targeting, imaging and drug delivery and photothermal therapy in a single system. Specifically, a modified seed-mediated method using binary surfactants was employed for AuNR synthesis. AuNRs were then covered by a mesoporous silica shell in a sol-gel and template removal process. Fine Au nanospheres were deposited on the outer surface of AuNR@mSiO₂ NPs via electrostatic absorption. The Au nanospheres formed copious hot spots for AuNR@mSiO₂@Au NPs for achieving strong SERS signals from Raman reporter molecules (rhodamine 6G) incorporated in the NPs. Furthermore, folic acid was conjugated onto AuNR@mSiO₂@Au NPs, allowing them to target folate receptor-overexpressed cancer cells. Doxorubicin hydrochloride (DOX) was loaded into mesopores of NPs as a model anticancer drug. Hela cells which overexpress folate receptor were used in in vitro investigations with MCF-7 cells for control studies. The morphology, structure and composition of AuNR@mSiO₂@Au NPs were studied. Drug release from mesoporous channels of AuNR@mSiO₂@Au NPs was investigated. Dark-field and fluorescent microscopies were used to assess targeting and imaging capabilities. In vitro SERS signals with targeted cells were measured using Raman spectroscopy. The combined chemo-photothermal effect by AuNR@mSiO₂@Au NPs was investigated after laser irradiation. The highly reduced cell viability after near-infrared laser irradiation suggested that the multifunctional nanoparticles could provide effective photothermal effect and control the release of loaded anticancer drug, thereby achieving chemo-photothermal therapies.

According to the statistics of the Centres for Disease Control and Prevention in the USA, more that 21 million people are diagnosed with diabetes in the USA, while more than 8 million are expected to be undiagnosed [1]. The situation is even worse in Europe, with 55 million people diagnosed, as claimed by the World Health Organisation [2].
Among the very important parameters for a successful treatment is the regularity of the injection of insulin. Although pumps are available on the market for many years, most people still use needles for the subcutaneous injection of insulin. However, the use of needles is painful for the patient, and may be a problem especially for children.
We report here the realization of a nanocomposite material composed of a hydrogel of biocompatible polymer matrix and double-walled carbon nanotubes (DWCNTs) added for enhancement of both mechanical and electrical properties. This nanocomposite is loaded with a solution of insulin and allows transdermal delivery through the skin by electro-stimulation, aiming at avoiding injections and thus improving the life quality of patients. Passive crossing of the skin is possible only for small molecules so in order to make possible the transdermal delivery of a large molecule such as insulin, the permeability of the skin must be increased. This is possible using electrical stimulation, known as electropermeabilisation. For such an application, a suitable material should be both electrically conducting and having a very large specific surface area (m²/g) in order to both store the insulin and be useful for electropermeabilisation. Carbon nanotubes (CNTs), and especially double-walled CNTs (DWNTs) [3] are an ideal candidate and combine a good electrical conductivity with a very high specific surface area (ca. 1000 m²/g) and good biocompatibility when included in a material or deposited on a surface [4]. Of course, toxicity aspects are largely included in in order to evaluate potential risks.
The preparation of the nanocomposite material as well as our first results of electrostimulated transdermal delivery using an ex vivo mouse skin model will be presented.

References:
[1] http://www.cdc.gov/diabetes/data/statistics/2014statisticsreport.html
[2] http://www.euro.who.int/en/health-topics/noncommunicable-diseases/diabetes/data-and-statistics
[3] E. Flahaut, R. Bacsa, A. Peigney, Ch. Laurent, "Gram-Scale CCVD Synthesis of Double-Walled Carbon Nanotubes", Chem. Commun., (2003), 1442-1443
[4] A. Béduer, F. Seichepine, E. Flahaut, I. Loubinoux, L. Vaysse, Ch. Vieu, "Elucidation of the role of carbon nanotube patterns on the development of cultured neuronal cells", Langmuir, 28, (50), (2012), 17363–17371

Gold nanoparticles (AuNPs) have a great potential in various biomedical applications such as imaging techniques, drug delivery, and gene therapeutic. They are also being explored as agents for radiofrequency ablation (delivering heat) to malignancies due to their stability, low toxicity and easiness of conjugation for targeted delivery. Unlike chemotherapeutic agents, AuNPs have no side effects because they are harmless unless activated inside of the tumor by the energy source, such as a near-infrared (IR) light or radiofrequency (RF) waves. Currently several types of IR-activated AuNPs are being tested in human clinical trials for the treatment of cancer. There are no clinical studies on RF-activated AuNPs, however, such particles have a significant advantage since RF waves do not interact with biological tissues and thus can penetrate deeper within the body than IR light.
Currently many research groups explore RF-activated AuNPs for cancer treatment, though there is no clear understanding what the exact mechanism of heat generation is. Some researchers presented evidences indicating that heat generation is a function of AuNPs size, another showed that it depends on ion concentration in the AuNPs colloidal suspension, others explain the phenomenon through the inductive, magnetic, or electrophoretic mechanisms.
In this study we investigated the energy absorption by AuNPs of different sizes, their supernatants, and different salts (AuNPs synthesis precursors) in the RF range 1-500 MHz. Our data indicates that in the colloidal AuNPs system supernatant accounts for most of the adsorption as compared to nanoparticles itself. In addition, effect of chemically vs. physically bound capping agent on absorption of AuNPs is also investigated. These findings provide an insight in the nature of RF-activated AuNPs colloids and considerations for RF-tumor ablation optimization.

Spiky gold nanoparticles (SGNPs) have gained wide interest for biomedical applications as their surface plasmon resonance (SPR) can be tuned to the near-infrared (NIR) region depending on the dimensions of the “spiky” branches. However, SGNPs typically exhibit photothermal instability; SGNPs can transform into more stable spherical structures and hence lose SPR in the NIR region upon excitation due to heat-mediated melting and reshaping of “spiky” branches. Here, we addressed this challenge by applying mussel-inspired polydopamine (PDA) coating on SGNPs. We report that the PDA outer shell on SGNPs prevents photothermal reshaping of SGNPs and sustains their NIR-responsiveness. PDA coating can be achieved using spontaneous polymerization of dopamine under a mild basic condition in the presence of SGNPs. The PDA layer thickness can be controlled in a range of 1 – 30 nm by varying dopamine concentration. After NIR excitation, uncoated SGNPs undergo shape transformation to a more spherical form in a laser power dependent manner, whereas SGNP/PDAs maintain spiky branches and structural integrity. The transition to spherical shape for uncoated SGNPs is accompanied by progressive blueshifts in SPR absorption causing decreased efficiency of photothermal heating under prolonged laser irradiation. In contrast, SGNP/PDAs show minimum absorption shift in an identical condition resulting in significantly higher temperature increase compared with uncoated SGNPs. We further demonstrate multiple combinational tumor therapeutic approach by co-administrating Doxorubucin (Dox) with SGNP/PDAs for chemo-immuno-photothermal therapy. The SGNP/PDA-mediated photothermal treatment effectively eliminates bulk tumor burden and Dox-mediated chemo-immunotherapy prevents relapse of treated tumors. In addition, the combination generates potent anti-tumor memory immune responses that decreases growth of metastatic tumors and confer protection against tumor re-challenge. Immuno-modulations in response to the treatments are analyzed by the population of immune cells in tumor microenvironment. We also discuss the mechanism of immune response based on the antibody-mediated depletion study. In conclusion, PDA coating is a simple and versatile method to produce stable NIR-responsive gold nanoparticles, and the combinational chemo-immuno-photothermal therapy using PDA-coated SGNPs and Dox permits efficient tumor treatment promising a new form of cancer therapy.

The problems arising from melanoma of skin has become a major public health issue in several countries. In the United States alone, it is estimated that 76,380 new cases will be diagnosed in 2016. Therefore, it is of prime importance to develop strategies to address this global issue. Herein, using a facile method, novel intrinsically therapeutic carbon nanoparticles (CNPs) are developed from Dunaliella Salina, a green microalgae bearing a high content of carotenoids. Carotenoid are terpenoid-based antioxidants with unique optical absorptive properties and benefit from preferential uptake by the cellular membrane due to their ability to be deeply fused with the lipid hydrophobic center. The water dispersible uniformly sized nanoparticles (2.6±0.5 nm) are derived through the hydrothermal synthetic approach and are extensively characterized for their chemical and physical properties. Transmission electron microscopy (TEM) and Atomic Force Microscopy (AFM) were utilized to observe the morphology of the CNPs. Subsequently, FT- IR spectroscopy revealed the presence of multiple hydrophilic surface moieties while Raman spectroscopy indicated the existence of well- established D and G bands of CNPs. Interestingly, although UV-Vis spectroscopy masked the contribution from carotenoid in the visible range (~530 nm), ¹H NMR spectroscopy properly confirmed the intact nature of carotenoid after transformation to nanoparticles, suggesting that these CNPs are ‘enclosing’ carotenoid molecules. Hence, it was hypothesized that CNPs could be efficiently utilized as nanoparticles with ‘embedded cargo’ for melanoma eradication under UV absorption. MTT assay in malignant human melanoma cell line demonstrated that the reactive oxygen species could be induced after 30 minutes UV exposure (wavelength=362 nm) leading to a decrease in cell viability compared to untreated control, agave based CNPs and free β- carotene. In addition, Fluorometric Intracellular ROS Kit was applied to indicate the generated ROS concentration over the period of 48 h. Inspection of the results revealed the enhanced amount of species in algal CNPs compared to control and agave based CNPs. Furthermore, the localization of the nanoparticles and their chemical features are made feasible using Raman imaging in C32 cells incubated with nanoparticles. Overall, these inexpensive naturally derived multifunctional nanoplatforms hold promise as the next generation phototherapeutic agents with intrinsic efficacy in cancer eradication.

Owing to their large range of properties which can be accurately tuned by the chemical composition, the shape and the dimensions, multifunctional nanoparticles appear as promising candidates for image-guided therapy.
In this context, we developed the synthesis of gadolinium chelate coated gold nanoparticles (Au@L-Gd with L a linear or macrocyclic polyaminocarboxylate chelator) which are designed for combining radiotherapy and multimodal imaging (magnetic resonance imaging (MRI), scintigraphy (SPECT after radiolabeling by indium-111) and X-ray imaging) [1-5]. The presence of gold element confers to the nanoparticles a radiosensitizing effect which led to a great increase in lifespan (ILS) of brain tumor bearing rats when they were treated by radiotherapy after intravenous injection of the gold nanoparticles. However the radiosensitizing effect appears to be underexploited owing to a too rapid renal clearance which limits their accumulation in solid tumor. Hence the exploitation of the potential of these gold nanoparticles for MRI guided radiotherapy requires the increase of the circulation time for reaching efficiently their specific target while preserving the ability for renal clearance.
For achieving an enhanced circulation time of the gold nanoparticles and therefore a greater accumulation in solid tumor, Au@L-Gd nanoparticles were immobilized onto large bioresorbable carriers (maghemite nanoflowers (nF)) [6-8]. The resulting golden nanoflowers are constituted of monocrystalline grains (11 nm) which are assembled in a flower-shaped structure and gold nanoparticles (Au@L-Gd). Such an association allows combining the imaging modalities and therapeutic activities of each part of the golden nanoflowers (T₁-weighted MRI, radiosensitization from the gold nanoparticles and T₂-weighted MRI, magnetic hyperthermia from iron oxide nanoflowers). In comparison to the case of Au@L-Gd nanoparticles, the circulation of golden nanoflowers after intravenous injection is longer-lasting: the presence of the golden nanoflower is observed 1 h after the injection whereas the content of gold nanoparticles is too low in the tumor for monitoring their accumulation by MRI 1h after the injection. This was mainly attributed to the larger size of the golden nanoflowers (30 nm vs 2.5 nm). Moreover the radiosensitizing effect of the golden nanoflowers provides for a same gold content in the injected suspension a higher ILS than in the case of Au@L-Gd. The immobilization of the gold nanoparticles Au@L-Gd onto nanoflowers allows therefore to better exploit the radiosensitizing effect of the gold nanoparticles.
References
[1] Alric et al., J. Am. Chem. Soc. 2008, 130, 5908
[2] Arifin et al., Radiology 2011, 260, 790
[3] Alric et al., Nanoscale 2013, 5, 5930
[4] Miladi et al., Small 2014, 10, 1116
[5] Laurent et al., Nanoscale 2016, 8, 12054
[6] Hugounenq et al., J. Phys. Chem. C 2012, 116, 15702
[7] Lartigue et al., ACS Nano 2012, 6, 10935
[8] Javed et al., Small 2014, 10, 3325

Fe and Fe-Au nanowires were synthesized by electrochemical deposition for use as contrast agents in magnetic resonance imaging (MRI). Contrast agents are often used in MRI to distinguish different tissues and help make diagnoses. The size, shape, surface, and magnetic moment of these agents each affect performance in T₁ or T₂ contrast. Here, Fe and Fe-Au nanowires of various lengths (0.5-2.7μm), diameters (32-110nm) were made from single electrolytes. Au was added to some electrolytes to make nanowires. The Au segments were used for thiol-functionalization and may be useful for future photo-thermal therapy, biomolecular targeting, and drug delivery. The nanowires are also useful for magnetic hyperthermia because of their high magnetic moments and anisotropies. In this work, these promising multifunctional electrodeposited Fe and Fe-Au nanowires were found to be comparable to commercial T₁ and T₂ contrast agents with r₁ = 2.0 mM^-1s^-1 and r₂ = 77 mM^-1s^-1. Values of r₁ were increased by increasing nanowire diameter, while r₂ values were increased by decreasing nanowires lengths. Gold-rich segments were coated with SH-PEG-COOH, which increased the r₂ by ~10X. Successful concentration-dependent contrast was seen in T₂-weighted images, which proves the feasibility of these multifunctional nanowires as active MRI contrast agents.

The aim of our work is to develop unprecedented methods to measure ultra-trace concentration of engineered nanoparticles in aqueous and environmental solution. Currently, nanoparticles release is a worrying environmental issue to consider, due to the lack of knowledge concerning their behavior and fate. This lack is clearly due to the urgent need to have an accurate method able to characterize the several physical and chemical parameters of nanomaterials (size, size distribution, shape, charge, etc.) within the context of ultra-diluted sample in natural media. From all the analytical techniques generally used to characterize nanomaterials such as light scattering, electronic microscopy, optical spectroscopy, chromatographic methods, none of them can access directly to the nanomaterial (NM) characterization in natural media at ultra-trace concentration. In our study, we present for the first time the potentiality of the Laser-induced breakdown detection technology for the characterization of nanoparticles in aqueous media for environmental application. The calibration of this tool was first optimized using polystyrenes nanospheres at different environmentally relevant concentrations. The aim of this optimization part is to carefully check all the
instrumental details from the quality of the measurement cell to the development of measurement protocol in order to get an accurate and relevant statistical distribution. Then, we applied and validated our methodology on fullerene nanoparticles in aqueous media using different preparation pathways. The preparation methods are developed in order to simulate both environmental and biological conditions using mechanical agitation and additives such as polyethylene glycol based molecules. Our first results show for the first time the ability to differentiate size population of nC60 at low concentrations according to their structures and dispersion state.

Biomedical applications of non-spherical nanoparticles such as photothermal therapy and molecular imaging require their efficient intracellular delivery, yet reported details on their interactions with the cell remain inconsistent. Here, the effects of nanoparticle geometry and receptor targeting on the cellular uptake and intracellular trafficking are systematically explored by using C166 (mouse endothelial) cells and gold nanoparticles of four different aspect ratios (ARs) from 1 to 7. When coated with poly(ethylene glycol) strands, the cellular uptake of untargeted nanoparticles monotically decreases with AR. Next, gold nanoparticles are functionalized with DNA oligonucleotides to target Class A scavenger receptors expressed by C166 cells. Intriguingly, cellular uptake is maximized at a particular AR: shorter nanorods (AR=2) enter C166 cells more than nanospheres (AR=1) and longer nanorods (AR=4 or AR=7). Strikingly, long targeted nanorods align to the cell membrane in a near-parallel manner followed by rotating by ~90° to enter the cell via a caveolae-mediated pathway. Upon cellular entry, targeted nanorods of all ARs predominantly traffic to the late endosome without progressing to the lysosome. Our studies yield important materials design rules for drug delivery carriers based on targeted, anisotropic nanoparticles.

Stimuli-responsive drug carriers have been intensively researched for controlled drug release because they can alter their physicochemical properties in response to specific cellular stimuli. For effective cancer therapy, it is desirable to use multi-stimuli responsive drug carriers because cancer exhibits various levels of stimuli at different tumor sites and tumor progression stages.[1] Multi-stimuli-responsive drug carriers are usually fabricated by emulsion method, droplet-based microfluidics, replication method, etc. Unfortunately, however, current methodologies have limitations to synthesize drug carriers which can independently respond to each cellular stimulus and then release suitable drugs in a controlled manner. For example, the synthesized drug carriers using these methods exhibit a burst drug release at complex stimuli without showing a controlled and sequential drug release responding to an each independent stimulus.[2,3]

Here, we introduce multi-sensitive drug carriers synthesized by stop flow lithography (SFL). The SFL is a promising platform to synthesize microparticles with complex geometries and chemical patterns.[4] Using this platform, we synthesized multi-compartmental drug carriers with chemically distinct compartments composed of different stimuli-responsive polymers. Each compartment of the drug carriers was synthesized with reducible disulfide-containing crosslinkers and acid-cleavable ketal-containing crosslinkers to selective release the independent drug in response to tumor miroenvironments (i.e., highly reductive and mildly acidic conditions). We successfully fabricated multi-compartmental particles where each compartment of the drug carriers encapsulates multiple drugs individually. The particles exhibited independently controlled drug release at different stimuli. We believe that multi-stimuli responsive drug carriers can be utilized for efficient treatment of tumor with heterogeneous tumor microenvironments.

[1] Wang, Jinqiang, et al. Advanced Materials 2013, 3670.

[2] Wu, Yanjuan, et al. RSC Advances 2015, 31972.

[3] Chen, Xin, and Zhongning Liu. Journal of Materials Chemistry B 2016, doi:10.1039/C6TB00694A.

[4] Hwang, Dae Kun, et al. Journal of the American Chemical Society 2009, 4499.

We have developed a novel photo-responsive, acidifying nanoparticle (paNP) that acutely restore pH of compromised lysosomes to rescue autophagic flux and cellular function in beta cells and hepatocytes under lipotoxicity. In pancreatic beta-cells (INS1) and liver hepatocytes (HepG2), chronic exposure to high levels of fatty acids (lipotoxicity) leads to an inhibition of autophagic flux and subsequent cellular dysfunction, which has been recently associated with impaired lysosomal acidification and elevated lysosomal pH. Therefore, restoration of lysosomal pH is essential in alleviating the block in autophagy and promote proper cellular function. Currently, there is a lack of approach to deliver “acid” into a specific subcellular compartment at a desired time. To modulate lysosomal pH and determine its effect on autophagy in a time-dependent manner, we have designed a novel photo-activated acidic nanoparticle (paNPs) that contains caged acid which can be uncaged by acute photo (UV)-activation of the nitrophenylethanol protecting group. Rhodamine labelled paNPs demonstrate dose dependent uptake into lysosomes of INS1 and HepG2 cells. Acute photo activation of paNPs for 5 minutes in INS1 and HepG2 cells exposed to lipotoxicity demonstrate controlled acidification of lysosomes and restored lysosomal pH with minimal cell cytotoxicity. Photo-activated paNPs also increased lysosomal cathepsin enzyme activity, and decreased both autophagic proteins LC3II and p62 levels, indicating both a rescue of lysosomal function and autophagic flux due to restoration of lysosomal acidity. Additionally, paNPs restored glucose-stimulated insulin secretion that is reduced in INS1 cells and mouse islets under lipotoxicity. These results indicate that acidifying lysosomes with paNPs improved lysosomal function and autophagic flux in INS1 and HepG2 cells under lipotoxicity, and are of therapeutic interest for pathologies associated with lysosomal acidity impairment such as type II diabetes and non-alcoholic fatty liver disease (NAFLD).

Brain imaging can measure the activity of large sets of neurons and analyze the brain circuits. The fluctuation of dopamine, an indispensable neurotransmitter, level in brain indicates a wide range of disease, including tumors of neural crest, drug addiction, and Parkinson disease. Particular interest focuses on dopaminergic tissue in different brain regions, and neural activity mapping is essential for early disease surveillance and therapy tracking. Near-infrared fluorescence (NIRF) imaging has been widely used to monitor therapy of cancer and other diseases in preclinical studies; however, this technology has not been applied smoothly to monitoring neural activity through dopamine. We design near-infrared fluorescence conjugated polymer with phenylboronic acid tags, which can self-assemble into biocompatible nanoparticles. Due to photo-induced charge transfer between the nanoparticles and the neurotransmitter, as well as optical signal amplification by molecular wire effect of the conjugated polymer, the nanoparticles can detect endogenous dopamine in living PC12 cells and variation in the neurotransmitter level distribution in mice brain.

The imminent threat of antibiotic resistance requires the development of new antibiotics to treat bacterial infections. We have repurposed existing penicillin antibiotics into polymer-penicillin conjugates, providing an effective method for the development of novel biocompatible antimicrobial polymers that exhibit enhanced activity in the presence of bacteria. Our novel conjugates display superior antibacterial efficacy compared to non-conjugated penicillins and prevent the attachment of bacteria.

We have used mild, aqueous colloidal carbodiimide chemistry (Manju 2011) to conjugate hyaluronic acid (HA) to common penicillin antibiotics via a labile ester bond sensitive to acidic pH, characteristic of infection. HA is degradable by hyaluronidase, an enzyme upregulated by common bacteria during infections (Ibberson 2014). Therefore, these HA-penicillin conjugates are bacteria-responsive, degrading in either acidic pH or hyaluronidase to form active penicillin-containing subunits. Our library of conjugates contains three narrow spectrum penicillins: carbenicillin, penicillin G, and ampicillin and two more recent penicillins effective against methicillin-resistant strains: nafcillin and oxacillin. FT-IR and size exclusion chromatography were used to confirm successful conjugation. The antibacterial efficacy of the products was examined using broth microdilution assays against gram-positive strains across a spectrum of penicillin resistance, including Staphylococcus aureus, methicillin-resistant S. aureus, and S. pyogenes, as well as gram-negative Pseudomonas aeruginosa. We determined that the conjugates were approximately 10 to 10³ times more effective than the unconjugated penicillin controls. For example, HA-oxacillin conjugates were approximately 3500 times more effective than oxacillin alone against one particular strain of MRSA, USA300. Animal studies using an invertebrate model, Galleria mellonella, demonstrated the same enhanced efficacy of the conjugates in vivo. Additionally, these HA-penicillin conjugates were developed into micron-scale self-defensive antibacterial coatings, and were characterized by atomic force microscopy, profilometry, and scanning electron microscopy. Bacteria were unable to attach to the conjugate coated glass at a surface concentration of 0.32 μg/mm², promising for preventing the development of biofilms. Finally, the conjugates were determined to be non-hemolytic at concentrations 10³ times greater than what is needed to eradicate bacteria. Our conjugates prolong the lifetime of existing therapeutics by reducing the required dose of the therapeutic, and thus lowering susceptibility to resistance, while demonstrating potential to be used in both injectable and topical therapies.

Nanoparticles exhibit unique physical properties, which make them suitable for imaging and imaging detection applications (e. g. bioimaging and contrast agents).^[1] For a sustainable use of nanoparticles in biological media, encapsulation is a powerful tool, which provides stable and well shielded nanoparticles in aqueous media. In the past, we presented encapsulation of nanocrystals (NCs) in the amphiphilic polymer poly-(isoprene-block-ethylene oxide) (PI-b-PEO).^[2,3] Furthermore we used seeded emulsion polymerization to build ultrasmall multifunctional magnetic nanohybrids.^[4] These results are summarized and a new polymer platform is presented. The encapsulation of nanomaterials with this new polymer yields polymer/nanocrystal structures with similar or even enhanced properties compared to the PI-b-PEO system as well as the nanohybrid system, however exhibiting much smaller hydrodynamic size. The polymer shell is analyzed with sensitive fluorescence quenching analysis. This new polymer platform may be beneficial for future applications of nanoparticles in nanomedicine, where hydrodynamic size is known to be important for cellular uptake, organ distribution and clearance from the body.

[1] Elmar Pöselt et al., ACS Nano 2012, 2, 1619-1624.
[2] Elmar Pöselt et al., ACS Nano 2012, 4, 3346-3355.
[3] Johannes Ostermann and Jan-Philip Merkl et al., ACS Nano 2013, 10, 9156-9167.
[4] Artur Feld and Jan-Philip Merkl et al., Angewandte Chemie 2015, 127, 12645-12648.

Membrane-active antimicrobial peptides and their synthetic mimics have been studied extensively as a new generation of antibiotics to fight against persistent bacteria infections that resist traditional antibiotic treatment. It has long been recognized that a delicate yet un-quantified balance between amphiphilicity and cationic charge is key to optimize the bactericidal efficiency and selectivity of these membrane-active antimicrobials (MAAs). The dilemma, however, is that the amphiphilic nature of MAAs that gives rise to their potency to disrupt microbial membranes is oftentimes detrimental to human cells. Hydrophilic antimicrobial peptides and polymers generally have good biocompatibility, but they have received much less attention due to their low antimicrobial activity. In this work, we investigate the roles of nanostructures on helping hydrophilic and cationic polymers (HCPs) gain high antimicrobial activity and selectivity. Silica nanoparticles of different sizes grafted with HCP (silica NP-HCPs) are synthesized, and their antimicrobial activity against different types of bacteria as well as their toxicity against human cells are evaluated and compared. We show that initially inactive HCPs, when grafted onto nanoparticles, can be turned into potent antimicrobial agents while maintaining a low level of toxicity, and that the antimicrobial activity and selectivity exhibit dependency on nanoparticle sizes. Using synchrotron small angle x-ray scattering, scanning electron microscopy, confocal microscopy, and model dye release experiments, we investigate the antimicrobial mechanism of silica NP-HCPs and reveal how they remodel microbial membranes to cause damages. Our results demonstrate that it is possible to utilize nanostructures to develop new families of MAAs with high antimicrobial activity, selectivity, and low toxicity to fight touch bacteria infections.

Hybrid materials, commonly referring to inorganic-organic composites, have found wide applications in various fields due to their complementary physiochemical properties. In practical case, the inorganic nanoparticles are usually required to be homogeneously blended with a continuous organic media. In order to retain the optimized characteristics, it is essential to achieve stable dispersion and avoid agglomeration. Through grafting or modifying with polymers (dendrite or linear) or organic molecules, the surface physiochemical properties of inorganic cores can be adjusted. Inspired by this, we propose a hybrid colloidal system of polymeric matrix embedded with inorganic nanoparticles. By manipulating chain length in outermost polymer layer, we not only realize fast and homogeneous dispersion of hybrid nanoparticles, but also verify that the aggregation and dispersion behaviors could be flexibly and reversibly tuned by changing the temperature.

In our experiment, the hybrid core consists polymer matrix and inorganic nanoparticles realized through mini-emulsion polymerization. Then, linear poly(metyl methacrylate-co-butyl acrylate) chains are grafted on the surface of the hybrid core through emulsion polymerization. In this design, the outermost linear polymer chains provide dynamic force to flexibly facilitate the dispersion and aggregation of the colloidal particles triggered by temperature, in co-solvents with different compositions.
Particularly, hybrid colloids with different chain-length in outermost polymer layer are prepared through introducing chain-transfer agent. The obtained colloids are dispersed in binary solvents composed of butyl acetate and ethanol. The results highlight the essential impact of chain-length on facilitating solubility, colloidal stability and reversible thermoresponsive behavior of hybrid nanoparticles. The shorter chain-length polymers show a relatively lower critical stabilization temperature (CST) at which aggregation or dispersion occurs upon continuously decreasing or increasing temperature. Under fast quenching below CST, we can even observe gelation of the dispersion, while increasing temperature, the dispersion state is recovered. This temperature-induced property could be utilized to controllably regulate the dynamic behavior of nanoparticles, potentially facilitating the recycling of inorganic fillers in painting, food or cosmetic industries.

Despite nearly universal incidence of dental caries worldwide, current dentist-recommended methods of caries diagnosis are unchanged in almost a century, as newer methods are expensive, require additional equipment, and generally fail to distinguish between active and inactive carious lesions. Active carious lesions have a characteristic white (when dried) roughened surface, indicating microscopic pores in the enamel, and a negative surface charge. Consequently, we designed a novel biopolymer nanomaterial which can: target and adhere to active caries lesions by sub-micron size and cationic charge; fluoresce when illuminated by a dental curing light; and degrade in the oral cavity into non-toxic compounds.
We measured the surface roughness and charge of active and inactive carious lesions using Scanning Probe Microscopy (SPM). We then prepared and characterized fluorescently-labeled cationic biopolymer nanoparticles, validated the degradation of the particles in saliva, and observed illumination of caries lesions macroscopically using computer image analysis and microscopically using two-photon microscopy.
AFM validated sub-micron porosity and negative surface charge of active carious lesions. Prepared nanoparticles were fluorescent, cationic (+5.8±1.2 mV), degradable in saliva, and able to penetrate early caries lesions (101±56 nm). Upon illumination with a dental curing light, these nanoparticles specifically illuminated active caries relative to anionic and uncharged controls (student’s t-test, n=15, p<10^-5), but did not illuminate remineralized (inactive) caries. Optical contrast was further improved using computer-aided image analysis to extract green-pixel images (student’s t-test, n=15, p<10^-6). Two-photon microscopy showed illumination of microscopic pores and may be useful for understanding the architecture of caries lesions.
These particles could be used for early cavity diagnosis in dental clinics, at home, or in developing countries and regions with limited access to dentists, or to monitor tooth mineralization in dental clinical trials. Similarly designed nanoparticles could be used to deliver fluoride, other remineralization aides, or antibacterial therapeutics directly into caries lesions.

The ability to detect and manipulate materials at the nanoscale has enabled extraordinary applications in disciplines that span from biology to engineering. There are many opportunities where nanotechnology can make an impact in oil and gas exploration and production technologies. In this talk, we will focus on exploring the concept of reservoir nanoagents, which has emerged in recent years as one of the promising areas for the application of nanotechnology. The combination of nano-sized and enhanced properties make these nano chemistry-based nanoagents very intriguing. Nanoagents are intended to roam the hydrocarbon reservoir, reporting about its properties, imposing a change by responding to natural or induced stimulus, supporting discovery procedures and oil recovery processes.

Use of known pharmaceutical drugs with different chemotherapeutic character in new area of medicine can be a clever approach to reduce the total time and cost spent on developing a drug molecule from scratch. Nifuroxazide, a nitro-furan anti-biotic has been recently identified as potent anti-cancer agent but physical limitations of low aqueous solubility and lack of targeted delivery reduces the extent of benefit could be achieved against cancer. To address this issue, we synthesized prodrug of nifuroxazide (Pro-nifuroxazide), enzymatically cleavable at Sn-2 site to release the drug with phospholipase A2, preferably in cancer cell membrane, and assembled as significantly stable pro-nifucelle. Pro-nifuroxazide were found to be better cell membrane interacting molecule than the nifuroxazide and in vitro studies in MDA-MB231, MCF-7 and C32 cells showed better cancer cell inhibition activity with 2-5-fold higher induced apoptosis. Pro-nifucelle was also found to be better by at least 2 fold in reducing CD44+ stem like cell population. It was also established that even after being in form of pro-nifucelle, cancer cell growth inhibition was always followed the STAT-3 inhibition pathway. Studies in nude mice xenografts with MCF-7 revealed the highly efficient calculated % effective growth inhibition to be more than 400 % for pro-nifucelle while H&E analysis showed significantly high nuclear fragmentation and retracted cytoplasm. Immuno-staining on tumor section showed significantly low level of pSTAT-3 by pro-nifucelle treatment establishing the inhibition of STAT-3 post pro-nifucelle by inhibition of STAT-3 phosphorylation. Thus, we could significantly improve the anti-cancer activity of repurposed nitro-furon antibiotic nifuroxazide to many folds with improved IC50, induced apoptosis, reduced CD44+ cell population, STAT-3 inhibition and reduced phosphorylation by using enzymatically trigerable pro-nifucelle.

Superparamagnetic nanocrystals have the ability to rapidly response to an external magnetic field and have been pursued as versatile carriers for diagnostic and therapeutic applications. Nanostructures assembled from inorganic nanoparticles (NPs) have also attracted numerous attentions due to their collective or advanced optical, electronic and magnetic properties. To combine these attractive features into one platform, we reported a unique system by assemble iron oxide NPs (IONPs) into vesicles as drug delivery vehicles. As the IONPs are densely packed inside the polymer membrane of the vesicles, the assemblies exhibited enhanced imaging signals and magnetic movement compared with individual IONPs. Furthermore, doxorubicin-loaded magnetic vesicles could be readily internalized into tumor cells and demonstrate comparable tumor inhibition efficiency to free drugs, suggesting the promising applications of the vesicles in imaging-guided drug delivery.

Bioresorbable polymers with varying lifetimes play a vital role in the development of implantable materials that are used in surgical procedures such as sutures, stents, wound dressings, bone plates; as controlled drug delivery systems; and as tissue engineering scaffolds that support three-dimensional tissue formation. The half-life of common bioresorbable polymers ranges from 3 to over 12 months and the slow bioresorption rates of these polymers restrict their use to a limited set of applications. The use of embedded enzymes was previously shown to decrease average degradation time to about 0.5 months. In this study, electromagnetic actuation of iron oxide magnetic nanoparticles embedded in an encapsulant polymer, poly(ethyleneoxide), PEO, was employed to initiate enzyme assisted degradation of bioresorbable polymer poly(caprolactone), PCL. Results indicate that the internal temperature of iron oxide doped PEO samples can be increased via an alternating magnetic field, and the temperature change depends strongly on nanoparticle concentration and magnetic field parameters. The temperature increase achieved was found to be sufficient to relax the PEO encapsulant and enable the diffusion of enzymes out of the PEO and into the surrounding PCL matrix. Current studies are directed at understanding the influence of various structural and process parameters on the degradation rate of PCL via released enzyme.

This material is based upon work supported by the National Science Foundation under Grant No. CMMI-1538730.

The design and development of multifunctional carriers for drug delivery based on silica nanoparticles have attracted intense interests. Ordinary spherical hollow nanoparticles (HNPs) were demonstrated to be promising candidates. However, the application of HNPs with special morphologies has rarely been reported. HNPs with sharp horns were expected to own higher endocytosis efficiencies than spherical counterparts. On the other hand, potential accumulation is a one of the key obstacles to limit the use of silica nanoparticles in cancer treatment. Herein, we designed two kinds of intelligent systems based on silica nanoparticles to realize the function of glutathione-induced drug/gene delivery. Novel star-shaped hollow silica nanoparticles (SHNPs) and DOX-loaded biodegradable silica nanoparticles (DOX@DS) were proposed as platforms respectively for the fabrication of redox-triggered multifunctional systems for synergy of gene therapy and chemotherapy.
The CD-PGEA gene vectors (consisting of β-CD cores and ethanolamine-functionalized poly(glycidyl methacrylate) (PGEA) arms) were introduced ingeniously onto the surfaces of SHNPs with plentiful disulfide bond-linked adamantine guests or disulfide bridged DOX@DS. The resulting supramolecular assemblies (SHNP-PGEA or DOX@DS-PGEA) possessed redox-responsive gatekeepers for loaded drugs of SHNP-PGEA or biodegradability of DOX@DS-PGEA. Meanwhile, they also demonstrated excellent performances to deliver genes. The gene transfection efficiencies, controlled drug release behaviors and synergistic antitumor effect of the as-developed silica-based carriers with different morphologies and sizes were investigated in details. Compared with ordinary spherical HNP-based or DS-PGEA counterparts, SHNP-PGEA carriers with six sharp horns and DOX@DS-PGEA with DOX loading were proven to be superior gene vectors and possess better efficacy for cellular uptake and antitumor effects. The present multifunctional carriers based on silica nanoparticles would have promising applications in drug/gene co-delivery and cancer treatment.

References:
[1] Zhao, N.; Lin, X.; Zhang, Q.; Ji, Z.; Xu, F. J. Small 2015, 11: 6467.
[2] Yan, P.; Wang, R.; Zhao, N.; Zhao, H.; Chen, D. F.; Xu, F. J. Nanoscale 2015, 7: 5281.
[3] Yan, P.; Zhao, N.; Hu, H.; Lin, X.; Liu, F.; Xu, F. J. Acta Biomater. 2014, 10: 3786.

The growing tide of bacterial resistance is one of the top 3 threats to global public health today. One significant challenge faced by nanomedicine translation is the contradiction between prolonged circulation time and enhanced, targeted delivery. Despite numerous studies on nanoparticle-based, circulation-prolonged and tumor-enhanced drug delivery, corresponding researches on anti-bacterial-infection studies have just really begun. Inspired by the recent pioneering studies in the feasibility of charge-switching polymeric nanoparticles for bacterial cell wall-targeted delivery of antibiotics, here we report on a charge-adaptive nanosystem for prolonged and enhanced in vivo antibiotic delivery. Our design is based on the following reasons: 1) PEG was the hydrophilic block of the polymer and the outer layer of nanoparticle to maintain its stability and enhance in vivo circulation; 2) polycaprolactone (PCL) and poly(β-amino ester) (PAE) are the hydrophobic core of nanoparticle at physiological environment, and PAE showed pH-tuning charge conversion from neutral to positive when pH decreases from 7.4 (physiological environment) to 6.5 (infection sites) because of its pH-responsiveness which could decrease protein adsorption during circulation and enhance particles-bacteria interaction at infection sites; and 3) vancomycin (Van) was a first-line antibiotic for gram-positive bacterial infections. PEG-b-PCL-b-PAE/Van nanoparticles were prepared at pH 7.4 by using modified emulsion/solvent evaporation techniques. Zeta potential studies showed that when pH decreased from 7.4 to 5.0, PEG-b-PCL-b-PAE/Van gradually became positively charged due to the protonation of PAE and became hydrophilic. In vitro Staphylococcus aureus (SA) targeting results indicated that PEG-b-PCL-b-PAE/Van showed significant increase in binding as pH decreased from 7.4. to 6.5. Subcutaneous inflammation model was used to evaluate the effect of charge conversion due to pH on antibiotic delivery in vivo. PEG-b-PCL-b-PAE/Van showed the highest fluorescence intensity 24h after injection, which was 2.7-folds and 1.8-folds of that of free Van and PEG-b-PCL/Van, respectively. We concluded that we developed a charge-adaptive nanocarrier, PEG-b-PCL-b-PAE, which can quickly realize acidity-sensitive charge alteration and extend antibiotic delivery and accumulation upon in vivo bacterial infection. Given the pH-dependent charge conversion characteristic of PAE, PEG-b-PCL-b-PAE/Van holds a negative charge and a compact nanoparticle at physiological environments, which is helpful for prolonged circulation. This then is converted to a positive charge when it reaches the acidic infection sites, which ultimately strengthens its combination with bacteria and enhances Van accumulation. We believed this PEG-b-PCL-b-PAE nanocarrier could be applied to other antibiotics and contribute to in vivo antibacterial therapy.

Quality of drug treatment can be improved significantly by controlled and targeted drug delivery, where changes of internal stimuli (such as pH, T, or redox-potential) induce a release of the drug at a given time and place. Drug dosage can be reduced and toxic side-effects can be minimized. Therefore, targeted drug delivery has an essential role in cancer therapy for promoting chemotherapeutic efficacy.
Among all novel researches done so far, the use of polymers which are intrinsically thermal responsive is the most popular choice and has been investigated extensively in the form of hydrogels and small capsules during the past decade. When temperature increases, these polymeric particles either swell or fracture so that the release of active is achieved. Lately, polymeric multilayer capsules synthesized with Layer-by-Layer (LbL) technique have opened exciting avenues for the design of thermal responsive carriers with very precise control over capsule size and morphology.
In this study, capsules made from poly(styrene sulfonate) (PSS) and poly(diallyldimethylammonium) (PDADAMAC) – PSS/PDADMAC polyelectrolytes were introduced. Spherical and uniform wall thickness of 50.5nm capsules with the size of 6.1 ± 1.2μm could be obtained. Heat treatment on these capsules resulted in shrinkage response of 54.6% and the critical temperature of this responsiveness was 45°C. Further investigation found that for PDADMAC-outmost capsules, temperature-induced expansion response can be promoted when the pH is monitored to 3 and below. Hence, in future development, thermal stable active drugs will be encapsulated and triggered delivery will be studied.

It is a widely accepted fact that environmental factors affect cells by modulating the components of subcellular compartments and altering metabolic enzymes. Factors (such as oxidative stress and heat-shock-induced proteins and heat shock factors, which upregulate stress-response related genes to protect affected cells) are commonly altered during changes in environmental conditions. Studies by our group and others have shown that nanoparticles (NPs) are able to efficiently attenuate oxidative stress by penetrating into specific tissues or organs. Such findings warrant further investigation on the effects of NPs on heat-shock-induced stress, specifically in cells in the presence or absence (pretreated) of NPs. Here, we examined the cytoprotective effects of two different NPs (cerium and selenium) on heat-induced cell death for a model cell using dermal fibroblasts. We report that both ceria and selenium NPs (at 500µg/mL) possess stress-relieving behavior on fibroblasts undergoing heat shock. Such results indicate the need to further develop these NPs as a novel treatment for heat shock.

In this paper, the preparation of superparamagnetic iron oxide nanoparticles(Fe₃O₄ NPs) with thermal decomposition was presented. And polyacrylic acid (PAA) functionalized iron oxide (Fe₃O₄@PAA) nanoparticles by ligand exchange were loaded into bridged silsequioxane precursor and then the magetic bridged silsequioxane aerogels were obtained by sol-gel method and supercritical CO₂ drying technique. The silsequioxane precursor was synthesis of vinyltriethoxysilane (VTES) and 2,2^‘-(ethylenedioxy)diethanethiol (EDDET) by a click reaction under ultraviolet light. The mechanism properties of the novel magnetic aerogel were characterized by scaning electron microscope (SEM), thermal conductivity, contact angle, adsorption/desorption isotherms (BET) and Fourier transforminfrared spectroscopy (FT-IR) and a series of adsorption experiments. The adsorption experiments showed that Fe₃O₄@PAA loaded aerogels had excellent adsorption property(94 mg/g for Cu²⁺, 250 mg/g for Pb²⁺) and recyclability for heavy metal ions. The adsorption principle and mechanism of the composite materials was discussed along with the Freundlich equation. Furthermore, the regeneration rate of this composite adsorbent had been investigated. As a consequence, it showed far more promise for application in wastewater treatment.

To achieve light triggered drug release in cancer chemotherapy multimodal titanium dioxide (TiO₂) nanocorals modified with methoxy polyethylene glycol (mPEG) were developed. TiO₂ nanocorals structure synthesized by optimizing solvothermal method and developed nanocorals structure can conjugate efficient chemotherapeutic drug over TiO₂ nanoparticles. The mPEG on the surface of multifunctional nanocorals could effectively conjugate the drug and coordinately improve the biocompatibility of nanocorals. Following UV light radiation, TiO₂ produced free radicals (OH^- and O2^-) are found to be effective for drug release in the cancer cells. Importantly, the amount of drug released from multimodal TiO₂ nanocorals can be regulated by the UV-light radiation time to further control the anti-cancer effect. This multimodal TiO₂ nanocorals exhibits a combination of light activated stimuli-triggered drug release and cancer cell targeting. The cytotoxicity, cellular uptake, and intracellular location of the formulations were evaluated in MCF7 cells. Our results showed that nanocorals-DOX exhibited a greater cytotoxicity toward MCF7 cells than free DOX. In addition, confocal laser scanning microscopy demonstrated that most of the nanocorals-DOX was predominantly distributed in the cytoplasm while DOX was located inside the nuclei. Thus, our work demonstrates that the therapeutic efficacy of TiO₂ nanocorals loaded DOX is strongly dependent on its loading mode and this provides significant break-through for the future applications of TiO₂ as a drug carrier in cancer chemotherapy.

Nowadays, the fabrication of nanostructured magnetic particles and their applications as recording media, magnetic sensors, catalysts, ferrofluids and nanomedicine, offers a challenging subject for scientific research. Magnetic particle hyperthermia for cancer treatment, drug delivery and contrast enhancement in magnetic resonance imaging (MRI) are only few of the wide world scale medical applications. Magnetic particle hyperthermia is based on the response of magnetic nanoparticles to a stimuli of an external AC magnetic field. In this work, a synthetic procedure of carbon encapsulated cobalt (Co@C) nanoparticles is presented. All samples were solvothermally prepared, via the wet chemical reduction of a cobalt complex or cobalt acetate tetrahydrated in the presence of different polyols, such as propylene glycol (PG), tetraethylene glycol (TEG) or triethylene glycol (TrEG) and sodium hydroxide (NaOH). Solvothermal is a quite simple and cheap method, while different samples may be prepared at the same time. In all cases, the reaction temperature, the heating rate and the reaction time were maintained immutable (200 ^oC, 2.5 ^oC/min and 24h respectively). The structural characterization of the samples was performed via X-ray diffraction (XRD) and scanning electron microscopy technique (SEM). X-ray diffraction revealed the fcc structure of cobalt and provided an approximate calculation of cell dimensions, while the presence of carbon was also indicated. The elemental analysis, illustrated by SEM, showed excessively presence of carbon, probably due to a core-shell structure effect. The presence of carbon was also evidenced by Raman spectroscopy, where peaks that correspond to G-band (1590 cm^-1) and D-band (1350 cm^-1) of carbon were revealed. TEM micrographs showed spherical or hexagonal nanoparticles with an average diameter of 200-300 nm forming islands. The magnetic properties of Co@C nanoparticles were determined by a vibrating sample magnetometer (VSM), where the external field reached to a maximum of 1 Tesla. The promising values of saturation magnetization (38.8 emu/g) and coercivity (274 Oe) at 1 Tesla and the relative large minor magnetization loop at 0.03 Tesla, led us to apply magnetic particle hyperthermia measurements, which were carried out at a frequency of 765 kHz. The observed values of specific loss power (SLP) at a concentration of 0.5 mg/mL in ethanol were 241.1 and 154.2 W/g at a field amplitude of 0.03 T and 0.025 T, respectively.

Oral biofilms are known to be contributing to the etiology of the diseases ranging from periodontal to cardiovascular complications. It is estimated that the annual financial burden of the associated diseases is ∼$81 billion in the U.S. alone. Herein, the large scale, cost effective synthesis of Hafnia (HfO_x) nanoparticles are demonstrated for the first time as a novel X- ray contrast media for dental computed tomography with the intrinsic bactericidal properties towards oral biofilm. The nanoparticles are derived through a confined sol- gel method in mciroemulsion using Hafnium ethoxide as the starting precursor. The surface of uniformly sized nanoparticles (3.6±0.6 nm) is further tuned through the hydrolysis and condensation of cationic and anionic Silanes to impart the nanoparticles with electrophoretic potentials in the range of -20 to +25 mV. Several characterization methods such as Transmission electron microscopy (TEM), Atomic force microscopy (AFM), Thermogravimetric analysis (TGA), and X-ray diffraction (XRD) were utilized to comprehensively characterize the nanoparticles. Selected area diffraction and XRD studies revealed that the majority of the nanoparticles are amorphous with small percentage of the crystalline features with d_spacing= ~3 °A and broad diffraction peaks at 2θ=15, 30° which could be assigned to monoclinic crystal system. Furthermore, solid state ²⁹Si NMR, ¹H NMR indicated the presence of T1 and T2 band as the chemical feature for successful Silane coupling while XPS analysis determined the formula as HfO_x. CT imaging was applied to show ~1.3 fold enhancement in the Hounsfield unit compared to clinically applied Iohexol while ex vivo studies on the extracted human tooth demonstrated striking CT attenuation in the nanoparticle vs. tooth. The cationic nanoparticles were further applied as intrinsic therapeutic agent for the eradication of dental biofilm via the strong electrostatic interaction with the negatively charged polysaccharide membrane of Streptococcus mutans (S.mutans), the primary culprit of dental caries and subacute
infective endocarditis. Fluorescent Live/ dead assay and bacterial growth kinetics studies on the planktonic bacteria after 2 h, signifies the improved efficacy of the nanaoparticles compared to bacitracin, an antibiotic agent, against S. mutans in a broad range of tested concentrations. TEM and SEM observation exhibited the mode of the interaction of nanoparticles with the bacteria membrane where they tend to “latch on” the bacteria and disrupt the outer bacterial sheath. Overall, these nanoparticles represent the first report on Hafnia based CT contrast agents with inherent theranostic potential for dental biofilm removal.

Early detection of small intravascular thrombus in coronary arties bears an importance in facilitating the diagnosis and risk assessment in patients with chest pain. A synthetic strategy is introduced in this work for developing vascularly constrained “soft” nanopartilces with hydrodynamic diameter of 180-220 nm. The rapid co- self- assembly of polystyrene-b-poly(acrylic acid) and polysorbate 80, a non-ionic surfactant, along with perfluorooctyl bromide (PFOB) occurs in water, resulting in colloidally stable micelles entrapping PFOB molecules. Subsequently, the nanoparticles are rigorously characterized for their physicochemical properties using TEM, AFM, SEM method. These nanoparticles are negatively charged (electrophoretic potential= -42±3 mV) and hence preserve their stability over extended period of time. The presence of PFOB endows the nanoparticles with the capability to be detected by ¹⁹F MRI imaging method. Due to the lack of the inherent fluorine signal in the body, ¹⁹F MRI is a promising technique in delineating the background from the tissue where nanoparticles reside. Moreover, the precise quantification is more feasible as a result of the elimination of the surrounding microenvironment water. In addition, the abundance of ¹⁹F isotope can ease the signal detection, on a level comparable to H nuclei. Subsequently, the nanoparticles are applied ex vivo to image carotid endarterectomy specimens as ¹⁹F MRI contrast agent where clear contrast enhancement is observed (3 Tesla). Preliminary Molecular dynamic simulation data indicated the Raman sensitivity potential of the nanoparticles; hence, making these particles as Raman active probes for the tissue level imaging. Overall, these multifunctional soft platforms offer multiscale imaging promise from tissue to anatomical level for sensitive, non-invasive Raman and MRI imaging of human thrombus.

Signal Transducer and Activator of Transcription Factor 3 (STAT-3) is known to be overexpressed in cancer stem cells. Poor solubility and variable drug absorption are linked to poor bioavailability, and ultimately affect the safety and efficacy of the agent. Many of the drugs regulating STAT-3 expression, lack aqueous solubility; hence hindering efficient bioavailability. A theranostics nanoplatform based on luminescent carbon nanoparticles decorated with cucurbit[6]uril is introduced for enhancing the solubility of niclosamide, a STAT-3 inhibitor. The host guest chemistry between cucurbit[6]uril and niclosamide makes the delivery of the hydrophobic drug feasible while carbon nanoparticles enhance cellular internalization. Extensive physicochemical characterizations confirm successful synthesis. Subsequently, the host guest chemistry of niclosamide and cucurbit[6]uril is studied experimentally and computationally. In vitro assessments in human breast cancer cells indicate ca. two-fold enhancement in IC₅₀ of drug. FT-IR and fluorescence imaging demonstrate efficient cellular internalization concluded from chemical signatures of the nanoparticle’s components. Furthermore, the catalytic biodegradation of the nanoplatforms occur upon exposure to the human myeloperoxidase in a short period. In vivo studies on athymic mice with MCF-7 xenograft indicate the size of the grown tumor in the treatment group is half of the controls after 40 days. Immunohistochemistry corroborates the downregulation of STAT-3 phosphorylation. Overall, the host guest chemistry on nanocarbon can be considered as a novel arsenal for STAT-3 inhibition.

Titanium dioxide, as many other oxides, is abundantly used in everyday products (paints, foods, cosmetic lotions) based on its biocompatibility, low-cost and optical properties (high refractive index and UV absorption). TiO₂ is also a semiconductor (band gap ca. 3.1 eV) with well-established photocatalytic properties. When near-UV photons impinge on the oxide, electron-hole pairs form and react easily with the surrounding medium, creating extremely reactive radicals [1].
Even if most of the TiO₂ powders used in market products are micro-structured, some specific applications require very small particles in the range of 20-50 nm, as is the case of transparent creams used for sun protection. Safety concerns raised that nanoparticles (NPs) of such size, once in contact with the human cells, can reach the cell nucleus acting as carcinogenic agent [2]. In principle, TiO₂ NPs should not be able to overpass the skin stratum basale and enter the vascular system, at least for intact epidermis. Nevertheless, NPs have been observed in the skin stratum corneum [3].
Stratum corneum protects the human skin from environmental hazards, such as UV radiations. Melanin, a polymeric biopigment situated in the stratum corneum as along all the epidermis, is well known to protect human skin from inflammation and oxidative stress caused by the energetic radiation. Such pigment protects the skin by means of light scattering and absorption, even if the barrier mechanism is not well understood yet [4]. It is possible that, if TiO₂ NPs reach the stratum corneum, TiO₂ and melanin experience chemical and, and under irradiation, photophysical interactions. As a matter of fact, indolic-OH groups of melanin sub-units can easily chemisorb at the oxide surfaces[5]. Such organic/inorganic hybrids are prone to show significant electronic interactions.
We report on the interaction between TiO₂ NPs and melanin in the skin (ex-vivo tests) by Raman micro-spectroscopy and confocal optical microscopy, in dark conditions and under irradiation, within the UV-Vis range. The TiO₂ NPs of different sizes and phase compositions are suspended in different emulsions (oil/water and water/oil) to simulate sunscreen, and eventually smeared on the skin. The structure of the TiO₂-melanin interface has been studied by electron microscopy whereas EPR is the key technique to reveal the electronic interactions, under different conditions of illumination. Preliminary results suggest that in the oxide-melanin hybrid there is the generation of a variety of free radicals, with respect to the bare melanin. Results are of primary importance for cosmetic as well as biomedical industries.

[1] Fujishima, A. et al., Surf. Sci. Rep. 2008, 63, 515-582;
[2] Serpone, N. et al., Inorgan. Chim. Acta, 2007, 360, 794-802;
[3] Adachi, K et al., Nanotoxicology, 2010, 4, 296-306;
[4] Santato, C. et al., Pigment Cell Melanoma Res., 2015, 28, 520-544;
[5] Sverjensky, D. A. et al., ACS Energ. Lett., 2012, 28, 17322-17330.

Active materials, like hydrogels, are continuously finding new applications in bioengineering. Researchers have successfully been able to exploit their swell-ability (expanding 10-20 times their initial de-swollen size) to achieve a variety of tasks on the microscale, like bio-sensing and drug delivery. In our work we employ mesoscale computational modeling to design an active micro-capsule capable of capturing nanoparticles from the surrounding solvent. We use a rigid perforated spherical shell that encloses a stimuli-sensitive microgel as our modelling system. The spherical shell has six holes that allow the swelling gel to expand outwards into the outer solvent when an appropriate external stimulus is introduced. The gel is functionalized to bind target nanoparticles enabling their capture while in the swollen phase. Upon removal of the external stimulus the gel retracts back to the microcapsule interior, bringing along with it the captured nanoparticles. We additionally decorate the shell with a polymer brush that prevents nanoparticles from uncontrollably diffusing into the microcapsule interior providing capturing selectivity. Periodic applications of the external stimulus cause the gel to continuously bring in more particles over time.

Functional nanostructures constructed from self-assembly of small aromatic molecules through weak non-covalent interactions such as hydrogen bonding, π-π interaction, and van der Waals forces are one of the most attractive subjects bridging supramolecular chemistry and materials science. In particular, developing stimuli-responsive nanoscale materials is of special interest because of their great potential in various application fields including catalyst, sensors, and drug delivery system. Recently, our group reported various self-assembled nanostructures including nanoparticles, nanofibers, and nanosheets constructed from a series of simple and wholly π-conjugated aromatic molecules bearing cyanostilbene and trifluoromethyl (CF₃) groups.¹ To give these peculiar nanostructured materials stimuli-responsive properties, we introduced specific stimuli-responsive moieties into the molecular structures.
First, a fluoride-responsive organogel compound AZ-TFBH carrying a salicylidene aniline moiety instead of the cyanostilbene linker is reported.² AZ-TFBH readily formed fluorescent organogels with entangled nanoribbon structures in a variety of organic solvents through the cooperative combination force of the π-π stacking, the electrostatic attraction, and the C-F…H-C interaction. The organogel showed a reversible gel-to-sol transition in response to fluoride ions, with considerable changes in optical properties including absorption and fluorescence colors. Mechanistic studies revealed that the triple-channel responses were attributed to the fluoride ion-induced deprotonation of the salicylidene aniline moiety.
Second, a highly sensitive and selective ascorbic acid (AA) probe is reported.³ NN-CN-TFFP, which bears a nitronyl-nitroxide moiety instead of the CF₃-containing aromatic ring, showed 260-fold fluorescence turn-on and diminished electron spin resonance (ESR) signal upon AA addition. The probe could detect AA over broad concentration range from as low as 1 μM up to 2 mM through a combination of fluorescence and ESR responses, which satisfies the needs in various analytic samples such as human blood plasma, serum, food and pharmaceuticals. In addition, the probe showed excellent selectivity over various antioxidants and acids including BHT, hydroxyquinone, Cys, Tyr, GSH, citric acid, CF₃COOH, and HCl. It is noteworthy that the AA concentrations in real samples including Vitamin drink and orange juices were successfully measured by the probe. Furthermore, we also prepared self-assembled nanoparticles and a sensor paper using the probe, and examined AA detection capabilities in aqueous condition and solid-state for bio-imaging applications and convenient detection kit, respectively.

References
1. An, B.-K.; Gierschner, J.; Park, S. Y. Accounts Chem. Res. 2012, 45, 544.
2. Lee, J.; Kwon, J. E.; You, Y.; Park, S. Y. Langmuir 2014, 30, 2842.
3. Nam, H.; Kwon, J. E.; Choi, M.-W.; Seo, J.; Shin, S.; Kim, S.; Park, S. Y. ACS Sens. 2016, 1, 392.

Magnetic-plasmonic hybrid nanoparticles (NPs) have gained much attention as promising bioprobes for simultaneous plasmonic imaging and magnetic manipulation. In fact, these dual functional NPs have a strong potential as an essential building block for next-generation NP-based biomedical technology such as magnetic separation of organelles, magnetic drug targeting techniques or magnetic immunodiagnostics. Great efforts have been made for synthesizing the hybrid NPs such as Ag@FeCo@Ag core-shell-shell NPs. However, there has been one severe problem for these NPs to be applied for biomedicine; that is the degradation of magnetic properties by the oxidation of their magnetic element. In this regard, Au@FeCo core-shell NPs have considerable promise as novel magnetic-plasmonic NPs which provide better oxidation resistivity resulting in more stable magnetic properties. This is because the oxidation of the FeCo shell is thought to be more suppressed in the Au@FeCo NPs than in the Ag@FeCo@Ag NPs. The reason is that the electron transfer from core to FeCo shell can be elucidated by a charge-compensation mechanism. More concretely, the net transferred charge from core atoms to shell atoms could be proportional to the difference in Pauling’s electronegativity (χ) between core element and shell element. The values of χ for Au and Ag are 2.54 and 1.93, respectively, and thus Au cores would be able to transfer larger amount of charge to the FeCo shell than Ag cores leading to the superior oxidation suppression capability to Ag@FeCo@Ag NPs. Therefore we synthesized Au@FeCo NPs by a modified polyol method followed by characterization using TEM-EDS, STEM-HAADF, UV-Vis, XRD, XPS, ICP-OES and SQUID. In this presentation, we mainly focus on characterization of the NPs and discuss magnetic-plasmonic properties for the future biomedical application.

Monodisperse temperature/pH sensitive poly(N,N-diethylacrylamide/acrylic acid) (P(DEA/AAc)) nanogels have been prepared. The resulting P(DEA/AAc) nanogels demonstrated very narrow size distribution with varying compositions of ionic monomer AAc up to 40 wt%. Monodisperse P(DEA/AA) nanogels were synthesized only at a very low concentration of initiator. The phase transition behavior of the nanogels in water can be tuned by pH and temperature. Due to low polydispersity, the nanogels self-assembled into colloidal crystals. The condensed color suspensions and colloidal crystals with different polymer concentration were obtained at different temperature. A sharp Bragg peak is evident in the crystal structure and the reflection peaks are significantly blue-shift with the increasing polymer concentration. In contrast, the crystals of the condensed suspensions with short-range periodic structures disappeared but still exhibited clear colors. Reflection spectra showed that the peak wavelength became a bit longer, much broader and the reflection intensity decreased. An elastic and color crosslinked nanogel networks made by one-pot and rapid light-initiated crosslinking method showed responses to pH and temperature. Furthermore, the interaction between the nanogels and peptide melittin was investigated. The results showed that the increasing content of AAc and larger particle size improved the neutralization activity. The DEA/AAc nanogels will promise to become an integral step for making a valuable naked-eye biosensor of simple, cheap and stable substitutes for antibody-based antidote.

Layered double hydroxides (LDHs) are a large family of chemical compounds exhibiting a wide composition variety which results in a huge number of possible applications such as catalysts, antacids or flame retardants. LDHs have a structure similar to that of brucite, where 2D layers are made of metals hydroxide octahedrons. As for brucite, all layers are neutral, for LDHs some divalent cations are substituted by trivalent ones which generates a positive charge of the layers. In order to ensure electrical neutrality, counter anions are intercalated between the layers and 3D framework is formed.
Low-toxicity, biocompatibility and anion exchange ability made Mg-Al LDH a subject of an intensive research in biological field, due to their potential use as drugs and genes delivery agents. Negatively charged molecules such as drugs or genes can be sandwiched between the LDHs layers which protects them against biological conditions. Moreover, hydroxide layers can be easily dissolved by the acidic media within the cells which results in drug/gene recovery. Those LDH-biomolecule nanohybrids have found several application and recently special interest has been placed on using gene therapy in tissue engineering.^[1]
The nanoparticles size is crucial in all biological applications, since it is directly related to the intercellular uptake and their distribution within the cell. For these reasons, the optimum nanopartciles size for clathrin-mediated endocytosis was established to be 50 - 200 nm.^[2] Nevertheless, another fundamental aspect which has to be considered in nanohybrid preparation is genes intercalation efficiency. It was proposed that the genes intercalation process proceeds in two steps: firstly, genes are adsorbed onto the nanoparticles surface and then, they are inserted into the LDH interlayer spacing.^[3] This implies that the same surface association results in higher degree of intercalation in the case of smaller particles. Regarding this, for those nanoparticles in the ideal size range, genes are mostly attached to the LDH surface, so they are not really protected, while smaller particles (< 50 nm), presenting higher effective surface area, showed much higher genes intercalation efficiency.^[3]
In the present work efficient methods to prepare Mg-Al LDH nanoplatelets with high control over size and shape containing easily exchangeable counter anion have been developed. Nanoplatelets with sizes varying from 25 to 100 nm were produced by a simple co-precipitation method and their geometrical attributes were changed by applying different aging conditions. Preliminary results of DNA-LDH nanohybrid observed inside the cell are very promising in tissue engineering and prospective bone repairing therapy.

[1] C. M. Curtin et al., Advanced Materials 2012, 24, 749-754.
[2] S.-J. Choi et al., Nanomedicine 2011, 6, 803-814.
[3] M. Chen et al., Journal of colloid and interface science 2013, 390, 275-281.

Ion channels are essential units which sustain life in all living cells as continuously transporting proper ions through plasma cell membranes. In addition, the vital functions of ion channels are fundamentally indispensable for sensory organ homeostasis. For decades, biological ion channels have led to much inspiration because of their unique and exquisite operational functions in living cells. Specifically, their extreme and dynamic sensing abilities can be realized by the combination of receptors and pores coupled together to build ion channel systems.
In this presentation, we demonstrate that artificial ion channel pressure sensors inspired by nature for detecting pressure are highly sensitive and wearable. Our ion channel pressure sensors basically consisted of receptors and nanopore membranes, enabling dynamic current responses to external forces for multiple applications. The ion channel pressure sensors had a sensitivity of ∼5.6 kPa⁻¹ and a response time of ∼12 ms at a frequency of 1 Hz. The power consumption was recorded as less than a few μW. Moreover, linear regression was performed in terms of temperature, which showed no significant variations, and there were no significant current variations with humidity. The patchable ion channel pressure sensors were then used to detect blood pressure in humans, and different signals were clearly observed for each person. Additionally, modified ion channel pressure sensors detected complex motions including pressing and folding in a high-pressure range.
In comparison to previous pressure sensors, our ion channel pressure sensors engaged sensory organs could provide feasible control of the bandwidth via selecting different electrolytes and unique signal shape according to the type of pressure receptor chosen, and the dynamic output signal may offer discrete information for an object. We expect that if the diverse functions of ion channels in nature could be further modified or mimicked for various applications, then artificial sensory systems such as the one described in this study would be tremendously greeted in a new era of sensors.

Chlorhexidine, as a broad spectrum antimicrobial agent, is widely used in dentistry. However, a lack a sustained and controllable release is always concerned with its applications, which may lead to repetitive infection and even failure of implants. Herein, we proposed a new method to fabricate spherical chlorhexidine particles and layer-by-layer encapsulation of the chlorhexidine particles to achieve a sustained release. Further functionalizing the particles with nanoparticles (gold nanorods and iron oxide nanoparticles) to enable an external stimuli responsive release.
Chlorhexidine particles with an average dimeter of 17.15 ± 1.99 µm were produced by precipitation of chlorhexidine diacetate and calcium chloride (CaCl₂). To form the homogeneous interconnected spherical drug crystals, the types of ions and the concentration were vital, and the size of particles could further be tuned by temperature. The formation of chlorhexidine particle was proved to be in an Ostwald Ripening mechanism. With the addition of gold nanorods or iron oxide nanoparticles in the system, the manner of chlorhexidine crystal growth was influenced as the nanoparticles acted as nuclei for crystal growth, which increased the final amount of chlorhexidine particles but reduced the mean diameter. Layer-by-layer deposition of polyelectrolytes on the surface of chlorhexidine particles produced capsules which stabilized the drug crystals and enabled a sustained release. The functionalization with gold nanorods and iron oxide nanoparticles enabled the chlorhexidine release responsive to the near-infrared (NIR) light and magnetic field. When the magnetic chlorhexidine particles were incorporated into UDMA-HEMA resin, chlorhexidine particles distribution and release kinetics could be changed by a magnetic field. The encapsulated chlorhexidine particles with gold nanorods could also be ruptured by a focused NIR light beam (840 nm, 100 mW) which was coupled with a microscope, and a stepwise burst chlorhexidine release was triggered by multiple cycles of NIR light illumination. The produced multifunctional chlorhexidine microparticles showed good stimuli responsive properties and will have great prospects in medicine and dentistry.

Self-assembly process of biomolecules has been widely applied to the fabrication of nano-biomaterials such as DNA origami because it can achieve low energy consumption in the fabrication process and fabrication of structures at a high spatial resolution which cannot be achieved by top-down approaches such as photolithography or soft lithography. In spite of these advantages, there are fundamental problems with the generation of defects in the self-assembly process. For in-situ modification of the defects, top-down control of self-assembly process at a nanometer scale is needed.
To achieve integration of top-down and bottom-up fabrication approaches, we have studied in-situ patterning of a virtual cathode in aqueous solutions using an electron beam (EB) [1-3]. In our system, a low energy EB is indirectly irradiated to liquid samples through a very thin silicon nitride (SiN) membrane [1]. Most of the scattered electrons are trapped within the membrane, and an electric field of the trapped electrons can be used as a virtual cathode. We previously reported the electric field around the EB-induced virtual cathode was strong enough to control electrophoresis and electroosmotic flow at the surface of the membrane [2], and the diameter of the virtual cathode was about 120 nm [3]. Therefore, our system can achieve in-situ and nanometer-scale control of the electric field in liquid samples.
In this work, we demonstrated electrical control of the self-assembled structures of DNA molecules in poly(ethylene glycol) (PEG) water solution by modification of ionic environment around the DNA molecules using an EB-induced virtual cathode. The method of the experiment was as follows: Bacteriophage T4 GT7 DNA was labeled with YOYO-1 dye, and then antioxidant, antifaders, and PEG4000 were added. The final concentrations of the DNA and PEG were 35 nMbp and 2.5% (w/v), respectively. 1 mL of the sample solution was dropped onto a 100-nm-thick SiN membrane and a 3.0 keV EB was irradiated in a rectangle region (10 μm x 10 μm) for 1 s.
The results were as follows: Before the EB irradiation, the DNA molecules were in a random coil state and the size of the random coil was about 2 μm. After 250 ms EB irradiation, the DNA strand around the EB irradiated area was elongated and the length of the strand became about 4 μm. This result meant that the self-assembled structure of the DNA molecule was transited from the random coil state to the elongated state. This transition would be due to the shear stress of electroosmotic flow and the electrophoretic force.
It is expected that the proposed method will be applied to the spatiotemporal control of the self-assembled structures of other biomaterials at a nanometer scale by patterning an EB-induced virtual cathode.
References
[1] T. Hoshino et al., Biochem Biophys Res Commun, 432(2), 345-349 (2013).
[2] H. Miyazako et al., Langmuir, 31(23), 6595-6603 (2015).
[3] T. Hoshino, H. Miyazako et al., Sens Actuators B (2016) (in press).

Nanocomposite based biomaterials have been increasingly studied for biomedical applications. Generally, the nanocomposite based biomaterials lack control over essential features such as stimuli responsiveness. Our study is aimed at formulating collagen immobilized Poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) film loaded with bovine serum albumin capped silver nanoparticles (Ag/BSA NPs) that will be stimuli responsive to changes such as pH especially when the pH variation is from physiological to acidic condition. The BSA shell of Ag/BSA nanoparticles will electrostatically interact with collagen grafted chains and loaded on PHBV matrix depending upon the pH and ionic strength of the medium. The nanoparticles loaded collagen immobilized PHBV film was characterized for its composition by X-ray Photoelectron Spectroscopy and Anodic Stripping Voltammetry. The extent of loading of Ag/BSA NPs on collagen immobilized PHBV film was found to depend on the chemistry of the functionalized PHBV film and the concentration of Ag/BSA NPs solution used for loading nanoparticles. Colony forming unit and optical density measurements showed broad antimicrobial activity towards E. coli, S. aureus, and P. aeruginosa at significantly lower concentration i.e., 0.19 and 0.31 µg/disc, compared to gentamicin and sulfamethoxazole trimethoprim while MTT assay showed that released nanoparticles from Ag/BSA NPs loaded collagen immobilized PHBV film has no impact on MCTC3-E1 cells viability. Studies are underway to trigger the release of nanoparticles from the hydrogel matrix by exploiting the interaction between BSA and hyaluronic acid of hydrogel for biomedical applications.
Acknowledgement : US Army Research Office Subcontract 5710004062

The use of waterborne acrylate latex coatings is widespread due to their excellent durability, toughness, optical clarity, UV stability, color retention and environmental friendliness as these are low volatile organic compounds (VOC) coatings. Hybrid nanocomposites consist of an organic polymeric matrix and inorganic fillers with dimensions at the nanometer scale. Waterborne acrylate/ metal oxide nanocomposites were synthesized by in situ emulsion polymerization using semi-batch process. Polymethyl methacrylate (PMMA), acrylic acid (AA) and butyl acrylate (BA) were copolymerized in the presence of alumina, silica and iron oxide nanoparticles. As cast coatings were optically transparent and TEM confirmed excellent dispersion of the nanoparticles throughout the acrylate matrix. SEM and EDS analysis further confirmed the dispersion of nanoparticles throughout the coatings. The nanoparticles induced significantly higher thermal stability and higher glass transition temperature, and these changes were dependent on the type of nanoparticle. Shear rheometry was applied to study the viscoelastic behavior of the coatings as a function of temperature and frequency of oscilaltion. The viscoelastic spectra showed a modification of macromolecular dynamics by the nanoparticles. Finally, the antimicrobial activity of the acrylate hybrid nanocomposite coatings was evaluated by formation of halo of inhibition for up to 7 days when exposed to Staphylococcus aureus bacteria medium.

Hydrogels are unique three-dimensional smart molecular networks made of hydrophilic molecules which can trap and hold large amounts of water or aqueous solutions. Thus, the use of hydrogels as biomaterials (drug delivery devices, scaffolds for tissue engineering, wound dressing systems), contact lenses, superabsorbents, molecular filtration, separation and bio-separation products, continue to increase. In this research, polyethylene glycol (PEG) diacrylate was photocured with polyhedral oligosisquioxane (POSS) and the morphology, thermal, viscoelastic and antibacterial properties were studied. The diacrylates of PEG of 3,350 g/mol and PCL of 10,000 g/mol were photocured in the presence of a crosslinker tetrathiol and photoinitiator. Through scanning electron microscopy (SEM), differential scanning calorimetry (DSC), and shear rheometry the influence of POSS on the physical properties of the hydrogel were investigated. Energy dispersive spectroscopy (EDS) elucidated the dispersion of silicon naocages in the hydrogels. The results showed that, at constant PEG molecular weight, POSS tuned the swelling rate, total swell and pore size of the hydrogels. Moreover, PEG crystal melting transition provided for an activation temperature for shape memory behavior. Furthermore, antimicrobial activity of silver loaded hydrogels was investigated through exposure to Pseudomona aeruginosa and Staphilococcus aureus with inoculation to a controlled cell density. These hybrid nanostructured biomaterials open up opportunities for intelligent substrates for antibacterial properties and drug delivery.

Nanoscale stress-sensing can be used across fields ranging from detection of incipient cracks in structural mechanics to monitoring forces in biological tissues. As premature failure of structural components invariably results from the initiation and incipient growth of small cracks, there is a vital need for auto-responsive structural materials that potentially self-detect and self-respond to environmentally-induced mechanical damage. Such materials have a built-in potential to prevent catastrophic failure in service applications. However, current technologies that can provide for the early self-detection of local stresses associated with incipient cracks are extremely limited. Mechanophoric dyes and piezoresistive materials, for example, are only effective at the millimeter length-scale with relatively low sensitivity; furthermore, such techniques are very challenging to implement “in the field”. In addition, many conventional sensing techniques adversely affect the properties of the host material^. A visible-light, nanoscale sensor with the ability to be embedded into a variety of “smart” structural materials without causing such degradation would be particularly appealing for the potential sensing of impending fractures in service. Furthermore, mechanical stresses exerted by biological tissues can be signatures of disease. Thus, such a sensor, if embedded into soft polymers, could also potentially be of significant use in biological applications such as sensing of stresses in cancer cell proliferation.

We demonstrate how tetrapod quantum dots (tQDs) embedded in block-copolymers act as sensors of tensile/compressive stress. Remarkably, tQDs can detect their own composite dispersion and mechanical properties, with a switch in optomechanical response when tQDs are in direct contact. Using experimental characterizations, atomistic simulations and finite-element analyses, we show that under tensile stress, densely-packed tQDs exhibit a photoluminescence peak shifted to higher energies (“blue-shift”) due to volumetric compressive stress in their core; loosely-packed tQDs exhibit a peak shifted to lower energies (“red-shift”) from tensile stress in the core. The stress-shifts result from the tQD’s unique branched morphology in which the CdS arms act as antennas that amplify the stress in the CdSe core. We show more than three orders of magnitude of tunability of the tQD stress response based on the polymer host matrix composition and mechanical properties as well as the interfacial chemical interactions between the tQD and the polymer. Our nanocomposites exhibit excellent cyclability and scalability with no degraded properties of the host polymer. Colloidal tQDs allow sensing in many materials to potentially enable auto-responsive, smart structural nanocomposites that self-predict impending fracture and self-report biomechanical stresses.

We have developed the colorimetric sensing materials with amyloid fibrils of a-synuclein and 10,12-pentacosadiynoic acid (PCDA) by using specific fatty acid interaction of a-synuclein and structural regularity of amyloid fibrils. PCDA interaction of a-synuclein resulted in proteins self-oligomerization and accelerated a-synuclein fibrillation with localizing PCDA molecules on amyloid fibrils. The PCDA-containing amyloid fibrils prepared by either co-incubating with a-synuclein (AF/PCDA) or mixing with the pre-made amyloid fibrils (AF+PCDA) turned to blue upon UV irradiation and became red with external stimuli of heat, pH and organic solvents. Interestingly, AF/PCDA and AF+PCDA on Whatman filter paper can also exhibit blue color with UV treatment and blue-to-red color transition with heat and organic solvents. The two types of paper sensors from AF/PCDA and AF+PCDA showed substantially different sensitivities toward temperature and solvents because of the molecular freedom of PCDA on amyloid fibrils which is dependent on the tightness of PCDA binding to amyloid fibrils. For example, AF/PCDA-containing paper sensors responded to the higher temperatures from 55^○C to 90^○C showing the blue-to-red color transition, but AF+PCDA on paper detected the lower temperatures from 25^○C to 60^○C. For this same reason, the AF/PCDA sensor showed color changes only after direct contact with the solvents in solution while the AF+PCDA sensor exhibited color transition against organic solvents with a brief exposure to their vapors. Additionally, AF+PCDA was capable of monitoring thermal history of materials by showing blue-to-red color changes in response to the increased temperatures and indicating an exact location of spot exposed to heat. Taken together, amyloid fibrils can provide adequate templates for PCDA to develop into paper-type sensors and then the PCDA-containing amyloid fibrils can be considered as novel polydiacetylene-based thermochromics and solvatochrmic sensors.

Abstract: In the current study, an optimized in vitro BBB model was established using mouse brain endothelial cells and astrocytes. In the meantime, superparamagnetic iron oxide nanoparticle (SPIONs) loaded thermosensitive liposomes (ferri-TSLs) were developed for better BBB penetration and selective targeting of the brain glioma under mild hyperthermia conditions. To specifically target the glioma cells, a new cell-penetrating peptide and an anti-glioma antibody were also conjugated onto the liposome surface. With the encapsulation of SPIONs and doxorubicin (DOX) inside the liposomes, a DOX drug release assay and cellular uptake studies were carried out and the in vitro BBB model was used to indicate the permeability of these samples.

Materials and Methods: Peptide 1-NS [1] and anti-Tenascin C antibody were conjugated onto mal-DSPE-mPEG2000 respectively by a well-established reaction and the ferri-TSLs were then prepared and encapsuled with SPIONs and DOX using the rehydration method [2-3]. TEM was used to assess the iron oxide inner core diameter as well as the liposome diameter. For the in vitro BBB model, astrocytes (ATCC CRL-2541) were seeded onto the bottom of the 24 well plates in complete DMEM media. After 24 hours of adhesion, endothelial cells (ATCC CRL-2299) were seeded onto the upper side of the inserts and the inserts were then placed in the 24-well plates containing astrocytes. The model was evaluated and confirmed using TEER and FITC-dextran transport [4]. After such confirmation, the permeability and DOX drug release efficiency from several functionalized ferri-TSLs were tested against the BBB model in an effort to increase BBB passage and deliver anti-tumor drugs for glioblastoma multiforme effectively while diminishing cytotoxicity. Each experiment was conducted in triplicate and differences between means were determined using ANOVA followed by student t-tests.

Results and Discussion: Results showed that all the functionalized ferri-TSLs had great permeability in the in vitro BBB model. In addition, drug release results indicated that compared to non-TSLs, functionalized TSL had significantly higher drug release efficiency and rate, as well as cellular uptake ability to tumor cells at its transition temperature (42 ^oC).

Conclusions: An in vitro model of BBB was established using a co-culture method. The results suggest a possibility to manipulate magnetic nanocarriers penetration across the BBB by modifying the surface chemistry. Moreover, among all the nanocarriers, functionalized ferri-TSL were found to be a good candidate for brain tumor drug delivery based on its good drug release results and BBB penetration results.

References: [1] Higa, M, et al. Biophys. Res. Commun. 2015;457: 206– 212; [2] Zahraa S. et al. J of Controlled Release. 2014;196:332-343; [3] Li L, et al. J of Controlled Release. 2013;168(2):142-150; [4] Shi D, et al. Nanotech. 2014; 25(7):075-101.

Magnetite (Fe₃O₄) is a favorite material to be used in biomedical applications owing to its remarkable magnetic properties and suitable biocompatibility & biodegradability. It has been widely studies as a material of choice for magnetic hyperthermia therapy, controlled drug delivery, magnetic separation and as a magnetic resonance imaging contrast agent [1-2]. Under an oscillating magnetic field magnetite nanoparticles generate heat due to Néel losses and Brownian rotations in physiological fluids [3]. Magnetite nanoparticles also respond to certain visible light wavelengths near infrared region and are excited upon irradiation. During de-excitation it releases extra energy into molecular vibration modes that transform into thermal energy [4].
Current study is about combining the magnetic hyperthermia and photothermal therapies into a single bimodal system for cancer therapy. The synthesized magnetite nanoparticles were superparamagnetic having appropriate colloidal stability due to being coated with polyethylene glycol. Cell viability and cytotoxicity were studied on HeLa cells using MTT assay and proteome apoptosis kit. They were subjected to an oscillating magnetic field of 375 kHz & 170 Oe for hyperthermia. For photothermal therapy, the system was irradiated with a laser emitting 808 nm wavelength which is a near infrared regime in electromagnetic spectrum. The two treatments were performed separately as well as in combination and the combined treatment was multifold more effective in treating the cancer cells as compared to separate sessions.

References
[1] K.M. Krishnan, IEEE Trans. Magn. 46, 2523 (2010).
[2] S.A. Shah, K.M. Krishnan, et al., Phys. Rev. B, 92, 094438 (2015).
[3] S.A. Shah, et al., Mater. Chem. Phys, 137, 365 (2012).
[4] M. Chu, Y. Shao, et al., Biomaterials, 34, 4078 (2013).

Due to its short plasma half-life, poor membrane penetrability, and lack of targeting ability, naked siRNA cannot be applied clinically. Nanoparticles (NPs) can serve as ideal delivery vehicles for siRNA because they can greatly enhance the siRNA’s stability, facilitate its cellular uptake, and offer tumor-targeting capabilities. However, NPs are taken up by cells through endocytosis. Thus, one major challenge associated with employing NPs as siRNA nanocarriers is their poor endosomal escape ability, thus leading to siRNA degradation and subsequently low gene silencing efficiency. Decomplexation of siRNA from polycationic polymer-based siRNA nanocarriers is also of vital importance in order to enhance gene silencing efficiency. Therefore, there is a need to develop smart polycationic polymer-based siRNA nanocarriers that can effectively deliver intact siRNA to cytosol.
In this study, a unique pH and redox dual-responsive cationic unimolecular NP containing histidine residues was developed for siRNA delivery. The cationic core was used for siRNA complexation and the PEG shell was used to provide the NPs with good water solubility and reduce their opsonization during blood circulation. The cationic polymer segments were conjugated onto the hyperbranched polymer (H40) via a pH-sensitive bond, which can be hydrolyzed at pH 5.0–6.5 to achieve cleavage of the nanocarriers after endocytosis, but is relatively stable at physiological conditions (pH 7.4). The histidine groups in the cationic segment had a pK_a of ∼6.0 and could be protonated in the acidic environment (pH 5.0–6.5) of the endosome, leading to osmotic swelling and endosome-membrane disruption. The cationic segment used for siRNA complexation contained reducible disulfide bonds. After the siRNA/catonic polymer complexes being released from the endosomes to the cytosol, the disulfide bonds underwent reduction owing to the presence of high concentrations of glutathione (GSH) (1–10 mM) in the cytosol, thereby facilitating the decomplexation of siRNA from the cationic polymer segments. GE11 peptide, which can efficiently bind to EFGR overexpressed in triple-negative breast cancer cells, was also conjugated onto the unimolecular NPs to achieve their active targeting ability. The gene silencing efficiencies of siRNA-complexed unimolecular NPs were studied in a GFP-MDA-MB-468 triple-negative breast cancer cell line. The GE11-conjugated GFP-siRNA-complexed NPs down-regulated the expression of GFP efficiently, while the NPs alone did not show any apparent cytotoxicity. These results suggest that this pH/redox dual-responsive unimolecular nanoparticle could be a promising nanoplatform for siRNA delivery.

Human dental pulp stem cells (DPSCs) can differentiate, showing potential for regenerative medicine. Designing artificial surfaces with properties appropriate for the initiation of extracellular matrix (ECM) adsorption and organization is a critical step in tissue engineering and can greatly impact protein adhesion. Sulfonated polystyrene (SPS), used as a scaffold for tissue development, stimulates protein adsorption due to the increased negative charge of sulfonate.
Graphene and graphene oxide (GO) sheets enhance stem cell growth and differentiation because they are soft membranes with “high in-plane” stiffness and have the potential to be a transferable and implantable platform. This research functionalized GO and reduced GO (RGO) with gold or silver nanoparticles, mixing with SPS to investigate their combined impact on DPSC differentiation and protein adsorption, hypothesizing that this combination supplies more charges to better absorb the proteins to the surface and stimulate differentiation.
Results indicate proteins of cells plated on the gold-RGO/SPS surfaces were the most highly adsorbed and most densely packed. Additionally, the cell moduli data indicated that the metal-rGO solutions substantially induced the odontoblastic differentiation because the cells plated on those surfaces were most rigid and therefore early stage differentiation was induced, showing promise of a new efficient scaffold for tissue engineering.

Mesoporous silica nanoparticles have attracted immense interest as drug carriers over the last years, and successful targeted drug delivery has been demonstrated in vitro and occasionally in vivo in small animal models. The flexibility in terms of particle size, shape, and surface chemistry makes it possible to fine-tune the physical and chemical properties to correspond to the requirements of a specific administration route. The presentation will critically discuss biodistribution issues found when moving from in vitro to in vivo studies, and relate these to the physicochemical properties of the nanoparticles controlling the extent of protein adsorption to the particles. Special focus will be put on the optimization of passive targeting of these particles to tumors. In the second part of the presentation approaches towards local administration of the particles for local therapies or tissue engineering applications will be discussed, with focus on organic scaffolds serving as hosts for the mesoporous silica nanoparticles. Efficient control of stem cell differentiation based on local, intracellular drug delivery will be demonstrated.

In this presentation, two examples will be introduced to demonstrate how functionalized nanoparticles can enhance the imaging-guide diagnostics and therapeutics of cancer. One will be presented for better seeing of tumor in early detection for targeted nanotherapy; the other will be addressed for deeper going of therapeutic activation in depth of tissue for selective nanotherapy.

In the first example, we will demonstrate the delivery strategy of targeted anti-cancer drug-nanoparticle conjugate using neural stem cell (NSC)-based carriers in an orthotopic human glioma xenograft model. HB1.F3.CD NSCs are FDA-approved for clinic trials utilizing local injection for treatment of recurrent high-grade glioma. We will first demonstrate that HB1.F3.CD cells could uptake MSN-Dox efficiently and that pH-controlled Dox release from the nanoparticle delayed drug-induced toxicity towards carrier cells, preserving their migratory function in vitro. Secondly, we will establish that MSN-Dox-loaded HB1.F3.CD cells could release MSN-Dox conjugates and cause significant toxicity to surrounding U87 glioma cells in vitro. Lastly, tumor-tropic migration of MSN-Dox-loaded HB1.F3.CD cells in an intracranial U87 xenograft mouse model will be illustrated, resulting in the induction of apoptosis within tumors and improvements in survival. Such local injection of the nano- particle-programmed NSCs enables these cells to disperse throughout the tumor via the tropic migration capacity of NSCs.

In the second part, we will report on the development and evaluation, both in vitro and in vivo, of silica-coated scintillating LaF₃:Tb nanoparticles that demonstrate not only enhanced photoluminescence efficiency but also the ability to generate substantial quantities of cytotoxic singlet oxygen and hydroxyl radical upon X-ray irradiation without the incorporation of any additional photosensitizers. In the absence of X-ray excitation, these scintillating nanoparticles exhibit both excellent biocompatibility and low cytotoxicity in vivo. These silica-coated scintillating LaF₃:Tb nanoparticles serving as photosensitizers hold great promise for X-ray PDT of deep tumors in vivo.

Cell organelles enclosed in membrane boundaries contain multiple enzymes to mediate signal transductions, negotiate metabolic reactions, and modify specific molecules to functional products. In a bio-inspired approach, encapsulating enzymes in hollow nanocarriers to create an optimal nanospace mimicking intracellular organelles would be very interesting and useful. This artificial organelle would be suitable for biomedical applications such as enzyme replacement therapy or controlling cell fate. In this talk, we present the approach of delivering hollow silica nanospheres with encapsulated cargos into biological cells for biological functions of catalysis or sensing. We report a method for synthesizing hollow porous silica nanospheres(HSN) by sol-gel templating water-in-oil (W/O) microemulsions. The formation pathway was studied in situ with small angle X-ray scattering (SAXS). Understanding the mechanism, the size of the HSN can be carefully controlled with synthesis conditions. Ship-in-bottle encapsulating of enzymes or nanoparticles can be easily accomplished during synthesis without harsh treatments. They are then applied as nanoreactors in biocatalysis or nanosensor in biomedicine. Four examples of applications will be presented:

1.Enzyme(HRP) encapsulated hollow silica nanospheres for intracellular biocatalysis: We demonstrated the intracellular catalytic activity of HRP by converting the prodrug indole-3-acetic acid into toxic free-radicals, which were able to reduce the cell viability of cancer cells.
2.Intracellular Implantation of Enzymes in Hollow Silica Nanospheres for Protein Therapy: Cascade System of Superoxide Dismutase and Catalase
3.A Broad-range fluorescent pH Sensor Based on Hollow Mesoporous Silica Nanoparticles with Surface Curvature Effect
4.Enzymatic Nanoreactors of HRP@Hollow Silica for Intracellular Sensing of Reactive Oxygen Species.

Pancreatic ductal adenocarcinoma (PDAC) is a fatal disease that is currently treated by two major drug regimens, namely gemcitabine, and a 4-drug combination known as FOLFIRINOX (oxaliplatin, 5-fluorouracil, Irinotecan, and leucovorin). In addition to the contributions of late diagnosis and early metastatic spread to mortality, a major treatment obstacle is the abundant dysplastic stroma, which provides a barrier to vascular access of chemotherapeutic agents as well as expression of cytidine deaminase, which leads to rapid destruction of gemcitabine. While FOLFIRINOX leads to better survival outcome, the high toxicity levels of irinotecan and oxaliplatin often prevent its use as a first-line treatment regimen. Recently, two therapeutic regimes making use of nanotechnology, was approved by FDA for the treatment of PDAC, namely the use of Abraxane, an albumin nanosphere capable of stromal suppression by the delivery of paclitaxel, as well as Onyvide, a liposomal formulation that delivers Irinotecan. In order to improve nanocarrier delivery of paclitaxel and irinotecan, we developed a mesoporous silica nanoparticle (MSNP) platform that improves drug delivery by increased loading capacity, ability to perform synergistic drug delivery and reduced toxicity due to the stability of the platform over liposomes. My talk will outline the use of lipid bilayer coating of the MSNP for the ratiometric combination of gemcitabine with paclitaxel, with the possibility to achieve synergistic drug delivery at orthotopic PDAC tumors sites, where suppression of the stroma allows increased uptake of activated gemcitabine. I will also demonstrate how the use of a lipid bilayer coated MSNP could be used for remote drug loading of irinotecan, with the ability to outperform a liposomal equivalent because of the increased drug loading capacity and stability, including reduced levels of liver, bone marrow and gastrointestinal tract toxicity. I will also discuss the use of the MSNP delivery system to develop synergistic perturbation of the immune system to attempt to link the improved chemotherapy cytotoxicity to improved tumor immunity.

Metal oxide nanoparticles are widely used in paint, food, sunscreens, cosmetics, plastics, ceramics, and as anti-bacterial agents. Titanium dioxide (TiO₂) nanoparticles are of particular importance as the most common metal oxide nanoparticle, used as photocatalysts, as well as the white pigment in foods, sunscreens, cosmetics, paints, and coatings. The high levels of use of these nanoparticles have raised the question of the effects of long-term exposure and subtle cellular effects. We have recently found that incubation of cells with low, non-cytotoxic, concentrations of TiO₂ nanoparticles, in the absence of UV light, produces a unique oxidative stress response. We used a PCR array to screen 84 oxidative stress-related genes following the incubation of cells with TiO₂ nanoparticles. At the concentrations used, standard measures of cell viability (MTT, LDH, and PI assays) showed no decrease in cell health. However, the PCR array showed that four members of the peroxiredoxin family of anti-oxidant enzymes were altered by ~50%. These enzymes, responsible for the clearance of peroxides from the cellular milieu, are essential to the oxidative stress response of cells. The changes observed for the peroxiredoxins were specific to TiO₂ nanoparticles: experiments with polystyrene nanoparticles showed no change in the peroxiredoxins. Current research is aimed at determining the chemical and biological relationship between metal oxide nanoparticles and cellular oxidative stress in the absence of UV light.

Porous Si nanoparticles can be simultaneously loaded and sealed using aqueous solutions of RNA in the presence of calcium chloride. The resulting core-shell nanostructures consist of an siRNA-loaded nanparticle core infiltrated with an insoluble shell of calcium silicate. The silicate in the shell derives from local dissolution of the pSi matrix, and Ca₂SiO₄ formation is self-limiting. The insoluble calcium silicate shell slows the degradation of the pSiNP skeleton and prolongs delivery of an siRNA payload, resulting in more effective gene knockdown in vitro. The calcium silicate shell increases the external quantum yield of photoluminescence from the porous silicon core due to the electronically passivating nature of the shell. Attachment of functional peptides impart neuronal targeting and cell penetration properrties to the constructs that show improved gene silencing in vitro and improved delivery in vivo. The intrinsic photoluminescence that derives from quantum confinement and surface defect states provides a non-toxic and biodegradable luminescent probe for in vivo and in vitro imaging. The relatively long (microseconds) excited state lifetime of this material can be harnessed for time-gated imaging, providing a theranostic potential.

Nanostructured materials have been developed for various medical and biological applications. They have been designed as stimuli-responsive drug delivery systems and sustained protein delivery systems. Nanocomposite systems have also been derived to provide simultaneous drug delivery and bioimaging functions as theranostic systems. Micellar nanocomplexes have been synthesized with green tea-based ingredients as unique carrier materials that offer synergistic therapeutic effects with the drugs to be delivered.

In addition, nanostructure processing has been employed in creating synthetic cell culture substrates for the expansion and controlled differentiation of stem cells. Nanostructured scaffolds have also been obtained for cell and tissue engineering.

The development of minimally invasive methods to modulate neuronal activity with high spatiotemporal resolution has been of particular interest for neurobiologists. Shapiro et al. and Carvalho-de-Souza et al. have demonstrated two methods by which neurons can be stimulated photothermally. Here, we explore a method by which neural activity can be elicited via a photoelectric effect. We demonstrate that individual gold atoms can be doped along the silicon side-walls during the gold nanoparticle catalyzed chemical vapor deposition of coaxial pin silicon nanowires. These gold atoms affect the surface potential of silicon, enabling the formation of axial diode components within a single nanowire structure, eliciting a photoelectric effect in conjunction with the radial p-n diode structure. Next, we have cultured these modified silicon nanowires with primary dorsal root ganglion rat neurons and showed that upon localized laser light stimulation, these nanowires can efficiently induce action potentials. These findings provide us with a novel method to optically and wirelessly modulate neuronal activity and thus a potential therapeutic strategy for patients suffering from neurodegenerative diseases.

Near-infrared guided switchable nanoparticles for enhanced targeted chemo/photothermal therapy against bladder cancer

Bladder carcinoma is one of the most common malignancies of the genitourinary system. Doxorubicin (DOX), together with methotrexate, vinblastine and cisplatin, is one of the standard chemotherapeutics for treating invasive bladder cancer in clinics. However, the applications of DOX and other chemotherapeutic species can cause severe detrimental side effects to patients, which eventually lead to a poor prognosis.
By taking advantages of enhanced permeation and retention (EPR) effect in tumor vasculature, the nanocarriers preferentially deliver the drugs to tumors with minimal unfavorable drug leakage, which is presumably ascribed to its inherent size and stealth properties. However, it is unlikely to translate the nanomedicines based solely on EPR effect to the clinic. The heterogenous tumor vasculature, such as difference in diameter and density of compressed blood vessels, may significant compromise the efficiency and targeting-specificity of drug delivery to tumors.
To circumvent this problem, we developed an external stimuli-responsive delivery system, based on chitosan/PNIPAAm nanogels encapsulating single-wall carbon nanotubes for chemo/photothermal combination therapy. Thereafter, the application of as-prepared nanocarriers in subcutaneous animal models of bladder cancer and orthotopic superficial bladder cancer, we showed that near-infrared (NIR) light-responsive size-switchable nanocarriers could considerably enhance the tumor-targeting of doxorubicin (DOX) in bladder tumor-bearing mice. Moreover, a synergistic effect of NIR-induced hyperthermia and DOX-mediated chemotherapy resulted in remarkable inhibition of tumor growth in mice. Histological results suggest that the change in morphology of tumor microvasculature together with the photo-induced size shrinkage accounted for enhanced extravasation and accumulation of the nanodrugs upon NIR irradiation. Together, these data suggest that this stimuli-responsive drug delivery system offers a safe and effective means of targeted chemo/photothermal therapy.

We report a fluorescent light-up nanoprobe with aggregation-induced emission (AIE) characteristics based on a fluorogen of (4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)oxy)benzylidene)-4-(1,2,2-triphenylvinyl)aniline (TPE-IPB) for highly specific and high-contrast in vivo imaging of elevated peroxynitrite (ONOO^-) generation in the inflammatory region. Through rational molecular design by introducing imine group to block the emission of tetraphenylethylene (TPE) moiety, the TPE-IPB is non-emissive; however, it is induced to emit intense fluorescence after reaction with ONOO^- at pH 7.0-7.4. It is demonstrated that the resultant reaction product is typically AIE-active and its great turn-on fluorescence is due to the formation of intramolecular hydrogen bonding. Formulation of TPE-IPB using lipid-PEG as the matrix yields uniform and stable AIE light-up nanoprobes. The in vivo studies reveal that the AIE light-up nanoprobe only selectively switches on its fluorescence in the inflammatory region in vivo with elevated generation of ONOO^-. Besides, the nanoprobe also shows a unique merit in monitoring in vivo treatment efficacy of anti-inflammatory agents. In addition, we designed and synthesized a quantum dot-sized organic dot with AIE characteristics as a promising cell tracker for noninvasive long-term tracking of adipose-derived stem cells (ADSCs) in living mice. We demonstrate that the AIE dots possess high fluorescence, strong photobleaching resistance, low in vivo toxicity, excellent retention in living ADSCs and negligible interference on the ADSC pluripotency and secretome. The AIE dots also exhibit far superior in vitro cell tracking capability as compared to the widely used commercial cell trackers of PKH26 and Qtracker 655. The in vivo studies using an ischemic hind limb-bearing mouse model reveal that the AIE dots can precisely and quantitatively report the fate of ADSCs and their therapeutic effect in vivo for 6 weeks. Moreover, we have also synthesized a far-red/near-infrared fluorescent AIEgen-based nanodot, which can efficiently generate reactive oxygen species (ROS) under light irradiation. It is found that the AIEgen-based nanodots are able to visualize tumor tissues and assess tumor margins during surgery in a tumor-bearing mouse model. More importantly, upon tumor removal followed by irradiation of the incision site with light, the AIE nanodots in the residual tumors will generate ROS, which has been demonstrated to successfully suppress the residual tumors and reduce the risk of tumor recurrence after debulking surgery.

One of the greatest challenges in current radiotherapy for cancer treatment is to provide a fatal dose only to a tumor within the tolerance of essential normal tissues. As gold nanoparticles have strong absorption coefficient, utilizing these nanoparticles as radiosensitizing agents have been demonstrated to increase the contrast between the dose absorbed by healthy and cancer cells. However clinical application of gold nanoparticles as radiosensitizing agents during radiotherapy requires detailed understanding of the mechanism mediated the biological effects of these nanoparticles. Reactive Oxygen Species (ROS) which typically include the super oxide radical (O^2-), hydrogen peroxide (H₂O₂), and the hydroxyl radical causes cellular damage including oxidation of lipids, proteins and DNA resulting in apoptotic and necrotic cell death due to mitochondrial dysfunction. It has been proposed that the main mechanism for radiation induced cell death is free radical mediated DNA damage predominantly by hydroxyl radicals (OH) formed due to radiolysis of water. It has been recently shown that hydroxyl radicals (OH) generated at the water-nanoparticle interface are responsible for 70% of DNA damage in cancer cells. Therefore, the surface of the nanoparticle plays a critical role on the OH generation yield during irradiation.
To furnish insight into effect of nanoparticles’ size on the OH generation yield during X-ray radiation, same concentration of gold nanoparticle solutions with different size have been separately added to 0.5 mM Coumarin solutions, followed by irradiation for various time duration which leads to different radiation dose. Quantifying the OH radical production yield through fluorescent measurements of Coumarin solution after irradiation show increase in OH generation with decrease in size of gold nanoparticles.

Acknowledgements: this work is supported by department of Chemical Engineering at Northeastern University.

Transdermal drug delivery is an attractive method for drug delivery compared to the traditional needle injection and oral administration methods. However, it is well known that stratum corneum of skin is a hydrophobic barrier which impedes delivery of hydrophilic macromolecules such as proteins. Many approaches such as microneedles, thermal ablation, iontophoresis and photomechanical waves have been studied in ordert to achieve the transdermal protein delivery. In our research, we developed a thermal ablation technique of stratum corneum mediated by photothermal effect of gold nanorods. Gold nanorods have strong absorption band at near-infrared region and can be heated by near-infrared light irradiation, and is so-called photothermal effect. Therefore, we hypothesized that it should be possible to heat only the stratum corneum layer by applying the gold nanorods directly onto the skin surface and then following the irradiation to this area with near-infrared light.
We prepared a transparent gel patch made of polysaccharides (gellan gum and chondroitin sulfate) with gold nanorods on the gel surface and FITC-labelled ovalbumin (FITC-OVA) therein as a model protein. The gel patch was put on mouse skin to be directly touched with the coated gold nanorods. It was then irradiated by a continuous-wave laser for 10 min at 150 mW. The laser irradiation heated the gold nanorods and the skin temperature increased to 43°C. In this condition, significant translocation of OVA into epidermis through the stratum corneum occurred. In contrast, no translocation of FITC-OVA into the skin was observed for the control gel without the gold nanorods. These results confirmed the capability of the transdermal protein delivery system to perforate the stratum corneum and thus facilitate the passage of proteins across the skin.