Localized cytotoxic T cell-associated antigen 4 and antioxidant islet encapsulation alters macrophage signaling and induces regulatory and anergic T cells to enhance allograft survival.

The loss of functional ß-cell mass is a hallmark of type 1 diabetes. Islet transplantation represents a promising alternative approach, but immune-mediated graft destruction remains a major challenge. We sought to use islet encapsulation technologies to improve graft survival and function without systemic immunosuppression. We hypothesized islet encapsulation with nanothin coatings consisting of tannic acid (TA), an antioxidant; poly(N-vinylpyrrolidone) (PVPON), a biocompatible polymer; and cytotoxic T cell-associated antigen 4 immunoglobulin (CTLA-4-Ig), an inhibitory immune receptor, will elicit localized immunosuppression to prolong islet allograft function and suppress effector T cell responses. In the absence of systemic immunosuppression, we demonstrated (PVPON/TA/CTLA-4-Ig)-encapsulated NOD.Rag islet grafts maintain function significantly longer than control IgG-containing (PVPON/TA/IgG) and nonencapsulated controls after transplantation into diabetic C57BL/6 mice. This protection coincided with diminished proinflammatory macrophage responses mediated by signal transducer and activator of transcription 1 signaling, decreased proinflammatory T cell effector responses, and CTLA-4-Ig-specific concomitant increases in anergic CD4+ T cells and regulatory T cells. Our results provide evidence that conjugation of CTLA-4-Ig to (PVPON/TA) coatings can suppress T cell activation, enhance regulatory T cell populations, prolong islet allograft survival, and induce localized immunosuppression after transplantation.

Direct Radiolabeling of Trastuzumab-Targeting Triblock Copolymer Vesicles with 89Zr for Positron Emission Tomography Imaging.

Radiolabeled drug nanocarriers that can be easily imaged via positron emission tomography (PET) are highly significant as their in vivo outcome can be quantitatively PET-traced with high sensitivity. However, typical radiolabeling of most PET-guided theranostic vehicles utilizes modification with chelator ligands, which presents various challenges. In addition, unlike passive tumor targeting, specific targeting of drug delivery vehicles via binding affinity to overexpressed cancer cell receptors is crucial to improve the theranostic delivery to tumors. Herein, we developed 89Zr-labeled triblock copolymer polymersomes of 60 nm size through chelator-free radiolabeling. The polymersomes are assembled from poly(N-vinylpyrrolidone)5-b-poly(dimethylsiloxane)30-b-poly(N-vinylpyrrolidone)5 (PVPON5-PDMS30-PVPON5) triblock copolymers followed by adsorption of a degradable tannin, tannic acid (TA), on the polymersome surface through hydrogen bonding. TA serves as an anchoring layer for both 89Zr radionuclide and targeting recombinant humanized monoclonal antibody, trastuzumab (Tmab). Unlike bare PVPON5-PDMS30-PVPON5 polymersomes, TA- and Tmab-modified polymersomes demonstrated a high radiochemical yield of more than 95%. Excellent retention of 89Zr by the vesicle membrane for up to 7 days was confirmed by PET in vivo imaging. Animal biodistribution using healthy BALB/c mice confirmed the clearance of 89Zr-labeled polymersomes through the spleen and liver without their accumulation in bone, unlike the free nonbound 89Zr radiotracer. The 89Zr-radiolabeled polymersomes were found to specifically target BT474 HER2-positive breast cancer cells via the Tmab-TA complex on the vesicle surface. The noncovalent Tmab anchoring to the polymersome membrane can be highly advantageous for nanoparticle modification compared to currently developed covalent methods, as it allows easy and quick integration of a broad range of targeting proteins. Given the ability of these polymersomes to encapsulate and release anticancer therapeutics, they can be further expanded as precision-targeted therapeutic carriers for advancing human health through highly effective drug delivery strategies.

Two-Dimensional and Three-Dimensional Ultrathin Multilayer Hydrogels through Layer-by-Layer Assembly.

Stimuli-responsive multilayer hydrogels have opened new opportunities to design hierarchically organized networks with properties controlled at the nanoscale. These multilayer materials integrate structural, morphological, and compositional versatility provided by alternating layer-by-layer polymer deposition with the capability for dramatic and reversible changes in volumes upon environmental triggers, a characteristic of chemically cross-linked responsive networks. Despite their intriguing potential, there has been limited knowledge about the structure-property relationships of multilayer hydrogels, partly because of the challenges in regulating network structural organization and the limited set of the instrumental pool to resolve structure and properties at nanometer spatial resolution. This Feature Article highlights our recent studies on advancing assembly technologies, fundamentals, and applications of multilayer hydrogels. The fundamental relationships among synthetic strategies, chemical compositions, and hydrogel architectures are discussed, and their impacts on stimuli-induced volume changes, morphology, and mechanical responses are presented. We present an overview of our studies on thin multilayer hydrogel coatings, focusing on controlling and quantifying the degree of layer intermixing, which are crucial issues in the design of hydrogels with predictable properties. We also uncover the behavior of stratified "multicompartment" hydrogels in response to changes in pH and temperature. We summarize the mechanical responses of free-standing multilayer hydrogels, including planar thin coatings and films with closed geometries such as hollow microcapsules and nonhollow hydrogel microparticles with spherical and nonspherical shapes. Finally, we will showcase potential applications of pH- and temperature-sensitive multilayer hydrogels in sensing and drug delivery. The knowledge about multilayer hydrogels can advance the rational design of polymer networks with predictable and well-tunable properties, contributing to modern polymer science and broadening hydrogel applications.

Dually Responsive Poly(N-vinylcaprolactam)-b-poly(dimethylsiloxane)-b-poly(N-vinylcaprolactam) Polymersomes for Controlled Delivery.

Limited tissue selectivity and targeting of anticancer therapeutics in systemic administration can produce harmful side effects in the body. Various polymer nano-vehicles have been developed to encapsulate therapeutics and prevent premature drug release. Dually responsive polymeric vesicles (polymersomes) assembled from temperature-/pH-sensitive block copolymers are particularly interesting for the delivery of encapsulated therapeutics to targeted tumors and inflamed tissues. We have previously demonstrated that temperature-responsive poly(N-vinylcaprolactam) (PVCL)-b-poly(dimethylsiloxane) (PDMS)-b-PVCL polymersomes exhibit high loading efficiency of anticancer therapeutics in physiological conditions. However, the in-vivo toxicity of these polymersomes as biocompatible materials has not yet been explored. Nevertheless, developing an advanced therapeutic nanocarrier must provide the knowledge of possible risks from the material's toxicity to support its future clinical research in humans. Herein, we studied pH-induced degradation of PVCL10-b-PDMS65-b-PVCL10 vesicles in-situ and their dually (pH- and temperature-) responsive release of the anticancer drug, doxorubicin, using NMR, DLS, TEM, and absorbance spectroscopy. The toxic potential of the polymersomes was evaluated in-vivo by intravenous injection (40 mg kg-1 single dose) of PVCL10-PDMS65-PVCL10 vesicles to mice. The sub-acute toxicity study (14 days) included gravimetric, histological, and hematological analyses and provided evidence for good biocompatibility and non-toxicity of the biomaterial. These results show the potential of these vesicles to be used in clinical research.

Xenotransplantation of tannic acid-encapsulated neonatal porcine islets decreases proinflammatory innate immune responses.

BACKGROUND: Islet transplantation with neonatal porcine islets (NPIs) is a promising treatment for type 1 diabetes (T1D), but immune rejection poses a major hurdle for clinical use. Innate immune-derived reactive oxygen species (ROS) synthesis can facilitate islet xenograft destruction and enhance adaptive immune responses. METHODS: To suppress ROS-mediated xenograft destruction, we utilized nanothin encapsulation materials composed of multilayers of tannic acid (TA), an antioxidant, and a neutral polymer, poly(N-vinylpyrrolidone) (PVPON). We hypothesized that (PVPON/TA)-encapsulated NPIs will maintain euglycemia and dampen proinflammatory innate immune responses following xenotransplantation. RESULTS: (PVPON/TA)-encapsulated NPIs were viable and glucose-responsive similar to non-encapsulated NPIs. Transplantation of (PVPON/TA)-encapsulated NPIs into hyperglycemic C57BL/6.Rag or NOD.Rag mice restored euglycemia, exhibited glucose tolerance, and maintained islet-specific transcription factor levels similar to non-encapsulated NPIs. Gene expression analysis of (PVPON/TA)-encapsulated grafts post-transplantation displayed reduced proinflammatory Ccl5, Cxcl10, Tnf, and Stat1 while enhancing alternatively activated macrophage Retnla, Arg1, and Stat6 mRNA accumulation compared with controls. Flow cytometry analysis demonstrated significantly reduced innate immune infiltration, MHC-II, co-stimulatory molecule, and TNF expression with concomitant increases in arginase-1+ macrophages and dendritic cells. Similar alterations in immune responses were observed following xenotransplantation into immunocompetent NOD mice. CONCLUSION: Our data suggest that (PVPON/TA) encapsulation of NPIs is an effective strategy to decrease inflammatory innate immune signals involved in NPI xenograft responses through STAT1/6 modulation without compromising islet function.

Shape Recovery of Spherical Hydrogen-Bonded Multilayer Capsules after Osmotically Induced Deformation.

The mechanical properties of microparticles intended for in vivo applications as drug delivery vehicles are among important parameters that influence their circulation in the blood and govern particle biodistribution. We report on the synthesis of soft but mechanically robust spherical capsules via a hydrogen-bonded multilayer assembly of (poly(N-vinylpyrrolidone), Mw = 10 000 g mol-1) with (poly(methacrylic acid) Mw = 100 000 g mol-1)) (PVPON/PMAA)n in methanol using 4 µm nonporous silica microparticles as sacrificial templates, where n = 5 and 10 and represents the bilayer number. The mechanical properties of (PVPON/PMAA)n spherical capsules were assessed using the osmotic pressure difference method and resulted in an elasticity modulus of 97 ± 8 MPa, which is in the range of Young's modulus for elastomeric networks. We also found that hydrogen-bonded (PVPON/PMAA)10 capsules demonstrated almost complete recovery from a concave buckled inward shape induced by the osmotic pressure difference from the addition of polystyrene sulfonate (PSS) to the capsule solution to their initial spherical shape within 12 h after the PSS solution was rinsed off. The permeability measurements through the capsule shell using fluorescently labeled dextran molecular probes revealed that the average mesh size of the hydrogen-bonded network assembled in methanol is in the range of 3 to 9 nm and is not permeable to FITC-dextran with a molecular weight of >40 000 g mol-1. Our study shows that physically cross-linked polyelectrolyte multilayer capsules are capable of withstanding large deformations, which is essential to the development of adaptable particles for controlled delivery.

Temperature-Responsive Polymersomes of Poly(3-methyl-N-vinylcaprolactam)-block-poly(N-vinylpyrrolidone) To Decrease Doxorubicin-Induced Cardiotoxicity.

Despite being one of the most potent chemotherapeutics, doxorubicin (DOX) facilitates cardiac toxicity by irreversibly damaging the cardiac muscle as well as severely dysregulating the immune system and impairing the resolution of cardiac inflammation. Herein, we report synthesis and aqueous self-assembly of nanosized polymersomes from temperature-responsive poly(3-methyl-N-vinylcaprolactam)-block-poly(N-vinylpyrrolidone) (PMVC-PVPON) diblock copolymers and demonstrate their potential to minimize DOX cardiotoxicity compared to liposomal DOX. RAFT polymerization of vinylpyrrolidone and 3-methyl-N-vinylcaprolactam, which are structurally similar monomers but have drastically different hydrophobicity, allows decreasing the cloud point of PMVCm-PVPONn copolymers below 20 °C. The lower critical solution temperature (LCST) of the PMVC58-PVPONn copolymer varied from 19.2 to 18.6 and to 15.2 °C by decreasing the length of the hydrophilic PVPONn block from n = 98 to n = 65 and to n = 20, respectively. The copolymers assembled into stable vesicles at room temperature when PVPON polymerization degrees were 65 and 98. Anticancer drug DOX was entrapped with high efficiency into the aqueous PMVC58-PVPON65 polymersomal core surrounded by the hydrophobic temperature-sensitive PMVC shell and the hydrophilic PVPON corona. Unlike many liposomal, micellar, or synthetic drug delivery systems, these polymersomes exhibit an exceptionally high loading capacity of DOX (49%) and encapsulation efficiency (95%) due to spontaneous loading of the drug at room temperature from aqueous DOX solution. We also show that C57BL/6J mice injected with the lethal dose of DOX at 15 mg kg-1 did not survive the 14 day treatment, resulting in 100% mortality. The DOX-loaded PMVC58-PVPON65 polymersomes did not cause any mortality in mice indicating that they can be used for successful DOX encapsulation. The gravimetric analyses of the animal organs from mice treated with liposome-encapsulated DOX (Lipo-DOX) and PMVC58-PVPON65 polymersomes (Poly-DOX) revealed that the Lipo-DOX injection caused some toxicity manifesting as decreased body weight compared to Poly-DOX and saline control. Masses of the left ventricle of the heart, lung, and spleen reduced in the Lipo-DOX-treated mice compared to the nontoxic saline control, while no significant decrease of those masses was observed for the Poly-DOX-treated mice. Our results provide evidence for superior stability of synthetic polymersomes in vivo and show promise for the development of next-generation drug carriers with minimal side effects.

Multilayer Hydrogel Capsules of Interpenetrated Network for Encapsulation of Small Molecules.

We report on a facile capsule-based platform for efficient encapsulation of a broad spectrum of hydrophilic compounds with molecular weight less than 1000 g mol-1. The encapsulated compounds extend from low-molecular-weight anionic Alexa Fluor 532 dye and cationic anticancer drug doxorubicin (DOX) to fluorescein isothiocyanate-dextrans with Mw ranging from 4000 to 40 000 g mol-1. The pH-sensitive hydrogel capsules with an interpenetrated network shell are synthesized by layer-by-layer assembly of poly(methacrylic acid) (PMAA, Mw = 150 000 g mol-1) and poly( N-vinylpyrrolidone) (PVPON, Mw = 1 300 000 g mol-1) on 5 µm silica microparticles followed by chemical cross-linking of the PMAA multilayers. Following core dissolution, the result is a hollow microcapsule with PVPON interpenetrated in the PMAA network. The capsules exhibit a reversible change in the diameter with a swelling ratio of 1.5 upon pH variation from 7.5 to 5.5. Capsules cross-linked for 4 h display high permeability toward molecules with molecular weight under 1000 g mol-1 at pH = 7.5 but exclude dextran molecules with Mw ≥ 40 000 g mol-1. Encapsulation of small molecules was achieved at pH = 7.5 followed by sealing the capsule wall with 40 000 g mol-1 dextran at pH = 5.5. This approach results in negatively charged molecules such as Alexa Fluor being entrapped within the capsule cavity, whereas positively charged molecules such as DOX are encapsulated within the negatively charged capsule shell. Considering the simple postloading approach, the ability to entrap both anionic and cationic small molecules, and the pH-responsiveness of the interpenetrated network in the physiologically relevant range, these capsules offer a versatile method for controlled delivery of multiple hydrophilic compounds.

Peptide-Functionalized Hydrogel Cubes for Active Tumor Cell Targeting.

Conjugation of bioactive targeting molecules to nano- or micrometer-sized drug carriers is a pivotal strategy to improve their therapeutic efficiency. Herein, we developed pH- and redox-sensitive hydrogel particles with a surface-conjugated cancer cell targeting ligand for specific tumor-targeting and controlled release of the anticancer drug doxorubicin. The poly(methacrylic acid) (PMAA) hydrogel cubes of 700 nm and 2 µm with a hepsin-targeting (IPLVVPL) surface peptide are produced through multilayer polymer assembly on sacrificial cubical mesoporous cores. Direct peptide conjugation to the disulfide-stabilized hydrogels through a thiol-amine reaction does not compromise the structural integrity, hydrophilicity, stability in serum, or pH/redox sensitivity but does affect internalization by cancer cells. The cell uptake kinetics and the ultimate extent of internalization are controlled by the cell type and hydrogel size. The peptide modification significantly promotes the uptake of the 700 nm hydrogels by hepsin-positive MCF-7 cells due to ligand-receptor recognition but has a negligible effect on the uptake of 2 µm PMAA hydrogels. The selectivity of 700 nm IPLVVPL-PMAA hydrogel cubes to hepsin-overexpressing tumor cells is further confirmed by a 3-10-fold higher particle internalization by hepsin-positive MCF-7 and SK-OV-3 compared to that of hepsin-negative PC-3 cells. This work provides a facile method to fabricate enhanced tumor-targeting carriers of submicrometer size and improves the general understanding of particle design parameters for targeted drug delivery.

Polyphenolic Polymersomes of Temperature-Sensitive Poly(N-vinylcaprolactam)-block-Poly(N-vinylpyrrolidone) for Anticancer Therapy.

We report a versatile synthesis for polyphenolic polymersomes of controlled submicron (<500 nm) size for intracellular delivery of high and low molecular weight compounds. The nanoparticles are synthesized by stabilizing the vesicular morphology of thermally responsive poly(N-vinylcaprolactam)n-b-poly(N-vinylpyrrolidone)m (PVCLn-PVPONm) diblock copolymers with tannic acid (TA), a hydrolyzable polyphenol, via hydrogen bonding at a temperature above the copolymer's lower critical solution temperature (LCST). The PVCL179-PVPONm diblock copolymers are produced by controlled reversible addition-fragmentation chain transfer (RAFT) polymerization of PVPON using PVCL as a macro-chain transfer agent. The size of the TA-locked (PVCL179-PVPONm) polymersomes at room temperature and upon temperature variations are controlled by the PVPON chain length and TA:PVPON molar unit ratio. The particle diameter decreases from 1000 to 950, 770, and 250 nm with increasing PVPON chain length (m = 107, 166, 205, 234), and it further decreases to 710, 460, 290, and 190 nm, respectively, upon hydrogen bonding with TA at 50 °C. Lowering the solution temperature to 25 °C results in a slight size increase for vesicles with longer PVPON. We also show that TA-locked polymersomes can encapsulate and store the anticancer drug doxorubicin (DOX) and higher molecular weight fluorescein isothiocyanate (FITC)-dextran in a physiologically relevant pH and temperature range. Encapsulated DOX is released in the nuclei of human alveolar adenocarcinoma tumor cells after 6 h incubation via biodegradation of the TA shell with the cytotoxicity of DOX-loaded polymersomes being concentration-dependent. Our approach offers biocompatible and intracellular degradable nanovesicles of controllable size for delivery of a variety of encapsulated materials. Considering the particle monodispersity, high loading capacity, and a facile two-step aqueous assembly based on the reversible temperature-responsiveness of PVCL, these polymeric vesicles have significant potential as novel drug nanocarriers and provide a new perspective for fundamental studies on thermo-triggered polymer assemblies in solutions.

Aqueous RAFT Synthesis of Glycopolymers for Determination of Saccharide Structure and Concentration Effects on Amyloid ß Aggregation.

GM1 ganglioside is known to promote amyloid-ß (Aß) peptide aggregation in Alzheimer's disease. The roles of the individual saccharides and their distribution in this process are not understood. Acrylamide-based glycomonomers with either ß-d-glucose or ß-d-galactose pendant groups were synthesized to mimic the stereochemistry of saccharides present in GM1 and characterized via 1H NMR and electrospray ionization mass spectrometry. Glycopolymers of different molecular weights were synthesized by aqueous reversible addition-fragmentation chain transfer (aRAFT) polymerization and characterized by NMR and GPC. The polymers were used as models to investigate the effects of molecular weight and saccharide unit type on Aß aggregation via thioflavin-T fluorescence and PAGE. High molecular weight (∼350 DP) glucose-containing glycopolymers had a profound effect on Aß aggregation, promoting formation of soluble oligomers of Aß and limiting fibril production, while the other glycopolymers and negative control had little effect on the Aß propagation process.

Small Angle Scattering for Pharmaceutical Applications: From Drugs to Drug Delivery Systems.

The sub-nanometer scale provided by small angle neutron and X-ray scattering is of special importance to pharmaceutical and biomedical investigators. As drug delivery devices become more functionalized and continue decreasing in size, the ability to elucidate details on size scales smaller than those available from optical techniques becomes extremely pertinent. Information gathered from small angle scattering therefore aids the endeavor of optimizing pharmaceutical efficacy at its most fundamental level. This chapter will provide some relevant examples of drug carrier technology and how small angle scattering (SAS) can be used to solve their mysteries. An emphasis on common first-step data treatments is provided which should help clarify the contents of scattering data to new researchers. Specific examples of pharmaceutically relevant research on novel systems and the role SAS plays in these studies will be discussed. This chapter provides an overview of the current applications of SAS in drug research and some practical considerations for selecting scattering techniques.

Melting of gelatin gels confined to silica nanopores.

Nanoconfinement is a way to create materials whose properties differ from the bulk. For the first time, this research explores the effect of nanoconfinement on the thermodynamics and kinetics of gel melting. Differential scanning calorimetry has been employed to study gelatin gels prepared inside 4, 6, 15, and 30 nm pores of a silica matrix. It has been found that with decreasing the pore size the heat of melting decreases from 3.5 J g-1 in bulk to 0.6 J g-1 in 6 nm pores, which is linked to a decrease in crosslinks formed via hydrogen bonding. Despite decreases in crosslink formation, the melting temperature for gels confined to 6 nm pores increased nearly 10 °C compared to bulk gel. In 4 nm pores, no gel melting was observed. Isoconversional kinetic analysis of the melting data has revealed that the increase in thermal stability is associated with a decrease in the pre-exponential factor that occurs upon nanoconfinement. The origins of the effect have been linked to diminished molecular mobility of the gelatin chains confined inside the nanopores, which leads to enhanced restoration of broken crosslinks.

Tuning assembly and enzymatic degradation of silk/poly(N-vinylcaprolactam) multilayers via molecular weight and hydrophobicity.

We report on enzymatically degradable nanothin coatings obtained by layer-by-layer (LbL) assembly of silk fibroin with poly(N-vinylcaprolactam) (PVCL) via hydrogen bonding and hydrophobic interactions. We found that both silk ß-sheet content, controlled through dipping and spin-assisted LbL, and PVCL molecular weight regulate film thickness, microstructure, pH-stability, and biodegradability with a nanoscale precision. Thickness of (silk/PVCL) films increased with increase in PVCL molecular weight and decrease in deposition pH. The impact of assembly pH on film growth was more dramatic for dipped films. These systems show a significant rise in thickness with increase in PVCL molecular weight at pH < 5 but become independent on polymer chain length at pH ≥ 5. We also found that spin-assisted films exhibited a greater stability at elevated pH and against enzymatic degradation as compared to their dipped counterparts. For both film types, the pH and enzymatic stability was improved with increasing PVCL length and ß-sheet content, indicating enhanced hydrophobic and hydrogen-bonded interactions between PVCL and silk. Finally, we fabricated spherical and cubical (silk/PVCL) LbL capsules of regulated permeability and enzymatic degradation. Our approach gives a unique opportunity to tune thickness, morphology, structure, and biodegradability rate of silk films and capsules by varying silk secondary structure and PVCL length. Accounting for all-aqueous fabrication and the biocompatibility of both polymers these biodegradable materials provide novel platforms for delivery systems and medical devices.

Encapsulation of anticancer drug by hydrogen-bonded multilayers of tannic acid.

Tannic acid (TA)-based multilayer assemblies have attracted increasing interest for biomedical applications. Here we explore properties of TA-poly(N-vinylpyrrolidone) (TA-PVPON) hydrogen-bonded multilayers for drug encapsulation and long-term storage. We demonstrate that the small molecular weight anticancer drug, doxorubicin (DOX), can be successfully loaded into (TA-PVPON) capsules with high encapsulation efficiency. We have also found that the encapsulated DOX can be efficiently stored inside the capsules for the pH range from pH = 7.4 to pH = 5. We show that the chemical and functional stability of TA at neutral and basic pH values is achieved through complexation with PVPON. The UV-vis spectrophotometry and in situ ellipsometry analyses of the hydrogen bonding interactions between TA and PVPON at different pH values reveal pH-dependent behavior of TA-PVPON capsules for the pH range from pH = 7.4 to pH = 5. Increasing deposition pH value from pH = 5 to pH = 7.4 leads to a 2-fold decrease in capsule thickness. However, this trend is reversed when salt concentration of the deposition solutions is increased from 0.01 M to 0.1 M NaCl. We have also demonstrated that the permeability of (TA-PVPON) capsules prepared using low salt deposition conditions and pH = 7.4 can be increased 2-fold by exposure of the capsules to 0.1 M NaCl salt solutions at the same pH. Our work opens new perspectives for design of novel polymer carriers for controlled drug delivery in cancer therapy.

Spatially controlled fabrication of a bright fluorescent nanodiamond-array with enhanced far-red Si-V luminescence.

We demonstrate a novel approach to precisely pattern fluorescent nanodiamond-arrays with enhanced far-red intense photostable luminescence from silicon-vacancy (Si-V) defect centers. The precision-patterned pre-growth seeding of nanodiamonds is achieved by a scanning probe 'dip-pen' nanolithography technique using electrostatically driven transfer of nanodiamonds from 'inked' cantilevers to a UV-treated hydrophilic SiO2 substrate. The enhanced emission from nanodiamond dots in the far-red is achieved by incorporating Si-V defect centers in a subsequent chemical vapor deposition treatment. The development of a suitable nanodiamond ink and mechanism of ink transport, and the effect of humidity and dwell time on nanodiamond patterning are investigated. The precision patterning of as-printed (pre-CVD) arrays with dot diameter and dot height as small as 735 nm ± 27 nm and 61 nm ± 3 nm, respectively, and CVD-treated fluorescent ND-arrays with consistently patterned dots having diameter and height as small as 820 nm ± 20 nm and, 245 nm ± 23 nm, respectively, using 1 s dwell time and 30% RH is successfully achieved. We anticipate that the far-red intense photostable luminescence (~738 nm) observed from Si-V defect centers integrated in spatially arranged nanodiamonds could be beneficial for the development of next generation fluorescence-based devices and applications.

Biocompatible shaped particles from dried multilayer polymer capsules.

We demonstrated a simple and facile approach to fabricate biocompatible monodisperse hollow microparticles of controlled geometry. The hemispherical, spherical, and cubical microparticles are obtained by drying multilayer capsules of hydrogen-bonded poly(N-vinylpyrrolidone)/tannic acid (PVPON/TA)n. Drying spherical capsules results in hemispherical particles if 15 < n < 20. This shape transformation is controlled by capsule stiffness, which is regulated by the layer number, capsule diameter, and PVPON molecular weight. Cubical and spherical hollow particles maintaining their three-dimensional shapes in the dry state are obtained if n ≥ 25.5. A 17-fold stiffness increase is required to lead from totally collapsed (PVPON/TA)5.5 to dried self-supporting (PVPON/TA)25.5 particles of 2 µm in dimensions. All hollow particles could be further resuspended in aqueous solutions while retaining their shapes upon rehydration. The cell growth and viability studies using human cancer cells revealed noncytotoxic properties of the (PVPON/TA) multilayer particles. Both spherical and hemispherical capsules were internalized by macrophages with the uptake of the hemispherical particles per cell two times more efficient. The method presented here allows for a robust preparation of biocompatible shaped particles whose shape and dimensions can be easily tuned by controlling capsule size and wall thickness. The reported structures can be potentially useful for biomedical applications such as shape-controlled cellular uptake and flow dynamics.

Hydrogel-Encapsulated Biofilm Inhibitors Abrogate the Cariogenic Activity of Streptococcus mutans.

We designed and synthesized analogues of a previously identified biofilm inhibitor IIIC5 to improve solubility, retain inhibitory activities, and to facilitate encapsulation into pH-responsive hydrogel microparticles. The optimized lead compound HA5 showed improved solubility of 120.09 µg/mL, inhibited Streptococcus mutans biofilm with an IC50 value of 6.42 µM, and did not affect the growth of oral commensal species up to a 15-fold higher concentration. The cocrystal structure of HA5 with GtfB catalytic domain determined at 2.35 Å resolution revealed its active site interactions. The ability of HA5 to inhibit S. mutans Gtfs and to reduce glucan production has been demonstrated. The hydrogel-encapsulated biofilm inhibitor (HEBI), generated by encapsulating HA5 in hydrogel, selectively inhibited S. mutans biofilms like HA5. Treatment of S. mutans-infected rats with HA5 or HEBI resulted in a significant reduction in buccal, sulcal, and proximal dental caries compared to untreated, infected rats.

Site-oriented conjugation of poly(2-methacryloyloxyethyl phosphorylcholine) for enhanced brain delivery of antibody.

Antibody therapeutics are limited in treating brain diseases due to poor blood-brain barrier (BBB) penetration. We have discovered that poly 2-methacryloyloxyethyl phosphorylcholine (PMPC), a biocompatible polymer, effectively facilitates BBB penetration via receptor-mediated transcytosis and have developed a PMPC-shell-based platform for brain delivery of therapeutic antibodies, termed nanocapsule. Yet, the platform results in functional loss of antibodies due to epitope masking by the PMPC polymer network, which necessitates the incorporation of a targeting moiety and degradable crosslinker to enable on-site antibody release. In this study, we developed a novel platform based on site-oriented conjugation of PMPC to the antibody, allowing it to maintain key functionalities of the original antibody. With an optimized PMPC chain length, the PMPC-antibody conjugate exhibited enhanced brain delivery while retaining epitope recognition, cellular internalization, and antibody-dependent cellular phagocytic activity. This simple formula incorporates only the antibody and PMPC without requiring additional components, thereby addressing the issues of the nanocapsule platform and paving the way for PMPC-based brain delivery strategies for antibodies.

Ultrathin polymeric coatings based on hydrogen-bonded polyphenol for protection of pancreatic islet cells.

Though transplantation of pancreatic islet cells has emerged as a promising treatment for Type 1 diabetes its clinical application remains limited due to a number of limitations including both pathogenic innate and adaptive immune responses. We report here on a novel type of multifunctional cytoprotective material applied to coat living pancreatic islets. The coating utilizes hydrogen-bonded interactions of a natural polyphenol (tannic acid) with poly(N-vinylpyrrolidone) deposited on the islet surface via non-ionic layer-by-layer assembly. We demonstrate that the coating is conformal over the surface of mammalian islets including those derived from rat, non-human primate (NHP), and human. In contrast to unmodified controls, the coated islets maintain their viability and ß-cell functionality for at least 96 hours in vitro. We also determine that the coating demonstrates immunomodulatory cytoprotective properties suppressing pro-inflammatory cytokine synthesis in stimulated bone marrow-derived macrophages and diabetogenic BDC-2.5 T cells. The coating material combines high chemical stability under physiologically relevant conditions with capability of suppressing cytokine synthesis, crucial parameters for prolonged islet integrity, viability, and function in vivo. Our study offers new opportunities in the area of advanced multifunctional materials to be used for a cell-based transplantation therapy.