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.

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.

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.

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.

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.

Poly(N-vinylpyrrolidone)-block-Poly(dimethylsiloxane)-block-Poly(N-vinylpyrrolidone) Triblock Copolymer Polymersomes for Delivery of PARP1 siRNA to Breast Cancers.

Nearly 20% of HER2-positive breast cancers develop resistance to HER2-targeted therapies requiring the use of advanced therapies. Silencing RNA therapy may be a powerful modality for treating resistant HER2 cancers due to its high specificity and low toxicity. However, the systemic administration of siRNAs requires a safe and efficient delivery platform because of siRNA's low stability in physiological fluids, inefficient cellular uptake, immunoreactivity, and rapid clearance. We have developed theranostic polymeric vesicles to overcome these hurdles for encapsulation and delivery of small functional molecules and PARP1 siRNA for in vivo delivery to breast cancer tumors. The 100 nm polymer vesicles were assembled from biodegradable and non-ionic poly(N-vinylpyrrolidone)14-block-poly(dimethylsiloxane)47-block-poly(N-vinylpyrrolidone)14 triblock copolymer PVPON14-PDMS47-PVPON14 using nanoprecipitation and thin-film hydration. We demonstrated that the vesicles assembled from the copolymer covalently tagged with the Cy5.5 fluorescent dye for in vivo imaging could also encapsulate the model drug with high loading efficiency (40%). The dye-loaded vesicles were accumulated in tumors after 18 h circulation in 4TR breast tumor-bearing mice via passive targeting. We found that PARP1 siRNA encapsulated into the vesicles was released intact (13%) into solution by the therapeutic ultrasound treatment as quantified by gel electrophoresis. The PARP1 siRNA-loaded polymersomes inhibited the proliferation of MDA-MB-361TR cells by 34% after 6 days of treatment by suppressing the NF-kB signaling pathway, unlike their scrambled siRNA-loaded counterparts. Finally, the treatment by PARP1 siRNA-loaded vesicles prolonged the survival of the mice bearing 4T1 breast cancer xenografts, with the 4-fold survival increase, unlike the untreated mice after 3 weeks following the treatment. These biodegradable, non-ionic PVPON14-PDMS47-PVPON14 polymeric nanovesicles capable of the efficient encapsulation and delivery of PARP1 siRNA to successfully knock down PARP1 in vivo can provide an advanced platform for the development of precision-targeted therapeutic carriers, which could help develop highly effective drug delivery nanovehicles for breast cancer gene therapy.

Anisotropic Particles through Multilayer Assembly.

The anisotropy in the shape of polymeric particles has been demonstrated to have many advantages over spherical particulates, including bio-mimetic behavior, shaped-directed flow, deformation, surface adhesion, targeting, motion, and permeability. The layer-by-layer (LbL) assembly is uniquely suited for synthesizing anisotropic particles as this method allows for simple and versatile replication of diverse colloid geometries with precise control over their chemical and physical properties. This review highlights recent progress in anisotropic particles of micrometer and nanometer sizes produced by a templated multilayer assembly of synthetic and biological macromolecules. Synthetic approaches to produce capsules and hydrogels utilizing anisotropic templates such as biological, polymeric, bulk hydrogel, inorganic colloids, and metal-organic framework crystals as sacrificial templates are overviewed. Structure-property relationships controlled by the anisotropy in particle shape and surface are discussed and compared with their spherical counterparts. Advances and challenges in controlling particle properties through varying shape anisotropy and surface asymmetry are outlined. The perspective applications of anisotropic colloids in biomedicine, including programmed behavior in the blood and tissues as artificial cells, nano-motors/sensors, and intelligent drug carriers are also discussed.

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.

Multilayer Microcapsules with Shell-Chelated 89Zr for PET Imaging and Controlled Delivery.

Radionuclide-functionalized drug delivery vehicles capable of being imaged via positron emission tomography (PET) are of increasing interest in the biomedical field as they can reveal the in vivo behavior of encapsulated therapeutics with high sensitivity. However, the majority of current PET-guided theranostic agents suffer from poor retention of radiometal over time, low drug loading capacities, and time-limited PET imaging capability. To overcome these challenges, we have developed hollow microcapsules with a thin (<100 nm) multilayer shell as advanced theranostic delivery systems for multiday PET tracking in vivo. The 3 µm capsules were fabricated via the aqueous multilayer assembly of a natural antioxidant, tannic acid (TA), and a poly(N-vinylpyrrolidone) (PVPON) copolymer containing monomer units functionalized with deferoxamine (DFO) to chelate the 89Zr radionuclide, which has a half-life of 3.3 days. We have found using radiochromatography that (TA/PVPON-DFO)6 capsules retained on average 17% more 89Zr than their (TA/PVPON)6 counterparts, which suggests that the covalent attachment of the DFO to PVPON provides stable 89Zr chelation. In vivo PET imaging studies performed in mice demonstrated that excellent stability and imaging contrast were still present 7 days postinjection. Animal biodistribution analyses showed that capsules primarily accumulated in the spleen, liver, and lungs with negligible accumulation in the femur, with the latter confirming the stable binding of the radiotracer to the capsule walls. The application of therapeutic ultrasound (US) (60 s of 20 kHz US at 120 W cm-2) to Zr-functionalized capsules could release the hydrophilic anticancer drug doxorubicin from the capsules in the therapeutic amounts. Polymeric capsules with the capability of extended in vivo PET-based tracking and US-induced drug release provide an advanced platform for development of precision-targeted therapeutic carriers and could aid in the development of more effective drug delivery systems.

Dampening Antigen-Specific T Cell Responses with Antigens Encapsulated in Polyphenolic Microcapsules.

Efficient T cell activation and effector responses require an antigenic peptide presented on the MHC complex to the TCR (signal 1), costimulatory molecule interactions between T cells and APCs (signal 2), and the synthesis of innate immune-derived proinflammatory cytokines and reactive oxygen species (signal 3). We previously demonstrated that the third signal dissipation impairs autoreactive T cell activation. In this study, we tested the hypothesis that encapsulation of Ag with an antioxidant-containing biomaterial would induce Ag-specific hyporesponsiveness. We cocultured bone marrow-derived dendritic cells with microcapsules composed of multilayer-assembled poly(N-vinylpyrrolidone) (PVPON) and the antioxidant tannic acid (TA). LPS-activated dendritic cells cocultured with (PVPON/TA) microcapsules displayed a decrease in TNF-α, IL-12p70, and CXCL10 synthesis. To study Ag-specific T cell responses, we incorporated chicken OVA into the (PVPON/TA) multilayers and stimulated OT-II splenocytes in a primary recall assay. Flow cytometric analysis demonstrated a significant inhibition of CD4 T cell activation markers, upregulation of CTLA-4 and PD-1, and blunted secretion of IL-2, IFN-γ, TNF-α, and CXCL10 by ELISA. To test microcapsule efficacy in vivo, we immunized OT-II mice with (PVPON/TA)-OVA microcapsules and performed an OVA recall assay. Immunization of OT-II mice with (PVPON/TA)-OVA microcapsules elicited a decrease in CD4 T cell differentiation and effector responses including IFN-γ, TNF-α, CCL3, and CCL5 by ELISA compared with OVA immunization alone. These data show that microcapsules composed of antioxidant and encapsulated Ags can effectively blunt innate immune-derived proinflammatory third signal synthesis necessary for Ag-specific effector T cell responses and present a prospective strategy for T cell-mediated autoimmunity.

Localized Immunosuppression With Tannic Acid Encapsulation Delays Islet Allograft and Autoimmune-Mediated Rejection.

Type 1 diabetes (T1D) is an autoimmune disease of insulin-producing ß-cells. Islet transplantation is a promising treatment for T1D, but long-term graft viability and function remain challenging. Oxidative stress plays a key role in the activation of alloreactive and autoreactive immunity toward the engrafted islets. Therefore, targeting these pathways by encapsulating islets with an antioxidant may delay immune-mediated rejection. Utilizing a layer-by-layer approach, we generated nanothin encapsulation materials containing tannic acid (TA), a polyphenolic compound with redox scavenging and anti-inflammatory effects, and poly(N-vinylpyrrolidone) (PVPON), a biocompatible polymer. We hypothesize that transplantation of PVPON/TA-encapsulated allogeneic C57BL/6 islets into diabetic NOD mice will prolong graft function and elicit localized immunosuppression. In the absence of systemic immunosuppression, diabetic recipients containing PVPON/TA-encapsulated islets maintained euglycemia and delayed graft rejection significantly longer than those receiving nonencapsulated islets. Transplantation of PVPON/TA-encapsulated islets was immunomodulatory because gene expression and flow cytometric analysis revealed significantly decreased immune cell infiltration, synthesis of reactive oxygen species, inflammatory chemokines, cytokines, CD8 T-cell effector responses, and concomitant increases in alternatively activated M2 macrophage and dendritic cell phenotypes. Our results provide evidence that reducing oxidative stress following allotransplantation of PVPON/TA-encapsulated islets can elicit localized immunosuppression and potentially delay graft destruction in future human islet transplantation studies.

Effect of temperature and hydrophilic ratio on the structure of poly(N-vinylcaprolactam)-block-poly(dimethylsiloxane)-block-poly(N-vinylcaprolactam) polymersomes.

Nanosized polymeric vesicles (polymersomes) assembled from ABA triblock copolymers of poly(N-vinylcaprolactam)-poly(dimethylsiloxane)-poly(N-vinylcaprolactam) (PVCL-PDMS-PVCL) are a promising platform for biomedical applications, as the temperature-responsiveness of the PVCL blocks enables reversible vesicle shrinkage and permeability of the polymersome shell at elevated temperatures. Herein, we explore the effects of molecular weight, polymer block weight ratios, and temperature on the structure of these polymersomes via electron microscopy, dynamic light scattering, small angle neutron scattering (SANS), and all-atom molecular dynamic methods. We show that the shell structure and overall size of the polymersome can be tuned by varying the hydrophilic (PVCL) weight fraction of the polymer: at room temperature, polymers of smaller hydrophilic ratios form larger vesicles that have thinner shells, whereas polymers with higher PVCL content exhibit interchain aggregation of PVCL blocks within the polymersome shell above 50 °C. Model fitting and model-free analysis of the SANS data reveals that increasing the mass ratio of PVCL to the total copolymer weight from 0.3 to 0.56 reduces the temperature-induced change in vesicle diameter by a factor of 3 while simultaneously increasing the change in shell thickness by a factor of 1.5. Finally, by analysis of the shell structures and overall size of polymersomes with various PVCL weight ratios and those without temperature-dependent polymer components, we bring into focus the mechanism of temperature-triggered drug release reported in a previous study. This work provides new fundamental perspectives on temperature-responsive polymersomes and elucidates important structure-property relationships of their constituent polymers.

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.

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.

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.

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.

Encapsulation and Ultrasound-Triggered Release of G-Quadruplex DNA in Multilayer Hydrogel Microcapsules.

Nucleic acid therapeutics have the potential to be the most effective disease treatment strategy due to their intrinsic precision and selectivity for coding highly specific biological processes. However, freely administered nucleic acids of any type are quickly destroyed or rendered inert by a host of defense mechanisms in the body. In this work, we address the challenge of using nucleic acids as drugs by preparing stimuli responsive poly(methacrylic acid)/poly(N-vinylpyrrolidone) (PMAA/PVPON)n multilayer hydrogel capsules loaded with ~7 kDa G-quadruplex DNA. The capsules are shown to release their DNA cargo on demand in response to both enzymatic and ultrasound (US)-triggered degradation. The unique structure adopted by the G-quadruplex is essential to its biological function and we show that the controlled release from the microcapsules preserves the basket conformation of the oligonucleotide used in our studies. We also show that the (PMAA/PVPON) multilayer hydrogel capsules can encapsulate and release ~450 kDa double stranded DNA. The encapsulation and release approaches for both oligonucleotides in multilayer hydrogel microcapsules developed here can be applied to create methodologies for new therapeutic strategies involving the controlled delivery of sensitive biomolecules. Our study provides a promising methodology for the design of effective carriers for DNA vaccines and medicines for a wide range of immunotherapies, cancer therapy and/or tissue regeneration therapies in the future.

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.

Resolution Agonist 15-epi-Lipoxin A4 Programs Early Activation of Resolving Phase in Post-Myocardial Infarction Healing.

Following myocardial infarction (MI), overactive inflammation remodels the left ventricle (LV) leading to heart failure coinciding with reduced levels of 15-epi-Lipoxin A4 (15-epi LXA4). However, the role of 15-epi LXA4 in post-MI acute inflammatory response and resolving phase is unclear. We hypothesize that liposomal fusion of 15-epi-LXA4 (Lipo-15-epi-LXA4) or free 15-epi-LXA4 will expedite the resolving phase in post-MI inflammation. 8 to 12-week-old male C57BL/6 mice were subjected to permanent coronary artery ligation. Lipo-15-epi-LXA4 or 15-epi-LXA4 (1 µg/kg/day) was injected 3 hours post-MI for (d)1 or continued daily till d5. 15-epi-LXA4 activated formyl peptide receptor (FPR2) and GPR120 on alternative macrophages but inhibited GPR40 on classical macrophages in-vitro. The 15-epi-LXA4 injected mice displayed reduced LV and lung mass to body weight ratios and improved ejection fraction at d5 post-MI. In the acute phase of inflammation-(d1), 15-epi-LXA4 primes neutrophil infiltration with a robust increase of Ccl2 and FPR2 expression. During the resolving phase-(d5), 15-epi-LXA4 initiated rapid neutrophils clearance with persistent activation of FPR2 in LV. Compared to MI-control, 15-epi-LXA4 injected mice showed reduced renal inflammation along with decreased levels of ngal and plasma creatinine. In summary, 15-epi-LXA4 initiates the resolving phase early to discontinue inflammation post-MI, thereby reducing LV dysfunction.

Temperature-responsive nanogel multilayers of poly(N-vinylcaprolactam) for topical drug delivery.

We report nanothin temperature-responsive hydrogel films of poly(N-vinylcaprolactam) nanoparticles (νPVCL) with remarkably high loading capacity for topical drug delivery. Highly swollen (νPVCL)n multilayer hydrogels, where n denotes the number of nanoparticle layers, are produced by layer-by-layer hydrogen-bonded assembly of core-shell PVCL-co-acrylic acid nanoparticles with linear PVPON followed by cross-linking of the acrylic acid shell with either ethylene diamine (EDA) or adipic acid dihydrazide (AAD). We demonstrate that a (νPVCL)5 film undergoes dramatic and reversible swelling up to 9 times its dry thickness at pH = 7.5, indicating 89v/v % of water inside the network. These hydrogels exhibit highly reversible ∼3-fold thickness changes with temperature variations from 25 to 50°C at pH = 5, the average pH of human skin. We also show that a (νPVCL)30 hydrogel loaded with ∼120µgcm-2 sodium diclofenac, a non-steroidal anti-inflammatory drug used for osteoarthritis pain management, provides sustained permeation of this drug through an artificial skin membrane for up to 24h at 32°C (the average human skin surface temperature). The cumulative amount of diclofenac transported at 32°C from the (νPVCL)30 hydrogel after 24h is 12 times higher than that from the (νPVCL)30 hydrogel at 22°C. Finally, we demonstrate that the (νPVCL) hydrogels can be used for multiple drug delivery by inclusion of Nile red, fluorescein and DAPI dyes within the νPVCL nanoparticles prior to hydrogel assembly. Using confocal microscopy we observed the presence of separate dye-loaded νPVCL compartments within the hydrogel matrix with all three dyes confined to the nanogel particles without intermixing between the dyes. Our study provides opportunity for development of temperature-responsive multilayer hydrogel coatings made via the assembly of core-shell nanogel particles which can be used for skin-sensitive materials for topical drug delivery.