Glutathione-Conjugated Hydrogels: Flexible Vehicles for Personalized Treatment of Bacterial Infections.

PURPOSE: Skin and soft tissue infections are increasingly prevalent and often complicated by potentially fatal therapeutic hurdles, such as poor drug perfusion and antibiotic resistance. Delivery vehicles capable of versatile loading may improve local bioavailability and minimize systemic toxicities yet such vehicles are not clinically available. Therefore, we aimed to expand upon the use of glutathione-conjugated poly(ethylene glycol) GSH-PEG hydrogels beyond protein delivery and evaluate the ability to deliver traditional therapeutic molecules. METHODS: PEG and GSH-PEG hydrogels were prepared using ultraviolet light (UV)-polymerization. Hydrogel loading and release of selected drug candidates was examined using UV-visible spectrometry. Therapeutic molecules and GST-fusion protein loading was examined using UV-visible and fluorescent spectrometry. Efficacy of released meropenem was assessed against meropenem-sensitive and -resistant P. aeruginosa in an agar diffusion bioassay. RESULTS: For all tested agents, GSH-PEG hydrogels demonstrated time-dependent loading whereas PEG hydrogels did not. GSH-PEG hydrogels released meropenem over 24 h. Co-loading of biologic and traditional therapeutics into a single vehicle was successfully demonstrated. Meropenem-loaded GSH-PEG hydrogels inhibited the growth of meropenem-sensitive and resistant P. aeruginosa isolates. CONCLUSION: GSH ligands within GSH-PEG hydrogels allow loading and effective delivery of charged therapeutic agents, in addition to biologic therapeutics.

Reducible Micelleplexes are Stable Systems for Anti-miRNA Delivery in Cerebrospinal Fluid.

Glioblastoma multiforme (GBM) and other central nervous system (CNS) cancers have poor long-term prognosis, and there is a significant need for improved treatments. GBM initiation and progression are mediated, in part, by microRNA (miRNA), which are endogenous posttranscriptional gene regulators. Misregulation of miRNAs is a potential target for therapeutic intervention in GBM. In this work, a micelle-like nanoparticle delivery system based upon the block copolymer poly(ethylene glycol-b-lactide-b-arginine) was designed with and without a reducible linkage between the lactide and RNA-binding peptide, R15, to assess the ability of the micelle-like particles to disassemble. Using confocal live cell imaging, intracellular dissociation was pronounced for the reducible micelleplexes. This dissociation was also supported by higher efficiency in a dual luciferase assay specific for the miRNA of interest, miR-21. Notably, micelleplexes were found to have significantly better stability and higher anti-miRNA activity in cerebrospinal fluid than in human plasma, suggesting an advantage for applying micelleplexes to CNS diseases and in vivo CNS therapeutics. The reducible delivery system was determined to be a promising delivery platform for the treatment of CNS diseases with miRNA therapy.

Pharmacokinetics and treatment efficacy of camptothecin nanocrystals on lung metastasis.

Cancer metastasis is difficult to treat, and its outcome becomes dreadful for a patient. Lung is a major metastatic site for many types of cancers, and the need for finding effective treatment for lung metastasis cannot be overemphasized. In a previous study, we showed that camptothecin nanocrystals demonstrated greater anticancer efficacy and achieved significantly higher concentration in lungs than a conventional, solution-based formulation. In this study, we further determined the pharmacokinetics of camptothecin nanocrystals in rats and investigated treatment efficacy in mice against metastatic lung tumors. The results show that camptothecin nanocrystals were capable of eliciting greater antimetastatic efficacy and achieve a longer survival time in the murine model compared with camptothecin salt solution. The study suggests that using engineered, solid nanoparticles may be a feasible approach in the treatment of lung cancer and lung metastatic cancer.

Toroidal-spiral particles for codelivery of anti-VEGFR-2 antibody and irinotecan: a potential implant to hinder recurrence of glioblastoma multiforme.

Heterogeneous toroidal-spiral particles (TSPs) were generated by polymer droplet sedimentation, interaction, and cross-linking. TSPs provide a platform for encapsulation and release of multiple compounds of different sizes and physicochemical properties. As a model system, we demonstrate the encapsulation and independently controlled release of an anti-VEGFR-2 antibody and irinotecan for the treatment of glioblastoma multiforme. The anti-VEGFR-2 antibody was released from the TS channels and its binding to HUVECs was confirmed by confocal microscopy and flow cytometry, suggesting active antibody encapsulation and release. Irinotecan, a small molecule drug, was released from the dense polymer matrix of poly(ethylene glycol) diacrylate (MW ~ 700 g/mol; PEGDA 700). Released irinotecan inhibited the proliferation of U251 malignant glioma cells. Since the therapeutic compounds are released through different pathways, specifically diffusion through the polymer matrix versus TS channels, the release rate can be controlled independently through the design of the structure and material of particle components.

In vivo investigation of hybrid Paclitaxel nanocrystals with dual fluorescent probes for cancer theranostics.

PURPOSE: To develop novel hybrid paclitaxel (PTX) nanocrystals, in which bioactivatable (MMPSense® 750 FAST) and near infrared (Flamma Fluor® FPR-648) fluorophores are physically incorporated, and to evaluate their anticancer efficacy and diagnostic properties in breast cancer xenograft murine model. METHODS: The pure and hybrid paclitaxel nanocrystals were prepared by an anti-solvent method, and their physical properties were characterized. The tumor volume change and body weight change were evaluated to assess the treatment efficacy and toxicity. Bioimaging of treated mice was obtained non-invasively in vivo. RESULTS: The released MMPSense molecules from the hybrid nanocrystals were activated by matrix metalloproteinases (MMPs) in vivo, similarly to the free MMPSense, demonstrating its ability to monitor cancer progression. Concurrently, the entrapped FPR-648 was imaged at a different wavelength. Furthermore, when administered at 20 mg/kg, the nanocrystal formulations exerted comparable efficacy as Taxol®, but with decreased toxicity. CONCLUSIONS: Hybrid nanocrystals that physically integrated two fluorophores were successfully prepared from solution. Hybrid nanocrystals were shown not only exerting antitumor activity, but also demonstrating the potential of multi-modular bioimaging for diagnostics.

Assessment of antibiotic release and antibacterial efficacy from pendant glutathione hydrogels using ex vivo porcine skin.

Acute bacterial skin and skin structure infections (ABSSSIs) confer a substantial burden on the healthcare system. Local antibiotic delivery systems can provide controlled drug release directly to the site of infection to maximize efficacy and minimize systemic toxicity. The purpose of this study was to examine the antibacterial activity of antibiotic-loaded glutathione-conjugated poly(ethylene glycol) hydrogels (GSH-PEG) against ABSSSIs utilizing an ex vivo porcine dermal explant model. Vancomycin- or meropenem-loaded GSH-PEG hydrogels at 3 different dose levels were loaded over 1 h. Drug release was monitored in vitro under submerged conditions, by the Franz cell diffusion method, and ex vivo utilizing a porcine dermis model. Antibacterial activity was assessed ex vivo on porcine dermis explants inoculated with Staphylococcus aureus or Pseudomonas aeruginosa isolates treated with vancomycin- or meropenem-loaded GSH-PEG hydrogels, respectively. Histological assessment of the explants was conducted to evaluate tissue integrity and viability in the context of the experimental conditions. A dose-dependent release was observed from vancomycin and meropenem hydrogels, with in vitro Franz cell diffusion data closely representing ex vivo vancomycin release, but not high dose meropenem release. High dose vancomycin-loaded hydrogels resulted in a >3 log10 clearance against all S. aureus isolates at 48 h. High dose meropenem-loaded hydrogels achieved 6.5, 4, and 2 log10 reductions in CFU/ml against susceptible, intermediate, and resistant P. aeruginosa isolates, respectively. Our findings demonstrate the potential application of GSH-PEG hydrogels for flexible, local antibiotic delivery against bacterial skin infections.

Stability of benzocaine formulated in commercial oral disintegrating tablet platforms.

Pharmaceutical excipients contain reactive groups and impurities due to manufacturing processes that can cause decomposition of active drug compounds. The aim of this investigation was to determine if commercially available oral disintegrating tablet (ODT) platforms induce active pharmaceutical ingredient (API) degradation. Benzocaine was selected as the model API due to known degradation through ester and primary amino groups. Benzocaine was either compressed at a constant pressure, 20 kN, or at pressure necessary to produce a set hardness, i.e., where a series of tablets were produced at different compression forces until an average hardness of approximately 100 N was achieved. Tablets were then stored for 6 months under International Conference on Harmonization recommended conditions, 25°C and 60% relative humidity (RH), or under accelerated conditions, 40°C and 75% RH. Benzocaine degradation was monitored by liquid chromatography-mass spectrometry. Regardless of the ODT platform, no degradation of benzocaine was observed in tablets that were kept for 6 months at 25°C and 60% RH. After storage for 30 days under accelerated conditions, benzocaine degradation was observed in a single platform. Qualitative differences in ODT platform behavior were observed in physical appearance of the tablets after storage under different temperature and humidity conditions.

In-house preparation of hydrogels for batch affinity purification of glutathione S-transferase tagged recombinant proteins.

BACKGROUND: Many branches of biomedical research find use for pure recombinant proteins for direct application or to study other molecules and pathways. Glutathione affinity purification is commonly used to isolate and purify glutathione S-transferase (GST)-tagged fusion proteins from total cellular proteins in lysates. Although GST affinity materials are commercially available as glutathione immobilized on beaded agarose resins, few simple options for in-house production of those systems exist. Herein, we describe a novel method for the purification of GST-tagged recombinant proteins. RESULTS: Glutathione was conjugated to low molecular weight poly(ethylene glycol) diacrylate (PEGDA) via thiol-ene "click" chemistry. With our in-house prepared PEGDA:glutathione (PEGDA:GSH) homogenates, we were able to purify a glutathione S-transferase (GST) green fluorescent protein (GFP) fusion protein (GST-GFP) from the soluble fraction of E. coli lysate. Further, microspheres were formed from the PEGDA:GSH hydrogels and improved protein binding to a level comparable to purchased GSH-agarose beads. CONCLUSIONS: GSH containing polymers might find use as in-house methods of protein purification. They exhibited similar ability to purify GST tagged proteins as purchased GSH agarose beads.

Stem cell-derived extracellular matrix enables survival and multilineage differentiation within superporous hydrogels.

Hydrophilic poly(ethylene glycol) diacrylate (PEGDA) hydrogel surfaces resist protein adsorption and are generally thought to be unsuitable for anchorage-dependent cells to adhere. Intriguingly, our previous findings revealed that PEGDA superporous hydrogel scaffolds (SPHs) allow anchorage of bone marrow derived human mesenchymal stem cells (hMSCs) and support their long-term survival. Therefore, we hypothesized that the physicochemical characteristics of the scaffold impart properties that could foster cellular responses. We examined if hMSCs alter their microenvironment to allow cell attachment by synthesizing their own extracellular matrix (ECM) proteins. Immunofluorescence staining revealed extensive expression of collagen type I, collagen type IV, laminin, and fibronectin within hMSC-seeded SPHs by the end of the third week. Whether cultured in serum-free or serum-supplemented medium, hMSC ECM protein gene expression patterns exhibited no substantial changes. The presence of serum proteins is required for initial anchorage of hMSCs within the SPHs but not for the hMSC survival after 24 h. In contrast to 2D expansion on tissue culture plastic (TCP), hMSCs cultured within SPHs proliferate similarly in the presence or absence of serum. To test whether hMSCs retain their undifferentiated state within the SPHs, cell-seeded constructs were cultured for 3 weeks in stem cell maintenance medium and the expression of hMSC-specific cell surface markers were evaluated by flow cytometry. CD105, CD90, CD73, and CD44 were present to a similar extent in the SPH and in 2D monolayer culture. We further demonstrated multilineage potential of hMSCs grown in the PEGDA SPHs, whereby differentiation into osteoblasts, chondrocytes, and adipocytes could be induced. The present study demonstrates the potential of hMSCs to alter the "blank" PEGDA environment to a milieu conducive to cell growth and multilineage differentiation by secreting adhesive ECM proteins within the porous network of the SPH scaffolds.

Biophysical Characterization of Interactions between Serum Albumin and Block Copolymer Micelles.

Block copolymer micelles have demonstrated great promise in the solubilization of hydrophobic drugs, but an understanding of the blood stability of the drug-laden micelles is needed for therapeutic advancement of micelle technologies. Following intravenous administration, mPEG-CL and mPEG-LA micelles have demonstrated quick release of their cargo and disassembly in blood, but the prevailing mechanisms of micelle disruption and key biomacromolecules driving this disruption have yet to be elucidated. Although protein interactions with solid polymeric nanoparticles have been characterized, not much is known regarding protein interactions with dynamic block copolymer micelles. Herein, we characterize the interaction of bovine and human serum albumins (BSA and HSA) with polymeric micelles, mPEG-CL and mPEG-LA, using protein fluorescence, isothermal titration calorimetry (ITC), and circular dichroism (CD) spectroscopy. We find that BSA and HSA have interactions with mPEG-CL, while only HSA is observed to weakly interact with mPEG-LA. Protein fluorescence suggests that binding of HSA to mPEG-CL and mPEG-LA is driven by electrostatic interactions. ITC suggests an interaction between serum albumin and mPEG-CL block copolymers driven by hydrogen bonding and electrostatic interactions in physiological MOPS-buffered saline, while mPEG-LA has no measurable interaction with either of the serum albumins. CD spectroscopy demonstrates that the protein secondary structure is intact in both proteins in the presence of mPEG-CL and mPEG-LA. Overall, BSA is not always predictive of polymeric interactions with HSA. Understanding of interactions between serum proteins and block copolymer micelles and the exact mechanisms of destabilization will direct the rational design of block copolymer systems for improving blood stability.

Hybrid nanocrystals: achieving concurrent therapeutic and bioimaging functionalities toward solid tumors.

Bioimaging and therapeutic agents accumulated in ectopic tumors following intravenous administration of hybrid nanocrystals to tumor-bearing mice. Solid, nanosized paclitaxel crystals physically incorporated fluorescent molecules throughout the crystal lattice and retained fluorescent properties in the solid state. Hybrid nanocrystals were significantly localized in solid tumors and remained in the tumor for several days. An anticancer effect is expected of these hybrid nanocrystals.

Toward understanding polymer micelle stability: Density ultracentrifugation offers insight into polymer micelle stability in human fluids.

Micelles, as a class of drug delivery systems, are underrepresented among United States Food and Drug Administration approved drugs. A lack of clinical translation of these systems may be due to, in part, to a lack of understanding of micelle interactions with biologic fluids following injection. Despite the limited clinical translation, micelles remain an active area of research focus and pre-clinical development. The goal of the present study was to examine the stability of amphiphilic block copolymer micelles in biologic fluids to identify the properties and components of biologic fluids that influence micelle stability. Micelle stability, measured via Förster resonance energy transfer-based fluorescent spectrometry, was complemented with density ultracentrifugation to reveal the colocalized, or dissociated, state of the dye cargo after exposure to human biologic fluids. Polymeric micelles composed of poly(ethylene glycol-block-caprolactone) (mPEG-CL) and poly(ethylene glycol-block-lactide) (mPEG-LA) were unstable in fetal bovine serum, human serum and synovial fluid, with varying levels of instability observed in ascites and pleural fluid. All polymeric micelles exhibited stability in cerebrospinal fluid, highlighting the potential for local cerebro-spinal administration of micelles. Interestingly, mPEG2.2k-CL3.1k and mPEG2k-LA2.7k micelles favored dissolution whereas mPEG5.4k-LA28.5k micelles favored stability. Taken together, our data offers both quantitative and qualitative evidence for micelle stability within human biologic fluids and offers evidence of polymer micelle instability in biologic fluids that is not explained by either total protein content or total unsaturated lipid content. The results help to identify potential sites for local delivery where stability is maintained.

In vitro evaluation of functional interaction of integrin alphavbeta3 and matrix metalloprotease-2.

Integrin alphavbeta3 and matrix metalloprotease-2 (MMP-2) are two established molecular targets of angiogenesis. Basic understanding of various forms of functional interaction of integrin alphavbeta3 and active MMP-2 may be used to develop therapeutic approaches. Based upon the idea that integrins are present on the surface of invasive cells and MMP-2 may be localized to this and other cell-surface receptors, we investigated the hypothesis that integrin binding will alter cleavage of MMP-2 substrates. To investigate this hypothesis, integrin-binding and MMP-2 cleavable motifs were combined in a single peptide, MMP-RGD, designed with fluorescent probes for monitoring peptide cleavage. MMP-RGD was bound to integrin alphavbeta3 with equal affinity compared to the integrin-binding motif and was cleaved with equal specificity by active MMP-2. MMP-RGD bound to human umbilical vein endothelial cells (HUVECs). MMP-2 from HUVECs cleaved MMP-RGD, but the cleavage was not altered due to integrin binding. Our results indicate that integrin alphavbeta3 and active MMP-2 may not be as functionally collaborative for substrate cleavage as expected based on the current knowledge of their cell surface colocalization.

Matrix metalloprotease selective peptide substrates cleavage within hydrogel matrices for cancer chemotherapy activation.

To utilize biologic mechanisms to elicit controlled release in response to disease, protease-sensitive devices have been created. Hydrogels were created with pendant peptide-drug complexes. For the matrix metalloproteases (MMPs) examined, a length of six amino acids greatly improved the specificity of the peptide (k(cat)/K(m) approximately 2.4+/-0.1x10(4)M(-1)s(-1)) over shorter sequences (k(cat)/K(m) approximately 4.4+/-0.2x10(2)M(-1)s(-1)). The peptides did not exhibit anti-proliferative effects upon cancer cells, and peptide-platinum complexes showed similar anti-proliferative effects upon the cancer cells compared to the free platinum drugs. Once the peptide-drug complex was incorporated into the hydrogels, the release was dependent upon the presence of MMP in the solution with approximately 35% of platinum released from hydrogels in the presence of MMP and only 10% without MMP in the week examined. The released drug exhibited the expected anti-proliferative activity over several days of incubation. The MMP selective drug delivery holds much potential for treatment of cancer and other diseases.

Hybrid superporous scaffolds: an application for cornea tissue engineering.

Engineering a cell-based keratoprosthesis often requires a struggle between two essential parameters: natural 3-D biological adhesion and mechanical strength. A novel hybrid scaffold of natural and synthetic materials was engineered to achieve both cell adhesion and implantable strength. This scaffold was characterized in terms of cell adhesion, cell migration, swelling, and strength. While the study was focused on engineering a biointegrable prosthetic skirt, a clear central core with an appropriate refractive index and light transmission was also incorporated into the design for potential functionality. The hybrid scaffold was tested in rat corneas. This uniquely designed scaffold was well tolerated and encouraged host cell migration into the implant. The hybrid superporous design also enhanced cell adhesion and retention in a superporous scaffold without altering the bulk mechanical properties of the hydrogel.

Understanding the adsorption mechanism of chitosan onto poly(lactide-co-glycolide) particles.

Polyelectrolyte-coated nanoparticles or microparticles interact with bioactive molecules (peptides, proteins or nucleic acids) and have been proposed as delivery systems for these molecules. However, the mechanism of adsorption of polyelectrolyte onto particles remains unsolved. In this study, cationic poly(lactide-co-glycolide) (PLGA) nanoparticles were fabricated by adsorption of various concentrations of a biodegradable polysaccharide, chitosan (0-2.4g/L), using oil-in-water emulsion and solvent evaporation techniques. The particle diameter, zeta-potential, and chitosan adsorption of chitosan-coated PLGA nanoparticles confirmed the increase of polyelectrolyte adsorption. Five adsorption isotherm models (Langmuir, Freundlich, Halsey, Henderson, and Smith) were applied to the experimental data in order to better understand the mechanism of adsorption. Both particle diameter and chitosan adsorption increased with chitosan concentration during adsorption. A good correlation was obtained between PLGA-chitosan nanoparticle size and adsorbed chitosan on the surface, suggesting that the increased particle size was primarily due to the increased chitosan adsorption. The zeta-potential of chitosan-coated PLGA nanoparticles was positive and increased with chitosan adsorbed until a maximum value (+55mV) was reached at approximately 0.4-0.6g/L; PLGA nanoparticles had a negative zeta-potential (-20mV) prior to chitosan adsorption. Chitosan adsorption on PLGA nanoparticles followed a multilayer adsorption behavior, although the Langmuir monolayer equation held at low concentrations of chitosan. The underlying reasons for adsorption of chitosan on PLGA nanoparticles were thought to be the cationic nature of chitosan, high surface energy and microporous non-uniform surface of PLGA nanoparticles.

The influence of natural deep eutectic solvents on bioactive natural products: studying interactions between a hydrogel model and Schisandra chinensis metabolites.

Natural Deep Eutectic Solvent (NADES) species can exhibit unexpected solubilizing power for lipophilic molecules despite their simple composition: hydrophilic organic molecules and water. In the present study, the unique properties of NADES species were applied in combination with a model polymer system: a hydrophilic chitosan/alginate hydrogel. Briefly, NADES species (e.g., mannose-dimethylurea-water, 2:5:5, mole/mole) formed matrices to 1) dissolve lipophilic molecules (e.g., curcumin), 2) load lipophilic molecule(s) into the hydrogel, and 3) spontaneously vacate from the system. NADES species ubiquitously occur in natural sources, and a crude extract is a mixture of the NADES species and bioactive metabolites. Based on these ideas, we hypothesized that the crude extract may also allow the loading of natural bioactive molecules from a natural NADES species into (bio)hydrogel systems. To evaluate this hypothesis in vitro, Schisandra chinensis fruit extract was chosen as a representative mixture of lipophilic botanical molecules and hydrophilic NADES species. The results showed that the NADES matrix of S. chinensis was capable of loading at least three bioactive lignans (i.e., gomisin A, gomisin J, and angeloylgomisin H) into the polymer system. The lipophilic metabolites can subsequently be released from the hydrogel. The outcomes suggest that a unique drug delivery mechanism may exist in nature, thereby potentially improving the bioavailability of lipophilic metabolites through physicochemical interactions with the NADES.

Enhanced osteoblast-like cell adhesion and proliferation using sulfonate-bearing polymeric scaffolds.

Orthopedic malfunction, degeneration, or damage remains a serious healthcare issue despite advances in medical technology. Proactive extracellular matrix (ECM)-mimetic scaffolds are being researched to orchestrate the activation of diverse osteogenic signaling cascades, facilitating osteointegration. We hypothesized that sulfonated functionalities incorporated into synthetic hydrogels would simulate anionic, sulfate-bearing proteoglycans, abundant in the ECM. Using this rationale, we successfully developed differentially sulfonated hydrogels, polymerizing a range of sulfopropyl acrylate potassium-acrylamide (SPAK-AM) mole ratios as monomer feeds under room temperature conditions. For anchorage-dependent cells, such as osteoblasts, adhesion is a critical prerequisite for subsequent osteointegration and cell specialization. The introduction of the sulfonated monomer, SPAK, resulted in favorable uptake of serum proteins with proportional increase in adhesion and proliferation rates of model cell lines, which included NIH/3T3 fibroblasts, MG-63 osteoblasts, and MC3T3-E1 subclone 4 preosteoblasts. In fact, higher proportions of sulfonate content (pSPAK75, pSPAK100) exhibited comparable or even higher degrees of adhesion and proliferation, relative to commercial grade tissue culture polystyrene in vitro. These results indicate promising potentials of sulfonated ECM-mimetic hydrogels as potential osteogenic tissue engineering scaffolds.

Matrix metalloproteases: underutilized targets for drug delivery.

Pathophysiological molecules in the extracellular environment offer excellent targets that can be exploited for designing drug targeting systems. Matrix metalloproteases (MMPs) are a family of extracellular proteolytic enzymes that are characterized by their overexpression or overactivity in several pathologies. Over the last two decades, the MMP literature reveals heightened interest in the research involving MMP biology, pathology and targeting. This review describes various strategies that have been designed to utilize MMPs for targeting therapeutic entities. Key factors that need to be considered in the successful design of such systems have been identified based on the analyses of these strategies. Development of targeted drug delivery using MMPs has been steadily pursued; however, drug delivery efforts using these targets need to be intensified and focused to realize the clinical application of the fast developing fundamental MMP research.

Activation of Macrophages in Response to Biomaterials.

Macrophages are the initial biologic responders to biomaterials. These highly plastic immune sentinels control and modulate responses to materials, foreign or natural. The responses may vary from immune stimulatory to immune suppressive. Several parameters have been identified that influence macrophage response to biomaterials, specifically size, geometry, surface topography, hydrophobicity, surface chemistry, material mechanics, and protein adsorption. In this review, the influence of these parameters is supported with examples of both synthetic and naturally derived materials and illustrates that a combination of these parameters ultimately influences macrophage responses to the biomaterial. Having an understanding of these properties may lead to highly efficient design of biomaterials with desirable biologic response properties.