Preparation,in vitro release,in vivo absorption and biocompatibility studies of insulin-loaded microspheres in rabbits

The purpose of this study was to develop a single-dose insulin delivery system based on poly (lactide-co-glycolide) (PLGA) microspheres to provide basal insulin level for a prolonged period. Insulin-loaded PLGA microspheres were prepared by water-in-oil-in-water double emulsion (batch A) and solid-in-oil-in-water emulsion (batch B) methods. Microspheres were characterized for physical characteristics and in vitro release. In vivo absorption of insulin and biocompatibility of insulin-loaded PLGA microspheres were performed in diabetic New Zealand white rabbits. Light and transmission electron microscopy were performed on the skin tissues excised from microspheres injected sites in order to study the biocompatibility. The burst release of insulin was high (47%) from batch B and low (5%) from batch A. Therefore, we mixed microspheres of batch A and B in ratio of 3:1 w/w, which produced desirable in vitro release profile. In vivo absorption study showed that insulin-loaded microspheres provided a serum insulin level of 20-40 microU/ml up to 40 days. Biocompatibility study provided evidence of normal inflammatory and foreign body reactions, which were characterized by the presence of macrophages, fibroblasts and foreign body giant cells. Neither necrosis nor tissue damage was identified. At the end of 12 weeks, no distinct histological differences were observed in comparison to the control tissue samples. In conclusion, insulin-loaded PLGA microspheres controlled the in vivo absorption of insulin to maintain the basal insulin level for longer period and the delivery system was biocompatible.     

Sustained local delivery of insulin for potential improvement of peri-implant bone formation in diabetes

Dental implantation is an effective standard treatment modality to restore missing teeth and maxillofacial defects. However, in diabetics there is an increased risk for implant failure due to impaired peri-implant osseous healing. Early topical insulin treatment was recently shown to normalize diabetic bone healing by rectifying impairments in osteoblastic activities. In this study, insulin/poly(lactic-co-glycolic acid) (PLGA) microspheres were prepared by a double-emulsion solvent evaporation method. Microspheres were then incorporated in fibrin gel to develop a local drug delivery system for diabetic patients requiring implant treatment. In vitro release of insulin from fibrin gel loaded with these microspheres was assessed, and sustained prolonged insulin release over 21 days ascertained. To assess the bioactivity of released insulin and determine whether slow release might improve impaired diabetic bone formation, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), alkaline phosphatase (ALP) activity, mineralized nodule formation, and ELISA (enzyme-linked immunosorbent assay) assays were performed. The insulin released from the drug delivery system stimulated cell growth in previously inhibited cells, and ameliorated the impaired bone-forming ability of human MG-63 cells under high glucose conditions. Fibrin gel loaded with insulin/PLGA microspheres shows potential for improving peri-implant bone formation in diabetic patients.     

Microencapsulation of insulin microcrystals

Insulin microcrystals were encapsulated (microcrystal/PLGA) within poly(lactide-co-glycolide) (PLGA 50:50) by the multiple emulsification solvent evaporation technique and compared with insulin solution microspheres (solution/PLGA) in terms of their morphology, size distribution, drug content, encapsulation efficiency, and stability of insulin during release.     

Alginate microparticles as novel carrier for oral insulin delivery

Alginate microparticles produced by emulsification/internal gelation were investigated as a promising carrier for insulin delivery. The procedure involves the dispersion of alginate solution containing insulin protein, into a water immiscible phase. Gelation is triggered in situ by instantaneous release of ionic calcium from carbonate complex via gentle pH adjustment. Particle size is controlled through the emulsification parameters, yielding insulin-loaded microparticles. Particle recovery was compared using several washing protocols. Recovery strategies are proposed and the influence on particle mean size, morphology, recovery yield (RY), encapsulation efficiency, insulin release profile, and structural integrity of released insulin were evaluated. Spherical micron-sized particles loaded with insulin were produced. The recovery process was optimized, improving yield, and ensuring removal of residual oil from the particle surface. The optimum recovery strategy consisted in successive washing with a mixture of acetone/hexane/isopropanol coupled with centrifugation. This strategy led to small spherical particles with an encapsulation efficiency of 80% and a RY around 70%. In vitro release studies showed that alginate itself was not able to suppress insulin release in acidic media; however, this strategy preserves the secondary structure of insulin. Particles had a mean size lower than the critical diameter necessary to be orally absorbed through the intestinal mucosa followed by their passage to systemic circulation and thus can be considered as a promising technology for insulin delivery.     

Conjugation of polyamidoamine dendrimers on biodegradable microparticles for nonviral gene delivery

We report on the preparation and characterization of poly(D, L-lactide-co-glycolide) (PLGA) microparticles with surface-conjugated polyamidoamine (PAMAM) dendrimers of varying generations. The buffering capacity and zeta-potential of the PLGA PAMAM microparticles increased with increasing generation level of the PAMAM dendrimer conjugated. Conjugation of the PAMAM dendrimer to the surface of the PLGA microparticle removed generation-dependent cytotoxicity in HEK293 and COS7 cell lines. PLGA PAMAM pDNA microparticles displayed similar cytotoxicity profiles to unmodified PLGA pDNA microparticles in COS7 cells. A generation three PAMAM dendrimer conjugated to PLGA microparticles significantly increased transfection efficiencies in comparison to unmodified PLGA microparticles.     

Propaedeutic study for the delivery of nucleic acid-based molecules from PLGA microparticles and stearic acid nanoparticles

We studied the mechanism governing the delivery of nucleic acid-based drugs (NABD) from microparticles and nanoparticles in zero shear conditions, a situation occurring in applications such as in situ delivery to organ parenchyma. The delivery of a NABD molecule from poly(DL-lactide-co-glycolide) (PLGA) microparticles and stearic acid (SA) nanoparticles was studied using an experimental apparatus comprising a donor chamber separated from the receiver chamber by a synthetic membrane. A possible toxic effect on cell biology, as evaluated by studying cell proliferation, was also conducted forjust PLGA microparticles. A mathematical model based on the hypothesis that NABD release from particles is due to particle erosion was used to interpret experimental release data. Despite zero shear conditions imposed in the donor chamber, particle erosion was the leading mechanism for NABD release from both PLGA microparticles and SA nanoparticles. PLGA microparticle erosion speed is one order of magnitude higher than that of competing SA nanoparticles. Finally, no deleterious effects of PLGA microparticles on cell proliferation were detected. Thus, the data here reported can help optimize the delivery systems aimed at release of NABD from micro- and nanoparticles.     

Controlled release of modified insulin glargine from novel biodegradable injectable gels

The objective of this study was to investigate the duration of biological effects of modified insulin glargine released from a novel biodegradable injectable gel in type II diabetic Zucker diabetic fatty (ZDF) rats. Modified insulin glargine was purified from the marketed formulation by process of dialysis followed by freeze-drying, and the purity was confirmed by the single peak, corresponding to insulin glargine in the HPLC chromatogram. To determine and to compare the biological activity of purified insulin glargine with marketed formulation, it was suspended in isotonic saline solutions and administered subcutaneously to ZDF rats at a dose of 10 IU/kg of insulin and the blood glucose levels were measured. The blood glucose levels of ZDF rats after a subcutaneous injection of a suspension of purified insulin glargine decreased below 200 mg/dL within 2 h and remained at this level up to 6 h, then steadily raised above 400 mg/dL in 12 h. Insulin glargine particles were loaded into a novel biodegradable injectable gel formulation prepared from a blend of polylactic-co-glycolic acid (PLGA) and biocompatible plasticizers. Approximately 0.1 mL of insulin glargine-loaded gel prepared with PLGA was administered subcutaneously to the ZDF rats, and blood glucose levels were measured. The PLGA gel formulations prepared with insulin glargine particles had duration of action of 10 days following a single subcutaneous injection. The addition of zinc sulfate to the formulations prepared with purified insulin glargine particles further slowed down the drop in blood glucose concentrations.     

Microparticle‐mediated gene delivery for the enhanced expression of a 19‐kDa fragment of merozoite surface protein 1 of Plasmodium falciparum

The 19 kDa carboxyl‐terminal fragment of merozoite surface protein 1 (MSP1₁₉) is a major component of the invasion‐inhibitory response in individual immunity to malaria. A novel ultrasonic atomization approach for the formulation of biodegradable poly(lactic‐co‐glycolic acid) (PLGA) microparticles of malaria DNA vaccines encoding MSP1₁₉ is presented here. After condensing the plasmid DNA (pDNA) molecules with a cationic polymer polyethylenimine (PEI), a 40 kHz ultrasonic atomization frequency was used to formulate PLGA microparticles at a flow rate of 18 mL h^?1. High levels of gene expression and moderate cytotoxicity in COS‐7 cells were achieved with the condensed pDNA at a nitrogen to phosphate (N/P) ratio of 20, thus demonstrating enhanced cellular uptake and expression of the transgene. The ability of the microparticles to convey pDNA was examined by characterizing the formulated microparticles. The microparticles displayed Z‐average hydrodynamic diameters of 1.50–2.10 μm and zeta potentials of 17.8–23.2 mV. The encapsulation efficiencies were between 78 and 83%, and 76 and 85% of the embedded malaria pDNA molecules were released under physiological conditions in vitro. These results indicate that PLGA‐mediated microparticles can be employed as potential gene delivery systems to antigen‐presenting cells in the prevention of malaria. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Wheat germ agglutinin functionalized complexation hydrogels for oral insulin delivery

Insulin was loaded into hydrogel microparticles after two hours with loading efficiencies greater than 70% for both poly(methacrylic acid-grafted-ethylene glycol) (P(MAA-g-EG)) and poly(methacrylic acid-grafted-ethylene glycol) functionalized with wheat germ agglutinin (P(MAA-g-EG) WGA). The pH-responsive release results demonstrated that the pH shift from the stomach to the small intestine can be used as a physiologic trigger to release insulin from P(MAA-g-EG) and P(MAA-g-EG) WGA microparticles, thus limiting release of insulin into the acidic environment of the stomach. Microplates were successfully treated with PGM to create a surface that allowed for specific binding between mucins and lectins. The 1% PGM treatment followed by a 2 h BSA blocking step gave the most consistent results when incubated with F-WGA. In addition, the PGM-treated microplates were shown to create specific interactions between F-WGA and the PGM by use of a competitive carbohydrate. The 1% PGM treated microplates were also used to show that adhesion was improved in the P(MAA-g-EG) WGA microparticles over the P(MAA-g-EG) microparticles. The interaction between the PGM-treated microplate and P(MAA-g-EG) WGA was again shown to be specific by adding a competitive carbohydrate, while the interaction between P(MAA-g-EG) and the PGM-treated microplate was nonspecific. Cellular monolayers were used as another method for demonstrating that the functionalized microparticles increase adhesion over the nonfunctionalized microparticles. This work has focused on improving the mucoadhesive nature of P(MAA-g-EG) by functionalizing these hydrogel carriers with wheat germ agglutinin (WGA) to create a specific mucosal interaction and then evaluating the potential of these carriers as oral insulin delivery systems by in vitro methods. From these studies, it is concluded that the addition of the WGA on the microparticles produces a specific adhesion to carbohydrate-containing surfaces and that P(MAA-g-EG) WGA shows great promise as an oral insulin delivery system.     

Characterization of porous PLGA/PLA microparticles as a scaffold for three dimensional growth of breast cancer cells

We have designed and evaluated biodegradable porous polymeric microparticles as a scaffold for cell growth. The hypothesis was that microparticles with optimized composition and properties would have better cell adhesion and hence cell growth into a tissue-like structure. Solvent-evaporation method was modified using sucrose as an additive to form large porous microparticles of poly(D,L-lactic-co-glycolic) (PLGA) and polylactide (PLA) polymers. Microparticles containing hydrophilic polymers (poly(vinyl alcohol) and chitosan) incorporated in their internal matrix structure were also formulated. Different formulations of microparticles were evaluated for physical properties, cell adhesion, and cell growth in culture. PLA microparticles containing poly(vinyl alcohol) (PVA) in the matrix structure (PLA-PVA) and treated with serum prior to cell seeding demonstrated better cell adhesion and cell growth than other formulations of microparticles. Cells were seen to grow into clumps, engulfing microparticles completely with time, and forming a 3-D tissue-like structure. Cell density of 1.5 x 10(6) cells per mg of microparticles was achieved in 9 days of culture, which was a 7-fold increase from the initial seeding cell density. The mechanism of better cell growth on PLA-PVA microparticles appears to be due to the PVA associated with the internal matrix structure of microparticles. These microparticles demonstrated better wetting in culture and also cell adhesion. In addition to tissue engineering applications, microparticles with cancer cells grown into a tissue-like structure in vitro can be potentially used as a model system for preclinical evaluation of the cytotoxic effect of anticancer agents.     

Continuous supercritical emulsions extraction: a new technology for biopolymer microparticles production

Supercritical emulsion extraction (SEE) was recently proposed for the production of biopolymer microparticles starting from oil‐in‐water emulsions. This technology can improve the product quality because of the fast and selective extraction of the dispersed oily phase by using supercritical carbon dioxide (SC‐CO₂). However, until now, SEE was proposed in batch configuration, sharing with the traditional processes an intrinsically discontinuous operation and problems of batches reproducibility and process yield. In this study, by using a countercurrent packed column, the SEE process was proposed in a continuous operating mode (SEE‐CM) for the production of poly‐lactic‐co‐glycolic acid (PLGA) microparticles. The new process design takes advantage of the large contact area between the SC‐CO₂ and emulsion allowing the production of PLGA microparticles with controlled and narrow size distributions in only few minutes. SEE‐CM operating parameters such as pressure, temperature, and flow rate ratios were analyzed and the process efficiency in terms of recovered material and its size distribution compared with SEE (batch mode operation) and conventional evaporation technology. PLGA microparticles showed a mean particle size between 1–3 µm (depending on the droplet sizes) with a SD that was always smaller than that associated with particles produced by discontinuous processes. Single and double emulsions were successfully treated and the microparticles physico‐chemical properties showed no morphological and structural differences between the SEE‐CM‐produced microparticles and the ones obtained by conventional evaporation technology. Biotechnol. Bioeng. 2011; 108:676–686. © 2010 Wiley Periodicals, Inc.     

Fabrication and Characterization of Polyelectrolyte Microparticles with Protein

The incorporation of proteins into microparticles fabricated by layer-by-layer adsorption of oppositely charged polyelectrolytes (dextran sulfate and protamine) on protein microaggregates was studied. Microaggregates with insulin were prepared by two different techniques: 1) formation of insoluble polyelectrolyte complex consisting of insulin and dextran sulfate (aggregate size of 7-20 micro m), or 2) salting out of insulin from solution by sodium chloride (aggregate size of 5-13 micro m). Microparticles varying in the number of cycles (from 1 to 8) of polyelectrolyte adsorption on protein aggregates were examined and compared. Morphology of the microparticles was studied by scanning electron and optical microscopy. It was shown that polyelectrolyte microparticles retained the shape and dimensions of the initial protein aggregates used as a template. Ultrasonication of microparticles obtained using salted out protein aggregates resulted in the formation of stable nanoparticles (100-200 nm). Regulation of protein release from the microparticles of both types by varying the number of polyelectrolyte adsorption cycles and pH of the medium was demonstrated. Insulin not bound to polyelectrolytes was released from the microparticles at pH values between 6 and 8, which corresponds to the pH of the human small intestine and ileum.     

Adjuvant Chemotherapy for Brain Tumors Delivered via a Novel Intra-Cavity Moldable Polymer Matrix

Introduction

Polymer-based delivery systems offer innovative intra-cavity administration of drugs, with the potential to better target micro-deposits of cancer cells in brain parenchyma beyond the resected cavity. Here we evaluate clinical utility, toxicity and sustained drug release capability of a novel formulation of poly(lactic-co-glycolic acid) (PLGA)/poly(ethylene glycol) (PEG) microparticles.

Methods

PLGA/PEG microparticle-based matrices were molded around an ex vivo brain pseudo-resection cavity and analyzed using magnetic resonance imaging and computerized tomography. In vitro toxicity of the polymer was assessed using tumor and endothelial cells and drug release from trichostatin A-, etoposide- and methotrexate-loaded matrices was determined. To verify activity of released agents, tumor cells were seeded onto drug-loaded matrices and viability assessed.

Results

PLGA/PEG matrices can be molded around a pseudo-resection cavity wall with no polymer-related artifact on clinical scans. The polymer withstands fractionated radiotherapy, with no disruption of microparticle structure. No toxicity was evident when tumor or endothelial cells were grown on control matrices in vitro. Trichostatin A, etoposide and methotrexate were released from the matrices over a 3-4 week period in vitro and etoposide released over 3 days in vivo, with released agents retaining cytotoxic capabilities. PLGA/PEG microparticle-based matrices molded around a resection cavity wall are distinguishable in clinical scanning modalities. Matrices are non-toxic in vitro suggesting good biocompatibility in vivo. Active trichostatin A, etoposide and methotrexate can be incorporated and released gradually from matrices, with radiotherapy unlikely to interfere with release.

Conclusion

The PLGA/PEG delivery system offers an innovative intra-cavity approach to administer chemotherapeutics for improved local control of malignant brain tumors.     

Oral bioavailability of insulin contained in polysaccharide nanoparticles

The pharmacological activity of insulin-loaded dextran sulfate/chitosan nanoparticles was evaluated following oral dosage in diabetic rats. Nanoparticles were mucoadhesive and negatively charged with a mean size of 500 nm, suitable for uptake within the gastrointestinal tract. Insulin association efficiency was over 70% and was released in a pH-dependent manner under simulated gastrointestinal conditions. Orally delivered nanoparticles lowered basal serum glucose levels in diabetic rats around 35% with 50 and 100 IU/kg doses sustaining hypoglycemia over 24 h. Pharmacological availability was 5.6 and 3.4% for the 50 and 100 IU/kg doses, respectively, a significant increase over 1.6%, determined for oral insulin alone in solution. Confocal microscopic examinations of FITC-labeled insulin nanoparticles showed adhesion to rat intestinal epithelium, and internalization of insulin within the intestinal mucosa. Encapsulation of insulin into dextran sulfate/chitosan nanoparticles was a key factor in the improvement of the bioavailability of its oral delivery over insulin solution.     

Preparation and characterization of poly(D,L-lactide-co-glycolide) microspheres for controlled release of human growth hormone

The purpose of this research was to assess the physicochemical properties of a controlled release formulation of recombinant human growth hormone (rHGH) encapsulated in poly(D,L-lactide-co-glycolide) (PLGA) composite microspheres. rHGH was loaded in poly(acryloyl hydroxyethyl) starch (acHES) microparticles, and then the protein-containing microparticles were encapsulated in the PLGA matrix by a solvent extraction/evaporation method. rHGH-loaded PLGA microspheres were also prepared using mannitol without the starch hydrogel microparticle microspheres for comparison. The detection of secondary structure changes in protein was investigated by using a Fourier Transfer Infrared (FTIR) technique. The composite microspheres were spherical in shape (44.6±2.47 μm), and the PLGA-mannitol microspheres were 39.7±2.50 μm. Drug-loading efficiency varied from 93.2% to 104%. The composite microspheres showed higher overall drug release than the PLGA/mannitol microspheres. FTIR analyses indicated good stability and structural integrity of HGH localized in the microspheres. The PLGA-acHES composite microsphere system could be useful for the controlled delivery of protein drugs.     

How autocatalysis accelerates drug release from PLGA-based microparticles: a quantitative treatment

The major aim of this study was to better understand the importance of autocatalysis in poly(lactic-co-glycolic acid) (PLGA)-based microparticles used as controlled drug delivery systems. Upon contact with biological fluids, PLGA is degraded into shorter chain alcohols and acids. An accumulation of the latter can lead to significant drops in micro-pH and subsequent accelerated polymer degradation. The system size, determining the diffusion path lengths, plays a crucial role for the occurrence/absence of autocatalytic effects. Using an oil-in-water solvent-extraction/evaporation process, different-sized drug-free and drug-loaded, PLGA-based microparticles were prepared and physicochemically characterized (SEM, DSC, SEC, optical microscopy, and UV-spectrophotometry) before and upon exposure to simulated biological fluids. Based on these experimental results, an adequate mathematical theory was developed describing the dominating mass transfer processes and chemical reactions. Importantly, a quantitative relationship could be established between the dimension of the device and the resulting drug release patterns, taking the effects of autocatalysis into account.     

Injectable Supramolecular Hydrogel from Insulin-Loaded Triblock PCL-PEG-PCL Copolymer and γ-Cyclodextrin with Sustained-Release Property

Supramolecular hydrogels formed by cyclodextrins and polymers have been widely investigated as a biocompatible, biodegradable and controllable drug delivery system. In this study, a supramolecular hydrogel based on biodegradable poly(caprolactone)–poly(ethylene glycol)–poly(caprolactone) (PCL-PEG-PCL) triblock copolymers and γ-cyclodextrin (γ-CD) was prepared through inclusion complexation as an injectable, sustained-release vehicle for insulin. The triblock copolymer PCL-PEG-PCL was synthesised by the ring-opening polymerisation method, using microwave irradiation. The polymerisation reaction and the copolymer structures were evaluated by nuclear magnetic resonance (NMR) and gel permeation chromatography (GPC). The supramolecular hydrogel was prepared in aqueous solution by blending an aqueous γ-CD solution with an aqueous solution of PCL-PEG-PCL triblock copolymer at room temperature. In vitro insulin release through the hydrogel system was studied. The relative surface hydrophobicity of standard and released insulin from the SMGel was estimated using 8-anilino-1-naphthalene sulfonic acid (ANS). Results of ¹HNMR and gel permeation chromatography revealed that microwave irradiation is a simple and reliable method for synthesis of PCL-PEG-PCL copolymer. Gelation occurred within a minute. The supramolecular hydrogel obtained by mixing 10.54% (w/v) γ-CD and 2.5% (w/v) copolymer had an excellent syringeability. Insulin was released up to 80% over a period of 20 days. Insulin kept its initial folding after formulating and releasing from SMGel. A supramolecular hydrogel based on complexation of triblock PCL-PEG-PCL copolymer with γ-cyclodextrin is a suitable system for providing sustained release of therapeutic proteins, with desirable flow behaviour.Key words: insulin, PCL-PEG-PCL, supramolecular hydrogel, triblock copolymer, γ-CD     

Effect of a freeze-dried CMC/PLGA microsphere matrix of rhBMP-2 on bone healing

The hypothesis of this research was that implants of poly(lactide-co-glycolide) (PLGA) microspheres loaded with bone morphogenetic protein-2 (rhBMP-2) and distributed in a freeze-dried carboxymethylcellulose (CMC) matrix would produce more new bone than would matrix implants of non-protein-loaded microspheres or matrix implants of only CMC. To test this hypothesis it was necessary to fashion microsphere-loaded CMC implants that were simple to insert, fit precisely into a defect, and would not elicit swelling. Microspheres were produced via a water-in-oil-in-water double-emulsion system and were loaded with rhBMP-2 by soaking them in a buffered solution of the protein at a concentration of 5.4 mg protein per gram of PLGA. Following recovery of the loaded microspheres by lyophilization matrices for implantation were prepared by lyophilizing a suspension of the microspheres in 2% CMC in flat-bottom tissue culture plates. Similar matrices were made with 2% CMC and with 2% CMC containing blank microspheres. A full-thickness calvarial defect model in New Zealand white rabbits was used to assess bone growth. Implants fit the defect well allowing for direct application. Six weeks postsurgery, defects were collected and processed for undecalcified histology. In vitro, 60% of the loaded rhBMP-2 released from devices or microspheres in 5 to 7 days. With the unembedded microspheres releasing faster than those embedded in CMC In vivo. the rhBMP-2 microspheres greatly enhanced bone healing, whereas nonloaded PLGA microspheres in the CMC implants had little effect. The results showed that a lyophilized device of rhBMP-2 PLGA microspheres in CMC was an effective implantable protein-delivery system for the use in bone repair. Published: October 7. 2001.     

In situ forming and rutin-releasing chitosan hydrogels as injectable dressings for dermal wound healing

An in situ gel-forming system composed of rutin- and tyramine-conjugated chitosan derivatives, horseradish peroxidase (HRP), and hydrogen peroxide (H(2)O(2)) was prepared and applied to dermal wound repair. Rutin was employed to enhance production and accumulation of extracellular matrix in the healing process. In vitro study demonstrates that released rutin significantly enhanced cell proliferation as compared with media without rutin. In vivo wound healing study was performed by injecting hydrogels on rat dorsal wounds with a diameter of 8 mm for 14 days. Histological results demonstrated that rutin-conjugated hydrogel exhibited enhancement of wound healing as compared with treatments with PBS, hydrogel without rutin, and a commercialized wound dressing (Duoderm). More specifically, rutin-conjugated hydrogels induced better defined formation of neo-epithelium and thicker granulation, which is closer to the original epithelial tissue. As a result, this study suggests that the in situ gel-forming system can be a promising injectable gel-type wound dressing.     

PLGA Nanoparticles Formed by Single- or Double-emulsion with Vitamin E-TPGS

Poly(lactic-co-glycolic acid) (PLGA) is a biocompatible member of the aliphatic polyester family of biodegradable polymers. PLGA has long been a popular choice for drug delivery applications, particularly since it is already FDA-approved for use in humans in the form of resorbable sutures. Hydrophobic and hydrophilic drugs are encapsulated in PLGA particles via single- or double-emulsion. Briefly, the drug is dissolved with polymer or emulsified with polymer in an organic phase that is then emulsified with the aqueous phase. After the solvent has evaporated, particles are washed and collected via centrifugation for lyophilization and long term storage. PLGA degrades slowly via hydrolysis in aqueous environments, and encapsulated agents are released over a period of weeks to months. Although PLGA is a material that possesses many advantages for drug delivery, reproducible formation of nanoparticles can be challenging; considerable variability is introduced by the use of different equipment, reagents batch, and precise method of emulsification. Here, we describe in great detail the formation and characterization of microparticles and nanoparticles formed by single- or double-emulsion using the emulsifying agent vitamin E-TPGS. Particle morphology and size are determined with scanning electron microscopy (SEM). We provide representative SEM images for nanoparticles produced with varying emulsifier concentration, as well as examples of imaging artifacts and failed emulsifications. This protocol can be readily adapted to use alternative emulsifiers (e.g. poly(vinyl alcohol), PVA) or solvents (e.g. dichloromethane, DCM).