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1.  Controlled release of vascular endothelial growth factor using poly-lactic-co-glycolic acid microspheres: In vitro characterization and application in polycaprolactone fumarate nerve conduits 
Acta biomaterialia  2011;8(2):511-518.
Vascular endothelial growth factor (VEGF) is a potent angiogenic stimulator. Controlled release of such stimulators may enhance and guide the vascularization process, and when applied in a nerve conduit may play a role in nerve regeneration. We report the fabrication and in vitro characterization of VEGF encapsulating poly-lactic-co-glycolic acid (PLGA) microspheres and the in vivo application of nerve conduits supplemented with VEGF-containing microspheres. PLGA microspheres containing VEGF were prepared by the double emulsion-solvent evaporation technique. This yielded 83.16% of the microspheres with a diameter < 53 µm. VEGF content measured by ELISA indicated 93.79 ±10.64% encapsulation efficiency. Release kinetics were characterized by an initial burst release of 67.6±8.25% within the first 24 hours, followed by consistent release of approximately 0.34% per day for 4 weeks. Bioactivity of the released VEGF was tested by human umbilical vein endothelial cell (HUVEC) proliferation assay. VEGF released at all time points enhanced HUVEC proliferation confirming that VEGF retained its bioactivity through the 4-week time period. When the microsphere delivery system was placed in a biosynthetic nerve scaffold, robust nerve regeneration was observed. This study established a novel system for controlled release of growth factors and enables in vivo studies of nerve conduits conditioned with this system.
doi:10.1016/j.actbio.2011.10.001
PMCID: PMC3972821  PMID: 22019759
microsphere; poly-lactic co-glycolic acid; vascular endothelial growth factor; bioactivity; biodegradation; nerve guide
2.  Pharmacokinetic and pharmacodynamic profiles of recombinant human erythropoietin-loaded poly(lactic-co-glycolic acid) microspheres in rats 
Acta Pharmacologica Sinica  2011;33(1):137-144.
Aim:
To characterize the pharmacokinetic and pharmacodynamic profiles of the recombinant human erythropoietin (rhEPO)-loaded poly(lactic-co-glycolic acid) (PLGA) microspheres in rats.
Methods:
The rhEPO-loaded microspheres were prepared using a solid-in-oil-in-water emulsion method. Pharmacokinetics and pharmacodynamics of the rhEPO-loaded microspheres were evaluated in male Sprague-Dawley rats. The serum rhEPO level was determined with ELISA. The level of anti-rhEPO antibody in the serum was measured to assess the immunogenicity of rhEPO released from the microspheres.
Results:
rhEPO was almost completely released from the PLGA microspheres in vitro, following zero-order release kinetics over approximately 30 d. After intramuscular injection (10 000 or 30 000 IU rhEPO/kg) in the rats, the serum rhEPO concentration reached maximum levels on d 1, then decreased gradually and was maintained at nearly steady levels for approximately 4 weeks. Furthermore, the release of rhEPO from the PLGA microspheres was found to be controlled mainly by a dissolution/diffusion mechanism. A good linear correlation (R2=0.98) was obtained between the in vitro and in vivo release data. A single intramuscular injection of the rhEPO-loaded PLGA microspheres (10 000 or 30 000 IU rhEPO/kg) in the rats resulted in elevated hemoglobin and red blood cell concentrations for more than 28 d. Moreover, the immunogenicity of rhEPO released from the PLGA microspheres was comparable with that of the unencapsulated rhEPO.
Conclusion:
The results prove the feasibility of using the PLGA-based microspheres to deliver rhEPO for approximately 1 month.
doi:10.1038/aps.2011.157
PMCID: PMC4010276  PMID: 22139004
recombinant human erythropoietin; poly(lactic-co-glycolic acid); microspheres; pharmacokinetics; pharmacodynamics
3.  Development and evaluation of a novel biodegradable sustained release microsphere formulation of paclitaxel intended to treat breast cancer 
Objective:
The objective of this study was to develop a novel 1 month depot paclitaxel (PTX) microspheres that give a sustained and complete drug release.
Materials and Methods:
PTX loaded microspheres were prepared by o/w emulsion solvent evaporation technique using the blends of poly(lactic-co-glycolic acid) (PLGA) 75/25, polycaprolactone 14,000 and polycaprolactone 80,000. Fourier transform infrared spectroscopy was used to investigate drug excipient compatibility. Compatible blends were used to prepare F1-F6 microspheres, the process was characterised and the optimum formulation was selected based on the release. Optimised formulation was characterised for solid state of the drug using the differential scanning calorimetry (DSC) studies, surface morphology using the scanning electron microscopy (SEM), in vivo drug release, in vitro in vivo correlation (IVIVC) and anticancer activity. Anticancer activity of release medium was determined using the cell viability assay in Michigan Cancer Foundation (MCF-7) cell line.
Results:
Blend of PLGA with polycaprolactone (Mwt 14,000) at a ratio of 1:1 (F5) resulted in complete release of the drug in a time frame of 30 days. F5 was considered as the optimised formulation. Incomplete release of the drug resulted from other formulations. The surface of the optimised formulation was smooth and the drug changed its solid state upon fabrication. The formulation also resulted in 1-month drug release in vivo. The released drug from F5 demonstrated anticancer activity for 1-month. Cell viability was reduced drastically with the release medium from F5 formulation. A 100% IVIVC was obtained with F5 formulation suggesting the authenticity of in vitro release, in vivo release and the use of the formulation in breast cancer.
Conclusions:
From our study, it was concluded that with careful selection of different polymers and their combinations, PTX 1 month depot formulation with 100% drug release and that can be used in breast cancer was developed.
doi:10.4103/2230-973X.119212
PMCID: PMC3807978  PMID: 24167783
Blends; complete release; microspheres; paclitaxel; polycaprolactone; polylactide-co-glycolide
4.  Release of a Wound-Healing Agent from PLGA Microspheres in a Thermosensitive Gel 
BioMed Research International  2013;2013:387863.
The purpose of this research was to develop a topical microsphere delivery system in a thermosensitive 20% poloxamer 407 gel (Pluronic F127) to control release of KSL-W, a cationic antimicrobial decapeptide, for a period of 4–7 days for potential application in combat related injuries. KSL-W loaded microsphere formulations were prepared by a solvent extraction-evaporation method (water-oil-water), with poly (D,L-lactic-co-glycolic acid) (PLGA) (50 : 50, low-weight, and hydrophilic end) as the polymeric system. After optimization of the process, three formulations (A, B, and C) were prepared with different organic to water ratio of the primary emulsion while maintaining other components and manufacturing parameters constant. Formulations were characterized for surface morphology, porous nature, drug loading, in vitro drug release, and antimicrobial activity. Microspheres containing 20% peptide with porous surfaces and internal structure were prepared in satisfactory yields and in sizes varying from 25 to 50 μm. Gels of 20% Pluronic F127, which were liquid at or below 24.6°C and formed transparent films at body temperature, were used as carriers for the microspheres. Rheological studies showed a gelation temperature of 24.6°C for the 20% Pluronic F127 gel alone. Gelation temperature and viscosity of formulations A, B, and C as a function of temperature were very close to those of the carrier. A Franz diffusion cell system was used to study the release of peptide from the microspheres suspended in both, phosphate-buffered saline (PBS) and a 20% Pluronic F127 gel. In vitro release of greater than 50% peptide was found in all formulations in both PBS and the gel, and in one formulation there was a release of 75% in both PBS and the gel. Fractions collected from the release process were also tested for bactericidal activity against Staphylococcus epidermidis using the broth microdilution method and found to provide effective antimicrobial activity to warrant consideration and testing in animal wound models for treating combat-related injuries.
doi:10.1155/2013/387863
PMCID: PMC3808721  PMID: 24224161
5.  Evaluation of protective efficacy using a nonstructural protein NS1 in DNA vaccine–loaded microspheres against dengue 2 virus 
Dengue virus results in dengue fever or severe dengue hemorrhagic fever/dengue shock syndrome in humans. The purpose of this work was to develop an effective antidengue virus delivery system, by designing poly (dl-lactic-co-glycolic) acid/polyethylene glycol (PLGA/PEG) microspheres using a double-emulsion solvent extraction method, for vaccination therapy based on locally and continuously sustained biological activity. Nonstructural protein 1 (NS1) in deoxyribonucleic acid (DNA) vaccine–loaded PLGA/PEG microspheres exhibited a high loading capacity (4.5% w/w), yield (85.2%), and entrapment efficiency (39%), the mean particle size 4.8 μm, and a controlled in vitro release profile with a low initial burst (18.5%), lag time (4 days), and continued released protein over 70 days. The distribution of protein on the microspheres surface, outer layer, and core were 3.0%, 28.5%, and 60.7%, respectively. A release rate was noticed to be 1.07 μg protein/mg microspheres/day of protein release, maintained for 42 days. The cumulative release amount at Days 1, 28, and 42 was 18.5, 53.7, and 62.66 μg protein/mg microspheres, respectively. The dengue virus challenge in mice test, in which mice received one dose of 20 μg NS1 protein content of microspheres, in comparison with NS1 protein in Al(OH)3 or PBS solution, was evaluated after intramuscular immunization of BALB/c mice. The study results show that the greatest survival was observed in the group of mice immunized with NS1 protein–loaded PLGA/PEG microspheres (100%). In vivo vaccination studies also demonstrated that NS1 protein–loaded PLGA/PEG microspheres had a protective ability; its steady-state immune protection in rat plasma changed from 4,443 ± 1,384 pg/mL to 10,697 ± 3,197 pg/mL, which was 2.5-fold higher than that observed for dengue virus in Al(OH)3 at 21 days. These findings strongly suggest that NS1 protein–loaded PLGA/PEG microspheres offer a new therapeutic strategy in optimizing the vaccine incorporation and delivery properties of these potential vaccine targeting carriers.
doi:10.2147/IJN.S49972
PMCID: PMC3753149  PMID: 23990724
dengue virus; adjuvant; NS1 protein–loaded PLGA/PEG microspheres; vaccination
6.  Nano-fibrous scaffold for controlled delivery of recombinant human PDGF-BB 
The localized and temporally controlled delivery of growth factors is key to achieving optimal clinical efficacy. In sophisticated tissue engineering strategies, the biodegradable scaffold is preferred to serve as both a three-dimensional (3-D) substrate and a growth factor delivery vehicle to promote cellular activity and enhance tissue neogenesis. This study presents a novel approach to fabricate tissue engineering scaffolds capable of controlled growth factor delivery whereby growth factor containing microspheres were incorporated into 3-D scaffolds with good mechanical properties, well-interconnected macroporous and nano-fibrous structures. The microspheres were uniformly distributed throughout the nano-fibrous scaffold and their incorporation did not interfere the macro-, micro-, and nanostructures of the scaffold. The release kinetics of platelet-derived growth factor-BB (PDGF-BB) from microspheres and scaffolds was investigated using poly(lactic-co-glycolic acid) (PLGA50) microspheres with different molecular weights (6.5 and 64kDa, respectively) and microsphere-incorporated poly(l-lactic acid) (PLLA) nano-fibrous scaffolds. Incorporation of microspheres into scaffolds significantly reduced the initial burst release. Sustained release from several days to months was achieved through different microspheres in scaffolds. Released PDGF-BB was demonstrated to possess biological activity as evidenced by stimulation of human gingival fibroblast DNA synthesis in vitro. The successful generation of 3-D nano-fibrous scaffold incorporating controlled-release factors indicates significant potential for more complex tissue regeneration.
doi:10.1016/j.jconrel.2006.01.011
PMCID: PMC2572756  PMID: 16516328
PDGF; Factor; Controlled delivery; Nano-fiber; Scaffold; Tissue engineering; Microspheres; Matrix; Polymer
7.  Controlled Release of Octreotide and Assessment of Peptide Acylation from Poly(D,L-lactide-co-hydroxymethyl glycolide) Compared to PLGA Microspheres 
Pharmaceutical Research  2011;29(1):110-120.
ABSTRACT
Purpose
To investigate the in vitro release of octreotide acetate, a somatostatin agonist, from microspheres based on a hydrophilic polyester, poly(D,L-lactide-co-hydroxymethyl glycolide) (PLHMGA).
Methods
Spherical and non-porous octreotide-loaded PLHMGA microspheres (12 to 16 μm) and loading efficiency of 60–70% were prepared by a solvent evaporation. Octreotide release profiles were compared with commercial PLGA formulation (Sandostatin LAR®); possible peptide modification with lactic, glycolic and hydroxymethyl glycolic acid units was monitored.
Results
PLHMGA microspheres showed burst release (~20%) followed by sustained release for 20–60 days, depending on the hydrophilicity of the polymer. Percentage of released loaded peptide was high (70–90%); > 60% of released peptide was native octreotide. PLGA microspheres did not show peptide release for the first 10 days, after which it was released in a sustained manner over the next 90 days; > 75% of released peptides were acylated adducts.
Conclusions
PLHMGA microspheres are promising controlled systems for peptides with excellent control over release kinetics. Moreover, substantially less peptide modification occurred in PLHMGA than in PLGA microspheres.
Electronic Supplementary Material
The online version of this article (doi:10.1007/s11095-011-0517-3) contains supplementary material, which is available to authorized users.
doi:10.1007/s11095-011-0517-3
PMCID: PMC3246586  PMID: 21744173
acylation; aliphatic polyester; controlled release; microspheres; octreotide; PLGA; stability
8.  Sustained-Release Delivery of Octreotide from Biodegradable Polymeric Microspheres 
AAPS PharmSciTech  2011;12(4):1293-1301.
The study reports on the drug release behavior of a potent synthetic somatostatin analogue, octreotide acetate, from biocompatible and biodegradable microspheres composed of poly-lactic-co-glycolic acid (PLGA) following a single intramuscular depot injection. The serum octreotide levels of three Oakwood Laboratories formulations and one Sandostatin LAR® formulation were compared. Three formulations of octreotide acetate-loaded PLGA microspheres were prepared by a solvent extraction and evaporation procedure using PLGA polymers with different molecular weights. The in vivo drug release study was conducted in male Sprague–Dawley rats. Blood samples were taken at predetermined time points for up to 70 days. Drug serum concentrations were quantified using a radioimmunoassay procedure consisting of radiolabeled octreotide. The three octreotide PLGA microsphere formulations and Sandostatin LAR® all showed a two-phase drug release profile (i.e., bimodal). The peak serum drug concentration of octreotide was reached in 30 min for all formulations followed by a decline after 6 h. Following this initial burst and decline, a second-release phase occurred after 3 days. This second-release phase exhibited sustained-release behavior, as the drug serum levels were discernible between days 7 and 42. Using pharmacokinetic computer simulations, it was estimated that the steady-state octreotide serum drug levels would be predicted to fall in the range of 40–130 pg/10 μL and 20–100 pg/10 μL following repeat dosing of the Oakwood formulations and Sandostatin LAR® every 28 days and every 42 days at a dose of 3 mg/rat, respectively.
doi:10.1208/s12249-011-9693-z
PMCID: PMC3225556  PMID: 21948321
in vivo drug release; pharmacokinetic simulation; PLGA microspheres; polypeptide/protein drug delivery; single depot injection
9.  Inhalable Large Porous Microspheres of Low Molecular Weight Heparin: In Vitro and In Vivo Evaluation 
This study tests the feasibility of large porous particles as long-acting carriers for pulmonary delivery of low molecular weight heparin (LMWH). Microspheres were prepared with a biodegradable polymer, poly(lactic-co-glycolic acid) (PLGA), by a double-emulsion–solvent-evaporation technique. The drug entrapment efficiencies of the microspheres were increased by modifying them with three different additives—polyethyleneimine (PEI), Span 60 and stearylamine. The resulting microspheres were evaluated for morphology, size, zeta potential, density, in vitro drug-release properties, cytotoxicity, and for pulmonary absorption in vivo. Scanning electron microscopic examination suggests that the porosity of the particles increased with the increase in aqueous volume fraction. The amount of aqueous volume fraction and the type of core-modifying agent added to the aqueous interior had varying degrees of effect on the size, density and aerodynamic diameter of the particles. When PEI was incorporated in the internal aqueous phase, the entrapment efficiency was increased from 16.22±1.32% to 54.82±2.79%. The amount of drug released in the initial burst phase and the release-rate constant for the core-modified microspheres were greater than those for the plain microspheres. After pulmonary administration, the half-life of the drug from the PEI- and stearylamine-modified microspheres was increased by 5- to 6-fold compared to the drug entrapped in plain microspheres. The viability of Calu-3 cells was not adversely affected when incubated with the microspheres. Overall, the data presented here suggest that the newly developed porous microspheres of LMWH have the potential to be used in a form deliverable by dry-powder inhaler as an alternative to multiple parenteral administrations of LMWH.
doi:10.1016/j.jconrel.2008.03.013
PMCID: PMC2556066  PMID: 18471921
Large porous particles; microspheres; Low molecular weight heparin; pulmonary delivery
10.  Improved peripheral nerve regeneration with sustained release nerve growth factor microspheres in small gap tubulization 
Objective: To evaluate the long-term results of the use of nerve growth factor (NGF)-loaded poly-D, L-lactide-co-glycolide (PLGA) microspheres for improve nerve regeneration with small gap tubulization. Methods: NGF microspheres were prepared by a modified W/O/W emulsion solvent evaporation method. Forty-eight male SD rats were separated into 4 groups and received a chitin conduit to bridge a sciatic nerve injury left a 2 mm gap. Saline (Group A), 20 ng/ml NGF solution (Group B), blank PLGA microspheres (Group C), or 40 ng/ml NGF-loaded microspheres (Group D) was injected in the gap. Each group had two study endpoints, 3 months subgroup and 1 year subgroup. Results: The myelinated fiber count at 2 mm distal to the conduit at 1 year was slightly less than at 3 months in all groups (P>0.05). However, the maturity of the myelinated nerves at 1 year was obviously improved. The fiber count, myelin sheath thickness, axon area of NGF microsphere group were significantly higher than the saline groups at 3 months (P=0.05, P<0.05, and P<0.05, respectively). The SFI was significantly improved in NGF microspheres group compared to the saline group and NGF solution group at 1 year (P<0.05, and P<0.05, respectively). Conclusions: The results demonstrated that the release of NGF microspheres in small gap tubulization benefit on peripheral nerve injury facilitated nerve regeneration histologically, especially for the maturity of early regenerative nerve fibers and also had an effect on functional recovery in the long term.
PMCID: PMC4113503  PMID: 25075258
Nerve growth factor (NGF); microsphere; nerve regeneration; small gap; tubulization
11.  Biodegradable Gelatin Microparticles as Delivery Systems for the Controlled Release of Bone Morphogenetic Protein-2 
Acta biomaterialia  2008;4(5):1126-1138.
This work evaluated gelatin microparticles and biodegradable composite scaffolds for the controlled release of bone morphogenetic protein-2 (BMP-2) in vitro and in vivo. Gelatin crosslinking (10 and 40 mM glutaraldehyde), BMP-2 dose (6 and 60 ng BMP-2 / mg dry microparticles), buffer type (phosphate buffered saline (PBS) and collagenase-containing PBS), and gelatin type (acidic and basic) were investigated for their effects on BMP-2 release. Release profiles were also observed using poly(lactic-co-glycolic acid) (PLGA) microparticles with varying molecular weights (8,300 and 57,500). In vitro and in vivo studies were conducted using radiolabeled BMP-2; the chloramine-T method was preferred over Bolton-Hunter reagent for radioiodination with this system. BMP-2 release from PLGA microparticles resulted in a moderate burst release followed by minimal cumulative release, while BMP-2 release from gelatin microparticles exhibited minimal burst release followed by linear release kinetics in vitro. Growth factor dose had a small effect on its normalized release kinetics probably because of an equilibrium between gelatin-bound and unbound BMP-2. Differences in release from acidic and basic gelatin microparticles may result from the different pretreatment conditions used for gelatin synthesis. The in vitro release kinetics for both gelatin microparticles alone and within composite scaffolds were dependent largely on the extent of gelatin crosslinking; varying buffer type served to confirm that controlled release relies on enzymatic degradation of the gelatin for controlled release. Finally, in vivo studies with composite scaffolds exhibited minimal burst and linear release up to 28 days. In summary, dose effects on BMP-2 release were found to be minimal while varying gelatin type and release medium can alter release kinetics. These results demonstrate a systematic control of BMP-2 delivery from gelatin microparticles can be achieved by altering the extent of basic gelatin crosslinking.
doi:10.1016/j.actbio.2008.04.002
PMCID: PMC3018807  PMID: 18474452
Controlled release; Growth factor delivery; Bone morphogenetic protein; Gelatin microparticles; Bone tissue engineering
12.  A Short Term Quality Control Tool for Biodegradable Microspheres 
AAPS PharmSciTech  2014;15(3):530-541.
Accelerated in vitro release testing methodology has been developed as an indicator of product performance to be used as a discriminatory quality control (QC) technique for the release of clinical and commercial batches of biodegradable microspheres. While product performance of biodegradable microspheres can be verified by in vivo and/or in vitro experiments, such evaluation can be particularly challenging because of slow polymer degradation, resulting in extended study times, labor, and expense. Three batches of Leuprolide poly(lactic-co-glycolic acid) (PLGA) microspheres having varying morphology (process variants having different particle size and specific surface area) were manufactured by the solvent extraction/evaporation technique. Tests involving in vitro release, polymer degradation and hydration of the microspheres were performed on the three batches at 55°C. In vitro peptide release at 55°C was analyzed using a previously derived modification of the Weibull function termed the modified Weibull equation (MWE). Experimental observations and data analysis confirm excellent reproducibility studies within and between batches of the microsphere formulations demonstrating the predictability of the accelerated experiments at 55°C. The accelerated test method was also successfully able to distinguish the in vitro product performance between the three batches having varying morphology (process variants), indicating that it is a suitable QC tool to discriminate product or process variants in clinical or commercial batches of microspheres. Additionally, data analysis utilized the MWE to further quantify the differences obtained from the accelerated in vitro product performance test between process variants, thereby enhancing the discriminatory power of the accelerated methodology at 55°C.
doi:10.1208/s12249-013-0052-0
PMCID: PMC4037492  PMID: 24519488
accelerated in vitro release; biodegradable microspheres; modified Weibull equation (MWE); QC tool
13.  IL-1ra delivered from poly(lactic-co-glycolic acid) microspheres attenuates IL-1β-mediated degradation of nucleus pulposus in vitro 
Arthritis Research & Therapy  2012;14(4):R179.
Introduction
Inflammation plays a key role in the progression of intervertebral disc degeneration, a condition strongly implicated as a cause of lower back pain. The objective of this study was to investigate the therapeutic potential of poly(lactic-co-glycolic acid) (PLGA) microspheres loaded with interleukin-1 receptor antagonist (IL-1ra) for sustained attenuation of interleukin-1 beta (IL-1β) mediated degradative changes in the nucleus pulposus (NP), using an in vitro model.
Methods
IL-1ra was encapsulated in PLGA microspheres and release kinetics were determined over 35 days. NP agarose constructs were cultured to functional maturity and treated with combinations of IL-1β and media conditioned with IL-1ra released from microspheres at intervals for up to 20 days. Construct mechanical properties, glycosaminoglycan content, nitrite production and mRNA expression of catabolic mediators were compared to properties for untreated constructs using unpaired Student's t-tests.
Results
IL-1ra release kinetics were characterized by an initial burst release reducing to a linear release over the first 10 days. IL-1ra released from microspheres attenuated the degradative effects of IL-1β as defined by mechanical properties, glycosaminoglycans (GAG) content, nitric oxide production and mRNA expression of inflammatory mediators for 7 days, and continued to limit functional degradation for up to 20 days.
Conclusions
In this study, we successfully demonstrated that IL-1ra microspheres can attenuate the degradative effects of IL-1β on the NP for extended periods. This therapeutic strategy may be appropriate for treating early-stage, cytokine-mediated disc degeneration. Ongoing studies are focusing on testing IL-1ra microspheres in an in vivo model of disc degeneration, as a prelude to clinical translation.
doi:10.1186/ar3932
PMCID: PMC3580573  PMID: 22863285
14.  Preparation and characterization of gelatin surface modified PLGA microspheres 
AAPS PharmSci  2001;3(2):14-24.
This study optimized conditions for preparing and characterizing gelatin surface modified poly (lactic-co-glycolic acid) (PLGA) copolymer microspheres and determined this systems interaction with fibronectin. Some gelatin microspheres have an affinity for fibronectin-bearing surfaces; these miscrospheres exploit the interaction between gelatin and fibronectin. PLGA copolymer microspheres were selected because they have reproducible and slowrelease characteristics in vivo. The PLGA microspheres were surface modified with gelatin to impart fibronectin recognition. Dexamethasone was incorporated into these microspheres because dexamethasone is beneficial in chronic human diseases associated with extra fibronectin expression (eg, cardiovascular disease, inflammatory disorders, rheumatoid arthritis). The gelatin surface modified PLGA microspheres (prepared by adsorption, conjugation, and spray coating) were investigated and characterized by encapsulation efficiency, particle size, in vitro release, and affinity for fibronectin. The gelatincoated PLGA microspheres had higher interaction with fibronectin compared with the other gelatin surface modified PLGA microspheres (adsorption and conjugation). Dexamethasone was released slowly (over 21 days) from gelatin surface modified PLGA microspheres.
doi:10.1208/ps030211
PMCID: PMC2779555  PMID: 11741261
Microspheres; Surface Modification; Gelatin; Fibronectin; PLGA; Dexamethasone
15.  Identification of chemically modified peptide from poly(D,L-lactide-co-glycolide) microspheres under in vitro release conditions 
AAPS PharmSciTech  2003;4(4):392-405.
The purpose of this research was to study the chemical reactivity of a somatostatin analogue octreotide acetate, formulated in microspheres with polymers of varying molecular weight and co-monomer ratio under in vitro testing conditions. Poly(D,L-lactide-co-glycolide) (PLGA) and poly(D,L-lactide) (PLA) microspheres were prepared by a solvent extraction/evaporation method. The microspheres were characterized for drug load, impurity content, and particle size. Further, the microspheres were subjected to in vitro release testing in acetate buffer (pH 4.0) and phosphate buffered saline (PBS) (pH 7.2). In acetate buffer, 3 microsphere batches composed of low molecular weight PLGA 50∶50, PLGA 85∶15, and PLA polymers (≤10 kDa) showed 100% release with minimal impurity formation (<10%). The high molecular weight PLGA 50∶50 microspheres (28 kDa) displayed only 70% cumulative release in acetate buffer with significant impurity formation (∼24%). In PBS (pH 7.4), on the other hand, only 50% release was observed with the same low molecular weight batches (PLGA 50∶50, PLGA 85∶15, and PLA) with higher percentages of hydrophobic impurity formation (ie, 40%, 26%, and 10%, respectively). In addition, in PBS, the high molecular weight PLGA 50∶50 microspheres showed only 20% drug release with ∼60% mean impurity content. The chemically modified peptide impurities inside microspheres were structurally confirmed through Fourier transform-mass spectrometry (FT-MS) and liquid chromatography/mass spectrometry (LC-MS/MS) analyses after extraction procedures. The adduct compounds were identified as covalently modified conjugates of octreotide with lactic and glycolic acid monomers within polymeric microspheres. The data suggest that due to steric hindrance factors, polymers with greater lactide content were less amenable to the formation of adduct impurities compared with PLGA 50∶50 copolymers.
doi:10.1208/pt040450
PMCID: PMC2750643  PMID: 15198545
somatostatin analogues; octreotide acetate; peptide acylation; peptide stability; poly(D,L-lactide-co-glycolide) (PLGA) microspheres
16.  Local Toxicity from Local Anesthetic Polymeric Microparticles 
Anesthesia and analgesia  2013;116(4):794-803.
Background
Local tissue injury from sustained release formulations for local anesthetics can be severe. There is considerable variability in reporting of that injury. We investigated the influence of the intrinsic myotoxicity of the encapsulated local anesthetic (lidocaine, low; bupivacaine, high) on tissue reaction in rats.
Methods
Cytotoxicity from a range of lidocaine and bupivacaine concentrations was measured in C2C12 myotubes over 6 days. Rats were given sciatic nerve blocks with 4 microparticulate formulations of lidocaine and bupivacaine: 10% (w/w) lidocaine poly-lactic-co-glycolic acid (PLGA), 10% (w/w) bupivacaine PLGA, 50% (w/w) lidocaine PLGA, and 50% (w/w) bupivacaine PLGA. Effectiveness of nerve blockade was assessed by a modified hotplate test and weight-bearing measurements. Myotoxicity was scored in histologic sections of injection sites. Bupivacaine and lidocaine release kinetics from the particles were measured.
Results
Median sensory blockade duration for 50% (w/w) lidocaine was 255 (90–540) min versus 840 (277–1215) min for 50% (w/w) bupivacaine (P=0.056). All microparticulate formulations resulted in myotoxicity. The choice of local anesthetic did not influence the severity of myotoxicity. Median myotoxicity scores for 50% (w/w) lidocaine compared to 50% (w/w) bupivacaine at 4 days was 3.4 (2.1–4.2) vs. 3.3 (2.9–3.5)(P=0.44) and at 14 days 1.9 (1.8–2.4) versus 1.7 (1.3–1.9)(P=0.23) respictively.
Conclusions
Lidocaine and bupivacaine PLGA microspheres resulted in similar degrees of myotoxicity, irrespective of drug loading. Intrinsic myotoxicity did not predict tissue injury from sustained release of these anesthetics. Caution is warranted in the use of such devices near muscle and nerve.
doi:10.1213/ANE.0b013e31828174a7
PMCID: PMC3606664  PMID: 23460564
17.  Effect of isopropyl myristic acid ester on the physical characteristics and in vitro release of etoposide from PLGA microspheres 
AAPS PharmSciTech  2000;1(4):49-54.
The purpose of this paper was to study the effect of the isopropyl myristic acid ester (IPM) on the physicochemical characteristics of etoposide-loaded poly(lactic-co-glycolic acid) (PLGA) microspheres-specifically, the effects on the size and drug loading of the microspheres, the polymer matrix and surface morphology, and the release of etoposide from the microspheres. The experiment was structured to examine 2 IPM concentrations (25% and 50%) and 1 control (no IPM) at 2 different etoposide-loading percentages (10% and 5%). The microspheres were prepared using a single-emulsion solvent-extraction procedure. Samples from each batch of microspheres were then analyzed for size distribution. drug-loading efficiency, surface characteristics, in vitro release, and in vitro microsphere degradation. The incorporation of 50% IPM significantly increased (P<05) the size of the microspheres when compared with the control and 25% IPM microspheres. However, incorporation of 25% or 50% IPM did not change (P>.05) the drug-loading efficiency in comparison with the microspheres prepared without IPM. The microspheres containing 50% IPM were shown to significantly increase (P<.05) the release of etoposide from the microspheres at both etoposide concentrations. The microspheres prepared incorporating 25% IPM and 5% etoposide increased the in vitro release (P<.05) in comparison with the microspheres prepared without IPM. The 5% etoposide-PLGA microspheres showed a smooth, nonporous surface that changed to a dimpled. nonporous surface after addition of 25% IPM. During the in vitro degradation study, the IPM-containing microspheres slowly became porous but retained their structural integrity throughout the experiment.
doi:10.1208/pt010432
PMCID: PMC2750456  PMID: 14727897
Etoposide; PLGA microspheres; Isopropyl myristic acid ester; In vitro release; Scanning electron microscopy
18.  Retention of in vitro and in vivo BMP-2 bioactivity in sustained delivery vehicles for bone tissue engineering 
Biomaterials  2008;29(22):3245-3252.
In this study, we investigated the in vitro and in vivo biologic activity of bone morphogenetic protein 2 (BMP-2) released from four sustained delivery vehicles for bone regeneration. BMP-2 was incorporated in 1) a gelatin hydrogel, 2) poly(lactic-co-glycolic acid) (PLGA) microspheres embedded in a gelatin hydrogel, 3) microspheres embedded in a poly(propylene fumarate) (PPF) scaffold and 4) microspheres embedded in a PPF scaffold surrounded by a gelatin hydrogel. A fraction of the incorporated BMP-2 was radiolabeled with 125I to determine its in vitro and in vivo release profiles. The release and bioactivity of BMP-2 were tested weekly over a period of 12 weeks in preosteoblast W20-17 cell line culture and in a rat subcutaneous implantation model. Outcome parameters for in vitro and in vivo bioactivity of the released BMP-2 were alkaline phosphatase (AP) induction and bone formation, respectively. The four implant types showed different in vitro release profiles over the 12-week period, which changed significantly upon implantation. The AP induction by BMP-2 released from gelatin implants showed a loss in bioactivity after 6 weeks in culture, while the BMP-2 released from the other implants continued to show bioactivity over the full 12-week period. Micro-CT and histological analysis of the delivery vehicles after 6 weeks of implantation showed significantly more bone in the microsphere/PPF scaffold composites (implant 3, p < 0.02). After 12 weeks, the amount of newly formed bone in the microsphere/PPF scaffolds remained significantly higher than in the gelatin and microsphere/gelatin hydrogels (p < 0.001), however there was no statistical difference compared to the microsphere/PPF/gelatin composite. Overall, the results from this study show that BMP-2 could be incorporated into various bone tissue engineering composites for sustained release over a prolonged period of time with retention of bioactivity.
doi:10.1016/j.biomaterials.2008.04.031
PMCID: PMC2577841  PMID: 18472153
19.  PLGA nanofiber membranes loaded with epigallocatechin-3-O-gallate are beneficial to prevention of postsurgical adhesions 
This study concentrates on the development of biodegradable nanofiber membranes with controlled drug release to ensure reduced tissue adhesion and accelerated healing. Nanofibers of poly(lactic-co-glycolic acid) (PLGA) loaded with epigallocatechin-3-O-gallate (EGCG), the most bioactive polyphenolic compound in green tea, were electrospun. The physicochemical and biomechanical properties of EGCG-releasing PLGA (E-PLGA) nanofiber membranes were characterized by atomic force microscopy, EGCG release and degradation profiles, and tensile testing. In vitro antioxidant activity and hemocompatibility were evaluated by measuring scavenged reactive oxygen species levels and activated partial thromboplastin time, respectively. In vivo antiadhesion efficacy was examined on the rat peritonea with a surgical incision. The average fiber diameter of E-PLGA membranes was approximately 300–500 nm, which was almost similar to that of pure PLGA equivalents. E-PLGA membranes showed sustained EGCG release mediated by controlled diffusion and PLGA degradation over 28 days. EGCG did not adversely affect the tensile strength of PLGA membranes, whereas it significantly decreased the elastic modulus and increased the strain at break. E-PLGA membranes were significantly effective in both scavenging reactive oxygen species and extending activated partial thromboplastin time. Macroscopic observation after 1 week of surgical treatment revealed that the antiadhesion efficacy of E-PLGA nanofiber membranes was significantly superior to those of untreated controls and pure PLGA equivalents, which was comparable to that of a commercial tissue-adhesion barrier. In conclusion, the E-PLGA hybrid nanofiber can be exploited to craft strategies for the prevention of postsurgical adhesions.
doi:10.2147/IJN.S68197
PMCID: PMC4149440  PMID: 25187710
nanofiber membrane; poly(lactic-co-glycolic acid); epigallocatechin-3-O-gallate; antiadhesion; tissue-adhesion barrier
20.  Microparticulate Based Topical Delivery System of Clobetasol Propionate 
AAPS PharmSciTech  2011;12(3):949-957.
Psoriasis is a chronic, autoimmune skin disease affecting approximately 2% of the world's population. Clobetasol propionate which is a superpotent topical corticosteroid is widely used for topical treatment of psoriasis. Conventional dosage forms like creams and ointments are commonly prefered for the therapy. The purpose of this study was to develop a new topical delivery system in order to provide the prolonged release of clobetasol propionate and to reduce systemic absorption and side effects of the drug. Clobetasol propionate loaded-poly(D,L-lactic-co-glycolic acid) (PLGA) microspheres were prepared by oil-in-water emulsion–solvent evaporation technique. Particle size analysis, morphological characterization, DSC and XRD analyses and in vitro drug release studies were performed on the microparticle formulations. Emulgel formulations were prepared as an alternative for topical delivery of clobetasol propionate. In vitro drug release studies were carried out from the emulgel formulations containing pure drug and drug-loaded microspheres. In addition, the same studies were performed to determine the drug release from the commercial cream product of clobetasol propionate. The release of clobetasol propionate from the emulgel formulations was significantly higher than the commercial product. In addition, the encapsulation of clobetasol propionate in the PLGA microspheres significantly delayed the drug release from the emulgel formulation. As a result, the decrease in the side effects of clobetasol propionate by the formulation containing PLGA microspheres is expected.
doi:10.1208/s12249-011-9661-7
PMCID: PMC3167258  PMID: 21748539
clobetasol propionate; emulgel; emulsion–solvent evaporation method; poly(D,L-lactic-co-glycolic acid) microparticles; psoriasis
21.  In Vitro Release of Vascular Endothelial Growth Factor From Gadolinium-Doped Biodegradable Microspheres 
A drug delivery vehicle was constructed that could be visualized noninvasively with MRI. The biodegradable polymer poly(DL-lactic-co-glycolic acid) (PLGA) was used to fabricate microspheres containing vascular endothelial growth factor (VEGF) and the MRI contrast agent gadolinium diethylenetriamine pentaacetic acid (Gd-DTPA). The microspheres were characterized in terms of size, drug and contrast agent encapsulation, and degradation rate. The PLGA microspheres had a mean diameter of 48 ± 18 μm. The gadolinium loading was 17 ± 3 μg/mg polymer and the VEGF loading was 163 ± 22 ng/mg polymer. Electron microscopy revealed that the Gd was dispersed throughout the microspheres and it was confirmed that the Gd loading was sufficient to visualize the microspheres under MRI. VEGF and Gd-DTPA were released from the microspheres in vitro over a period of ∼6 weeks in three phases: a burst, followed by a slow steady-state, then a rapid steady-state. Biodegradable Gd-doped microspheres can be effectively used to deliver drugs in a sustained manner, while being monitored noninvasively with MRI.
doi:10.1002/mrm.20092
PMCID: PMC2396272  PMID: 15170848
gadolinium; vascular endothelial growth factor; contrast media; drug delivery
22.  Use new PLGL-RGD-NGF nerve conduits for promoting peripheral nerve regeneration 
Background
Nerve conduits provide a promising strategy for peripheral nerve injury repair. However, the efficiency of nerve conduits to enhance nerve regeneration and functional recovery is often inferior to that of autografts. Nerve conduits require additional factors such as cell adhesion molecules and neurotrophic factors to provide a more conducive microenvironment for nerve regeneration.
Methods
In the present study, poly{(lactic acid)-co-[(glycolic acid)-alt-(L-lysine)]} (PLGL) was modified by grafting Gly-Arg-Gly-Asp-Gly (RGD peptide) and nerve growth factor (NGF) for fabricating new PLGL-RGD-NGF nerve conduits to promote nerve regeneration and functional recovery. PLGL-RGD-NGF nerve conduits were tested in the rat sciatic nerve transection model. Rat sciatic nerves were cut off to form a 10 mm defect and repaired with the nerve conduits. All of the 32 Wistar rats were randomly divided into 4 groups: group PLGL-RGD-NGF, group PLGL-RGD, group PLGL and group autograft. At 3 months after surgery, the regenerated rat sciatic nerve was evaluated by footprint analysis, electrophysiology, and histologic assessment. Experimental data were processed using the statistical software SPSS 10.0.
Results
The sciatic function index value of groups PLGL-RGD-NGF and autograft was significantly higher than those of groups PLGL-RGD and PLGL. The nerve conduction velocities of groups PLGL-RGD-NGF and autograft were significantly faster than those of groups PLGL-RGD and PLGL. The regenerated nerves of groups PLGL-RGD-NGF and autograft were more mature than those of groups PLGL-RGD and PLGL. There was no significant difference between groups PLGL-RGD-NGF and autograft.
Conclusions
PLGL-RGD-NGF nerve conduits are more effective in regenerating nerves than both PLGL-RGD nerve conduits and PLGL nerve conduits. The effect is as good as that of an autograft. This work established the platform for further development of the use of PLGL-RGD-NGF nerve conduits for clinical nerve repair.
doi:10.1186/1475-925X-11-36
PMCID: PMC3465232  PMID: 22776032
RGD peptide; Nerve growth factor; Peripheral nerve; Nerve conduits; Nerve regeneration
23.  Incorporation of Chitosan Microspheres into Collagen-Chitosan Scaffolds for the Controlled Release of Nerve Growth Factor 
PLoS ONE  2014;9(7):e101300.
Background
Artifical nerve scaffold can be used as a promising alternative to autologous nerve grafts to enhance the repair of peripheral nerve defects. However, current nerve scaffolds lack efficient microstructure and neurotrophic support.
Methods
Microsphere–Scaffold composite was developed by incorporating chitosan microspheres loaded with nerve growth factor (NGF–CMSs) into collagen-chitosan scaffolds (CCH) with longitudinally oriented microchannels (NGF–CMSs/CCH). The morphological characterizations, in vitro release kinetics study, neurite outgrowth assay, and bioactivity assay were evaluated. After that, a 15-mm-long sciatic nerve gap in rats was bridged by the NGF–CMSs/CCH, CCH physically absorbed NGF (NGF/CCH), CCH or nerve autograft. 16 weeks after implantation, electrophysiology, fluoro-gold retrograde tracing, and nerve morphometry were performed.
Results
The NGF–CMSs were evenly distributed throughout the longitudinally oriented microchannels of the scaffold. The NGF–CMSs/CCH was capable of sustained release of bioactive NGF within 28 days as compared with others in vitro. In vivo animal study demonstrated that the outcomes of NGF–CMSs/CCH were better than those of NGF/CCH or CCH.
Conclusion
Our findings suggest that incorporation of NGF–CMSs into the CCH may be a promising tool in the repair of peripheral nerve defects.
doi:10.1371/journal.pone.0101300
PMCID: PMC4077743  PMID: 24983464
24.  Formulation of anastrozole microparticles as biodegradable anticancer drug carriers 
AAPS PharmSciTech  2006;7(3):E38-E46.
The purpose of this study was to develop poly(d,1-lactic-coglycolic acid) (PLGA)-based anastrozole microparticles for treatment of breast cancer. An emulsion/extraction method was used to prepare anastrozole sustained-release PLGA-based biodegradable microspheres. Gas chromatography with mass spectroscopy detection was used for the quantitation of the drug throughout the studies. Microparticles were formulated and characterized in terms of encapsulation efficiency, particle size distribution, surface morphology, and drug release profile. Preparative variables such as concentrations of stabilizer, drug-polymer ratio polymer viscosity, stirring rate, and ratio of internal to external phases were found to be important factors for the preparation of anastrozole-loaded PLGA microparticles. Fourier transform infrared with attenuated total reflectance (FTIR-ATR) analysis and differential scanning calorimetry (DSC) were employed to determine any interactions between drug and polymer. An attempt was made to fit the data to various dissolution kinetics models for multiparticulate systems, including the zero order, first order, square root of time kinetics, and biphasic models. The FTIR-ATR studies revealed no chemical interaction between the drug and the polymer. DSC results indicated that the anastrozole trapped in the microspheres existed in an amorphous or disordered-crystalline status in the polymer matrix. The highest correlation coefficients were obtained for the Higuchi model, suggesting a diffusion mechanism for the drug release. The results demonstrated that anastrozole microparticles with PLGA could be an alternative delivery method for the long-term treatment of breast cancer.
doi:10.1208/pt070361
PMCID: PMC2750503  PMID: 17025242
Breast cancer; microencapsulation; biodegradation; anastrozole; PLGA
25.  Suitable carriers for encapsulation and distribution of endostar: comparison of endostar-loaded particulate carriers 
Background
Particulate carriers are necessary to control the release of endostar and prolong its circulation in vivo. The purpose of this study was to identify a suitable carrier for the capsulation and delivery of endostar.
Methods
We prepared a series of poly (DL-lactide-co-glycolide) (PLGA) and poly (ethylene glycol) (PEG)-modified PLGA (PEG-PLGA) particulate carriers, and then characterized them according to their ability to prolong the circulation of endostar, their physicochemical properties, endostar-loading content, and in vitro and in vivo particulate carrier release profiles.
Results
All the particulate carriers had spherical core shell structures. The PEG-PLGA material and nanosize range appeared to enable the carriers to encapsulate more endostar, release endostar faster in vitro, and accumulate more endostar in vivo. The drug loading capacity of PEG-PLGA and PLGA nanoparticles was 8.03% ± 3.41% and 3.27% ± 5.26%, respectively, and for PEG-PLGA and PLGA microspheres was 15.32% ± 5.61% and 9.21% ± 4.73%. The cumulative amount of endostar released from the carriers in phosphate-buffered saline over 21 days was 23.79%, 20.45%, 15.13%, and 10.41%, respectively. Moreover, the terminal elimination half-life of endostar in the rabbit was 26.91 ± 7.93 hours and 9.32 ± 5.53 hours in the PEG-PLGA group and the PLGA nanoparticle group. Peak endostar concentration was reached at day 7 in the group treated with subcutaneous injection of PEG-PLGA microspheres and at day 14 in the group receiving subcutaneous injection of PLGA microspheres. Endostar was detectable in vivo in both groups after injection of the particulate carriers.
Conclusion
PEG-PLGA nanoparticles might be better than other nanoparticulate carriers for encapsulation and distribution of endostar.
doi:10.2147/IJN.S21881
PMCID: PMC3152471  PMID: 21845043
poly(DL-lactide-co-glycolide); nanoparticle; microsphere; endostar; peptide delivery

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