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The Preparation Of GM-1-PLGA Microspheres And Evaluation Of The Release Property In Vitro

Posted on:2011-02-21Degree:MasterType:Thesis
Country:ChinaCandidate:Q C JiangFull Text:PDF
GTID:2144360305450028Subject:Pharmaceutical Engineering
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Objective:GM-1, one of the most important gangliosides, may promote the growth, differentiation, regeneration of nerve cell in vitro, exert adjustment on the nerve growth factor, and promote the proliferation of neuronal axons and dendrites, lateral protrusion formation, inhibition of cell degeneration, enhance nerve nutrition, reduce the death of damaged cells of body,promote neuronal regeneration and functional recovery. The injection has been listed, mainly for the treatment of vascular or traumatic lesions of the central nervous system including brain trauma, spinal cord trauma, stroke, hypoxic ischemic encephalopathy, white matter injury of the brain in premature children, Parkinson's disease, etc.The present clinical practice was mainly via intramuscular injection or intravenous infusion of drugs in large doses and long period for treatment of central nervous system diseases. But the common GM-1 preparations must be repeated in multiple doses to maintain the effective concentration in cerebrospinal fluid, patient compliance is poor.In recent years, the research on the drugs controlled release system such as liposomes, microemulsions, microspheres, nanocapsules, nanoparticles formulated by the emulsion encapsulation techniques, becomes more and more popular. These preparations would be administrated by one dose per severay days, even months, can significantly reduce the dose frequency, enhance the safety and the efficiency, and improve the patient compliance. Amongst these controlled release preparations, the biodegradable long-acting injectable microspheres is becoming one of the research focus during the recent 30 years in pharmaceutical science.Lactic acid-glycolic acid (PLGA) by glycolic acid and lactic acid polymerization [4,5], is one of the most acceptable synthetic biodegradable polymers. The degradation of PLGA in vivo through the hydrolysis reaction could be controlled, the degradation intermediates are lactic acid and glycolic acid, the final degradation products are carbon dioxide and water, all are normal metabolites in vivo. Thus, PLGA is non-toxic, non-irritating, and has good biocompatibility. In the United States, PLGA has been approved by FDA as drug carrier material, and the application of domestically-made PLGA as pharmaceutical excipients are being processed. The cost of the production of PLGA based controlled release system will be reduced greatly after the application of PLGA is approved in China. Thus, intramuscular injectable GM-1 loaded sustained-release PLGA microspheres, which might reduce the administration frequency, alleviate the suffering of patients and increase patient compliance, should providing us good social and economic benefits.Methods:GM-1-PLGA microspheres were prepared by the emulsion-solvent evaporation method. The formulation and the process parameters were optimized by orthogonal experimental design based on the results of the single factor experiments using the entrapment efficiency and the particle size as the indexes. Microspheres morphology was observed by optical microscope. Microspheres diameter and size distribution were detected by the arithmetic mean method; Microspheres and free drug were separated by ultracentrifugation. The GM1 concentration was determined by HPLC. The method for the determination of the entrapment efficiency and drug loading was established. The release in vitro of the GM1-PLGA-MS in PBS (pH7.4) containing 1%Tween -80,0.05% sodium azide, was studied through dynamic membrane dialysis method. The release of drug in vitro from the GM1 loaded PLGA microspheres fits to Weibull equation. GM-1-PLGA microspheres initial stability was investigated by accelerated testing method.Results:The HPLC method of determination of GM-1 concentration is specific, simple, sensitive, and good for the determination of the entrapment efficiency. There was good linearity in the concentration range of 4~40μg/mL, the linear regression equation was A= 11462C+8347.7, r= 0.9991; the intra- and inter-day precision RSD were both less than 2%, the average recoveries were between 99%~101%.The major factors that affect the entrapment efficiency and size of particle are volume ratio of water phase and organic solvent phase (W1:O), volume ratio of colostrum and the external phase (O:W2), content of PLGA organic solvent, vortex speed and time, concentration of the external phase emulsifier (PVA), shear rate and time; Factors optimized by single factor tests are the following:the vortex time was 90s, vortex speed was 2800r/min, shear rate was 15000r/min, and the shearing time was 30s. On this basis, orthogonal design was carried out using entrapment efficiency as the main index, and the PLGA concentration, PVA concentration, volume ratio of in aqueous and organic solvent (W1:O), volume ratio of colostrum and the external phase (O:W2) were investigated. The best formulation and the process parameters were as follows:PLGA concentration was100mg/mL, PVA concentration was 3%,W1: O (V:V) was 1:15, and O:W2 (V:V) was 1:25. Three batches GM-1-PLGA microspheres were obtained under the optimized process, which had the appearance of white powder, good fluidity and no adhesion. The microspheres was a form of spherical ball or like, observed under light microscope; the average diameter of the GM-1-PLGA microspheres was (8.2±6.0)μm; entrapment efficiency was (84.17±1.545)%, and drug loading was (17.86±0.723)%.The in vitro release of GM1 from GM-1-PLGA microspheres showed sustained release property, and the in vitro release was consistent with Weibull equation model, equation could be described as lg[-ln(1-Q)]=0.50761gt-0.5565, r=0.9968。No significant changes in form and drug content were observed after storing under 40℃for 10d in the accelerated test. Due to limited time, it can not predict the term of validity of this preparation.Conclusion:This project has successfully developed a GM-1-PLGA microspheres preparation. The preparation technology is simple, feasible, with good reproducibility, high entrapment efficiency, good stability and sustained release property. The GM-1-loaded PLGA microspheres would reduce the dose frequency and increase patient compliance, it might be a promising sustained release preparation for GM1 adiminstration.
Keywords/Search Tags:Monosialoteterahexosyl ganglioside (GM-1), Poly(lactic-co-glycolic acid)(PLGA), Microspheres, the emulsion-solvent evaporation method
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