【作者】 宋凤兰；

【导师】 杨帆；

【作者基本信息】 广东药学院 ， 药剂学， 2007， 硕士

【摘要】 本研究以干扰素α（IFNα）为模型药物,以聚乳酸—乙醇酸（PLGA）为载体材料,采用W/O/W复乳溶剂挥发法制备了干扰素微球,并对制得微球的缓释效果及相关性质进行了评价。在W/O/W复乳溶剂挥发法制备微球的研究中,首先以微球粒径、圆整度为指标,考察了微球制备过程中的各种影响因素,初步确定了干扰素微球的制备工艺条件。然后通过L9（3⁴）正交试验设计考察了PLGA浓度、聚乙烯醇（PVA）浓度、油水相比、稳定剂种类与用量对微球质量的影响,对W/O/W法制备干扰素微球的工艺进行了优化,确定了制备干扰素微球的最佳工艺条件。在微球缓释效果的研究中,首先对微球的体外缓释效果进行了考察。采用自行设计的体外释药方法对0.17dL/g、0.39dL/g、0.60dL/g、0.89dL/g、1.13dL/g五个不同黏度,5%、10%、15%、20%、25%五个不同浓度的PLGA制备的干扰素微球的体外缓释效果进行了比较性研究,得出了不同黏度、不同浓度的PLGA微球缓释时间的长短,进一步完善了微球的最佳制备工艺。结果表明15%的0.89dL/g PLGA干扰素微球的缓释效果较好,其体外释放能很好的符合缓释制剂的特征,Higuchi方程为Q=2.2034t^1.2+25.605,r=0.9023,15天的累积释放量约为75%。对此微球的体外释放液中的干扰素的生物学活性进行考查,发现其一周的体外释放液都有很好的生物学活性。以大鼠为研究对象,对干扰素微球的体内缓释效果进行考察,干扰素微球具有明显的缓释作用,生物利用度比干扰素粉针提高了2倍。本研究还对15%的0.89dL/g PLGA微球的理化性质与生物学性质进行评价。结果干扰素微球的球形圆整,不黏连,粒径分布较均匀,平均粒径为65.84μm。干扰素微球的堆密度为0.1856g/ml;休止角为29.42°,流动性较好:吸湿性非常小,在90%相对湿度的环境中,吸湿增重比不大于1%,但是湿度会使干扰素的含量下降,所以干扰素微球应该密封干燥保存。DSC分析结果表明干扰素被包裹在PLGA微球中,而不是黏附在微球表面。顶空—气质联用色谱法测定了微球中残留的二氯甲烷量,得出微球有机溶剂残留量符合药典规定。微球的生物学性质研究主要是指生物降解性和生物相容性研究。在微球的生物降解性研究中,对其体内、外降解情况都进行了研究,结果微球在体内降解速率比在体外降解速率要快,在体内6周可以完全降解,不会在体内造成蓄积。对微球的体内组织相容性进行研究,大鼠肌肉注射一定的微球,在6周内观察,注射部位肌肉无充血,无水肿,触之无包块,对机体无明显刺激性反应,说明微球具有良好的生物相容性。本研究为多肽、蛋白质类药物新制剂的开发开辟了道路,为微球制剂的研究提供了理论技术指导。根据目前的研究结果,如再予以更深入细致的研究,干扰素微球剂有望成为临床治疗乙肝、丙肝等疾病的一种新剂型。更多还原

【Abstract】 Interferon-alpha（IFNα）was chosen as a model drug and poly（lactic acid-glycolic acid） （PLGA）was used as the carrier to prepare Interferon-alpha sustained-release microspheres by W/O/W double emulsions solvent evaporation method.Furthermore,the drug release behaviors and the properties of IFNαmicrospheres were deeply studied.During the process of IFNαmicrospheres preparation,the effect of various factors were studied at first with the particle’s size and roudness of the microspheres as the criteria.Then the optimum formulation of IFNαmicrospheres prepared by W/O/W double emulsions solvent evaporation method was determined by employing L9（3⁴）orthogonal test design,including the effect of PLGA concentration,polyvinyl alcohol（PVA） concentration,oil/water ratio,and choice of stabilizers and quantity used.In the sustained-release study of IFNαmicrospheres,the in vitro release behaviors of IFNαmicrospheres were studied firstly by an improved release method.We compared the in vitro release profiles of IFNαmicrospheres,which prepared by five different viscosities, 0.17dL/g、0.39dL/g、0.60dL/g、0.89dL/g、1.13dL/g and five different concentrations,5%、10%、15%、20%、25%.The optimum formulation of IFNαmicrospheres preparation was also further perfected by the results of the in vitro release.In vitro release profile of IFNαmicrospheres prepared by 15%0.89dL/g PLGA met the criteria of sustained release formulation preferably and it was able to prolong drug action.Higuchi equation was Q=2.203t^1/2+25.605,r=0.9023.The accumulated amount of IFNαreleased was 75%in 15 days.Moreover,the biological activity of IFNαwas measured by Cytopathic Inhibition Assay suggesting that bioactive IFNαhad been firmly released from microsphere for more than 7 days.The pharmacokinetic experiments of this microspheres showed an obvious sustained fashion,and the relative bioavailability of IFNαmicrospheres to IFNαwas about 200%.The IFNαmicrospheres prepared by optimum formulation were white powder with fine fluidity.The surface morphology of microspheres had a spherical shape with a smooth and porous surface.Particle size of IFNαmicrospheres were nearly normality and the average diameter of microspheres was 65.84μm.The bulk density and the angel of repose of microspheres were 0.1856g/ml and 29.42°respectively.The moisture absorption of microspheres was low for their hygroscopic ratio less than 1%under the condition of 90% relative humidity.However,IFNαmicrospheres should be stored in airproof and dry container since humidity could accelerate IFNαdegradation.The DSC curve of microspheres confirmed that IFNαwas loaded within the microspheres,rather than adsorbed on their surfaces.For dichloromethane was used to prepare microspheres,the residual quantity of dichloromethane in microspheres should be measured.The amount determined by gas chromatography and mass spectra was lower than the criteria of Chinese Pharmacopeia.The biodegradability of PLGA microspheres was investigated by an improved method in vitro and by intramuscular injection in rats in vivo.The biodegradable speeds of IFNαmicrospheres in vivo were faster than those in vitro.After six weeks,the degradable percentage of IFNαmicrospheres was 80%in vitro and 100%in vivo,so that IFNαmicrospheres would not be accumulated in body.Biocompatibility of microspheres had been observed for six weeks by intramuscular injection in rats.It showed that IFNαmicrospheres were biocompatible for only slight inflammatory response but no pathological change at the site of injection.The results of the above study provided a useful exploration for the experimental methods and theories of peptide and protein microspheres and a scientific basis for developing of IFNαmicrospheres as a new drug to cure hepatitis.更多还原