摘要
背景:已往研究表明,神经生长因子微球对脊髓损伤的修复有促进作用。然而,传统复乳法制备微球过程中诸多因素会严重影响生物大分子药物的生物活性,如何改善制备工艺提高微球的缓释性能至关重要。目的:课题提出改良复乳法制备神经生长因子微球的方法,并考察和验证其一般性质和体外释药特征。设计、时间及地点:随机对照细胞学实验,于2008-05/2009-05在温州医学院药学院及温州医学院附属第二医院骨科分子生物学实验室完成。材料:重组人神经生长因子(美国R&D公司),乳酸/羟基乙酸共聚物(聚乳酸/聚羟基乙酸75/25,Mw=20000,黏度=0.025L/g)(山东省医疗器械研究所);聚乙烯醇(日本可乐丽公司);聚乙二醇400(德国Appli Chem公司)。方法:以乳酸/羟基乙酸共聚物为载体材料,对传统复乳溶剂挥发法进行改进,采用全循环一体机装置制备神经生长因子缓释微球,对缓释微球的物理表观性质进行检测,然后通过ELISA-Kit法和PC12细胞共培养法来检测微球中神经生长因子的活性。主要观察指标:以微球形态、粒径、包封率等为评价指标,并通过体外释放率和PC12细胞活性考察其体外释药性质。结果:改良法和传统法制备的微球粒径分别为(22.61±3.94)μm和(21.32±4.82)μm,包封率分别为(92.08±4.39)%和(89.17±3.74)%。改良组的神经生长因子微球可以持续分泌有生物活性的神经生长因子长达7周,累积释放率为85.7%,而传统组的微球只能释放4周,累积释放率为60.8%,且第4周时释放的神经生长因子活性明显低于改良组,差异有显著性意义(P<0.01)。结论:改良法制备的神经生长因子微球粒径适宜、包封率高,较传统复乳溶剂挥法制备的微球有更好的缓释性能。
BACKGROUND： Studies have shown that nerve growth factor microspheres can promote repair of spinal cord injury. However, various factors of traditional double emulsion method to prepare microspheres greatly affect bioactivity of biomacromolecule medicine. Therefore, to modify preparation technology is very important to improve microsphere delayed release. OBJECTIVE： To explore optimal preparation technology and to investigate the general properties and in vitro drug release characteristics of recombinant human nerve growth factor （rhNGF） microspheres. DESIGN, TIME AND SETTING： Randomized, controlled cytology. The experiment was performed at Molecular Biology Laboratory, Department of Orthopedics, Second Affiliated Hospital and School of Pharmacy of Wenzhou Medical College from May 2008 to May 2009. MATERIALS： rhNGF （R＆D, USA）, lactic acid/glycolic acid copolymer （poly（lactic-co-glycolic acid, PLGA 75/25, Mw= 20 000, viscosity=0.025 L/g, Shandong Institute of Medical Instruments）; polyvinyl alcohol （Kuraray, Japan）; polyoxyl 400 （AppliChem, Germany）. METHODS： PLGA was utilized as delivery material to prepare the rhNGF microspheres via Total Recirculation One-Machine System （TORMS） and traditional double emulsion solvent evaporation method, respectively. The physical appearance of microspheres were detected, and NGF activity in the microspheres was evaluated by ELISA quantitative determination and a PC-12 cell neurite outgrowth assay. MAIN OUTCOME MEASURES： The morphology, particle size, and encapsulation efficiency were used as evaluating index. The rhNGF release from microspheres were evaluated by ELISA quantitative determination and a PC-12 cell neurite outgrowth assay. RESULTS： The diameters of microspheres in the improved and traditional groups were （22.61±3.94） μm and （21.32±4.82） μm respectively; the encapsulation efficiency were （92.08±4.39）% and （89.17±3.74）%, respectively. Microspheres prepared using TORMS released 85.7% of the total protein content within 7 weeks. A sustained release was observed for four weeks in the traditional group, and the accumulative release was 60.8%. Moreover, the bioactivity of NGF at fourth week was significantly lower in traditional group than improved group （P 〈 0.01）. CONCLUSION： The NGF-PLGA microspheres prepared by improved method has suitable size, high encapsulation efficiency and better control release properties compared with traditional double emulsion method.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2009年第42期8286-8290,共5页
Journal of Clinical Rehabilitative Tissue Engineering Research
基金
浙江省教育厅资助项目(20070904)
温州市科技局资助项目(H20080031)~~
