缓释成骨生长肽聚乳酸-聚羟乙酸共聚物微球的制备及其体外释放实验

背景:既往动物实验证实,局部或全身应用成骨生长肽,能够促进骨折愈合。但存在着半衰期短及口服生物利用率低等缺点,限制了其在临床上的应用。目的:用可吸收性生物材料包裹成骨生长肽于微球中,观察成骨生长肽在体外释放的过程及其结构变化,为控制释放系统选取合适的载体材料。设计:分组观察对比实验。单位:西安交通大学生命科学院实验室。材料:成骨生长肽由西安蓝晶生物科技公司按照Fmoc系统合成。质谱分析其纯化后纯度超过98%,Mr1523650符合理论Mr1523750),其序列分析符合理论序列。聚乳酸-聚羟乙酸共聚物(PLGA)(50∶50,Mr30000;75∶25Mr80000)由山东医疗器械研究所提供。方法:应用两种不同Mr的PLGA,用复乳溶剂挥发法包裹成骨生长肽,制备成骨生长肽PLGA微球。利用扫描电镜观察微球的表面结构及形态。应用激光粒度计数仪测量微球的粒径分布。高效液相色谱法检测成骨生长肽的包裹率、缓释时间及制备过程对多肽的结构稳定性的影响。结果:①成功制备了较均匀的圆形成骨生长肽微球。PLGA50∶50微球的平均粒径为(19.6±4.5)μm,包裹率为(83.9±4.2)%,载药率为(83.9±4.2)%;PLGA75:25微球的平均粒径为(35.8±3.6)μm,包裹率为(65.6±6.8)%,载药率为(65.6±6.8)%。②高效液相色谱法结果显示,成骨生长肽在制备过程没有发生化学结构改变及凝集,与制备前的结构一致。两种微球均有突释现象,但成骨生长肽-PLGA75∶25微球突释较重,成骨生长肽-PLGA50∶50微球能够缓释成骨生长肽56d,且累计缓释效果良好,成骨生长肽-PLGA75∶25缓释70d。成骨生长肽-PLGA50∶50微球35d的成骨生长肽累计缓释率低于成骨生长肽-PLGA75∶25,差异有显著性意义(P<0.05)。结论:与成骨生长肽-PLGA75∶25缓释微球相比,成骨生长肽-PLGA50∶50缓释微球具有较好的控制释放效果,且缓释时间能够满足骨折或骨缺损愈合局部应用需要。

Preparation and characterization of poly (lactic-co-glycolic acid) microspheres for controlled release of osteogenic growth peptide

BACKGROUND:Previous animal studies have revealed that osteogenic growth peptide (OGP) applied locally or systemically could promote fracture healing. But the disadvantages of short in vivo half-life and low oral bioavailability limit its clinical application.OBJECTIVE: To study the encapsulation and delivery of synthetic OGP (sOGP) from biodegradable polymeric microspheres in vitro so as to choose better carrier for the future study.DESIGN: Grouping observation and comparative trail.SETTING: Laboratory of School of Life Science and Technology, Xi'an Jiaotong University.MATERIALS: sOGP was synthesized by Xi'an Langene Bio-science Co., Ltd. with Fmoc system. The purity of sOGP after purification was over 98 % identified by reverse phase high performance liquid chromatography, and the molecular weight of sOGP was 1 523 650, which was consistent with the theoretical value (Mr 1 523 750); the result of whole sequence analysis of sOGP was consistent with the theoretical sequence of OGP. Poly (lactic-co-glycolic acid) (PLGA,50:50, Mr 30 000; 75:25 Mr 80 000) was obtained from Shandong Medical Instrumental Institute (Ji'nan, China)METHODS: PLGA with a 50:50 or 75:25 lactide to glycolide ratio was used for microsphere preparation using a modified double emulsion solvent extraction Water-in-oil-in-water (w/o/w) technique. The surface structure and appearance of microsphere was observed under scanning electron microscope; particle size distribution of microsphere was counted by laser diffraction particle sizer; efficiency of encapsulation, release time and the structural integrity of sOGP released from PLGA were assessed using high performance liquid chromatography (HPLC).RESULTS: ①Spherical microspheres of sOG-PLGA were formulated successfully. The average particle diameter of the PLGA 50:50 microsphere was (19.6±4.5) μm, efficiency of encapsulation (83.9±4.2)% with (83.9±4.2) % drug-loading efficiency, while the PLGA 75:25 microspheres showed an average size of (35.8±3.6) μm, efficiency of encapsulation (65.6±6.8)% with (65.6±6.8)% drug-loading efficiency. ②HPLC results indicated that sOGP were not chemically altered,physically aggregated but presented a intact structure as the original sOGP. An initial burst release was observed for both PLGA microspheres, especially from PLGA 75:25. sOGP was released from PLGA 50:50 microsphere for 56 days,and from PLGA 75:25 microspheres for over 70 days. The cumulative release of sOGP from PLGA 50:50 for 35 days was significantly lower than from PLGA 75:25 (P ＜ 0.05).CONCLUSION: The controlled release of sOGP encapsulated within PLGA 50:50 is better than the delivery from PLGA 75:25. Moreover, the release time could meet the requirements for fracture or bone defect site.