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(英文)

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

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。

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 appiication. OB.3EETIVE： To study the encapsulation and delivery of synthetic OGP （sOGP） from biodegradable polymeric microspheres in vftro 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 encapsulat（on, 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.