载血小板复合生长因子明胶缓释微球制备及其体外缓释作用研究

Study on preparing gelatin microspheres which carry and release platelet compound growth factors and examining their releasing effect in vitro

目的制备载血小板复合生长因子明胶缓释微球,观察其一般特性,检测其体外缓释效果。方法以明胶为原料,戊二醛为交联剂,采用改良的乳化冷凝聚合法制备明胶微球,以微球粒径为参考指标,通过单因素实验对明胶微球的制备条件进行优化。用光学显微镜、扫描电镜(SEM)对其大小结构形貌等一般特性进行表征;以血管内皮生长因子(vascular epithelial growth factor,VEGF)的含量为代表,酶联免疫吸附测定(ELISA)法检测其含量,计算微球吸附生长因子率、载生长因子量,测定体外缓释效果。结果当明胶溶液浓度为15%、Span8加入量为1.5 m L,搅拌速度为(750-855)r/min时可获得最优的明胶微球制备方案,获得的明胶微球平均粒径为15.95±3.79μm,其中7-20.5μm的微球占88.23%。制备的PG上清中VEGF的含量为(694.45±1.13)pg/m L,微球吸附生长因子率为87.7%,载VEGF量为6.09 pg/mg,体外缓释7 d累计释放量占总载药率的75.73%。结论制备的明胶缓释微球外观圆整,粒径均匀,分散度良好,吸附生长因子率、载生长因子量及体外缓释效果良好,初步实现了载血小板复合生长因子明胶缓释微球冻干制品的研制。

Objective To prepare gelatin microspheres which carry composite growth factors that form platelet-rich plasma and slow release them,to observe the general characteristics and to examine the releasing effect in vitro. Methods Gelatin was used as raw materials and glutaraldehyde was used as crosslinking agent. A modified emulsion cold condensation method was used to prepare gelatin microspheres. With the size of the microspheres as the reference,the preparation conditions of gelatin microspheres were optimized by single factor experiments. Its general characteristics were determined with optical microscopy and scanning electron microscopy（ SEM）,such as their size,structure and morphology. The levels of VEGF was used as representative. The content of growth factor was tested with ELISA kit. The adsorption rate of growth factor and levels of loading growth factor were estimated,and the slow releasing effect in vitro was evaluated. Results When the concentration of gelatin solution approached 15%,the dose of Span8 was 1. 5 m L,stirring speed was 750 - 855 r / min,the optimized gelatin microspheres preparation program was formed. The average particle size of gelatin microspheres was15. 95 ± 3. 79 μm. The microspheres within 7 μm-20. 5 μm accounted for 88. 23%. The concentration of VEGF in the prepared supernatant of PG was 694. 45 ± 1. 13 pg / m L. The microsphere adsorption of growth factor was 87. 7%. The amount of loading VEGF was 6. 09 pg / mg,In seven days,a total of sustained release in vitro was 75. 73%. Conclusion The general characteristics of gelatin microspheres are satisfactory. The growth factor adsorption rate,the amount of growth factor content and the releasing effect in vitro of prepared microspheres are ideal. The initial development of freeze-dried gelatin microspheres products which carry PRP growth factor mixture followed by slow release them is realized.