载VEGF/VAN多层海藻酸盐-壳聚糖缓释微球的制备

Preparation of multilayer alginate chitosan microspheres loading VEGF and vancomycin

目的:制备载有血管内皮生长因子(VEGF)和万古霉素(VAN)的多层海藻酸盐-壳聚糖缓释微球,探讨其体外释放特性。方法:采用乳化交联和层层自组装技术制备微球;正交实验设计考察海藻酸钠浓度、氯化钙(CaCl2)浓度、油水比及span80浓度对VEGF和VAN包封率(EE)和载药量(DL)的影响,以优化制备工艺;扫描电子显微镜(SEM)观察多层微球的表面形貌和粒径;傅里叶变换红外光谱仪(FTIR)检测海藻酸与壳聚糖的自组装情况;分别采用ELISA双抗体夹心法和紫外分光光度法检测VEGF和VAN的EE、DL和体外释放量并绘制累积释放曲线。结果:所制备微球呈黄褐色粉末状;SEM观察,微球呈圆球形,表面光滑,分散性较好,平均粒径约为50μm。制备缓释微球时,海藻酸钠浓度为1.0g·mL^(-1)、CaCl2浓度为8g·mL^(-1)、油水比为3∶1及span80浓度为2%时为最佳配方,VEGF和VAN的EE分别达49.63%和16.67%,体外累计释放时间分别为16.5和12.5d,释放量可达95%。结论:本研究通过优化制备工艺,制备了粒径较小、EE较高、缓释效果较好的载VEGF/VAN多层海藻酸盐-壳聚糖缓释微球。

Objective： To prepare the multilayer alginate chitosan microspheres loading vascular endothelial growth factor （VEGF） and vancomycin （VAN）, and to study in vitro release characteristics. Methods： The mierospheres were prepared by emulsion cross-linking and self-assembly techniques. The effects of sodium alginate concentration, calcium chloride concentration, oil/water ratio and spanS0 concentration on the entrapment efficiency （EE） and drug loading （DL） of VEGF and VAN were investigated by orthogonal experimental design to optimize the preparation process. The surface morphology and particle size of microspheres were observed by scanning electron microscope （SEM）. Self-assembly was detected by Fourier transform infrared spectroscope （FTIR）. The EE, DL and in vitro release of VEGF and VAN were detected by ELISA double antibody sandwich method and ultraviolet spectrophotometry, and the cumulative release curve was drawn. Results： The prepared microspheres were yellowish brown powder. The SEM results showed that the microspheres were spherical, the surface was smoothy, and the dispersity was better. The average particle size was about 50 μm. Sodium alginate concentration of 1.0 g · mL 1, CaC12 concentration of 8 g · mL^-1, oil to water ratio of 3 ： 1, and spanS0 concentration of 2% were the best formula. The EE of VEGF and VAN were 49.63% and 16.67%, respectively. In vitro, the cumulative release last 16.5 d and 12.5 d respectively and the amount reached up to 95%. Conclusion： The multilayer alginate chitosan microspheres loading VEGF and VAN present several advantages, such as smaller particle size, higher EE and better controlled release.