重组人骨形态发生蛋白-2缓释凝胶微球的研制及其溶胀、降解、载药、释药特征

Preparation of recombinant human bone morphogenetic protein-2 sustained release hydrogel microspheres, and in vitro studies of their swelling, biodegradation, drug loading and release

目的制备载人骨形态发生蛋白-2(rhBMP-2)的甲基丙烯酸缩水甘油酯右旋糖酐(dex-GMA)凝胶微球并初步考察其体外溶胀、降解、载药与释药特征。方法以液体石蜡为油相,Span-80为乳化剂,采用乳化化学交联技术制备载rhBMP-2的凝胶微球(BMP-HMs)并通过正交设计法优化其制备工艺;观察BMP-HMs形态和粒径,测定其包封率与载药量;用微球的吸水能力表示微球的溶胀率(Rs),扫描电镜观察微球的体外降解,动态观察体外释药特征及其与微球溶胀、降解的关系。结果所制备的BMP-HMs形态规整,粒径40～50μm,分布均匀;rhBMP-2载药量(10.6±4.8)%,包封率(88.9±1.0)%,BMP-HMs冻干剂4℃以下存放6个月性能稳定,但在磷酸盐缓冲液(PBS)中20～40d内可以完全降解。微球Rs随反应促进剂四甲基乙二胺(TEMED)用量的增大而减小,0.3mlTEMED制备的BMP-HMs体外释药实验表明80%的rhBMP-2在前20d左右释放。结论BMP-HMs对rhBMP-2具有确定的缓释作用,并可以通过制备工艺的改变控制其释药。

Objective To investigate the preparation method of recombinant human bone morphogenetic protein-2 （rhBMP-2） loaded dextran-based hydrogel microspheres （BMP-HMs） and evaluate their characteristics in swelling and biodegradation, drug loading and release. Methods By using liquid olefin as the oil phase and Span -80 as the emuifier, BMP-HMs were prepared by emulsion-chemical crosslink technique. The process of the recombination preparation was optimized by orthogonal factorization method. Then, the characteristics of BMP-HMs were evaluated by routine procedure. Swelling behavior was expressed by swelling ratio （Rs） and the size of BMP-HMs determined by electronic microscopy. The rhBMP-2 loading and encapsulation rate of BMP-HMs and their biodegradation characteristic were also studied. Dynamic dialysis was used to determine the release characteristic of BMP-HMs in vitro. Resuits BMP-HMs were homogenous and stable with the average diameter of 40-50 μm. rhBMP-2 loading and encapsulation rate was （ 10.6 ±4.8） % and （88.9 ± 1.0） %, respectively. Rs of BMP-HMs varied from 76% to 89% according to the amount of TEMED that was added at preparation of BMP-HMs. The in vitro experiment showed that 80% of rhBMP-2 could be released during the first 20 days, which could be controlled by Rs. Freeze-dried BMP-HMs could be kept under 4℃ for more than six months but be completely degradable in about 40 days in phosphate buffer solution （PBS）. Conclusion As a new sustained release system of rhBMP-2, BMP-HMs exert definitive controlled release effect that can be controlled through change of the preparation technique.