静电纺丝纳米纤维的制备及其蛋白药物缓释体系

发布时间：2018-10-10 11:31

【摘要】：细胞因子蛋白复杂脆弱的高级构象极易因组织工程支架制备过程中的苛刻条件变性而失活,甚至引起有害的抗体反应。目前有关组织工程支架材料的研究方法尚未实现以天然构象长期缓释细胞因子蛋白。针对这个难题,本研究运用水相-水相冷冻相分离蛋白颗粒制备法,将蛋白药物血管内皮生长因子(VEGF_(165))保护性载入葡聚糖纳米颗粒中,进而将载有蛋白的多糖颗粒混悬于作为缓释基质的聚乳酸-羟基乙酸共聚物(PLGA)中,通过高压静电纺丝,制得长效缓释VEGF_(165)的组织工程纤维膜。一系列物理化学表征,包括扫描电镜(SEM)、接触角、强度试验等,表明上述方法制得了表面形态与机械强度较理想的组织工程纤维。对制备过程中各个阶段的蛋白进行回收,并采用CCK-8法进行活性检测证明VEGF_(165)的生物活性在多糖颗粒制备阶段保持在97%以上,在最终的组织工程纤维膜阶段保持在87%以上,远大于w/o型乳液法制备得到的纤维(低于55%)。体外试验证明颗粒复合纤维能抑制蛋白突释且达到长效缓释效果,蛋白在初始5 d内的释放总量不到35%,到25 d时释放量超过92%。而w/o型乳液法制得的纤维在初始5 d内的释放总量超过53.12%,到25 d时释放量为84.52%。最后将纤维膜植入下肢缺血的大鼠体内,进行免疫组化观察,结果表明颗粒复合PLGA纤维的再生血管数量明显高于w/o型乳液法制备得到的纤维。
[Abstract]:The complex and fragile high conformation of cytokine proteins is easily inactivated due to the severe denaturation of tissue engineering scaffolds and even causes harmful antibody reactions. At present, the research methods of tissue engineering scaffold materials have not been achieved with the natural conformation of long-term sustained release cytokine protein. In order to solve this problem, the preparation of protein particles in aqueous phase freezing phase was studied. The protein drug vascular endothelial growth factor (VEGF_ _ (165) was protected into dextran nanoparticles. Then the polysaccharides containing protein were mixed in the poly (lactic acid-glycolic acid) copolymer (PLGA) as the sustained-release matrix, and the tissue engineering fiber membrane of long-lasting sustained release VEGF_ _ (165) was prepared by high-voltage electrostatic spinning. A series of physicochemical characterizations, including SEM (SEM), contact angle, strength test and so on, show that the surface morphology and mechanical strength of the tissue engineering fibers are ideal. The protein was recovered from each stage of the preparation and the bioactivity of VEGF_ _ (165) was determined by CCK-8 method. The bioactivity of VEGF_ _ (165) was over 97% in the preparation stage of polysaccharides and more than 87% in the final phase of tissue engineering fiber membrane. It is much larger than the fiber (less than 55%) prepared by w / o emulsion method. In vitro experiments showed that the granular composite fiber could inhibit the sudden release of protein and achieve long-term sustained release effect. The total amount of protein released within 5 days was less than 35%, and at 25 days the release amount exceeded 92%. In the first 5 days, the total release amount of the fibers obtained by the w / o emulsion method was more than 53.12, and at 25 days, the release amount was 84.52. Finally, the fibrous membrane was implanted into the rats with lower limb ischemia. The results showed that the number of regenerated vessels of granular composite PLGA fibers was significantly higher than that of the fibers prepared by w / o emulsion method.
【作者单位】：上海交通大学药学院;
【分类号】：R943

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