PLGA纳米粒缓释系统的建立及其对rhFGF-2和rhBMP-2生物学活性的维持

发布时间：2018-09-03 08:47

【摘要】：研究背景及目的:在节段性骨缺损修复、四肢关节重建、脊柱融合等临床治疗中,常需要进行骨移植。自体骨和同种异体骨等骨修复材料因自身固有缺陷而在临床应用中受到极大限制。人工骨因其可大批量生产、便于储存运输等特点,成为近年来骨修复材料领域的研究热点。目前已有多种类型的人工骨应用于临床,以磷酸钙骨水泥、羟基磷灰石和可降解有机材料等为主,主要用于非结构性植骨。但这些人工骨的骨修复效果有限或不佳,远逊于自体骨或异体骨,其主要原因是不具备骨诱导活性。生长因子可以促进间充质干细胞的增殖、分化,可有效促进新骨的形成,将其复合到人工骨支架上,将有望赋予人工骨有效的诱导成骨活性。但生长因子的半衰期很短,直接使用将很快被降解或稀释,不能在骨修复局部形成有效持久的药物浓度。不仅使用量大,经济成本高,而且随血液分布到机体其他部位,可能出现并发症。因此,如何构建一种有效的生长因子缓释系统成为该领域发展需解决的一大关键难题。聚(乳酸-乙醇酸)(PLGA)纳米粒在药剂学方面的快速发展为骨科医生带来了启示。PLGA是一种人工合成的高分子聚合物,可作为多种药物的载体而实现体内的持久释放。如果使用PLGA作为载体对生长因子进行负载,则有望实现生长因子在体内的缓慢释放,从而在骨修复局部维持足够的浓度,充分发挥它们的促成骨功能。将这个缓释系统同人工骨主体支架复合,则有望制备出具有较高成骨活性的复合人工骨,来满足临床对理想骨修复材料的需求。本课题组拟筛选一种PLGA作为主体材料建立纳米粒缓释系统,对制备条件进行探索优化,使之更高效地包封蛋白质类物质,能有效地保护蛋白质的生物学活性,并可稳定释放这些物质达一个月以上,为制备出高活性的人工骨修复材料打下基础。方法:(1)查阅相关文献,根据对缓释系统的实际需求和生长因子的性质,最终选择分子量较低、乳酸与乙醇酸单体比例为50:50的PLGA作为纳米粒制备的主体材料。采用复乳溶剂挥发法(W/O/W)制备纳米粒,以牛血清白蛋白(BSA)为模型蛋白,探索PLGA浓度、BSA与PLGA质量比、内水相与油相体积比、内水相溶剂类型等因素对制备PLGA纳米粒的影响,通过测定蛋白的包封率并兼顾载药率来寻找最优制备条件。(2)通过优化条件制备纳米粒,分别研究其对BSA、重组人骨形态发生蛋白2(rh BMP-2)、重组人碱性成纤维细胞生长因子(rh FGF-2)的体外释放动力学,评价其对蛋白质的缓释效果。(3)与间充质干细胞共培养,用CCK-8法和livedead染色来证实PLGA纳米粒的生物相容性。(4)利用细胞增殖实验来验证rh FGF-2/PLGA纳米粒的生物学活性,利用碱性磷酸酶(ALP)染色、ALP活性检测、骨钙素(OCN)含量检测、成骨相关标志物的表达等来验证rh BMP-2/PLGA纳米粒的生物学活性。结果:(1)成功制备了PLGA纳米粒并优化了制备条件,当PLGA浓度为200mg/m L、BSA与PLGA质量比为1:40、水油体积比1:10、内水相使用去离子水时可获得对BSA最理想的包封率和载药率。(2)制备的纳米粒呈规则球形,粒径在300 nm左右,对BSA、rh BMP-2、rh FGF-2的包封率分别为(70.0±1.3)%、(68.2±1.7)%、(66.8±2.9)%。(3)纳米粒可实现蛋白类物质的持久缓慢释放,长达一个月以上,突释现象不明显。(4)纳米粒的生物相容性良好,与间充质干细胞共培养3天,细胞存活力好。(5)rh FGF-2/PLGA纳米粒对细胞有明显促增殖作用,共培养7天后其促增殖作用显著强于rh FGF-2溶液组(p0.05)。ALP染色、ALP活性检测、骨钙素含量检测、成骨相关标志物表达结果证实rh BMP-2/PLGA纳米粒对C2C12细胞具有促成骨分化作用,且显著强于rh BMP-2溶液组(p0.05)。结论:以优化条件制备的PLGA纳米粒呈规则球形,对蛋白质的包封效率高,能实现一个月以上的缓慢释放,具有良好的生物相容性,利用PLGA纳米粒负载的rh BMP-2、rh FGF-2仍具有良好的生物学活性,且表现出比直接使用相同剂量的相应生长因子溶液更优效的作用。PLGA纳米粒适宜作为生长因子的缓释载体,为赋予人工骨高成骨诱导活性提供了新的选择。
[Abstract]:BACKGROUND AND OBJECTIVE: Bone transplantation is often needed in the clinical treatment of segmental bone defect repair, limb joint reconstruction, spinal fusion and so on. Many types of artificial bone have been used in clinic, mainly calcium phosphate cement, hydroxyapatite and biodegradable organic materials, mainly for non-structural bone grafting. However, the effect of bone repair of these artificial bone is limited or poor, far inferior to that of autogenous bone or allogeneic bone, and the main reason is that these artificial bone grafts are mainly used in non-structural bone grafting. Growth factors can promote the proliferation and differentiation of mesenchymal stem cells and promote the formation of new bone effectively. When they are combined with artificial bone scaffolds, they are expected to endow artificial bone with effective osteogenic activity. As a result, how to construct an effective growth factor sustained-release system has become a key problem in this field. Poly (lactic acid-glycolic acid) (PLGA) nanoparticles are used in pharmaceuticals. PLGA is a synthetic polymer that can be used as a carrier for many drugs to achieve sustained release in vivo. If PLGA is used as a carrier to load growth factors, it is expected to achieve slow release of growth factors in vivo, thus maintaining adequate local bone repair. It is hoped that the composite artificial bone with high osteogenic activity can be prepared to meet the clinical demand for ideal bone repair materials. Our team is going to select a kind of PLGA as the main material to establish nanoparticle sustained-release system for preparation. Conditions were optimized to encapsulate protein substances more efficiently, protect the biological activity of protein effectively, and release these substances steadily for more than one month, laying a foundation for the preparation of highly active artificial bone repair materials. PLGA with low molecular weight and 50:50 ratio of lactic acid to glycolic acid monomer was chosen as the main material for the preparation of nanoparticles. (2) The in vitro release kinetics of PLGA nanoparticles to BSA, recombinant human bone morphogenetic protein-2 (rh BMP-2) and recombinant human basic fibroblast growth factor-2 (rh FGF-2) were studied by optimizing the preparation conditions. To evaluate the sustained-release effect of rh-FGF-2/PLGA nanoparticles. (3) The biocompatibility of PLGA nanoparticles was confirmed by co-culture with mesenchymal stem cells, CCK-8 method and livedead staining. (4) The biological activity of rh-FGF-2/PLGA nanoparticles was verified by cell proliferation test, alkaline phosphatase (ALP) staining, ALP activity detection, osteocalcin (OCN) content detection, osteogenesis. Results: (1) PLGA nanoparticles were successfully prepared and the preparation conditions were optimized. When the concentration of PLGA was 200 mg/ml, the mass ratio of BSA to PLGA was 1:40, the volume ratio of water to oil was 1:10, and the deionized water was used in the inner water phase, the best encapsulation efficiency and drug loading rate of BSA were obtained. The encapsulation efficiency of BSA, RH BMP-2 and Rh FGF-2 was (70.0 6550 After co-culture for 3 days, the cell viability was good. (5) Rh-FGF-2/PLGA nanoparticles could promote the proliferation of C2C12 cells significantly. After co-culture for 7 days, the proliferation-promoting effect of rh-FGF-2 nanoparticles was significantly stronger than that of rh-FGF-2 solution group (p0.05). ALP staining, ALP activity detection, osteocalcin content detection, osteogenesis-related markers expression results confirmed that rh-BMP-2/PLGA nanoparticles could promote the proliferation of C2C12 cells. CONCLUSION: PLGA nanoparticles prepared under the optimized conditions are regular spheres with high encapsulation efficiency for protein, can achieve slow release for more than a month, and have good biocompatibility. RhBMP-2 and rh-FGF-2 loaded with PLGA nanoparticles still have good biological activity. PLGA nanoparticles are suitable for sustained-release carrier of growth factors, which provides a new choice for endowing artificial bone with high osteogenic induction activity.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R687;R318.17

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