同轴静电纺丝法制备神经生长因子纳米纤维缓释载体

Preparation of coaxial electrospinning nanofibers for nerve growth factor delivery

背景:传统的缓释药物的制备过程,常需要把蛋白质类药物与有机溶剂混合,降低了蛋白质的活性,同轴静电纺丝法减少了蛋白质与有机溶剂的接触,有望提高蛋白质的活性,提供新型的缓释载体。目的:探讨应用同轴静电纺丝技术制备蛋白质类药物神经生长因子"壳-芯"结构纳米纤维缓释载体的可行性。设计、时间及地点:对比细胞学实验,于2007-07/12在东华大学生物科学与技术研究所完成。材料:乳酸己内酯共聚物(50∶50,Mw=378,839g/mol,Mw/Mn=2.7324)由东华大学生物科学与技术研究所提供;β-神经生长因子为R&D Systems公司产品;牛血清白蛋白为Sigma-Aldrich公司产品;大鼠肾上腺嗜铬细胞瘤细胞PC12细胞由中国科学院细胞库提供。方法:应用同轴静电纺丝技术制备以乳酸己内酯共聚物为壳,神经生长因子和牛血清蛋白为芯的复合纳米纤维;然后进行体外缓释8周,将不同时间点缓释液加入到无血清RPMI培养基中,培养大鼠肾上腺嗜铬细胞瘤细胞(PC12细胞)。根据芯层溶液流速的不同分为0.10,0.15,0.25mL/h3组纳米纤维。主要观察指标:通过扫描电镜和透射电镜对纳米纤维形貌特征进行表征,应用图像分析软件Image-J计算纳米纤维的直径分布范围;PC12细胞在神经生长因子的诱导下可以向神经元细胞分化,通过观测其分化率检测神经生长因子的生物活性。结果:成功制备了具有壳芯结构的乳酸己内酯共聚物/牛血清蛋白/神经生长因子纳米纤维,纳米纤维的平均直径和直径分布范围随着芯层溶液流速的增加而增大。当芯层溶液流速为0.10mL/h时或0.15mL/h,纺丝稳定,所得纤维平均直径较小,直径分布范围较窄,当芯层溶液流速为0.25mL/h,纺丝不稳定,有大量"串珠"出现。在各个时间点乳酸己内酯共聚物/牛血清蛋白/神经生长因子缓释上清液能够诱导PC12细胞分化神经元细胞,生长轴突,说明神经生长因子保持了一定程度的生物活性至少8周。结论:应用同轴静电纺丝技术可制备蛋白质类药物神经生长因子"壳-芯"结构纳米纤维缓释载体。

BACKGROUND： Protein drugs are commonly admixed with organic solvents in traditional fabrication process of sustained release system, which denatures the proteins. Coaxial electrospinning method may reduce the contact between proteins and organic solvents, and is expected to improve the bioactivity of proteins and become a new type drug release system. OBJECTIVE： To investigate the feasibility of encapsulating protein drug of nerve growth factor （NGF） into the core-shell nanofibers through coaxial electrospinning for sustained release. DESIGN, TIME AND SETTING： Comparative observation experiments were performed in the Institute of Biological Sciences and Biotechnology, Donghua University （Shanghai, China） from July to December in 2007. MATERIALS： Poly（L-lactide-co-epsilon-caprolactone） [P（LLA-CL）, 50：50, Mw=378, 839 g/mol, Mw/Mn=2.732 4] was supplied from Institute of Biological Sciences and Biotechnology, Donghua University （China）. Recombinant rat β -NGF was supplied from R＆D Systems （USA）. Bovine serum albumin （BSA） was purchased from Sigma-Aldrich lnc （USA）. Rat pheochromocytoma cell line （PC 12 cells） was obtained from the Cell Bank of Chinese Academy of Sciences （China）. METHODS： The core-shell structured composite nanofibers with P（LLA-CL） as a shell and BSA/NGF as a core were fabricated through coaxial electrospinning technology. The nanofibers were released in vitro for 8 weeks, and at various time points, the NGF supernatant from the P（LLA-CL）/BSA/NGF fibers was added into serum-free RPMI medium to culture PC12 cells. The nanofibers were divided into three groups according to the core solution flow rate： 0.10, 0.15 and 0.25 mL/h. MAIN OUTCOME MEASURES： Through the images of scanning electron microscopy and transmission electron microscopy, we obtained morphological characters of the nanofibers. The diameter range of the fabricated nanofibers was measured using an image visualization software Image-J. PC12 cells, which differentiated to neuronal phenotype in the presence of bioactive NGF, were used to test the bioactivity of the NGF released from the electrospun fibers. RESULTS： The core-shell structure of P（LLA-CL）/BSA/NGF nanofibers was achieved by coaxial electrospirming, and the average diameter and diameter range of the nanofibers were increased with the increase of the core solution flow rate. When the core solution flow rate was low as 0.10 or 0.15 mL/h. P（LLA-CL）/BSA/NGF compound had better electrospinnability than the rate was 0.25 mL/h, obtaining the stable and completely ＂bead-free＂ nanofibers. PC12 cells could differentiate into neurons and outgrowth neurites in the NGF supernatant from P（LLA-CL）/BSA/NGF nanofibers. It indicated that the released NGF retained some degree of bioactivity for at least 8 weeks. CONCLUSION： It is feasible to encapsulate protein drug NGF into core-shell nanoflbers through coaxial electrospinning technology for sustained release.