制备载mngf的plga纳米粒子及其在磁场引导下修复大鼠坐骨神经损伤的初步探究.doc

制备载 mNGF 的 PLGA 纳米粒子及其在磁场引导下修复大鼠坐骨神经损伤的初步探究 海米提·阿布都艾尼 李立军 倪东馗 宗强 天津医科大学研究生院 天津医科大学第二医院骨科 滨州医学院烟台附属医院创伤骨科 摘 要： 目的 制备载鼠神经生长因子 (mNGF) 的聚乳酸-羟基乙酸共聚物 (PLGA) 磁性纳米微粒, 评价其体外释放行为, 通过 MRI 显像研究大鼠左坐骨神经损伤后, 在外加磁场的引导下, 磁性纳米粒子在损伤部位的聚集情况方法 以 mNGF 为模型药物, 采用超声乳化法, 制成磁性 mNGF-PLGA 纳米粒子, 分别用透射电子显微镜、激光粒度分析仪等对纳米粒子进行表征, 用酶联免疫吸附分析 (ELISA) 法绘制 mNGF 标准曲线, 测包封率和载药量及体外释放用 SD 大鼠制作坐骨神经损伤模型, 通过尾静脉注射药物磁性 mNGF-PLGA 纳米粒子, 分别扫描未注射药物大鼠、尾静脉注射磁性 mNGFPLGA 纳米粒子溶液大鼠及尾静脉注射磁性mNGF-PLGA 纳米粒子溶液后使左下肢固定在 1.0 T 外磁场引导 2 h 的大鼠, 分别记录左、右下肢 T2*值结果 所得磁性 mNGF-PLGA 样品为棕色混悬液, 大小均匀, 平均粒径为 (205.9±1.2) nm, 粒径分散均一, 透射电子显微镜证实纳米粒子形态为球型, 其内包裹了大量 mNGF;药物包封率和载药率分别为 (69.43±2.80) %和 (3.11±3.27) %。

体外释放实验显示, 磁性 mNGF-PLGA 纳米粒子持续缓释 mNGF, 前 12 h 释放率为 30%, 第 5 天累积释放率为 92%MRI显像显示注射药物前左右下肢 T2*值都较高, 分别为 312.68、314.74, 注射药物后其有所下降, 但左右两侧值差距不明显, 分别为 264.43、263.78而在外在磁场下引导 2 h 后, T2*值进一步下降, 并且左下肢明显低于右下肢, 分别为150.90、233.54结论 (1) 通过单乳化溶剂挥发法能制备出粒径小、分布窄、包封率和载药率较高的磁性 mNGFPLGA 纳米粒子 (2) 该磁性纳米粒子体外释放效果良好, 具有可控的、持续时间较长的缓释效应 (3) 载 mNGF 的 PLGA 磁性纳米粒子能够有效降低 T2*值, 在外在磁场下有明显聚集性, 为进一步研究磁靶向修复周围神经损伤奠定良好基础关键词： 神经生长因子; 磁性氧化铁; 周围神经损伤; 聚乳酸-羟基乙酸共聚物; 纳米粒子; 磁共振成像 (MRI) ; 作者简介：海米提·阿布都艾尼 (1990-) , 男, 新疆乌鲁木齐市人 (维吾尔族) , 硕士研究生, 主要从事骨折研究。

022-88328518E-mail:511134378@.com作者简介：倪东馗 (1963-) , 男, 河北邢台市人, 硕士, 主任医师, 主要从事临床骨科工作022-88328518E-mail:ndkui@收稿日期：2017-08-31基金：天津市卫生和计划生育委员会科技基金资助项目 (2015KZ094) Preparation of PLGA nanoparticles loaded with mNGF and its repair of sciatic nerve injury in rats under magnetic field guidanceHai Miti Abuduaini LI Li-jun NI Dong-kui ZONG Qiang Graduate School of Tianjin Medical University; Department of Orthopedics, The Second Hospital of Tianjin Medical University; Department of Traumatology, Yantai Affiliated Hospital of Binzhou Medical University; Abstract： Objective To prepare the poly (lactide-co-glycolide) (PLGA) nanoparticles (NP) of mNGF and evaluate their release behavior, and observe the aggregation of NP in rats under the guidance of external magnetic field after left sciatic nerve injury by MRI. Methods The mNGF as model drug were prepared mNGF-PLGA NP by phacoemulsification. The NP were characterized by transmission electron microscopy and laser particle size analyzer, and mNGF standard curve was drawn by enzyme linked immunosorbent assay (ELISA) , the encapsulation efficiency, drug loading and in vitro release were measured. The sciatic nerve injury model was made by SD rats, and then drug-loaded mNGF-PLGA NP were injected into tail vein. Scanned noninjected drug rats, intravenous injection of magnetic mNGF-PLGA NP rats and left lower limb was fixed in l.0 T external mag-netic field for 2-hour rats after injection of magnetic mNGF-PLGA NP, the left and right lower extremity T2*values were recorded. Results The mNGF-PLGA NP sample was brown suspension in mean size of (205.9 ± 1.2) nm with distribute uniformly. The transmission electron microscope confirmed that NP were spherical and contained lots of mNGF. The encapsulation efficiency and drug loading rate was (69.43 ± 2.80) % and (3.11 ± 3.27) %, respectively. In vitro release experiments showed that magnetic mNGF-PLGA NP sustained releaseof mNGF, the 12-hour release rate was 30 %, and cumulative release rate was92 % at 5-day. The MRI showed that T2*values of left and right lower limbs were 312.68 and 314.74, then decreased after injection, the left and right sides were 264.43 and 263.78, the difference was no obviously. After 2-hour guidance of external magnetic field, T2*values further declined, and left lower limb was significantly smaller than that of right lower limb, the left and right lower limbs were 150.90 and 233.54. Conclusion It is demonstrated that emulsification method could prepare magnetic mNGF-PLGA NP with small particle size, narrow distribution, high entrapment efficiency and drug loading rate, which showed good release effect in vitro with controllable and slow-released. The mNGF-PLGA NP could effectively reduce T2*value and aggregate under external magnetic field, and lays the good foundation for further study of magnetic targeted repair of peripheral nerve injuries.Keyword： nerve growth factor; magnetic iron particles; peripheral nerve injury; poly (lactic-co-glycolic acid) ; nanoparticles; magnetic resonance imaging (MRI) ; Received： 2017-08-31周围神经损伤的治疗已成为世界性难题, 由于神经的特殊结构, 周围神经再生过程缓慢且通常不完整。

近年来, 神经组织工程学的迅速发展为临床治疗周围神经损伤带来新的希望周围神经损伤后神经功能缺损和促进神经再生的治疗策略已成为临床研究的热点[1]Lundborg G[2]对周围神经损伤再生修复进行了深入的研究, 描述了“神经再生室”存在的局部微环境这种微环境包含完整的神经通道、有活力的雪旺细胞、多种神经营养因子参与[3]、局部良好的血液供应因而, 改善周围神经损伤的微环境, 提供神经轴突再生所需的多种神经营养因子, 是促进周围神经的再生、修复的有效方法周围神经损伤微环境中包含多种神经营养因子, 包括神经生长因子 (nerve growth factor, NGF) 、脑源性神经营养因子 (brain derived neurotrophic factor, BDNF) 、神经营养因子 3 (neurotrophins-3, NT-3) 等, 其中 NGF 研究最为广泛自 20 世纪 50 年代 LeviMontalcini R 和 Cohen S 发现并纯化了NGF, 并在 60 年代获得 NGF 抗体以来, 有关 NGF 对中枢和外周神经系统研究迅速发展NGF 作为第一个和最综合的神经营养因子[4], 相对分子质量约为 140 000, 由 α、β 和 Υ3 肽键的非共价键结合构成[5], 它在调节神经元的存活、生长、分化、再生和功能维持等方面发挥重要作用。

大量体外研究表明[6], NGF 能诱导多种神经干细胞、胚胎干细胞、间充质干细胞, 分化成不同种类的特定神经元如果身体失去 NGF, 这些神经元可能会凋亡研究表明, NGF。