Objective: To evaluate the feasibility of the chitosan-poly (lactide-co-glycolide) (PLGA) double-walled microspheres for sustained release of bioactive nerve growth factor (NGF) in vitro.
Methods: NGF loaded chitosan-PLGA double-walled microspheres were prepared by emulsion-ionic method with sodium tripolyphosphate (TPP) as an ionic cross-linker. The double-walled microspheres were cross-linked by different concentrations of TPP [1%, 3%, 10% ( W/ V)]. NGF loaded PLGA microspheres were also prepared. The outer and inner structures of double-walled microspheres were observed by light microscopy, scanning electron microscopy, confocal laser scanning microscopy, respectively. The size and distribution of microspheres and fourier transform infra red spectroscopy (FT-IR) were analyzed. PLGA microspheres with NGF or chitosan-PLGA double-walled microspheres cross-linked by 1%, 3%, and 10%TPP concentration (set as groups A, B, C, and D respectively) were used to determine the degradation ratio of microspheres in vitro and the sustained release ratio of NGF in microspheres at different time points. The bioactivity of NGF (expressed as the percentage of PC12 cells with positive axonal elongation reaction) in the sustained release solution of chitosan-PLGA double-walled microspheres without NGF (set as group A1) was compared in groups B, C, and D.
Results: The chitosan-PLGA double-walled microspheres showed relative rough and spherical surfaces without aggregation. Confocal laser scanning microscopy showed PLGA microspheres were evenly uniformly distributed in the chitosan-PLGA double-walled microspheres. The particle size of microspheres ranged from 18.5 to 42.7 μm. The results of FT-IR analysis showed ionic interaction between amino groups and phosphoric groups of chitosan in double-walled microspheres and TPP. In vitro degradation ratio analysis showed that the degradation ratio of double-walled microspheres in groups B, C, and D appeared faster in contrast to that in group A. In addition, the degradation ratio of double-walled microsphere in groups B, C, and D decreased when the TPP concentration increased. There were significant differences in the degradation ratio of each group ( P<0.05). In vitro sustained release ratio of NGF showed that when compared with PLGA microspheres in group A, double-walled microspheres in groups B, C, and D released NGF at a relatively slow rate, and the sustained release ratio decreased with the increase of TPP concentration. Except for 84 days, there was significant difference in the sustained release ratio of NGF between groups B, C, and D ( P<0.05). The bioactivity of NGF results showed that the percentage of PC12 cells with positive axonal elongation reaction in groups B, C, and D was significantly higher than that in group A1 ( P<0.05). At 7 and 28 days of culture, there was no significant difference between groups B, C, and D ( P>0.05); at 56 and 84 days of culture, the percentage of PC12 cells with positive axonal elongation reaction in groups C and D was significantly higher than that in group B ( P<0.05), and there was no significant difference between groups C and D ( P>0.05).
Conclusion: NGF loaded chitosan-PLGA double-walled microspheres have a potential clinical application in peripheral nerve regeneration after injury.
目的: 评估壳聚糖-聚乳酸-聚乙醇酸共聚物［poly（lactide-co-glycolide），PLGA］双壁微球在体外持续缓释具有生物活性的 NGF 的可行性。.
方法: 应用乳化-离子交联方法制备包埋 NGF 的、具有不同三聚磷酸钠（sodium tripolyphosphate，TPP）交联浓度［1%、3%、10%（ W/ V）］的壳聚糖-PLGA 双壁微球，同时制备包埋 NGF 的 PLGA 微球。通过光镜、扫描电镜、激光共聚焦显微镜观察双壁微球的表面及内部形态，并行双壁微球的粒径分析和红外光谱分析。取包埋 NGF 的 PLGA 微球或 1%、3%、10%TPP 浓度交联的包埋 NGF 的壳聚糖-PLGA 双壁微球（分别设为 A、B、C、D 组），于不同时间点行体外微球降解率测定、微球中 NGF 缓释率检测；以未包埋 NGF 的壳聚糖-PLGA 双壁微球缓释液（设为 A1 组）作为对照，比较 A1、B、C、D 组体外微球缓释液中 NGF 的生物活性［以缓释液中具有阳性轴突延长反应的大鼠肾上腺嗜铬细胞瘤（PC12） 细胞百分比表示］。.
结果: 包埋 NGF 的壳聚糖-PLGA 双壁微球呈球形结构，具有相对粗糙的外表面；激光共聚焦显微镜观察示 PLGA 微球均匀分布于壳聚糖-PLGA 双壁微球内；双壁微球的粒径范围为 18.5～42.7 μm；红外光谱分析结果说明双壁微球中壳聚糖的氨基与 TPP 中的磷酸基发生了静电反应。体外降解率测定显示，B、C、D 组双壁微球降解速度明显快于 A 组，并随着 TPP 浓度的增加而逐渐减慢，培养各时间点各组间微球降解率比较差异均有统计学意义（ P<0.05）。体外 NGF 缓释率测定显示，与 A 组 PLGA 微球相比，B、C、D 组双壁微球以相对较慢的速度缓释 NGF，且缓释速度随 TPP 浓度增加而减慢。除 84 d 外，各时间点 B、C、D 组间比较 NGF 总缓释率差异均有统计学意义（ P<0.05）。体外 NGF 的生物活性评估显示，培养各时间点 B、C、D 组缓释液中具有阳性轴突延长反应的 PC12 细胞百分比均显著高于 A1 组（ P<0.05）。培养 7、28 d，B、C、D 组间比较差异无统计学意义（ P>0.05）；56、84 d 时，C、D 组缓释液中具有阳性轴突延长反应的 PC12 细胞百分比显著高于 B 组（ P<0.05），C、D 组间差异无统计学意义（ P>0.05）。.
结论: 包埋 NGF 的壳聚糖-PLGA 双壁微球在周围神经损伤后的再生方面具有临床应用潜力。.
Keywords: Chitosan; microspheres; nerve growth factor; nerve injury; poly (lactide-co-glycolide).