乳铁蛋白修饰的壳聚糖磁性纳米粒的制备、表征及海马神经细胞摄取

Synthesis and characterization of lactoferrin modified chitosan magnetic nanoparticles and its uptake by HT-22 hippocampal cells

目的制备、表征载四氧化三铁（Fe3O4）的乳铁蛋白修饰的两亲性壳聚糖（Lf-QMC）磁性纳米粒,研究其对海马神经细胞的亲和力及磁场介导下的体内脑靶向性。方法以Lf-QMC纳米粒为基础,采用溶剂挥发法将油酸（OA）改性的磁性Fe3O4（OA-Fe3O4）包载,形成载OA-Fe3O4的Lf-QMC纳米粒,载药量为1.5%;通过傅里叶红外光谱（FTIR）、透射电镜（TEM）、动态光散射（DLS）、X射线衍射（XRD）和振动磁强计（VSM）对其进行表征;刃天青法检测载OA-Fe3O4的Lf-QMC纳米粒对HT-22海马细胞活性的影响;TEM考察该纳米粒被HT-22海马神经细胞摄取的情况;荧光分析法检测载该纳米粒的体内脑靶向性。结果成功构建了载OA-Fe3O4的Lf-QMC纳米粒,其饱和磁化强度为1.2 emu·g^-1;该磁性纳米粒在2-200μg·mL^-1对HT-22海马细胞无显著毒性;TEM观察显示该磁性纳米粒对HT-22海马细胞有较好的亲和性并能通过有效的途径进入细胞;在磁场的介导下,OA-Fe3O4-Lf-QMC纳米粒可以实现更好的脑靶向效果。结论载OA-Fe3O4的Lf-QMC纳米粒有望在磁场和乳铁蛋白的双重靶向作用下透过血脑屏障向神经细胞递送药物。

Objective To synthesize and characterize Fe3O4 loaded lactoferrin modified amphiphilic chitosan（Lf-QMC） magnetic nanoparticles（NPs）, and to determine their affinity to HT-22 hippocampal cells and the in vivo brain targeting induced by magnetic field. Methods Oleic acid（OA） modified Fe3O4（OA-Fe3O4） were loaded into Lf-QMC NPs by solvent evaporation method to obtain OA-Fe3O4-Lf-QMC NPs. The drug loading content of OA-Fe3O4-Lf-QMC NPs was 1.5%. The characterization of OA-Fe3O4-Lf-QMC was measured by FITR, TEM, DLS, XRD and VSM. Resazurin was used to ascertain the cytotoxicity of OA-Fe3O4-Lf-QMC Nps on HT-22 hippocampal cells. The uptake of OA-Fe3O4-Lf-QMC NPs by HT-22 hippocampal cells was investigated by TEM. Fluorescence analysis was used to examine the brain targeting of OA-Fe3O4-Lf-QMC NPs in vivo. Results OA-Fe3O4-Lf-QMC NPs were developed successfully with the saturation magnetic induction intensity of 1.2 emu·g^-1. These magnetic NPs had no distinct cytotoxicity on HT-22 hippocampal cells at 2- 200 μg·mL^-1. In addition, they had good affinity with HT-22 hippocampal cells and could efficiently enter these cells. Under the guidance of magnetic field, OA-Fe3O4-Lf-QMC NPs exhibited more desirable brain targeting than Lf-QMC NPs in vivo. Conclusion OA-Fe3O4-Lf-QMC NPs are expected to deliver drug to nerve cells through blood-brain barrier due to the double targeting effect of magnetic field and lactoferrin.