乳铁蛋白结合聚乙二醇聚酸纳米粒脑内递药特性的体内外研究.docVIP

乳铁蛋白结合聚乙二醇聚乳酸纳米粒脑内递药特性的体内外研究 （复旦大学药学院药剂教研室，上海 200032） 摘要：目的 构建一种新型可生物降解脑内递药系统-乳铁蛋白结合聚乙二醇聚乳酸纳米粒（Lf-NP），并对其脑内递药特性进行体内外评价。 方法 采用马来酰亚胺基聚乙二醇-聚乳酸和甲氧基聚乙二醇-聚乳酸通过复乳溶剂挥发法制备纳米粒（NP）,将其与巯基化的乳铁蛋白（Lf）共价连接制得Lf-NP。采用透射电镜技术，免疫电镜技术，动态光散射分析，X-射线光电子能谱分析及酶联免疫吸附试验对Lf-NP的形态,粒径和表面Lf的连接进行鉴定和表征。以香豆素-6为荧光探针定性及定量的研究了小鼠脑毛细血管内皮细胞bEnd.3对Lf-NP的摄取情况。采用CCK-8Lf-NP对细胞活力的影响。利用荧光显微技术和冷冻切片技术观察小鼠尾静脉注射载香豆素-6的Lf-NP后的脑内分布情况并进行了药动学研究。 结果 制得的Lf-NP形态圆整，粒径在110 nm左右。免疫电镜技术，X-射线光电子能谱分析及酶联免疫吸附试验证实了Lf-NP表面共价连接有Lf，且每个Lf-NP表面的Lf数约为55。bEnd.3对Lf-NP的摄取在37℃，不同浓度和不同孵育时间条件下均显著高于未结合纳米粒（NP）。静脉注射载香豆素-6的纳米粒后，Lf-NP组小鼠脑内香豆素-6的量约为NP组的3倍。3 mg/ml的Lf-NP对bEnd.3细胞的活力无显著影响。结论 Lf-NP能显著增加药物的脑内递送且具有较好的生物相容性，有望作为一种新型的脑内递送系统输送蛋白多肽和基因等大分子药物入脑。 关键词：乳铁蛋白；血脑屏障；香豆素-6；生物可降解纳米粒；脑内递送 中图分类号：R9 Lactoferrin-conjugated PEG–PLA nanoparticles with improved brain delivery: in vitro and in vivo evaluations HU Kai-li, LI Jing-wei, SHEN Ye-hong, LU Wei, GAO Xiao-ling, Jiang Xin-guo (Department of Pharmaceutics, School of Pharmacy, Fudan University, Shanghai 200032) ABSTRACT：OBJECTIVE To construct the lactoferrin (Lf) conjugated poly (ethyleneglycol) –poly (lactide) (PEG–PLA) nanoparticle (Lf–NP) as a novel biodegradable brain drug delivery system and evaluate its in vitro and in vivo delivery properties. METHODS Lf was thiolated and conjugated to the distal maleimide functions surrounding on the pegylated nanoparticles prepared by double emulsion and solvent evaporation procedure to form the Lf–NP. Dynamic light scattering analysis, transmission electron micrograph (TEM), X-ray photoelectron spectrometry analysis and ELISA were used to characterize the Lf-NP. A fluorescent probe, coumarin-6 was incorporated into Lf–NP and its uptake by a mouse brain capillary endothelial cell line, bEnd.3 were compared to that of unconjugated nanoparticle (NP). The brain distribution of Lf-NP following an intravenous administration was evaluated quantitatively by pharmacokinetic studies and qualitatively by fluorescent microscopy. The influence of Lf-NP on cell viabili