介孔硅脂质囊纳米粒的研制及其细胞摄取机制研究

Preparation and Cellular Uptake Mechanism Evaluation of Liposomal Coating-cationic Mesoporous Silica Nanoparticles

采用改良Stober法一步合成氨基修饰的阳离子型介孔硅纳米粒(CMSN),结合薄膜水化法和高速离心法制备介孔硅脂质囊纳米粒(LP-CMSN)。所得LP-CMSN呈圆整球形、粒径分布均一、无团聚、具有明显的核-壳结构,粒径为(114.5±3.7)nm,ζ电位为(9.3±2.5)mV,多分散系数(PDI)为0.17±0.05。细胞水平上,LP-CMSN在0~100μg/ml范围内对Hela和A549细胞均无明显细胞毒性。以LP-CMSN为载体,选择多柔比星为模型药,采用饱和溶液吸附法制得了载药量为(15.58±3.25)%的载药纳米粒,有效改善了CMSN中药物易突释缺点。选择异硫氰酸荧光素(FITC)为荧光探针,合成了标记率为0.52%的荧光纳米粒。通过流式细胞仪和激光共聚焦显微镜考察LP-CMSN在Hela细胞的摄取机制和入胞途径。结果显示,CMSN通过网格蛋白介导的内吞途径摄取进入细胞,而LP-CMSN主要经过膜融合途径进入细胞,最终经溶酶体逃逸后释放药物发挥药效。因此,LP-CMSN具有良好的生物相容性和缓控释特性,并能通过无损害的膜融合途径摄取进入细胞,有望成为新型纳米递药载体。

In this study, we adopted the modified Stober method in one-pot mode to synthesize cationic mesoporous silica nanoparticles （CMSN）, and combined with thin film hydration method and high-speed centrifugation to prepare liposomal coating-cationic mesoporous silica nanoparticles （LP-CMSN）. The prepared LP-CMSN displayed quite uniform distribution of size with better polydispersion index of 0.17±0.05, and core-shell structural morphology without agglomeration. The average particle size and the potential of the LP-CMSN were （114.5±3.7）nm and （9.3± 2.5）mV. Within the concentration of 100μg/ml, LP-CMSN had no obvious toxicity on Hela and A549 cells. Taking use of saturated solution adsorbing method, the model drug doxorubicin was highly encapsulated into LP-CMSN with drug loading capacity of （15.58±3.25）% . In drug release experiment, LP-CMSN presented excellent sustained- release properties with cumulative release amount of 85.6% in 48 h and, more importantly, inhibition of initial burst release compared with CMSN. By using the fluorescein isothiocyanate-labeled LP-CMSN in 0.52 % marking ratio, we investigated the cellular uptake mechanism and pathway of LP-CMSN with flow cytometry and laser scanning confocal microscopy. The results showed that CMSN could be taken into cells by clathrin-mediated endocytosis, whereas, the cellular uptake pathway of LP-CMSN was mostly by membrane fusion which could not be blocked by a series of endocytosis inhibitors. So, LP-CMSN resulted in lysosome escape to exert therapeutic effect without the influence of endocytosis inhibitions and low temperature. In conclusion, LP-CMSN with excellent biocompatibility, outstanding sustained-release properties and novel harmless membrane fusion cellular uptake pathway would he a promising drug delivery carrier in future.