Department of Chemical and Biomolecular Engineering, The Hong Kong University of Science and Technology, Hong Kong, China.
J Pharm Pharmacol. 2014 Apr;66(4):564-73. doi: 10.1111/jphp.12134. Epub 2013 Aug 25.
We aim to quantify the effect of size and degree of folate loading of folate-decorated polymeric nanoparticles (NPs) on the kinetics of cellular uptake and the selection of endocytic pathways in retinal pigment epithelium (RPE) cells.
In this study, methoxy-poly(ethylene glycol)-b-polycaprolactone (mPEG-b-PCL) and folate-functionalized PEG-b-PCL were synthesized for assembling into nanoparticles with sizes ranging from 50 nm to 250 nm. These nanoparticles were internalized into ARPE-19 (human RPE cell line) via receptor-mediated endocytosis. A two-step endocytosis process mathematical model was adopted to quantify binding affinity and uptake kinetics of nanoparticles in RPE cells in uptake and inhibition studies.
Nanoparticles with 100% folate loading have highest binding affinity and uptake rate in RPE cells. Maximum uptake rate (Vmax) of nanoparticles increased as the size of particles decreased from 250 nm to 50 nm. Endocytic pathway study was studied by using chlorpromazine and methyl-β-cyclodextran (MβCD), which are clathrin- and caveolae-mediated endocytosis inhibitors, respectively. Both chlorpromazine and MβCD inhibited the uptake of folate-decorated nanoparticles. Inhibition constant (Ki) and maximum uptake rate (Vmax) revealed that 50 nm and 120 nm folate-decorated nanoparticles were found to be internalized via both clathrin- and caveolae-mediated endocytosis. The 250 nm folate-decorated nanoparticles, however, were only internalized via caveolae-mediated pathway.
Increased uptake rate of folate-decorated NPs into RPE cells is observed with increasing degree of folate modification. These NPs utilize both clathrin- and caveolae-mediated receptor-mediated endocytosis pathways to enter RPE cells upon size variation. The 50 nm NPs are internalized the fastest, with clathrin-mediated endocytosis as the preferred route. Uptake of 250 nm particles is the slowest and is dominated by caveolae-mediated endocytosis.
我们旨在量化叶酸修饰的聚合物纳米粒子（NPs）的大小和叶酸加载程度对视网膜色素上皮（RPE）细胞摄取动力学和内吞途径选择的影响。
在这项研究中，甲氧基聚（乙二醇）-b-聚己内酯（mPEG-b-PCL）和叶酸功能化的 PEG-b-PCL 被合成用于组装成尺寸范围为 50nm 至 250nm 的纳米粒子。这些纳米粒子通过受体介导的内吞作用进入 ARPE-19（人 RPE 细胞系）。采用两步内吞过程数学模型来量化摄取和抑制研究中 RPE 细胞中纳米粒子的结合亲和力和摄取动力学。
叶酸负载 100%的纳米粒子在 RPE 细胞中具有最高的结合亲和力和摄取率。纳米粒子的最大摄取速率（Vmax）随着粒子尺寸从 250nm 减小到 50nm 而增加。通过使用氯丙嗪和甲基-β-环糊精（MβCD）研究内吞途径，氯丙嗪和 MβCD 分别是网格蛋白和小窝蛋白介导的内吞作用抑制剂。氯丙嗪和 MβCD 均抑制叶酸修饰纳米粒子的摄取。抑制常数（Ki）和最大摄取速率（Vmax）表明，50nm 和 120nm 的叶酸修饰纳米粒子被发现通过网格蛋白和小窝蛋白介导的内吞作用被内化。然而，250nm 的叶酸修饰纳米粒子仅通过小窝蛋白介导的途径被内化。
随着叶酸修饰程度的增加，观察到叶酸修饰的 NPs 进入 RPE 细胞的摄取速率增加。这些 NPs 在尺寸变化时通过网格蛋白和小窝蛋白介导的受体介导的内吞作用途径进入 RPE 细胞。50nm 的 NPs 被内化的速度最快，以网格蛋白介导的内吞作用为主。250nm 颗粒的摄取最慢，主要由小窝蛋白介导的内吞作用主导。
