Size-dependent internalisation of folate-decorated nanoparticles via the pathways of clathrin and caveolae-mediated endocytosis in ARPE-19 cells

Abstract

Objectives 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. Methods 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 50nm to 250nm. 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. Key findings 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 250nm to 50nm. 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 50nm and 120nm folate-decorated nanoparticles were found to be internalized via both clathrin- and caveolae-mediated endocytosis. The 250nm folate-decorated nanoparticles, however, were only internalized via caveolae-mediated pathway. Conclusions 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 50nm NPs are internalized the fastest, with clathrin-mediated endocytosis as the preferred route. Uptake of 250nm particles is the slowest and is dominated by caveolae-mediated endocytosis. Graphical Abstract © 2013 Royal Pharmaceutical Society.