Enhanced Therapeutic Efficacy of iRGD-Conjugated Crosslinked Multilayer Liposomes for Drug Delivery
Figure 3
Clathrin-mediated internalization of iRGD-cMLVs and caveolin-dependent endocytosis of cMLVs. ((a), (b)) HeLa cells were incubated with DiD-labeled cMLV nanoparticles (red, (a)) or DiD-labeled iRGD-cMLVs particles (red, (b)) for 30 min at 4°C to synchronize internalization. The cells were then incubated at 37°C for 15 min, fixed, permeabilized, and immunostained with anti-clathrin (green) or anti-caveolin-1 antibody (green). The nucleus of cells was counterstained with DAPI. Scale bar represents 10 μm. ((c), (d)) Quantification of cMLV and iRGD-cMLV particles colocalized with clathrin (c) or caveolin-1 signals (d) after 15 min of incubation. Overlap coefficients were calculated using Manders’ overlap coefficients by viewing more than 30 cells of each sample using the Nikon NIS-Elements software. Error bars represent the standard deviation of the mean from analysis of multiple images (). (e) Inhibition of clathrin-dependent endocytosis by chlorpromazine (CPZ, 25 μg/mL) and caveolin-dependent internalization by Filipin (10 μg/mL). The uptake of DiD-labeled cMLV and DiD-labeled iRGD-cMLV nanoparticles was determined by measuring DiD fluorescence via flow cytometry. Error bars represent the standard deviation of the mean from triplicate experiments (, ).
