Figure 8.

Effect of free FA on FA-PPSu-PEG-Rho NPs cellular uptake. HeLa K, T47D, MCF7 and MDA-MB-231 cells were pre-incubated for 1 h with 3mM free folic acid, followed FA-PPSU-PEG-Rhodamine nanoparticle addition at a low concentration (2.2 μg/mL polymer). NPs internalization was monitored for 4 h, and images were acquired every 10 min. (A) Representative images of HeLa K, T47D, MCF7 and MDA-MB-231 cell lines, upon addition of the FA-PPSu-PEG-Rho NPs (control), or 60 and 240min post NPs addition, in the presence (+FA) or absence of free folic acid (-FA). Cell nuclei were stained with Hoechst 33342 and shown in blue, whereas Rhodamine-labeled NPs are shown in red. Scale bar denotes 5 μm. (B–E) Graphs demonstrating cellular uptake of the FA-PPSu-PEG-Rho NPs for the four cell lines in the presence (+FA) or absence of FA. Relative fluorescence intensity is shown in Arbitrary Units (A.U) and fluorescence for each cell line is calculated as fold of its own maximum intensity at 240 minutes after NPs’ addition. The effect of FA on NPs’ internalization was monitored for 4 h and expressed as fold-change, relative to fluorescence values measured upon NPs addition. The fluorescent intensity of at least 100 cells per cell line was measured. For each cell line, nanoparticles uptake after FA treatment was plotted against the uptake of NPs per se. (F) SDS PAGE analysis showing the expression of FOLR1 protein in the four different cell lines: HeLa K, T47D, MCF7 and MDA-MB-231. α-tubulin serves as a loading control.