Figure 2. In vitro toxicity and transfection efficiency of mRNA NPs in PC3 cells.
Cells were treated with various mRNA concentrations (at 0.062, 0.125, 0.250, 0.500 μg/mL) of EGFP-mRNA-PGCP NPs for 16h and further incubated for 24h in standard cell culture incubation conditions. (a) AlamarBlue cytotoxicity assay; cell viability was normalized with the untreated control group. Error bars represent the s.d. (n=6). (b) Transfection efficiency (%GFP positive cells) was determined using flow cytometry (****p<0.0001). Error bars represent the s.d. (n=4) and significance was determined using One-Way ANOVA test. (c) Transfection efficiency was also analyzed with the histograms for the respective groups using Flowjo software. (d) Fluorescence microscopy images of PC3 cells transfected with naked EGFP mRNA, EGFP-mRNA-PGCP NP, and Lipofectamine 2000 (L2K)-EGFP-mRNA (magnification at 20×). (e) Mechanism of cellular uptake and endosomal escape of mRNA NPs in PC3 cells. The cells were pre-incubated for 30 min in serum-free medium containing inhibitors (Filipin, CPZ, EIPA, and Baf A1 were used as the inhibitor for caveolae-mediated endocytosis, clathrin-mediated endocytosis, macropinocytosis, and intracellular proton pump effects, respectively) or combinations of inhibitors (Filipin, CPZ, or EIPA mixed with Baf A1) prior to transfection with EGFP-mRNA PGCP NPs at mRNA concentration of 0.250 μg/ml. Transfection efficiency (%GFP positive cells) was determined using flow cytometry (***p<0.001 and ****p<0.0001). Error bars represent the s.d. (n=5) and significance was determined using One-Way ANOVA test. (f) The mechanism of cellular uptake and intracellular transport of the hybrid mRNA NPs is schematically illustrated.