The Effect of Charge on Cellular Uptake and Phototoxicity of Polylysine Chlorine6Conjugates

Abstract— The effect of charge on the cellular uptake, localization and phototoxicity of conjugates between chlorin_e6 ( c _e6) and poly-L-lysine was studied in vitro. These conjugates (average MW35–55 kDa) were synthesized to have polycationic, polyanionic or neutral charges. Two human cell lines (A431 epidermoid carcinoma cells and EA.hy926 hybrid endothelial cells) were studied and the cellular uptake of c _e6 delivered by the conjugates of varying charge and free c _e6 was measured at conjugate c _e6 equivalent concentrations up to 0.4 μM. Uptake was time and concentration dependent and temperature dependent in the case of neutral and anionic conjugates. Relative uptake at 6 h for A431 cells was 73:15:4:1 and for EA. hy926 cells was 63:11:3:1 for cationic, anionic, neutral and free c _e6, respectively, but EA. hy926 cells took up 1.5-2 times as much c _e6 from all the conjugates as A431 cells. Localization as studied by fluorescence microscopy indicated that the cationic conjugate was in aggregates bound to the plasma membrane, while the other forms were internalized in organelles and membranes. Phototoxicity was measured by 3-(4,5-dimethylthiazol-2-yl)-diphenyltetra-zolium bromide (MTT) assay after irradiation with5–20 J cm^?2of 666 nm light. In contrast to the uptake, the order of phototoxicity for both cell types per mole of c _e6 uptake per cell was neutral ? anionic > cationic > free c _e6. Polymeric c _e6 conjugates bearing positive, negative and neutral charges may have different tissue-localizing properties and could play a role in photodynamic therapy.