Synthesis of two folate conjugates and their targeting effect in vitro

The aim of this study was to study the synthesis of two folate conjugates and their application in the preparation of folate targeted liposome, and to investigate their targeting effect in hepatocellular carcinoma HepG2 cell line in vitro. In this study, Folate-PEG-Cholesteryl hemisuccinate(Folate-PEG₂₀₀₀-CHEMS and Folate-PEG₄₀₀₀-CHEMS)were synthesized by linking folate and cholesterol succinate with two kinds of PEG materials. Structures of Folate-PEG₂₀₀₀-CHEMS and Folate-PEG₄₀₀₀-CHEMS were characterized by ¹H NMR and ultra-high resolution mass spectrometry. Calcein was selected as the model drug, and calcein liposomes FA-PEG₂₀₀₀-L and FA-PEG₄₀₀₀-L were prepared by film dispersion method using Folate-PEG₂₀₀₀-CHEMS and Folate-PEG₄₀₀₀-CHEMS, respectively. The particle size and Zeta potential of FA-PEG₂₀₀₀-L and FA-PEG₄₀₀₀-L were measured by laser particle size analyzer. The drug delivery effect of FA-PEG₂₀₀₀-L and FA-PEG₄₀₀₀-L was evaluated by cellular uptake experiment in HepG2 cell line in vitro. Flow cytometry and laser confocal scanning microscope were used to determine fluorescence in HepG2 cells in vitro. The results showed that the average particle size of calcein liposome was (205.8 ± 10.2) nm, and the Zeta potential of calcein liposome was -(1.19 ± 0.31) mV.There was no significant difference in particle size and Zeta potential between FA-PEG₂₀₀₀-L and FA-PEG₄₀₀₀-L. The fluorescence intensity of FA-PEG₄₀₀₀-L liposome group was about 3.6 and 3.1 times higher than that of non-targeted liposome group and FA-PEG₂₀₀₀-L liposome group, with statistically significant difference (P < 0.01). The drug delivery efficiency of FA-PEG₄₀₀₀-L group in HepG2 cells was higher than that in FA-PEG₂₀₀₀-L and non-targeted groups, and the results indicated that Folate-PEG₄₀₀₀-CHEMS can promote the uptake of liposome by HepG2 cells in vitro. All in all, Folate-PEG₄₀₀₀-CHEMS could be applied in the preparation of folate targeted liposome, which could promote the uptake of liposome by HepG2 cells.