Study On The Fatty Acid Modification Of R-Lycosin-I And Lycosin-I And Its Anticancer Activities

R-lycosin-I was a cationic peptide with a linear alpha-helix conformation,obtained by amino acid substitution from Lycosin-I.Lycosin-I was isolated from the venom of the spider lycosa singoriensis.Althought they have anticancer activities,their susceptibility to hydrolysis by proteases and poor stability limit their further application.The use of chemical modification can effectively make up for the shortcomings.Compared to structural modification,PEGylation and glycosylation,fatty acid modification can not only increase stability,enhance biological activity,but also improve the fat solubility and membrane permeability of peptide drugs.Therefore,the purpose of this study is to obtain analogues of R-lycosin-I and Lycosin-I with high stability,high activity and long-acting by fatty acid modification.In order to improve the anticancer activity of R-lycosin-I,fatty acids with different chain length with C₁₂,C₁₄,C₁₆,C₁₈,C₂₀ were introduced to the N-terminal of R-lycosin-I to yield five lipopeptides(named as R-C₁₂,R-C₁₄,R-C₁₆,R-C₁₈,R-C₂₀).The average sizes,zeta potential,morphology and secondary structures of the five lipopeptides were determined by DLS,TEM and circular dichroism spectroscopy,and its anticancer activity,hemolytic activity and serum stability were investigated.The DLS results confirmed that most of the lipopeptides showed a decrease in average size relative to R-lycosin-I,and positive charges.The TEM results showed that all of the peptides displayed homogeneous spherical nanoparticles.CD spectrum revealed the secondary structures of lipopeptides exhibited a random coil stricture in phosphate buffer solution.However,under the condition of 50%TFE,lipopeptides exhibited anα-helical structure,especially,the helical content of R-lycosin-I（35.5%helix）was significantly lower than that of lipopeptides（from 62.99%to 100.00%helix）.The cytotoxic activity of lipopeptides was evaluated with serum-containing and serum-free media,respectively,and the results showed that their anticancer activities were all increased.The cytotoxic activity of R-C₁₆6 was 4-fold higher than that of original R-lycosin-I under serum-containing media,and also was the strongest among them of all the five lipopeptides.The hemolytic activity results revealed that both R-lycosin-I and five lipopeptides displayed dose-dependent hemolytic effects.The serum stability results demonstrated that the inhibitory activity of lipopeptides was about 70%at 48 h.In contrast,the inhibitory activity of R-lycosin-I on A549 cells was approximately 10%after 48 h.SEM and LDH leakage assay results indicated that R-C₁₆ could either induce the disruption of cell membrane or promote apoptosis to exert anticancer activity.The dodecanoic acid was covalently coupled to theε-amino group of Lys residue of Lycosin-I which produced eight different positional lipopeptides.The average sizes,zeta potential and secondary structures of the eight lipopeptides were determined by DLS and circular dichroism spectroscopy,and studied on its anticancer activity,hemolytic activity and serum stability.The DLS results showed that the zeta potential of all lipopeptides were increased,but the particle size was decreased.The CD results revealed that all of peptides adopted random coil conformation in PBS buffer but alpha-helical conformation in 50%TFE.The CCK-8 assay results proved that the anticancer activity of all lipopeptides were increased.Compared to Lycosin-I,the colony formation assay results showed that L-C₁₂ could effectively inhibit cancer cell proliferation.The serum stability results demonstrated that the anticancer activity of lipopeptides was not change at 48 h while the anticancer activity of Lycosin-I was decreased.SEM and LDH leakage assay results indicated that lipopeptides exert their anticancer activity by acting on cell membranes.In summary,the fatty acid modified the N-terminal of R-lycosin-I and the?-amino group of Lys residue of Lycosin-I can effectively improve the anticencer activity,serum stability and other biological activities,and lay a ground for anticancer peptides chemical modification.