| Branched and linear polyethylenimine (PEI) are promising molecules for gene delivery. However, like other non-viral gene carriers, the problem of PEI lies in relatively low transfection efficiency. Melittin is a 26 amino acids peptide from bee venom. The structure of melittin is similar to lipid compounds: the first 20 residues form a hydrophobic"tail"and the positively charged residue 21-24 (KRKR) form the positively charged"head". It was reported that melittin could form secondaryα-helical structure and twoα-helix molecules could interact with each other, resulting membrane permeability and cytolysis. Ogris et al. covalently attached melittin to PEI and found that the membrane lytic activity of melittin enabled efficient release of the PEI/DNA into the cytoplasm and increased its nuclear localization. In our previous experiment, we first verified that the first 20 residues of melittin (termed as MT20), mixed with PEI, could enhance PEI-mediated gene transfection in HeLa cells. Then, the homologous peptide named florae was also found to have the similar characteristics with melittin in our study.Objective: To study the mechamism of cell-penetrating peptides and its role in enhancing branched polyethylenimine (BPEI) mediated gene transfection efficiency in different kinds of cells.Methods: Melittin,florae and their hydrophobic core MT20 and FL20 were synthesized and the secondary structure of them were analysized with circular dichroism spectra scanning. Their relative membrane penetration activity were evaluated by detecting the haemolysis level in different pH buffer at different concentration. The distribution of calcein in HeLa cells before and after peptides treatment was observed; The effect of these peptides on the DNA condensation ability of PEI were evaluate by agarose gel electrophoresis. Size andζ-potential of the complexes (peptide/PEI/DNA) were evaluated by Zeta PALS analyzer; PEI and melittin,florae,MT20 or FL20 were mixed at different ratios before binding with Luciferase plasmids and the transfection efficiency was assayed by detecting Luciferase activity in 293FT,B16F10 and CHO-K1 cells after gene transfection; The cytotoxicity was analyzed by MTT assay at 48 hours after transfection.Results: In the mimetic membrane environment (methanol solution), melittin and MT20 presented 53% and 29%α-helix respectively, florae and FL20 presented 52% and 34%α-helix respectively.In the presence of MT20 or FL20, certain degree of hemolytic activity in the acidic buffer (pH 5.4~5.0) could be detected, and when peptides concentration was 12μM, the hemolytic degree MT20 and FL20 was 41% and 53% respectively at pH 5.4. When the pH value was above 5.4, hemolytic activity of MT20 and FL20 increased considerably with increasing pH, reaching 73% and 81% respectively at pH 7.4. At pH 7.4,, the hemolytic activity of MT20 and FL20 increased with the increasing concentration of MT20 and FL20, and reached 98% and 91% respectively at 24μM. However, in the presence of melittin and florae, no hemolytic activity was detected in the acidic buffer (pH 5.4~5.0). The hemolytic activity of melittin and florae were 80% and 82% respectively at pH 7.4 (12μM). With the increasing concentration at pH 7.4, the hemolytic activity of melittin and florae increased, reaching 90% and 96% respectively at 24μM.In the absence of peptides, small amount of membrane-impermeable calcein fluorophore entered cells and was restricted within intracellular vesicles appearing as bright punctuate structures. When the cells were treated with 45μg/mL melittin/florae, a large number of punctuate spots were observed in the whole cells together with some amount of diffuse staining, and some were located in the nucleoli. Treated with 45μg/mL MT20/FL20 resulted in a stronger diffuse staining compared to melittin/florae groups. Moreover, disperse and uniform fluorescence distribution throughout the whole cells was observed in MT20/FL20 groups, with disappearance of bright punctuate vesicles . The agarose gel electrophoresis results showed that whether peptides were present or not, BPEI has a strong DNA binding ability. When the weight ratio of CPP/BPEI/DNA was 1.5:1:1 (N/P=7.5) or 3:2:1 (N/P=15), PEI could condense DNA into nanosized complexes with the size range of about 180~648 nm. When the weight ratio of CPP/PEI/DNA was 6:4:1 (N/P=30), the size of the polymers reaches 310~10~96nm. All complexes presented net positive charges and the mean value ofζ-potential was 4~26 mV in different weight ratios.All of four peptides could enhance gene transfection of BPEI in 293FT,B16F10 and CHO-K1 cells and MT20/FL20 groups make higher enhancement. In 293FT cells, when the N/P ratio was 7.5, the transfection efficiency of MT20 and FL20 group were 3.34×10~9 and 3.17×10~9 RLU/mg protein respectively, which were 2.5 folds and 2.4 folds of BPEI group, 2.2 folds and 2.1 folds of Lipofectamine 2000 group respectively. In B16F10 cells, when the N/P ratio was15, the transfection efficiency of MT20 and FL20 group were 1.73×10~9 and 2.03×10~9 RLU/mg protein respectively, which were 2.2 folds and 2.5 folds of BPEI group, 2 folds and 2.3 folds of Lipofectamine 2000 group respectively. In CHOK-1 cells, when the N/P ratio was 30, the transfection efficiency of MT20 and FL20 group were 3.98×10~9 and 3.66×10~9 RLU/mg protein respectively, which were 4.3 folds and 4 folds of BPEI group, 27.6 folds and 25.4 folds of Lipofectamine 2000 group respectively.The results of MTT assays indicated that addition of peptides did not increase cytotoxicity. In 293FT cells, the cell viability of MT20 and FL20 groups were 86% and 83% versus 71% (melittin group), 85% (florae group), 71% (BPEI group) and 83% ( Lipofectamine 2000 group) respectively. In B16F10 cells, the cell viability of MT20 and FL20 groups were 68% and 74% versus 68% (melittin group), 71% (florae group), 62% (BPEI group) and 57% ( Lipofectamine 2000 group) respectively. In CHO-K1 cells, the cell viability of MT20 and FL20 groups were 93% and 95% versus 87% (melittin group), 90% (florae group), 86% (BPEI group) and 48% ( Lipofectamine 2000 group) respectively.Conclusion: MT20/FL20 enabled efficient vesicular escape and enhanced nuclear access of nonviral gene delivery vectors (BPEI), consequently enhancing gene transfection efficiency. It is concluded that hydrophobic core of melittin/florae (MT20/FL20) may be a potential enhancer of PEI which will be used to improve gene transfection in difficult to transfect cells. |
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