| Non-viral gene carrier has been a very important direction of gene therapy study in recent years. As comparing to lipid, cationic polymers showed lower cytotoxicity, higher compatibility. Polyethylenimine (PEI) is the most common used cationic polymer in gene delivery study. It can bind plasmid DNA with high affinity and condense it into nano-scale polyplexes. PEI showed high gene delivery efficiency in both in vitro, and in vivo animal model. However, in some cases, the transfection efficiency mediated by PEI is much lower then that of cationic lipids, such as Lipofectamine 2000. Cationic lipid molecules pose positively charged ?head? and hydrophobic ?tail?. Its hydrophobic tail shows high affinity to cell membrane and facilitates cellular uptaking and endosomal releasing, while cationic polymers don?t have hydrophobic structure. We speculated that it maybe the main reason why its transfection efficiency is lower than lipofectamine 2000 in some cell lines.It was found that cell penetration peptide (CPP) could improve PEI transfection efficiency. Melittin is one of CPP peptide from bee venom which contains 26 amino acids (GIGAVLKVLTTGLPA LISWIKRKRQQ-NH2)..The structure of melittin is similar to lipid compounds: the 1st to 20th amino acids forms a hydrophobic"tail"and the 21st to 24th amino acids (KKRK) forms positive charged"head". Ogris reported that conjugation of Melittin to PEI could enhance the gene transfection efficiency mediated by PEI in HeLa cells.Our previous work has demonstrated that the hydrophobic tail of Melittin could increase gene delivery efficiency mediated by BPEI. The aim of this study was to investigate the mechanism of enhancing transfection efficiency of branched polyethylenimine(BPEI) in HeLa cells by hydrophobic tail of Melittin.The first 20 hydrophobic amino acids melittin was synthesized and termed as MT20. As compare to melittin, MT20 is lack of 6 amino acids (included 4 positively charged Arg or Lys).Its membrane permeable activity of MT20 was evaluated by hemolysis test. The peptide was mixed with BPEI and DNA, and the effect of MT20 on the DNA binding affinity of BPEI was analyzed by DNA gel retardation assay. The physicochemical characteristics of MT20/BPEI/DNA was studied by particle size and zeta potential determination. Green fluorescent protein gene (GFP) was used as a reporter gene to determine the transfection efficiency in HeLa cells.The cytotoxicity of the mixture was detected by MTT assay at 24 hours after transfection.The results showed that low concentration of MT20 did?nt showed significant hemolysis, however the hemolysis rate significantly increased with the increasing concentration of MT20. When the concentration of MT20 was 20.0μmol/L,the 82% of RBCs were lysized in PBS buffer while about 59% RBCs were lysized at PCS (pH5.6) buffer. The results indicated that MT20 could induce hemolysis both at neutral and acidic condition, and the hemolysis activity in neutral condition was higher than that in acidic condition. There was no significant change in the electrophoresis? pattern of BPEI/DNA complex after MT20 was added. The results suggested that MT20 would not affect the DNA binding affinity of BPEI. The physicochemical results showed MT20 reduced zeta potential of BPEI/DNA complexes,while particle size slightly increased when the MT20/BPEI ratio was 2:1. However, the particle size and zelta potential was in the optimal range for gene transfection. The transfection assay showed that the peptide could significantly improve the transfection efficiency of BPEI, and the GFP fluorescent density gradually increased when different amount of MT20 was mixed with BPEI (75 ng/well) before gene transfection.When the amount of MT20 was 112.5 ng/well, GFP fluorescent density was 4821.3 RFU, about 4 times of that in the BPEI/DNA group,similar to that of transfected cells mediated by Lipofectamine 2000(4719RFU) .The results indicate that MT20 could significantly enhance BPEI mediated transfection efficiency. In the optimal condition, cell survival fraction was 73.81% in BPEI mediated transfection,while the cell viability was above 94.44% in the MT20 enhancing group. It indicated that the cytotoxicity of MT20 enhancing group was very low.HeLa cell is commonly used in cell biology experiment, especially in tumor biological experiments. But the transfection efficiency mediated by BPEI is very low (only 5-10% ) in HeLa cell. Since MT20 could significantly enhance BPEI mediated transfection efficiency, another objective of the study is to transfect siRNA expression plasmid targeting to ATR gene into HeLa cells using MT20 and BPEI. After G418 selection, the positive clone was obtain. The ATR protein in siRNA transfected HeLa cells (siRNA+HeLa) and control was detected by western bloting. The cellular sensitivity to alkylating agents (antitumor drug) was analyzed by MTT assay. The result showed that siRNA expression plasmid targeting to ATR gene was effectively transfected into HeLa cells by using MT20 and BPEI, ATR gene expression was greatly inhibited in siRNA+ HeLa cell. In drug resistant study, both HeLa cells and siRNA+ HeLa cells were resisted to Nimustine hydrochloride (ACNU), the ID50 were more than 100?g/ml,however the siRNA+ HeLa cells were more resisted to ACNU than HeLa cells. Streptozocin(STZ) could increase the cellular sensitivity to ACNU, and when the cells were pretreated with 10 mmol/L STZ, the MGMT activity was significantly inhibited. Both of the cells became very sensitive to ACNU,and ID50 of both cells were less then 10?g/ml and siRNA+HeLa was more sensitive than HeLa cells. The results indicated that inhibition of ATR protein could affect the tumor sensitivity to alkylating antitumor drugs and provide evidence for us to understanding the mechanism of alkylating reagent in killing tumor cells.In a word, it is concluded that hydrophobic tail of melittin may be a potential enhancer to improve transfection efficiency mediated by cationic polymers in difficult to transfect cells and this method will be powerful tool in gene function study in the near future.
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