Construction Of Poly(Amino Amine)s Modified By Pyrrolidone Gene Delivery System And Evaluation Of Its Biological Properties

Poly(amino amine)s(PAAs),as a class of non-viral gene vectors,has attracted extensive attention from researchers in recent years.This type of gene vector is a peptide-like polymer,synthesized from two kinds of compounds with amide monomer and amine monomer through Michael addition reaction in one step.Among them,the PAAs,structure of amide monomer with disulfide bond and amine monomer,has more advantages,which is also known as poly(disulfide amine)(SS-PAAs).p(CBA-DAH),with N,N'-cystaminebisacrylamide(CBA)as amide monomer and N-Boc-1,6-hexanediamine(N-Boc-1,6-DAH)as amine monomer,is a representative polymer in SS-PAAs family,and shows high transfection ability.Objective:In this study,polyvinylpyrrolidone(PVP)with serum tolerance and non-toxic effect was modified to the side chain amino group of p(CBA-DAH)in a certain proportion,aiming to reduce the cytotoxicity of p(CBA-DAH)to cells and improve its tolerance to serum.N-(3'-aminopropyl)-2-pyrrolidinone(APP)containing pyrrolidinone group was used as amine monomer to synthesize new SS-PAAs containing pyrrolidinone group on the main chain,so as to evaluate the differences in gene carrier performance between the two kinds of SS-PAAs containing pyrrolidinone group.Methods:(1)p(CBA-DAH)and p(CBA-APP)were synthesized by Michael addition reaction,and PVP-COOH with functional groups determined by molecular weight was synthesized by RAFT polymerization reaction.p(CBA-DAH)-g-PVP was synthesized by the reaction of p(CBA-DAH)and PVP-COOH,and the structures of the polymers were characterized by IR and ~1H-NMR.(2)The ability of p(CBA-DAH),p(CBA-APP)and p(CBA-DAH)-g-PVP to compress and release DNA were evaluated by gel electrophoresis experiment and particle size measurement,the buffering capacity of each polymers was evaluated by p H meter.(3)The cytotoxicity of different concentrations of each polymers and different weight ratios of each polymers/DNA complexes on Bel-7402,He La,Hep G2and NIH3T3 cells was investigated by MTT assay.The apoptosis of Bel-7402 cells was examined by DNA Ladder and Annexin V-FITC/PI double staining.The transfection ability of polymers to DNA was investigated by inverted fluorescence microscope and flow cytometry in different weight ratios,cell lines and serum.The mechanism of cell delivery of target DNA to Bel-7402 cells was investigated by using cell channel inhibitors and fluorescence labeling staining methods.Results:p(CBA-DAH),p(CBA-APP)and p(CBA-DAH)-g-PVP were successfully synthesized and characterized.p(CBA-DAH),p(CBA-APP),p(CBA-DAH)-g-PVP were formed with DNA,and the DNA was compressed completely when the polymers/DNA weight ratio was 3:1,2:1and 3:1,and the DNA was released by glutathione and sodium dodecyl sulfonate.The introduction of pyrrolidone group in the main chain and side chain structure greatly reduced the toxicity of the polymer to the cells,and the toxicity of the polymer to the cells was in the order of p(CBA-APP)<p(CBA-DAH)-g-PVP<p(CBA-DAH)<PEI 25 k Da,and all polymers could not induce apoptosis of Bel-7402 cells.Both p(CBA-APP)and p(CBA-DAH)-g-PVP can transfer DNA to Bel-7402,He La,Hep G2 and NIH3T3cells.The transfection efficiency of p(CBA-DAH)-g-PVP in Bel-7402 cells was 36.63%when p(CBA-DAH)-g-PVP/p DNA weight ratio was 9:1,and there was no significant difference between the transfection efficiency of PEI25 k Da(37.91%)when PEI 25 k Da/DNA weight ratio was 1:1.And then,p(CBA-DAH),p(CBA-APP),and p(CBA-DAH)-g-PVP can transfer target DNA to Bel-7402 cells through clathrin-dependent endocytosis pathway,and then escape into the nucleus through lysosomes in the cells.Conclusion:p(CBA-DAH),p(CBA-DAH)-g-PVP and p(CBA-APP)have good physical and chemical properties and biological properties.Compared with p(CBA-DAH),p(CBA-DAH)-g-PVP and p(CBA-APP)showed better DNA compression ability and lower cytotoxicity.In particular,p(CBA-DAH)-g-PVP was introduced into the side chain with pyrrolidone group.Although the introduction of PVP reduced the DNA transfection ability of p(CBA-DAH),p(CBA-DAH)-g-PVP/DNA complex with the optimal weight ratio(9:1)showed similar DNA transfection ability to that of PEI 25k Da in Bel-7402 cells line.In conclusion,p(CBA-DAH)-g-PVP can be used as a good gene carrier for further study.