| Poly(β-amino ester)s(PβAEs)contains tertiary amine backbones and biodegradable ester bonds,making them highly biocompatible,biodegradable and p H-responsive.Meanwhile,PβAEs are simple to synthesize,easy to obtain raw materials and can be easily adapted to meet the different performance needs of gene carriers by adjusting the monomer type,monomer ratio and reaction time.Therefore,PβAEs are promising material for non-viral gene carriers.PβAEs is usually synthesized by Michael addition reaction,using acrylates as branching unit,diacrylate as basic unit,primary alcohol as side chains and end sealers diamine as monomers.Objective:This study synthesized four PβAEs by adopting trimethylolpropanetriacrylate(TMPTA)and2-methyl-1,5-Diaminopentane(MPMD)as branched units,reacting with base monomer 1,4-butanediol diacrylate(C4)and side chain monomer5-amino-1-pentanol(S5),capping with Spermine(SPE)or MPMD.The physicochemical and biological properties of PβAEs with different branched monomers and end capping agents were investigated,and the optimal weight ratio(Weight ratio,w/w)of polymer/pDNA with good transfection performance was screened.Methods:(1)Polymer MPMD-SPE,MPMD-MPMD,TMPTA-SPE and TMPTA-MPMD were synthesized by Michael addition reaction.And their structural characterizations were determined Fourier Transform Infrared Spectrometer(FTIR)and Proton Nuclear Magnetic Resonance(~1H-NMR).(2)Gel electrophoresis was used to investigate the ability of four polymers to compress and dissociate pDNA.Particle size analyzer was used to investigate the particle size of the polymer/pDNA complexes.And p H meter was used to investigate the buffering ability of each polymer.(3)MTT assay was used to investigate the cytotoxicity of different concentrations of polymers and complexes with different polymer/pDNA weight ratios in NIH 3T3,Hela,Hep G2 and Bel-7402 cells.(4)The transfection ability of each polymer at different polymer/pDNA weight ratios in NIH 3T3,Hela,Hep G2 and BEL-7402 cells was investigated by inverted fluorescence microscopy and flow cytometry.Results:(1)Polymer of MPMD-SPE,MPMD-MPMD,TMPTA-SPE and TMPTA-MPMD were successfully synthesized,and their structure characterizations were basically determined by FTIR and ~1H-NMR spectrum.(2)The polymer of MPMD-SPE,MPMD-MPMD,TMPTA-SPE and TMPTA-MPMD can completely compress pDNA at the weight ratio of 0.5:1,0.7:1,0.5:1 and 1:1,respectively.At the weight ratio of 1:1-100:1,sodium dodecyl sulfonate successfully dissociated pDNA from polymer/pDNA complexes.Four PβAEs can control the particle size of the polymer/pDNA complexes at<200 nm at the appropriate weight ratio range.Titration curves showed that the polymers of MPMD-SPE,MPMD-MPMD,TMPTA-SPE and TMPTA-MPMD had certain proton buffering capacity.(3)Four PβAEs all had good biocompatibility.The cytotoxicity of four polymers in NIH 3T3,Hep G2 and Bel-7402 cells were much lower than that of PEI 25k,and were similar with that of PEI 25k in Hela cells.The complexes by four PβAEs and pDNA still showed 50%cell viability at weight ratio=15:1.(4)The complexes of MPMD-SPE,MPMD-MPMD,TMPTA-SPE/pDNA had good transfection ability in NIH3T3 and Bel-7402 cells,and strong green fluorescence could be observed.The transfection efficiency of MPMD-SPE/pDNA,MPMD-MPMD/pDNA and TMPTA-SPE/pDNA with the optimal w/w=12:1 in NIH 3T3 cells was 43.27%,19.36%and 40.60%,respectively,all of that had significant difference with PEI 25k at w/w=1:1 which was 9.96%.The transfection efficiency of MPMD-SPE/pDNA,MPMD-MPMD/pDNA and TMPTA-SPE/pDNA with the optimal respectively w/w at 11:1,9:1,9:1 in Bel-7402 cells was 40.15%,25.97%and 13.13%,respectively.The transfection efficiency of the former complexes was significantly different from PEI 25k at w/w=1:1 with 11.28%.Among the four polymers,MPMD-SPE had the best transfection performance,and had good transfection ability in NIH 3T3 and Bel-7402 cells.MPMD-MPMD and TMPTA-SPE showed selectivity to Bel-7402 cells and NIH 3T3 cells,respectively,and the transfection effect of the two polymers was relatively superior in the corresponding cells.Conclusion:The polymer of MPMD-SPE,MPMD-MPMD,TMPTA-SPE and TMPTA-MPMD all have good physicochemical properties,all of which have good pDNA compression ability and low cytotoxicity.Among them,the polymer of MPMD-SPE,MPMD-MPMD and TMPTA-SPE all had certain transfection ability,and the transfection efficiency with the optimal w/w and cell type was significantly better than that of positive control PEI 25k.In summary,the polymer of MPMD-SPE,MPMD-MPMD and TMPTA-SPE is a potential non-viral gene vector material. |
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