Study On The Delivery Mechanism Of Two Bioresponsive Polymer Gene Vectors (Agmatine/Arginine With N,N-Bis-Acryloyl Cystamine)

Objective: Poly(amido amine)s with multiple disulfide linkages(SS-PAAs)have been widely used as a novel vector of gene therapy,but there are still some problems.Therefore,in order to explore its transfection efficiency,cytotoxicity and other vector performance,we introduced guanidine group on these compounds and then prepared the guanidinylated Poly(amido amine)s with multiple disulfide linkages(Gua-SS-PAAs)vectors.It set a foundation for the research and application of this kind of Gua-SS-PAAs vectors.Methods: In order to evaluate the performance of the constructed polymer vectors as the vector of gene therapy route of DNA delivery,the prepared Gua-SS-PAAs(agmatine with N,N-bis-acryloyl cystamine(AGM-CBA),arginine with N,N-bis-acryloyl cystamine(ARG-CBA))polymer vectors encapsulated enhanced green fluorescent protein plasmid(pIRES2-EGFP).And Lipofectamine 2000 and Polyethyleneimine(PEI)and were selected as positive control vectors,which was the classical cationic liposome and cationic polymer,respectively.Firstly,the ability of plasmid encapsulation and release in polymer vectors was studied.And dynamic light scattering(DLS)was used to study the particle size,Zeta potential of the gene carrier complex.And then in SK-OV-3 cells,MTT,fluorescence microscope(FM),FACScan flow cytometer(FCM)and other methods were used to study the cell viability and transfection efficiency of the gene carrier complex in vitro.Then laser scanning confocal microscopy(LSCM)was used to study some characteristics of pDNA/Gua-SS-PAAs,such as the cell uptake process,endosome escape process and nuclear localization effect.FACScan flow cytometer was used to study the transport mechanism of gene carrier complex in vitro.And lactate dehydrogenase(LDH)release assay with cell membrane integrity assay were used to indirectly and directly evaluate the cell membrane integrity and explore the transport mechanism of gene carrier complex.Results: 1.Compared with the positive control vectors(PEI,Lipo),two kinds of polymer vectors(pDNA/AGM-CBA,pDNA/ARG-CBA)showed good abilities in encapsulating pDNA,and release pDNA rapidly in the intracellular reduction environment.2.The particle size of two kinds of polymer carriers(AGM-CBA,ARG-CBA)encapsulated pDNA was less than 70 nm.The results of Zeta potential showed that the polymers had similar Zeta potential with the positive control PEI in a certain condition of N/P(pDNA/polymer)ratio.3.The cell viability experiments of two kinds of polymer vectors(pDNA/AGM-CBA,pDNA/ARG-CBA)showed that,compared with the positive control vectors,they showed controllable cytotoxicity in a certain range of N/P ratio.Under the optimal N/P ratio,the cell viability of two vectors was higher than that of the PEI,Lipo.4.In vitro transfection efficiency of two kinds of polymer vectors showed that,compared with positive control vectors,the transfection efficiency of the vectors increased with the decrease of N/P ratio.Under the optimal N/P ratio,the transfection efficiency of two vectors was significantly higher than that of positive control vectors,reaching a higher level(more than 65%).After comprehensive analysis,the optimal N/P ratio was 1/48.5.The results of cell uptake experiments showed that the speed and quantity of polymers(pDNA/AGM-CBA,pDNA/ARG-CBA)into the cells were significantly higher than those of the two positive control vectors.6.Moreover,the transmembrane transport of Gua-SS-PAAs polymer vectors showed a unique mechanism,pDNA/AGM-CBA entered cells partly through clathrin-mediated endocytosis.pDNA/ARG-CBA entered cells partly through pathway of megacytosis.And the two complexes were more likely to enter the cell through other non-classical routes.7.The results of endosomal escape experiment showed that there was an obvious endosomal escape process in pDNA/AGM-CBA and pDNA/ARG-CBA.And this process was faster than that of Lipo and PEI,and their escape ability was stronger.8.The results of nuclear localization experiments showed that the cells were transfected with pDNA/AGM-CBA and pDNA/ARG-CBA for 4 h,and then pDNA were concentrated in the nucleolar region.9.LDH release test and cell membrane integrity test showed that pDNA/Gua-SS-PAAs had a certain damage to cell membrane,which was higher than that of Lipo,but lower than that of PEI.Conclusion: Gua-SS-PAAs(AGM-CBA,ARG-CBA)polymer vectors could effectively deliver pDNA(pIRES2-EGFP)at the optimal N/P ratio condition(1/48)in SK-OV-3 cells,and its cytotoxicity was still in a controllable range.They could effectively encapsulate negatively charged exogenous genes and other materials through electrostatic interaction,so that the materials could cross the cell membrane,escape from the endosome and concentrate in the nucleolar region.The guanidine structure could produce the nuclear localization effect of the peptidomimetics.The two polymeric vectors(AGM-CBA,ARGCBA)will have potential prospects in clinical applications.