Synthesis And Characterization Of Bioreducible Cationic Polymers For Efficient Gene Delivery

Gene therapy delivers an auspicious pattern for the handling of numerous acquired or inherited diseases,such as cystic fibrosis,severe combined immunodeficiency,cancer,diabetes and transmittable diseases.In gene delivery techniques,genes are shifted into definite human tissues or cells to change defective genes,substitute missing genes,silence undesirable gene expression or introduce new cellular biofunctions.In gene delivery process,genetic materials initially must be shifted to target tissue cells securely at a good level of efficacy,and then perform their therapeutic role.The naked DNA can be degraded easily by different enzymes in the blood and in cellular compartments i.e.,endosomes and lysosomes within very short time.For the purpose to develop efficient gene delivery,DNA is generally condensed by a gene vector to form nanoparticles and then transported to the target tissue cells.Glutathione(GSH)is low molecular weight compound produced in the cytosol of the cell.The GSH levels in intracellular environment are 2 to 3 orders higher than those of the GSH levels in extracellular environment.Therefore,disulfide bonds containing polymers have been commonly used for reductive design in gene delivery.The disulfide bonds show higher stability in extracellular environment while presented quick degradation in the intracellular reducing fluids,could smartly resolve the inconsistent needs for an efficient non-viral gene transfer vectors,i.e.,admirable binding ability and safety of nucleic acids in extracellular fluids and efficient release of nucleic acids inside the cells.Encouraged by the success of disulfide bond,four types of gene delivery systems have been synthesized,i.e,CBA-AEP-His,PEI-SS-PEG-SS-PEI,m PEG-PLGA-SS-PEI and p(CBA-CDH).CBA-AEP-His copolymer possesses disulfide linkages,which enable it with redox-responsivity to the intracellular environment.This polymer efficiently condenses p ZNF580 into complexes with the size of 160?±?4?nm to 280?±?5?nm and positive zeta potential of 20?±?0.3 m V to 30?±?0.4 m V.CBA-His-AEP had good buffering ability and competed with the buffering capacity of PEI 25 k Da.The gel electrophoresis experiment was presented that CBA-AEP-His was able to completely load p ZNF580 at the w/w ratio of 1/1 and higher.The gene transfection efficiency of CBA-AEPHis/p ZNF580 complexes at mass ratio from 40/1 to 100/1 was determined in EA.hy926 cells.It was noted that CBA-AEP-His polymer can efficiently transfect p ZNF580 into EA.hy926 cells at high mass ratio(≥ 60/1).The MTT assay indicates that CBA-AEP-His and its complexes exhibit lower cytotoxicity than PEI 25 k Da.The wound healing assay was performed to evaluate the migration of the transfected cells.The results suggested that the migration of transfected cells by CBA-AEP-His/p ZNF580 complexes was higher than that of PEI 25 k Da/p DNA complexes.In the second part,the synthesis of disulfide linked block copolymer(PEI-SS-PEG-SS-PEI)have been reported.The experimental results indicated that the buffer capacity of PEI-SS-PEGSS-PEI was higher than the PEI 1.8 k Da but lower than the PEI 10 k Da.The size and zeta potential of PEI-SS-PEG-SS-PEI/p ZNF580 polyplexes were determined at different mass ratios of 10/1,20/1,30/1,40/1,50/1,60/1,70/1,80/1,90/1 and 100/1 by zeta sizer.The particle size of polyplexes decreases from 370 ± 4 nm to 240 ± 3 nm when the mass ratio increases from 10/1 to 100/1.Similarly,the zeta potential of polyplexes increases from 8 mV to +12 mV.The biological experiments were shown that PEI-SS-PEG-SS-PEI have good cellular uptake,high transfection efficiency and low cytotoxicity as compared to PEI 10 k Da.In the third part,m PEG-PLGA-SSPEI have been synthesized as an intracellular reductive polymer for gene delivery.These polymers show good buffering ability.Their polyplexes have nano sized with positive zeta potential.The biological assays of m PEG-PLGA-SS-PEI were performed and show that these polymers having high transfection efficiency with low cytotoxicity as compared to PEI 10 k Da.In the final part,intracellular reductive low molecular weight cationic polymers p(CBA-CDH)was synthesized.All p(CBA-CDH)polyplexes having nano sized with positive zeta potential.The biological assay indicated that p(CBA-CDH)has good transfection with low cytotoxicity as compared to PEI 600 Da.In conclusion,CBA-AEP-His,PEI-SS-PEG-SS-PEI,mPEG-PLGA-SS-PEI and p(CBACDH)possess high advantages over the hydrolytically degradable polymers because their polyplexes are unstable for long circulation of time and escape their DNA cargo before cell internalization.This issue may be avoided by using these synthesized polymers,i.e.,CBA-AEPHis,PEI-SS-PEG-SS-PEI,m PEG-PLGA-SS-PEI and p(CBA-CDH)that possess biodegradability properties due to the presence of bioreducible disulfide linkages in their chain.these experimental results proposed that these biocompatible and bioreducible polymers are very efficient,which may assist as a strong candidate for gene delivery.