Synthesis And Investigation Of Bioreducible Polymers For Efficient Gene Delivery

Gene therapy is concerned with the transfer of complement genes to functionally defective cells for the treatment of most challenging diseases in a safe and directed manner.The overall success of gene therapy is associated with the development of an efficient gene carrying system,which is used to introduce a therapeutic gene into target cells or tissue.However,the development of an efficient and safe gene carrier has been a major challenge in the clinical application of both viral and non-viral gene therapy.Recently,synthetic non-viral vectors have been extensively investigated for gene delivery owing to their promising applications in gene therapy and considered a suitable alternative to viral gene carriers because of several advantageous properties.Even though synthetic gene carriers have produced modest gene expression.However,the clinical applications of synthetic gene carriers have been severely hindered by different challenges like high cytotoxicity,low transfection efficiency,low cell recognition ability,and cellular uptake.These challenges must be overcome to ensure the successful gene delivery into the target cell and achieve good gene expression.Among the non-viral gene carrier's cationic polymers based gene carriers is the center of interest due to facile synthesis,diversity in chemical structure and ease in structural modifications.Polyethyleneimines(PEI)is the most efficient gene carrier because of good p DNA condensation,good cellular uptake and successful endosomal escape of the p DNA.All these properties are advantageous for efficient gene delivery.However,the non-biodegradable nature of PEI caused cell death due to their high cytotoxicity which discourages the clinical applications of PEI.The most efficient and safe gene delivery appeal the development of biodegradable and biocompatible synthetic polymers based gene carriers to maintain high cell viability and achieve good transfection efficiency.Recently the smart polymers which can respond to some physical or biological changes occurring in the biological environment are appealing the interest of researcher for the development of smart gene carriers.Therefore,the difference in physiological redox environment inside and outside of the cell can be utilized for the synthesis of redox-responsive cationic polymers.The disulfide bond is highly dynamic in nature which selectively responds to the reducing environment of the cytoplasm due to the presence of reducing agent glutathione(GSH).The disulfide bonds containing polymers are enough smart to discriminate the redox gradient while moving from an extracellular environment to the intracellular environment of the cell.The reducible polymers would form stable polyplexes,which would have long circulation time during blood circulation.Moreover,the presence of disulfide linkages in the reducible polymers formed smart polyplexes will allow the on-demand release of p DNA in the response to reducing environment of cytoplasm.To accomplish these goals herein,we reported the synthesis of a bioreducible poly(ethyleneglycol)-b-poly(disulfide-L-lysine)cationic polymer(denoted as PEG-SSL).The polymer consisted of PEG block which improved biocompatibility,enhances circulation time by providing colloidal stability to polymer/p DNA complexes.PEG would also improve cellular uptake of the polymer/p DNA complexes via enhanced permeability and retention effect.Poly(disulfide-L-lysine)consisted of repeating units of L-lysine which are bonded together via disulfide bond containing moiety cystamine bisacrylamide.This exhibited dual function,which not only condensed p DNA due to cationic charge of L-lysine but would also allow trigger p DNA release due to cleavage of disulfide bonds in the reducing environment of the cytoplasm.PEG-b-poly(disulfide-L-lysine)polymer was successfully synthesized via Michael addition between acryloyl functionalized PEG[PEG(Ac)₄]and the terminal amine group of poly(disulfide-L-lysine).The molecular weight of the PEG-SSL polymer was found to be 27.8 k Da.PEG-SSL efficiently condensed plasmid ZNF580 gene(p ZNF580)and formed nano-sized polyplexes with positive zeta potential.The particles size was found in the range of 155±4 to 285±3 nm with a surface charge from 1.9±0.1 to 26.7±0.4 m V at polymer/DNA weight ratios of 5/1 to 80/1.Gel-electrophoresis result demonstrated that PEG-SSL successfully retarded p ZNF580 at smaller polymer/DNA weight ratio of 10/1 and higher.The stability of polymer/DNA complexes was confirmed by incubating polymer/DNA complexes in 20?M of DTT solution.These results demonstrated that no significant effect was observed on p DNA release,particularly at a higher weight ratio of PEG-SSL in polymer/DNA complexes.However,when polymer/DNA complexes were incubated in 5?M of DTT solution and then electrophoresed.The trigger release of p ZNF580 DNA was confirmed.The results exhibited that PEG-SSL unable to retard p DNA even at higher polymer/DNA weight ratios in the presence of 5 m M DTT.These result demonstrated that PEG-SSL polymer would effectively hold p DNA in the extracellular environment,but efficiently release p DNA due to the cleavage of the disulfides bonds in the polymer when exposed to the reducing environment of the cytoplasm.Similarly,the colloidal stability of the PEG-SSL polyplexes was investigated by incubating complexes at different weight ratios of 30/1,50/1 and 80/1 in phosphate(20 m M,p H 7.4)buffer with 150 m M Na Cl solution at 37°C and the particle size was measured after different time intervals(0,4,12,24 hr).The result demonstrated that the increase in particle size was negligible after 4 and 12 hr incubation,but a little increased in particle size was observed after 24 hr incubation.These results demonstrated that PEG-SSL/p DNA complexes are stable and would not aggregate to formed large particles.The DNase stability of was investigated by incubating naked p ZNF580 and PEG-SSL/p ZNF580 complexes at different weight ratios(10/1 and 20/1)for different times(0,20,40,60 and 120 min)in a buffer solution containing DNase I.The results exhibited that PEG-SSL/p ZNF580 complexes exhibited good stability against DNase I and efficiently protected p DNA from degradation of DNase.In vitro transfection and cytotoxicity were investigated in EA.hy926 cells.The results showed that p ZNF580 was successfully delivered into the cells with less cytotoxicity compared to PEI25k Da.The transfection efficiency increased with increasing the weight ratio of PEG-SSL/p ZNF580 from 20/1 to 60/1 and then declined.Similarly,the cytotoxicity was investigated in EA.hy926 cell,the result exhibited that both PEG-SSL polymer and their polyplexes exhibited high cell viability than PEI25k DA.The cellular uptake of PEG-SSL complexes was measured with the help of flow cytometry.The results indicated that PEG-SSL/Cy5-oligonucleotide complexes exhibited good cellular uptake.Confocal laser scanning microscopy was used to investigate the subcellular localization of polyplexes.results indicated that naked p DNA could hardly be internalized into the cells.However,PEG-SSL polyplexes exhibited not only good cellular uptake but also efficiently escape from the endosomes and accumulated in the perinuclear region.In the second part,we have reported the synthesis of reducible disulfide conjugated low molecular weight polyethyleneimines(denoted as SS-PEI 1.8 k Da).SS-PEI were synthesized via poly-condensation reaction and evaluated as a non-viral gene carrier.The reducible conjugated PEI consisted of LWM-PEI conjugated via disulfide bond contained cross-linker.The polycondensation reaction was carried out by treating LMW-PEI(1.8k Da)with bis(p-nitrophenyl)-3,3-dithiodipropionate.Physio-chemical characteristics of polymer/DNA complexes were analyzed by gel electrophoresis,particle size,and zeta-potential measurements.It was concluded that disulfide conjugated PEI was able to efficiently condense p DNA into nano-sized particles.These nano-sized polymer/DNA complexes were in size range from 170±1.5 to 255±1.6 nm and positive zeta potential of 3±0.4 to 17±0.9 m V.The SS-PEI polymer exhibited comparable buffer capacity to PEI25k DA.A good buffer capacity would ensure the protection of p DNA from DNase degradation.Gel electrophoresis results indicated that it successfully retarded p ZNF580 at small polymer/DNA complexes at a weight ratio of 1/1 and higher.This exhibited good electrostatic interaction between the reducible conjugated PEI and p DNA.The p DNA released in the reducing environment of cytoplasm was confirmed by treating polymer/DNA complexes with DTT.The trigger released of the p DNA was observed when complexes were exposed to 5 m M DTT solution,which provides reducing environment mimetic to the intracellular environment.SS-PEI polymer also exhibited good buffer capacity and effectively protected p ZNF580 from DNase I degradation.The transfection efficiency was investigated in EA.hy926 cells.The results demonstrated that SS-PEI exhibited comparable transfection efficiency to PEI25k Da.Moreover,MTT results demonstrated that SS-PEI exhibited low cytotoxicity than PEI25k DA.Confocal laser scanning microscopy was used to confirm the location of SS-PEI/p DNA complexes composed of Cy5-labeled p DNA.The result indicated that SS-PEI polymer efficiently delivered p DNA into the cells,accumulated around the nucleus and finally internalized into the nucleus of the cell.These results indicated that both PEG-SSL and SS-PEI efficiently delivered p DNA into EA.hy926 cells,while maintaining high cell viability.In conclusion,these results suggest that these bioreducible polymers are efficient,biocompatible polymers,which may serve as a promising candidate for gene delivery.