Construction And Transfection Of Low Molecular Weight PEI-Grafted Soybean Protein As Gene Carriers

A growing number of studies have found that abnormal and defected gene can lead to a series of diseases,icluding cancer,cardiovascular disease,genetic disease,endangering the life and health of human beings.We further found that gene therapy was concerned as a new type of treatment.The molecular means for gene therapy can correct abnormal gene are safe,accurate and effective.Gene carrier plays a vital role that how to accurately carry the purpose gene to the target cells.Moreover to construct safe and effective gene carriers are also currrent research key and difficulties points in the research.Polyethyleneimine(PEI)is applied as one of the most used polycation,which is attributable to its high charge density after the protonation and good proton buffering capacity.Branched(25 KDa)has become a benchmark for its strong DNA condensation ability and high gene transfection efficiency.On the one hand,linear PEI with low molecular weight(LMW,MW<2 KDa)has low cytotoxicity but shows poor transfection efficiency.On the other hand,high molecular weight of PEI has high transfection efficiency but displays high cytotoxicity and nonbiodegradability.It is a major consideration for the design of new gene vectors with enhancing transfection efficiency and minimizing the toxicity.To circumvent dilemma of PEI one rational strategy has been propsed to combine low molecular weight PEIs with degradable linkage to achieve the high transfection with low toxicity.Various types of copolymers have been designed by grafting LMW PEIs with natural products.A type of new gene delivery vectors(SP-PEI600,SP-PEI1200,SP-PEI1800)have been prepared by acylation reaction between soybean protein(SP)and low molecular weight PEI(LMW PEI).This design has not been reported elsewhere yet.To confirm the design,the first structural properties of SP-PEIs polymers were analyzed.The target soybean protein was acquired by the centrifugation and dialyzing of enzymatic hydrolysis of SP(15-17 KDa).The FTIR spectrum and Zeta potential experiments prove that the PEIs are grafted onto the SP.the grafting efficiency of PEI in SPI-PEI600,SPI-PEI1200,SPI-PEI1800 is 11.3%,18.0%,19.3%examined by elemental analysis,respectively.The grafting efficiency of PEI in SPI-PEI6C0,SPI-PEI1200,and SPI-PEI1800 is 12%,20%,22%examined by gravimetric method,respectively.The buffer capabilities of SP-PEIs are lower than that of PEI 25 KDa whereas the buffer capabilities of SP-PEI1800 are higher than those other SP-PEIs.Protein BSA adsorption assay was used to study serum-tolerant capacity of SP-PEIs.The SP-PEIs/DNA polyplexesshow appeared significant superiority in suppressing protein adsorption,compared to branched PEI(25 KDa).SPI-PEIs polyplexes showed strong serum-tolerant capacity,which is favorable to deliver plasmid DNA into cells.The proper size and zeta-potential of polymers/DNA polyplexes could effectively improve the stability of polyplexes and their ability of enter into cells,therefore,increasing transfection.At pH 7.4,the diameter of SP-PEIs/DNA polyplexesin aqueous buffer is about 150 nm at N:P(w/w)rations ranging from 1/8 to 24 by dynamic light scattering.With increased weight ratio of SP-PEIs/DNA complexes,average particle size is stabilized around 150 nm,and gradually decreases.In addition,the zeta-potential of SP-PEIs/DNA polyplexeswas detected as one of the requirements for ideal polymeric vectors.The zeta-potential of SP-PEIs/DNA polyplexeswere given as an increasing trend along with the increasing weight ratio of N:P.The size and count rate of SP-PEIs/DNA polyplexes are nearly unchanged at pH 7.4.However,the SP-PEIs/DNA polyplexes display an acid-triggered decomposition and significant increase in particle size under the mild acid conditions.In this study,the DNA condensation ability of SP-PEIs polymers were evaluated by agarose gel retardation and DNase I degradation experiments.SP-PEI1800 displays a slightly stronger DNA condense ability when compared to the other SP-PEIs,which might be attributed to the higher mount of amine groups.The DNase I degradation and heparin displacement experiments further confirm that SP-PEI1800 can condense DNA more effectively than SP-PEI1200 and SP-PEI600.At the cellular level,low cytotoxicity is known as the premise of a candidate polycationic gene vectors for effectively gene transfection.The cytotoxicity of SP-PEIs at various concentrations was evaluated on 293T cells and SH-SY5Y cells by using MTT assay.Compared to branched PEI(25 KDa),the cell viability of SP-PEIs were distinctly higher at all concentrations on 293T cells and SH-SY5Y cells.Especially,more than 70%cell viability was obtained in 293T cells and SH-SY5Y cells at concentration ranging from 1 to 50 mg/mL of SP-PEI1800.In summary,all SP-PEIs display low toxicity and biocompatibility.It is an important standard for gene transfection experiment to select suitable carrier concentration by MTT.In vitro transfection efficiency of SP-PEIs/DNA polyplexes in vitro in 293T and SH-SY5Y cells was assessed by the flow cytometry and fluorescence microscope.Flow cytometry showed that the SP-PEIs/DNA polyplexes have better transfection efficiencies in the absence of serum and in the presence of 10%serum.For SH-SY5Y cell,the transfection efficiency of SP-PEI1800/DNA polyplexes at weight ratio of 24(1.33%)is about 3.4 folds higher than that of PEI 25 KDa(0.396%)in the presence of 10%serum.Likewise,the transfection efficiency of SP-PEI1800/DNA polyplexes at weight ratio of 24(5.27%)is about 2.7 folds higher than that of 25KDa PEI(1.94%)in the absence of serum.As for 293T cell,the SP-PEI1800/DNA polyplexes perform the highest transfection efficiency of 15.1%(w/w=24),which is closed to that of the branced PEI(w/w=1.4,16.6%)in absence of serum.In addition,SP-PEI1800/DNA polyplexes display the highest transfection efficiency(8.19%,w/w = 24),which give slightly higher than that of the branced PEI(w/w=1.4,7.22%).In conclusion,SP-PEIs polymers(SP-PEI600,SP-PEI1200,SP-PEI1800)were successfully preparated by the acylation reaction between SP and different low molecular weight PEI.SP-PEIs can not only reduce cytotoxicity as gene carriers but also reduce the tolerance on serum,and improve the transfection effiency.These biomaterials may become useful gene carrier in vivo by further optimization.