Cationic Modification Of Bombyx Mori Silk Fibroin As A Gene Delivery Carrier Of ING4-IL-24

Lung cancer is one of the most harmful forms of cancer which threatens the health of human beings.Gene therapy represents a new and promising therapeutic modality for various types of cancer.Inhibitor of growth 4（ING4）and interleukin-24（IL-24）are the most ideal cytokines in gene therapy,which can specifically recognize various types of tumor cells and induce to apoptosis.However,their half-lives in vivo are short and easy to be inactivated or degraded.A suitable gene delivery can effectively pack and condense plasmid DNA（p DNA）containing cytokine genes,mediating p DNA transfection in situ and expression to stimulate the inhibition of cell growth and the formation of vasculature network,inducing tumor cell apoptosis.Bombyx mori silk fibroin has good biocompatibility,controllable biodegradation,contains a mass of polar amino acids with ionized side chains,making silk fibroin more chemical reaction sites.Surface charge of silk fibroin after chemical modification can turn negative into positive,and silk fibroin may be a gene delivery carrier for lung cancer therapy.In this paper,silk fibroin was modified by low weight polyethyleneimine（PEI）,making surface charge of silk fibroin turn negative into positive;then used the cationic silk fibroin（PSF）packed and condensed p DNA containing ING4 and IL-24 dual gene simultaneous expression plasmid to form positively charged PSF/p DNA complexes at various w/w ratios which protected p DNA from enzymatic degradation,creating a new ING4-IL-24 coexpression gene delivery carrier.In in vitro experiment,the cationic silk fibroin/p DNA complexes transfected A549 cells and WI-38 cells to study the influences of the complexes at various w/w ratios on gene transfection efficiency and cell viability,respectively.First,Bombyx mori silk fibroin was modified by 1800 Da PEI.EDC/NHS activated the hydroxyl of silk fibroin side chain,and positively charged PEI covalently reacted with the side chain of silk fibroin.The effects of the various weight ratios of PEI to modify silk fibroin were studied by measuring the zeta potential,isoelectric point（p I）,XPS and FTIR.The results show that the zeta potential of cational modified silk fibroin from-11.8 m V to+10～+12 m V,the isoelectric point increased from 3.68 to 8.82;the primary amino contents increased from（12.5±0.1）×10^-2 mmol/m L to（15.3±0.2）×10^-2 mmol/m L,N element content increased from 13.4%to 18.47%,and O element content from 27.47%to 18.93%.These results showed polyethyleneimine could effectively react with silk fibroin.Secondly,the PSF was packaged p DNA through electrostatic interaction and prepared PSF/p DNA with different mass ratio.Then particle size,zeta potential,surface morphology and physical properties of the complexes were measured by Malvern zerasizer nano ZS90and SEM.The results show that the zeta potential values of PSF/p DNA complexes increased from-8.2 m V to+11.3 m V,the particle sizes decreased from 260 nm to 122 nm.The results of SEM and agarose gel electrophoresis showed that with the increase of the PSF mass,the ability of silk fibroin coating p DNA was enhanced,and the p DNA could be completely encapsulated.Finally,the effects of PSF/p DNA complex on the apoptosis of A549 and the cytotoxicity of WI-38 cells were studied.Using laser scanning confocal microscope and fluorescence microscope to observe cell transfection efficiency and cell morphology,using CCK-8 reagent to observe cell viability and the flow cytometry was used to detect the transfection efficiency.The results showed that compared with PEI/p DNA complex,the ability of apoptosis of PSF/p DNA complexes was significantly enhanced,and WI-38 cells was no obvious apoptosis.The transfection efficiency of 128μg/2μg和196μg/2μg were 58%and 47.5%,respectively.These results indicated the cationic silk fibroin could be a gene delivery carrier of ING4-IL-24 to induce apoptosis in lung cancer cells.In this paper,Bombyx mori silk fibroin was modified by low weight PEI to obtain the cationic silk fibroin for the first time,and cationic silk fibroin was used as ING4-IL-24gene delivery carrier can induce of A549 cells apoptosis effectively and had no obvious side effect on WI-38 cells.This may provide an new gene delivery carrier for lung cancer targeted gene therapy.