Lung Cancer Targeting Silk Fibroin/ING4-IL-24 Dual-gene Co-expression Adenovirus Complex

Lung cancer is one of the most serious diseases threatening human health.Gene therapy for malignant tumors has attracted much attention due to its advantages of strong pertinence,low toxicity and good curative effect.Inhibitor of growth 4(ING4)and interleukin-24(IL-24)genes can inhibit tumor cell proliferation and induce tumor cell apoptosis from both intracellular and extracellular pathways,respectively.As a gene vector,adenovirus can efficiently deliver and express target genes,but its clinical widely application is limited due to its deficiency in immunogenic risk,cytotoxicity at high titer and orientation to cells with innate receptors.Antheraea pernyi silk fibroin has excellent biocompatibility and low immunogenicity,and it contains a large number of polar amino acid residues,which endue it rich chemical reactivity and modification sites.To further improve the efficiency of adenovirus infection and reduce its effective use titer to reduce the side effects,and to protect adenovirus from the neutralization of adenovirus antiserum,this study developed a novel cationic Antheraea pernyi silk fibroin/ING4-IL-24 dual-gene co-expression adenovirus complex targeting lung cancer.By in vitro studies and animal experiments,the targeted infection,growth inhibition and apoptosis promotion effects of this complex on lung cancer cells were studied,as well as its toxic and side effects on normal tissue cells,to effectively inhibit lung cancer with low titer adenovirus and avoid the potential toxic and side effects of high titer adenovirus.In order to realize this goal,the low-molecular-weight polyethylamine was used to modify Antheraea pernyi silk fibroin,and the specific oligopeptide C9(CSNIDARAC)in lung cancer cells was grafted onto the side chains of cationic silk fibroin.The targeted and cationic Antheraea pernyi silk fibroin was coated on the surface of ING4-IL-24 dual-gene co-expressed adenovirus to obtain the dual-gene co-expression complex.The targeting effect,infection efficiency,inhibition of proliferation and induction of apoptosis of the complex on human large cell lung cancer cells H460 were studied in vitro,and the growth inhibition and tumor inhibition mechanism of the complex on transplanted tumor of H460 lung cancer in mice were investigated in vivo.Meanwhile,in vitro and in vivo experiments were conducted to analyze and evaluate the toxic and side effects of the complex on normal human umbilical vein endothelial cells HUVEC and normal tissue.Firstly,the ING4-IL-24 co-expression adenovirus vector was constructed.After homologous recombination of ING4-IL-24 co-expression transfer plasmid and adenovirus skeleton plasmid,a double-gene co-expression adenovirus vector(Ad)was obtained through multiple rounds of infection and amplification in human embryonic kidney cells QBI-293A.The results of Western blotting and ELISA showed that Ad could mediate the expression of exogenous ING4 and IL-24 genes in human embryonic kidney cells QBI-293 A cells.Secondly,under the mediation of carbodiimide,the amino groups of low-molecular-weight polyethylenimine coupled with the carboxyl groups of Antheraea pernyi silk fibroin(ASF)to form amide bonds,thus the cationic ASF(CASF)was obtained.When PEI accounted for 4%of ASF,the surface Zeta potential of the modified silk fibroin reached+12.03 mV,and the graft rate was about 86.34%.Then,with N-succinimidyl-3-(2-pyridyldithiol)propionate as crosslinking agent,the lung cancer cells targeting peptide C9 was grafted to the side chain of CASF in the form of disulfide bond,and the lung cancer cells targeting Antheraea pernyi silk fibroin(CASF-C9)was obtained.The experimental results showed that the surface Zeta potential of CASF-C9 decreased compared with CASF.To initially evaluate the ability of CASF-C9 to carry genes and its biological effects,CASF-C9 were used to package plasmid DNA(pDNA)to prepare CASF-C9/pDNA complexes of different mass ratios.The experimental results indicating when the mass ratio of material to pDNA was 64/2,128/2 and 256/2,plasmid DNA could be completely enveloped.The green fluorescence expression quantity and transfection efficiency after transfection of lung cancer cells H460 by CASF-C9/pDNA complex were significantly higher than that of CASF/pDNA complex in the control group,and the higher the quality of grafted peptide C9,the higher the fluorescence expression quantity and transfection efficiency of cells,and the greater the inhibitory effect on H460 cell proliferation.On that basis,the lung cancer targeting silk fibroin/ING4-IL-24 dual-gene co-expression adenovirus CASF-C9/Ad was prepared by coating adenovirus vector with CASF-C9 by electrostatic interaction.The results of morphology,surface Zeta potential and particle size distribution showed that the complex was spherical or globular-like particles,and the surface Zeta potential and particle size increased with the increase of the concentration of CASF-C9 used to coat Ad.Next,CASF-C9 was labeled with FITC,respectively,to study the targeting recognition effect of peptide C9 on H460 cells.According to the results of laser confocal focusing,material CASF-C9 and the complex CASF-C9/Ad,both were grafted with peptide C9,showed specific recognition and adhesion effect on lung cancer cells H460.Furthermore,lung cancer cells H460 and umbilical vein endothelial cells HUVEC were infected with CASF-C9/Ad complex in vitro,respectively.The fluorescence expression and infection efficiency were detected,and the results showed that the fluorescence expression intensity and infection efficiency of H460 cells and HUVEC cells infected with CASF-C9/Ad were significantly higher than the naked Ad in control group.In addition,the growth of H460 cells infected with CASF-C9/Ad was significantly inhibited,and the survival rate of H460 cells infected with CASF-C9/Ad was significantly lower than the naked Ad and CASF/Ad in control groups.The target genes ING4 and IL-24 in CASF-C9/Ad complex could induce apoptosis of lung cancer cells after effective expression in H460 cells.The apoptosis rate of H460 cells infected with CASF-C9/Ad was significantly higher than the naked Ad and CASF/Ad in control groups.Totally different from infection of lung cancer cells H460,after HUVEC cells were infected with complex CASF-C9/Ad,the cell morphology and cell proliferation activity of HUVEC cells were not significantly affected,which proved that CASF-C9/Ad complex had obvious targeting and inhibiting proliferation and promoting apoptosis effects on lung cancer cells,but had no obvious toxicity on normal cells.Finally,the ING4-IL-24 co-expression CASF-C9/Ad complex was used to treat transplanted tumor of H460 lung cancer in mice.From the results of tumor volume changes,tumor weights and tumor inhibition rates,CASF-C9/Ad complex could significantly inhibit the growth of transplanted tumor,and the tumor inhibition rate of CASF-C9/Ad complex was significantly higher than that of the naked Ad and CASF/Ad.After treatment with complex CASF-C9/Ad,large areas of necrosis were found in tumor tissues,tumor cells were disintegrated,cell nucleus staining disappeared,tumor cells presented swollen and vacuolated structure,and cell membrane continuity was destroyed.The degree of tumor tissue necrosis after treatment with complex CASF-C9/Ad was more evident than the naked Ad and CASF/Ad in control groups.The dual-gene co-expression CASF-C9/Ad complex could efficiently deliver the target genes ING4 and IL-24 to lung cancer cells,and inhibited the proliferation of lung cancer cells by down-regulating the expression of Ki 67 and Bcl-2,and promoted apoptosis of lung cancer cells by up-regulating expression of C Caspase-3 and Bax as well as blocked tumor angiogenesis by down-regulating the expression of VEGF and CD31.The CASF-C9/Ad complex achieved tumor suppressive effect on transplanted tumor of H460 lung cancer in mice through multiple ways.In this paper,the oligopeptide CSNIDARAC specific to lung cancer cells was grafted to the side chain of Antheraea pernyi silk fibroin to construct a cationic silk fibroin/ING4-IL-24 dual-gene co-expression adenovirus complex CASF-C9/Ad that could target and infect lung cancer cells.This complex could not only deliver the target gene to lung cancer cells,but also reduce the effective use titer of adenovirus.It could also shield the direct action of adenovirus and host,protect adenovirus from the neutralization of serum antibody,and reduce the immunogenicity risk of adenovirus.Meanwhile,it showed no obvious toxicity to normal cells.Animal experiments proved that the CASF-C9/Ad complex inhibited the proliferation of lung cancer cells,promoted apoptosis of lung cancer cells,and blocked tumor angiogenesis,achieving the suppressive effect on transplanted tumor of H460 lung cancer in mice in multiple ways.