Targeted Peptide And Polyethylenimine Co-modified Antheraea Pernyi Silk Fibroin As A Gene Delivery Vector

Liver cancer is one of the most common malignancies in China,its morbidity and mortality are very high.Traditional treatments such as surgical resection,radiotherapy,and chemotherapy are not effective for patients with advanced liver cancer.A gene therapy technique that uses a substance having a high degree of specificity for a tumor as a carrier and targetedly delivers a tumor cell suppressor gene to tumor cells provides a new approach for the treatment of malignant tumors.Inhibitor of growth 4?ING4?and interleukin-24?IL-24?genes are ideal cytostatic factors in gene therapy of liver cancer.Their synergistic effects not only selectively inhibit tumor cell growth and new angiogenesis,which can induce apoptosis of tumor cells and have no toxic effect on normal cells.However,cytokines have a short half-life in vivo and are easily inactivated or degraded.Therefore,it is the key to cancer gene therapy to search for a safe and efficient gene carrier,to target the genes of cytokines,to deliver them to the cells stably and efficiently and to induce apoptosis of tumor cells.The Antheraea pernyi silk fibroin?ASF?has good biocompatibility,low epidemiology,biodegradability,etc.The amino acid sequence of ASF contains a large number of arginine-glycine-aspartic acid tripeptide sequences which can specifically recognize and bind to the surface of malignant cells and the high expression of?_v?₃ in tumor proliferation neovascular endothelial cells.And the ASF contains a large number of amino acid residues with polar and side groups that can be ionized,and these amino acid residues can be used as chemical modification sites for ASF.FQHPSFI heptapeptide?HCBP1?is a small molecule peptide with high specificity to hepatoma cells.It can differentiate between normal cells and hepatoma cells in vitro and in vivo,and it is a promising ligand for hepatoma with development and application prospects.In this paper,chemical modification of ASF with low molecular weight polyethylenimine?1.8 kDa?resulted in a positive charge on the surface of ASF,which was then specifically recognized on the surface grafting of cationized Antheraea pernyi fibroin?CASF?.The HCBP1 peptide of hepatocellular carcinoma cells encapsulated the pDNAs which was encoded by the dual genes of ING4 and IL-24 to form a complex.And that could protect,compress the pDNA,and make the surface positively charged.The above steps were for the preparation of a plasmid complex which was co-expressed with CASF modified by a hepatoma cell targeting peptide and the ING4-IL-24 dual gene?CASFP/pDNA?.Hepatoma cells HepG2 and normal liver cells L-02 were transfected with the complexes respectively,and the effects of different mass ratios on the transfection efficiency and cytotoxicity of hepatoma cells and normal cells were studied.Firstly,the carboxyl group on the side chain of ASF reacted with the amino group on polyethylenimine to form a new amide bond mediated by carbodiimide,thereby cationizing the Antheraea pernyi silk fibroin.It was shown that the Zeta potential of polyethylenimine-modified Antheraea pernyi silk fibroin was inverted from a negative value to a positive value compared with the unmodified Antheraea pernyi silk fibroin.With the increase of the mass ratio of polyethylenimine and the Antheraea pernyi silk fibroin protein,the Zeta potential of modified Antheraea pernyi silk fibroin was increased.When the mass ratio of polyethyleneimine and the Antheraea pernyi silk fibroin was 4%,the Zeta potential value could achieve+11.33±0.38 mV.At the same time,the isoelectric point of the modified Antheraea pernyi silk fibroin was increased from 4.31 to 9.38,indicating that the modified Antheraea pernyi silk fibroin has a large number of positive charges on its surface.The results of ¹H-NMR and FT-IR tests showed that polyethylenimine was covalently attached to the side chain of the Antheraea pernyi silk fibroin,which achieved the purpose of cationizing the the Antheraea pernyi silk fibroin.Secondly,the amino group on the side chain of the cationized Antheraea pernyi silk fibroin?CASF?reacted with the thiol group of HCBP1 under the mediation of3-?2-pyridyldithio?propionic acid N-hydroxysuccinimide ester,and HCBP1 was attached to the Antheraea pernyi silk fibroin?CASF?side chain in the form of a disulfide bond to prepare the cationized Antheraea perny silk fibroin?CASFP?modified by HCBP1 peptide.The experimental results showed that the Zeta potential of the Antheraea pernyi silk fibroin?CASF?modified by HCBP1 peptide was reduced.With the increase of the mass ratio of HCBP1/CASF,the Zeta potential of the modified Antheraea pernyi silk fibroin?CASF?gradually decreased.When the mass ratio of HCBP1/CASF was 1/400,the Zeta potential value was+8.22±0.62 mV.The results of ¹H-NMR and FT-IR tests showed that the amino group on the side chain of the Antheraea pernyi silk fibroin?CASF?reacted with the thiol group of HCBP1 to covalently attach HCBP1 to the side chain of CASF.Thirdly,CASFP/pDNA complexes with different mass ratios were prepared by electrostatic adsorption.The result of agarose gel electrophoresis showed that CASFP could completely coat pDNA.The surface zeta potential of CASFP/pDNA complexes increased with the increase of the mass ratio of CASFP/pDNA.The particle size of the composite was 300-600 nm,and the particle size decreased with the increase of CASFP/pDNA mass ratio.The observation and analysis of the composites by SEM,TEM,fluorescence inverted microscope and EDS showed that the complexes were uniform in morphology and compact in structure,further demonstrating that CASFP could encapsulate and compress the pDNA to form hundreds of nanometer spherical particles.Finally,HepG2 cells were transfected with the CASFP/pDNA complex in vitro.After24 hours of transfection,the shape of the cells became round,the volume became small er,and they showed apoptotic characteristics such as separation from other cells.When the mass ratio of CASFP/pDNA was 256/2,transfection efficiency of HepG2 cells reached13.03%after 24 hours of transfection,which was close to 15.51%of 25 kDa PEI.After transfecting L-02 cells with CASFP/pDNA complexes for 24 hours,the cells were able to adhere to the wall and grow normally with good morphology,and the cell viability was significantly higher than that of cells transfected with 25 k Da PEI,indicating that CASFP/pDNA complexes had no significant effect on the growth of normal hepatocytes.These results indicated that the CASFP/pDNA complexes not only could transfect hepatoma cells,inhibit the growth of hepatocellular carcinoma cells,induce the apoptosis of hepatoma cells,but also had no obvious toxicity to normal cells.The plasmid complex CASFP/pDNA was co-expressed with the cationized Antheraea pernyi silk fibroin modified by a hepatoma cell targeting peptide and the ING4-IL-24 dual gene in the article,which provided a new gene therapy system for liver cancer and had the potential for development and clinical application.