Cleavable Polyethylene Glycol Branched Chain Modified Antheraea Pernyi Silk Fibroin As A Gene Delivery Carrier

It is an important subject in the field of cancer gene therapy to construct a gene delivery carrier that can effectively mediate the expression of the target genes,has low toxicity to normal tissue cells,and can resist non-specific adsorption of plasma proteins.Growth inhibitory factor 4(ING4)and interleukin-24(IL-24)genes can inhibit the growth of tumor cell from two ways,inside and outside the cell,respectively.Antheraea pernyi silk fibroin(ASF)has good biocompatibility and can be biodegraded.Polyethylene glycol(PEG)has the ability to resist non-specific adsorption of plasma proteins.After grafting the matrix metalloproteinase sensitive peptide(MMP2 sensitive peptide,the sequence is GPLGIAGQC)on its branches,the PEG chain can be broken under the action of highly expressed MMP2 outside the tumor cells,thereby enhancing the internalization of the complex particles by the tumor cells.The purpose of this article is to construct a new ING4-IL-24 double gene co-expression plasmid delivery vector based on Antheraea pernyi silk fibroin.In order to reduce non-specific adsorption and prolong the in vivo circulation time of the carrier,the surface of the carrier was grafted with PEG-MMP2 sensitive peptide branches.When the carrier reached the surface of tumor cells with high expression of MMP2,the PEG chain on the surface of the carrier could be cleaved under the action of MMP2.To achieve the above goals,this article used branched low molecular weight polyethyleneimine(PEI)to chemically modify Antheraea pernyi silk fibroin to obtain cationized Antheraea pernyi silk fibroin(CASF),so that the surface of the ASF was reversed from a negative charge to a large number of positive charges.Then,PEG-MMP2 sensitive peptide was covalently grafted onto the side chain of CASF to prepare cationized Antheraea pernyi silk fibroin grafting with PEG-MMP2 sensitive peptide(CASFMP).CASFMP was used to coat the ING4-IL-24 dual-gene co-expression pDNA to prepare the CASFMP/pDNA complexes.CASFMP/pDNA complexes with different mass ratios were used to transfect human fibrosarcoma cell HT1080 with high expression of matrix metalloproteinase(MMP2)and human normal embryonic kidney cells HEK293 to study the complex's ability to transfect and suppress growth of malignant tumor cells,and its toxicity to normal cells.Firstly,after being activated under the action of EDC,-COOH on the side chain of ASF undergone amidation reaction with-NH2 on the branched low molecular weight PEI to obtain a cationized Antheraea perhyi silk fibroin,which reversed the ASF surface charge from negative to positive.The Zeta potential of CASF increased with the increase of the mass ratio of PEI/ASF.When the mass ratio of PEI/ASF was 4%,the Zeta potential on the surface of ASF was +10.76±0.40 mV,the isoelectric point was 9.38,and the number of free amino groups on its side chains increased significantly.FT-IR and 1H-NMR indicated that PEI was grafted to the side chain of ASF.Secondly,the PEG-MMP2 cleavage peptide was coupled to the CASF side chain by the action of the bifunctional cross-linking agent 3-(2-pyridine dimercapto)propionate N-hydroxysuccinimide ester(SPDP)to prepare PEG-MMP2 cleavage peptide-CASF(CASFMP).When the PEG-MMP2 sensitive peptide/CASF mass ratio were 1/500,1/400,1/300,the surface Zeta potential of CASFMP increased slightly as the PEG-MMP2 sensitive peptide/CASF mass ratio increased.When the PEG-MMP2 sensitive peptide/CASF mass ratio were 1/200,1/100,1/50,the surface Zeta potential of CASFMP decreased as the PEG-MMP2 sensitive peptide/CASF mass ratio increased.FT-IR and 1H-NMR showed that the PEG-MMP2 sensitive peptide was connected to the CASF side chain in the form of disulfide bonds under the action of SPDP.Gel protein electrophoresis showed that the PEG chain could be effectively cleaved from PEG-MMP2 sensitive peptide-CASF under the action of matrix metalloproteinase MMP2.Then,the positively charged CASFMP encapsulated and compressed negatively charged plasmid DNA by electrostatic adsorption to form a CASFMP/pDNA complex.Agarose gel electrophoresis showed that when the CASFMP/pDNA mass ratio were 64/2,128/2,196/2,the three ratios of CASFMP could effectively encapsulate plasmid DNA.The surface zeta potentials of the three ratio composites were all positive.The composites all formed dense,spherical-like nanoparticles,and were evenly dispersed in the aqueous solution and serum environment,without mutual aggregation,indicating that the PEG chain coupled to the CASFMP/pDNA complex could effectively shield the interaction between the complex particles and serum proteins.Finally,HT1080 cells expressing MMP2 and HEK293 cells were transfected with CASFMP/pDNA complexes.After transfection of HT1080 cells with the complex for 24 h,the cells began to turn round and some cells began to float,and the cell survival rate was significantly lower than that of the blank control group,indicating that CASFMP/pDNA complex could inhibit the proliferation of HT1080 cells.When the C50ASFMP/pDNA mass ratio was 128/2,the efficiency of HT 1080 cells transfected with the complex was higher than that of HEK293 cells transfected with the complex,indicating that PEG branched chains in CASFMP could be cleaved under the action of MMP2,thus enhancing the transfection efficiency of the complex to HT 1080 cells.After transfection of HEK293 cells with the complex for 24 h,the cells were fully extended and the cell survival rate was above 95%,which was significantly higher than that of 25 kDa PEI/pDNA complex,indicating that compared with 25 kDa PEI/pDNA complex,CASFMP/pDNA complex was less toxicity to HEK293 cells.The ING4-IL-24 dual-gene co-expression plasmid delivery system based on cationized Antheraea pernyi silk fibroin with cleavable polyethylene glycol is constructed in this paper.It could effectively inhibit the growth of human fibrosarcoma cell HT1080,had no obvious toxicity to normal tissue cells,and had non-specific adsorption capacity of anti-plasma proteins.Experiments have shown that the PEG on the CASFMP branch could be cleaved under the action of MMP2,which helped to improve the transfection efficiency of the gene loaded on malignant tumor cells that highly express MMP2.The research in this paper provides an efficient and safe gene delivery carrier for gene therapy of malignant tumors.