A Mannosylated PEI-CPP Hybrid For TRAIL Gene Targeting Delivery For Colorectal Cancer Therapy

ObjectiveGene therapy is an ideal method for the treatment of tumor and other gene diseases by transferring therapeutic gene drugs to specific targeted location in tumor cells to change gene expression in order to treat disease.But the implication of gene therapy is limited for lacking safe and efficient gene vectors.The purpose of this study is to design a safe and efficient non viral vector,which can carry nucleic acid drugs to construct a gene transfer system for the treatment of colon cancer.This system may provide new research ideas for the gene therapy of colon cancer.MethodsPolyethyleneimine(Polyethylenimine 5000,PEI_5K),with its good biocompatibility,was used as a vector,but its transfection effect is poor.We synthesize Man-PEI_5K by the reaction between isothiocyanate group of MPITC with the amino group of PEI_5K.Then through the amino groups on Man-PEI_5KK react with the sulfhydryl group of low molecular weight protamine,the vector Man-PEI_5K-CPP was successfully synthesized.The vector,which has strong positive charge,can carry nucleic acid drugs by electrostatic action,forming gene transfer system Man-PEI_5K-CPP/pDNA.The particle size and potential of this system are measured by Malvin laser particle size analyzer,and the transmission electron microscopy（Transmission Electron Microscope,TEM）is used to observe the morphology of this system.In the in vitro evaluation part of the gene system,the cell uptake study and the tumor spheroid uptake were evluated.The toxicity of the vector’s material itself and the selective killing effect on the tumor cells were investigated by the cell proliferation inhibition experiment;The Mannose receptor（MR）expression or the protein expreesion of therapeutic gene on tumor cells was investigated by Western Blot technology,the transfection efficiency of the vector was examined by cell transfection assay.In vivo evaluation,the mice bearing HCT116 tumors were used to investigate the biodistribution of this system by in vivo tumor imaging.The anti-tumor effect of the system was investigated through pharmacodynamic experiments,and the biosafety of the gene delivery system was evaluated.Results and ConclusionWe found that Mannose receptor（MR）is overexpressed in the colon cancer cell line HCT116,aparting from in macrophages and liver sites.We design a vector,which is based on biocompatible polyethyleneimine 5000 (Polyethylenimine 5000,PEI_5K),to improve the targeting of gene transfer system.In order to inmprove the transfection efficicency,we have modified mannose and penetrating peptides on the gene vector.We use the reaction of isothiocyanate group of isothiocyanate（alpha-D-Mannopyranosylphenyl isothiocyanate,MPITC）with amino group on PEI_5K to synthesize Man-PEI_5K,then the residual amino group on Man-PEI_5K is reacted with the sulfhydryl group of low molecular weight protamine（Low Molecular Weight Protamine,LMWP）.The final vector is called Man-PEI_5K-CPP.The vetor was characterized by Fu Liye transform infrared spectroscopy and ¹H-NMR.The plasmid DNA was loaded with Man-PEI_5K-CPP,a new mannose and transmembrane peptide modified gene delivery system Man-PEI_5K-CPP/pDNA was successfully constructed.This system is a uniformly dispersed spherical nanoparticle with positive charge.We use the tumor necrosis factor related apoptosis inducing ligand gene（Tumor necrosis factor-related apoptosis-inducing ligand,TRAIL）as a model nucleic acid drug,and construct the gene transfer system Man-PEI_5K-CPP/pTRAIL.This system can induce apoptosis of tumor cells,but do not induce normal cell apoptosis.PEI_25K is a common non-viral vector,which is a"gold standard"for gene transfection.However,its therapeutic application is hampered by the high cytotoxicity and poor in vivo transfection efficiency.The toxicity of the vector Man-PEI_5K-CPP itself is examined by MTT assay,and its toxicity is less than PEI_25K.Then PEI_25K/pTRAIL was used as positive control,through cytotoxicity test,apoptosis assay,we detected anti tumor effect of Man-PEI_5K-CPP/pTRAIL,it was found that the anti tumor effect of Man-PEI_5K-CPP/pTRAIL in vitro was better than PEI_25K/pTRAIL.Man-PEI5kIL.L in-1-labeled DNA was constucted to investigate the targeting and penetrating effects of the gene delivery system.Using Enhanced Green Fluorescent Protein（EGFP）as the reporter gene,it was found that this system had the highest transfection efficiency.The results of the above experiments showed that Man-PEI_5K-CPP/pEGFP transfection,uptake and tumor penetration were better than PEI_25K/pEGFP.Although the transfection efficiency of PEI_25K in vitro is high,its effect in vivo is not satisfactory.Therefore,the in vivo evaluation of gene delivery system with good transfection efficiency is very important.In this chapter,the mice model bearing HCT116 tumors was established to investigate distribution and efficacy of Man-PEI_5K-CPP/pTRAIL,and the toxicity in vivo was evaluated.The results of in vivo distribution experiments showed that Man-PEI_5K-CPP/pTRAIL had better effect on circulating time and targeting tumor sites than PEI_25K/pTRAIL in vivo.The results of pharmacodynamic experiments showed that Man-PEI_5K-CPP/pTRAIL had good anti-tumor effect in vivo and less toxicity in vivo.In conclusion,the constructed Man-PEI_5K-CPP/pDNA has a good anti-tumor effect and can provide a new idea for the treatment of colon cancer.