Synthesis Of A Novel Gene Vector CMCS-PEI₆₀₀-CyD And Its Application In Immunotherapy

Objective（s）:Gene therapy,as a promising emerging technology,can treat many complex diseases,and the development of safe and effective nucleic acid delivery vectors is the key to realize gene therapy.Polyethyleneimine PEI is the most widely studied cationic gene vector,and its transfection efficiency is proportional to its molecular weight,however,high molecular weight PEI has high cytotoxicity,which largely limits its application.Chitosan can also be used as a carrier for gene therapy because of its better biocompatibility,however,chitosan alone has poor solubility and low transfection efficiency.In this study,a novel gene nanocarrier CMCS-PEI₆₀₀-CyD was synthesized by modifying carboxymethyl chitosan CMCS with low molecular weight PEI(PEI₆₀₀),andβ-cyclodextrin（β-CyD）as a backbone,and its application as a gene carrier and in immunotherapy was investigated at the cellular level.Methods:1.Chitosan was sequentially depolymerized and carboxymethylated using chemical synthesis to obtain carboxymethyl chitosan CMCS,the nanocarrier PEI-CyD was synthesized,and PEI-CyD was coupled with CMCS to form CMCS-PEI₆₀₀-CyD nanocarrier;2.CMCS-PEI₆₀₀-CyD/pDNA nanocomplexes were prepared using electrostatic interaction,and the complexes were examined for The morphology,particle size,surface charge and DNA wrapping ability of the complexes were investigated;3.EGFP green protein was used as a reporter gene to compare the transfection effects of different mass ratios and different vectors on human renal epithelial cell line 293T and human dendritic cells h DCS;4.The transfection efficiency of different vectors on human renal epithelial cell line 293T and human dendritic cells h DCS was quantified;5.The cytotoxicity of different vectors on human renal epithelial cell line 293T and mouse bone marrow-derived dendritic cells DC2.4 was detected by CCK-8 assay;6.CMCS-PEI₆₀₀-CyD/pDNA nanocomplex was transfected in DC2.4 cells,and the cytokines such as IL-10,IL-6,TNF-α,IL-1β,etc.were detected by q PCR in DC2.4 cells,IL-1βand other cytokines at the m RNA level;7.Transfection of CMCS-PEI₆₀₀-CyD/pDNA nanocomplex in DC2.4 cells and detection of the expression of IL-6,IL-12,TNF-α,IL-1βand other cytokine proteins in the supernatant of the cells after nanocomplex transfection using ELISA technique;8.Transfection of DC2.4 cells with After CMCS-PEI₆₀₀-CyD/pDNA nanocomplex transfection,the expression of DC2.4 cell surface molecules（e.g.CD80,CD86,MHC class II molecules）was detected by flow cytometry as a marker of cell maturation.Results:1.Transmission electron microscopy revealed that CMCS-PEI₆₀₀-CyD nanocarrier（CPC for short）can wrapDNA well into spherical particles;the particle size of nanocomplex CMCS-PEI₆₀₀-CyD/pDNA is about 118 nm and the surface charge is about 23 m V;the agarose gel electrophoresis results showed that CPC can wrapDNA well into spherical particles when the nanocomplex CMCS-PEI₆₀₀CyD/pDNA at a mass ratio of 0.5:1,the CPC can completely wrap the plasmid DNA;2.Cell transfection experiments revealed that the CPC/p GFP nanocomplex reached the best transfection efficiency when the mass ratio was 20:1;3.Quantitative experiments demonstrated that the CPC/p Luc had the best transfection efficiency on both 293T cells and h DC cells when the mass ratio was 20:1 and the transfection efficiency was better than that of carboxymethyl chitosan CMCS,PEI₆₀₀and PEI-CyD;4.The cell viability of CMCS-PEI₆₀₀-CyD/pDNA nanocomplexes 293T and DC2.4 was determined by CCK-8 method,and it was found that the nanocarrier CPC had lower cytotoxicity;5.CMCS-PEI₆₀₀-CyD/pluc nanocomplex transfection promoted the expression of cytokines such as IL-10,IL-6,TNF-α,IL-1βat m RNA level;6.CMCS-PEI₆₀₀-CyD/pluc nanocomplex transfection promoted the expression of immune factors IL-6,IL-12,TNF-α,IL-1βand DC2.4 surface molecules CD80,CD86,and MHCⅡupregulation,which in turn promoted cell maturation.Conclusion（s）:The nanovector CMCS-PEI₆₀₀-CyD is a novel vector with high transfection efficiency and low toxicity;it can promote efficient gene transfection on human renal epithelial 293T and human dendritic cell h DC;the vector can also promote the maturation of immune factors and DC cells and play a potential immune activation role.