Nanocarrier-mediated PiggyBac Transposon System For Generation Of CAR-NK Cells

BackgroundAdoptive immunotherapy(AIT)is a rapidly developing cancer treatment method in recent years.Chimeric antigen receptor(CAR)has made remarkable achievements in modifying immune cells.The key of cell immunotherapy is to introduce the target gene into the cell based on the safe and efficient gene transfer method.There are many methods of gene transfer,such as lentivirus,retrovirus and electroporation,but the limited efficiency and low biosafety limit its development.Cationic polymer nanomaterials have good biocompatibility,easy to modify,simple preparation and good stability,they come into people's attention as a potential gene carrier.In this study,the gene transduction method of chimeric antigen recepter(CAR)was used to modify natural killer(NK)cells by using a novel cationic polymer nanocarrier mPEG-P(Asp-AED-g-HFB)(PAEF)and PiggyBac transposon systerm,which achieved high gene transfer efficiency and provided experimental basis for tumor cell immunotherapy.ObjectiveTo explore the gene transduction method of chimeric antigen receptor(CAR)to modify natural killer(NK)cells by using a novel cationic polymer nanocarrier PAEF and PiggyBac transposon system,providing a new strategy for immunotherapy of cancer cells.Methods1.PAEF/DNA(transposase+transposon)complex were prepared.The particle size and zeta potential were measured with Nano-ZSE Dynamic Light Scattering System.2.The DNA binding ability of nanoparticles was evaluated by agarose gel retardation assay,the condation of the PAEF/DNA complex to enter NK cells were optimized.3.The cell cytotoxicity of PAEF/DNA complexes under different N/P ratios wasanalysed by CCK-8 cytotoxicity test.4.Transduction efficiency of NK cells were evaluat by fluorescence microscopy and Flow cytometry.Results1.PAEF can encapsulate DNA to form nanocomposites with the diameter of 100-150 nm,which is situtable to mediate DNA entry into cells.2.When the N/P ratio was 20,naked DNA could be completely wrapped in PAEF.In the presence of dithiothreito(DTT),PAEF had a good ability to release DNA.3.The PAEF/DNA complexes formed at the N/P ratio of 80 attained the high cell viability(72.50±3.9)%,which significantly higher than lipofectamine(64.03±1.8)%(P<0.05).4.Under the condition of N/P value of 80,fluorescence microscopic observation showed more RFP-positive cells,Flow cytometry also showed the highest transfection efficiency(24h,83.4%,P<0.05).Conclusion1.In this study,a new cationic polymer nanocarrier PAEF and DNA composite was successfully prepared.Particle size and Zeta potential measurement,DNA gel electrophoresis encapsulation and release experiments showed that PAEF was feasible as a gene carrier.2.The cytotoxicity of PAEF/DNA complex to NK-92 cells was significantly lower than that of liposome transfection group,indicating good biocompatibility.Fluorescence microscopy and flow cytometry showed that the transfection efficiency was higher,and fluorescence microscopy showed that fluorescence was still highly expressed after subculture.In this study,we established a gene transfer method of NK cells mediated by piggyBac transposon based on nanocarriers,which achieved high gene transfer efficiency and provided experimental basis for gene transfer of adoptive immunotherapy.