Study Of Self-assembled Nanoparticles For T Lymphocytes Gene Transfection

With a deep understanding of the mechanisms of cancer and the development of cancer-related technologies,new cancer strategies have been proposed and developed rapidly.Among them,a single-chain antibody able to recognize a tumor-associated antigen and a signal transduction domain capable of promoting T-cell to activate are fusionally expressed on the surface of autologous T-cells through genetic engineering techniques,thereby enables the T-cell to have long-lasting and tumor-targeting killing effects.Chimeric antigen receptor modified-T cell（CAR-T）,which is capable of expanding,is one of the most rapidly developing and clinically promising therapeutic treatments.However,there still exist some problems in the clinical application of this technology,such as the separation of patient’s T cells,in vitro culture and expansion and virus-based CAR gene transfection leading to the highly cost and risk of this technology.Based on our previous research on self-assembled nanoparticles,the purpose of the study is to symstematically synthesize new self-assembled molecular modules and optimize the formulation through the evaluation of reporter gene transfected to Jurkat cells.Hopefully we can develop a gene-loaded self-assembled nanoparticle that can be directly used for the transfection of T cells in vivo.Based on the above objectives,firstly we synthesized the self-assembled molecular moieties include adamantane-polyethylene glycol(Ad-PEG₅₀₀₀)and adamantane-polyamidoamine with different generations(Ad-PAMAM_G1/G3/G5),β-cyclodextrin-modified polyethyleneimine with different molecular weights(CD-PEI_{800/2000/10000})and each product was confirmed by nuclear magnetic resonance spectroscopy.Subsequently,we mixed Ad-PEG,plasmid DNA,Ad-PAMAM with different generations and CD-PEI with different molecular weights in a certain concentration,dosage and order by the host-guest interaction of Ad withβ-CD and the electrostatic interaction of plasmid DNA with PEI/PAMAM to gain the self-assembled nanoparticles(DNA@SNP^x/y,where x is the generation of PAMAM and y is the molecular weight of PEI).The particle size,zeta potential and stability of the respective assembled nanoparticles were characterized using a Malvern nanosizer,and the ability of the nanoparticles to encapsulate DNA was verified by agarose gel electrophoresis.Experiment results showed that the self-assembled nanoparticles we prepared can effectively load the reporter gene with a good stability.Further,we carried out the transfection experiments of Jurkat cells with various p GL3@SNP^x/y loaded with luciferase reporter gene and found out that p GL3@SNP^G1/800 has the highest transfection efficiency to Jurkat cells.Then,we continued to systematically optimize the formulation through a series of experiments.Firstly,for the four molecular modules of DNA@SNP^G1/800,we chose three concentrations respectively for the orthogonal design of four factors and three levels（L3⁴）.The self-assembled nanoparticles formed at different concentrations of each component were examined.The optimal preparation for each component to transfect Jurkat cells is Ad-PEG 0.815nmol,Ad-PAMAM_G10.735nmol,p GL3 0.690nmol.Since the N/P ratio of the nanoparticles has a great influence on the transfected cells,we investigated the transfection ability of the self-assembled nanoparticles formed at different N/P ratio to Jurkat cells and found out the best N/P ratio for the preparation is 20.DNA@SNP^G1/800（N/P 20）,which can encapsulate DNA well,has a particle size of 90 nm,zeta potential of 20+m V,and transmission electron microscopy showed it has a uniform particle size and a regular shape.We further studied the interaction between the preparation and the cells after the fluorescent labeling of components of the self-assembled nanoparticle.Results showed that the nanoparticles can bind well to Jurkat cells and enter through endocytosis.PEI and PAMAM in the preparation may promote the release of nanopartilces from lysosomes in Jurat cells through proton sponge effect,thus leads to a high level of gene expression.Self-assembled nanoparticles(p EGFP@SNP^G1/800)loaded with green fluorescent protein（p EGFP）gene was confirmed to have a high protein expression level by the transfection to Jurkat cells.CCK-8 test showed that DNA@SNP^G1/800 has little toxicity to Jurkat cells at the experimental concentration.Through the above studies,we developed and prepared DNA@SNP^G1/800with a uniform shape and good stablity,which has a good gene encapsulation,a high level of transfection and little cytotoxicity to Jurkat cell,therefore laid a good foundation for the gene loading of CAR-T and the study of transfection to T cells in vivo.