Specific Capture,release,and Transfection Of T Lymphocytes Based On Sgc8 Modified Si-Nanowire And Self-assemble Nanoparticle

With the continuous understanding of cancer diseases,tumor immunotherapy methods have developed and matured.Adoptive cell transfer therapy(ACT)as one of the important methods for tumor immunotherapy has achieved good results both in the laboratory and in clinical studies.Therefore,more and more researchers have paid attention to it.In recent years,chimeric antigen receptor(CAR)genetically modified T cell therapy has shown outstanding potential in adoptive cell transfer therapy.To improve the targeting,killing,and proliferation by transforming T lymphocytes to express functional proteins under conditions that are not restricted by the immunogen,thereby enabling the killing of tumor cells and opening up new strategies for tumor immunotherapy.However,chimeric antigen receptor T lymphocyte immunotherapy still faces various problems in the process of tumors treatment.For example,T lymphocytes need to be isolated in vitro during the application process;the biosafety of the viral vector system;inflammatory response,etc.Based on these problems,we designed and developed a system for the specific capture and release of T lymphocytes and a non-viral vector self-assembly nanoparticle system with high transfection ability to transfect T lymphocytes with CAR plasmids.In this study,the third generation of chimeric antigen receptor EGFR variant Ⅲ-CD28-4-1BB-CD3ζwas designed and synthesized.EGFR variant Ⅲ epidermal growth factor receptor mutant is a tumor-specific antigen with a high expression rate in various types of malignant tumors,it is a ideal molecular target for tumor targeted therapy.Targeting of tumor antigen was achieved by using sc Fv of EGFR variant Ⅲ.Costimulatory molecule CD28 enhanced T lymphocytes activity;CD3ζ-activated T lymphocytes toxic reaction.The aim of this study is to identify,capture and release T lymphocytes through Si-Nanowire modified by Sgc8 aptamer.Using self-assembled nanoparticles to delivery plasmid DNA into T lymphocytes to express as CAR~+T lymphocytes.We first prepared silicon nanowires(Si NWs)using metal-catalyzed chemical etching and continued exploration of experimental conditions and reaction parameters to prepare Si NWs of different heights for subsequent biological experiments.According to literature reports,aptamer Sgc8,which can specifically bind to T lymphocytes.We synthesized the aptamer Sgc8 and determined the binding activity of this aptamer to T lymphocytes by flow cytometry.Subsequently,the Sgc8-Si NWs were obtained by modifying the Sgc8aptamer on the silicon nanowire array through bioorthogonal reaction between biotin and streptavidin,and then the specific capture of T lymphocytes was successfully achieved using Sgc8-Si NWs.And the release of T lymphocytes was achieved by the exonucleotide degrading effect on the Sgc8 aptamer under the condition of ensuring the cell activity.Furthermore,we developed a self-assembled nanoparticle system for CAR gene transfection of captured T lymphocytes.First,we synthesize three self-assembled molecular modules for gene-loading self-assembly nanoparticle systems:β-cyclodextrin-modified low molecular weight polyethyleneimine(CD-PEI800),and adamantane-terminated polyethylene glycol(Ad-PEG-Biotin)and adamantane-modified polyamide-amine(Ad-PAMAM,G1)were used to characterize the chemical structure of each module molecule by ~1H-NMR.At the same time,we constructed a third-generation chimeric antigen receptor EGFR variant Ⅲ-CD28-4-1BB-CD3 targeting EGFR variant Ⅲ with high expression of glioma cells.Subsequently,we used the above three self-assembled molecular modules and CAR plasmid vectors for the preparation of DNA-loaded self-assembling nanoparticles(DNA@SNPs)based on molecular recognition and charge interaction,and through agarose gel electrophoresis,laser scattering particle size analytical and transmission electron microscopy analysis or characterization of DNA@SNPs DNA packaging capabilities,particle size,morphology and stability,found that the DNA@SNPs can well wrapped DNA,showing a uniform spherical structure,particle size 100nm,good stability.Next,we used reporter gene plasmid DNA(luciferase reporter and green fluorescent protein)loaded self-assembled nanoparticles to transfect T cells to verify that DNA@SNPs can achieve high levels of transfection of T cells by the carried genes.On this basis,we used self-assembling nanoparticles to introduce EGFR variant Ⅲ-CD28-4-1BB-CD3ζplasmids into T lymphocytes and validated CAR expression using flow cytometry and Western Blot protein imprinting.The experimental results showed that the EGFR variant Ⅲ-CD28-4-1BB-CD3ζwas successfully expressed on the T cell membrane,and the expressed EGFR variant Ⅲ-CD28-4-1BB-CD3ζspecifically recognizes and binds to the EGFR variant Ⅲ highly expressed Huh7 tumor cells.