The Research Of Tumor Endothelial Cell Targeted Delivery System Chemo-attracting Cytotoxic T Lymphocytes In The Treatment Of Cancer

Background:Tumor antigen-specific cytotoxic T lymphocytes(cytotoxic T lymphocytes,CTLs)has become a hot tumor biological treatment.Due to the limited number of lymphocytes infiltrating the tumor and the inhibition of tumor microenvironment,most of the killer cells lose their function in the process of conventional adoptive immunotherapy,which cannot completely and efficiently remove the tumor cells.Therefore,to improve the efficacy of adoptive immunotherapy,it is important to increase the concentration of tumor antigen-specific CTLs and improve their anti-tumor activities in tumor tissues.Objective:to construct an aptamer-modified targeted liposome nano-carrier system(ENG-Apt/mIP-10-LP)that targets mouse tumor vascular endothelial cells(mTEC)and recruit the tumor antigen-specific CTLs to the tumor site.Methods:The liposome which was loaded with IP-10 plasmid DNA(LP-mIP-10)was prepared by the ethanol and calcium coprecipitation method.To obtain ENG-Apt/mIP-10-LP,the DSPE-PEG2000-ENG-Aptamer was connected to the surface of LP-mIP-10 by Post-insertion method.The material structure characteristics of ENG-Apt/mIP-10-LP were examined by dynamic light scattering instrument(DLS)and transmission electron microscope(TEM).And the biophysical and cell transfection properties of ENG-Apt/mIP-10-LP were determined by a series of in vitro experiments.The TRP2-specific CD8+ T cells were induced by artificial antigen presenting cells(H-2Kb-Ig:TRP2-aAPC)and enriched by magnetic beads.The melanoma tumor-bearing C57BL/6 mice were treated by ENG-Apt/mIP-10-LP,or ENG-Apt/mIP-10-LP and TRP2-specific CD8+ T cells via the tail vein injection.In vivo optical imaging was used to track the distribution and metabolism of nanoparticles in tumor-bearing mice.Flow cytometry was used to measure the number of CXCR3+ CD8+ T cells and TRP2-specific CD8+ T cells in tumor,the number of myeloid-derived suppressor cells(MDSCs),as well as the number of T-regulatory cells(Tregs).The in situ tetramer staining(ISTS)was used to observed the infiltration of TRP2-specific CD8+ T cells in tumor.Enzyrme-linked immunospot assay(ELISPOT)was used to measure the level of interferon-gamma(IFN-y).Immunohistochemistry was used to detect IP-10 expression,tumor vessel density,cell proliferation and apoptosis.In addition,the mice were monitored daily for survival and tumor volume was calculated according to the formula.Results:The ENG-Apt/mIP-10-LP of which diameter was about 150 nm was successfully synthesized and distributed evenly with low cytotoxicity and high stability in serum.ENG-Apt/mIP-10-LP significantly inhibited the growth of tumor,prolonged the survival of mice in melanoma-bearing mice,increased in the number of CXCR3+ CD8+ T cells and TRP2-specific CD8+ T cells in tumor,increase the number of IFN-y-positive cells,decreased the number of MDSCs and Tregs in the tumor-besaring mice,inhibited the proliferation of tumor cells,and induced the apoptosis of cancer cells.Conclusion:The ENG-Apt/mIP-10-LP delivery system provide a promising strategy for cancer therapy.