Study On The Enhancement Of Antitumor Immunotherapy By Cationic Liposomes/DNA Complexes

Background and objective:Immunotherapy has brought new hope for cancer patients.However,the clinical research found that the response and therapeutic effect of most patients to tumor immunotherapy were not ideal,the main reason was that the immune tolerance mechanism was established in the process of tumor development,so as to evade the surveillance of the immune system and resist immunotherapy.Tumor-specific CD8⁺cytotoxic T lymphocytes?CTLs?can recognize and kill tumor cells,and play an important role in the anti-tumor immune response.Antigen presenting cells,especially dendritic cells?DC_S?,cross present tumor antigens to CD8⁺T cells to activate antigen-specific CTL.Recent studies have shown that immunogenic death of tumor cells can significantly activate dendritic cells and increase the number of CTLs.Therefore,inducing tumor cells to produce immunogenic death in vivo will be one of the effective strategies to improve the effect of tumor immunotherapy.Nano drug delivery system can enhance the enrichment of drug molecules in tumor tissue and improve the therapeutic effect of tumor.Although cationic liposomes have the ability of carrying nucleic acid drugs efficiently,they have strong interaction with the negative cell membrane,which often leads to obvious cytotoxicity and hinders their application in drug delivery.However,the cytotoxic effect of cationic liposomes is expected to improve the immunogenic death of tumor cells and enhance the anti-tumor immune response,and no relevant research has been reported.In order to study the feasibility and mechanism of cationic liposomes carrying plasmid DNA to enhance the immunotherapeutic effect of tumor,we used cholesterol,DOTAP and DSPE-mPEG2000 or DSPE-PEG2000-NH₂ to prepare liposomes?CLN and CLN-NH₂?containing cationic methoxy group?PEG-OCH₃?or amino group?PEG-NH3⁺?respectively,and further combined them with plasmid DNA to form cationic lipid/DNA recombination Complexes:CLN/DNA and CLN-NH₂/DNA.The effects of CLN,CLN-NH₂,CLN/DNA and CLN-NH₂/DNA in vitro and in vivo on the induction of immunogenic death of tumor cells,the enhancement of DCs activation and the enhancement of anti-tumor effect of the immune system were studied.Methods:Two kinds of cationic liposomes with different PEG end modifications were prepared by film hydration method,and the corresponding cationic liposome/DNA complex was obtained.Firstly,the particle size and potential of the four nanoparticles were characterized,and their morphology was observed by TEM.The apoptosis level of CT26 tumor cells treated with different nanoparticles and the effect of DC_S activation were detected.We also examined the activation of DC_S induced by four nanoparticles.Using CT26 subcutaneous tumor model,we detected the inhibition of tumor growth by injecting different nanoparticles into the tumor,and evaluated the changes of tumor infiltrating immune cells by flow cytometry.Finally,we studied the effect of different nanoparticles on the formation and growth of distal tumors.Results:1.We have prepared the potential of cationic liposome/DNA complexes with high cytotoxicity in tumor immunotherapy by the thin-film hydration method.The cationic lipid/DNA composite nanoparticles with the potential of tumor immunotherapy were successfully prepared by thin-film hydration method.2.In vitro,compared with the other three nanoparticles,CLN/DNA significantly promoted the immunogenic death of CT26 cells.The tumor cells treated with CLN/DNA can be engulfed by BMDCs,which can improve the maturation and activation of DC_S.It has been proved that PEG end modification and DNA loading are the key factors that influence the effect of CLN/DNA on the immunogenic death of tumor cells.3.There was a strong interaction between CLN/DNA injected into tumor and CD45 tumor cells,and the activation level of DC_S in draining lymph nodes was increased.4.Intratumoral injection of CLN/DNA significantly increased the proportion of tumor infiltrating CD8⁺T cells and enhanced the anti-tumor immune response.At the same time,this treatment strategy also improves the tumor immunosuppressive microenvironment by reducing the percentage of tumor infiltrating Treg cells and increasing the proportion of M1/M2tumor-related macrophages.5.Intratumoral injection of CLN/DNA successfully induced systemic anti-tumor immune protection,effectively inhibited tumor growth in situ and distant tumor formation.This study provides an effective tumor immunotherapy strategy.Conclusion:In this paper,cationic lipid/DNA nanocomposites with the potential of tumor immunotherapy were prepared.We found that the nanoparticles can induce the immunogenic death of tumor cells,enhance DC activation,and enhance the anti-tumor immune response mediated by CD8⁺T cells.Moreover,the nanoparticles can also activate DCs cells.It is important that the injection of CLN/DNA into the tumor can produce systemic anti-tumor immune response and inhibit the injection site and distal tumor.