Study On MRNA Preparation Based On Lysate Of Lactobacillus Reuteri In The Treatment Of Mouse Colon Cancer

Objective: Colorectal cancer(CRC)is a common malignant tumor with a high incidence and fatality rate.Gene therapy provides a new means for tumor therapy by delivering therapeutic nucleic acid molecules to complement,silence and edit specific gene targets.mRNA based immunogene therapy can effectively induce anti-tumor immune response and tumor killing,which is a potential new treatment strategy for colon cancer.Lactobacillus reuteri is a member of the gut microbiota.Lactobacillus reuteri is directly related to the immune response and oxidative stress of tumors,and has potential anti-tumor immune stimulation ability.As a member of the interleukin family,IL-23 A can promote the proliferation of T cells,promote the production of IFN-γ by T cells and antigen-presenting cells,regulate the co-stimulatory function of dendritic cells,and play an important role in anti-tumor and anti-metastasis in treatment.In this study,based on non-viral nanocarrier DMP,we intend to co-deliver Lactobacillus reuteri lysates and IL-23 A mRNA to prepare an IL-23 A mRNA gene preparation that can stimulate local and systemic anti-tumor immune responses,and achieve a highly effective immune gene therapy effect on mouse colon cancer tumor models.In addition to characterizing the physicochemical properties and mRNA delivery ability of DMP-Lac,we will study its in vitro and in vivo immune stimulation ability and mechanism of action,and evaluate its therapeutic ability and safety on mouse colon cancer at the cellular and animal levels,so as to provide a new strategy for mRNA-based immune gene therapy for colon cancer.Methods: DMP-Lac was prepared by self-assembly and its physicochemical properties were characterized.The ability of DMP-Lac nanoparticles to deliver mRNA,lysosome escape,stimulate antigen-presenting cells and have antitumor effects in vitro were studied at the cellular level.After the ability of DMP-Lac nanoparticles to stimulate local and systemic immunity was verified in vivo experiments,a variety of mouse tumor models were established to verify the anti-tumor ability of DMP-Lac/IL-23 A complex in vivo,and the mechanism of DMP-Lac/IL-23 A complex to enhance immune response was further studied by protein chip technology.Results: In this study,cationic nanoparticles coated with Lactobacillus reuteri lysate were successfully constructed,and the encapsulation rate reached 34.44%.Cell experiments showed that nano granulocytes were less toxic,had higher mRNA delivery efficiency and protective ability,and entered cells mainly through lipid raft mediated and gridprotein mediated endocytosis pathway,and could effectively escape lysosome uptake.In vitro and in vivo immunostimulation experiments and anti-tumor experiments proved that DMP-Lac can effectively mobilize local and systemic immunity and inhibit tumor cell growth.DMP-Lac/IL-23 A complex can effectively inhibit tumor growth and promote cancer cell apoptosis through abdominal and lung metastasis models of colon cancer in mice,thus achieving the therapeutic purpose.Conclusion: In this study,DMP-Lac vector coated with lytic solution of Lactobacillus reuteri was successfully constructed,which could achieve co-delivery of bacterial lytic solution and IL-23 A mRNA,and was verified to have the ability to improve immunity and anti-tumor activity by in vitro and in vivo experiments.The mRNA vaccine based on Lactobacillus reuteri lysate has significant anti-tumor effect.It provides a new strategy for immunogene therapy of colon cancer.