Interfacial Modified Salmonella For Enhanced Microbial Immunotherapy Of Tumor

Background and ObjectivesThe anaerobic and facultative anaerobes exhibit unique advantages for tumor immunotherapy owing to their excellent tumor-targeting ability and comprehensive immune regulation.They can effectively overcome the drawbacks of the current preparations,including poor tumor targeting,limited tumor penetration and incomplete immune regulation.Among the bacteria used for tumor therapy,Salmonella（Sal）stands out on account of the excellent chemotaxis effects towards tumor,convenience for genetic engineering and multiple anti-tumor mechanisms.The anti-tumor effect has been demonstrated at animal level but failed in clinical trials.The studies have shown that the immune clearance mediated by anti-infection immunity of host and immune barrier formed by neutrophils recruited by Sal are crucial factors that restrict its clinical efficacy.This study aims to break through the above bottleneck problems in the clinical application of Sal,make a new attempt for enhanced tumor immunotherapy and also provide reference for the development and clinical translation of tumor microbial therapy.Methods and ResultsTo address the above problems,“smart”bacteria was constructed by interfacial modification of Sal with tumor microenvironment-responsive materials,thus achieving“stealth”in the circulation,overcoming the immune clearance and then realizing tumor homing and in-situ activation.The shackle of immune barrier could be broken through by the loaded immune agonists,thus optimizing the in vivo application of Sal and realizing enhanced microbial immunotherapy.In the second chapter,Sal was coated with p H-sensitive degradable material polyserotonin（PST）based on the oxidative self-polymerization of 5-hydroxytryptamine（5-HT）under alkaline conditions,and the functional nuclei acids Dz MN composed of immune agonist PD-L1DNAzyme（Dz）were further adsorbed through multiple forces to form microbe/nanomedicine assembly system Sal@PST/Dz MN.It could protect Sal from rapid elimination in the circulation and achieve synergistic targeted delivery under the chemotactic effects of Sal.The PST was degraded in the slightly acidic tumor microenvironment,with Sal released,reactivated and reached the central region of tumor to exert their anti-tumor effects.The released Dz MN downregulated the immune checkpoint PD-L1 in the tumor periphery and blocked the immune escape of tumor.In view of the complementary and synergistic effect of Sal and Dz MN on the activation of anti-tumor immune responses,Sal and Dz MN showed a combined anti-tumor effect in B16F10 tumor-bearing mice model with good biosafety.In the third chapter,considering the multiple mechanisms of tumor immunosuppression,Sal was modified with Ca CO₃ through in-situ mineralization of Ca Cl₂ and Na HCO₃,with A23187 co-loaded to construct Sal@Ca CO₃/A23187 with multiple immune regulation effects.It could achieve“stealth”in the circulation,but release Ca²⁺in the slightly acidic tumor microenvironment,which flowed into immune cells under the mediation of A23187 and induced DCs maturation,macrophage polarization and T cells activation.Accompanied by tumor acidity neutralization by Ca CO₃ degradation and the immune regulation effects of Sal,the tumor immunosuppressive microenvironment could be reshaped from multiple dimensions.The robust anti-tumor immune responses were induced,which effectively broke through the shackle of immune barrier and realized in-situ immune sensitization.The combined anti-tumor effects of Sal,Ca²⁺influx and tumor acidity neutralization were found in B16F10 tumor-bearing mice model,which effectively inhibited the tumor growth and metastasis and showed good biosafety.ConclusionsIn this study,Sal was successfully engineered through interfacial modification,which shielded their surface antigen and reduced immune clearance in vivo.The immune agonists were targeted delivered to tumors capitalized on the chemotaxis effects of Sal,which reshaped the tumor immunosuppressive microenvironment from multiple dimensions,broke through the shackle of immune barrier and realized enhanced microbial immunotherapy.This study provided a potential solution and experimental evidence for the bottleneck problems in the clinical application of Sal.There are 111 figures,6 tables and 101 references in this thesis.