Ti₃C₂-MXene-Au Combining With αOX40 Based Photothermal/Catalysis/Immunotherapy For Triple-negative Breast Cancer

Background:Breast cancer remains one of the most common malignant tumors worldwide,the latest global data shows that breast cancer ranks first in morbidity of female malignant tumors.Breast cancer is molecularly heterogeneous and categorized into four molecular subtypes:Luminal-A、Luminal-B、HER2 and triple-negative breast cancer.Triple-negative breast cancer has the worst prognosis among all subtypes of breast cancer,the main treatments for which are surgery,radiotherapy,chemotherapy and targeted therapy.However,surgery has brought great trauma to women’s physiology and psychology.Chemotherapy treatment regimes with poor response and high toxicity,and the use of small-molecule drug targeted therapy also has certain limitations.Therefore,it is urgent to develop safe and effective targeted tumor therapy strategies.Immunotherapy holds great promise to cure tumors by mobilizing the body’s immune system and enhancing antitumor immunity.Immunotherapy has shown good therapeutic prospects and become a hot spot in tumor research.However,the low immunogenicity and immunosuppressive tumor microenvironment（TME）severely limit the efficacy of immunotherapy.The unique properties of PTT,such as inducing immunogenic death and activating antitumor immunity,provide new insights for tumor treatment.In the present study,we take advantage of the feature that photothermal therapy can induce immunogenic death,further combine it with immunotherapy to treat malignant tumors to explore more favorable and effective treatment strategy for triple-negative breast cancer.Photothermal therapy（PTT）is a promising emerging treatment that converts nanomaterials from light to heat energy and has shown immense prospects in preclinical and clinical trials.The two-dimensional transition metal carbon（nitrogen）compound（MXene）has the distant advantages of efficient photothermal conversion efficiency,thermal conductivity,hydrophilicity,large specific surface area and abundant surface modification groups and is widely used as photothermal agents to achieve efficient treatment.Nano-enzyme catalyzed therapy triggered by tumor-specific endogenous stimulus is a new tumor treatment method,showing a high application prospect in the process of tumor treatment.Nano-enzyme with peroxide-like activity has attracted much attention due to its ability to effectively produce reactive oxygen species.The gold nanoparticles have been proved to have the characteristics of peroxidase,with good photothermal conversion efficiency and chemical inertness,and have been widely used in diagnosis,imaging and therapy.Collectively,in this topic,we choose gold nanoparticles to modify the surface of MXene,which can play the photothermal effect and also play the peroxidase activity,so as to realize the strategy of photothermal synergistic enzyme therapy of tumor cells.In addition,in order to fully mobilize the body’s immune cells to exert anti-tumor effects,we introduce anti-OX40 antibody signaling molecules on the basis of photothermal synergistic enzyme therapy to further support T cells expansion and increase cytokines secretion,thus achieving effective treatment of triple-negative breast cancer.Purpose:To verify the synergistic therapeutic effect of photothermal/catalysis/immune therapy on triple-negative breast cancer in mice and provide a solid theoretical and reliable experimental basis for developing triple-negative breast cancer therapy.Method:（1）Ti₃C₂-MXene-Au was prepared and characterized by its physical and chemical properties.The photothermal conversion efficiency,stability,biocompatibility and enzyme-like activity of Ti₃C₂-MXene-Au were analyzed.（2）The killing effect of Ti₃C₂-MXene-Au nanocomposites on tumor cells by photothermal combined catalytic therapy and possible mechanisms were explored through experiments such as cell uptake,cell killing,reactive oxygen species and cell apoptosis.（3）The effect of Ti₃C₂-MXene-Au combined with antibody OX40 on tumor growth,biocompatibility,biological safety,targeting ability and the immune system was verified through a mouse orthotopic breast cancer model in vivo.Results:（1）The morphology,structure and zeta potential of Ti₃C₂-MXene-Au were characterized by TEM,SEM,UV-Vis and Zeta Sizer,which proved the successful preparation of Ti₃C₂-MXene-Au nanocomposites.（2）Stability test and hemolysis experiments showed that Ti₃C₂-MXene-Au had excellent stability,biocompatibility and safety.（3）Ti₃C₂-MXene-Au was verified as a photothermal agent for its high photothermal conversion efficiency of up to 55%,combined with concentration and power-dependent heating performance and photothermal stability.（4）Enzyme catalysis experiments confirmed that Ti₃C₂-MXene-Au had peroxidase-like characteristics,which could increase reactive oxygen species and apoptosis of tumor cells.（5）The ability of Ti₃C₂-MXene-Au to induce immunogenic death of 4T1 cells under 808 nm irradiation was confirmed by detecting immunogenic death-related molecules CRT,HMGB1 and ATP.（6）The great tumor-targeting ability of Ti₃C₂-MXene-Au was confirmed by photoacoustic imaging and photothermal imaging in vivo experiments.（7）The combination of photothermal,enzyme catalysis and immune therapy effectively inhibited tumor growth,activated antigen-presenting cells,stimulated the expansion and infiltration of effective T cells,and reduced the proportion of MDSCs through flow cytometry analysis after injection of Ti₃C₂-MXene-Au and OX40antibody in intraperitoneal.（8）H&E staining was used to detect the organ damage of the heart,liver,spleen,lung and kidney through venous blood collected from tumor-bearing mice,which verified that Ti₃C₂-MXene-Au has excellent biological safety.Conclusion:In summary,Ti₃C₂-MXene-Au has photothermal conversion properties and peroxidase-like characteristics,leading to immunogenic death and apoptosis of tumor cells.On this basis,the combination of OX40 antibody further strengthened the proliferation and activation of DC and T cells and reduced the myeloid-derived suppressor cells（MDSCs）,additionally reversed the immunosuppressive microenvironment and induced a significant anti-tumor immune response,which is a potential therapeutic strategy for triple-negative breast cancer.