| Objective:Osteosarcoma is the most common primary malignant bone tumor with high malignancy and poor prognosis.At present,the traditional treatment of surgery combined with chemotherapy makes the average 5-year survival rate of osteosarcoma patients about 60%.Once recurrence or metastasis occurs,the 5-year survival rate drops significantly,and the long-term survival rate is less than 20%.There have been many studies on chemotherapy drugs and regimens in the past 20years,but no breakthrough has been achieved so far.Therefore,it is imperative to explore new treatment modalities for osteosarcoma therapy,especially for those with metastatic and recurrent tumors.Previous studies have shown that photothermal therapy（PTT）combined with immunotherapy based on nanomaterials is very promising in antitumor therapy in the future.In this study,Ti3C2 nanosheets,a photothermal agent,was modified with polyethylene glycol（PEG）and used as nanoplatforms loaded with OVA protein and manganese ions(Mn2+)to prepare the two-dimensional（2D）nanocomposites Ti3C2-PEG-OVA-Mn2+（TPOM）,possessing photothermal effect and immunogenicity.Further,we study on the effect and mechanism of this photothermal-triggered immunotherapy for osteosarcoma based on the TPOM nanocomposites.Methods:（1）Preparation of TPOM nanocomposite and its physicochemical properties.First,the Ti3C2 nanosheets were modified with polyethylene glycol with one amino group（PEG-NH2）,and the OVA protein was thiolated to adsorb Mn ions.Then the carboxyl groups on the OVA protein were activated by NHS/EDC,and then amide reaction occurred between carboxyl groups on the OVA and amino groups on the surface of Ti3C2-PEG-NH2 to synthesis TPOM nanocomposite.Morphology,structures,sizes and chemical properties of TPOM nanocomposite were confirmed by transmission electron microscopy（TEM）,atomic force microscopy（AFM）,X-ray diffraction（XRD）,particle size analysis,Zeta potential,X-ray photoelectron spectroscopy（XPS）,elemental mappings,magnetic resonance imaging（MRI）and Fourier transform infrared spectroscopy（FTIR）.The loading and release of Mn ions and OVA proteins in TPOM nanocomposite were detected by inductively coupled plasma mass spectrometry（ICP-MS）and BCA Protein Quantification Kit;The photothermal performance of TPOM nanocomposites were also studied by in vitro photothermal experiments and thermal imaging techniques.（2）In vitro study of cytotoxicity of TPOM nanocomposites and their activation of immune responses.The phagocytosis of TPOM nanocomposites by DCs was investigated by immunofluorescence staining（IF）.The in vitro cytotoxicity of TPOM nanocomposites and its antitumor effects under NIR irradiated were investigated by CCK8 assay and Living/Dead Staining Kit.Real-time quantitative polymerase chain reaction（RT-q PCR）and IF were performed to explore the effect of TPOM nanocomposites-mediated photothermal therapy on the release of mitochondrial DNA（mt-DNA）from tumor cells.Flow cytometry（FCM）,Western blot（WB）and enzyme-linked immunosorbent assay（ELISA）were used to explore the effect and specific mechanism of TPOM nanocomposites-mediated photothermal therapy on promoting maturation of DCs in vitro.（3）In vivo photothermal imaging and early activation of immune response by TPOM nanocomposites.First,a unilateral subcutaneous tumor model was constructed,and the photothermal therapy combined with immunotherapy was given only once.The in vivo photothermal effect and imaging ability of TPOM were studied by photothermal experiments in vivo and thermal imaging technology.The effects of TPOM-mediated photothermal therapy on promoting DCs maturation and T cell infiltration in vivo were further explored by FCM,ELISA and immunohistochemical staining（IHC）.Tumor growth inhibition and tumor apoptosis were explored by HE staining and TUNEL staining.（4）Inhibition of primary tumors and metastatic tumors by TPOM-mediated PTT combined with immunotherapy.First,a bilateral subcutaneous tumor model was constructed（tumors on the left side as the primary tumor,and tumors on the right which were delayed injected as the metastatic tumors）,and the combination therapy was given twice to the tumor on the left side.Then the inhibitory effect of tumor growth was assessed by recording the size of bilateral tumors.The infiltration of cytotoxic T cells（CTLs）and T helper cells（Th）in the tumor site were analyzed by FCM and IHC.The infiltration of NK cells in the tumor site was explored by immunofluorescence staining.（5）The potential side effect of TPOM nanocomposites to major organs was evaluated by blood biochemistry,blood routine examination and HE staining of major organs.Besides,the pharmacokinetic changes of TPOM in vivo were also studied by ICP-MS.Results:（1）The as-prepared Ti3C2 has a single-layer sheet-like structure and has good dispersibility in aqueous solution.The zeta potential of TPOM nanocomposites was-2.27,and the particle size was about 200 nm.Different characterizations were performed to confirm the loading of OVA protein and Mn ions to TPOM nanocomposites.And the TPOM nanocomposites retained the excellent photothermal properties of Ti3C2,with ideal photothermal conversion efficiency and photothermal stability.（2）The pure TPOM nanocomposites showed good biocompatibility that even if the concentration reached 400μg m L-1,there is no significant cytotoxicity to be observed（co-culture for 24 hours）.A significant antitumor effect was observed when TPOM combined with Near-infrared（NIR）laser irradiation,and the antitumor effect was in proportion to the concentration of TPOM,NIR power density and time of irradiation.It was shown in phagocytosis experiments that DCs could uptake significantly more nanoparticles which were irradiated by NIR before.It was also observed that TPOM-mediated PTT could promote the release of mt-DNA of tumor cells to cytoplasm and extracellular matrix.On the one hand,adaptive immunity is activated by OVA and tumor antigen;on the other hand,innate immunity is activated by up-regulating the STING pathway by mt-DNA and Mn2+,which ultimately promoted the maturation of DCs and the release of IL-6 and TNF-α.（3）Photothermal-triggered immunotherapy mediated by TPOM nanocomposites could effectively activate the early antitumor immune response,which is mainly manifested in the increased proportion of mature DCs in mouse lymph nodes and the expression of IL-6,IL-12 and TNF-αin the circulation.Hence,the CTLs and Th infiltrating in tumor site increased significantly.Besides,tumor growth was obviously inhibited and the proportion of apoptosis was significantly increased.（4）Photothermal-triggered immunotherapy mediated by TPOM nanocomposites not only effectively inhibited the primary tumor,but also had a significant inhibitory effect on distant tumors,and the infiltration of CTLs and Th cells in the distant tumors were significantly enhanced.Furthermore,the infiltration of NK cells,the representative of innate immunity,in tumor sites was also significantly increased.（5）No significant liver and kidney toxicity was observed,and there were no obvious damages to major organs from H&E staining.Besides,TPOM nanocomposites could be eliminated in time in vivo,indicating great biosafety and clinical application potentialConclusion:This study proposed a novel photothermal-triggered immunotherapy that could induce the release of mt-DNA from tumor cells,as well as the release of OVA protein and Mn2+from TPOM.The tumor antigens and OVA could significantly enhance adaptive immune response.In the meantime,mt-DNA and Mn2+synergistically up-regulated the expression of STING pathway-related proteins to activate the innate immune response,which ultimately promoted the maturation of DCs,ultimately increasing the infiltration of CTLs and Th cells in tumor sites.The proposed photothermal-triggered immunotherapy mediated by TPOM not only effectively inhibited the primary tumor,but also had a significant inhibitory effect on distant tumors.In conclusion,the combinational therapy mediated by TPOM nanocomposites has important scientific importance and great application prospects in the treatment of osteosarcoma. |
