ES-MIONs Loaded With Sorafenib And Anti-TGF-β Antibody Induce Ferroptosis And Synergistically Improve The Tumor Microenvironment

The complex tumor microenvironment play a crucial role in growth and therapy of tumor.Studies have shown that ferroptosis is beneficial to tumor antigen presentation,and immunotherapy drugs such as anti-TGF-β antibodies can improve the function of immune cells in the tumor microenvironment.Therefore,we hypothesized that the combination of both may further improve the efficacy of tumor therapy.However,TGF-β is widely distributed in the body,how to accurately and effectively use ferroptosis and anti-TGF-β antibody synergistic improvement of tumor microenvironment to improve tumor therapeutic efficacy is still a thorny problem.With the development of nanomedicine,the application of nanomaterials in tumor diagnosis and treatment has achieved good results.There are currently few reports on therapeutic diagnostic nanomaterials.Therefore,it is necessary to construct novel therapeutic diagnostic nanomaterials to modify the tumor microenvironment in order to improve anti-tumor efficacy and clarify the mechanism.In this study,Fe3O4/Gd2O3 hybrid nanoparticles(FeGd-HN)were synthesized based on extremely small magnetic iron oxide nanoparticles(ES-MIONs),and successfully loaded with sorafenib by electrostatic interaction and grafted anti-TGF-βmonoclonal antibody and RGD peptide dimer(RGD2)utilizing EDC/NHS,which activate carboxyl groups to react with primary amine group,by which a novel type of tumor diagnostic treatment nanoparticle FeGd-HN@Sf@αTGF-β/RGD2 was successfully constructed.It is proposed to use a variety of molecular biology methods to clarify the therapeutic effect and mechanism of FeGd-HN@Sf@αTGF-β/RGD2 on tumors and the effect of FeGd-HN@Sf@αTGF-β/RGD2 on the tumor microenvironment by in vitro cell experiments and in vivo animal experiments.The T1-weighted MRI imaging capability of FeGd-HN@Sf@αTGF-β/RGD2 in vivo and in vitro was examined by nuclear magnetic resonance imaging(MRI).The main results are obtained as follows:1.FeGd-HN@Sf@αTGF-β/RGD2 is relatively stable and could target the cancer cellsFeGd-HN@Sf@αTGF-β/RGD2 was identified as a round nanometer particle with a diameter of about 10nm by transmission electron microscopy and DLS,and could be stably preserved in saline and water,suggesting that FeGd-HN@Sf@αTGFβ/RGD2 has good stability.After analyzing the expression of integrin αvβ3 in MC38,B16F10 and MFC,it was confirmed that FeGd-HN@Sf@αTGF-β/RGD2 could only be taken up by MC38 and B16F10 which have high expression of integrin αvβ3,showing good targeting.2.FeGd-HN@Sf@αTGF-β/RGD2 inhibit tumor growthIt was confirmed by MTT assay that FeGd-HN@Sf@αTGF-β/RGD2 could inhibit tumor growth in vitro,and that FeGd-HN@Sf@αTGF-β/RGD2 could inhibit tumor growth in vivo in subcutaneous tumor-bearing mouse model and lung metastasis mouse model.3.FeGd-HN@Sf@αTGF-β/RGD2 induce ferroptosis in cancer cellsBy MTT assay and mouse model,it was confirmed that inhibitors of ferroptosis can protect cancer cells from FeGd-HN@Sf@αTGF-β/RGD2 cytotoxicity in vitro and in vivo.After the uptake of FeGd-HN@Sf@αTGF-β/RGD2 nanoparticles,the volume of mitochondrial decreased,the density of bilayer membrane increased,and the mitochondrial crest decreased.The result of flow cycle and immunofluorescence showed that FeGd-HN@Sf@αTGF-β/RGD2 could increase the level of ROS and lipid peroxidation,accumulate lipid peroxidation products and decrease GSH level in cancer cells.We further demonstrated that FeGd-HN@Sf@αTGF-β/RGD2 inhibited the expression of cystine glutamate anti-transporters SLC7A11 and GPX4 in tumors in both cellular and animal levels and the process could be blocked by inhibitors of ferroptosis.4.FeGd-HN@Sf@αTGF-β/RGD2 can enhance the functions of DCs and T cells in TIME,and improve the tumor microenvironmentIn the in vitro co-culture model of tumor cells and DCs,tumor cells treated with FeGd-HN@Sf@αTGF-β/RGD2 are phagocytosed by DCs more easily and activate DCs more effectively.In the mice with subcutaneous tumor,the expression of HMGB1 was increased after FeGd-HN@Sf@αTGF-β/RGD2 treatment,DCs infiltrating in tumor microenvironment were activated,and the expression of antitumor molecules in T cells was up-regulated.5.FeGd-HN@Sf@αTGF-β/RGD2 can be used as T1-weighted MRI contrast agent for tumor imagingIn 3.0T and 7.0T imaging systems,FeGd-HN and FeGd-HN@Sf@αTGFβ/RGD2 are superior to existing contrast agents at T1-weighted MRI imaging.FeGdHN@Sf@αTGF-β/RGD2 can also be used as T1-weighted MRI contrast agent to image tumor sites at animal level.Main conclusions and significance:In this study,a sorafenib-containing ES-MIONs,FeGd-HN@Sf@αTGFβ/RGD2 were successfully constructed for the diagnosis and treatment of tumors.FeGd-HN@Sf@αTGF-β/RGD2 inhibits the SLC7A11-GSH-GPX4 signal axis,resulting in an imbalance in the intracellular oxidation-antioxidant system and accumulation of lipid peroxidation products,ultimately leading to ferroptosis in cancer cells.At the same time,HMGB1 released by cancer cells and the anti-TGF-βantibodies delivery to tumor,synergistically promote DCs maturation,recruit and activate T cells,realize the synergy between ferroptosis and immunotherapy,improve the tumor microenvironment,and provide a new and effective means for the simultaneous diagnosis and treatment of tumors.