Construction Of Functionalized Inorganic Nano-drug Carrier And Their Activation Of Tumor Immune Response And Ferroptosis

Malignant tumor is one of the most important diseases threatening the life and health of all human beings.So far,the basic and clinical research on the treatment of malignant tumor still faces enormous challenges.TIME is the main cause of tumor immune failure.Therefore,the release of immunosuppression is the main method to improve immunotherapy.Ferroptosis is a novel tumor cell death mode,and induction of ferroptosis has a promoting effect on tumor immunotherapy.Therefore,how to efficiently induce ferroptosis in tumor cells is expected to overcome the limitations of traditional treatments.Benefiting from the enhanced osmotic retention effect（EPR）of solid tumors,nanomaterials have been developed as nanodrug delivery systems with long blood circulation time and tumor targeting.Inorganic nanomaterials have an intrinsic regulatory effect on the tumor immunosuppressive microenvironment due to their own immunomodulatory properties,and the unique physical catalytic activity of inorganic nanoparticles can regulate the intracellular ferroptosis signaling pathway and induce ferroptosis in tumor cells.In addition,inorganic nanoparticles are relatively stable and can be designed into responsive drug delivery systems for specific tumor microenvironments,which have obvious advantages in releasing immunosuppression,activating immune responses and ferroptosis.In summary,based on the part of the inorganic nanoparticles itself superior ability,immune regulation and induced ferroptosis has prepared the 5 kinds of inorganic nano-drug carrier,respectively,from the simple immunotherapy,immunotherapy combined with chemotherapy,ferroptosis,ferroptosis combined with immunotherapy,used in regulating tumor immunosuppression microenvironment,activating the immune response and inducing ferroptosis and then nanoparticle-related tumor immunological effects were evaluated.The main contents and findings of this paper are as follows:（1）Construction of hollow mesoporous Fe₃O₄ nanodrug carriers for tumor immunotherapy activation.Based on the immunomodulatory activity of Fe₃O₄ nanoparticles,we synthesized hollow mesoporous Fe₃O₄ nanoparticles（PHNPs）and loaded with PI3Kγsmall molecule inhibitors（3-MA）.Bovine serum albumin（BSA）was used to block PHNPs mesoporous structures,and carbonyl mannose was used to modify PHNPs to target macrophages.At the same time,considering the high GSH characteristics of macrophages,disulfide bonds were introduced into the nanomaterials during the modification process to achieve GSH responsive release of 3-MA.The results indicate that we have successfully prepared the hollow mesoporous Fe₃O₄ controlled release vector for nanomaterials,which can target the mannose molecules to macrophages,and then release the small molecule inhibitor 3-MA in response to the high concentration of GSH in macrophages.Western Blotting experiments showed that the nano-drug carrier could inhibit PI3Kγexpression and activate NF-κB P65 protein with 3-MA,while Fe₃O₄ nanoparticles could also activate NF-κB P65 protein.Flow cytometry（FCM）detection results showed that the drug carrier could regulate the above signaling pathways,and polarize the macrophages phenotype to M1-type.In vivo experiments on animal subcutaneous tumors demonstrated that the nanodrug carrier could repolarize the TAMs phenotype in vivo and,by activating immunotherapy,slow the growth of tumor cells and prolong the survival time of tumor bearing mice.（2）Construction of cascade-responsive nano-drug platform for combined anti-tumor therapy with chemotherapy and immunotherapy.To improve the killing effect on tumor cells,we have developed a combined anti-tumor strategy of chemotherapy and immunotherapy.Based on the tumor microenvironment,we synthesized a GSH-responsive hollow mesoporous silica-based Nanocarrier for loading HCPT drug and further immobilizing BSA on its surface for efficient encapsulation.To load the siRNA that inhibits the expression of MCT4,the surface of the BSA-encapsulated nanocarriers was modified with strongly positively charged PEI through amide bond coupling.Furthermore,we reacted HCPT/siRNA-loaded nanoplatforms with weakly acid-responsive PEG fragments through a similar reaction pathway to enhance their stability during blood circulation and endow the nanodrug carriers with specific charge-reversal ability to promote tumor cell uptake.In vitro cell and co-culture experiments,FCM and CLSM experiments confirmed that the nanocarrier platform can achieve repolarized TAMs phenotype and potent killing effect on tumor cells by inhibiting lactate efflux.Finally,the effects of the nanocapheresis platform on inhibiting lactic acid efflux,repolarization of TAMs phenotype and removal of tumor immunosuppressive microenvironment were revealed in a small animal subcutaneous tumor model,and the effects of the nanocapheresis platform on tumor growth through chemotherapy and immunotherapy were verified in vivo.（3）Construction of functionalized tumor-targeting nanosheets and study on the activation of tumor ferroptosis.Based on the resistance of malignant tumor cells to traditional death modes,we developed a new cell death mode-ferroptosis.How to efficiently induce ferroptosis in tumor cells has become an urgent problem to be solved.We developed a way to efficiently activate ferroptosis from both classical and non-canonical pathways.A surfactant-assisted approach was used to improve on previous studies,and two-dimensional（2D）nanosheets（Cu-Hemin）were formed through the synergistic effect of the carboxyl groups on Cu（Ⅱ）and Hemin.The nanosheets possess the ability of acid-induced degradation and GSH depletion.Furthermore,the tumor-targeting nanosheets exhibited excellent targeting ability to HepG2 cells in vitro.After uptake by HepG2 cells,acid degradation of Cu-hemin-PEG-LA in lysosomes released Hemin and Cu（Ⅱ）.In vitro cellular experiments confirmed that Cu（Ⅱ）and Hemin can efficiently induce ferroptosis from two modes by confocal,flow and Western Blotting experiments:the classical mode,where Cu（Ⅱ）depletes intracellular GSH by converting it to GSSG content.Depletion of intracellular GSH content can further downregulate the expression of GPX4 protein,thereby inducing ferroptosis.In a non-classical mode,released Hemin significantly upregulated the expression of HMOX1 protein and exhibited intracellular Fe²⁺overload.A small animal hypodermic tumor model was used to reveal the effect of the nanosheets on activating ferroptosis,validating the antitumor growth and biocompatibility effects of the nanosheets in vivo.（4）Construction of multifunctional bioenzyme nanosheet drug carrier and study on activation of tumor ferroptosis.To improve the sensitivity of tumor cells to ferroptosis,we developed a method to destroy tumor cells GPX4/GSH and FSP1/Co Q10 signaling pathway,sensitizes ferroptosis.The nano-drug delivery system used Cu（Ⅱ）and MESO-tetrakis（4-carboxyphenyl）porphine ferric chloride（TCPP（Fe））to synthesize MOF nanosheets as substrate materials,and is synthesized by in situ synthesis of Au NPs and viaπ-πstacking was loaded with the ferroptosis inducer RSL3,and then the nanosheet surface was modified with PEG fragments with long blood circulation time and tumor-targeted iRGD ligands（Au/Cu-TCPP（Fe）@RSL3-PEG-iRGD）.In vitro cell experiments confirmed that after the targeted composite nanosheets were taken up,the nanosheets could affect the GPX4 and FSP1 ferroptosis regulation pathways in tumor cells by consuming intracellular glucose and GSH by FCM,CLSM and related kit detection.In addition,the nanozyme drug delivery system can release RSL3 throughπ-πstacking in acidic lysosomes,which bind to the catalytic selenocysteine residue of GPX4,further destroying its anti-ferroptosis ability.The in vivo small animal subcutaneous tumor model revealed the effect of the biomimetic bioenzyme nanosheet drug delivery system in activating ferroptosis in vivo,and verified its antitumor efficacy and biocompatibility in vivo.（5）Construction of tumor-targeted nanoreactor drug carrier and study on activation of tumor immunotherapy and ferroptosis.Activation of ferroptosis alone lacks long-term efficacy in malignancies,so we developed a combined anti-tumor strategy of ferroptosis and immunotherapy.We developed a nanoreactor Cu_2-xSe that overcomes tumor cell hypoxia,sensitizes ferroptosis and repolarizes the TAMs phenotype.The reaction of Cu⁺and H₂O₂ is used to generate O₂ and Cu²⁺,and at the same time,Cu²⁺reacts with GSH to generate Cu⁺and GSSG,so that the mutual conversion of Cu elements between Cu⁺and Cu²⁺is used to realize amplifying the activity of catalyzing the generation of O₂ and the consumption of GSH.Finally,the ability to relieve tumor hypoxia and induce ferroptosis was achieved.In addition,to increase the ability of tumor cells to undergo ferroptosis,we modified ZIF-8 coating on the outer layer of Cu_2-xSe and loaded the ferroptosis activator Erastin for induction of ferroptosis.ferroptosis,while increasing the expression of intracellular NOX4 protein and increasing the content of endogenous H₂O₂ for the catalyzed production of O₂ by Cu_2-xSe.In order to prolong the blood circulation time and tumor specificity of the nanoreactor,we modified PEG and FA molecules on the surface of Cu_2-xSe/ZIF-8@Era,and completed the construction of tumor-targeted nanoreactor drug carrier,Cu_2-xSe/ZIF-8@Era-PEG-FA.In vitro cell experiments,co-culture experiments and Western Blotting experiments confirmed that Erastin can activate the expression of intracellular NOX4 protein and increase the H₂O₂ content of tumor cells.At the same time,Cu+can generate Cu²⁺and O₂ under the catalysis of H₂O₂,and Cu²⁺can consume intracellular GSH.Generate GSSG and Cu+to increase O₂content and consume GSH content.Increased O₂ content can reduce the expression of HIF-1α,reduce lipid ROS consumption caused by HIF-1α,and inhibit the expression of intracellular PTEN/PI3Kγin TAMs,and repolarize the phenotype of TAMs to M1-type.The small animal subcutaneous tumor model revealed the effect of nanoreactors on repolarizing the TAMs phenotype,releasing the immunosuppressive microenvironment,increasing the infiltration of IFNγ⁺CD8⁺T cells,and verifying its antitumor immune response and biocompatibility in vivo.