Autophagy Cascade Regulating Nanoparticles For Boosting Oxaliplatin-Induced Anti-Tumor Immunity

Some specific chemotherapeutic drugs(such as anthraquinone drugs,oxaliplatin and cyclophosphamide)can trigger immunogenic cell death(ICD)of tumor cells and cause the release of tumor antigen and damage associated molecular patterns(DAMPs),thus activating anti-tumor immune system to kill residual cancer cells,which present a new way for the optimization of chemotherapy.However,the monotherapy of chemotherapeutic agent can only induce weak immune responses,and new methods are needed to enhance their immune stimulation effect.Autophagy plays an important roles in promoting the chemotherapy-induced immunogenic death of tumors.In dying tumor cells,autophagy isolates tumor-associated proteins and delivers them into lysosomes,where the tumor components are processed into antigen peptides and then loaded onto major histocompatibility complex-Ⅰ(MHC-Ⅰ).The antigen-MHC-Ⅰcan then be presented to CD8~+T cells.In addition,autophagy is also involved in the extracellular release of damage associated molecular patterns such as ATP from dying tumor cells.The released ATP can bind to P2RY2 receptors on the surface of dendritic cells and recruit them into the tumor site.Therefore,in the process of tumor immunogenic death and activation of immunity,activation of autophagy is conducive for tumor antigens processing and immune cell recruitment.However,for a specific tumor cell,there is a contradiction in autophagy activation.When autophagy is“mildly”activated,the“mildly activated autophagy”will not cause tumor death.On the contrary,it will clear chemotherapy-induced damaged proteins and organelles,thus assist tumor cells to resist drug therapy and play a protective role.However,when autophagy is over activated and exceeds the critical point,the“over-activated autophagy”will cooperate with chemotherapeutics to kill the tumor,and also participate in the antigen presentation process of dying cells.Unfortunately,chemotherapeutic drugs are not powerful autophagy activators,and can only stimulate autophagy to the"mildly activated"stage rather than the"over-activated"stage,makeing autophagy to exhibit its cyto-protective roles instead of cytotoxic roles.Therefore,when OXA is adopted to induce the immunogenic death of tumor cells,timely and accurately leading autophagy level to the over activated stage could not only kill tumor cells more efficiently by inducing the autophagic death,but also enhance the subsequent antigen processing and DAMPs exposing behaviours.In order to solve this problem,we proposed the"on-demand"autophagy cascade amplification strategy and constructed the tumor GSH and autophagy level dual-responsive"core-shell"nanoparticle ASN.The core of ASN is autophagy-sensitive amphiphilic block micelle(C-TFG micelle),whose hydrophobic fragment is cholesterol and hydrophilic fragment is TFG peptide(sequence is GTFGFRRRRRRRR),a specific substrate of autophagy enzyme(Atg4).The autophagy-inducing drug STF-62247 was loaded into the core of the C-TFG micelle.The shell of ASN is hyaluronic acid-oxaliplatin prodrug(HA-OXA),which is adsorbed on the surface of C-TFG micelle through electrostatic adsorption.When ASN enters tumor cells,the HA-OXA prodrug shell is first stripped by hyaluronidase,and free OXA is released due to high concentration of reductive GSH in tumor cells.OXA can cause tumor cells immunogenic death and slightly activate autophagy.Then,C-TFG micelles can respond to the OXA-stimulated autophagy level and disintegrate,realizing the"on-demand"release of autophagy inducer STF-62247.The release of STF-62247 further activates autophagy and leading tumor cells to the stage of autophagic death,as well as enhancing the exposure of tumor antigens from dyingcells.In contrast,in normal cells with low autophagy levels,C-TFG micelles keep stable,thus allowing it to penetrate into another target tumor cells.In CT26 tumor-bearing mice,ASN group recruited the highest percent of immature dendritic cells into tumors site,with 2.19 and 1.53-fold of that of free OXA and OXA/STF groups,respectively.ASN group also caused the highest number of CD4~+T cells and CD8~+T cells in spleen than other treatment groups.ASN not only triggers the immunogenic death of tumor cells,but also promotes the release of immune-stimulating molecules of dying tumor cells through the precise regulation of autophagy level in tumor cells.However,ASN also has its disadvantages,which are mainly reflected in the fact that ASN only modulate tumor cells,but lacks the regulation of immune cells.As we know,in the process of chemoimmunotherapy,tumor fragments and DAMPs such as CRT,HMGB1 and ATP released from tumors are only the first step of immune initiation.Subsequently,dendritic cells(DCs)are required to recruit to tumor site,engulf tumor fragments,and present tumor antigen to T cells.Therefore,when we use OXA to kill tumor cells,timely stimulating the antigen-presenting ability of dendritic cells is expected to activate more potent anti-tumor immunity from multiple dimensions.Dendritic cells are the most powerful antigen presenting cells,and autophagy plays a key role in their uptake,processing,and presentation of tumor antigens.After dendritic cells take up exogenous tumor antigens,autophagy is involved in wrapping tumor-associated proteins and delivering them to lysosomes,processing tumor components into antigens,and binding antigen peptides to MHC molecules.Studies have shown that autophagy can promote the presentation of antigen-MHC-Ⅱcomplex to CD4~+T cells.Therefore,moderate activation of autophagy can enhance anti-tumor immunity.After tumor formation,the first site of metastasis was tumor-draining lymph nodes(TDLNs).TDLN is the first place where immune cells such as dendritic cells migrate.Here,dendritic cells engulf tumor antigens,process the antigen into peptides and finally present them to CD4~+and CD8~+T cells with the assistance of MHC-Ⅱmolecules.Therefore,for the purpose of accurately enhance autophagy of anti-tumor related immune cells,it is necessary to deliver the autophagy activator to TDLN.Endogenous albumin in tumor microenvironment usually flows back to TDLN through lymphatic vessels,while some lipid materials(such as DSPE,etc.)can be adsorbed on endogenous albumin and thus flow to TDLN.This physiological feature provides an important method for TDLN targeting.Based on the above analysis,in the second part of this project we constructed POR/DT micelle which is consist of two prodrugs that could not only deliver chemotherapy drugs to tumor sites and induce tumor immunogenic death,but also deliver autophagy inducer to tumor draining lymph nodes and activate immunity.The micelle system consists of two block copolymers,one is the PBAE-OXA-R8d GR copolymer(named POR),and the other is DSPE-PEG-Trehalose(named DSPE-PEG-TRE or DT).POR/DT micelles could reach the tumor site passively through EPR effect.Upon arrival at the pH 6.8 tumor microenvironment,the hydrophobic fragment PBAE of PBAE-OXA-R8d GR was protonated and changed from hydrophobic to hydrophilic,resulting in the rapid disintegration of the POR/DT micelles.After the disintegration of the POR/DT,on the one hand,the released PBAE-OXA-R8d GR could actively target to tumor cells through R8d GR which can recognize integrin receptor,and then release OXA to induce tumor cells immunogenic death.At the same time,the exposed DSPE-PEG-TRE prodrug can be adsorbed onto hydrophobic area of endogenous albumin,thus flow to draining lymph nodes.In TDLN,trehalose can gently activate autophagy of DC and thus promote their tumor antigen presenting ability.To sum up,the first part of this project designed"on-demand"autophagy cascade amplification nanoparticle ASN.Through accurately over-activating tumor autophagy,ASN not only increased the OXA-induced tumor death,but also promoted the release of immune stimulating molecules of dying cancer cells.In the second part of this project,we propose therapeutic strategies of synergistically inducing tumor immunogenic death and immune system autophagy.Based on EPR effect,tumor microenvironment and lymphatic reflux characteristics of tumor endogenous albumin,oxaliplatin and trehalose were innovatively delivered to tumor cells and tumor draining lymph nodes respectively through POR/DT micelle.POR/DT micelle can not only cause the release of tumor cell antigens,but also promote the antigen-presenting ability of dendritic cells in the tumor drainage lymph nodes by elevate their autophagy level.This strategy provides a new therapeutic strategy and drug delivery system for enhancing the immune stimulation ability of chemotherapy drugs,which has important research value and potential clinical significance.