Controllable Autophagy Cascade Amplification Polymeric Nanoparticles For Enhanced Tumor Immunotherapy

Due to tumor immunotherapy’s unique ability to stimulate or mobilize immune response,it has shown excellent performance in tumor treatment and received increasingly keen attention.The key of immunotherapy is to enhance the immunogenicity of tumors and improve the recognition ability of the body’s immune system to tumor cells.Immunogenic cell death（ICD）can improve the immunogenicity of tumors,promote the maturation of antigen presenting cells（APC）,activate cytotoxic T cells（CTLs）,and mediate antitumor immune response by enhancing the secretion of endogenous antigens.Autophagy is the phenomenon of cells’engulfing themselves,which is a metabolic process inherent in cells.It shows a unique double-edged sword effect in tumor treatment.Mild autophagy can protect tumor cells and resist external damage to tumor cells.However,when the level of autophagy exceeds the critical point,severe autophagy will lose its cyto-protective function,thus leading to autophagic cell death（ACD）and inhibiting tumor growth.There is a close relationship between autophagy and ICD.Epirubicin（EPI）,a commonly used chemotherapeutics in clinical practice,can not only kill tumor cells,but also lead to the ICD effect by inducing autophagy.And the level of autophagy is positively correlated with ICD effect.The problem is that EPI can only induce weakly protective autophagy,which reduces the efficacy of chemotherapy,and the ICD-mediated immune response is also difficult to meet the therapeutic needs.Based on this,a new anti-tumor drug delivery system was designed in this paper.It enhanced the EPI-induced autophagy and triggered ACD by adding autophagy inducer,so as to achieve the dual purpose of breaking through protective autophagy and enhancing ICD level for better anti-tumor effect.It was composed of hyaluronic acid（HA）.By the cysteamine containing disulfide bond,HA was connected to arginine（Arg）,polyethyleneglycol-polycaprolactone（PEG-PCL）and EPI.The polymer pro-drug AHPPE（Arg-HA-PEG-PCL/EPI）had triple functions:active tumor targeting,enhanced cellular uptake and sensitive REDOX response.The autophagy inducer STF-62247（STF）was wrapped into it to form the final STF@AHPPE nanoparticles.The HA coat could not only prevent protein adsorption and reduce hemolytic toxicity,but also bind to the CD44receptor highly expressed on the tumor cells’surface for active tumor targeting.After reaching the tumor site,Arg could assist the vehicle to enter tumor cells to promote effective carrier internalization and drug accumulation.Due to the high glutathione（GSH）concentration,the disulfide bond broke,and led to the release of EPI and STF@PP secondary nanoparticles.EPI induced mild autophagy and a certain degree of ICD.STF@PP secondary nanoparticles gradually released STF to increase autophagy level,induce ACD and enhanced ICD for stronger immunotherapy effect.The intermediates and final products of polymer materials were characterized by nuclear mangnetic resonance（¹H-NMR）and gel permeation chromatography（GPC）.The chemical structure,composition and molecular weight of the target molecule were confirmed,and the grafting rates of cysteamine,Arg,PEG-PCL and EPI were calculated.Finally,the successful synthesis of the target polymer AHPPE was proved.The STF@AHPPE drug-loaded nanoparticles were prepared by dialysis method and characterized by dynamic light scattering（DLS）,transmission electron microscopy（TEM）and ultraviolet–visible pectrophotometer（UV-Vis）.The particle size was 175.20±1.10nm,and the zeta potential was-14.90±0.20 m V.Their morphology was spherical and uniform under TEM.The drug loading efficiency of STF was 2.75%±0.01%.Meanwhile,they had good stability,REDOX sensitivity and drug release performance.Hemolytic experiments further proved their good biocompatibility.In vitro experiments,the half maximal inhibitory concentrations(IC₅₀)of AHPPE and STF@AHPPE on mouse colon cancer cell CT26 were 1.89μg/m L and 0.08μg/m L,respectively based on MTT assay.It was proved that the addition of autophagy inducer can significantly enhance antitumor effect of prodrug nanoparticles.The results of flow cytometry（FCM）,confocal laser scanning microscope（CLSM）and western blot（WB）showed that cell uptake increased with the extension of incubation time,and STF@AHPPE can effectively trigger ACD,and significantly amplifying ICD effect.In vivo experiments,mouse subcutaneously ectopic tumor model was established.In vivo imaging results showed that STF@AHPPE was effective enrichment in tumor sites and had good tumor targeting ability.Hematoxylin and eosin（H&E）staining result proved that it had good biosafety.It was obvious that STF@AHPPE had the best induction ability of autophagy,ICD and apoptosis,and the best tumor inhibition ability due to tumor sections’H&E staining,and immunohistochemical tests of TUNEL,Ki67,etc.Results of FCM and enzyme linked immunosorbent assay（ELISA）further demonstrated that STF@AHPPE can significantly stimulate immune response in mice.In conclusion,this study successfully constructed a novel anti-tumor drug delivery system STF@AHPPE,which integrated multiple strategies including tumor active targeting,REDOX response and autophagy cascade amplification,and provided a new method for enhanced tumor immunotherapy.