Investigation On The Construction Of Intelligent Honeycomb-like Melanin Nanoassemblies With Variable Particle Size To Enhance Antitumor Chemoimmunotherapy

Currently,chemo-immunotherapy based on immunogenic cell death（ICD）has attracted much attention,which can promote the release of relevant antigens from tumor cells and activate the antitumor immune response.However,due to the influence of the tumor immunosuppressive microenvironment and the tumor extracellular stromal barrier,the delivery of ICD inducers to tumors alone cannot achieve the ideal immunotherapy effect.In tumor microenvironment（TME）,cancer-associated fibroblasts（CAFs）can not only secrete a variety of cytokines to promote the formation of immunosuppressive tumor microenvironment.Moreover,it can form a dense fibrotic interstitial barrier with other matrix components such as collagen,which affects the delivery of nanodrugs and tumor infiltration of immune effector cells,resulting in poor immunotherapy effects.It has been reported that fibroblast activation protein-α（FAP-α）,which is highly expressed by CAFs,has been used as an endogenous response signal to construct a nanodelivery system with particle size control to improve the delivery efficiency of nanodrugs.However,it is still insufficient in overcoming the immunosuppression of TME.Here,with CAFs as therapeutic targets,the development of a nanosystem with intelligent particle size controllable and multiple drug co-delivery for targeted delivery of TME modulators and ICD inducers is expected to produce better immunotherapy effects.In this study,the melanin nanoparticles（MNP）were modified with polyethylene glycol（PEG）and aminoethyl anisamide（AEAA）as the carrier to obtain nanoparticles APM with particle size of～20 nm.At the same time,doxorubicin（DOX）and trivalent ferric ion(Fe³⁺)were loaded on APM surface byπ-πconjugation and metal ion chelation,respectively,to obtain APM@Fe/DOX.Finally,the FAP-αspecifically severable polypeptide chain（Asp-Ala-Thr-Gly-Pro-Ala,Pep）was used as the linkage arm to chemically cross-link APM@Fe/DOX,and Losartan（LOS）was encapsulated in the mesopore formed by cross-linking.The honeycomb-like nanoassembly APM@Fe/DOX-LOS with a particle size of～90 nm was obtained.Once the nano-assembly is in contact with CAFs in TME,the Pep can be broken to achieve particle size shrinkage and convert to APM@Fe/DOX with smaller particle size.At the same time,LOS was released programmatically near blood vessels to degrade the collagen matrix and reshape TME.Subsequently,APM@Fe/DOX can penetrate deep into the tumor,release Fe3+and generate reactive oxide species（ROS）through fenton like reaction,and combine with DOX to enhance the ICD induction ability of tumor cells,promote the maturation of dendritic cells（DCs）,and trigger the body’s enhanced anti-tumor immune response.The results of FT-IR,UV-Vis,HPLC and GPC confirmed the successful construction of the carrier system Pep-APM and the successful loading of DOX,Fe³⁺and LOS.The results of transmission electron microscopy（TEM）and laser particle size analyzer showed that the particle size of APM was～20 nm.The Pep-APM@Fe/DOX-LOS obtained by Pep cross-linking were uniformly dispersed,with a particle size of～90 nm and a potential of+2.01±2.16 m V.The results of the BET results showed that the specific surface area of Pep-APM was 84.1 m²g^-1 and the average pore diameter was 8.88 nm.However,after LOS loading,the specific surface area and pore size decreased to 1.57 m²g^-1 and 6.33 nm,respectively.These results indicated that LOS was successfully loaded into the mesopore formed by Pep cross-linking.The results proved that this nanocluster could provide enough space binding sites for drug loading and had excellent drug loading capacity.The responsiveness of nanoformulations to FAP-α,the ability to penetrate tumor cells and the ability to induce ICD were investigated in vitro.The results of enzyme sensitivity test in vitro showed that the large-size Pep-APM nanocarrier could respond to FAP-αto achieve particle size shrinkage and convert to small-size APM.The results of drug release showed that Pep-APM@Fe/DOX-LOS could release LOS,DOX and Fe³⁺in a programmed manner under TME conditions.The results of permeability experiments on multicellular tumor spheres showed that Pep-APM@Fe/DOX-LOS can enhance the drug delivery capacity through particle size shrinkage and collagen degradation.Calreticulin（CRT）assay results showed that DOX and ROS could cooperatively enhance the expression of CRT on the surface of tumor cells,which would provide sufficient immune stimulation for the activation of anti-tumor immune response.Using 4T1 tumor-bearing mice as a model,the targeting property of nanoassemblies to tumor tissue was investigated in real time by small animal imaging technology.The results showed that the Pep-APM carrier system could prolong the half-life of blood circulation,and had strong tumor targeting and retention.Confocal laser scanning microscope（CLSM）was used to observe the penetration ability of nano-assembly into tumor tissue.It was found that Pep-APM could penetrate into deep tumor regions far from blood vessels.In addition,the flow cytometry results of Pep-APM distribution in different cells of tumor tissue showed that this nano-assembly has a preferential targeting ability to CAFs and tumor cells in tumor tissue.The above results proved that Pep-APM can anchor CAFs around tumor blood vessels to decompose into small-size nanoparticles,which can improve the penetration ability of drugs into deep tumor tissues.The results of pharmacodynamics evaluation of anti-tumor immunotherapy in 4T1tumor-bearing mice showed that Pep-APM@Fe/DOX-LOS could significantly inhibit tumor growth and also significantly prolonged the survival time of mice.The results of H&E staining and TUNEL staining showed that the apoptosis and necrosis of tumor cells were most obvious after Pep-APM@Fe/DOX-LOS treatment,and the apoptosis rate reached 89.3%.The changes of immune cells in TME after administration were detected by flow cytometry and immunofluorescence staining.The results showed that the Pep-APM@Fe/DOX-LOS treatment promoted the recruitment and maturation of DCs,increased the infiltration of cytotoxic T lymphocytes（CTLs）,CD4⁺T cells and natural killer cells（NKs）in TME,and decreased the proportion of immunosuppressive regulatory T cells（Tregs）and myeloid-derived suppressor cells（MDSCs）.These results suggest that Pep-APM@Fe/DOX-LOS therapy can enhance the induction of ICD of tumor cells,release a large number of tumor-associated antigens,and then induce the maturation of DCs,enhance the invasion and attack of immunoeffector cells to tumor,and inhibit the immunosuppressor cells,and produce a significant anti-tumor immunotherapy effect.The results of lung metastasis in mice showed that Pep-APM@Fe/DOX-LOS could induce systemic anti-tumor immune response and significantly inhibit lung metastasis from tumor lesions.Finally,the safety evaluation results of the nanopreparation proved that Pep-APM@Fe/DOX-LOS has good biocompatibility and safety.The remodeling effect of Pep-APM@Fe/DOX-LOS on TME was investigated by investigating the expression of cytokines and related proteins in TME of mice after treatment.The results showed that Pep-APM@Fe/DOX-LOS group could up-regulate the expression of interferon-γ（IFN-γ）,interleukin-6（IL-6）and tumor necrosis factor-α（TNF-α）in tumor tissues.The results showed that Pep-APM@Fe/DOX-LOS group could reverse the immunosuppressive TME by enhancing the secretion of immunoeffecting cytokines.Immunofluorescence staining results showed that the Pep-APM@Fe/DOX-LOS group raised the expression of the CRT and reduce the expression of CAFs,Collagen I,hypoxia inducing factor 1α（HIF-1α）and vascular endothelial growth factor（VEGF）in TME.These results indicate that Pep-APM@Fe/DOX-LOS can not only enhance the ICD induction ability of tumor cells,but also degrade the tumor interstitial barrier and improve tumor hypoxia to reshape TME.Western Blot results showed that Pep-APM@Fe/DOX-LOS could inhibit the activation of CAFs and inhibit the formation of immunosuppressive TME by reducing the expression of TGF-β.In conclusion,Pep-APM@Fe/DOX-LOS has sensitive responsiveness of FAP-α,which can not only enhance tumor penetration by achieving particle size shrinkage,but also inhibit the expression of CAFs,disrupt the extracellular stromal barrier and remodeling TME to promote the penetration and accumulation of nano-drugs in deep tumor sites.At the same time,Pep-APM@Fe/DOX-LOS can improve the induction of tumor ICD,promote the maturation of DCs,increase the tumor infiltration of T lymphocytes,and then play a killing effect on tumor cells,and effectively inhibit the growth and metastasis of tumor.