Engineered Highly Immunogenic Tumor Cell Vaccine Integrated Dissolving Microneedle Patch In Combination With Autophagy Regulation For Robust Antitumor Immunotherapy

Tumor immunotherapy refers to the application of immunological principles and methods,by activating immune cells in the body and enhancing the antitumor immune response,specifically removing the minimal residual tumor lesions,inhibiting tumor growth,and breaking immune tolerance.Tumor immunotherapy involves the process of anti-tumor immune cycle,in which tumor antigens and antigen presentation are the key to start effective anti-tumor immune cycle.However,the tumor immunosuppressive microenvironment severely limits the immunogenicity of tumor cells and the antigen presentation ability of antigen-presenting cells(APCs),resulting in the low efficiency of anti-tumor immunity.Tumor cell vaccines(TCVs)express a comprehensive set of characterized and uncharacterized tumor-associated antigens(TAAs),have been proved to show obvious advantages in tumor immunotherapy.Photothermal therapy(PTT)can eliminate pathogenicity and enhance the immunogenicity of tumor cells by inducing immunogenic cell death(ICD),which is a simple and feasible strategy to prepare highly immunogenic TCVs for starting effective anti-tumor immune cycle.Dendritic cells(DCs)are professional antigen-presenting cells with the strongest antigen-presenting ability in vivo,and have long been the key target of tumor immunotherapy research.The maturity and antigen-presenting ability of infiltrating DCs in the tumor microenvironment are significant for antitumor immune responses.Studies have shown that high levels of endocytic and lysosomal activity are required for antigen presentation by DCs,both of which are closely related to autophagy.Regulating the level of autophagy in DCs is expected to relieve the inhibition of the tumor microenvironment on the function of DCs and enhance their antigen-presenting ability.Dissolving microneedles(DMNs)for the delivery of vaccines in a painless and minimally invasive transdermal administration method have several advantages:(1)The skin contains a large number of immune cells,and DMNs can directly deliver the loaded drugs to immune cells,thereby effectively activating the immune response.(2)Deep vaccine delivery in superficial skin tumors can be achieved,overcoming the problem of poor penetration of solid tumors.(3)DMNs can maintain the stability and effectiveness of the drug during storage and delivery,simplifying the supply chain.Herein,a novel highly immunogenic dissolving microneedle(TCV-DMNs)patch was designed as a cancer vaccine to synergistically elicit a robust immune response by the intralesional co-delivery of highly immunogenic TCVs,granulocyte-macrophage colony-stimulating factor(GM-CSF),and autophagy promoter(Tat-beclin 1).After being directly inserted into the skin,the encapsulated cargoes were gradually released into the skin site accompanied by the dissolving of DMNs.This system worked first by recruiting DCs to the administration site through GM-CSF.Subsequently,the highly immunogenic TCVs were effectively phagocytosed,processed,and presented to T cells,thus inducing an antitumor immune response in the body.Tat-beclin 1 further promoted the antigen presentation ability of DCs by upregulating the level of autophagy.To prepare an innovative DMNs vaccine patch,melanoma cells with high expression of TAAs and complete cell structure,namely TCVs,were induced by photosensitizer IR780micelles combined with NIR irradiation.m Cherry-e GFP-LC3 plasmid transfection,Western blot,and transmission electron microscopy showed that the autophagy level of DCs was significantly upregulated after being treated by TCVs combined with Tat-beclin 1.The transport process of TCVs in DCs was tracked by laser scanning confocal microscope.The results showed that DCs ingested TCVs through phagocytosis,and then presented TCVs through lysosome,endoplasmic reticulum,and Golgi apparatus,which was consistent with the antigen cross-presentation pathway of DCs.The co-localization of TCVs and autolysosome showed that autophagy was involved in the antigen cross-presentation process of DCs.The results of flow cytometry showed that TCVs and Tat-beclin 1 synergistically stimulated the maturation of DCs and upregulated the antigen cross-presentation ability of DCs.In this study,TCVs,Tat-beclin 1,and GM-CSF were co-loaded into DMNs by a micromold method,and TCV-DMNs with hyaluronate acid(HA)as needle tip material and PVP K90 as base material were prepared.Per TCV-DMNs prepared contained 3～4×10~5TCVs.In vitro rat skin penetration and dissolution experiments showed that TCV-DMNs had a good mechanical property and solubility.SDS-PAGE results showed that DMNs could maintain the stability of TAAs of TCVs after loaded in TCV-DMNs.The results of transmission electron microscopy showed that the TCVs produced after the dissolution of TCV-DMNs still had a complete cell structure.The local accumulation and biological distribution of TCVs after TCV-DMNs pricking were explored through the small animal imaging experiments.TCVs first gathered at the administration site,then gradually spread and migrated to lymph nodes.TCV-DMNs successfully recruited DCs to the administration site and activated DCs at the same time.The in vivo prophylactic experiments showed that TCVs in synergy with Tat-beclin 1 could effectively activate the immune system and resist the subsequent melanoma attack by promoting the maturation of DCs,upregulating the antigen presentation ability of DCs,and activating T lymphocytes,with the tumor control rate as high as 92%.In vivo antitumor experiments showed that this synergistic strategy led to regression of large and established melanoma,followed by a relapse-free survival of more than 40 days via activating DCs,upregulating antigen presentation ability of DCs,promoting secretion of immunostimulatory cytokines,and increasing tumor infiltration of CD8~+T cells.In addition,compared with in situ PTT induced TCVs,direct intratumoral delivery of TCVs using DMNs had greater advantages in inducing strong antitumor immunity and ensuring bio-safety.To sum up,TCV-DMNs loaded with TCVs,Tat-beclin 1,and GM-CSF with HA as the tip material and PVP K90 as the base material were constructed in this research.It was intended to initiate an effective Cancer-Immunity Cycle and induced the body to produce a strong antitumor immune response by solving the problems of insufficient immunogenicity of tumor cells and low antigen presentation ability of DCs.The ICD effect of melanoma cells was induced by PTT to produce highly immunogenic TCVs,which aimed to effectively start the Cancer-Immunity Cycle.In addition,the autophagy strategy was used to upregulate the antigen cross-presentation ability of DCs and further promote the occurrence of the Cancer-Immunity Cycle.Overall,this work provided a versatile,generalizable framework for employing DMNs-mediated vaccination to initiate robust antitumor immunity,which was attributed to the enhanced immunogenicity of TCVs,elevated antigen cross-presentation ability of DCs by upregulating autophagy,and the superior ability of transdermal MNs that directly targeted vaccines to immune cells.