A Study On Nir-responsive Upconverting Nanoparticles Mediated Apoptosis And Ferroptosis For Enhanced Melanoma Immunotherapy

Backgroud:Immunotherapies have greatly improved the survival rate of melanoma patients.However,patient response rates for tumor vaccines and oncolytic viruses remain low.High-dose interleukin-2 and adoptive T-cell therapy are poorly targeted and bring serious systemic side effects.Immune checkpoint blockades,as the preferred treatment for patients with metastatic melanoma,also suffer from drug resistance and a high incidence of immune-related adverse events.Although little is known about the mechanisms of immunotherapy resistance,available evidence points to a local lack of immune cells infiltration in melanoma.The"immune desert-type"inhibitory micro-environment is associated with low immunogenicity and insufficient activation of antigen-presenting cells.Therefore,it is urgent to develop a safe and efficient new strategy to improve the efficacy of melanoma immunotherapy.Photodynamic therapy（PDT）has been used for clinical treatment of skin tumors due to its features of less systemic toxicity,precise targeting of lesions,and less trauma.However,the limited penetration depth of visible light,melanin filtering,and resistance to apoptosis limit the application of PDT in melanoma.In recent years,ferroptosis is a new form of non-apoptotic cell death,which provides a new target for apoptosis-resistant tumor therapy.More importantly,ferroptosis is more immunogenic than apoptosis.In this study,combining the advantages of upconversion nanoparticles（UCNPs）excited by near-infrared（NIR）light to improve tissue penetration and reduce melanin light absorption,we synthesized UCNPs loaded with photosensitizer Ce6 and ferroptosis inducer BSO,named UCNP@m Si O₂@lipsome-Ce6-BSO nanoparticles（NPs）.By characterizing and identifying its physicochemical properties,evaluating its therapeutic effects,immune response effects,mechanisms of action and safety in vitro and in vivo,the aim is to provide new ideas for safe and efficient melanoma immunotherapy.Methods:In this study,core-shell upconversion nanoparticles were prepared by thermal decomposition method,while the DPBF indicator was used to evaluate their ability to generate reactive oxygen species（ROS）.First,the uptake of melanoma cells was detected by confocal microscopy and flow cytometry,and its dark toxicity and phototoxicity were determined by CCK-8 method and live dead cell staining.Subsequently,the ratio of apoptosis,the expression levels of apoptosis-related proteins,the content of glutathione（GSH）,the level of ROS,the degree of lipid peroxidation,the degree of glutathione peroxidase 4（GPX4）expression,the effect of inhibitors on cell viability and cell survival were compared in each group.Submicroscopic structure to explore the effect of UCB NPs-mediated PDT on the form of cell death.A melanoma mouse model was constructed,tumor tissue was collected for immunofluorescence and immunohistochemical staining at the end of the experiment to evaluate the anti-tumor effect and forms of cell death.Further,the degree of dendritic cells（DCs）maturation,T lymphocyte infiltration and activation in vitro and in vivo were compared.Subsequently,changes of damage-associated molecular patterns（DAMPs）were detected by western blot and flow cytometry.Finally,whole blood and serum of mice treated with UCB NPs for 2 weeks were collected.Blood routine,liver and kidney function and pathological changes of major organs were determined to evaluate the biological safety of UCB NPs.Results:UCB NPs have photoconversion ability,their emission peaks match the UV-vis absorption peaks of Ce6.UCB NPs efficiently generate ROS after being irradiated with 980nm light.The endocytosis of UCB NPs by melanoma cells was positively correlated with co-incubation time.UCB NPs had no obvious toxicity to fibroblasts and melanoma cells when not illuminated,but PDT effect was obvious after illuminated.Compared with the control group,the apoptosis ratio of UCB NPs treated with PDT increased,and expression of apoptosis-related proteins also up-regulated.At the same time,the content of GSH decreased,the production of reactive oxygen species increased,the degree of lipid peroxidation increased,and the expression of GPX4 down-regulated.In addition,the cell survival rate increased after ferroptosis inhibitor treatment,and mitochondria also shrunk under electron microscope.After 14 days of UCB NPs PDT treatment,the melanoma volume was the smallest,tumor weight was the lowest,and local TUNEL expression was the highest,while GPX4 expression was the lowest.In vitro and in vivo experiments showed that UCB NPs PDT treated tumor cells with increased high mobility group box 1（HMGB1）protein release and calreticulin（CRT）exposure,which promoting DCs maturation,increasing cytotoxic T lymphocytes infiltration and activation,enhancing anti-melanoma immune responses.In addition,compared with the control group,UCB NPs showed no significant difference in liver and kidney function,blood cell level and tissue H&E staining.Conclusion:UCB NPs are biocompatible nanocomposites that can greatly inhibit melanoma in vitro and in vivo.The drug-loaded platform utilized NIR light to penetrate deep into lesions with minimized light filitration of melanin,realizing the PDT treatment of melanoma.More importantly,Ce6-mediated PDT promoted ROS production,and BSO depletion of GSH downregulated GPX4-induced ferroptosis.The two interacted,boosting ferroptotic and apoptotic cell death.Effective tumor killing increased the exposure of DAMPs such as HMGB1 and CRT,improving immunogenicity.Further,it promoted the maturation of DCs,CTL infiltration and activation.This strategy of combining ferroptosis and apoptosis with PDT provides a new strategy for melanoma immunotherapy.