A nano-immunomodulator F@PDA@I and its application in tumor immunotherapy

Abstract

The present invention belongs to the field of tumor immunotherapy, and specifically relates to a nano-immunomodulator F@PDA@I and its application in tumor immunotherapy, wherein the nano-immunomodulator integrates the activities of superoxide dismutase (SOD), catalase (CAT), peroxidase (POD) and glutathione oxidase (GSHOx). Photodynamic therapy (PDT) and photothermal therapy (PTT) are used to achieve tumor immunotherapy. The multi-enzyme-like activity not only contributes to the generation of oxygen to relieve hypoxia and promotes the polarization of M2 to M1 macrophages, but also consumes GSH in the TME to promote PDT and induce ferroptosis. As a result, a large amount of necrotic debris is released, which ultimately promotes the maturation of dendritic cells and the infiltration of cytotoxic T lymphocytes, thereby reversing the immunosuppressive TME. In in vivo experiments, F@PDA@I is combined with a Toll-like receptor agonist for synergistic immunotherapy against primary and distal tumors.

Description

Technical Field

[0001] The present invention belongs to the field of tumor immunotherapy, and specifically relates to a nano-immunomodulator F@PDA@I and its application in tumor immunotherapy. Background Art

[0002] Tumor immunotherapy has become one of the most promising treatment modalities and has been successfully applied in various advanced and refractory tumors. Photodynamic therapy (PDT) can induce the generation of cytotoxic ROS, leading to apoptosis and necrosis of tumor cells. These therapies also promote the exposure of tumor-associated antigens (TAAs) to immune infiltrates, thereby enhancing the antitumor effect. However, the efficacy of ROS generated by PDT is limited by the TME. Insufficient oxygen levels and overexpression of cellular GSH reduce the efficiency of PDT and weaken immune activation by reducing ROS production. As another PDT, photothermal therapy (PTT) is also a promising tumor treatment method that can induce local hyperthermia and enhance damage-associated molecular patterns (DMAPs) to trigger immune responses under hypoxic conditions. However, conventional PTT still faces challenges such as insufficient tumor tissue damage and insufficient antigen presentation. Therefore, it is necessary to develop drug structures to overcome the limitations of the TME and enhance the immune response to specific antigens. In recent years, nanozymes have attracted much attention due to their biomimetic catalytic activity. These nanozymes mimic natural enzymes such as SOD, CAT, GPx, POD, and GSHOx. Superoxide anion (O2 •- ) is converted into H2O2 by SOD, and then the generated H2O2 is catalyzed by CAT and POD to produce O2 and •OH, exerting cytotoxicity. Based on these properties, H2O2 homeostasis regulators can be designed to increase its production in immunosuppressive TME. In addition, after GSH is eliminated by GSHox, the expression of the antioxidant GPX4 is reduced, thereby triggering ferroptosis. Ferroptosis and higher H2O2 concentrations will promote the corresponding catalytic decomposition to produce •OH and O2. During PDT, O2 can be converted into 1 O2. As well as the heat generated by PTT, a sufficient number of tumor cell fragments are presented, leading to the activation and infiltration of CTLs in the TME and inducing the maturation of dendritic cells (DCs). However, there is currently no outstanding photodynamic nanozyme that can achieve the above-mentioned cascade catalysis and significantly enhance the presentation of tumor cell apoptotic fragments for effective immunotherapy. The present invention discloses an innovative ROS homeostasis regulator F@PDA@I for synergistic PTT, PDT and efficient immunotherapy. In this system, the multifunctional F@PDA@I nanozyme produces H2O2, O2 and •OH, while consuming GSH, thereby changing the immunosuppressive TME. In addition, the generation of oxygen leads to 1The increased O2 levels enhanced the efficacy of PDT. The combined application of PTT, PDT, and R848 promoted the release of a large number of TAAs, activated CTLs, and promoted their infiltration into tumors, thereby achieving immunotherapy and ultimately inhibiting the growth of primary and distal tumors. This innovative photodynamic nanozyme provides a promising design strategy to simultaneously enhance antigen presentation and reverse the immunosuppressive TME. Summary of the invention

[0003] The present invention provides a nano-immunomodulator F@PDA@I, characterized in that the nano-immunomodulator F@PDA@I is prepared by the following method: under stirring, a tannic acid solution is added to a PDA@I aqueous dispersion, and after continuing to stir for 5-15 minutes, a ferrous salt solution is added, and after continuing to stir for 5-15 minutes, a precipitate is collected by centrifugation, and the precipitate is washed with water, and then freeze-dried to obtain a black-green (dark green) solid powder. The concentration of the tannic acid solution is preferably 40 mg / mL, and the amount is preferably 40-80 μL (preferably 60 μL). The concentration of the PDA@I aqueous dispersion is preferably 5 mg / mL, and the amount is preferably 2-5 mL (preferably 3 mL); the ferrous salt is preferably ferrous chloride or its hydrate, ferrous sulfate or its hydrate, ferrous nitrate or its hydrate, or a mixture of several thereof. The concentration of the ferrous salt solution is preferably 40 mg / mL, and the amount is preferably 40-80 μL (preferably 60 μL); the number of precipitate washings is preferably 1-3 times; and the freeze-drying medium is preferably water.

[0004] The PDA@I is prepared by the following method: a dopamine polymer PDA solution is mixed with an I-Cy7 solution, the mixture is stirred thoroughly in the dark, and the precipitate is collected by centrifugation to obtain PDA@I. Preferably, the loading amount of I-Cy7 in the obtained PDA@I is above 70% (preferably 72.8%). The preferred preparation scheme is as follows: 5 ml of dopamine polymer PDA (10 mg / ml) is mixed with 50 μl of I-Cy7 (10 mg / ml); after stirring in the dark at 25°C for 24 hours, the mixture is centrifuged (10,000 g, 10 minutes) and the precipitate is collected, which is the PDA@I.

[0005] Dopamine polymer (PDA) can be purchased from commercial sources or prepared by the following method: take 180 mg of dopamine hydrochloride, add an appropriate amount of water, heat to 50°C until completely dissolved, add 760 μl of NaOH (1M) solution, and after the reaction is complete (about 5 hours), centrifuge the reaction mixture for 15 minutes (12000 rpm), discard the supernatant and collect the precipitate below; repeat the centrifugation and precipitate collection process 1-3 times using distilled water; then, ultrasonically disperse the precipitate, centrifuge at 4000 rpm for 5 minutes, remove large particle precipitates, and collect the well-dispersed upper suspension (i.e., dopamine polymer PDA solution).

[0006] The I-Cy7 structure is as follows:

[0007] The synthesis method comprises the following steps: adding acetic anhydride and sodium acetate to compound 1 and compound 2, heating to 50-70° C. for reaction, and obtaining I-Cy7. The molar amount of compound 1 is 2.0-2.2 times that of compound 2, and the molar amount of sodium acetate is the same as that of compound 1. The amount of acetic anhydride is excessive, and those skilled in the art can reasonably select and adjust it according to the actual reaction situation.

[0008] Compound 1 is prepared by the following method: 5-iodo-2,3,3-trimethyl-3H-indole and 4-iodobenzyl bromide are reacted in an organic solvent to obtain compound 1. The molar amount of 4-iodobenzyl bromide is 3-5 times that of 5-iodo-2,3,3-trimethyl-3H-indole. The reaction is preferably carried out at 40° C. to reflux temperature, and the organic solvent is preferably chloroform, acetonitrile, etc.

[0009] Another embodiment of the present invention provides the above-mentioned I-Cy7 structural compound or its tautomer.

[0010] Another embodiment of the present invention provides the use of the above-mentioned I-Cy7 structure compound in the preparation of the nano-immunomodulator F@PDA@I.

[0011] Another embodiment of the present invention provides the use of the above compound 1 in the preparation of I-Cy7 structure compounds.

[0012] Another embodiment of the present invention provides the use of the above-mentioned nano-immunomodulator F@PDA@I in the preparation of tumor immunotherapy drugs.

[0013] Another embodiment of the present invention provides a pharmaceutical composition, characterized in that the pharmaceutical composition uses the above-mentioned nano-immunomodulator F@PDA@I as an active ingredient. The pharmaceutical composition may also include other anti-tumor drugs (preferably Toll-like receptor agonists, and more preferably lecitramod (R848)). The pharmaceutical composition may also include pharmaceutically acceptable excipients. The dosage form is preferably a solid preparation, a liquid preparation or a semi-solid preparation.

[0014] Compared with the prior art, the advantages of the present invention are as follows: (1) The present invention discloses a photothermal active multi-enzyme-like enhanced nano-immunomodulator F@PDA@I, which integrates the activities of superoxide dismutase (SOD), catalase (CAT), peroxidase (POD) and glutathione oxidase (GSHOx). Photodynamic therapy (PDT) and photothermal therapy (PTT) are used to achieve tumor immunotherapy. The multi-enzyme-like activity not only contributes to the generation of oxygen (O2) to relieve hypoxia and promotes the polarization of M2 to M1 macrophages, but also consumes GSH in the TME to promote PDT and induce ferroptosis. As a result, a large amount of necrotic debris is released, which ultimately promotes the maturation of dendritic cells and the infiltration of cytotoxic T lymphocytes (CTLs), thereby reversing the immunosuppressive TME. In in vivo experiments, F@PDA@I was combined with the Toll-like receptor agonist relizumab (R848) for synergistic immunotherapy against primary and distal tumors. The design and corresponding mechanism of the cascade multienzyme-based nanoplatform provide a potential avenue for effective tumor immunotherapy. (2) In vitro and in vivo experiments confirmed that the ingenious design of the F@PDA@I structure enabled efficient antigen presentation and successful induction of immune cell death (ICD). Due to the significant GSHox-like enzyme activity of F@PDA@I, downregulation of GPX4 expression was detected, thereby inducing ferroptosis. In addition, F@PDA@I could enhance ROS production through SOD-like, CAT-like, and POD-like activities. A series of enzymatic reactions produced O2, thereby reversing the hypoxic tumor microenvironment. Further combined with photothermal therapy and photodynamic therapy, a synergistic effect was observed, leading to significant ablation of tumor cells in vivo. In this process, M2 macrophages were polarized toward the M1 phenotype. Sufficient levels of ROS and heat-induced tumor-associated antigens accelerated the maturation of dendritic cells and the infiltration of CTLs into the tumor. Finally, further introduction of R848 led to complete inhibition of primary and distant tumors in a bilateral tumor-bearing model. This study presents a promising strategy to simultaneously reverse the immunosuppressive tumor microenvironment, promote ROS production, and induce significant hyperthermia to trigger effective tumor immunotherapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is a schematic diagram of the preparation of the nano-immunomodulator F@PDA@I of the present invention and the TME activated enzyme cascade catalysis enhanced immunotherapy. After uniformly targeting tumor tissue, the multi-enzyme mimetic activity of F@PDA@I can reverse the immunosuppressive TME, thereby synergistically enhancing immunotherapy through photodynamic therapy (PDT), photothermal therapy (PTT) and chemodynamic therapy (CDT).

[0016] Figure 2a is the transmission electron microscopy image of F@PDA@I and the corresponding element distribution map (scale: 50 μm); b is the particle size distribution map of F@PDA@I; c is the Zeta potential map of PDA, PDA@I and F@PDA@I; d is the XPS full spectrum of PDA and F@PDA@I; e is the Fe 2p XPS analysis map of F@PDA@I and PDA; f is the SOD-like activity map of F@PDA@I; g is the CAT-like activity map of F@PDA@I; h is the ·OH generation map under different concentrations of F@PDA@I detected by MB determination; i is the GSH depletion map caused by F@PDA@I detected by DTNB probe under acidic conditions (pH = 6.5); j is the GSH depletion map under 808 nm laser irradiation (1 W / cm 2 ) under the conditions of time-dependent DPBF degradation of F@PDA@I with or without H2O2 (10 mM); k is the time-dependent DPBF degradation curves detected by ABDA assay under 808 nm laser irradiation (1 W / cm 2 ) 1 O2 generation diagram; l is the detection of O2 generated from I-Cy7 using DHR123 1 O2 diagram; m is the detection of the F@PDA@I using DHR123 1 O2 picture.

[0017] Figure 3 a is a representative merged CLSM image of 4T1 cells treated with 0.5 μM ER-Tracker Green and 1 μM I-Cy7 or F@PDA@I (red) for 2 h; b and c are the Pearson correlation coefficients of I-Cy7 and F@PDA@I / ER-Green-Tracker signals in CLSM images; for ER-Tracker Green, the emission wavelength is in the range of 500-600 nm (excitation wavelength is 490 nm); for I-Cy7 or F@PDA@I probes, the emission wavelength is in the range of 750-800 nm (excitation wavelength is 640 nm); d is the DCFH-DA probe used to evaluate the emission wavelength of ER-Tracker Green after different treatments (808 nm, 1 W / cm 2Figure 5 ROS production in 4T1 and MCF7 cells after different treatments (5 min, 5 min); the emission wavelength of the DCFH-DA probe was in the range of 500-600 nm (excitation wavelength was 502 nm); e is the evaluation of ·OH production in 4T1 and MCF7 cells after different treatments using the HPF probe; the emission wavelength of the HPF probe was in the range of 500-600 nm (excitation wavelength was 502 nm); f is a live-dead staining image of cells; for Calcein-AM and PI probes, the emission light was collected in the range of 505-545 nm (excitation wavelength was 494 nm) and 600-700 nm (excitation wavelength was 535 nm), respectively, and the scale bar is 200 μm; g and h represent the quantitative results of DCFH-DA fluorescence intensity detected by FCM in 4T1 and MCF7 cells after different treatments, respectively; i and j represent the quantitative results of HPF fluorescence intensity detected by FCM in 4T1 and MCF7 cells after different treatments, respectively.

[0018] Figure 4 a and d represent the analysis of γH2AX protein expression in 4T1 and MCF7 cells after different treatments using CLSM. For γH2AX, the emission wavelength is in the range of 500-600 nm (excitation wavelength is 488 nm); b and e represent the quantification of γH2AX protein expression in 4T1 and MCF7 cells after different treatments using flow cytometry; c and f represent the phototoxicity of I-Cy7 and F@PDA@I to 4T1 and MCF7 cells under 808 nm laser irradiation; g represents the analysis of 53BP1 protein expression in 4T1 and MCF7 cells after different treatments using CLSM. For 53BP1, the emission wavelength is in the range of 600-700 nm (excitation wavelength is 561 nm).

[0019] Figure 5Figure a shows the co-localization experiment using endoplasmic reticulum green fluorescent probe and F@PDA@I in 4T1 cells. For ER-Tracker Green, the emission wavelength was collected at 500-600 nm (excitation wavelength was 490 nm), and for F@PDA@I probe, the emission wavelength was collected at 750-800 nm (excitation wavelength was 640 nm); b shows the Pearson correlation coefficient between the green and red fluorescence channels; c, d, and i show the immunostaining analysis of the relevant indicators of cells treated with I-Cy7 or F@PDA@I after near-infrared light irradiation (808 nm, 1 W / cm2, 5 min). For the green probe, the emission wavelength was collected at 500-600 nm (excitation wavelength was 490 nm), and for the DAPI probe, the emission wavelength was collected at 400-500 nm (excitation wavelength was 350 nm) were collected, and the changes of CRT, HSP70, HMGB1 and GRP78 were evaluated by flow cytometry using markers e, f, g and h; j is the immunofluorescence staining of GPX4 in 4T1 cells treated with F@PDA@I. For GPX4, the emission wavelength was collected at 500-600 nm (excitation wavelength was 490 nm), and for DAPI probe, the emission wavelength was collected at 400-500 nm (excitation wavelength was 350 nm); k is the Western blot analysis of GPX4 expression in 4T1 cells under different treatment conditions; l is the quantitative analysis of GPX4 protein; m is the expression of GPX4 in 4T1 cells after near-infrared light irradiation (808 nm, 1 W / cm 2 , 5 min), detection of external ATP content in cells with different treatments; n is flow cytometry analysis of 4T1 cells treated with AnnexinV-FITC and PI, scale bar = 50 μm.

[0020] Figure 6 a is the detection of immune molecule expression; b is the representative flow cytometric image of mature DCs in the primary tumor 16 days after light irradiation of mice; d is the representative flow cytometric image of CD3+ CD8+ T cells in the primary tumor 16 days after light irradiation of mice; f is the representative flow cytometric image of CD3+ CD4+ T cells in the primary tumor 16 days after light irradiation of mice; h is the representative flow cytometric image of Treg cells in the primary tumor 16 days after light irradiation of mice; c, e, g and i represent the quantitative analysis of DC cells, CD3+ CD8+ T cells, CD3+ CD4+ T cells and Treg cells, respectively; j is the immunofluorescence staining of HMGB1 and CRT in tumor tissues of different experimental groups, DAPI was used to stain cell nuclei, scale bar: 100 μm.

[0021] Figure 7In the figure, a is a schematic diagram of the mouse model generation process; b is the potential mechanism that triggers immune cell death; c is the relative volume of the mouse primary tumor; d is the weight of the mouse primary tumor; e is the image of the mouse primary tumor; f is the relative volume of the mouse distal tumor; g is the weight of the mouse distal tumor; h is the image of the mouse distal tumor; i and j are the concentrations of cytokines (TNF-𝛼, IFN-𝛾) in serum after various treatments; k is the immunofluorescence staining of mouse tumor tissue, including CD3 molecules, CD8 molecules and FOXP3 molecules; l, m, n and o represent the quantitative analysis of CD3, CD8, FOXP3 and CD4 fluorescence intensities, respectively.

[0022] Figure 8 It is the UV absorption spectra of I-Cy7, PDA, and F@PDA@I.

[0023] Fig. 9 It is the measurement result diagram of encapsulation efficiency of I-Cy7, where a is the UV-visible absorption peak diagram of I-Cy7 at different concentrations, and b is the linear fitting data diagram of absorbance.

[0024] Fig.10 This is a picture of histological staining experiments on mouse heart, liver, spleen, lung and kidney tissues.

[0025] Fig.11 4T1 tumor-bearing mice were injected with F@PDA@I or PBS and then exposed to 808 nm, 1 W / cm 2 , infrared thermal image after 5 minutes of irradiation.

[0026] Fig.12 This is an immunofluorescence staining analysis of GPX4 in distant tumor tissues.

[0027] Fig.13 is compound 1 1 H NMR spectrum (400 MHz, CDCl3).

[0028] Fig.14 This is the high-resolution mass spectrum of compound 1.

[0029] Fig.15 I-Cy7 1 H NMR spectrum (400 MHz, DMSO-d6).

[0030] Fig.16 I-Cy7 13 C NMR spectrum (100 MHz, DMSO-d6).

[0031] Fig.17 This is the high-resolution mass spectrum of I-Cy7. DETAILED DESCRIPTION

[0032] Materials: Dopamine hydrochloride and methylene blue (MB) were obtained from Shanghai Aladdin Biochemical Technology Co., Ltd. Cu / Zn-SOD and Mn-SOD enzyme activity detection kits (including WST-8) were purchased from Beyotime Biotechnology (S0103). 5,5'-Dithio(2-nitrobenzoic acid) (DTNB) and 1,3-diphenylisobenzofuran (DPBF) were provided by Bid Pharmaceutical Technology Co., Ltd. (Shanghai, China). All solvents used were of analytical grade. Rhodamine B (RB) and 9,10-anthracene diester-bis(methylene)dimalonic acid (ABDA) were obtained from Shanghai Aladdin Biochemical Technology Co., Ltd. ER-Tracker Green, Lyso-Tracker Green, Golgi-Tracker Green, Hoechst 33342, JC-1 Mitochondrial Membrane Potential Detection Kit, Annexin V-FITC Apoptosis Detection Kit, and Cytotoxicity Detection Kit were purchased from Beyotime Biotechnology Co., Ltd. TNF-α ELISA kit and IFN-γ were obtained from Shanghai Sorabi Biotechnology Co., Ltd. PE anti-mouse CD86 antibody [GL-1], FITC anti-mouse CD80 antibody [16-10A1], FITC anti-mouse CD3 antibody [17A2], PE anti-mouse CD8 antibody [53-6.7], PE anti-mouse CD4 antibody [RM4-5], FITC anti-mouse CD25 antibody [PC-61.5.3], PE anti-mouse Foxp3 antibody [3G3], and FITC anti-mouse CD206 / MMR antibody [C068C2] were obtained from Proteintech Biotechnology Co., Ltd. ATP detection kit and lactate dehydrogenase detection kit were obtained from Beyotime Biotechnology Co., Ltd. MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide) was obtained from Energy Chemical Co., Ltd. Hydroxyphenylfluorescein (HPF) was obtained from Shanghai Maokang Biotechnology Co., Ltd. Calcein-AM / PI detection kit, GSH and GSSG detection kits, GPX4 rabbit polyclonal antibody, and HRP-labeled goat anti-rabbit IgG (H+L) were obtained from Beyotime Biotechnology Co., Ltd. C11 BODIPY 581 / 591 lipid peroxide sensor was obtained from Maokang Biotechnology Co., Ltd. (Shanghai, China). Mouse breast cancer cells (4T1) and Michigan Cancer Foundation-7 (MCF-7) were obtained from the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences. Tannic acid (TA) and FeCl2·4H2O were obtained from Bid Pharmaceutical Technology Co., Ltd.

[0033] Methods: 1H-NMR and 13C-NMR spectra were measured using a Bruker Avance II 400 spectrometer. The mass spectrometry (ESI-MS) data were detected using an Ultimate 3000 (Thermo Scientific) instrument. Fluorescence images were captured using a TCS SP8 confocal laser scanning microscope (Lecia). Cell apoptosis was detected by FCM (BECKMAN COULTER), and cytotoxicity experiments were performed using a multi-function microwell reader (SpectraMax i3x). The morphology of F@PDA@I was observed using a transmission electron microscope (FEI Company, USA). Dynamic light scattering (DLS) and zeta potential measurements were performed using a ZS Nanohybrid Analyzer (Malvern, UK). The ultraviolet-visible-near-infrared (UV–vis–NIR) absorption spectra of different samples were recorded using an Evolution 220 UV–vis spectrophotometer (UV-2700i, Japan). The values ​​of iron in different valence states were experimentally analyzed using XPS (ESCA Lab 250, ThermoFisher Scientific, USA).

[0034] Example 1

[0035] 1. Preparation and characterization of nanoimmunomodulator F@PDA@I

[0036] Figure 1 The design process of F@PDA@I is summarized. Under alkaline conditions, dopamine polymer PDA is formed by dopamine polymerization. Subsequently, photosensitizer (PSs) I-Cy7 is loaded into PDA via electrostatic adsorption and capillary force to form PDA@I. 2+ A pH-responsive metal-polyphenol network shell can be formed to obtain F@PDA@I. When the nanoparticles are internalized by tumor cells, the shell will act as a SOD-like catalyst to convert O2 •- Then, H2O2 will pass through Fe 3+ and Fe 2+ ions catalyze the production of O2 and •OH. 3+ ions will consume GSH. Due to the reduced GSH and abundant O2, the PDT efficiency of I-Cy7 is improved. Combining the PTT activity of PDA and I-Cy7, F@PDA@I will cause a large amount of DAMP release and trigger ICD, promote the infiltration of DC cells and T lymphocytes, and ultimately achieve anti-tumor immune response. The detailed synthesis process and structural characterization results of these compounds are described in detail in Example 2.

[0037] The morphology of F@PDA@I was analyzed by transmission electron microscopy (TEM) and was found to be similar to that of PDA. Figure 2 The energy spectrum analysis in a shows that Fe is evenly distributed on the surface of F@PDA@I, indicating that the drug component is well supported on PDA. According to the laser particle size test, the particle size of nano F@PDA@I is estimated to be 133.95 nm ( Figure 2 b). In addition, the high uncharged potential suggests the good dispersibility and stability of F@PDA@I ( Figure 2 c). The particle size changes of F@PDA@I in different media were further monitored over seven days, and the results showed that the particle size changes were minimal, an observation that emphasizes the excellent stability of F@PDA@I.

[0038] In order to identify the surface element state, X-ray photoelectron spectroscopy (XPS) analysis was performed. Based on the full XPS spectrum, the presence of Fe in F@PDA@I was confirmed ( Figure 2 d). Previous studies have shown that the enzyme activity of iron-based nanomaterials originates from various oxidation states of Fe ions. XPS analysis of Fe 2p confirmed that Fe 2+ (710.1 eV) and Fe 3+ (715.1 eV), providing the enzyme catalytic performance of F@PDA@I ( Figure 2 e). UV-visible absorption spectroscopy quantitatively confirmed that I-Cy7 was successfully loaded onto PDA, with a loading rate of 72.8% ( Figure 8 and 9 ). The multi-enzyme-like activity of F@PDA@I was then evaluated. To this end, the SOD-like activity of F@PDA@I was evaluated using Cu / Zn-SOD and Mn-SOD assay kits based on WST-813. Basically, WST-8 reacts with O2 generated by xanthine oxidase to produce •− The reaction generates an azo dye with strong UV-vis absorption at 450 nm. After the addition of F@PDA@I, the UV-vis absorption intensity decreases rapidly with the increase of F@PDA@I concentration, confirming the concentration-dependent SOD-like activity of F@PDA@I ( Figure 2 f).

[0039] Subsequently, endogenous H2O2 in tumors is degraded into O2 and H2O through catalase (CAT)-like activity, which helps relieve tumor hypoxia and promotes the reversal of the tumor immunosuppressive microenvironment. Therefore, the CAT-like activity of F@PDA@I was determined using a dissolved O2 meter. F@PDA@I (50 μg / mL) was mixed with H2O2 (50 mM), and the O2 gas concentration increased over time ( Figure 2g), indicating that F@PDA@I contributes to the alleviation of tumor hypoxia through CAT-like activity. After incorporation of I-Cy7 into F@PDA@I, a photodynamic therapy effect was induced. Subsequently, the UV-vis absorption decay of 1,3-diphenylisobenzofuran (DPBF) under 808 nm laser irradiation was monitored. 1 O2 generation. Under laser irradiation, the UV-vis-NIR intensity of the F@PDA@I plus H2O2 (10 mM) group decreased significantly, which was attributed to the CAT-like activity of F@PDA@I producing a large amount of O2 ( Figure 2 j). In addition, H2O2, which is abundant in tumors, can be catalyzed into •OH by peroxidase-like enzymes for dynamic tumor chemotherapy. Therefore, the peroxidase-like activity was monitored using methylene blue (MB), and MB would be degraded by •OH. As expected, there was no difference in MB UV-vis-NIR absorption between F@PDA@I and H2O2. However, after mixing F@PDA@I with H2O2 (10 mM) in NaHCO3 solution (25 mM) for 30 min, the MB UV-vis-NIR absorption decreased sharply, indicating that the Fenton-like reaction mediated by F@PDA@I produced •OH ( Figure 2 h). Although F@PDA@I generated a large amount of ROS, overexpression of GSH in tumors (≈10 mM) helped prevent damage. The GSHox-like activity of F@PDA@I on GSH elimination ability was evaluated using 5,5'-dinitrobenzodisulfate (DTNB) when high-valent iron ions were present. DTNB is a glutathione indicator that reacts with glutathione to produce a colored product. After adding F@PDA@I to the mixture of GSH and DTNB, the UV-vis absorption intensity at 412 nm decreased with increasing F@PDA@I dose ( Figure 2 i), confirming the strong GSHOx-like activity of F@PDA@I.

[0040] After confirming the enzymatic properties of F@PDA@I, the activity of I-Cy7 was determined using Rose Bengal as a standard. 1 The O2 quantum yield was 27.4%. Excellent ROS generation was observed in F@PDA@I within five minutes using ABDA and DHR123 probes ( Figure 2k, 2l, 2m). After obtaining the above experimental results, the photothermal conversion performance of F@PDA@I under different conditions was evaluated. Compared with the PBS control group, the temperature of F@PDA@I reached 61.3 °C after five minutes of irradiation. It was then found that the temperature increase was proportional to the F@PDA@I concentration at the same irradiation intensity. In addition, the temperature increase was measured using different laser intensities, and a linear relationship between the laser intensity and the temperature increase was found. The ROS generated by F@PDA@I can cause severe oxidative damage, thereby enhancing the local immune response inside the tumor. These findings confirm the excellent multi-enzyme activity of F@PDA@I, including SOD-like, CAT-like, POD-like, and GSHox-like enzyme activities. F@PDA@I is not only able to self-supply sufficient H2O2 and O2, but also produces a large amount of ROS and depletes GSH, thus having the potential to overcome tumor resistance and immunosuppressive microenvironment defense.

[0041] 2. F@PDA@I has outstanding photodynamic performance in vitro

[0042] Thanks to the excellent ROS generation ability of F@PDA@I, its in vitro performance was further verified by cell experiments. Initially, the cellular uptake of I-Cy7 and F@PDA@I in 4T1 cells was analyzed. It was observed that the fluorescence intensity of F@PDA@I increased significantly after 30 minutes of co-incubation with cells, which was similar to the results of co-incubation with I-Cy7 alone, indicating that nanoencapsulation did not affect drug uptake. Since the loaded I-Cy7 photosensitizer has cationic properties, it is conducive to localization in negatively charged subcellular organelles. By using a green fluorescent probe, the colocalization coefficient of I-Cy7 and F@PDA@I with the endoplasmic reticulum was evaluated after 2 hours of co-incubation.

[0043] The colocalization coefficient between F@PDA@I and the endoplasmic reticulum reached 0.97 ( Figure 3 a and 3c), while the coefficient for I-Cy7 was 0.93 ( Figure 3 a and 3b). This indicates the excellent localization of F@PDA@I within the endoplasmic reticulum. Subsequently, the colocalization coefficients of I-Cy7 and F@PDA@I with mitochondria were measured using the same method. F@PDA@I exhibited a colocalization coefficient of 0.92 within mitochondria, while I-Cy7 showed a coefficient of 0.90, indicating effective localization within mitochondria. Subsequently, colocalization analysis of lysosomes and Golgi apparatus was performed, and the results showed that the colocalization coefficients of F@PDA@I with these two organelles were relatively low. Similarly, I-Cy7 showed similarly low colocalization coefficients in lysosomes and Golgi apparatus.

[0044] In a series of subcellular localization experiments, F@PDA@I showed significant localization ability in the endoplasmic reticulum and mitochondria. The ROS production in cells of different treated tissues was evaluated using the DCFH-DA probe. It is worth noting that the F@PDA@I + NIR group showed the highest ROS production in both 4T1 cells and MCF7 cells ( Figure 3 d). Flow cytometry analysis ( Figure 3 g and 3h). This trend was observed continuously. F@PDA@I exhibits POD enzyme activity, which is able to convert intracellular H2O2 into cytotoxic ·OH. Therefore, we investigated the production of ·OH in cells. The HPF probe is specific for ·OH and emits strong green fluorescence after binding ( Figure 3 e). It is worth noting that the F@PDA@I + NIR group showed significant ·OH generation in both 4T1 cells and MCF7 cells, which was further confirmed by flow cytometry ( Figure 3 i and 3j). In addition, consistent results were observed using the O27 probe to detect ·OH. These experiments together verified that F@PDA@I exhibited a large amount of ROS production under near-infrared irradiation. After near-infrared irradiation, the dopamine component caused a significant photothermal effect. The combined action of abundant photothermal effect and ROS jointly promoted immune cell death. Cell viability after various treatments was evaluated using Calcein-AM and PI staining. Specifically, F@PDA@I was co-incubated with 4T1 cells and then subjected to near-infrared irradiation (808 nm, 1 W / cm 2 , 5 min) resulted in significant cell death ( Figure 3 f). Similar phenomenon was also observed in MCF7 cells.

[0045] It has been reported that excessive ROS generation and photothermal effects can significantly damage the cell nucleus, leading to the release of a large amount of nuclear material20. After nuclear damage, a large amount of HMGB1 protein translocates to the cell membrane surface, acting as an “eat me” signal to recruit free dendritic cells. Therefore, it is crucial to detect nuclear damage, and analysis of phosphorylated histone H2AX (γH2AX) and p53 binding protein 1 (53BP1) was performed. Cells were treated with PBS, near-infrared radiation (808 nm, 1 W / cm 2 , 5 min) and F@PDA@I. The cells were immunostained with γH2AX antibody and 53BP1 antibody after exposure to light under normal oxidative conditions. Notably, the nuclear density spots increased significantly after F@PDA@I + NIR treatment compared with the other groups. After incubation with green fluorescent secondary antibody, the γH2AX fluorescence intensity was significantly enhanced in the F@PDA@I + NIR group ( Figure 4a and 4d), showing a significant increase in the density of spots in the nucleus.

[0046] Subsequently, flow cytometry was used to evaluate the expression of γH2AX protein in different treatment groups, and the results were consistent with the CLSM observations ( Figure 4 b and 4e). In addition, immunostaining of 53BP1 using a red fluorescent secondary antibody showed strong green fluorescence in the F@PDA@I + NIR group, indicating significant DNA damage ( Figure 4 g). Under dark conditions, when the concentration of I-Cy7 or F@PDA@I reached 100 μg / mL, the cell viability exceeded 95%, indicating that the cytotoxicity of PS was very low. However, under light irradiation conditions ( Figure 4 c), the survival rate of 4T1 cells dropped sharply to 22% (I-Cy7) and 6% (F@PDA@I). The calculated half maximal inhibitory concentration (IC 50 ) showed that the IC50 of F@PDA@I was significantly lower than that of I-Cy7 (IC 50 = 10.2 μg / mL compared to the IC of I-Cy7 50 = 18.6 μM). These trends were consistent in experiments performed with RAG and HepG2 cancer cells, confirming the growth inhibitory potential of all tested photosensitizers.

[0047] In addition, the IC of F@PDA@I on MCF7 cells 50 Conduct an assessment ( Figure 4 f). Without light irradiation, very little cell death was observed in the F@PDA@I treated group. However, under near-infrared light (808 nm, 1 W / cm 2 , 5 min) irradiation, cell death increased significantly, and about half of the cells showed death when the drug concentration was about 9.4 μg / mL.

[0048] 3. Detection of immune cell death and ferroptosis induced by combined photodynamic therapy and photothermal therapy

[0049] After intracellular uptake, F@PDA@I was exposed to near-infrared light (808 nm, 1 W / cm 2, 5 min) to induce a significant hyperthermia response. Subsequently, I-Cy7, which has the dual targeting ability to the endoplasmic reticulum and mitochondria, is released. This released I-Cy7 can generate ROS inside the endoplasmic reticulum and mitochondria, triggering the release of multiple DAMPs. Severe endoplasmic reticulum stress causes a large amount of CRT protein to translocate to the cell membrane surface, while mitochondrial damage causes a large amount of ATP to flow out of the cell membrane and produce a large amount of peroxide to damage the cell nucleus. The cell nucleus then releases HMGB1 protein and migrates to the cell membrane surface, while a large amount of heat generation leads to upregulation of intracellular HSP70 expression and translocation to the cell membrane. These conditions jointly trigger ICD, releasing a large number of DAMPs molecules to the cell membrane surface, which, as a signature "eat me" signal, recruits a large number of immune effector substances in the body, thereby enhancing the host immune response to tumors. In addition, F@PDA@I has properties similar to GSHox, promoting the conversion of reduced GSH to oxidized GSSG. Therefore, this process reduces the expression of GPX4, thereby triggering ferroptosis.

[0050] First, the targeting efficiency of I-Cy7 released from F@PDA@I under different illumination times was evaluated. Surprisingly, regardless of the illumination time, I-Cy7 consistently exhibited robust targeting to the ER ( Figure 5 a and 5b). Subsequently, markers associated with ICD were evaluated. After co-incubation of F@PDA@I with 4T1 cells, ICD was significantly induced by near-infrared light irradiation. Specifically, GRP78 expression was significantly upregulated ( Figure 5 c and 5h), CRT gradually translocates from the endoplasmic reticulum to the extracellular space ( Figure 5 d and 5e), HMGB1 protein is completely released from the nucleus to the extracellular space ( Figure 5 i and 5g, and activate HSP70, which is then translocated to the extracellular space ( Figure 5 f). In addition, ROS-induced mitochondrial damage leads to the release of a large amount of ATP into the extracellular space ( Figure 5m). These validated ICD-associated markers prompted further mechanistic investigations into cell death. It was hypothesized that F@PDA@I could induce ferroptosis by inhibiting GPX4 expression. To explore this, groups pre-treated with ferroptosis inhibitors were used, of which the F@PDA@I + NIR group, which was pre-treated with ferroptosis inhibitors, showed significant recovery of cell survival, suggesting that ferroptosis is indeed a cell death pathway. Notably, lipid peroxidation (LPO) is a key signal of ferroptosis. To detect LPO in F@PDA@I-treated 4T1 cells, the C11-BODIPY probe was first employed. Only the F@PDA@I + NIR group showed green fluorescence of C11-BODIPY in an oxidized state, and this fluorescence was extinguished after Fe-1 pretreatment, indicating that F@PDA@I-mediated light treatment caused a large amount of LPO accumulation. According to the Western blot and CLSM immunofluorescence results ( Figure 5 j, 5k and 5l), it can be found that the F@PDA@I + NIR group significantly demonstrated performance similar to that of the GSHox enzyme, thereby depleting GPX4. Then, the F@PDA@I + NIR group further showed a decrease in cellular GSH concentration and loss of mitochondrial membrane potential. These findings undoubtedly indicate that F@PDA@I can effectively induce cellular ferroptosis by generating ROS and reducing GSH. Subsequently, we used flow cytometry to quantitatively analyze cell apoptosis. Obviously, compared with the PBS group, cells in the F@PDA@I + NIR group showed significant apoptosis, with an apoptosis rate of 42.18% ( Figure 5 n). This further demonstrated the ability of F@PDA@I to significantly induce multi-pathway cell death.

[0051] 4. In vitro and in vivo immune activation of F@PDA@I

[0052] A key element of the immunosuppressive tumor microenvironment (TME) is the infiltration of M2 macrophages into “cold” tumors. In cancer immunotherapy, a promising strategy is to counteract immunosuppression by driving macrophages to switch from the M2 to the M1 phenotype. Previous studies have shown that elevated ROS production leads to oxidative stress. Therefore, inspired by the ability of F@PDA@I to generate ROS, we performed immune activation experiments using 4T1 cells. RAW264.7 macrophages were exposed to IL-4 to establish the M2 macrophage population, confirming the polarization of tumor-associated macrophages (TAMs) induced by F@PDA@I. The proportion of M2 and M1 macrophages was analyzed by flow cytometry (FCM) by co-culturing RAW264.7 macrophages with differently treated 4T1 cells in a transwell insert system. Compared with the control group, the proportion of M2 macrophages in the I-Cy7 + NIR and F@PDA@I + NIR groups decreased from 46.77% to 37.75% and 29.07%, respectively, while the proportion of M1 macrophages increased from 31.69% to 48.25% and 60.80%, respectively.

[0053] The levels of IL-10 secreted by M2 macrophages and IL-12 secreted by M1 macrophages were quantified by ELISA kits. Similarly, the trends of IL-10 and IL-12 levels in the I-Cy7 + NIR and F@PDA@I + NIR groups were similar to those of M2 and M1 macrophages. These findings suggest that F@PDA@I effectively promotes the differentiation of M2 macrophages into M1 macrophages, thereby reversing the immunosuppression in the tumor microenvironment. Recent studies have shown that inducing tumor necrosis by combining photothermal therapy (PTT) and photodynamic therapy (PDT) can effectively trigger a systemic antitumor immune response in the host. This suggests that the synergistic application of PTT and PDT not only leads to the eradication of irradiated tumors, but also has potential for the treatment of non-irradiated tumors. In addition, R848, a small molecule Toll-like receptor 7 / 8 agonist, has been shown to activate innate immune cells, promote the secretion of IFN-γ, activate CTLs, and enhance the maturation of dendritic cells. Therefore, combining F@PDA@I with R848 can further stimulate its potential in anti-tumor immune response. Figure 6 As shown in a, a mouse model bearing bilateral 4T1 tumors was established, with primary and distal tumors engineered on each side 7 and 5 days before treatment, respectively. 24 hours later, F@PDA@I was administered, followed by 808nm laser irradiation of the primary tumor. Subsequently, mice received R848 treatment on days 3, 7, and 10. Tumors from different treatment groups were isolated into single-cell suspensions and stained for immune-related cells using various antibodies for flow cytometric analysis.

[0054] Compared with the PBS group, the number of M2 macrophages in the F@PDA@I + NIR-treated primary tumor group was significantly reduced (from 41.11% to 12.28%), a trend consistent with the findings in the distal tumor group (from 44.44% to 14.25%). In contrast, the proportion of M1 macrophages in the F@PDA@I + NIR-treated primary tumor group was significantly increased, consistent with the observations in the distal tumor group. This shift in M1 macrophage polarization suggests that F@PDA@I + NIR effectively reshapes the immunosuppressive tumor microenvironment to an immune-supportive state. Subsequently, the expression of immune molecules in primary and distal tumors of mice was evaluated. Compared with the PBS group, the F@PDA@I + NIR group exhibited a 50.90% dendritic cell maturation rate at the primary tumor site ( Figure 6 b and 6c). Combined R848 treatment followed by NIR irradiation (808 nm, 1 W / cm 2 , 5 minutes), the maturation rate of dendritic cells increased to 74.51%. Similar trends were observed in the evaluation of distal tumors. CTLs play a vital role in antitumor immunity. In the primary tumors of the F@PDA@I + R848 + NIR group, the proportions of CD3+ CD8+ T cells (from 20.06% to 48.49%) and CD3+ CD4+ T cells (from 7.47% to 28.35%) increased significantly, which was significantly higher than that of the PBS group ( Figure 6 d, 6e, 6f, and 6g). Similar results were consistently obtained in distal tumor assessments.

[0055] Treg cells are a subset of immunosuppressive T cells and can be used as a marker of the host's immunosuppressive state. In the primary tumors of the F@PDA@I + NIR group, the number of CD25+ FOXP3+ T cells was significantly reduced (from 35.57% to 9.85%) compared with the PBS group (see Figure 6 h and 6i). Moreover, the proportion of CD25+ FOXP3+ T cells was significantly reduced after co-administration with R848, with consistent results in distal tumors. Together, these findings highlight the potent immunomodulatory effects of F@PDA@I under NIR irradiation, thereby improving the immunosuppressive tumor microenvironment. Furthermore, co-administration with R848 not only increased the proportion of cytotoxic T cells but also reduced the number of suppressive T cells, thereby promoting antitumor immunity. This potent immunostimulatory response correlated with the degree of ICD induced by PTT combined with PDT. Immunohistochemical analysis of tissue samples from different treatment groups confirmed that CRT expression was significantly upregulated and HMGB1 was released into the blood in tumors treated with F@PDA@I + NIR + R848. These results are consistent with earlier cell experiments (see Figure 6j), highlighting the potential of this combination therapy to elicit a potent anti-tumor immune response.

[0056] 5. Evaluation of in vivo immunotherapy effects

[0057] Inducing a systemic immune response to eliminate local tumors and prevent tumor metastasis is an ideal anti-tumor therapy. The ability of F@PDA@I to generate heat, enhance ROS production, and ultimately stimulate host immune responses in mice bearing bilateral 4T1 tumors was used to further evaluate its anti-tumor efficacy. A brief schematic diagram of the animal experiment is shown in Figure 7 Due to its potential to induce tumor-specific immune responses, the immunostimulatory properties of F@PDA@I can be further confirmed by evaluating dendritic cell maturation and T cell infiltration ( Figure 7 b).

[0058] Compared with the PBS group, mice injected with F@PDA@I showed significantly increased temperature at the tumor site, as confirmed by photothermal imaging ( Fig.11 ). However, antigenicity of immune cell death induced by PTT alone is usually insufficient. In addition, F@PDA@I can produce 1 O2 and ·OH, synergistically with their photothermal effect, enhanced the anti-tumor immune response. Subsequently, the mice were randomly divided into six groups (n = 5): PBS group, PBS + NIR group, F@PDA@I group, F@PDA@I + NIR group, R848 group, and F@PDA@I + R848 +NIR group. R848 was used to increase the proportion of cytotoxic cells. In the PBS group, after 14 days of treatment, both primary and distal tumors showed rapid growth, with the average volume increasing from 101 to 820 mm3 (an increase of 8.1 times) and from 80 to 489 mm3 (an increase of 6.11 times), respectively ( Figure 7 c and 7f).

[0059] Compared with the PBS group, the F@PDA@I + NIR group showed a significant inhibitory effect, with the primary and distal tumor volumes increasing by 3.86-fold and 3.47-fold, respectively, within 14 days, indicating a significant tumor inhibition level ( Figure 7 c and 7f). R848, a Toll-like receptor agonist, effectively stimulated CTLs. In the F@PDA@I + R848 + NIR group, tumor growth was significantly inhibited, demonstrating enhanced antitumor immunotherapy through the synergistic effect of F@PDA@I and R848. Among these groups, mice in the F@PDA@I + R848 + NIR group showed the smallest tumor mass ( Figure 7d, 7e, 7g, and 7h). In all groups, the average body weight of mice did not change significantly, indicating the high safety and low toxicity of our material. Histological analysis of the heart, liver, spleen, lung, and kidney of each group of mice was performed by H&E staining. Compared with the PBS group, the F@PDA@I + R848 + NIR group showed no obvious organ damage, indicating the absence of drug-induced organ toxicity ( Fig.10 ).

[0060] In all groups, tumor tissue examination showed dense tumor tissue without obvious damage in the PBS group. However, in the F@PDA@I + NIR group, obvious tissue damage and sparse tissue were seen. Similarly, in the F@PDA@I + R848 + NIR group, tumor tissue showed obvious damage, characterized by nuclear condensation and sparse tissue distribution ( Figure 7 k). The degree of immune response to tumors is directly correlated with the number of CTLs and helper T cells. Considering that the immune response to distant tumors reflects the primary tumor and systemic immune activation, we used immunofluorescence staining to evaluate the infiltration of CD3+ T cells, CD4+ T cells, and CD8+ T cells in distant tumors. Although the PBS group lacked immune cells, both the F@PDA@I + NIR group and the F@PDA@I +R848 + NIR group showed moderate fluorescence, indicating that CD3+ T cells, CD4+ T cells, and CD8+ T cells infiltrated into the tumor, suggesting a significant reversal of immunosuppression within the tumor microenvironment ( Figure 7 k, 7l, 7m).

[0061] Subsequently, immunofluorescence staining analysis of suppressive T cells in distal tumors showed that the fluorescence of the F@PDA@I+ R848 + NIR group was significantly reduced, indicating that the immune microenvironment was significantly reversed in anti-tumor immunotherapy by combining photothermal therapy and photodynamic therapy ( Figure 7 k and 7n). Effector T cells are widely present in lymphoid and non-lymphoid tissues, and provide timely immune protection against secondary invasions of the same pathogen by secreting multiple cytokines such as TNF-α and IFN-γ. It is worth noting that compared with the PBS group, the levels of TNF-α and IFN-γ in the blood of mice treated with F@PDA@I + R848 + NIR were significantly increased ( Figure 7i and 7j). F@PDA@I exhibited multifaceted nanozyme activities, as evidenced by pronounced GSHO-like activity in in vitro experiments. F@PDA@I induced ferroptosis by upregulating GSSG synthesis and subsequently downregulating GPX4 expression. In vitro experiments highlighted the ability of F@PDA@I to induce cellular ferroptosis. Immunohistochemical analysis was used to evaluate the expression of GPX4 in the F@PDA@I + NIR group, revealing a significant decrease in GPX4 expression compared to the PBS group ( Fig.12 ).

[0062] 6. Conclusion

[0063] In vitro and in vivo experiments confirmed that the clever design of the F@PDA@I structure enabled efficient antigen presentation and successful induction of immune cell death (ICD). Downregulation of GPX4 expression was detected due to the significant GSHox-like enzymatic activity of F@PDA@I, thereby inducing ferroptosis. In addition, F@PDA@I could enhance ROS production through SOD-like, CAT-like, and POD-like activities. A series of enzymatic reactions produced O2, thereby reversing the hypoxic tumor microenvironment. Further combined with photothermal therapy and photodynamic therapy, a synergistic effect was observed, leading to significant ablation of tumor cells in vivo. During this process, M2 macrophages polarized toward the M1 phenotype. Sufficient levels of ROS and heat-induced tumor-associated antigens accelerated the maturation of dendritic cells and the infiltration of CTLs into tumors. Finally, further introduction of R848 resulted in efficient suppression of primary and distant tumors in a bilateral tumor-bearing model. This study proposes a promising strategy that can simultaneously reverse the immunosuppressive tumor microenvironment, promote ROS production, and induce significant hyperthermia for effective tumor immunotherapy.

[0064] Example 2

[0065] 1. Synthesis of compound 1

[0066]

[0067] Dissolve 5-iodo-2,3,3-trimethyl-3H-indole (6 mmol) and 4-iodobenzyl bromide (24 mmol) in 20 mL acetonitrile, heat to 50°C and react overnight. TLC shows that the reaction is complete. Evaporate the acetonitrile, add a small amount of dichloromethane to completely dissolve, then add a large amount of n-hexane to produce a precipitate, filter and dry to obtain a light red solid (3.21 g, i.e., compound 1); 1H NMR (400 MHz, CDCl3) δ 7.93 – 7.82 (m, 1H), 7.71 (dd, J = 21.7, 7.8 Hz, 2H), 7.14 (d, J =7.9 Hz, 1H), 6.98 (d, J = 8.0 Hz, 1H), 6.00 (s, 1H), 4.42 (d, J = 2.1 Hz,1H), 3.10 (s, 1H), 1.70 (s, 3H), 1.26 (s, 3H), 0.84 (s, 3H). HRMS (ESI): m / zcalc. for [C 18 H 18 I2N + ] - 501.9524, found 501.9508 [M].

[0068] 2. Synthesis of I-Cy7

[0069]

[0070] Take compound 1 (1 mmol) and compound 2 (0.5 mmol), add 10 mL of acetic anhydride and sodium acetate (1 mmol), heat to 70°C and stir to react. TLC detection shows that the reaction is complete. After conventional post-treatment, I-Cy7 (389 mg) is obtained; 1 H NMR (400MHz, DMSO) δ 8.23 ​​(d, J = 13.9 Hz, 2H), 8.09 (s, 2H), 7.81 – 7.64 (m, 6H), 7.23 (d, J = 8.4 Hz, 2H), 7.08 (d, J = 7.9 Hz, 4H), 6.37 (d, J = 14.0 Hz, 2H), 5.77 (t, J = 1.5 Hz, 8H), 5.48 (s, 4H), 1.71 (s, 10H). 13 C NMR (101 MHz, DMSO) δ 172.60, 143.80, 142.59, 138.22, 135.15, 129.44, 128.18, 102.99,94.51, 90.47, 55.41, 49.63, 27.85. HRMS (ESI): m / z calc. for [C 44 H 40 ClI4N2 + ] 1138.9054, found 1138.90125 [M] + .

[0071] 3. Synthesis of F@PDA@I

[0072] (1) Preparation of PDA@I: 5 ml of dopamine polymer PDA (10 mg / ml) was mixed with 50 μl of I-Cy7 (10 mg / ml). After stirring at 25°C in the dark for 24 h, the mixture was centrifuged (10,000 g, 10 min) and the precipitate was collected to obtain PDA@I. The loading amount of I-Cy7 in PDA was determined using UV-visible-near infrared absorption spectroscopy.

[0073] The dopamine polymer (PDA) can be purchased from commercial sources or prepared by the following method: take 180 mg of dopamine hydrochloride, add an appropriate amount of water, heat to 50°C until completely dissolved, add 760 microliters of NaOH (1M) solution, and after the reaction is complete (about 5 hours), centrifuge the reaction mixture for 15 minutes (12000 rpm), discard the supernatant and collect the precipitate below; repeat the centrifugation and precipitate collection process 1-3 times with distilled water; then, ultrasonically disperse the precipitate, centrifuge at 4000 rpm for 5 minutes, remove the large particle precipitate, and collect the well-dispersed upper suspension (i.e., the dopamine polymer PDA solution). Note: This method starts with high-speed centrifugation and collection of the precipitate in order to remove water-soluble impurities. The last low-speed centrifugation, collecting the upper suspension and discarding the precipitate is to separate the good product with small particles and good dispersion from the poor product with large particles and poor dispersion.

[0074] (2) Preparation of F@PDA@I: 60 μL of tannic acid TA solution (40 mg / mL) was added to the PDA@I aqueous dispersion (5 mg / mL, 3 mL) under stirring. After stirring for 10 min, 60 μL of FeCl2·4H2O solution (40 mg / mL) was added and stirred for another 10 min. The mixture was centrifuged at 15,000 rpm to collect the dark green precipitate. The precipitate was washed twice with distilled water and freeze-dried using water as the medium to obtain a dark green solid powder, namely F@PDA@I.

[0075] Superoxide dismutase (SOD)-like activity of F@PDA@I

[0076] Superoxide dismutase (SOD)-like activity was assessed using Cu / Zn-SOD and Mn-SOD enzyme activity assay kits with WST-8. Briefly, WST-8 reacts with O2 generated by xanthine oxidase to •− When interacting with nanomaterials that exhibit SOD-like enzyme activity, O2•− was converted into water and O2, thereby hindering the production of toluene. Specifically, F@PDA@I with different concentrations was incubated with WST and xanthine oxidase at 37°C for 30 min. Subsequently, the UV-Vis absorbance at 450 nm was measured. The percentage inhibition rate was calculated using the following formula: Inhibition rate (%) = [(A0-A i ) / A0] × 100%, where A0 and A i represent the absorbance of control and sample, respectively.

[0077] ·OH generation triggered by the peroxidase (POD)-like activity of F@PDA@I

[0078] In a typical experiment, MB (5 μg / mL), H2O2 (10 mM), and F@PDA@I (100 μg / mL) were mixed in NaHCO3 (25 mM) solution and incubated at 37°C for 30 min. The degradation of MB caused by ·OH was evaluated by monitoring the change in absorbance at 665 nm. The concentration-dependent POD-like activity of F@PDA@I was evaluated using UV-vis-NIR spectroscopy.

[0079] Effect of catalase (CAT)-like activity of F@PDA@I on O2 generation capacity

[0080] The catalase-like activity of F@PDA@I was followed by using a dissolved oxygen meter at 37°C. 50 μL of H2O2 (2 M) was added to 2 mL of F@PDA@I solution (50 μg / mL). After various reaction times, the concentration of dissolved O2 (mg / L) was quantitatively determined.

[0081] F@PDA@I 1 O2 generation capacity

[0082] Using 1,3-diphenylisobenzofuran (DPBF) as 1 The detection probe of O2 was used to evaluate the F@PDA@I solution under different irradiation times. 1 O2 generation. Specifically, F@PDA@I samples were dispersed in 5 mL of DPBF aqueous solution with or without H2O2 (1 mM). The mixture was exposed to a power density of 1 W / cm 2 The 808 nm laser was irradiated at different time intervals and then evaluated by UV-VIS spectrophotometer. 1 The production of O2.

[0083] Cell culture

[0084] MCF7 cells, 4T1 cells, RAG cells, and HepG2 cells were cultured in a medium containing 10% fetal bovine serum and 1% antibiotics (penicillin / streptomycin, 100 U mL -1 The cells were cultured in DMEM medium containing 5% dMEM and maintained at 37°C in an atmosphere of CO2 / air (5% / 95%).

[0085] Cytotoxicity test

[0086] 4T1 cells were cultured at 1×10 4 The cells were seeded into 96-well plates at a density of 100 cells / well, and 100 μL of culture medium was added to each well and cultured at 37°C for 24 hours. The original culture medium was aspirated, and different concentrations (0-100 μg / mL) of F@PDA@I and I-Cy7 were added to the wells and further incubated in a constant temperature incubator for 2 hours. Subsequently, the cells were illuminated under 808 nm light (1 W / cm 2 , 5 minutes). Continue to incubate in the constant temperature incubator for 24 hours. Subsequently, 20 μL of MTT (5 mg / mL) was added to each well, and the cells were incubated for another 4 hours. The old medium was aspirated and 150 μL of dimethyl sulfoxide was added to each well. Then, shake for 10 minutes. The absorbance at 570 nm was measured using a microplate reader (SpectraMax i3x). The cell viability was calculated using the following formula.

[0087]

[0088] To determine the darkness of F@PDA@I, the illumination condition was removed and the other steps remained the same. 4T1 cells were measured in the same way.

[0089] Cellular uptake assay

[0090] The cellular uptake of F@PDA@I at different time points was evaluated. The cells were dispersed in a laser confocal culture dish and 1 mL of culture medium was added to reach a cell density of 1×10 5 They were cultured in a constant temperature incubator for 24 h. Subsequently, they were treated with 50 μg / mL of F@PDA@I at different time intervals. After treatment, the cells were washed three times with PBS and observed under a confocal laser microscope.

[0091] Western Blot analysis of 4T1 cells treated with F@PDA@I

[0092] The experiment started with seeding cells in 6-cm dishes, in which F@PDA@I (50 μg / mL) was added to the culture medium. After incubation in the dark for 2 h, the cells were irradiated with 808 nm light for 5 min. Subsequently, after an additional 6-h incubation, the cells were trypsinized (0.25% trypsin) and then washed three times with PBS. Then, the cells were lysed using cell lysis buffer containing protease and phosphatase inhibitors and kept on ice for 30 min. After protein quantification by Bradford method, equal proteins were loaded into each slot of SDS-PAGE gel for electrophoresis. Subsequently, the proteins were transferred to polyvinylidene fluoride (PVDF) membranes. Then, the membranes were blocked with 5% bovine serum albumin and incubated with specific primary antibodies overnight at 4 °C. Subsequently, peroxidase-labeled goat anti-rabbit HRP secondary antibodies were applied and incubated for 1 h at room temperature. Finally, immunoblots were visualized using GPX4 (Shanghai Barrett Biotechnology Co., Ltd.).

[0093] Intracellular 1 O2 Detection

[0094] The cells were seeded into laser confocal microplates at a concentration of 1 × 10 cells / ml. 5 The culture dish was then placed in a cell culture incubator and cultured for 24 hours until the cells were fully attached. The seeded cell culture dishes were then divided into six groups: PBS group, PBS + NIR group, I-Cy7 group, I-Cy7 + NIR group, F@PDA@I group, and F@PDA@I + NIR group. After incubation in the cell culture incubator for 1 hour, DCFH-DA (10 μM) green fluorescent probe was added. The incubation was extended for another hour. Subsequently, the PBS + NIR group, I-Cy7 + NIR group, and F@PDA@I + NIR group were subjected to 808 nm NIR light (power density of 1 W / cm 2 ) for 5 min. Afterwards, the old medium was removed and the cells were washed three times with PBS. They were then observed under a confocal laser microscope.

[0095] Detection of intracellular OH

[0096] Detection using HPF probe 1 The procedure for O2 is the same as that for the DCFH-DA probe, except that the DCFH-DA probe is replaced by the HPF probe.

[0097] Subcellular localization

[0098] The cells were seeded into laser confocal culture dishes and cultured in a constant temperature incubator for 24 hours. Fresh culture medium containing F@PDA@I (50 μg / mL) was added and incubated for another 2 hours. After incubation, the purchased subcellular localization probe was added to the laser confocal culture dish according to the instructions and incubated for 1 hour. The cells were washed three times with PBS, fresh culture medium was added, and then observed under a confocal laser microscope.

[0099] Cell viability test

[0100] First, cells were extracted from 25T culture flasks and seeded into laser confocal culture dishes to reach 1 × 10 cells per ml. 5 The culture dishes containing cells were placed in a cell culture incubator and incubated for 24 hours to ensure that the cells were completely attached to the bottom surface. According to the experimental protocol, the culture dishes were divided into six groups: PBS group, PBS + NIR group, I-Cy7 group, I-Cy7 + NIR group, F@PDA@I group, and F@PDA@I + NIR group. Subsequently, the incubation of cells in the cell culture incubator continued for 6 hours. Then, the PBS + NIR group, I-Cy7 + NIR group, and F@PDA@I + NIR group were exposed to NIR light at 808 nm and 1 W / cm2 intensity for 5 minutes. After illumination, the cells were further incubated in the cell culture incubator for 6 hours, and then the corresponding reagents in the cell viability detection kit were added. After constant temperature incubation for 2 hours, they were washed three times with PBS. Finally, the culture dishes were examined using a confocal laser microscope.

[0101] Apoptosis detection

[0102] Cells were extracted from 25T culture flasks at 1 × 10 per ml. 5 The cells were seeded into a 6-well plate at a density of . Subsequently, the plate was placed in a cell culture incubator and incubated for 24 hours until the cells were fully attached. According to the experimental protocol, the cells were divided into six groups: PBS group, PBS + NIR group, I-Cy7 group, I-Cy7 + NIR group, F@PDA@I group, and F@PDA@I + NIR group. I-Cy7 and F@PDA@I (50 μg / mL) were added accordingly and continued to be incubated in the cell culture incubator for 2 hours. Subsequently, the PBS + NIR group, I-Cy7 + NIR group, and F@PDA@I + NIR group were exposed to 808 nm and 1 W / cm 2 The cells were exposed to NIR light of 1000 nm for 5 minutes. After illumination, the cells were further incubated in a cell culture incubator for 12 hours. Enzymatic digestion was performed, and then appropriate detection reagents were added and the samples were tested by machine.

[0103] Expression of GRP78 protein

[0104] The cells in the 25T flask were cultured at 1 × 10 cells / ml. 6 The density of cells was cultured in laser confocal culture dishes. Subsequently, the culture dishes were placed in a cell culture incubator and incubated for 24 hours until the cells were completely attached to the surface. According to the experimental requirements, the cells were divided into six groups: PBS group, PBS + NIR group, I-Cy7 group, I-Cy7 + NIR group, F@PDA@I group, and F@PDA@I + NIR group. After treatment with F@PDA@I and I-Cy7 (50 μg / mL), the cells were further incubated in the incubator for 2 hours and then exposed to NIR light (808 nm, 1 W / cm 2 ) for 5 minutes. After illumination, the old medium was aspirated and the cells were fixed overnight with immunostaining fixative solution. After fixation, the cells were incubated with GRP78 primary antibody (1:100) for 4 hours. Subsequently, the primary antibody was removed, the culture dish was washed with PBS, and then incubated with fluorescent secondary antibody for another 4 hours. After incubation, the secondary antibody (1:500) was removed and the culture dish was washed with PBS. The samples were then observed under a confocal laser microscope.

[0105] Expression of HSP70 protein

[0106] The same protocol as described above was followed, with the only exception that the GRP78 primary antibody was replaced with the HSP70 primary antibody.

[0107] Expression of HMGB1 protein

[0108] The same protocol as described above was followed, with the only exception that the HMGB1 primary antibody was replaced with the HSP70 primary antibody.

[0109] Expression of CRT protein

[0110] The same protocol as described above was followed, with the only exception that the CRT primary antibody was replaced with the HSP70 primary antibody.

[0111] GPX4 protein expression

[0112] Cells were cultured from a 25T flask at 1 × 10 cells per ml. 6 The density of cells was cultured in laser confocal culture dishes. Subsequently, the culture dishes were placed in a cell culture incubator and incubated for 24 hours until the cells were completely attached to the surface. According to the experimental requirements, the cells were divided into four groups: PBS group, PBS + NIR group, F@PDA@I group, and F@PDA@I + NIR group. After treatment with F@PDA@I (50 μg / mL), the cells were further incubated in the incubator for 2 hours and then exposed to near-infrared light (808 nm, 1 W / cm 2) for 5 minutes. After illumination, the old medium was aspirated and the cells were fixed overnight with immunostaining fixative solution. After fixation, the cells were incubated with GPX4 primary antibody (1:100) for 4 hours. Subsequently, the primary antibody was removed, the culture dish was washed with PBS, and then incubated with fluorescent secondary antibody for another 4 hours. After incubation, the secondary antibody (1:500) was removed and the culture dish was washed with PBS. The samples were then observed under a laser confocal microscope.

[0113] Establishment of bilateral tumor model in mice

[0114] All procedures followed the Guidelines for the Protection and Utilization of Laboratory Animal Resources and were approved by the Experimental Animal Ethics Committee of Shandong Second Medical University with approval number 2023SDL238. Six-week-old Balb / c mice were purchased from Beijing Vittel Laboratory Animal Technology Co., Ltd. Initially, the mice adapted to the new environment for one week. Subsequently, the tumor was implanted in the left axillary region. When the left tumor grew to the third day, the tumor was implanted on the right side using the same method. According to the experimental requirements, the mice were divided into four groups: PBS group, PBS + NIR group, F@PDA@I group, F@PDA@I + NIR group, R848 group, and F@PDA@I + NIR + R848 group. Treatment was performed on the left tumor. When the volume of the left tumor reached 100 mm 3 At 10 ℃, mice received intratumoral injection of F@PDA@I (1 mg / kg) and R848. Four hours after injection, mice received near-infrared (808 nm, 1 W / cm 2 ) irradiation. After treatment, the weight and bilateral tumor volume of the mice were recorded and observed every day. The formula for calculating the tumor volume of mice is as follows:

[0115] V represents the tumor volume, a and b represent the major and minor diameters, respectively.

[0116] Detection of surface molecule expression on mouse tumors

[0117] After extracting mouse tumor cells, the tumor tissue was minced into fine pieces using surgical scissors. Subsequently, the minced tissue was digested in collagenase at 37 degrees Celsius for 30 minutes. After digestion, the cells were suspended in PBS, and the cell suspension was filtered through a 300-mesh filter. The resulting cell suspension was centrifuged at 1500 rpm for 5 minutes. Then, the precipitate was initially suspended in buffer, and 600 μL of buffer was added to each tube. Subsequently, a small portion of 30 μL was taken for staining, and the machine test was performed after the staining procedure was performed for one hour.

Claims

1. A nano-immunomodulator F@PDA@I, characterized in that The nanoimmunomodulator F@PDA@I is prepared by the following method: Under stirring, the tannic acid solution is added to the PDA@I aqueous dispersion, and the stirring is continued for 5-15 minutes. Then, the ferrous salt solution is added, and the stirring is continued for 5-15 minutes. The precipitate is collected by centrifugation, and the precipitate is washed with water and freeze-dried to obtain a black-green solid powder, which is the nano-immunomodulator F@PDA@I. The concentration of the tannic acid solution is 40 mg / mL, and the dosage is 40-80 μL; the concentration of the PDA@I solution is 5 mg / mL, and the dosage is 2-5 mL; The ferrous salt is selected from one or a mixture of ferrous chloride or its hydrate, ferrous sulfate or its hydrate, ferrous nitrate or its hydrate; the concentration and dosage of the ferrous salt solution are the same as those of the tannic acid solution; The PDA@I is prepared by the following method: mixing a dopamine polymer PDA solution with an I-Cy7 solution, stirring and mixing in a dark environment, and collecting the precipitate by centrifugation to obtain the PDA@I; the loading amount of I-Cy7 in the PDA@I is more than 70%; The I-Cy7 structure is as follows: 。 2. The use of compound I-Cy7 in the preparation of the nano-immunomodulator F@PDA@I according to claim 1, wherein the structure of the compound I-Cy7 is as follows: 。 3. Use of the nano-immunomodulator F@PDA@I according to claim 1 in the preparation of tumor immunotherapy drugs, wherein the tumor is selected from 4T1 and MCF7.

4. A pharmaceutical composition, characterized in that The pharmaceutical composition uses the nano-immunomodulator F@PDA@I described in claim 1 as an active ingredient.

5. The pharmaceutical composition according to claim 4, characterized in that The pharmaceutical composition also includes other anti-tumor drugs.

6. The pharmaceutical composition according to any one of claims 4 to 5, characterized in that The pharmaceutical composition also includes pharmaceutically acceptable excipients.

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