A novel aggregation-induced emission substance targeted delivery for tumor chemotherapy, radiotherapy and immunotherapy triple therapy

Abstract

This invention discloses a novel aggregation-induced luminescent substance targeted delivery method for a triple therapy combining chemotherapy, radiotherapy, and immunotherapy for tumors. This invention relates to the field of biotechnology, specifically to a novel aggregation-induced luminescent substance targeted delivery method for a triple therapy combining chemotherapy, radiotherapy, and immunotherapy for tumors. This invention provides a drug with inhibitory or anti-tumor activity against tumor cells. The active ingredient of the drug contains LNC@AIEgen, which is composed of inactivated cancer cells and Complex 1 loaded onto the inactivated cancer cells. The nanoplatform LNC@AIEgen designed in this invention is used for synergistic chemotherapy-radioimmunotherapy. This system fully utilizes the advantages of the delivery carrier, enhancing the anti-tumor therapeutic effect and acting as an anti-cancer vaccine. This simple yet effective strategy enables large-scale clinical treatment of cancer and contributes to personalized medicine.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a novel aggregation-induced luminescent substance targeted delivery method for a triple therapy combining chemotherapy, radiotherapy, and immunotherapy for tumors. Background Technology

[0002] Impairment of the immune system, leading to a loss of immune surveillance, is one of the mechanisms that promotes tumor progression. Therefore, reversing this damage and activating the suppressed immune system can inhibit or completely eliminate cancer cell growth. Previous studies have developed several methods to enhance immune surveillance and immune responses, including local chemotherapy and radiation therapy (RT), immunotherapy, and tumor vaccines. Notably, recent research has demonstrated that immunotherapy is an effective treatment strategy due to its high specificity. Immunotherapy can induce an immune response, activate the immune system, thereby eliminating tumor cells, preventing tumor recurrence, and inhibiting metastasis. Currently, tumor immunotherapy encompasses cytokine therapy, immune checkpoint blockade (ICB, monoclonal antibody) therapy, adoptive cell transfer therapy, and tumor-specific vaccines, and has achieved significant breakthroughs in the treatment of certain types of tumors. Generally, acquired immunity (primarily mediated by T cells) plays a central role in anti-tumor immune responses by gradually increasing the number of potent cytotoxic T lymphocytes (CTLs) in tumor tissue. Therefore, one approach to ICB therapy is to directly target T cells. However, due to the immunosuppressive effects of the tumor microenvironment and the inhibitory effects of the biological barrier, the response rate of immunotherapy (20-30%) is far lower than expected.

[0003] Previous in-depth studies of immune mechanisms have revealed the importance of innate immunity in anti-tumor therapy. Activation of innate immunity not only promotes acquired immunity but also establishes immune memory, thereby preventing long-term tumor recurrence. Therefore, targeted immunotherapy based on innate immunity can provide an ideal "fulcrum" for systemic tumor immunotherapy. Furthermore, widely used local therapies in clinical practice, such as chemotherapy and radiotherapy, can also achieve multi-target immune stimulation to enhance immune activation. These approaches can eliminate primary tumors and further induce immunogenic cell death (ICD). Therefore, initiating a systemic anti-tumor immune response can also eliminate distant tumors and act as an in situ vaccine. These data suggest that powerful local therapies that stimulate adaptive and innate immunity to further improve treatment efficiency may be an effective tumor treatment strategy.

[0004] Over the past few decades, with the rapid development and innovation of nanomedicine, many nanomaterial-based therapeutic strategies have been developed to induce ICD more effectively than traditional treatments. In recent years, inactivated tumor cell-mediated anti-tumor therapy has attracted increasing attention due to its inherent tumor-targeting ability (through homologous protein recognition) and multiple immune activation properties. More importantly, after these inactivated cells enter tumor tissue, they can activate immune responses in multiple dimensions and alter the tumor immune microenvironment. Simultaneously, after being digested in living cells, inactivated tumor cells can release damaged DNA fragments or other components, thereby inducing activation of the cGAS-STING signaling pathway, upregulating type I interferon and other cytokines, and further amplifying both innate and adaptive immunity. Therefore, these inactivated cells can play a supportive role in anti-tumor immune responses. Summary of the Invention

[0005] The technical problem to be solved by this invention is how to improve the effect of inactivated tumor cells in anti-tumor immune response and improve treatment efficiency.

[0006] To address the above problems, the present invention provides a drug that inhibits tumor cell activity or has anti-tumor activity.

[0007] The present invention provides a drug having inhibitory or antitumor activity against tumor cells, wherein the active ingredient of the drug contains LNC@AIEgen, wherein LNC@AIEgen is composed of inactivated cancer cells and Complex1 loaded on the inactivated cancer cells, wherein Complex1 is a compound with the structural formula of Formula 1.

[0008]

[0009] Formula 1.

[0010] Complex1 is a compound with the structural formula of Formula 1: molecular weight: 1041.3942, molecular formula: C57H60AuN4+

[0011] The detailed synthesis of Complex 1 is as follows: AgNO3 (22 mg, 0.13 mmol) was suspended in 5 mL of anhydrous ethanol via a ligand exchange reaction. This was then added to 10 mL of dichloromethane solution containing Complex 2 (81 mg, 0.13 mmol). The mixture was stirred for 10 min, and the precipitate was filtered. TBP (60 mg, 0.13 mmol) and NH4PF6 (21 mg, 0.13 mmol) were then added to the filtrate. The mixture was stirred at room temperature for 1 h, and then 30 mL of diethylether was added to precipitate the product. The precipitate was filtered and washed with hexane to obtain a red solid product.

[0012] The obtained red solid product was identified by mass spectrometry, and the results are as follows: ¹H NMR (400MHz, THF- d 8), δ (ppm): 8.42 (d, J = 8Hz, 2H), 8.22 (m, 1H), 8.13 (d, J = 8Hz, 2H),8.00-7.95 (m,3H),7.90(s, 2H), 7.60(t, J = 8Hz, 2H), 7.45(d, J = 8Hz, 4H), 7.28(t, J = 8Hz, 4H),7.15-7.12 (m, 6H), 7.05 (t, J = 4Hz, 2H), 2.69-2.63 (m, 4H), 1.42 (d, J = 8Hz, 12H), 1.29 (d, J =8Hz, 12H).13CNMR (100MHz,THF- d 8), δ (ppm): 167.7, 154.0, 150.4,147.1,145.6, 145.5, 136.7, 133.5, 133.3, 131.4, 131.1, 131.0, 130.2, 129.4,126.2, 125.2,124.8, 124.7, 124.6, 124.4, 123.7, 122.2, 28.9, 24.8, 24.0. HRMS(MALDI-TOF): m / z [MH-PF6]+ calculated for C57H60AuN4+: 1041.3953; found: 1041.3942., The molecular weight of Complex1 is 1041.3942, and its molecular formula is C57H60AuN4+.

[0013] In the aforementioned drug, the inactivated cancer cells are inactivated cells obtained by treating cancer cells with liquid nitrogen.

[0014] The cancer cells may originate from mammals, such as humans or mice. The cancer cells may be mouse skin melanoma cells.

[0015] The inactivated cancer cells can be prepared by a method including the following steps: suspending the cancer cells in a non-programmed cell cryopreservation solution to obtain a cell culture medium, rapidly cooling the cell culture medium in liquid nitrogen and then immersing it for more than 12 hours to obtain the inactivated cancer cells.

[0016] The present invention also provides a method for preparing the above-mentioned drug.

[0017] The method for preparing the above-mentioned drug provided by the present invention includes mixing inactivated cancer cells and a prepared Complex1 to obtain LNC@AIEgen, and using the LNC@AIEgen as the active ingredient of the drug to obtain the drug; wherein the LNC@AIEgen is composed of inactivated cancer cells and Complex1 loaded on the inactivated cancer cells, and the Complex1 is a compound with the structural formula of Formula 1, the molecular weight of the Complex1 is 1041.3942, and the molecular formula is C57H60AuN4+.

[0018] Formula 1.

[0019] In the above method, the inactivated cancer cells are inactivated cells obtained by treating cancer cells with liquid nitrogen.

[0020] The cancer cells may originate from mammals, such as humans or mice. The cancer cells may be mouse skin melanoma cells.

[0021] The inactivated cancer cells can be prepared by a method including the following steps: suspending the cancer cells in a non-programmed cell cryopreservation solution to obtain a cell culture medium, immersing the cell culture medium in liquid nitrogen for more than 12 hours to obtain the inactivated cancer cells.

[0022] The present invention also provides a product for treating tumors, the product comprising the aforementioned drug and an isotope that generates X-rays.

[0023] The isotope that generates X-rays can be Au.

[0024] The present invention also provides the use of Complex1 described above in the preparation of a drug that enhances the antitumor cell activity of the inactivated cancer cells described above, wherein the cancer cells are the aforementioned cancer cells.

[0025] This article also provides the application of the inactivated cancer cells derived from the tumor cells described above in the preparation of a drug that enhances the antitumor cell activity of the Complex1 material described above, wherein the tumor cells are the aforementioned cancer cells.

[0026] This invention also provides the use of the aforementioned drug in the preparation of products that enhance the efficacy of tumor radiotherapy.

[0027] The radiotherapy is X-ray therapy. The product may contain an isotope that generates X-rays. The X-ray-generating isotope may be Au.

[0028] This invention also provides the application of the Complex1 material described above in the preparation of products that enhance the effect of radiotherapy.

[0029] The use of Complex1, inactivated cancer cells, or LNC@AIE as described above in the preparation of drugs with inhibitory or antitumor activity is also within the scope of protection of this invention.

[0030] In this invention, we utilize inactivated cancer cells LNC and novel Complex1 to generate an effective imaging-guided triple therapy platform for chemotherapy-radiation-immunotherapy, LNC@AIE. This platform provides multi-level immune stimulation, effectively activating both adaptive and innate immunity.

[0031] First, a novel Complex1 material was loaded into inactivated cancer cells (liquid nitrogen-treated cancer cells, LNCs) using a simple and efficient co-incubation method. The inactivated vector carrying Complex1 (LNC@AIEgen) specifically targets the tumor site due to homologous protein recognition between LNCs and living cancer cells, eliminating the primary tumor through the immunomodulatory effects of LNCs and chemotherapy. Furthermore, DNA fragments generated by inactivated cancer cells and RT further activate the cGAS-STING pathway, promoting systemic immune induction against the tumor. Chemotherapy and radiotherapy induce ICDs, further activating the immune response by releasing endogenous antigens from dead cancer cells, promoting cross-presentation of tumor-specific antigens and acquired immunity. In addition, the radiotherapy and chemotherapy of LNC@AIEgen produce an effect similar to a cancer cell vaccine, potentially inhibiting tumor formation and growth. This study demonstrates the great potential of the pleiotropic immune-activating function of inactivated cancer cell vectors and the potential for using these vectors in cancer therapy. Attached Figure Description

[0032] Figure 1 The synthetic route for the aggregation-induced luminescent material Complex1 is described. Figure 2 The synthesis route for LNC@AIEgen.

[0033] Figure 3 Images of B16F10 cells and inactivated LNC cancer cells. The left image shows B16F10 cancer cells (Livecell), and the right image shows inactivated cancer cells (LNC cells).

[0034] Figure 4Characterization of LNC@AIEgen materials. a) Molecular structure of the prepared Complex1; b) Transmission electron microscopy images of Complex1, LNC cells, and LNC@AIEgen; c) UV-Vis spectra of nanoparticles; Insert image: Images of LNC and LNC@AIEgen solutions after centrifugation; d) Photoluminescence (PL) spectra of Complex1 in ddH2O-DMSO mixtures of different proportions; e) Relative emission intensity (I / I0) curves at 632 nm with ddH2O-DMSO mixtures containing Complex1, where I0 represents the emission intensity of Complex1 in pure dimethyl sulfoxide solution; fg) DLS analysis of the prepared...

[0035] f) Nanoparticle size and zeta potential g); h) Electron spin resonance spectra of •OH generated by Complex1 (20 μg / mL) in ddH2O under different doses of X-ray irradiation, using DMPO as a spin trap; i) Decomposition rate of MB induced by •OH generated by Complex1 (20 μg / mL) in relation to the indicated formulation under different doses of X-ray irradiation; j) Decomposition rate of DPBF caused by 1O2 generated by Complex1 (20 μg / mL) in relation to the indicated formulation under different doses of X-ray irradiation; k) Laser confocal scanning electron microscope image of LNC@AIEgen.

[0036] Figure 5 Typical fluorescence images of calmodulin AM / PI staining for B16F10 cancer cells and LNC cells.

[0037] Figure 6 Annexin V-FITC / PI staining was used to analyze LNCs. Typical flow cytometry analysis was performed on control and LNT B16F10 cells using Annexin-V / PI.

[0038] Figure 7 The CCK-8 assay was used to analyze the survival rate of B16F10 live cells and LNC cells.

[0039] Figure 8 Total protein in B16F10 live cells and LNC cells was analyzed by SDS-PAGE electrophoresis. Figure 9 CLSM image of B16F10 cells loaded with Complex1.

[0040] Figure 10 DLS analysis of nanoparticles.

[0041] Figure 11 The graph shows the Complex1 content in different numbers of LNC cells.

[0042] Figure 12 This study investigates the subcellular localization and biosafety of LNC@AIEgen in in vitro experiments. a) CLSM images of LNC@AIEgen subcellular localization based on different organelle probes: ER / ER-Tracker Green, Lyso / Lyso-Track

[0043] Green, Mito / MitoTracker Red CMXRos, DAPI, nuclear probe; b) Fluorescence images of ROS probes of different types of materials, where “-RT” rows indicate no X-ray irradiation and “+RT” rows indicate X-ray irradiation; c) Hemolysis assays of LNC@AIE at different concentrations (n=4); d)-f) Relative survival rates of L929, GL261 and B16F10 cells after incubation with different types and concentrations of nanomaterials for 24 hours (n=6).

[0044] Figure 13 MFI for ROS probes.

[0045] Figure 14 Fluorescent images of 1O2 probe staining, where the "-RT" row indicates no X-ray irradiation and the "+RT" row indicates X-ray irradiation.

[0046] Figure 15 Fluorescent images of O2- probe staining, where the "-RT" row indicates no X-ray irradiation and the "+RT" row indicates X-ray irradiation.

[0047] Figure 16 illustrates the enhancement of radiotherapy efficacy based on Complex1-mediated ICD. a) Cell viability of B16F10 cells after incubation with different nanoparticles and irradiation with different doses of X-rays (n=4); b) H2A.X (S139) staining of B16F10 cells after different treatments.

[0048] c) Image of colony formation obtained from different treatment groups; d) CRT immunofluorescence staining after different treatments; ef) Levels of ATP e) and cGAMP f) released after different treatments (n=4); gj) Levels of interferon-γ, tumor necrosis factor α, IL-2, and IL-12 in B16F10 tumor cells after different treatments (n=4). Data are expressed as mean ± scanning electron microscopy. P-values ​​were calculated using one-way ANOVA. p<0.05, where “-RT” rows indicate no X-ray exposure and “+RT” rows indicate X-ray exposure.

[0049] Figure 17 shows the effect of different nanoparticles combined with X-ray irradiation on calcein-AM / PI staining of B16F10 tumor cells, where the "-RT" row indicates no X-ray irradiation and the "+RT" row indicates X-ray irradiation.

[0050] Figure 18 Quantitative analysis of γ-H2A.X(S139) in different groups.

[0051] Figure 19 Quantitative analysis of colony formation rate in different groups.

[0052] Figure 20 The average fluorescence intensity of CRT immunofluorescence staining.

[0053] Figure 21 Western blotting analysis of γ-H2A.X (S139), cGAS-STING pathway proteins, and high mobility group 1 (HMGB1).

[0054] Figure 22 Determination of the targeting and biodistribution of LNC@AIEgen in vivo. a) Typical fluorescence images at different time points in mice treated with Complex1 or LNC@AIEgen; b) Fluorescence images of major organs and tumors 12 h after injection, He: heart,

[0055] Lv: liver, Sp: spleen, Lu: lung, Ki: kidney, Tu: tumor; c) radiation efficiency corresponding to a) (n=3); d) circulation of Complex1 and LNC@AIEgen in vivo; e) fluorescence radiation efficiency corresponding to b) different organs of B16F10 tumor-bearing mice at different time points.

[0056] Figure 23 To evaluate antitumor immunotherapy in vivo. a) Tumor volume growth curves; b) Body weight curves; c) Survival analysis of mice after different treatments; d) Tumor weight analysis; e) Tumor images obtained on day 14 after treatment, x: death; f)-i) Levels of f) interferon-γ, g) tumor necrosis factor α, h) IL-2, and i) IL-12 in B16F10 tumor-bearing mice after different treatments (n=4); j) Representative fluorescent staining images of different immune cell subsets. Data are presented as mean ± scanning electron microscopy. P-values ​​were calculated using one-way ANOVA. p<0.05, p<0.01, p<0.001, ns indicates no significant difference.

[0057] Figure 24 Images of tumor sections. a) Representative images of ROS levels in different groups (red: ROS, blue: DAPI); b) TUNEL-stained images of apoptotic fluorescence (red: apoptotic cells, blue: DAPI); c) H&E-stained tumor sections after different treatments; d) IHC-stained tumor sections after different treatments. IHC staining was used to detect changes in the expression of apoptosis-related proteins e) Bcl-2 and f) Caspase-3.

[0058] Figure 25 These are representative fluorescence images of CD4+ and CD8+ T cells in the spleen.

[0059] Figure 26 For biosafety analysis. a) Hematological parameters after 14 days of various treatments: WBC, RBC, HGB, MCV, MCHC, PLT, HCT. b) Blood biochemical parameters after 14 days of various treatments: ALT, AST, TP, GLB, TBIL, UREA, CREA, ALB. In the horizontal axis, 1 is control, 2 is LNC, 3 is Complex1, 4 is LNC@AIEgen, 5 is control + RT, 6 is LNC + RT, 7 is Complex 1 + RT, and 8 is LNC@AIEgen + RT.

[0060] Figure 27 H&E staining was used to evaluate the in vivo toxicology of major organs such as heart, liver, spleen, lung and kidney in mice after different treatments. The "+RT" row indicates X-ray irradiation.

[0061] Figure 28 To investigate the effect of different doses of X-ray irradiation on the viability of B16F10 tumor cells using the CCK-8 assay.

[0062] Figure 29 This illustrates the in vivo preventive effect of LNC@AIEgen combined with RT. a) Schematic diagram of the in vivo preventive effect of LNC@AIEgen combined with RT

[0063] Figure 1; b) Tumor images 28 days after treatment; o: No tumor; c) Tumor volume curve; d) Body weight curve; e) Tumor weight; f) Individual growth kinetics of tumors in different groups; g) HE staining results of tumor tissue sections; h) Fluorescence imaging of different immune cell subsets, namely M1 / M2 macrophages: CD80+ / CD86+, CD4 T cells: CD4+, CD8 T cells: CD8+, and NK cells: CD3- / CD49b+. Data are expressed as mean ± scanning electron microscopy. P-values ​​were calculated using one-way ANOVA. p<0.05 and p<0.01.

[0064] Figure 30 To enable the delivery of Complex1 to inactivated cancer cells for use in triple therapy of chemotherapy-radiotherapy-immunotherapy and tumor vaccination strategies. Detailed Implementation

[0065] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0066] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0067] Unless otherwise specified, all quantitative experiments in the following examples are performed in triplicate. Experimental materials:

[0068] B16F10 cancer cells were purchased from Shanghai Fuheng Biotechnology Co., Ltd. (Shanghai, China), catalog number FH0361. Non-programmed cell cryopreservation solution was purchased from Cyagen (NCRC-10001-50).

[0069] Cell counting kit (CCK-8) was purchased from MCE. Reactive oxygen species assay kit, Annexin V-FITC / PI apoptosis assay kit, ATP assay kit, ER Tracker Green kit, Mito-Tracker Red CMXRos kit, Lyso-Tracker Green kit, Calcein-AM kit, and PI staining kit were all purchased from Beyotime.

[0070] Biotechnology.

[0071] PBS (pH 7.4) and fetal bovine serum (FBS) were purchased from Gibco Life Technologies (AG, Switzerland). DMEM, trypsin-EDTA, and penicillin-streptomycin (PS) were purchased from Corning. Immunoblotting and fluorescent staining sections describe antibody information.

[0072] All other chemicals used in this invention are analytical reagent grade and require no further purification. All solutions were prepared using ultrapure water (18.25 MΩ·cm, 25°C).

[0073] The experimental results of this invention are expressed as mean ± standard deviation (SEM). Statistical analysis was performed using one-way ANOVA, and the graph-based honesty test for significant differences was conducted using GraphPad PRIMILE 8.0 (GraphPad software). p<0.05, p<0.01 and p<0.001, ns indicates no significant difference.

[0074] Example 1: Preparation of inactivation vector carrying Complex1 (LNC@AIE)

[0075] The preparation process of the inactivation vector LNC@AIEgen carrying Complex1 (LNC@AIEgen for short) is as follows: Figure 2 As shown. The specific steps are as follows:

[0076] 1. Preparation of aggregation-induced emission material Complex1

[0077] Complex1 was prepared via a ligand exchange reaction. The Complex1 compound was prepared according to... Figure 1 The synthesis route was used to prepare the product, with an overall yield of 81%.

[0078] N , N The synthesis method of ⁻diphenyl-4-(7-(pyridin-4-yl)benzo[c][1,2,5]thiadiazol-4-yl)aniline (TBP) is given in the reference: L. Wang, X. Yang, J. Zhao, F. Zhang, X. Wang, L. Sun, Efficient organic saturators with pyridine-N-oxide as an anchor group for ye-sensitized solar cells. Chem Sus Chem, 7 (2014) 2640-2646. Complex2 was purchased from Aladdin Chemical Reagents, catalog number: C284031.

[0079] The detailed synthesis of Complex 1 is as follows: AgNO3 (22 mg, 0.13 mmol) was suspended in 5 mL of anhydrous ethanol via a ligand exchange reaction. This was then added to 10 mL of dichloromethane solution containing Complex 2 (81 mg, 0.13 mmol). The mixture was stirred for 10 min, and the precipitate was filtered. TBP (60 mg, 0.13 mmol) and NH4PF6 (21 mg, 0.13 mmol) were then added to the filtrate. The mixture was stirred at room temperature for 1 h, and then 30 mL of diethylether was added to precipitate the product. The precipitate was filtered and washed with hexane to obtain a red solid product.

[0080] The obtained red solid product was identified by mass spectrometry, and the results are as follows: ¹H NMR (400MHz, THF- d 8), δ (ppm): 8.42 (d, J = 8Hz, 2H), 8.22 (m, 1H), 8.13 (d, J = 8Hz, 2H),8.00-7.95 (m,3H),7.90(s, 2H), 7.60(t, J = 8Hz, 2H), 7.45(d, J = 8Hz, 4H), 7.28(t, J = 8Hz, 4H),7.15-7.12 (m, 6H), 7.05 (t, J = 4Hz, 2H), 2.69-2.63 (m, 4H), 1.42 (d, J = 8Hz, 12H), 1.29 (d, J =8Hz, 12H).13CNMR (100MHz,THF- d 8), δ (ppm): 167.7, 154.0, 150.4,147.1,145.6, 145.5, 136.7, 133.5, 133.3, 131.4, 131.1, 131.0, 130.2, 129.4,126.2, 125.2,124.8, 124.7, 124.6, 124.4, 123.7, 122.2, 28.9, 24.8, 24.0. HRMS(MALDI-TOF): m / z[MH-PF6]+ calculated for C57H60AuN4+: 1041.3953; found: 1041.3942., The molecular weight of Complex1 is 1041.3942, and its molecular formula is C57H60AuN4+.

[0081] All intermediates and products of the synthesis reaction were characterized using nuclear magnetic resonance (NMR) and high-resolution mass spectrometry (HRMS). The structure of the prepared Complex1 is shown below. Figure 4 As shown in Figure a), the prepared antigen contains TBP-2 active units and monovalent gold, which respectively exhibit AIE effect and enhance radiotherapy efficacy.

[0082] Complex1 was dissolved in DMSO to obtain a final concentration of 2 mg / mL for later use. 2. Preparation of LNC cells.

[0083] First, B16F10 cancer cells were cultured in DMEM + 10% FBS + 1% PS medium, collected, and counted. Then, 2 × 10⁵ cells were transplanted into the dorsal side of the right hind limb of a mouse. When the tumor volume reached 150-200 mm³, the mouse was anesthetized, and tumor tissue was collected. The tissue was washed with pre-cooled PBS buffer (0.01 M, pH 7.4) to remove blood cells, cut into small pieces, and digested with trypsin to form a single-cell suspension. The cells were counted by trypan blue staining, and 1 × 10⁶ cells were suspended in 1 mL of unprogrammed cell cryopreservation medium to obtain a cell-containing culture medium. This cell-containing culture medium was then immersed in liquid nitrogen for 12 h to obtain liquid nitrogen-treated cancer cells (LNC cells). Figure 2 As shown, this is for further experiments. Figure 3 In the middle, the left image shows B16F10 cancer cells (Livecell), and the right image shows inactivated cancer cells (LNC cell).

[0084] 3. Complex1 was loaded onto LNC cells to obtain the nanotherapy platform LNC@AIEgen.

[0085] The LNC cells obtained in step 2 were thawed in a 37°C water bath and centrifuged at 600g for 5 min. After washing three times with PBS solution (pH 7.4), 1×10⁵ LNC@AIEgen cells were resuspended in 0.5 mL of PBS buffer, and 200 μg of Complex1 was added. The mixture was rotary mixed at 30 rpm for 3 h to obtain Complex1-loaded inactivated cancer cells (referred to as LNC@AIEgen, nanotherapy platform). The precipitate was collected by centrifugation, and this precipitate was identified as LNC@AIEgen cells. The LNC@AIEgen cells were resuspended in PBS buffer, the drug loading was measured, and the cells were stored at 4°C for further experiments.

[0086] 4. Characterization of Complex1 and LNC@AIEgen materials

[0087] Images were captured using a JEM-3200FS transmission electron microscope. UV-Vis-NIR absorption spectra were recorded using a SPARK10M (TECAN, USA). Zeta potential and particle size analysis of the nanomaterials were performed using a ZETASIZER (NanoSeries).

[0088] Measurements were performed using a Malvern microscope. Fluorescence intensity was measured using a Spark 10M (TECAN, USA). Fluorescence images were acquired using a laser confocal microscope (TCS SP8, Lecia, Germany). Small animal imaging systems (IVIS Spectrum) were used.

[0089] The distribution within the organism was detected using PerkinElmer (USA). Radiation therapy was performed using XRAD160 (Percision X-ray, USA).

[0090] Complex1's absorption spectrum has two typical absorption peaks at 311 and 480 nm. Figure 4 (c)). After excitation with a 480 nm laser, a distinct fluorescence emission peak is observed at 696 nm in dimethyl sulfoxide (DMSO) solution. Figure 4 (d).

[0091] Based on transmission electron microscope images ( Figure 4 In section b), the prepared Complex1 cells exhibited a typical punctate morphology, indicating that Complex1 loading did not cause significant changes in LNC cell structure during LNC@AIEgen preparation. Laser confocal microscopy (…) Figure 4 (k) and UV-Vis spectral analysis ( Figure 4 c) and bright-field imaging ( Figure 4 The illustration in (c) verifies the successful preparation of LNC@AIEgen.

[0092] The fluorescence properties of Complex1 in a water-DMSO mixture are as follows: Figure 4As shown in d), Complex1 exhibits very low luminescence intensity in pure DMSO. When the water-DMSO mixture contains 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, and 90% water content, the intramolecular free motion of non-radiative decay is suppressed with increasing water content. Increasing the water content in the water-DMSO mixture (from 0% to 40%) leads to a loss of polarity and a slight increase in luminescence intensity and a blue shift (from 696 nm to 632 nm). Figure 4 (d) and (e)). When the water content is greater than 50%, the luminescence intensity of Complex1 is significantly enhanced due to the aggregation of Complex1, indicating the presence of Complex1 features.

[0093] 5. Characterizing LNC cells

[0094] Cell viability analysis was performed using calcein AM / PI staining. After staining, LNC cells were placed in confocal culture dishes and analyzed under a confocal microscope. Cell death analysis was performed according to the instructions using the Annexin V-FITC / PI apoptosis kit, followed by flow cytometry analysis.

[0095] In the cell proliferation assay, B16F10 cancer cells (live cells) and LNC cells were suspended in cell culture medium (DMEM + 10% FBS + 1% PS, phenol red-free) and added to 48-well plates at a density of 1×10⁴ cells / well. The cells were cultured at 37°C and 5% CO₂. At different time points (0.5h, 6h, 12h, 24, 48h), 20 μL of LCK-8 reagent was added to each well. After 1.0h of incubation, the absorbance at 450 nm was measured using a TECAN microplate reader.

[0096] Bright-field confocal microscopy revealed that LNC cells exhibited distinct cellular structures compared to untreated B16F10 cancer cells (live cells). LNC cell viability was negligible compared to untreated B16F10 cancer cells (live cells) as analyzed by calmodulin AM / PI staining, flow cytometry, and CCK-8 cell counting. Almost all LNC cells were labeled with propidium iodide (PI, indicating dead cells), but no significant Calcein AM signal (indicating live cells) was observed. Annexin V-FITC / PI staining and flow cytometry confirmed LNC cell death. Figure 6 Furthermore, CCK-8 assays showed that LNC cells did not exhibit proliferative activity like live cancer cells. Figure 7 These results indicate that LNC cells lost their activity, suggesting they are safe in vivo.

[0097] The expression of proteins that affect tumor targeting was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE).

[0098] Total protein from LNC cells and untreated B16F10 cancer cells (live cells) was analyzed by SDS-PAGE protein electrophoresis. Equal numbers (1 × 10⁶ cells) of LNC cells and untreated B16F10 cancer cells (live cells) were added to 60 μL of SDS loading buffer containing protease inhibitors and boiled at 100 °C for 10 min. 20 μL of the prepared protein sample was then electrophoresed on a 12% SDS-PAGE gel at 120 V for 1.5 h. The resulting gel was stained with Coomassie blue for 2 hours, washed overnight with ddH₂O, and then imaged.

[0099] The results are as follows Figure 8 As shown, most of the proteins expressed by B16F10 cancer cells (live cells) that were not treated with liquid nitrogen were retained in LNC cells.

[0100] Inactivated B16F10 cancer cells loaded with Complex1 were obtained by rotational mixing of Complex1 with LNC cells. Complex1 was then loaded into live cells (…). Figure 9 ) and LNC cells ( Figure 4 No significant changes were observed in the complex1 loading. Furthermore, DLS analysis showed that complex1 loading had no significant effect on LNC cell size, with an average of 9 μm. Figure 4 f), Figure 10 This indicates that LNC cells remained intact after being loaded with Complex1. In aqueous solution, the Zeta potential of Complex1 was positive (15.96 mV), and the Zeta potential of LNC cells did not change significantly after loading with Complex1 (from -10.53 mV to -10.26 mV). Figure 4 The positive charge on Complex1 enhances their affinity for the outer membrane of LNC cells.

[0101] Complex1 was mixed with LNC cells by rotation, and the supernatant was collected after centrifugation. The fluorescence intensity of the supernatant was measured, and the content of the remaining Complex1 material in the supernatant was calculated based on the fluorescence intensity to obtain the loading rate. Loading rate = (initial amount - remaining amount) / initial amount × 100%. When LNC cells were suspended in 0.5 mL PBS containing 200 μg Complex1, the estimated loading rate was 99.65% (Figure 10).

[0102] Example 2, The ability of radiotherapy Complex 1 to generate ROS 1. Study on the efficiency of radiotherapy Complex 1 in generating •OH

[0103] The specific steps are as follows: Complex1 and LNC@AIE (20 μg / mL) and methylene blue MB (15 μg / mL) were added to an aqueous solution. After irradiation with different doses (0, 2, 4, 6, 8, 10 Gy) of X-rays, the absorbance of the above solution at 665 nm was measured, and the degradation of MB was recorded. The degradation of MB under oxidative conditions could be detected by the generation of •OH. In addition, the generation of •OH was evaluated by ESR spectroscopy when 5,5-dimethyl-1-pyrrolline-N-oxide (DMPO) was used as a free radical scavenger.

[0104] 2. Study on the efficiency of singlet oxygen (1O2) production by radiotherapy combined with Complex1

[0105] The generation of 1O2 after X-ray radiotherapy was detected using 1,3-diphenylisobenzofuran (DPBF) as an indicator.

[0106] The specific steps are as follows: Add 1 μL (80 μM) of DPBF to a 48-well plate containing 20 μg / mL Complex1 or PBS, with 2 wells per group. Irradiate with different doses (0, 2, 4, 6, 8 and 10 Gy) of X-rays and record the absorbance at 450 nm using UV-Vis spectroscopy to obtain the attenuation rate (Tecan).

[0107] Effective ROS generation capability is a fundamental prerequisite for ICD inducers based on radiotherapy and is important for improving radiotherapy outcomes. The generation of hydroxyl radicals (•OH) and 1O2 in the prepared Complex1 nanomaterials was detected using 5-dimethylpyrrolidone-N-oxide (DMPO), methylene blue (MB), and 1,3-diphenylisobenzofuran (DPBF). Figure 4 As shown in h), with

[0108] With increasing irradiation dose, the characteristic ESR peak of •OH / DMPO increased significantly, indicating that the prepared antigen can effectively generate •OH. Simultaneously, MB and DPBF assays also showed a significant increase in the characteristic signal intensity of •OH and 1O2. Figure 4 In i) and j), it is shown that Complex1 effectively generates ROS.

[0109] In summary, these results demonstrate that the Complex1-loaded inactivated cancer cell delivery vector prepared in Example 1 can effectively generate a variety of ROS under X-ray irradiation, showing great potential in RT-based antitumor therapy.

[0110] Example 3. Effects of LNC@AIEgen material on in vitro cells 1. Detection of cytotoxicity of LNC@AIEgen material

[0111] L929 and GL261 (Shanghai Fuheng Biotechnology Co., Ltd., catalog numbers FH0534 and FH1097 respectively) B16F10 cells were cultured in 96-well plates (5×103 cells / well) in DMEM (Gibco) + 10% FBS + 1% PS at 37℃, 5% CO2, and 95% humidity. Once the cells reached 60-65% confluence, 200 μL of complete culture medium (DMEM + 10% FBS + 1% penicillin and streptomycin) containing different concentrations of nanoparticles (0, 5, 10, 20, 50, and 100 μg / mL) was used to replace the original culture medium. Four cell groups were established for each concentration.

[0112] Cell viability was determined using the CCK-8 assay: Cells were first treated with nanoparticles for 6 hours, then washed three times with different doses (0, 2, 4, 6, 8, 10 Gy) of PBS (please adjust the concentration and pH of the buffer solution), and cultured for 12 hours. Then, 10 μL of CCK-8 and 90 μL of serum-free culture medium were added to each well, and the cells were incubated at 37°C for 1 hour. Cell viability was evaluated by measuring the absorbance at 450 nm.

[0113] 2. Sublocalization of LNC@AIEgen

[0114] Complex1 and LNC@AIEgen were incubated with B16F10 cancer cells for 6 hours, followed by incubation with Mito-Tracker,

[0115] The new complete culture media of Lyso-Tracker and ER-Tracker were cultured at 37°C for 30 min, washed three times with PBS, and then imaged on a laser confocal microscope. The colocalization correlation parameters were further analyzed using ImageJ software and used as Pearson correlation coefficients.

[0116] Subcellular localization of organelle-specific probes was assessed by evaluating the overlap between organelle-specific probes and Complex1 in B16F10 cancer cells. Laser confocal microscopy (CLSM) was used to evaluate this subcellular localization. Figure 12As confirmed in Figure a), after B16F10 cancer cells were incubated with Complex1 or LNC@AIEgen at 37°C for 6 hours, Complex1 was found to be mainly distributed in the endoplasmic reticulum, showing fluorescence overlap with ER-Tracker Green (Pearson's R values ​​were 0.63 and 0.60, respectively). This overlap was higher than that of Lyso-Track Green (Pearson's R values ​​were 0.33 and 0.30, respectively), MitoTracker Red CMXRos (Pearson's R values ​​were 0.05 and 0.02, respectively), and the nuclear detector (DAPI, Pearson's R values ​​were 0.01 and 0.04, respectively). The results indicate that the prepared Complex1 failed to be strictly immobilized on specific organelles.

[0117] 3. Assessment of ROS generation in B16F10 cancer cells

[0118] B16F10 cells were incubated for 6 h with different concentrations of Complex1 (final concentration 20 μg / mL), LNC (final concentration 5 × 10⁴ cells / mL), LNC@AIEgen (final concentration equivalent to 20 μg / mL Complex1), and control (PBS) in 48-well plates (200 μL). After incubation, the cells were irradiated with X-rays (4 Gy) for 2 h and then cultured in fresh serum-free medium containing 5 μM DCFH-DA, singlet oxygen sensor reagent, and dihydroethidine for 30 min. The production of ROS, ¹O₂, and O₂⁻ was detected, and ROS analysis was performed using laser confocal microscopy.

[0119] 2,7-Dichlorodihydrofluorescein diacetate (DCF-DA), singlet oxygen sensor reagent, and ethidium dihydrogen phosphate were used as indicators of ROS, 1O2, and O2- to test whether the prepared Complex1 could generate ROS in cancer cells. Results are as follows: Figure 12 (b) and Figure 13 As shown, B16F10 cancer cells exhibited high intracellular green fluorescence after incubation with Complex1 and irradiation with low-dose X-rays (4 Gy), due to the reaction of DCF-DA and ROS. This was attributed to the effect of 1O2 on the oxygen sensor reagent (…). Figure 14 The effect of O2- on dihydroethidium ( Figure 15 Bright green and red fluorescence were also observed within the cells. These results demonstrate Complex1's ability to effectively generate ROS in cancer cells after X-ray irradiation. This abundant ROS production may...

[0120] It can damage the structure and function of the endoplasmic reticulum and further induce cancer cell death. Figure 12In section b), the "-RT" row indicates no X-ray (4Gy) exposure, and the "+RT" row indicates X-ray (4Gy) exposure.

[0121] 4. B16F10 cell colony formation assay

[0122] Six thousand B16F10 cells were seeded into 12-well plates and cultured for 16 hours. The cultured cells were then divided into four groups: each group was treated with different amounts of Complex1 (final concentration 20 μg / mL) and LNC (final concentration 5 × 10⁴ cells / mL).

[0123] LNC@AIEgen (final concentration in the system equivalent to 20 μg / mL Complex1) and control group (PBS) (200 μL) were incubated for 6 h, then irradiated with X-rays (4 Gy) and cultured for 2 h. Cells were then collected and re-seeded into 6-well plates at a density of 100 cells / well. The cells were cultured for 1 week, and fresh complete culture medium was replaced on time. After 1 week, the cells were fixed with pre-cooled methanol for 5 min, washed, stained with crystal violet and then imaged.

[0124] 5. Hemolysis detection

[0125] Blood samples used in the experiment were obtained from C57BL / 6J mice (GemPharmatech Co., Ltd.). Normally, 1 mL of blood sample was diluted with 5 mL of PBS, and then red blood cells (RBCs) were separated from the serum by centrifugation (2000 rpm, 10 min). After washing repeatedly with PBS to completely remove plasma, the RBCs were diluted with 10 mL of PBS. Different final concentrations of LNC@AIE (5, 10, 20, ...) were used.

[0126] Equal numbers of erythrocytes were treated with 50 and 100 μg / mL solutions, with ddH2O and PBS used as positive and negative controls, respectively, to evaluate hemolytic activity. After incubation at 150 rpm for 3 h in a shaker at 37 °C, the cells were then centrifuged at 10,000 rpm for 10 min. Finally, the absorbance of the supernatant at 570 nm was recorded using a TECAN microplate reader to calculate the hemolysis rate.

[0127] 6. In vitro experimental detection of antitumor efficacy

[0128] B16F10 cells (1x10⁵) were seeded in 24-well plates and co-cultured for 6 h with Complex1 (final concentration 20 μg / mL), LNC (final concentration 2 × 10⁴ cells / mL), LNC@AIEgen (final concentration equivalent to 20 μg / mL Complex1), and control (PBS). After irradiation with 4 Gy of X-rays, the cells were cultured for another 12 h. The antitumor effect was evaluated using the CCK-8 Assay, live cell staining, and dead cell staining methods described above.

[0129] Biocompatibility is a fundamental requirement for the clinical translation of bio-nanomedicines. Therefore, we used hemolysis and CCK-8 assays to determine the biocompatibility and toxicity of the prepared nanoparticles. The results are as follows: Figure 12 As shown in Figure c), LNC@AIE exhibits good biocompatibility in vitro. Even at a Complex1 concentration of 100 μg / mL, the hemolysis rate remains below 5%. Different concentrations of...

[0130] Complex1 and LNC@AIEgen were incubated with different cell lines (L929, GL261, and B16F10) for 24 h. CCK-8 analysis showed that Complex1 and LNC@AIEgen were effective against normal cells (L929, GL261, and B16F10). Figure 12 The drug (d) showed no significant cytotoxic effect, but it was effective against tumor cells (GL261 and B16F10). Figure 12 The inhibitory effects of e) and f) were significant. At 20 μg / mL, the inhibition rates were 36.61% and 36.14%, respectively, and at 50 μg / mL, the inhibition rates were 46.91% and 45.00%, respectively, suggesting selective toxicity to cancer cells. These data indicate that the prepared LNC@AIEgen can serve as a good therapeutic platform for anti-tumor radiotherapy and chemotherapy.

[0131] 7. ATP level detection

[0132] B16F10 cells were seeded at a density of 6 × 10⁴ cells / well in 12-well plates and cultured for 16 h (to reach 80–85% confluence). Then, the cells were cultured for 6 h in fresh DMEM medium containing different concentrations of Complex1 (final concentration 20 μg / mL), LNC (final concentration 1 × 10⁴ cells / mL), LNC@AIEgen (final concentration equivalent to 20 μg / mL Complex1), and control (PBS). After irradiation with 4 Gy X-rays, the cells were cultured for 12 h. The cell cultures were then collected, and the ATP release content was determined using an adenosine triphosphate (ATP) assay kit according to the manufacturer's instructions.

[0133] In vitro cytokine release assay: First, B16F10 cells were seeded at a density of 1×10⁵ cells per well in 6-well plates. When cell confluence reached 60-65%, the cells were grouped according to the following treatment methods: Complex1 (final concentration 20 μg / mL), LNC (final concentration 0.5×10⁴ cells / mL), LNC@AIEgen (final concentration equivalent to 20 μg / mL Complex1), and control group (PBS buffer), as well as whether or not X-ray irradiation was performed.

[0134] After the above treatment, B16F10 cells were collected and the following cytokines were detected by ELISA: IFN-γ (BioLegend430807), TNF-α (BioLegend430904), IL-2 (BioLegend431007) and IL-12 (BioLegend433604).

[0135] 8. Western blotting detection of whole-cell protein lysis products

[0136] Whole-cell protein lysates were diluted with 5×SDS protein loading buffer, boiled for 10 min, and then added to SDS-PAGE gels of different concentrations (8-15%) for electrophoresis. After electrophoresis, the lysates were transferred to PVDF membranes and blocked with Beyotime blocking buffer.

[0137] The membrane was blocked for 1 hour with Biotechnology (CAT.no. P0023B) and incubated overnight at 4°C with the following primary antibodies: cGAS (cat.no: ab224144, Abcam), STING (cat.no. 13647S, CST), Calreticulin (CRT, cat.no: ab92516, Abcam), HMGB1 (cat.no: ab79823, Abcam), γ-H2A.X (S139) (cat.no: ab81299, Abcam), and β-actin (cat.no. AC026, Abclonal), with β-actin used as an internal control. The membrane was washed three times for 5 minutes each in a mixture of PBS and Tween-20 (PBST), then incubated for 2 hours with HRP-conjugated anti-rabbit IgG (cat. no. 7074; CST) and washed again three times for 5 minutes each in PBST. The PVDF film obtained from the above steps was treated with a developer (cat.no. 32132;Life) and then imaged using a ChemiScope Series 6000 Touch (Clinx Science Instruments Co., Ltd.).

[0138] 9. Cell immunofluorescence staining

[0139] B16F10 cell samples were fixed with 4% paraformaldehyde for 10 min at room temperature. The samples were then infiltrated with 0.1% Triton X-100 PBS for 5 min, followed by blocking with blocking buffer containing 5% bovine serum albumin (BSA) for 2 h at room temperature. After blocking, the samples were incubated overnight at 4°C with primary antibody (CRT, cat no:ab92516, Abcam; γ-H2A.X (S139), cat no:ab81299) at an appropriate dilution, followed by washing three times with PBS. They were then incubated with secondary antibody for 2 h at room temperature. Finally, cell or section samples were stained with DAPI, washed with PBS, and imaged using a Leica CLSM (Leica, SP8).

[0140] To investigate the ability of Complex1 to induce ICD in vitro through radiosensitization and toxicity, B16F10 tumor cells were incubated with different nanoparticles for 6 hours and irradiated with different doses of X-rays. Figure 16As shown in Figure a), incubation with LNC did not result in any significant cancer cell death compared to untreated cancer cells. However, when B16F10 cells were incubated with 20 μg / mL Complex1 or LNC@AIEgen, the cancer cell death rate increased significantly, even without X-ray irradiation. The death rate of B16F10 cancer cells increased dramatically with increasing X-ray dose. For example, at 4 Gy, the mortality rate was approximately 63%, significantly higher than the control group (23%) that received only X-ray irradiation.

[0141] Cancer cell survival was assessed using calmodulin AM and PI dual fluorescence staining. Results showed that B16F10 cancer cells were effectively killed after incubation with 20 μg / mL Complex1 or LNC@AIEgen. Figure 17 Therefore, Complex1, used in this study, is a good candidate for enhancing the combination of chemotherapy and radiotherapy.

[0142] In target cells, RT can directly damage macromolecules such as lipids, proteins, and DNA, leading to cell cycle arrest and potential necrotizing, apoptotic, or eosinophilic cell death in cancer cells, both directly and indirectly. Therefore, fluorescence staining and clonogenic analysis were subsequently used to determine tumor cell death following Complex1 combined with RT therapy.

[0143] First, B16F10 cancer cells were co-incubated with LNC, Complex1, or LNC@AIEgen for 6 hours, followed by irradiation with X-rays (4 Gy). The results were as follows... Figure 16 As shown in b), the RT group, LNC+RT group, Complex1+RT group, and LNC@AIEgen+RT group all showed obvious fluorescent foci, while the other treatment groups without X-ray irradiation did not, suggesting that RT has a significant DNA damaging effect on B16F10 tumor cells. The DNA damage was particularly severe in the Complex1+RT group and the LNC@AIEgen+RT group, indicating...

[0144] Complex1 enhanced the induction of DNA damage by radiotherapy. Immunoblotting experiments also confirmed enhanced expression of γ-H2A.X (S139) protein in the Complex1+RT group and the LNC@AIEgen+RT group. Figure 18 ).

[0145] Simultaneously, the inhibitory effect of Complex1 as a radiotherapy enhancer on tumor cells was evaluated using a clonogenic assay. Results are as follows: Figure 16 c) and Figure 19 As shown, after tumor cells were incubated with LNC, Complex1, and LNC@AIEgen, the clonal shape...

[0146] There were no significant differences in the control group. However, X-ray irradiation significantly inhibited colony formation, especially in the groups treated with Complex1 or LNC@AIEgen. This indicates that Complex1 or LNC@AIEgen can significantly increase the X-ray sensitivity of B16F10 cancer cells.

[0147] RT-induced ICD effectively activated the immune response and induced immunomodulatory effects against the tumor. We found that...

[0148] Complex1 accumulates in the endoplasmic reticulum and lysosomes, and can effectively induce ICD when the endoplasmic reticulum and lysosomes are damaged. Inactivated cancer cells release DNA, which combines with RT-induced DNA damage to induce damage-associated molecular pattern molecules (DAMPs) from the cell nucleus and subsequent DNA damage repair (DDR).

[0149] Furthermore, lysosomes targeted by Complex1 can be damaged by its toxicity and RT-induced ROS, leading to disruption of lysosomal membrane permeability and subsequently inducing ICD. We hypothesized that Complex1-based RT could elicit an ICD-induced immune response through direct or indirect pathways, and investigated whether the Complex1 radiosensitizer prepared in this paper could induce ICD after X-ray irradiation. Figure 16 (d) and Figure 20 As shown, Complex1 combined with X-ray irradiation significantly enhanced the expression of CRT on the surface of B16F10 cancer cells, suggesting that this combination has a good ICD-inducing ability. Complex1 chemotherapy can also enhance the expression of CRT on the surface of B16F10 cancer cells, leading to stronger ICD.

[0150] Immunoblotting showed increased CRT expression in both the RT-only group and the Complex1+RT group. Figure 21 In addition to CRT, ICD also highly expresses high-mobility group box 1 (HMGB1), secreted cGAMP, adenosine triphosphate (ATP), and cytokines, including IFN-γ.

[0151] IL-2 and IL-12. This is also associated with enhanced cGAS-STING pathway activity.

[0152] Western blotting results showed that after Complex1 radiosensitization, the expression of HMGB1, cGAS, and STING in B16F10 cancer cells was enhanced. Figure 21 Using adenosine triphosphate (ATP) detection kit ( Figure 16 Quantitative analysis by ELISA and other methods also confirmed elevated levels of adenosine triphosphate (ATP), cGAMP, interferon-γ, tumor necrosis factor α, IL-2, and IL-12. Figure 16 (f)-j) Importantly, ICD-associated cytokines can further activate the immune system by acting on different immune cell populations. Based on the detection of ICD markers, it is reasonable to conclude that RT with the prepared antigen can effectively induce ICD.

[0153] Example 3. Effects of Complex1 and LNC@AIEgen materials on in vivo cells 1. Construction of animal tumor models

[0154] Female C57BL / 6J mice (5–6 weeks old, weight: 18–20 g) were purchased from GemPharmatech Co., Ltd. and housed in the SPF laboratory of the Animal Center of Shenzhen People's Hospital, at a temperature of 22±1℃, humidity of 40–50%, a 12-hour light / dark cycle, and free access to water and standard experimental food. All surgeries were approved by the Animal Experimentation Ethics Committee and performed in accordance with the procedures of Shenzhen People's Hospital (Second Clinical Medical College of Jinan University, First Affiliated Hospital of Southern University of Science and Technology). Each mouse was subcutaneously injected with 1 × 10⁵ B16F10 cancer cells (1 × 10⁵ cells suspended in 100 μL PBS) in the right abdomen to establish a tumor-bearing model. Tumors were allowed to grow to 150–170 mm³ for further use.

[0155] 2. In vivo experiments to verify the biodistribution of tumor-targeting genes.

[0156] When the tumor volume of B16F10 mice in C57BL / 6J reached 150–170 mm³, they were randomly divided into two groups: the Complex1 group and the LNC@AIEgen group, with 3 mice in each group. Each mouse in the Complex1 group received a tail vein injection of 100 μL of Complex1 liquid (the liquid obtained by dissolving Complex1 in DMSO and then resuspending it in physiological saline), resulting in a dosage of 5 mg Complex1 / kg body weight. Each mouse in the LNC@AIEgen group received a tail vein injection of 100 μL of LNC@AIEgen liquid (the liquid obtained by resuspending LNC@AIEgen in physiological saline), resulting in a dosage of 5 mg LNC@AIEgen / kg body weight. The mice were then placed in an IVIS imaging system, and fluorescence images at default times (0, 6, and 12 h) were observed (Ex: 480 nm, Em: 632 nm).

[0157] Twelve hours later, mice were anesthetized, and tumors and major organs (liver, heart, lung, spleen, and kidney) were collected for fluorescence analysis. Fluorescence intensities of the heart, liver, spleen, lung, kidney, and tumor were collected at 12 hours. Simultaneously, 15 μL of blood was collected from the tail vein at different time points.

[0158] The fluorescence intensity of Complex1 was measured to evaluate the blood circulation and metabolism of the nanoparticles.

[0159] The results of targeting ability detection using the IVIS spectroscopy system are shown in the figure. Both the Complex1 group and the LNC@AIEgen group showed high fluorescence intensity within the tumor, and this intensity increased over time. Figure 22 (a), (c)). Moreover, the fluorescence intensity of mice injected with LNC@AIEgen was higher than that of mice injected with Complex1 alone, indicating that inactivated cancer cells can enhance the targeting of Complex1.

[0160] Twelve hours after injection of nanoparticles, mice were anesthetized and euthanized, and in vitro fluorescence imaging was performed to examine the tumor targeting and biodistribution of the nanoparticles. Results are as follows: Figure 22 As shown in b) and d), the fluorescence intensity in tumor tissues of mice injected with LNC@AIEgen was higher than that in other organs of the animals and in tumor tissues of mice injected with Complex1. This indicates that LNC cells can specifically and effectively target tumor tissues.

[0161] The biometabolism of nanoparticles was evaluated by collecting 15 μL of whole blood at different time points after tail vein injection. The circulation patterns of Complex1 and LNC@AIEgen were assessed by detecting fluorescence intensity. Results are as follows: Figure 22 As shown in Figure e), the circulating half-life and elimination half-life of Complex1 and LNC@AIEgen were 0.72 and 12.58 h, and 1.72 and 21.19 h, respectively. These results indicate that the inactivated cancer cell delivery platform LNC@AIEgen significantly reduced the clearance of antigens in vivo.

[0162] 3. In vivo antitumor effects study

[0163] The in vivo therapeutic efficacy of LNC@AIEgen, a radiotherapy platform based on Complex1, was measured.

[0164] A B16F10 tumor-bearing model was established in C57BL / 6J mice by subcutaneous injection of cancer cells into the dorsal side of the right hind limb. When the tumor volume reached 170 mm³, the B16F10 tumor-bearing C57BL / 6J mice were randomly divided into 8 groups (n=6 per group):

[0165] G1: Control group (injected with 100 μL PBS); G2: LNC (injected with 100 μL: 1 × 10⁴ cells); G3: Complex1 (5 mg / kg Complex1);

[0166] G4: LNC@AIEgen (Injection volume: 100μL, please specify the content of the ingredient).

[0167] G5: Control group (injected with 100 μL PBS) + local X-ray irradiation (4 Gy) 12 h after injection. G6: LNC (injection volume: 100 μL) + local X-ray irradiation (4 Gy) 12 h after injection.

[0168] G7: Complex1 (injection volume: 100 μL) + local X-ray irradiation (4 Gy) 12 h after injection; G8: LNC@AIEgen (injection volume: 100 μL) + local X-ray irradiation (4 Gy) 12 h after injection.

[0169] Irradiation was performed using X-RAD 160, Percision X-ray, USA, X-ray, Metal Ceramic, Fixed Anode, and Watercooled.

[0170] On day 0, each mouse was intravenously injected with 100 μL of PBS or prepared NPs (1 × 10⁴ cells or 5 mg / kg Complex 1). Mouse body weight and tumor size were measured every 2 days. Tumor volume was calculated as length × width 2 × 1 / 2. HE staining was used for analysis.

[0171] The expression of Caspase-3, Bcl2 and Ki-67 in tumor tissues was detected by TUNEL staining and immunohistochemistry, and the apoptosis index was analyzed.

[0172] Tumor tissue sections were treated with an in situ end-labeling assay kit (Beyotime Biotechnology, catalog number C1089). In this experiment, apoptotic cells were stained with red and analyzed under a fluorescence microscope.

[0173] The effectiveness of treatment was assessed based on factors such as tumor size, body weight, and survival time, which were recorded every two days for 14 days. Tumor weight was measured after 14 days of the experiment. The results are as follows: Compared with the control group G1 (1008.94 mm3), groups G2, G3, and G4 showed no significant tumor suppression. Figure 23 (a)). However, groups G5 to G8, which received X-ray irradiation, showed significantly stronger inhibitory effects than group G1. The inhibitory effect of Complex1 on tumors was significantly enhanced, with the highest level observed in group G8. This is because targeted delivery increased the number of Complex1 cells in the tumor tissue, thus demonstrating the best therapeutic outcome. Figure 23 (a)). During the treatment period (day 0 to day 14), there were no significant differences in body weight between the groups. Figure 23 (b)). Natural deaths were observed only in groups G1 and G2. Figure 23(c) This indicates that even though Complex1 is toxic to cancer cells, the injection dose and the autologous...

[0174] I also ensured that the nanoparticles would not cause significant toxicity in vivo. All mice were anesthetized and euthanized on day 14, and tumor tissue was isolated and weighed. Figure 23 (d) and (e)). Tumor images ( Figure 23 (e) and weight analysis ( Figure 23 As shown in Figure d), RT significantly inhibited tumor growth, and the prepared Complex1, being a good radiosensitizer, further suppressed tumor growth in vivo. These findings are consistent with tumor volume measurements. Therefore, Complex1 and LNC@AIEgen exhibit good anti-tumor radiochemotherapy effects in vivo.

[0175] ROS levels and apoptosis indices in collected tumor sections were analyzed to further examine the efficacy of Complex1-enhanced radiotherapy. Figure 24 As shown in (a), significant ROS production was observed in the RT groups (G5-G8), but the highest ROS production levels were observed in G7 and G8. Similarly, TUNEL staining showed that the apoptosis rate in the G5-G8 groups was higher than that in the G1-G4 groups. The fluorescence signal enhancement was most pronounced in the G7 and G8 groups. Figure 24 (b)). Other indicators of antitumor efficacy include H&E staining ( Figure 24 IHC staining of c) and apoptosis proteins, such as Ki67 ( Figure 24 (d) ), Bcl2 ( Figure 24 (e) and Caspase-3 ( Figure 24 (f) further validated the therapeutic effect of Complex1-enhanced radiotherapy and chemotherapy.

[0176] 4. In vivo experiments to detect cytokine release

[0177] First, tumor tissues from different groups were collected from tumor-bearing mice, homogenized with RIPA lysis buffer, and centrifuged to obtain tumor tissue lysates. Various cytokines, including IFN-γ, TNF-α, IL-2, and IL-12, were detected according to the instructions of the cytokine ELISA kit (company and catalog number).

[0178] 5. Immune cell analysis

[0179] A series of immune cells were analyzed by fluorescent staining with classic labeled antibodies.

[0180] The specific steps are as follows: On day 14, mice in 5 groups (n=5 per group, initial weight 19-20g) were anesthetized and sacrificed. Tumor tissue and spleen were fixed with 4% paraformaldehyde at room temperature for 30 min. Then, the samples were infiltrated with 0.1% Triton X-100 PBS for 10 min and incubated in blocking buffer containing 5% BSA at room temperature for 2 h. After that, the specimens were incubated overnight with specific primary antibodies: DCs: MHC II+ / CD86+; M1 macrophages: Tregs: CD4+ / Foxp3+; T cells: CD3+ / CD4+ / CD8+; cytotoxic T lymphocytes: CD3+ / CD8+; tumor-infiltrating NK cells: CD49b+ / CD45+; M1 macrophages: F4 / 80+ / CD80+; M2 macrophages: F4 / 80+ / CD206+.

[0181] To verify whether the prepared Complex1-based nanoparticles effectively induced ICD in cancer cells, the following tests were conducted.

[0182] Whether Complex 1 and RT treatment induce an immune response. After treatment, tumor tissue homogenates were collected and sectioned. The levels of cytokines interferon-γ, tumor necrosis factor-α, interleukin-2, and interleukin-12 were detected by enzyme-linked immunosorbent assay (ELISA). At the same time, specific markers of immune cells were detected in tumor sections.

[0183] The results are as follows Figure 23 As shown in f)-i), enzyme-linked immunosorbent assay (ELISA) results indicate that in vivo RT treatment significantly upregulated the aforementioned cytokines. Combined chemotherapy and radiotherapy also caused an increase in these cytokines, but the levels were lower than with radiotherapy alone. The increase in cytokines helps remodel the tumor environment and activates the immune system through the cGAS-STING signaling pathway. Therefore, ICDs and cytokines can act as activators of the immune response, thereby transforming the tumor from a "cold" to a "hot" state, ultimately leading to tumor elimination.

[0184] RT or Complex1 / LNC@AIEgen treatment increased the intratumoral abundance of dendritic cells (DCs, MHC II+ / CD86+) and M1 macrophages (F4 / 80+ / CD80+), while decreasing the abundance of M2 macrophages (CD11b+ / F4 / 80+ / CD86+). The G8 group showed an increase in M1 macrophages and a decrease in M2 macrophages, suggesting that LNC@AIEgen+RT promotes antitumor immune activation by inducing macrophage polarization from the M2 phenotype to the M1 phenotype, thereby enhancing antigen cross-presentation. Furthermore, the abundance of immunosuppressive immune cells, namely Tregs (CD4+ / Foxp3+), was decreased in the Complex1 / LNC@AIEgen+RT group. Furthermore, the G5-G8 groups had a higher abundance of effector CD4+ / CD8+ T cells, cytotoxic T cells (CTLs, CD3+ / CD8+), and tumor-infiltrating NK cells (CD49b+ / CD45+) than the groups that did not receive RT, but the increases were lowest in the G3 and G4 groups. In addition, LNC@AIEgen+RT was more effective than other treatment combinations. Figure 23 (j).

[0185] In addition, considering that the spleen is a major source of immune cells in the body, the increase in CD4+ and CD8+ T cells in the spleen at the end of treatment was also measured. The results are as follows: Figure 25 The fluorescence intensity of G5 through G8 was greater than that of G1 through G4. However, due to chemotherapy-induced cancer cell death, there was a slight increase in CD4 and CD8 positive T cells in the G3 and G4 groups. LNC cells alone only induced a mild immune response and tumor suppressor effect, possibly because fewer LNC cells were used than in previous studies. The data confirm that the prepared LNC@AIEgen material and Complex1-based nanoparticles can produce a potent antitumor effect through radiotherapy, chemotherapy, and immunotherapy.

[0186] 7. Safety studies of tumor vaccines

[0187] The drug was administered (LNC, Complex1, or LNC@AIEgen, with a final concentration of 20 μg / mL, and divided into five groups: Control, Control+RT, LNC + RT, Complex1+RT, and LNC@AIEgen+RT, as described in step 3 of Example 3).

[0188] Mice were sacrificed 14 days later, and major tissues (heart, liver, spleen, lung, kidney, and tumor) were extracted for histological sectioning and image analysis. Complete blood count (CBC) and biochemical analysis were also performed. CBC measurements included white blood cell count (WBC), red blood cell count (RBC), hemoglobin (HGB), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet count (PLT), and hematocrit (HCT). Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), total protein (TP), globulin (GLb), total bilirubin (TBIL), blood urea nitrogen (BUN), creatinine (CREA), and albumin (ALB) were measured.

[0189] Biotoxicity is an important indicator for the clinical translation of nanomedicines. Figure 23 As shown in (b), the body weight of mice in all groups remained relatively stable. The systemic biosafety of the nanomedicine was investigated by routine blood tests, blood chemistry evaluation, and H&E staining of major organs (heart, liver, spleen, lungs, and kidneys).

[0190] The results are as follows Figure 26 As shown in a) and b), the blood routine and biochemical test results of the two groups are comparable. H&E staining showed ( Figure 27 No significant damage or destruction of organs was observed in any of the mouse groups. Therefore, the prepared Complex1 and LNC@AIEgen nanoparticles exhibit good biocompatibility in vivo.

[0191] 8. Study on the preventive efficacy of tumor cell vaccines based on LNT@AIEgen and RT

[0192] To effectively stimulate ICD and ensure that cancer cells lose their tumorigenicity, cell viability was first tested under different X-ray irradiation doses (specifically 0 Gy, 2 Gy, 4 Gy, 8 Gy, 10 Gy, 20 Gy, 40 Gy, 80 Gy, and 100 Gy) without Complex1.

[0193] The results are as follows Figure 28 As shown, cell viability rapidly decreased with increasing X-ray irradiation dose. After a single irradiation with 80 Gy X-rays, CCK-8 assays revealed that B16F10 cancer cells completely lost their viability.

[0194] B16F10 tumor cells were washed with PBS and irradiated with 80 Gy X-rays to completely kill them, and then cultured at 37°C and 5% CO2 for 12 hours to induce ecto-CRT in LNC@AIEgen.

[0195] The specific steps for studying the preventive efficacy of tumor cell vaccines based on LNT@AIEgen and RT are as follows ( Figure 29(a)): 1) Five- to six-week-old C57BL / 6J mice (average weight 18-20g) were randomly divided into the following five groups, with five mice in each group: G1: Control group; injected with an equal volume of PBS

[0196] G2: Control + RT group: Injected with an equal volume of PBS and received local X-ray irradiation (4 Gy) G3: LNC + RT group: Injected with 1 × 10⁴ LNC cells and received local X-ray irradiation (4 Gy) G4: Complex1 + RT group: Injected with Complex1 cells and received local X-ray irradiation (4 Gy)

[0197] G5: LNC@AIEgen+RT group: injection equivalent to 20 mg / μL Complex1, followed by local X-ray irradiation (4 Gy).

[0198] 2) B16F10 cancer cells were treated with 20 μg / mL Complex1 or LNC@AIEgen and an equal volume of LNC cells for 6 h, washed with PBS, irradiated with X-rays, and cultured for another 12 h. Mice were then immunized at a density of 2 × 10⁶ cells / mouse on -14 days and -7 days, with the injection site being the dorsal side of the right hind limb. The G1 group was injected with an equal volume of PBS, while the other groups underwent the corresponding treatments shown in 1).

[0199] 3) On day 0, untreated live B16F10 cells were injected into the dorsal side of the left hind limb of mice at a density of 1 × 10⁵ cells / mouse. Tumor formation and body weight were then monitored every 2 days for 28 days. Tumor volume was measured to monitor the tumor growth in the five groups of mice.

[0200] Tumor growth was monitored. Tumor volume (V) was calculated as V = W² × L / 2, where W and L are the width and length of the tumor, respectively. Survival was also monitored throughout the study.

[0201] The results are as follows Figure 29 As shown in (b), quantitative tumor volume analysis revealed that tumor formation and growth were significantly inhibited in the LNC@AIEgen group, with an average tumor volume of 161.13 mm³. Figure 29 The values ​​in the control groups (b), e), and f) were approximately 11.67 times smaller than those in the control group (1867.14 mm3). This value was also significantly lower in the Control+RT, LNC+RT, and Complex1+RT groups than in the control group (b), e), and f). Figure 29 (b), (e), and (f)). The weight of isolated tumors also confirms the preventive efficiency of this new strategy. Figure 29 (d)

[0202] Furthermore, 60% and 20% of mice treated with the tumor cell vaccine in the Complex1+RT group and LNC@AIEgen+RT group, respectively, showed no tumor growth. Its anti-tumor effect was verified by H&E staining. Figure 29 (g)). Due to the large tumor volume in the control group, the preventive efficacy of the tumor cell vaccine based on LNT@AIEgen and RT was not detected until 28 days after the live cancer cell challenge. During the monitoring period, the body weight of the mice remained unchanged. Figure 29 (c) This indicates that the prevention strategy of using a combination of LNT@AIE and RT is a good safety measure.

[0203] Furthermore, to elucidate the mechanism by which tumor cell vaccines induce anti-tumor immunity, immune cells are identified in tumor tissues based on specific standardized biomarkers. For example... Figure 29 As shown in Figure h), the abundance of M1 macrophages, CD4+ T cells, CD8+ T cells, and NK cells in immunized mice was significantly increased, while the abundance of M2 macrophages was significantly decreased. Therefore, Complex1 combined with RT can serve as a useful tumor cell vaccine and elicit a fairly strong anti-tumor immune response to prevent tumor development in vivo.

[0204] This invention uses liquid nitrogen-treated dead cancer cells as a target Complex1 vector and an anti-tumor vaccine for chemotherapy-radioimmunotherapy. Figure 30 The results showed that dead cancer cells (LNTs) exhibited good targeting, circulation capacity, and safety, and helped reduce the toxicity of Complex1. LNTs demonstrated high loading efficiency and caused Complex1 to accumulate extensively in the endoplasmic reticulum of cancer cells. When LNC@AIEgen was combined with RT, it induced ICD and activated immune responses by promoting ROS production and simultaneously disrupting the structure and function of ER, lysosomes, and DNA, thus demonstrating good radiotherapy efficacy. Data from this study showed that the combination of LNC@AIEgen and RT significantly inhibited tumor growth. Furthermore, dead cells were also able to independently induce immune responses. Finally, the tumor cell vaccine based on LNT@AIEgen combined with RT can inhibit tumor formation in vivo.

[0205] In summary, this invention designs a nanoplatform, LNT@AIEgen, for synergistic chemotherapy-radioimmunotherapy, using dead cancer cells as a carrier and Complex1 as a radiosensitizer. This system fully leverages the advantages of the delivery carrier, enhancing anti-tumor therapeutic effects and acting as an anti-cancer vaccine. Results show that this simple yet effective strategy enables large-scale clinical treatment of cancer and contributes to personalized medicine.

[0206] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A product for treating melanoma in mouse cutaneous tissue, characterized in that: The product contains LNC@AIEgen and isotopes that produce X-rays; The LNC@AIEgen comprises inactivated cancer cells and Complex 1 loaded onto the inactivated cancer cells. The inactivated cancer cells are inactivated cells obtained by treating cancer cells with liquid nitrogen. The cancer cells are mouse skin melanoma cells. Complex 1 is a compound with the structural formula of Formula 1.

2. Application of LNC@AIEgen in the preparation of drugs that enhance the radiotherapy effect of mouse skin melanoma; The radiotherapy mentioned is X-ray therapy; The LNC@AIEgen comprises inactivated cancer cells and Complex 1 loaded onto the inactivated cancer cells. The inactivated cancer cells are inactivated cells obtained by treating cancer cells with liquid nitrogen. The cancer cells are mouse skin melanoma cells. Complex 1 is a compound with the structural formula of Formula 1.

3. Application of Complex 1 in the preparation of drugs that enhance the radiotherapy effect of mouse skin melanoma; The radiotherapy mentioned is X-ray therapy; Complex 1 is a compound with the structural formula of Formula 1.

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