A gallium nanoparticle formulation, its preparation method and application

Abstract

This invention belongs to the field of biomedical technology and relates to a gallium nanoparticle formulation, its preparation method, and its application. The gallium nanoparticle formulation, by weight, comprises: (a) albumin: 80-100 parts; (b) manganese dioxide: 2-8 parts; (c) gallium ions: 0.5-3 parts; wherein the manganese dioxide is in situ mineralized on albumin to form composite particles, and the gallium ions are loaded on the surface or interior of the composite particles, or the gallium ions are loaded on both the surface and interior of the composite particles. The gallium nanoparticle formulation of this invention, while inducing ferroptosis, simultaneously downregulates PD-L1 and CD47 endogenously through the mitochondrial / AMPK / c-MYC axis, achieving a dual functional integration of cell death and immune desuppression, thereby solving the problem of immune tolerance in tumor ferroptosis therapy.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to a gallium nanoparticle formulation, its preparation method, and its application. Background Technology

[0002] Ferroptosis is a type of iron-dependent regulated cell death, mainly manifested as lipid peroxidation accumulation, glutathione system imbalance, and mitochondrial ultrastructural alterations. In recent years, ferroptosis has become a hot topic in cancer treatment due to its potential to enhance tumor therapy sensitivity, release damage-related molecules, and mobilize the immune microenvironment.

[0003] However, preclinical studies and clinical practice have gradually revealed a key obstacle: traditional ferroptosis induction therapy, while killing tumor cells, also induces adaptive immune tolerance in these cells. Specifically, this manifests as follows: Compensatory upregulation of PD-L1: Cellular stress, interferon signaling, or activation of certain transcription factors accompanying ferroptosis can lead to a significant increase in the expression of programmed death-ligand 1 (PD-L1) on the surface of tumor cells. After PD-L1 binds to PD-1 on T cells, it inhibits the activation, proliferation, and killing function of T cells, thereby weakening the ferroptosis-induced anti-tumor immune response and even leading to immune escape.

[0004] Compensatory upregulation of CD47: Simultaneously, conventional ferroptosis induction therapy also increases the expression of CD47 on tumor cell membranes. CD47 transmits inhibitory signals by binding to SIRPα on the surface of macrophages, preventing macrophages from phagocytizing and clearing tumor cells. This prevents the effective removal of dead cell debris and surviving cells after ferroptosis, further contributing to an immunosuppressive tumor microenvironment.

[0005] Therefore, simply inducing ferroptosis often leads to limited therapeutic effects, easy relapse, and even promotes tumor immune escape and progression.

[0006] To address these issues, researchers have attempted to develop various nanoplatforms. For example, Chinese patent application CN111870705A discloses a microenvironment-responsive nanomaterial for in situ gliomas, using albumin as a carrier to load manganese dioxide (MnO2) nanocrystals and protoporphyrin (PpIX) via biomineralization, for use in tumor microenvironment-responsive magnetic resonance imaging and sonodynamic therapy. This material utilizes the interaction between MnO2 and H+ in the tumor microenvironment. + It reacts with H2O2 to produce oxygen, improving the hypoxic microenvironment of tumors, and generates reactive oxygen species to kill tumor cells under ultrasound stimulation. However, the MnO2 in this patent application is mainly used as an MRI contrast agent and oxygen generator, and its therapeutic function depends on external ultrasound stimulation, and it cannot solve the problem of immune tolerance associated with ferroptosis therapy.

[0007] Gallium ions share certain biochemical similarities with iron ions and can act as non-redox iron mimics to interfere with iron metabolism, ribonucleotide reductase activity, and mitochondrial function. Public studies have shown that some gallium compounds possess antitumor or ferroptosis-inducing potential; however, existing gallium formulations (such as gallium nitrate) suffer from drawbacks such as poor targeting, toxicity to normal tissues, and inability to synergistically regulate immune checkpoints.

[0008] For example, Chinese patent application CN120647616A discloses gallium ion-quercetin coordination nanoparticles (GQNPs) for treating Parkinson's disease through multidimensional anti-ferroptosis. These nanoparticles utilize gallium ions (Ga... 3+ This patent application uses gallium ions to competitively bind to transferrin, reducing intracellular iron levels, and simultaneously leverages the antioxidant capacity of quercetin to achieve multidimensional inhibition of ferroptosis. However, the technical approach of this patent application is to inhibit ferroptosis through gallium ions, rather than inducing it; and it also fails to address the issue of immune tolerance in tumor ferroptosis therapy. Summary of the Invention

[0009] The purpose of this invention is to provide a gallium nanoparticle formulation capable of reversing immune tolerance, its preparation method, and its application.

[0010] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A gallium nanoparticle formulation, by weight, comprises: (a) Albumin: 80-100 parts; (b) Manganese dioxide: 2-8 parts; (c) Gallium ions: 0.5-3 parts; The manganese dioxide is mineralized in situ on albumin to form composite particles, and the gallium ions are loaded on the surface or inside the composite particles, or the gallium ions are loaded on both the surface and inside the composite particles.

[0011] This invention demonstrates through numerous experiments that the gallium nanoparticle formulation of this invention can effectively induce tumor ferroptosis and avoid or reverse ferroptosis-related PD-L1 / CD47 dual upregulation immune tolerance; it possesses tumor microenvironment response release capability and can form a stable synergy with radiotherapy; it can enhance local tumor suppression and improve distant and anti-metastatic effects.

[0012] According to embodiments of the present invention, the present invention can be further optimized, and the optimized technical solution is as follows: In one preferred embodiment, the manganese dioxide is 2-5 parts by weight; preferably 3-5 parts.

[0013] In one preferred embodiment, the gallium ions are 0.5-2 parts by weight; preferably 1-2 parts.

[0014] In one preferred embodiment, the albumin is bovine serum albumin, human serum albumin, or recombinant albumin, wherein the recombinant albumin is expressed by gene recombination technology and has the same or at least 95% homology with natural human serum albumin or bovine serum albumin.

[0015] Typical recombinant albumins include recombinant human serum albumin (rHSA), which can be obtained through Pichia pastoris or Escherichia coli expression systems.

[0016] In one preferred embodiment, the average hydrated particle size of the gallium nanoparticles is 50-200 nm, preferably 80-150 nm.

[0017] In one preferred embodiment, the gallium nanoformulation has a zeta potential of approximately -18 mV to -5 mV.

[0018] Based on the same inventive concept, this invention also claims protection for a method for preparing the gallium nanoparticle formulation, comprising the following steps: S1. Add a manganese source to the albumin solution to obtain an intermediate solution; S2. Add a gallium source to the intermediate solution to load gallium ions onto the intermediate; S3. Alternately add manganese and gallium sources, and perform multiple cycles; S4. Add manganese source dropwise for final mineralization and purification to obtain the gallium nanoparticle formulation; The gallium source contains gallium ions; the manganese source is a manganese-containing compound that can generate manganese dioxide in situ under an albumin template.

[0019] More preferably, the gallium source is an inorganic gallium salt; and the manganese source is a permanganate.

[0020] This invention employs a unique stepwise alternating mineralization-loading process, introducing gallium ions in stages during the intermittent formation of MnO2 from the manganese source mineralization. Unlike preparation methods that mix all raw materials at once, the alternating operation of this invention achieves: Suppressing explosive nucleation: The addition of manganese source in stages avoids the explosive generation of MnO2, so that the mineralization process is always under the control of albumin template, thereby obtaining nano-formulations with narrower particle size distribution (PDI<0.2) and higher colloidal stability.

[0021] Achieving high co-localization among components: Through multiple cycles, gallium ions are continuously embedded in the newly formed MnO2 layer, rather than simply adsorbed on the particle surface. This results in a uniform distribution of Ga and Mn elements throughout the nanoparticles, which is the structural basis for achieving synergistic functions (simultaneously inducing ferroptosis and downregulating immune checkpoints).

[0022] In one preferred embodiment, the permanganate includes one or both of potassium permanganate and sodium permanganate; potassium permanganate is preferred.

[0023] In one preferred embodiment, the inorganic gallium salt includes one or more of gallium nitrate, gallium chloride, and gallium sulfate.

[0024] In one preferred embodiment, the concentration of the albumin solution is 1-20 mg / mL, preferably 5-10 mg / mL; more preferably 5-8 mg / mL.

[0025] In one preferred embodiment, the concentration of the manganese source is 1-10 mg / mL, preferably 5-10 mg / mL.

[0026] In one preferred embodiment, the concentration of the gallium source is 1-10 mg / mL, preferably 4-8 mg / mL, and more preferably 4 mg / mL.

[0027] In one preferred embodiment, in step S1, the volume ratio of the manganese source to the albumin solution is 0.2-2.0:50-200, preferably 0.5-1.0:50-200.

[0028] In one preferred embodiment, in step S2, the volume ratio of the gallium source to the albumin solution is 0.2-2.0:50-200, preferably 0.5-1.0:50-200.

[0029] In one preferred embodiment, in step S4, the volume ratio of the manganese source to the albumin solution is 0.5-5.0:50-200, preferably 1.0-3.0:50-200.

[0030] In one preferred embodiment, in step S3, the alternation cycle is repeated 3-10 times, preferably 3-5 times.

[0031] In one preferred embodiment, in step S4, the purification is performed using ultrafiltration or dialysis, preferably using an ultrafiltration device equipped with a 30-50 kDa ultrafiltration membrane.

[0032] Based on the same inventive concept, this invention also claims protection for the use of the above-described gallium nanoparticles or gallium nanoparticles prepared by the above-described preparation method in the preparation of drugs that reverse tumor immune tolerance.

[0033] In one preferred embodiment, the drug reverses tumor immune tolerance by activating AMPK and reducing c-MYC.

[0034] In one preferred embodiment, the drug is able to endogenously downregulate PD-L1 expression and CD47 expression in tumor cells.

[0035] In one preferred embodiment, the tumor is a solid tumor, preferably bladder cancer, breast cancer, lung cancer, liver cancer, colorectal cancer, pancreatic cancer, glioma, or melanoma.

[0036] In one preferred embodiment, the drug further includes pharmaceutically acceptable excipients.

[0037] In one preferred embodiment, the drug is an injection, a lyophilized powder for injection, or an intravenous infusion preparation.

[0038] Based on the same inventive concept, this invention also claims protection for the use of the above-described gallium nanoparticles or gallium nanoparticles prepared by the above-described preparation method in the preparation of drugs for reversing tumor immune tolerance in combination with radiotherapy.

[0039] Compared with the prior art, the beneficial effects of the present invention are: The gallium nanoparticle formulation of this invention releases gallium ions in response to the tumor microenvironment. While inducing ferroptosis in tumor cells, it simultaneously downregulates PD-L1 and CD47 endogenously, achieving integrated synergistic effects of cell death and immune desuppression. This fundamentally overcomes the immune tolerance defect of traditional ferroptosis therapy, thus breaking the dilemma of traditional ferroptosis therapy killing tumors but inducing tolerance.

[0040] This invention achieves endogenous dual checkpoint downregulation through gallium ion release, without relying on exogenous antibodies, and completes multiple regulation with a single nanomedicine, significantly reducing the complexity and risk of toxic side effects of combination therapy.

[0041] This invention utilizes the tumor microenvironment response characteristics of MnO2 (acid / pH response release of Mn) 2+ (Relieves tumor hypoxia) and has a natural synergistic effect with radiotherapy: Mn 2+ It can catalyze the production of oxygen from H2O2 and enhance radiosensitization; radiotherapy-induced immunogenic cell death can further activate anti-tumor immunity, forming a positive feedback with the immune tolerance reversal effect of the present invention, and enhancing the systemic anti-tumor immune response.

[0042] The gallium nanoparticle formulation of the present invention has good biocompatibility and low immunogenicity; it has strong tumor enrichment ability; the preparation process is mild and controllable, which is conducive to scale-up production and freeze-drying preservation.

[0043] This invention employs albumin-mediated biomineralization and positive and negative charge adsorption methods, which can be completed under mild conditions. By alternately adding manganese and gallium sources, the degree of MnO2 mineralization and gallium ion loading can be precisely controlled, resulting in products with uniform particle size and minimal batch-to-batch variation.

[0044] The gallium nanoparticle formulation provided by this invention can simultaneously achieve: (a) inducing tumor ferroptosis; (b) endogenously downregulating PD-L1 and activating T cells; (c) endogenously downregulating CD47 and enhancing macrophage phagocytic capacity; and (d) relieving hypoxia and sensitizing radiotherapy. This multi-effect significantly simplifies clinical medication regimens and improves patient compliance.

[0045] This invention, through the core mechanism of reversing immune tolerance, has broad-spectrum anti-tumor potential and unique advantages against cold tumors resistant to traditional immunotherapy. Attached Figure Description

[0046] Figure 1 This is a schematic diagram of the synthesis route of Ga@MnO2@Alb nanoparticles in the embodiment.

[0047] Figure 2 This is a dynamic light scattering (DLS) detection image of Ga@MnO2@Alb nanoparticles.

[0048] Figure 3 These are the results of high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) analysis of Ga@MnO2@Alb nanoparticles; among which... Figure 3 Image 'a' in the image is a high-angle annular dark-field scanning transmission electron microscope image. Figure 3 In this diagram, 'b' represents the gallium (Ga) elemental mapping. Figure 3 In the diagram, 'c' represents the oxygen (O) elemental mapping. Figure 3 In the diagram, d represents the elemental mapping of manganese (Mn).

[0049] Figure 4 This is a graph showing the change in hydration particle size of Ga@MnO2@Alb nanoparticles after incubation at different temperatures (25℃, 37℃) and in different media (PBS, serum) for 48 hours.

[0050] Figure 5 This is a bar graph showing the change in hydrated particle size of Ga@MnO2@Alb nanoparticles after 6 months of storage at 4℃.

[0051] Figure 6 This is an in vitro release curve of Ga ions from Ga@MnO2@Alb nanoparticles under different pH conditions (pH 5.5, pH 7.4) and different H2O2 concentrations.

[0052] Figure 7This is a graph showing the particle size distribution of DLS after Ga@MnO2@Alb nanoparticles were incubated with different concentrations of H2O2.

[0053] Figure 8 This is a diagram showing the results of an in vitro hemolysis experiment using Ga@MnO2@Alb nanoparticles. The top group is the group without red blood cells, and the bottom group contains red blood cells.

[0054] Figure 9 This is a Western blot result of the expression levels of CD47, PD-L1, c-MYC, and GPX4 proteins in MB49 cells after treatment with different concentrations of the ferroptosis inducer FIN56 in Example 2.

[0055] Figure 10 This is a Western blot result of the expression levels of CD47 and PD-L1 proteins in T24 cells after treatment with different concentrations of ferric ammonium citrate (FAC) in Example 2.

[0056] Figure 11 This is a Western blot result of the expression levels of CD47, PD-L1, and c-MYC proteins in MB49 cells after treatment with different concentrations of cisplatin in Example 2.

[0057] Figure 12 This is a Western blot result of the expression levels of CD47, PD-L1, and c-MYC proteins after MB49 cells were treated with different doses of radiotherapy (RT) in Example 2.

[0058] Figure 13 This is a Western blot result of the expression levels of CD47, PD-L1, and GPX4 proteins after MB49 bladder cancer cells were treated with different types of inorganic gallium salts (gallium nitrate, gallium sulfate, gallium chloride, and trigallium salt) for 24 hours in Example 3.

[0059] Figure 14 The figure shows the Western blot results of the expression levels of c-MYC, CD47, and PD-L1 proteins in MB49 cells of different treatment groups in Example 4. In the figure, 1 is the control group, 2 is the MnO2@Alb group, 3 is the gallium nitrate group, and 4 is the Ga@MnO2@Alb group.

[0060] Figure 15 This is an immunofluorescence staining image of PD-L1 (green) and CD47 (red) expression in MB49 cells of different treatment groups in Example 4; in the image, 1 is the control group, 2 is the MnO2@Alb group, 3 is the gallium nitrate group, and 4 is the Ga@MnO2@Alb group.

[0061] Figure 16Example 4 shows the detection effect of CD8 in the co-culture system. + The figure shows the killing effect of T cells on MB49 cells in different treatment groups; in the figure, 1 is the control group, 2 is the MnO2@Alb group, 3 is the gallium nitrate group, and 4 is the Ga@MnO2@Alb group.

[0062] Figure 17 This is the flow cytometry result of the phagocytic activity of macrophages on MB49 cells in different treatment groups in Example 4; whereby... Figure 17 In the image, 'a' represents the result of flow cytometry analysis. Figure 17 In the figure, b represents the statistical analysis graph; in the graph, 1 is the control group, 2 is the MnO2@Alb group, 3 is the gallium nitrate group, and 4 is the Ga@MnO2@Alb group.

[0063] Figure 18 This is a confocal laser scanning microscope image of the phagocytic activity of RAW264.7 macrophages on MB49 cells in Example 4.

[0064] Figure 19 The intracellular ferrous ions (Fe) in MB49 cells from different treatment groups in Example 5 2+ The concentration detection results are shown in a bar chart. In the chart, 1 is the control group, 2 is the MnO2@Alb group, 3 is the gallium nitrate group, and 4 is the Ga@MnO2@Alb group.

[0065] Figure 20 This is a bar chart showing the relative content of malondialdehyde (MDA) in MB49 cells from different treatment groups in Example 5. In the chart, 1 is the control group, 2 is the MnO2@Alb group, 3 is the gallium nitrate group, and 4 is the Ga@MnO2@Alb group.

[0066] Figure 21 This is a bar chart showing the relative content of oxidized glutathione (GSSG) in MB49 cells of different treatment groups in Example 5. In the figure, 1 is the control group, 2 is the MnO2@Alb group, 3 is the gallium nitrate group, and 4 is the Ga@MnO2@Alb group.

[0067] Figure 22 These are transmission electron micrographs of the mitochondrial ultrastructure of MB49 cells from different treatment groups in Example 5. The white arrows indicate shrunken mitochondria. In the figures: Figure 22 In the image, 'a' is a transmission electron microscope image of the mitochondrial ultrastructure of the control group cells (scale bar 1 μm). Figure 22 b in the image is a transmission electron microscope image of the mitochondrial ultrastructure of Ga@MnO2@Alb group cells (scale bar 1 μm). Figure 22 In the image, 'c' is a transmission electron microscope image of the ultrastructure of mitochondria in the control group cells (scale bar 500 nm). Figure 22In the image, d represents the ultrastructure of mitochondria in Ga@MnO2@Alb group cells as a transmission electron microscope image (scale bar 500 nm).

[0068] Figure 23 These are morphological observation images of MB49 cells from different treatment groups in Example 5; in the figures: Figure 23 In the figure, 'a' represents the morphological observation of cells in the 0µMGa@MnO2@Alb group (scale bar 100μm). Figure 23 b in the figure represents the morphological observation of cells in the 0µM Ga@MnO2@Alb group (scale bar 50μm). Figure 23 c in the figure represents the morphological observation of cells in the 5µM Ga@MnO2@Alb group (scale bar 100μm). Figure 23 In the figure, d represents the morphological observation of cells in the 5µM Ga@MnO2@Alb group (scale bar 50μm). Figure 23 In the figure, 'e' represents the morphological observation of cells in the 10µMGa@MnO2@Alb group (scale bar 100μm). Figure 23 f in the figure represents the morphological observation of cells in the 10µM Ga@MnO2@Alb group (scale bar 50μm). Figure 23 In the figure, g represents the morphological observation of cells in the 10µM Ga@MnO2@Alb+Fer-1 group (scale bar 100μm). Figure 23 h in the figure represents the morphological observation of cells in the 10µM Ga@MnO2@Alb+Fer-1 group (scale bar 50μm).

[0069] Figure 24 This shows the tumor growth in mice from different treatment groups in Example 6; in the figure: Figure 24 In the figure, 'a' represents the tumor growth curves of mice in different treatment groups. Figure 24 In the figure, b represents the actual tumor images of mice in different treatment groups; among them, 1 is the control group, 2 is the MnO2@Alb group, 3 is the gallium nitrate group, and 4 is the Ga@MnO2@Alb group.

[0070] Figure 25 This is a bar chart showing the final tumor weight statistics of mice in different treatment groups in Example 6. Among them, 1 is the control group, 2 is the MnO2@Alb group, 3 is the gallium nitrate group, and 4 is the Ga@MnO2@Alb group.

[0071] Figure 26 This is a bar chart showing the serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), creatine kinase (CK), and creatinine (CREA) levels in mice from different treatment groups in Example 6; The chart shows: Figure 26 In Example 6, 'a' represents a bar chart showing the serum alanine aminotransferase (ALT) levels in mice from different treatment groups. Figure 26In Example 6, b is a bar chart showing the serum aspartate aminotransferase (AST) levels in mice from different treatment groups. Figure 26 A bar chart showing the serum urea nitrogen levels in mice from different treatment groups in Example 6 (c). Figure 26 A bar graph showing the serum creatine kinase levels in mice from different treatment groups in Example 6; Figure 26 The bar graph shows the serum creatinine levels of mice in different treatment groups in Example 6.

[0072] Figure 27 These are H&E staining images of the major organs (heart, liver, spleen, lung, and kidney) of mice in different treatment groups in Example 6.

[0073] Figure 28 This is a graph showing the quantitative analysis results of PD-L1 and CD47 protein expression levels in tumor tissues of mice in different treatment groups in Example 6. In the graph, 1 is the control group, 2 is the MnO2@Alb group, 3 is the gallium nitrate group, and 4 is the Ga@MnO2@Alb group.

[0074] Figure 29 This is an immunohistochemical staining image of PD-L1 and CD47 protein expression in tumor tissues of mice in different treatment groups in Example 6.

[0075] Figure 30 CD3 infiltrating in tumor tissues of mice in different treatment groups in Example 6 + CD4 + T cells and CD3 + CD8 + The result chart shows the proportion of T cells.

[0076] Figure 31 This shows the tumor growth in mice from different treatment groups in Example 7; in the figure: Figure 31 In the figure, 'a' represents the tumor growth curves of mice in different treatment groups. Figure 31 In the figure, b represents the actual tumor images of mice in different treatment groups; in the figure: 1 is the control group, 2 is the Anti-PD-L1+Anti-CD47 dual immune checkpoint blockade group, 3 is the Ga@MnO2@Alb monotherapy group, 4 is the RT radiotherapy group, 5 is the Anti-PD-L1+Anti-CD47+RT immunotherapy combined with radiotherapy group, and 6 is the Ga@MnO2@Alb+RT nano-formulation combined with radiotherapy group.

[0077] Figure 32This is a bar chart showing the final tumor weight statistics of mice in different treatment groups in Example 7; in the figure: 1 is the control group, 2 is the Anti-PD-L1+Anti-CD47 dual immune checkpoint blockade group, 3 is the Ga@MnO2@Alb monotherapy group, 4 is the RT radiotherapy group, 5 is the Anti-PD-L1+Anti-CD47+RT immunotherapy combined with radiotherapy group, and 6 is the Ga@MnO2@Alb+RT nano-formulation combined with radiotherapy group. Detailed Implementation

[0078] This invention is not limited to the specific embodiments listed below. Those skilled in the art can implement this invention using various other specific embodiments based on the content disclosed herein. Any modifications or alterations made to the design structure and concept of this invention fall within the protection scope of this invention. It should be noted that, unless otherwise specified, the embodiments and features described in this invention can be combined with each other.

[0079] Terminology Explanation: In this invention, the manganese source refers to a manganese-containing compound that can generate manganese dioxide (MnO2) in situ through a redox reaction in the presence of albumin and under mild reaction conditions (e.g., room temperature). In the product thus formed, manganese element exists in the form of MnO2.

[0080] Gallium sources refer to sources that can dissociate into free gallium ions (Ga) in water. 3+ Gallium-containing compounds. The gallium source includes inorganic gallium salts, such as gallium nitrate (Ga(NO3)3), gallium chloride (GaCl3), and gallium sulfate (Ga2(SO4)3). Gallium nitrate or gallium chloride is preferred because of its good water solubility, complete dissociation, and lack of introduction of harmful impurities. It should be noted that water-insoluble gallium compounds (such as gallium oxide Ga2O3) are not suitable for this invention because they cannot release a sufficient concentration of Ga under mild conditions. 3+ .

[0081] Example 1

[0082] according to Figure 1 The schematic diagram of the synthesis route shows that albumin solution was used as the template system, and a manganese source and oxidation / mineralization conditions were introduced to form composite particles of manganese dioxide on the albumin template, yielding the MnO2@Alb precursor. Subsequently, an inorganic gallium salt solution was added to the MnO2@Alb precursor, allowing the gallium component to be loaded onto the particle surface or interior through electrostatic adsorption, surface complexation, or ionic interaction. If necessary, free ions were removed by ultrafiltration, centrifugation, dialysis, etc., to obtain Ga@MnO2@Alb nanoparticles. The specific process is as follows: First, under stirring conditions at room temperature (25℃), 600 μL of potassium permanganate (KMnO4, 5 mg / mL) solution was slowly added dropwise to 50 mL of 6 mg / mL human serum albumin solution. After standing for 5 minutes to complete the initial reaction stage, 600 μL of gallium nitrate (Ga(NO3)3) solution with a concentration of 4 mg / mL was slowly added dropwise to the resulting MnO2@Alb solution under continuous stirring. Subsequently, KMnO4 and Ga(NO3)3 solutions were added alternately every 5 minutes for a total of 5 cycles. Finally, 1 mL of KMnO4 solution was added dropwise to the Ga@MnO2@Alb intermediate under stirring, followed by the removal of free gallium ions using an ultrafiltration device equipped with a 30 kDa ultrafiltration membrane, ultimately forming Ga@MnO2@Alb nanoparticles.

[0083] The resulting nanoparticle solution was a clear, brownish-red solution with good aqueous dispersibility. Dynamic light scattering (DLS) analysis showed the following results: Figure 2 As shown in the inset, the morphology is obtained from scanning electron microscopy (SEM). The results show an average hydrated particle size of 126.4 ± 1.4 nm. The zeta potential is approximately -18 mV. High-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) analysis yielded the following results: Figure 3 As shown, where Figure 3 Image 'a' in the image is a high-angle annular dark-field scanning transmission electron microscope image. Figure 3 In this diagram, 'b' represents the gallium (Ga) elemental mapping. Figure 3 In the diagram, 'c' represents the oxygen (O) elemental mapping. Figure 3 In the figure, d represents the manganese (Mn) elemental mapping. The results show that the Ga@MnO2@Alb nanoparticles are spherical or near-spherical in shape, with a relatively uniform particle distribution and no obvious agglomeration, exhibiting good dispersibility and uniform morphology. Gallium ions were successfully loaded onto the nanoparticles and uniformly distributed, without obvious surface enrichment or core-shell separation. Gallium (Ga), manganese (Mn), and oxygen (O) elements are highly co-localized in individual nanoparticles, and the signal distribution profile perfectly matches the particle morphology. The co-localization of Mn and O signals is consistent with the expected elemental composition of MnO2. Combined with the reaction pathway of KMnO4 reduction by albumin during the preparation process, this indicates that manganese exists in the form of MnO2. The high overlap between Ga and Mn signals indicates that gallium ions have been successfully loaded onto the MnO2@Alb composite particles and are uniformly distributed, without obvious phase separation or surface enrichment. These results demonstrate that this invention successfully constructed Ga@MnO2@Alb nanoparticles with a uniform structure.

[0084] Ga@MnO2@Alb nanoparticles were placed in PBS and culture medium containing 10% fetal bovine serum (FBS), respectively, and incubated at 25℃ or 37℃ for 48 h. The results are as follows: Figure 4As shown in the figure. The results showed no significant change in particle size. After the formulation was freeze-dried into powder and stored at 4°C for 6 months, the results were as follows. Figure 5 As shown in the illustration, the lyophilized powder and the reconstituted solution are photographs. The results show that the lyophilized powder retains its original particle size and dispersibility when reconstituted in ultrapure water, with no aggregation.

[0085] The in vitro release behavior of Ga ions from Ga@MnO2@Alb nanoparticles was detected by dialysis. Four release systems were set up: pH 7.4 PBS (simulating normal physiological environment), pH 7.4 PBS + 100 μM H2O2, pH 5.5 PBS (simulating the acidic tumor microenvironment), and pH 5.5 PBS + 100 μM H2O2 (simulating the tumor microenvironment). The results are as follows: Figure 6 As shown, the results are as follows: In a tumor microenvironment simulation system with pH 5.5 and 100 μM H2O2, Ga ions exhibited significant responsive and sustained release, with a cumulative release rate of approximately 80% over 48 hours.

[0086] In an acidic system with pH 5.5 and no H2O2, the cumulative release rate of Ga ions is only about 25% after 48 hours.

[0087] In a normal physiological environment at pH 7.4, Ga ions showed only very low levels of non-specific leakage, regardless of whether H2O2 was added, with a cumulative release rate of <10% over 48 hours.

[0088] After incubation with 100 μM H2O2 in a Ga@MnO2@Alb solution, the results are as follows: Figure 7 As shown in the inset, the solution was photographed before and after incubation. The results showed that the solution color gradually changed from brownish-yellow to light yellow, and DLS analysis revealed a significant reduction in particle size, further confirming that the nanostructure has a specific response to H2O2 in the tumor microenvironment.

[0089] Within the test concentration range of 10-200 μM, after co-incubation of Ga@MnO2@Alb nanoparticles with mouse erythrocytes for 2 hours, the results were as follows: Figure 8 As shown in the figure. In vitro hemolysis test results showed no obvious hemolysis, and the hemolysis rate was <5%, which meets the safety requirements of intravenous injection preparations.

[0090] Example 2

[0091] MB49 bladder cancer cells (purchased from Oricell) were treated with various ferroptosis-related induction conditions to verify the correlation between ferroptosis and immune checkpoint expression. The specific steps are as follows: Ferroplasmosis Inducer Treatment: Cells were treated with different concentrations (0, 1.25, 2.5, 5, 10, 20 μM) of the GPX4 inhibitor FIN56 (MCE, HY-103087) for 24 h, followed by Western blot analysis. Results are shown below. Figure 9 As shown in the figure, the results indicate that GPX4 expression gradually decreased with increasing FIN56 concentration, while the expression of c-MYC, PD-L1, and CD47 proteins increased in a concentration-dependent manner.

[0092] Iron overload treatment: MB49 cells were treated with different concentrations (0, 125, 250, 500, 1000 μM) of ferric ammonium citrate (FAC) for 24 h, and then the cell lines were analyzed by Western blot. Results are as follows: Figure 10 As shown in the figure. The results showed that the expression of PD-L1 and CD47 proteins gradually increased with increasing iron concentration.

[0093] Chemotherapy treatment: MB49 cells were treated with different concentrations (0, 2.5, 5, 10, 20 μM) of cisplatin for 24 h, and then the cell lines were analyzed by Western blot. Results are as follows: Figure 11 As shown, the results indicate that the expression of c-MYC, PD-L1, and CD47 proteins increased in a concentration-dependent manner.

[0094] Radiotherapy treatment: MB49 cells were irradiated with different doses (0, 2, 4, 6, 8 Gy) of X-rays. Western blot analysis was performed on the treated cell lines 24 hours later. Results are as follows: Figure 12 As shown, the results indicate that the expression of PD-L1 and CD47 proteins gradually increases with increasing radiotherapy dose.

[0095] The above results indicate that regardless of the method used to induce ferroptosis, it is accompanied by the upregulation of c-MYC-mediated PD-L1 and CD47 expression, suggesting that traditional ferroptosis therapy may limit its immunotherapeutic effect due to the compensatory increase of immune checkpoints.

[0096] Example 3

[0097] MB49 cells were treated for 24 h with different concentrations of gallium nitrate (400 μM), gallium sulfate (80 μM), gallium chloride (120 μM), gallium tris(tris) (200 μM, Aladdin, 108560-70-9), gallium phthalocyanine chloride (150 μM, Gallium(III)-PhthalocyanineChloride), and the organogallium compound gallium(III) porphyrin (150 μM). Western blot analysis was then performed on the treated cell lines. Results are as follows: Figure 13 As shown in the figure, the results indicate that the protein levels of both PD-L1 and CD47 decrease in a concentration-dependent manner as the concentration of gallium ions in the inorganic gallium salt increases; while the inhibitory effects of strongly coordinated organic gallium compounds (gallium phthalocyanine chloride and porphyrin gallium) on PD-L1 and CD47 are not significant, suggesting that the ability to release free gallium ions is a key factor in achieving this regulatory effect.

[0098] Example 4

[0099] MB49 cells (mouse bladder transitional cell carcinoma line) were treated with gallium nitrate, MnO2@Alb, and Ga@MnO2@Alb at the same concentration (400 μM) for 24 h. Western blot analysis was then performed on the treated cells. Results are as follows: Figure 14 As shown in the figure: 1 is the control group, 2 is the MnO2@Alb group, 3 is the gallium nitrate group, and 4 is the Ga@MnO2@Alb group. The results showed that MnO2@Alb treatment alone had no significant inhibitory effect on the protein expression of c-MYC, CD47, and PD-L1; gallium nitrate alone could slightly downregulate the expression of c-MYC and CD47 to a certain extent; while Ga@MnO2@Alb showed the strongest inhibitory effect on all three proteins (c-MYC, CD47, and PD-L1), indicating that Ga@MnO2@Alb can effectively reduce the expression of oncogenes and immune checkpoint proteins in tumor cells simultaneously, and has the potential to synergistically inhibit tumor immune escape.

[0100] The treated cells were then subjected to immunofluorescence staining. The results are as follows: Figure 15 As shown in the figure: 1 is the control group, 2 is the MnO2@Alb group, 3 is the gallium nitrate group, and 4 is the Ga@MnO2@Alb group. The results show that immunofluorescence directly validated the conclusions of the Western blotting at the cellular level: Ga@MnO2@Alb significantly reduced the expression of PD-L1 and CD47 on the cell surface, while MnO2@Alb alone had no significant effect, and gallium nitrate alone only partially inhibited CD47 expression. PD-L1 and CD47 are key immune checkpoints for tumor cells to evade T cell and macrophage killing; their downregulation suggests that Ga@MnO2@Alb can effectively relieve the immunosuppressive microenvironment of tumors.

[0101] Detection effect of CD8 on treated cells + The cytotoxic effect of T cells, combining T24 cells (human bladder transitional cell carcinoma line) from different treatment groups with activated CD8... + Effector T cells were co-cultured for 24 hours, and the results were as follows: Figure 16As shown in the figure: 1 is the control group, 2 is the MnO2@Alb group, 3 is the gallium nitrate group, and 4 is the Ga@MnO2@Alb group. The results show that Ga@MnO2@Alb treatment significantly enhances the killing effect of T cells on tumor cells, while MnO2@Alb treatment alone cannot enhance the killing effect of T cells, and gallium nitrate alone can only partially enhance the killing effect. This result is highly consistent with the effect of immune checkpoint inhibition at the protein level, indicating that Ga@MnO2@Alb effectively relieves the immunosuppression of T cells by tumor cells by downregulating the expression of PD-L1 and CD47, thereby enhancing the T cell-mediated anti-tumor immune response.

[0102] Flow cytometry was used to detect the phagocytic effect of macrophages on MB49 cells in different treatment groups. MB49 cells and RAW264.7 macrophages in different treatment groups were co-cultured for 2 hours (MB49 cells: RAW264.7 macrophages = 1:5). The results are as follows: Figure 17 As shown, where Figure 17 In the image, 'a' represents the result of flow cytometry analysis. Figure 17 In the figure, b represents the statistical analysis. In the figure: 1 is the control group, 2 is the MnO2@Alb group, 3 is the gallium nitrate group, and 4 is the Ga@MnO2@Alb group. The results showed that MnO2@Alb alone did not significantly promote the tumor phagocytic ability of macrophages; gallium nitrate alone could partially relieve the inhibition of macrophages by tumor cells and improve phagocytic efficiency; compared with the control group, the phagocytic ability of macrophages against tumor cells was strongest after Ga@MnO2@Alb treatment, indicating that it can effectively block CD47-mediated signaling on the surface of tumor cells, relieve immunosuppression, and promote the recognition and phagocytosis of tumor cells by macrophages. This result corroborates the previous results of CD47 protein downregulation, further demonstrating that Ga@MnO2@Alb can enhance the innate immune response by targeting CD47 and exert a significant anti-tumor effect.

[0103] The phagocytosis of MB49 cells (green, WGA-labeled) by RAW264.7 macrophages (red, F4 / 80 labeled) was observed using confocal laser scanning microscopy, following the same method as flow cytometry for phagocytic efficiency assay. Results are as follows: Figure 18 As shown in the figure, the results indicated that in the control group, tumor cells highly expressed CD47 on their surface, which inhibited macrophage phagocytosis through signaling, resulting in almost no phagocytosis. However, after treatment with Ga@MnO2@Alb, CD47 expression in tumor cells was significantly downregulated, and the phagocytic inhibition signal of macrophages was relieved, enabling them to effectively recognize and phagocytose tumor cells, as evidenced by abundant red-green fluorescence co-localization. This result is highly consistent with the quantitative results of flow cytometry phagocytosis experiments, further demonstrating at the cellular level that Ga@MnO2@Alb can enhance the innate anti-tumor immune function of macrophages by blocking CD47 signaling.

[0104] Example 5

[0105] MB49 cells were treated for 24 h with gallium nitrate, MnO2@Alb, and Ga@MnO2@Alb at the same concentration (400 μM), where the Ga equivalent was 16 μM. The intracellular ferrous ion concentration (Fe2+) in MB49 cells from different treatment groups was measured according to the kit instructions. 2+ The relative concentrations of malondialdehyde (MDA) (Solepro, BC5415), oxidized glutathione (GSSG) (Beyotime, S0053), and their respective results are as follows: Figure 19 , Figure 20 , Figure 21 As shown in the figure; 1 is the control group, 2 is the MnO2@Alb group, 3 is the gallium nitrate group, and 4 is the Ga@MnO2@Alb group. The results show that intracellular ferrous ions (Fe...) 2+ The concentration was higher than that of the control group (see figure). The levels of malondialdehyde (MDA), a lipid peroxidation end product, were significantly higher than those in the control group (Figure 1). (The figure indicates a significant difference compared to the control group), oxidized glutathione (GSSG) levels increased (as shown in the figure). The symbol indicates a significant difference compared to the control group.

[0106] The treated cells were observed using transmission electron microscopy, and the results are as follows: Figure 22 As shown in the figure: Figure 22 In the image, 'a' is a transmission electron microscope image of the mitochondrial ultrastructure of the control group cells (scale bar 1 μm). Figure 22 b in the image is a transmission electron microscope image of the mitochondrial ultrastructure of Ga@MnO2@Alb group cells (scale bar 1 μm). Figure 22 In the image, 'c' is a transmission electron microscope image of the ultrastructure of mitochondria in the control group cells (scale bar 500 nm). Figure 22 In the image, 'd' represents a transmission electron microscope (TEM) image (scale bar 500 nm) of the mitochondrial ultrastructure in Ga@MnO2@Alb group cells. The results show that the mitochondria in the Ga@MnO2@Alb group cells exhibit significant shrinkage, cristae breakage, or disappearance, consistent with the typical ultrastructural characteristics of ferroptosis.

[0107] MB49 cells were pretreated with the ferroptosis-specific inhibitor Ferrostatin-1 (Fer-1) for 12 hours, followed by treatment with 10 µM Ga@MnO2@Alb for 24 hours. Cell morphology was observed using an optical microscope. The results were compared with the control group (0 µM Ga@MnO2@Alb) and the groups treated with only (5 µM, 10 µM) Ga@MnO2@Alb. Figure 23 As shown in the figure: Figure 23In the figure, 'a' represents the morphological observation of cells in the 0µM Ga@MnO2@Alb group (scale bar 100μm). Figure 23 b in the figure represents the morphological observation of cells in the 0µM Ga@MnO2@Alb group (scale bar 50μm). Figure 23 c in the figure represents the morphological observation of cells in the 5µM Ga@MnO2@Alb group (scale bar 100μm). Figure 23 In the figure, d represents the morphological observation of cells in the 5µM Ga@MnO2@Alb group (scale bar 50μm). Figure 23 In the figure, 'e' represents the morphological observation of cells in the 10µM Ga@MnO2@Alb group (scale bar 100μm). Figure 23 f in the figure represents the morphological observation of cells in the 10µM Ga@MnO2@Alb group (scale bar 50μm). Figure 23 In the figure, g represents the morphological observation of cells in the 10µM Ga@MnO2@Alb+Fer-1 group (scale bar 100μm). Figure 23 In the figure, h represents the morphological observation of cells in the 10µM Ga@MnO2@Alb+Fer-1 group (scale bar 50μm). The results showed that the abnormal cell morphology and cell death induced by Ga@MnO2@Alb were significantly reversed by Fer-1, confirming that the cell death induced by this nano-formulation was ferroptosis.

[0108] Example 6

[0109] MB49 bladder cancer cells were injected subcutaneously into mice to construct a mouse model of subcutaneous MB49 bladder cancer xenografts. The tumors were allowed to grow to approximately 50 mm in size. 3 Mice were randomly divided into 4 groups (n=6): control group (blank xenograft mouse model), MnO2@Alb group, gallium nitrate group, and Ga@MnO2@Alb group. The drugs were administered via tail vein injection every 3 days for a total of 5 times.

[0110] 6.1 In vivo antitumor efficacy Tumor volume was measured every two days during treatment, and the results were as follows: Figure 24 As shown in the figure: Figure 24 In the figure, 'a' represents the tumor growth curves of mice in different treatment groups. Figure 24 In the figure, b represents actual tumor images of mice in different treatment groups; in the figure, 1 is the control group, 2 is the MnO2@Alb group, 3 is the gallium nitrate group, and 4 is the Ga@MnO2@Alb group. The results showed that MnO2@Alb alone had no significant inhibitory effect on tumor growth; gallium nitrate alone could partially delay tumor growth; while Ga@MnO2@Alb showed a very strong in vivo anti-tumor effect, with a tumor growth inhibition rate of over 90%, almost completely inhibiting tumor progression.

[0111] Mice were euthanized after treatment, and tumors were collected and weighed. Results were as follows: Figure 25 As shown in the figure, 1 is the control group, 2 is the MnO2@Alb group, 3 is the gallium nitrate group, and 4 is the Ga@MnO2@Alb group. The results showed that the average tumor weight of the Ga@MnO2@Alb group was significantly lower than that of the other three groups (P<0.001).

[0112] After treatment, peripheral blood was collected from mice in the control group and the Ga@MnO2@Alb treatment group. Serum was separated, and the levels of liver function indicators (alanine aminotransferase ALT, aspartate aminotransferase AST), kidney function indicators (blood urea nitrogen BUN, creatinine Cr), and myocardial injury indicators (creatine kinase CK) were detected using a fully automated biochemical analyzer.

[0113] The results are as follows Figure 26 As shown in the figure: Figure 26 In Example 6, 'a' represents a bar chart showing the serum alanine aminotransferase (ALT) levels in mice from different treatment groups. Figure 26 In Example 6, b is a bar chart showing the serum aspartate aminotransferase (AST) levels in mice from different treatment groups. Figure 26 A bar chart showing the serum urea nitrogen levels in mice from different treatment groups in Example 6 (c). Figure 26 A bar graph showing the serum creatine kinase levels in mice from different treatment groups in Example 6; Figure 26 The results of serum creatinine levels in mice from different treatment groups in Example 6 are shown in the bar graph. The results showed that compared with the control group, there were no statistically significant differences in the levels of alanine aminotransferase, aspartate aminotransferase, blood urea nitrogen, creatine kinase, and creatinine in the Ga@MnO2@Alb treatment group. All indicators were within the normal physiological range, and no indicators related to liver, kidney, or myocardial damage were elevated.

[0114] After treatment, the mice were euthanized, and the main organs, such as the heart, liver, spleen, lungs, and kidneys, were dissected and separated. After fixation with 4% paraformaldehyde, paraffin embedding, and sectioning, the organs were stained with hematoxylin and eosin (H&E) and the histomorphological changes of each organ were observed under an optical microscope.

[0115] The results are as follows Figure 27 As shown, the results indicated that the heart, liver, spleen, lungs, and kidneys of both the control group and the Ga@MnO2@Alb treatment group exhibited intact tissue structure and normal cell morphology. No obvious pathological damage changes such as inflammatory cell infiltration, tissue necrosis, edema, or fibrosis were observed. The histological characteristics of each organ were not significantly different from those of the control group. This confirms that the nano-formulation has good in vivo biocompatibility; there was no significant difference in body weight among the groups during treatment, and no obvious toxic side effects were observed.

[0116] 6.2 Immune activation in vivo Tumor tissue was taken after treatment and Western blot analysis was performed. The results were as follows: Figure 28 As shown in the figure. 1 represents the control group, 2 represents the MnO2@Alb group, 3 represents the gallium nitrate group, and 4 represents the Ga@MnO2@Alb group. The results showed that the protein expression of PD-L1 and CD47 in tumor tissues of the Ga@MnO2@Alb group was downregulated by approximately 70% compared to the Vehicle group.

[0117] Immunohistochemical staining was performed on tumor tissue, and the results were as follows: Figure 29 As shown in the figure. The results showed that Ga@MnO2@Alb significantly downregulated the protein expression of PD-L1 and CD47 in tumor tissues in vivo.

[0118] The tumor tissue was ground and separated into single cells, and the infiltration of immune cells in the tumor tissue was detected by flow cytometry. The results are as follows: Figure 30 As shown in the figure. The results showed that the CD4 counts in the control group and the MnO2@Alb group were significantly lower. + The proportion of T cells was low; the proportion was slightly higher in the gallium nitrate group; the proportion of CD4 in the Ga@MnO2@Alb group was lower. + The proportion of T cells was significantly higher in the group than in the other three groups, and CD3 was observed in the flow cytometry scatter plot. + CD4 + The proportion of double-positive cells increased from 0.82% in the control group to 4.45%. The CD8+ of the control group and MnO2@Alb... + The proportion of T cells was extremely low; only a slight increase was observed in the gallium nitrate group; CD8+ of Ga@MnO2@Alb was significantly reduced. + The proportion of T cells increased significantly, from 0.45% in the control group to 4.48%. This indicates that the tumor tissue of the Ga@MnO2@Alb group showed a higher proportion of CD3+ cells. + CD4 + T cells and CD3 + CD8 + The proportion of T cells was significantly increased, including CD8 cells. + The proportion of effector T cells increased by about 3 times compared to the control group.

[0119] Example 7

[0120] MB49 bladder cancer cells were injected subcutaneously into mice to construct a mouse model of subcutaneous MB49 bladder cancer xenografts. The tumors were allowed to grow to approximately 50 mm in size. 3Mice were randomly divided into 6 groups (n=6): control group (blank xenograft mouse model), Anti-PD-L1+Anti-CD47 dual immune checkpoint blockade group (injected with PD-L1 mAb 5 mg / kg (Cat. #BE0101, BioXCell) and CD47 mAb 5 mg / kg (Cat. #BE0270, BioXCell)), Ga@MnO2@Alb monotherapy group (Ga: 4.0 mg / kg, Mn: 7.77 mg / kg), RT radiotherapy group, Anti-PD-L1+Anti-CD47+RT immunotherapy combined with radiotherapy group, and Ga@MnO2@Alb+RT nanoparticles combined with radiotherapy group. Each group received the corresponding treatment drugs via tail vein injection on days 0, 3, 6, and 9. The radiotherapy group received local radiotherapy to the tumor site on days 1, 4, 7, and 10, with a single dose of 3 Gy and a dose rate of 6 Gy / min.

[0121] In vivo anti-tumor efficacy Tumor volume was measured every two days during treatment, and the results were as follows: Figure 31 As shown in the figure: Figure 31 In the figure, 'a' represents the tumor growth curves of mice in different treatment groups. Figure 31 In the figures, b represents actual tumor images of mice in different treatment groups; 1 represents the control group, 2 represents the Anti-PD-L1+Anti-CD47 dual immune checkpoint blockade group, 3 represents the Ga@MnO2@Alb monotherapy group, 4 represents the RT radiotherapy group, 5 represents the Anti-PD-L1+Anti-CD47+RT immunotherapy combined with radiotherapy group, and 6 represents the Ga@MnO2@Alb+RT nanoparticle combined with radiotherapy group. The results showed that the treatment with Anti-PD-L1+Anti-CD47 dual antibodies alone (Group 2) only showed a partial inhibitory effect on tumor growth; the treatment with Ga@MnO2@Alb alone (Group 3) could significantly delay tumor growth; radiotherapy alone (Group 4) had a certain inhibitory effect on tumor growth; while the anti-tumor effect of the group with Anti-PD-L1+Anti-CD47+RT immunotherapy combined with radiotherapy (Group 5) was better than that of each single treatment group; the group with Ga@MnO2@Alb+RT nanoparticles combined with radiotherapy (Group 6) showed a very strong synergistic anti-tumor effect, almost completely inhibiting tumor progression, and the tumor growth inhibition rate was significantly higher than that of all other groups, with statistical differences.

[0122] Mice were euthanized after treatment, and tumors were collected and weighed. Results were as follows: Figure 32As shown in the figure: 1 is the control group, 2 is the Anti-PD-L1+Anti-CD47 dual immune checkpoint blockade group, 3 is the Ga@MnO2@Alb monotherapy group, 4 is the RT-only radiotherapy group, 5 is the Anti-PD-L1+Anti-CD47+RT immunotherapy combined with radiotherapy group, and 6 is the Ga@MnO2@Alb+RT nanoparticle combined with radiotherapy group. The results showed that the mean tumor weight of the Ga@MnO2@Alb+RT nanoparticle combined with radiotherapy group (group 6) was significantly lower than that of all other treatment groups, and significantly lower than that of the Ga@MnO2@Alb monotherapy group (group 3) and the Anti-PD-L1+Anti-CD47+RT immunotherapy combined with radiotherapy group (group 5) (P<0.001), confirming that the combined treatment of nanoparticles and radiotherapy can achieve the optimal in vivo anti-tumor effect.

[0123] It should be noted that the above embodiments are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. Those skilled in the art can make other variations or modifications based on the above description. It is impossible to exhaustively list all possible implementations here. All obvious variations or modifications derived from the technical solutions of this invention are still within the scope of protection of this invention.

Claims

1. A gallium nanoparticle formulation, characterized in that, By weight, it contains: (a) Albumin: 80-100 parts; (b) Manganese dioxide: 2-8 parts; (c) Gallium ions: 0.5-3 parts; The manganese dioxide is mineralized in situ on albumin to form composite particles, and the gallium ions are loaded on the surface or inside the composite particles, or the gallium ions are loaded on both the surface and inside the composite particles.

2. The gallium nanoparticle formulation according to claim 1, characterized in that, The manganese dioxide content is 2-5 parts; the gallium ions content is 0.5-2 parts; the albumin is bovine serum albumin, human serum albumin, or recombinant albumin, wherein the recombinant albumin is a recombinant albumin expressed by gene recombination technology and has the same or at least 95% homology with natural human serum albumin or bovine serum albumin.

3. The gallium nanoparticle formulation according to claim 1, characterized in that, The average hydrated particle size of the gallium nanoparticles is 50-200 nm.

4. The method for preparing gallium nanoparticles according to any one of claims 1-3, characterized in that, Includes the following steps: S1. Add a manganese source to the albumin solution to obtain an intermediate solution; S2. Add a gallium source to the intermediate solution to load gallium ions onto the intermediate; S3. Alternately add manganese and gallium sources, and perform multiple cycles; S4. Add manganese source dropwise for final mineralization and purification to obtain the gallium nanoparticle formulation; The gallium source contains gallium ions; the manganese source is a permanganate.

5. The preparation method according to claim 4, characterized in that, The permanganate includes one or both of potassium permanganate and sodium permanganate; the concentration of the manganese source is 1-10 mg / mL.

6. The preparation method according to claim 4, characterized in that, The gallium source is an inorganic gallium salt; the inorganic gallium salt includes one or more of gallium nitrate, gallium chloride, and gallium sulfate; the concentration of the gallium source is 1-10 mg / mL.

7. The preparation method according to claim 4, characterized in that, The concentration of the albumin solution is 1-20 mg / mL; in step S1, the volume ratio of the manganese source to the albumin solution is 0.2-2.0:50-200; in step S2, the volume ratio of the gallium source to the albumin solution is 0.2-2.0:50-200; in step S4, the volume ratio of the manganese source to the albumin solution is 0.5-5.0:50-200.

8. The preparation method according to claim 4, characterized in that, In step S3, the alternating cycle is repeated 3-10 times.

9. The use of the gallium nanoparticle formulation according to any one of claims 1-3 or the gallium nanoparticle formulation prepared by the preparation method according to any one of claims 4-8 in the preparation of drugs to reverse tumor immune tolerance.

10. The use of the gallium nanoparticle formulation according to any one of claims 1-3 or the gallium nanoparticle formulation prepared by the preparation method according to any one of claims 4-8 in the preparation of a drug for reversing tumor immune tolerance in combination with radiotherapy.

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