Polyethylene glycol modified gold-manganese dioxide nanoparticles as well as preparation method and application thereof

Abstract

The invention discloses a polyethylene glycol modified gold and manganese dioxide nano particle and a preparation method and application thereof, and belongs to the technical field of biological medicine, the polyethylene glycol modified gold and manganese dioxide nano particle (GMCN and PEG) has good biocompatibility under neutral physiological conditions, can relieve tumor hypoxia and increase generation of active oxygen in acidic TME, and has a good anti-tumor effect. The radiotherapy sensitivity is improved; and immune cell death is induced. Meanwhile, a cGAS-STING signal channel can be activated, dendritic cell maturation, macrophage M1 polarization and T cell infiltration are promoted, and the immunosuppression state in TME is counteracted. In addition, the GMCN-coated PEG also has an MR-CT bimodal imaging enhancement function, and integration of diagnosis and treatment is realized. The nano sensitizer disclosed by the invention has huge potential in the aspects of improving the radiotherapy curative effect, remodeling the tumor microenvironment, promoting the anti-tumor immunity, enhancing the biomedical imaging and the like.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a nanosensitizer for enhancing the effect of radioimmunotherapy and having a dual-modality imaging function, and in particular to polyethylene glycol-modified gold@manganese dioxide nanoparticles (GMCN@PEG) and a preparation method and application thereof. Background Art

[0002] Radiotherapy is one of the main treatments for malignant solid tumors. It kills cancer cells by directly inducing DNA breakage or indirectly increasing the production of reactive oxygen species (ROS) in cancer cells. However, in actual clinical applications, the efficacy of radiotherapy is restricted by many factors. On the one hand, the rapid growth of cancer cells in solid tumors and abnormal vascular structure cause hypoxia in tumor tissues. This hypoxic state gives tumor cells a stronger ability to repair radiotherapy-induced DNA damage, which in turn induces drug resistance and ultimately leads to radiotherapy failure. On the other hand, problems such as insufficient energy deposition, microenvironment hypoxia, and low radiation energy absorption coefficient result in insufficient ROS generation during radiotherapy, significantly reducing the killing effect of radiotherapy on cancer cells. Therefore, improving the hypoxic state of the tumor microenvironment and increasing the level of ROS in tumor cells have become the key to improving the efficacy of radiotherapy.

[0003] At the same time, although radiotherapy can regulate the immune response of tumors, such as inducing the release of tumor antigens, activating the cGAS-STING pathway, and enhancing the anti-tumor activity of T cells, the tumor microenvironment (TME) itself has immunosuppressive properties and limited radiation absorption. Radiotherapy alone is often difficult to induce a systemic anti-tumor immune response and cannot effectively inhibit distant metastasis and recurrence of tumors.

[0004] In recent years, nanoparticles have attracted widespread attention in the field of tumor radioimmunotherapy. However, most related studies only enhance anti-tumor immunity from a single pathway, and fail to fully utilize the potential advantages of radioimmunotherapy. At present, most nanoparticles used for radiosensitization can only enhance the sensitivity of tumor cells to radiation in a single way. For example, although some nanomaterials can increase energy deposition, they cannot effectively improve the hypoxic microenvironment of tumors. In terms of immune regulation, existing nanoparticles can usually only activate a single immune cell or signaling pathway, and it is difficult to fully reshape the tumor microenvironment in an immunosuppressive state. For example, although some nanoparticles can activate T cells, their promoting effects on dendritic cell maturation and macrophage polarization are relatively limited. In terms of imaging function, the imaging effect of existing nanomaterials is significantly affected by the tumor microenvironment, and it is difficult to stably present clear tumor images under different physiological conditions. Therefore, the development of a new type of nanoparticle that can reshape the immunosuppressive TME, enhance the effect of tumor radioimmunotherapy through multiple pathways, and have other auxiliary functions (such as imaging function) has become an important issue to be solved in the current field of tumor treatment. Summary of the Invention

[0005] In view of the many deficiencies in radiotherapy in the prior art, the present invention provides a nano-sensitizer for enhancing radioimmunotherapy, its preparation method and applications.

[0006] The first aspect of the present invention:

[0007] It relates to polyethylene glycol-modified gold@manganese dioxide nanoparticles (GMCN@PEG), including a gold nanoparticle (GNP) core with a diameter of 10 - 50 nm;

[0008] A manganese dioxide (MnO2) shell with a thickness of 10 - 20 nm;

[0009] A polyethylene glycol (PEG) modification layer with a molecular weight of 1000 - 5000 Da;

[0010] Among them, the polyethylene glycol-modified gold@manganese dioxide nanoparticles decompose responsively in an acidic tumor microenvironment (pH 5.5 - 6.5), releasing Mn 2+ and exposing the gold core, achieving integrated diagnosis and treatment through the following functions:

[0011] (a) Alleviating tumor hypoxia and increasing ROS generation through the peroxidase-like (POD-like) and catalase-like (CAT-like) activities of MnO2;

[0012] (b) Inducing immunogenic cell death (ICD) by activating the cGAS-STING signaling pathway, promoting dendritic cell maturation, macrophage M1 polarization, and T cell infiltration;

[0013] (c) Enhancing the T1-weighted magnetic resonance imaging (MRI) signal through the released Mn 2+ and enhancing the CT imaging contrast through the gold core.

[0014] In the above technical solution, the thickness of the manganese dioxide shell is 15 ± 2 nm; the mass ratio of the gold core to the manganese dioxide shell is 1:1.5 - 1:3; the grafting density of the PEG modification layer is 0.5 - 2 PEG molecules / nm 2 .

[0015] In the above technical solution, under acidic conditions (pH 6.0), the T1 longitudinal relaxation rate of the polyethylene glycol-modified gold@manganese dioxide nanoparticles is 5.0 - 6.5 mM -1 s -1 ; under neutral conditions (pH 7.4), the T1 longitudinal relaxation rate is 0.5 - 1.0 mM -1 s -1; The CT value is increased by 20% - 30% compared to the bare gold core at the same concentration.

[0016] The second aspect of the present invention: relates to a preparation method of the above-mentioned polyethylene glycol-modified gold@manganese dioxide nanoparticles, and the specific steps are as follows:

[0017] (1) Synthesize gold nanoparticles (GNP) by the polyol reduction method, control the reaction temperature at 200 - 220 °C, and the reaction time at 1 - 3 hours;

[0018] (2) Incubate GNP with a citric acid solution (5 - 20 mM) for 10 - 14 hours, and remove the unadsorbed citric acid by centrifugation;

[0019] (3) In the presence of potassium oxalate (0.05 - 0.2 M), reduce and deposit potassium permanganate (0.01 - 0.05 M) on the surface of GNP to form a manganese dioxide shell, with the reaction temperature at 50 - 70 °C and the reaction time at 1 - 3 hours;

[0020] (4) Conjugate the product of step (3) with PEG (molecular weight 1000 - 5000 Da), and monitor the changes in particle size and Zeta potential by dynamic light scattering until the particle size is stable at 80 - 120 nm and the Zeta potential is -15 to -30 mV.

[0021] The third aspect of the present invention:

[0022] Relates to the application of the above-mentioned nano-sensitizer in the preparation of radiopharmaceuticals for radioimmunotherapy and biomedical imaging reagents. The polyethylene glycol-modified gold@manganese dioxide nanoparticles enhance the radiotherapy efficacy through the following mechanisms in the tumor microenvironment:

[0023] (a) By catalyzing the decomposition of H2O2 into O2, the oxygen partial pressure in the tumor tissue is increased by 40% - 60%;

[0024] (b) By generating hydroxyl radicals (·OH) through peroxidase-like activity, the intracellular ROS level is increased by 3 - 5 times;

[0025] (c) Through the radiation energy deposition effect of the gold core, the DNA double-strand break rate in tumor cells is increased by 2 - 4 times.

[0026] In the above technical solution, the polyethylene glycol-modified gold@manganese dioxide nanoparticles activate anti-tumor immunity through the following pathways:

[0027] (a) Induce the exposure of calreticulin (CRT) on the surface of tumor cells and the release of HMGB1, and promote the maturation of dendritic cells (CD 80 CD 86 cell ratio ≥ 80%);

[0028] (b) By releasing Mn 2+ Activates the cGAS-STING pathway, increasing the secretion of IFN-β by 5-10 times;

[0029] (c) Promotes an increase in the proportion of tumor-infiltrating CD8 + T cells to 30%-50%, and inhibits the proportion of regulatory T cells (Tregs) to ≤5%.

[0030] In the above technical solution, the application of the polyethylene glycol-modified gold@manganese dioxide nanoparticles in the preparation of MR-CT dual-modal imaging contrast agents. At the tumor site, the MRI signal intensity of the polyethylene glycol-modified gold@manganese dioxide nanoparticles is increased by 2-3 times compared with normal tissues, the CT value is increased by 20%-30% compared with the bare gold core, and the imaging contrast remains stable within 24 hours after injection.

[0031] In the above technical solution, for the polyethylene glycol-modified gold@manganese dioxide nanoparticles, after intravenous injection, its blood half-life (t 1 / 2 ) is 6-8 hours, the tumor-targeting accumulation amount ≥15% ID / g, mainly metabolized through the kidneys, and the total clearance rate ≥90% within 72 hours.

[0032] In the above technical solution, when the dose ≤20 mg / kg, there is no significant effect on hematological parameters (white blood cell count, hemoglobin, platelet) and liver and kidney function indexes (ALT, AST, BUN, Cr) (P>0.05), and the hemolysis rate ≤5%.

[0033] In the above technical solution, the radioimmunotherapy kit of the polyethylene glycol-modified gold@manganese dioxide nanoparticles. The kit also includes a radiotherapy dose guidance module, recommending a single X-ray irradiation dose of 2-10 Gy, the total dose ≤30 Gy, and the time interval between GMCN@PEG injection and radiotherapy is 12-24 hours.

[0034] Beneficial effects:

[0035] (1) Enhances the efficacy of radiotherapy: GMCN@PEG can effectively relieve tumor hypoxia and generate a large amount of ROS in the acidic TME, significantly improve the radiotherapy sensitivity, enhance the killing effect of radiotherapy on tumor cells, and inhibit tumor growth.

[0036] (2) Regulates the immune response: By inducing ICD and activating the cGAS-STING signaling pathway, GMCN@PEG promotes DC maturation, macrophage M1 polarization and T cell infiltration, remodels the immunosuppressive TME, enhances the body's anti-tumor immune response, and effectively inhibits distant metastasis and recurrence of tumors.

[0037] (3)Theranostic integration: GMCN@PEG has MR-CT dual-modal imaging function, which can provide strong support for the early diagnosis and precise treatment of tumors, realize the organic combination of diagnosis and treatment, and improve the clinical treatment effect.

[0038] (4)High biosafety: In vivo experiments have shown that GMCN@PEG has good biosafety in vivo. The results of hematological and biochemical analyses, histological examinations of major organs, and hemolysis experiments on mice show that it has no significant effect on hematological parameters, liver function, and renal function, and does not induce obvious hemolysis, providing a safety guarantee for its clinical application. Brief Description of the Drawings

[0039] Figures 1A - 1J : Synthesis and characterization diagrams of GMCN.

[0040] Figures 2A - 2I : Enzyme-like activity diagrams of GMCN.

[0041] Figures 3A - 3H : Radiosensitization effect diagrams of GMCN@PEG in vitro.

[0042] Figures 4A - 4F : Effect diagrams of GMCN@PEG enhancing radioimmunotherapy in vitro.

[0043] Figures 5A - 5G : Biosafety and biocompatibility diagrams of GMCN in vivo.

[0044] Figures 6A - 6F : Diagrams of the enhancing effect of GMCN@PEG on radiotherapy for primary tumor-bearing mice.

[0045] Figures 7A - 7G : Diagrams of the enhancing effect of GMCN@PEG on radioimmunotherapy in vivo.

[0046] Figures 8A - 8H : Enhanced effect diagrams of magnetic resonance-computed tomography (MR-CT) dual-modal imaging of GMCN@PEG. Detailed Embodiments

[0047] The present invention will be described in detail below in conjunction with the accompanying drawings and specific embodiments. However, the following embodiments are only for explaining the present invention, and the protection scope of the present invention should include all the contents of the claims. Moreover, through the description of the following embodiments, those skilled in the art can fully implement all the contents of the claims of the present invention.

[0048] Embodiments

[0049] 1. Preparation of GMCN@PEG

[0050] (1)Preparation of GNP: The synthesis of GNP was carried out using a previously reported method. First, 30 μL of 1 M HAuCl4, 1.2 mL of PDDA, and 12 μL of 0.1 M AgNO3 aqueous solution were sequentially added to a glass vial containing 60 mL of ethylene glycol. Then the mixture was vigorously stirred at room temperature to ensure uniform distribution. Subsequently, the prepared solution was heated in an oil bath at 220 °C for 2 hours, during which the color of the solution changed from yellow to red. The nanoparticles were obtained by centrifugation at 10000 rpm, washed repeatedly with ultrapure water, and then sonicated to remove residual ethylene glycol and PDDA. Finally, the volume of the nanoparticle solution was adjusted to 5 mL.

[0051] (2)Preparation of GMCN: The prepared GNP solution was incubated with 25 mL of 10 mM citric acid (CA) solution for 12 hours. After incubation, the CA-modified GNP was obtained by centrifugation at 10000 rpm, washed thoroughly with ultrapure water, and then sonicated to remove the excess citric acid. The volume of the GNP solution was adjusted to 30 mL. Subsequently, 0.8 mL of 0.1 M KMnO4 and 4 mL of 0.1 M potassium oxalate (K2C2O4) were added to the solution. The mixture was sonicated for 1 minute to ensure uniform distribution. Then the solution was heated in a water bath at 60 °C for 2 hours. After the reaction was completed, the product was collected by centrifugation at 8000 rpm, washed repeatedly with ultrapure water and sonicated to remove the residual KMnO4 and K2C2O4. Finally, the volume of the nanoparticle solution was adjusted to 5 mL to obtain GMCN.

[0052] (3)Preparation of GMCN@PEG: The previously prepared GNP / GMCN solution was mixed with 150 mg of PEG2000 and added to 50 mL of ultrapure water. The mixture was sonicated for 30 minutes to ensure uniform dispersion, and then stirred with a magnetic stirrer for 6 hours to promote the interaction between the components. After the stirring period ended, the product was collected by centrifugation at 10000 rpm, washed repeatedly with ultrapure water and sonicated to remove the supernatant containing excess PEG. The changes in particle size and zeta potential during the reaction were monitored by dynamic light scattering (DLS) technology to determine whether the PEGylation process was successful.

[0053] (4)Characterization of GMCN

[0054] The morphology of the final product was characterized using a field emission scanning electron microscope (SEM, ZEISS, ΣIGMA). Transmission electron microscopy (TEM, JEOL, JEM -1400) Characterize the products. For TEM analysis, the prepared solution was dropped on a carbon-coated copper grid and then evaporated at room temperature. EDS mapping and high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) analysis were performed using Talos F200X and JEOL Arm-200F electron microscopes. X-ray photoelectron spectroscopy (XPS) analysis was carried out on an ESCALAB 250 X-ray photoelectron spectrometer using Al Kα radiation. The ultraviolet-visible (UV-vis) absorption spectrum was recorded using a UV -1 601 spectrophotometer. The molar ratio and mass ratio of Au and manganese (Mn) in GMCN were determined using inductively coupled plasma mass spectrometry (ICP-MS, Thermo, icapQc). In addition, the zeta potential, hydrodynamic diameter, and polydispersity index (PDI) of the samples were measured using a Malvern Zetasizer Nano ZS90.

[0055] 2. Performance testing and applications of GMCN@PEG

[0056] 2.1 Materials and methods

[0057] (1) Enzyme-like activity test

[0058] a. POD-like activity

[0059] The concentration dependence of the POD-like activity of GMCN was determined by adding different concentrations (0, 5, 10, 20, and 40 μg mL -1 ) of GMCN and 1 mM TMB to a 100 mM H2O2 solution at pH 6.0. The final absorbance of the mixture at 652 nm was detected using a UV-vis spectrophotometer. To study the effect of pH on the POD-like activity of GMCN, 40 μg mL -1 of GMCN and 1 mM TMB were added to 100 mM H2O2 solutions at different pH values. The absorbance was measured as described above. Using H2O2 as a substrate, the steady-state kinetics of GMCN was determined by adding 40 μg mL -1 of GMCN and 1 mM TMB to H2O2 solutions at different concentrations (50, 100, 150, 200, 250, and 300 mM). Then, according to the Beer-Lambert law, the absorbance values obtained at 652 nm were converted to the concentration of the TMB-derived oxidation product. The Michaelis constant was determined from the Michaelis saturation curve.

[0060] b. ·OH radical

[0061] A capillary tube containing 20 μL of water or GMCN solution was placed into an electron spin resonance (ESR) apparatus (Bruker EMXplus 10 / 20). The hydroxyl radical spin adduct (DMPO / ·OH) was used to detect the characteristic peak (1:2:2:1) of GMCN-catalyzed H2O2 to generate ·OH at different pH values. The content of ·OH was quantitatively estimated by the ESR signal intensity.

[0062] c. CAT-like activity

[0063] By adding GMCN at different concentrations (0, 5, 10, 20, and 30 μg mL -1 ) to a 200 mM H2O2 solution at pH 6.0, the concentration dependence of GMCN-like CAT activity was determined. The solubility of the generated O2 was measured after different reaction times. Using H2O2 as the substrate, steady-state kinetics of GMCN was determined by adding 20 μg mL -1 of GMCN to H2O2 solutions at different concentrations (50, 100, 150, 200, 250, and 300 mM). The solubility of the generated O2 was monitored at different reaction times. The Michaelis constant was determined from the Michaelis saturation curve.

[0064] (2) In vitro radiotherapy sensitization effect test

[0065] a. Cell viability

[0066] 4T1 cells were seeded in a 96-well plate at a density of 5000 cells per well and incubated for 24 hours. Then, the medium in each well was aspirated, and the cells were exposed to a series of GNP@PEG / GMCN@PEG concentrations (0, 12.5, 25, 50, 100 μg mL -1 ) in a medium at pH 6.0 or pH 7.4 for 8 hours, followed by irradiation with 10 Gy of X-rays. After irradiation, the cells were incubated for another 24 hours. Subsequently, the medium was replaced with 100 μL of fresh medium containing CCK-8 reagent, and the cells were incubated for another 2 hours. Subsequently, the optical density (OD) at 450 nm was measured using a spectrophotometer.

[0067] b. Calcein-AM / Propidium Iodide (PI) live / dead cell double staining

[0068] Live / dead cell double staining was performed using a calcein-AM / PI staining kit. 4T1 cells were seeded into a 12-well plate at a density of 100000 cells per well and incubated for 12 hours. Then, the cells were treated with a concentration of 100 μg mL -1The cells were treated with GNP@PEG / GMCN@PEG for 8 hours. After treatment, the cells were irradiated with X-rays at 10 Gy and further incubated for 24 hours. At the end of the post-irradiation incubation, the culture medium was aspirated and the cells were washed twice with PBS. Subsequently, the cells were stained with a solution containing 2 μM calcein-AM and 4.5 μM PI and incubated for 30 minutes. After staining, the cells were washed three times with PBS and observed under a fluorescence microscope.

[0069] c. Colony formation assay

[0070] 4T1 cells were seeded in 6-well plates at a density of 1000 cells per well and cultured for 12 hours. After the initial incubation, the cells were treated with 50 μg / mL -1 of GNP@PEG / GMCN@PEG for 2 hours and then irradiated with X-rays. Then the cells were cultured for 14 days and the culture medium was changed every 3 - 4 days. At the end of the incubation period, the cells were fixed with 4% paraformaldehyde (PFA) and stained with 10% crystal violet dye. After counting the cells using ImageJ software, the sensitivity to radiotherapy was quantified by calculating the survival fraction (SF) and the dose modification factor (DMF).

[0071] d. DNA damage and ROS

[0072] 4T1 cells were seeded into 12-well plates at a density of 100000 cells per well. After 24 hours of incubation, the cells were subjected to the same treatment process as described above. After that, the cells were fixed with 4% paraformaldehyde for 1 hour. Subsequently, the samples were blocked with 5% bovine serum albumin (BSA) at 37 °C for 1 hour. Then the samples were co-incubated with anti-γ-H2AX antibody diluted 1:1000 and FITC-labeled goat anti-mouse IgG secondary antibody also diluted 1:1000. Finally, observation was performed using a fluorescence microscope.

[0073] The generation of intracellular ROS was evaluated using the DCFH-DA fluorescent probe. 4T1 cells were seeded into 12-well plates at a density of 100000 cells per well. After 24 hours of incubation, the cells were subjected to the same treatment process as described above. Subsequently, the cells were stained with a solution containing 30 μM [Ru(dpp)3]Cl2 and incubated for 30 minutes. After staining, the cells were washed three times with PBS and observed under a fluorescence microscope.

[0074] e. Hypoxia alleviation

[0075] Evaluate the generation of intracellular O2 using the [Ru(dpp)3]Cl2 fluorescent probe. Seed 4T1 cells into a 12-well plate at a density of 100,000 cells per well. After incubation for 24 hours, the cells are subjected to the same treatment process as described above. Subsequently, the cells are stained with a solution containing 30 μM [Ru(dpp)3]Cl2 and incubated for 30 minutes. After staining, the cells are washed three times with PBS and observed under a fluorescence microscope.

[0076] (3)In vitro test for enhancing the effect of radioimmunotherapy

[0077] Detect the expression of CRT on the surface of 4T1 cells by immunofluorescence staining to evaluate the effect of GMCN@PEG on radiotherapy-induced ICD. Specifically, 4T1 cells in different treatment groups are subjected to immunofluorescence staining, and the expression of CRT is observed under a fluorescence microscope and compared with the control group, GNP@PEG group, GMCN@PEG group, IR alone group, and IR + GMCN@PEG group.

[0078] Detect the concentration of HMGB1 in the supernatant of 4T1 cells in different treatment groups by ELISA analysis to further verify the enhancing effect of GMCN@PEG on ICD. Collect the supernatants of cells in different treatment groups and detect them according to the operation instructions of the ELISA kit, and compare the concentration differences of HMGB1 in each group.

[0079] Evaluate the secretion of type I interferon IFNβ in DCs and macrophages in different treatment groups to study the ability of GMCN@PEG to promote the activation of the STING signaling pathway during radiotherapy. Treat DCs and macrophages separately, and after culturing for a period of time, collect the cell culture supernatants and detect the secretion amount of IFNβ by methods such as ELISA, and compare the differences between different treatment groups.

[0080] Use flow cytometry to detect CD 80 and CD 86 expression, and analyze the effects of combined treatment with IR and GMCN@PEG on the maturation of DCs and the M1 polarization characteristics of macrophages. After treating DCs and macrophages differently, use a flow cytometer to detect the expression levels of CD 80 and CD 86 on the cell surface, and explore the synergistic effect of combined treatment with IR and GMCN@PEG in regulating the immune microenvironment by analyzing the proportion of positive cells.

[0081] (4)In vivo performance test

[0082] a. Biosafety

[0083] Inject mice intravenously with PBS as a control, or inject at a concentration of 200 μL 1 mg / mL-1 GMCN@PEG solution. Quantitative analysis of blood routine and blood biochemical indexes was performed at different time points after injection (24 hours, 72 hours, and 120 hours). When performing blood biochemical analysis, 200 μL of blood was collected from each mouse and placed in a test tube containing 0.15% (mass / volume) potassium ethylenediaminetetraacetate dihydrate (EDTA-K2·2H2O). Then the blood samples were centrifuged at 3500 revolutions per minute (rpm) at 4 °C for 8 minutes, and the supernatant was collected for analysis.

[0084] The main organs of the mice (heart, liver, spleen, lung, and kidney) were collected and fixed in 4% PFA overnight. Then the tissues were dehydrated in 30% sucrose and cut into 20-μm sections for staining. The cell nuclei were stained with 1% eosin (Biosharp, BL702B) for 10 minutes, and the cytoplasm was stained with hematoxylin (Biosharp, BL703B) for 2 minutes. To selectively remove the excess dye, the sections were soaked in 1% acidic alcohol for a few seconds and then sealed with neutral resin.

[0085] b. In vivo biodistribution evaluation

[0086] At 24, 72, and 120 hours after injection of GMCN@PEG, the main organs (heart, liver, spleen, lung, and kidney) and tumors of the treated mice were collected. Then these organs and tumors were dissolved in aqua regia and incubated overnight. After incubation, all samples were centrifuged at 12000 revolutions per minute (rpm) for 20 minutes and diluted 100 times, and the gold concentration was measured using inductively coupled plasma (ICP) analysis.

[0087] c. Half-life of GMCN@PEG in vivo circulation

[0088] To study the half-life of GMCN@PEG in vivo circulation, all mice were intravenously injected with the solution (200 μL, dose of 10 mg / kg). Blood samples (10 μL) were collected at different time points after injection (2, 8, 15, 30 minutes and 1, 2, 4, 12, 24, and 48 hours). Each blood sample was mixed with 10 μL of an anticoagulant solution containing 0.15% (mass / volume) EDTA-K2·2H2O. The blood samples were dissolved in nitric acid overnight and then diluted 100 times. The gold content was estimated using inductively coupled plasma optical emission spectrometry (ICP-OES). The two-compartment pharmacokinetic model was used to determine the circulation half-life (τ 1 / 2 ) of GMCN@PEG in the blood. The elimination rate curve was plotted by fitting the natural logarithm (ln(Cp)) of the plasma concentration against time. The slope of this curve represents the elimination rate constant.

[0089] d. In vivo antitumor study

[0090] To investigate the effects of GMCN@PEG on enhancing the radiosensitivity of primary tumors and stimulating immune responses, BALB / c mice bearing primary subcutaneous 4T1 tumors were randomly divided into six experimental groups, with 10 mice in each group: G1: control group; G2: GNP@PEG group; G3: GMCN@PEG group; G4: radiotherapy group (IR); G5: radiotherapy + GNP@PEG group; G6: radiotherapy + GMCN@PEG group. GNP@PEG, GMCN@PEG or PBS was directly injected into the tumor tissue. Twelve hours after injection, groups 4 - 6 received X-ray irradiation at the tumor site. A total of three treatments were carried out. The start of the treatment was marked as day 0, and the mice were weighed daily. Using the formula:

[0091] V == L × W 2 / 2;

[0092] The tumor volume was measured, where V is the tumor volume, L is the tumor length, and W is the tumor width.

[0093] To further investigate the effects of GMCN@PEG on enhancing the radiosensitivity and immune responses of metastatic tumors, BALB / c mice bearing metastatic subcutaneous tumors were randomly divided into six experimental groups, with 10 mice in each group. PBS, GNP or GMCN@PEG was directly injected into the tumor tissue. Twelve hours after injection, groups 4 - 6 received X-ray irradiation at the primary tumor site. The body weight and survival status of the mice were monitored daily. When the volume of the right tumor reached 1500 mm³, the mice were considered to have reached the study endpoint. The dimensions of the bilateral tumors were measured and recorded every other day for 14 days.

[0094] After the experiment, histopathological examinations were performed on the tumor tissues. Hematoxylin and eosin (H&E) staining was used to observe the morphological changes of tumor cells and judge cell necrosis; TdT-mediated dUTP nick end labeling (TUNEL) staining was used to detect the apoptosis of tumor cells, and the number of apoptotic cells was observed through a fluorescence microscope; immunofluorescence analysis was carried out, and an antibody against HIF -1 A was used to detect the expression of HIF -1 A to evaluate the improvement of tumor hypoxia; the ROS level was detected using the DCFH-DA fluorescent probe to verify the POD-like activity of GMCN@PEG, and comprehensively evaluate the effect of GMCN@PEG on enhancing the radiotherapy effect of primary tumor-bearing mice and the role of enhancing radioimmunotherapy in vivo.

[0095] A 4T1 bilateral tumor mouse model was established by sequentially injecting 4T1 tumor cells into the right and left abdominal regions of the mice. The left abdominal region served as the model for distant metastasis. During the 14-day observation period, the tumor progression and body weight changes of the mice were closely monitored. Histological analysis of the metastatic lesions was performed, and H&E and TUNEL staining were used to observe the cell morphology and apoptosis in the metastatic lesions; immunofluorescence and flow cytometry were performed to analyze the number and activity of CD8 + T cells in the metastatic lesions and evaluate the anti-tumor immune activation in the metastatic sites; the survival time of the mice was recorded through survival studies, and survival curves were plotted; ELISA experiments were performed to detect the level of IFNγ in the tumors, and the inhibitory effect of GMCN@PEG on metastatic tumors was comprehensively judged.

[0096] (5) Imaging performance test of GMCN@PEG

[0097] Aqueous solutions of GMCN@PEG with different [Mn] concentrations and sample concentrations were prepared, and T1-weighted magnetic resonance imaging and CT imaging were performed under acidic (pH 6.0) and neutral (pH 7.4) conditions, respectively. The effect of [Mn] concentration on the T1 signal and the effect of sample concentration on the CT value were analyzed to evaluate the imaging effect of GMCN@PEG under different conditions.

[0098] (6) Statistical analysis

[0099] All graphs and statistical analyses were performed using GraphPad Prism 9.3 and OriginLab Origin 2022 software. Unpaired t-tests were used for comparison of differences between two groups, and one-way analysis of variance (one-way ANOVA) was used for comparison of differences among multiple groups. The values obtained from the analysis were expressed as mean ± standard deviation (SD). P < 0.05 was considered statistically significant.

[0100] 3. Results analysis

[0101] 3.1 Characterization of GMCN@PEG

[0102] Figure 1A And Figure 1B respectively show scanning electron microscopy (SEM) and transmission electron microscopy (TEM) images, clearly showing that GMCN has a uniformly dispersed spherical morphology and an obvious core-shell structure. The core is composed of gold nanoparticles, surrounded by a manganese dioxide shell layer with a thickness of approximately 15 nanometers. Energy-dispersive X-ray spectroscopy (EDS) mapping of GMCN further confirmed its core-shell structure ( Figures 1C - 1D ).

[0103] X-ray photoelectron spectroscopy (XPS)-derived spectra of GMCN were obtained. The full-range scan of the XPS spectrum showed the presence of Au, Mn, and O elements in GMCN (Figure 1E ). Figures 1F - 1H The high-resolution spectra of Au4f, Mn2p, and O1s are shown. The two peaks of the Au4f spectrum are located at 84.06 eV and 87.73 eV, corresponding to Au4f5 / 2 and Au4f7 / 2 ( Figure 1F ). Mn2p also exhibits two distinct peaks, located at 641.96 eV and 653.72 eV, which are attributed to the Mn2p1 / 2 and Mn2p3 / 2 spin-orbit ( Figure 1G ). The positions of these peaks are consistent with the characteristic peak positions of MnO2, indicating that the Mn in GMCN is MnO2. In addition, the O1s spectrum was detected, revealing three peaks ( Figure 1H ). The main peak located at 521.97 eV belongs to the lattice oxygen bound to Mn. The shoulder peak observed at 531.45 eV may be due to surface-adsorbed oxygen species, while the weakest peak at a higher binding energy of 532.70 eV may be attributed to the O in adsorbed H2O 2- .

[0104] The ultraviolet-visible-near-infrared spectrum shows that GMCN has a characteristic absorption peak at 577 nm, which can be attributed to the redshift peak of GNP in the formed GMCN ( Figure 1I ). In addition, zeta potential measurements found that after the MnO2 coating process, the surface charge of GNP changed from negative to positive, thus forming GMCN ( Figure 1J ). These values highlight the excellent physical stability and dispersibility of the synthesized material. The increase in particle size, the change in ultraviolet-visible-near-infrared spectral properties, and the decrease in zeta potential together constitute strong evidence for the successful synthesis of GMCN.

[0105] 3.2 Performance Detection

[0106] (1) Enzyme-like activity test

[0107] The peroxidase-like activity of GMCN was evaluated using the TMB colorimetric method. As Figure 2A shown, as the concentration of GMCN increases, the color of the blue solution deepens significantly, which is correlated with the increase in absorbance at 652 nm. Given that tumor tissues and cells are usually more acidic than normal tissues, the peroxidase-like activity of GMCN under different pH conditions was analyzed, and the results are as Figure 2B shown. GMCN exhibits moderate peroxidase-like activity under weak alkaline conditions (pH 7.4), while its enzyme activity increases significantly under weak acidic conditions (pH 6.0).

[0108] In addition, the detection results of electron spin resonance (SER) are as Figure 2CAs shown, under weakly acidic conditions (pH 6.0), the electron spin resonance (ESR) signal intensity of ·OH (a quartet signal of 1:2:2:1) is stronger than that under weakly basic conditions (pH 7.4), indicating that the peroxidase-like activity of gold-manganese composite nanocages (GMCN) is pH-dependent. In addition, steady-state catalytic kinetic analysis was performed to systematically evaluate the peroxidase-like catalytic performance of gold-manganese composite nanocages (GMCN) ( Figure 2D and Figure 2E ). Subsequently, the initial reaction rate (v0) was calculated according to the Beer-Lambert law, and V max (the maximum reaction rate when the enzyme is saturated with the substrate) and K m (the affinity of the enzyme for its substrate) values were determined as described above. It was found that the V max and K m of the peroxidase-like activity of gold-manganese composite nanocages (GMCN) were 2.88×10 -8 Ms -1 and 520.77 mM ( Figure 2D ), respectively.

[0109] In addition, a dissolved oxygen meter was used to detect the generation of O2. As Figure 2F shown, the catalytic efficiency of GMCN showed an obvious concentration dependence. Further steady-state kinetic analysis. After adding gold-manganese composite nanocages (GMCN, 20 μg mL -1 ) and different concentrations of H2O2 to an acidic phosphate buffer solution (PBS, pH 6.0), the time dependence, H2O2 concentration dependence of the reaction, and the amount of O2 generated were recorded. The initial reaction rate (v0) at different H2O2 concentrations was evaluated ( Figure 2G ). Subsequently, the reaction rate was plotted against the corresponding H2O2 concentration and fitted using a Michaelis saturation curve ( Figure 2H and Figure 2I ). After calculation, the V max and K m of gold-manganese composite nanocages (GMCN) were 4.85 mg L -1 min -1 and 45.36 mM ( Figure 2H ), respectively.

[0110] (2) In vitro radiotherapy sensitization effect test

[0111] It can be clearly seen from the bar chart of the cell viability detection results in Figures 3A - 3D that when the pH value is 6.0 and the concentration of GMCN@PEG is appropriate, the cell viability decreases most significantly, indicating that the radiotherapy sensitization effect is the best at this time. At the same time, the calcein-AM / PI staining method was used to observe the cell survival situation through a fluorescence microscope, and apoptotic cells were labeled with red fluorescence to visually evaluate the cell apoptosis situation.

[0112] In addition, Figure 3E The cell images under the fluorescence microscope clearly showed the apoptosis of cells in different treatment groups, further verifying the radiotherapy sensitization effect of GMCN@PEG.

[0113] Using the colony formation assay, cells were irradiated with different doses of X-rays. After culturing for a certain period of time, the number of cell colonies formed was observed and counted to further verify the radiosensitization characteristics of the cells. From Figure 3F the results of the colony formation assay, it can be seen that as the concentration of GMCN@PEG increased, the number of cell colonies formed decreased, indicating that GMCN@PEG enhanced the killing effect of radiotherapy on cells.

[0114] Using fluorescence imaging technology, DNA damage sites were labeled with γ-H2AX antibody, and the ROS level was detected with DCFH-DA probe to evaluate the changes in DNA damage and ROS level after radiotherapy. Figure 3H The fluorescence imaging in showed that in the cells treated with radiotherapy in the presence of GMCN@PEG, the DNA damage sites labeled with γ-H2AX antibody increased, and the ROS level detected by DCFH-DA probe increased significantly, proving that GMCN@PEG promoted DNA damage and ROS production.

[0115] The oxygen-sensitive fluorescence probe [Ru(dpp)3]Cl2 was used to detect the ability of GMCN@PEG to relieve tumor hypoxia. By observing the change in the fluorescence intensity of the probe, the change in the oxygen content in the tumor microenvironment was judged. From Figure 3G the fluorescence intensity change curve, it can be seen that GMCN@PEG can effectively relieve tumor hypoxia, creating more favorable conditions for radiotherapy.

[0116] (3)In vitro test for enhancing the effect of radioimmunotherapy

[0117] As Figure 4A shown, the CRT expression in the IR+GMCN@PEG group was significantly higher than that in other groups, indicating that GMCN@PEG combined with radiotherapy can significantly induce ICD. From Figure 4B the column chart of HMGB1 concentration comparison, it can be seen that the HMGB1 concentration in the IR+GMCN@PEG group increased significantly, further confirming the enhancing effect of GMCN@PEG on ICD. Figures 4C - 4D The column chart of IFNβ secretion in showed that the IFNβ secretion in the GMCN@PEG combined with radiotherapy group was significantly higher than that in other groups, indicating that GMCN@PEG can effectively activate the STING signaling pathway. From Figures 4E - 4F the results of flow cytometry detection, it can be seen that the CD 80 and CD 86The proportion of positive cells increased significantly, indicating that this combined treatment promoted the maturation of DCs and the M1 polarization of macrophages, effectively regulating the immune microenvironment.

[0118] (4)In vivo performance testing

[0119] As Figures 5A - 5B shown by the hematological and biochemical analysis data, after injecting GMCN@PEG, all indicators fluctuated within the normal range, indicating that GMCN@PEG had no obvious adverse effects on the blood system and liver and kidney functions of mice. Histological examinations were performed on the main organs (heart, liver, spleen, lung, kidney, etc.). The organs were made into tissue sections, stained with H&E, and the tissue sections were observed under a microscope to check for abnormal signs such as inflammation, bleeding, and necrosis.

[0120] From Figure 5C the tissue section images, it can be seen that the organizational structures of the main organs were normal and no obvious abnormalities occurred. An incubation experiment of nanoparticles with mouse red blood cells was carried out. GMCN@PEG was incubated with mouse red blood cells under suitable conditions for 12 hours, the hemoglobin content in the supernatant was detected, the hemolysis situation was evaluated, and its blood compatibility was judged.

[0121] Figure 5D As shown by the hemolysis experiment results in

[0122] GMCN@PEG was injected through the tail vein. Mice were sacrificed at different time points, and the main organs (liver, kidney, spleen, lung, heart, etc.) were collected. ICP-OES was used to quantitatively analyze the gold content in each organ to study the biodistribution of GMCN@PEG in the main organs. From Figure 5E the column chart of gold content distribution, it can be seen that GMCN@PEG was distributed more in the liver and kidney, providing a basis for further studying its metabolic and excretion pathways.

[0123] A two-compartment model was used to simulate the circulation of GMCN@PEG in the blood. By analyzing the data of the change in GMCN@PEG concentration in blood samples over time, pharmacokinetic parameters such as the circulation half-life and elimination rate constant were calculated, as Figures 5F - 5G .

[0124] The schematic diagram of in vivo treatment of primary 4T1 tumors is as Figure 6A shown. From Figures 6B - 6E the tumor growth curve, it can be seen that the tumor growth in the IR+GMCN@PEG group was significantly inhibited and the volume increased slowly, indicating that GMCN@PEG combined with radiotherapy could effectively inhibit the growth of primary tumors. Figure 6FThe histopathological images therein showed the necrosis and apoptosis of tumor cells. The immunofluorescence analysis images showed a decrease in HIF-1A expression, and the DCFH-DA fluorescent probe detected an increase in ROS levels, fully demonstrating the therapeutic effect of GMCN@PEG in vivo.

[0125] From Figures 7A - 7G the histological images of metastatic foci, the detection results of immunofluorescence and flow cytometry, the survival curves, and the IFNγ level data, it can be seen that GMCN@PEG combined with radiotherapy significantly inhibited the growth of metastatic tumors, prolonged the survival time of mice, and activated the anti-tumor immune response at the metastatic sites.

[0126] (5)Imaging performance test of GMCN@PEG

[0127] As Figures 8A - 8H shown, under acidic conditions (pH = 6.0), as the [Mn] concentration increased, the T1-weighted magnetic resonance imaging signal increased significantly; at the same time, when the sample concentration increased, the CT value also increased accordingly, and the image became clearer. By intravenously injecting GMCN@PEG into tumor-bearing mice, at different time points before and after injection (such as 0 h, 0.5 h, 1 h, 2 h, 24 h, etc.), T1-weighted magnetic resonance imaging and CT images of the tumor site were collected using a magnetic resonance imaging instrument and a CT scanner. Observe the change in the signal intensity of the tumor area, analyze the signal enhancement effect and the signal decay time, determine the imaging performance of GMCN@PEG in vivo, and verify its MR-CT dual-modal imaging ability.

[0128] From Figures 8A - 8H the imaging diagrams at different time points, it can be seen that after injecting GMCN@PEG, the signal intensity in the tumor area increased significantly in both T1-weighted magnetic resonance imaging and CT imaging, and remained at a high level for a certain period of time, demonstrating that GMCN@PEG has good MR-CT dual-modal imaging ability and can provide clear image information for tumor diagnosis.

[0129] In this invention, gold@manganese dioxide shell nanoparticles modified with polyethylene glycol, GMCN@PEG, were successfully prepared. It has good biocompatibility under neutral physiological conditions and exhibits various excellent properties in the acidic tumor microenvironment. GMCN@PEG can effectively relieve tumor hypoxia, increase ROS production, induce ICD, activate the cGAS-STING pathway, enhance the effect of radioimmunotherapy, and at the same time has an MR-CT dual-modal imaging enhancement function, realizing the combination of precise diagnosis and efficient treatment of tumors. The in vitro and in vivo experimental results fully verified the great potential of GMCN@PEG in the field of tumor treatment, and it is expected to provide an innovative and effective treatment method and diagnostic tool for clinical tumor treatment.

[0130] The above are only specific embodiments of the present application, enabling those skilled in the art to understand or implement the present application. Various modifications to these embodiments will be obvious to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application will not be limited to these embodiments shown herein, but rather will be accorded the widest scope consistent with the principles and novel features claimed herein.

Claims

1. Polyethylene glycol-modified gold@manganese dioxide nanoparticles, characterized in that, Comprising: A gold nanoparticle core with a diameter of 10 - 50 nm; A manganese dioxide shell with a thickness of 10 - 20 nm; A polyethylene glycol modification layer with a molecular weight of 1000 - 5000 Da; Among them, the polyethylene glycol-modified gold@manganese dioxide nanoparticles decompose in response to the acidic tumor microenvironment with a pH of 5.5 - 6.5, releasing Mn 2+ and exposing the gold core, achieving the integration of diagnosis and treatment through the following functions: (a) Alleviating tumor hypoxia and increasing ROS generation through the peroxidase - like and catalase - like activities of MnO2; (b) Inducing immunogenic cell death by activating the cGAS - STING signaling pathway, promoting dendritic cell maturation, macrophage M1 polarization, and T cell infiltration; (c) By released Mn 2+ Enhance the T1-weighted magnetic resonance imaging signal and enhance the CT imaging contrast by the gold core.

2. The polyethylene glycol-modified gold@manganese dioxide nanoparticles according to claim 1, wherein: The thickness of the manganese dioxide shell is 15 ± 2 nm; The mass ratio of the gold core to the manganese dioxide shell is 1:1.5 - 1:3; The grafting density of the PEG modification layer is 0.5 - 2 PEG molecules / nm 2 .

3. The polyethylene glycol-modified gold@manganese dioxide nanoparticles according to claim 1, characterized in that: Under acidic conditions at pH 6.0, the T1 longitudinal relaxation rate of the polyethylene glycol-modified gold@manganese dioxide nanoparticles is 5.0 - 6.5 mM -1 s -1 ; under neutral conditions at pH 7.4, the T1 longitudinal relaxation rate is 0.5 - 1.0 mM -1 s -1 ; the CT value is increased by 20% - 30% compared to the bare gold core at the same concentration.

4. A method for preparing the polyethylene glycol - modified Au@MnO2 nanoparticles according to any one of claims 1 - 3, comprising the following steps: (1) Synthesizing gold nanoparticles by the polyol reduction method, controlling the reaction temperature at 200 - 220 °C and the reaction time at 1 - 3 hours; (2) Incubating GNPs with a 5 - 20 mM citric acid solution for 10 - 14 hours, and removing the unadsorbed citric acid by centrifugation; (3) In the presence of 0.05 - 0.2 M potassium oxalate, reducing and depositing 0.01 - 0.05 M potassium permanganate on the surface of GNPs to form a manganese dioxide shell, with a reaction temperature of 50 - 70 °C and a reaction time of 1 - 3 hours; (4) Conjugating the product of step (3) with PEG having a molecular weight of 1000 - 5000 Da, and monitoring the changes in particle size and Zeta potential by dynamic light scattering until the particle size is stable at 80 - 120 nm and the Zeta potential is - 15 to - 30 mV.

5. An application of the polyethylene glycol - modified Au@MnO2 nanoparticles according to any one of claims 1 - 3 in the preparation of a radiosensitizer, characterized in that: The polyethylene glycol - modified Au@MnO2 nanoparticles enhance the radiotherapy efficacy through the following mechanisms in the tumor microenvironment: (a) By catalyzing the decomposition of H2O2 into O2, increasing the oxygen partial pressure in tumor tissues by 40% - 60%; (b) Generating hydroxyl radicals through peroxidase - like activity, increasing the intracellular ROS level by 3 - 5 times; (c) Through the radiation energy deposition effect of the gold core, increasing the DNA double - strand break rate in tumor cells by 2 - 4 times.

6. The application according to claim 5, wherein: The polyethylene glycol - modified Au@MnO2 nanoparticles activate anti - tumor immunity through the following pathways: (a) Induce the exposure of calreticulin on the surface of tumor cells and the release of HMGB1, promote the maturation of dendritic cells, and the proportion of CD 80 CD 86 cells ≥ 80%; (b) By releasing Mn 2+ Activates the cGAS-STING pathway, increasing the secretion of IFN-β by 5-10 times; (c) Promote the infiltration of CD8 + T cells, increasing the proportion to 30%-50% and inhibiting the proportion of regulatory T cells to ≤5%.

7. An application of the polyethylene glycol - modified Au@MnO2 nanoparticles according to any one of claims 1 - 3 in a radioimmunotherapy kit, characterized in that: At the tumor site, the MRI signal intensity of the polyethylene glycol - modified Au@MnO2 nanoparticles is 2 - 3 times higher than that of normal tissues, the CT value is 20% - 30% higher than that of the bare gold core, and the imaging contrast remains stable within 24 hours after injection.

8. The polyethylene glycol-modified gold@manganese dioxide nanoparticles according to any one of claims 1-3, characterized in that: After intravenous injection, its blood half - life is 6 - 8 hours, the tumor - targeting accumulation amount is ≥ 15% ID / g, mainly metabolized through the kidneys, and the total clearance rate is ≥ 90% within 72 hours.

9. The polyethylene glycol-modified gold@manganese dioxide nanoparticles according to any one of claims 1-3, characterized in that: When the dose is ≤ 20 mg / kg, it has no significant effect on hematological parameters and liver and kidney function indicators, and the hemolysis rate is ≤ 5%.

10. A radioimmunotherapy kit comprising the polyethylene glycol-modified gold@manganese dioxide nanoparticles according to any one of claims 1-3, characterized in that: The kit further includes a radiotherapy dose guidance module, which recommends a single X-ray irradiation dose of 2-10 Gy, a total dose ≤ 30 Gy, and the time interval between GMCN@PEG injection and radiotherapy is 12-24 hours.

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