Nuclide labeled cell membrane coated nano material as well as preparation method and application thereof

Abstract

The invention discloses a nuclide labeled cell membrane coated nano material as well as a preparation method and application thereof. The nuclide-labeled cell membrane-coated nano material is obtained by loading beta nuclide on a manganese-based monatomic nano enzyme coated with a tumor cell membrane, and the manganese-based monatomic nano enzyme has hollow mesopores. The invention also discloses an application of the nuclide labeled cell membrane coated nano material in preparation of a drug targeting carrier or a tumor treatment drug. The nuclide-labeled cell membrane-coated nano-material can be efficiently targeted to a tumor site, has self-cascade catalytic enzyme-like activity, and can effectively exert an anti-tumor effect.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and in particular relates to a radionuclide-labeled cell membrane-coated nanomaterial, a preparation method thereof, and an application thereof. Background Art

[0002] Tumor immunotherapy is considered to be one of the most successful methods in the treatment of malignant tumors in recent years. Despite its rapid development, the treatment response rate is not high. How to overcome the therapeutic resistance of immunotherapy and further improve its efficacy is the current challenge facing the clinic. With the in-depth exploration of radiation biology, ionizing radiation has been revealed to exert anti-tumor effects through immune pathways, which is of great significance for enhancing immunotherapy. However, the effect of internal radionuclide irradiation therapy (Targeted Radionuclide Therapy, TRT) is usually limited by endogenous / exogenous factors, such as cell apoptosis resistance, hypoxia / immunosuppressive tumor microenvironment, etc. Therefore, TRT is usually combined with other therapies, such as chemotherapy, immunotherapy, etc., to improve tumor radiosensitivity. The development of precise radionuclide carriers to ensure tumor targeting and maximize the synergistic tumor treatment effect of radiation is of vital importance for radionuclide-based internal irradiation therapy.

[0003] Nanoparticle-based radionuclide targeted delivery systems have demonstrated unique advantages, such as good biocompatibility, ease of functionalization with targeting molecules, and high stability, low immunogenicity, rapid clearance, and minimal nonspecific binding to other tissues. These nanomaterials passively target tumors through the inherent enhanced permeability and retention (EPR) effect of the tumor vasculature. In recent years, cell membrane-inspired nanomaterials have gradually demonstrated their application advantages due to their excellent biocompatibility and low immunogenicity. They can avoid capture by the reticuloendothelial system / mononuclear phagocyte system, achieving long-term circulation in the body and passive targeting to tumor sites. For example, the surface of tumor cell membranes contains highly specific isotypes that can be used to identify their own species, facilitating the enrichment of homologous tumor cells in tumor tissues and achieving active targeted drug delivery. Therefore, using tumor cell membrane-coated nanocarriers as radionuclide targeting carriers can achieve precise and safe tumor targeting through the dual effects of "enhanced permeability and retention effect + homologous targeting effect."

[0004] In recent years, various single-atom nanozymes have been designed with metalNx active sites similar to those of natural metalloenzymes. Manganese, as an essential element in biology, plays a key role in regulating redox reactions due to its multivalent and high-spin properties. Manganese is a key cofactor for multiple metalloenzymes, playing an important regulatory role in various metalloenzymes such as Mn superoxide dismutase (MnSOD), glutamine synthetase (GS), pyruvate carboxylase, and glutathione S-transferase, thereby affecting the metabolism and redox homeostasis of life. Based on this property, there have been many reports on Mn single-atom nanozymes. For example, Zhu et al. constructed a PEGylated Mn-based SAE, which exhibited significant therapeutic efficacy through the production of multiple ROS and photothermal activity stimulated by the tumor microenvironment (TME). However, whether β-rays can synergize with Mn-based single-atom nanozymes to achieve efficient anti-tumor effects remains unknown. Summary of the Invention

[0005] In order to solve the deficiencies in the prior art, the present invention aims to provide a radionuclide labeled cell membrane coated nanomaterial and its preparation method and application. The present invention adopts a biomimetic carbonization method to construct a Mn / SAE nanomaterial, and then coats the tumor cell membrane on its surface to obtain a biomimetic nanomaterial Mn / SAE@M, and then uses a chloramine T labeling method to label the 131 I is connected with the nanomaterial Mn / SAE@M to obtain 131 I-Mn / SAE@M. achieves this through homologous active targeting and EPR passive targeting effects. 131 Precise targeting and treatment of I. This invention innovatively synthesizes a new type of radioactive drug with tumor targeting ability, which can stably load 131 I and precisely target the tumor site.

[0006] The specific technical solutions are as follows:

[0007] The present invention provides a radionuclide-labeled cell membrane-coated nanomaterial, which is obtained by loading β-nuclide on a manganese-based single-atom nanozyme wrapped in a tumor cell membrane, and the manganese-based single-atom nanozyme has hollow mesopores.

[0008] Furthermore, the manganese-based single-atom nanozyme has a hollow nanocube structure.

[0009] Furthermore, the size of the manganese-based single-atom nanozyme is 100-200 nm.

[0010] Furthermore, the tumor cell membrane is a non-small cell lung cancer cell membrane.

[0011] Furthermore, the β nuclide is 131 I.

[0012] The present invention also provides a method for preparing the radionuclide-labeled cell membrane-coated nanomaterial, comprising the following steps:

[0013] (1) ZIF-8 nanocubes were etched with tannic acid solution to produce hollow structures, and ZIF-8 nanoparticles with hollow mesopores were obtained. Then, Mn 2+ absorption to obtain Mn-ZIF-8HNC;

[0014] (2) Under argon protection, Mn-ZIF-8HNC was pyrolyzed at 900 °C to obtain manganese-based single-atom nanozymes;

[0015] (3) The tumor cell membrane was wrapped on the manganese-based single-atom nanozyme to obtain the nanomaterial Mn / SAE@M;

[0016] (4) The chloramine T labeling method is used to load β-nuclides on the nanomaterial Mn / SAE@M, thereby obtaining the nuclide-labeled cell membrane-coated nanomaterial.

[0017] Furthermore, the step of preparing the Mn-ZIF-8HNC in step (1) includes: dissolving Zn(NO3)2·6H2O and CTAB in water, and then adding them to water containing 2-methylimidazole under vigorous stirring at room temperature to obtain ZIF-8 nanocubes; centrifuging the obtained precipitate, washing it with water, and vacuum drying it; then dispersing the ZIF-8 nanocubes in water under ultrasound at room temperature; after forming a uniform dispersion, adding a tannic acid aqueous solution to the mixture, and then stirring it at room temperature to obtain;

[0018] Preferably, 20 mg of ZIF-8 nanocubes are dispersed in 3 mL of water, the concentration of the tannic acid aqueous solution is 25 mg / mL, the amount of the tannic acid aqueous solution is 3 mL, and the tannic acid aqueous solution is added to the mixture and stirred at room temperature for 2 h.

[0019] Furthermore, in step (2), Mn-ZIF-8HNC was pyrolyzed at 900 °C for 2 h;

[0020] In step (3), the tumor cell membrane is wrapped in the manganese-based single-atom nanozyme using the membrane extrusion method.

[0021] The present invention also provides the use of the radionuclide-labeled cell membrane-coated nanomaterial in the preparation of drug targeting carriers or tumor therapeutic drugs.

[0022] The present invention also provides a pharmaceutical composition comprising the radionuclide-labeled cell membrane-coated nanomaterial.

[0023] The beneficial effects of the present invention are:

[0024] The present invention synthesizes manganese-based single-atom nanozyme (Mn / SAE) nanoparticles based on ZIF-8 nanoparticles with hollow mesopores, then coats them with tumor cell membranes and loads β-nuclides ( 131 I) to construct nanomaterials 131 I-Mn / SAE@M. This nanomaterial can be efficiently targeted to the tumor site and has self-cascading catalytic enzyme-like activity, which can effectively exert anti-tumor effects. 131 I-Mn / SAE@M can play the role of β-nuclides 131 I also exhibits catalase and oxidase activities to alleviate the hypoxic conditions in the TME, thereby catalyzing the production of excessive ROS. 131 I-Mn / SAE@M can also interfere with the lipid metabolism homeostasis of tumor cells, induce immunogenic ferroptosis, and then exert a strong anti-tumor immune effect. The results show that Mn / SAE is a potential TRT carrier that can synergize with β-nuclides to improve the efficacy of tumor treatment. 131 I-Mn / SAE@M is a new type of TME-responsive radionuclide drug. Specifically:

[0025] 1) 131 I is easily accessible, has a purer decay chain, and can decay to release beta particles for treatment and gamma particles for imaging. The energy generated by this "high dose rate" beta nuclide decay can be rapidly deposited into the tumor, and the off-target toxicity caused by daughter nuclide recoil and daughter nuclide redistribution is much smaller. In addition, 131 The gamma rays released during the decay of I can be detected by SPECT / CT, which is expected to achieve dynamic imaging in the body and has great potential in the integration of diagnosis and treatment.

[0026] 2) 131 I-MnSAE@M improves anti-tumor immunity by simulating cascade reactions through enzymes. Enzyme property tests show that 131 I-Mn / SAE@M has catalytic activities similar to catalase (CAT), oxidase (OXD), peroxidase (POD), and GSH oxidase (GSHOx), which can reprogram tumor lipid metabolism through cascade catalytic reactions and further elucidate that it can activate a potent anti-tumor immune response. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 a) Mn / SAE@M and 131Schematic diagram of the formation of I-Mn / SAE@M. b) Scanning electron microscope image of Mn / SAE. c) TEM image of Mn / SAE. d) HAADF STEM image of Mn / SAE, showing dispersed single iron atoms as bright spots (indicated by red circles). e) Atomic mapping image of C, Mn, and O in Mn / SAE. f) XRD pattern of Mn / SAE. g) XPS spectrum of Mn / SAE. h) XPS spectrum of Mn2p. i) ESR spectrum of Mn / SAE@M. j) SDS-PAGE gel analysis of the protein content of Mn / SAE nanoparticles, LLC cell membranes, and Mn / SAE@M nanoparticles. k) TEM image of Mn / SAE@M. l) 131 Radiolabeled stability analysis of I-Mn / SAE@M after incubation in PBS and FBS (37°C) for 4 h, 12 h, 24 h, and 48 h.

[0028] Figure 2 a) Schematic diagram of multi-enzyme biocatalytic process simulation. b) 131 The dissolved oxygen level of I-Mn / SAE@M in H2O2 solution was used to monitor the CAT mimetic activity. c) The absorbance of DPBF at 420 nm was bleached by H2O2 (2 mM) to measure the -O2- generation efficiency. d) TMB assay was used to determine the 131 I-Mn / SAE@M oxidase-like activity at different Mn / SAE concentrations (1.0 mM TMB, pH = 5.0, 2.5 mM H2O2). e) TMB assay at different TMB concentrations (100 μg / mL, Mn / SAE, 2.5 mM H2O2, pH = 5.0). 131 POD-like activity of I-Mn / SAE@M. f) 5,5'-Dithiobis-(2-nitrobenzoic acid) (DTNB) and 131 UV-visible spectra after co-incubation of I-Mn / SAE@M were used to evaluate GSHox-like activity. g) Intracellular Mn accumulation in LLC cells at different time points after treatment with Mn / SAE or Mn / SAE@M was measured by ICP-MS. h) CLSM was used to observe the intracellular Mn accumulation in LLC cells at different time points. 131 I-Mn / SAE@M absorption. i, j) Fluorescence images and corresponding mean fluorescence intensity (MFI) of ROS in LLC cells stained with DCFH-DA after different treatments. k, l) Levels of GSH and GSSG in LLC cells after different treatments. m) Viability of LLC cells treated with Mn / SAE in the absence or presence of H2O2 (2.5 mM). n) Cell counting kit-8 (CCK-8) assay. 131 I, Mn / SAE, 131Cytotoxic effects of I-Mn / SAE@M gradient activity on LLC cells for 24 h (n = 4 per group). o,p) Flow cytometry diagram (o) and data (p) of LLC cells stained with propidium iodide (PI) and annexin V-FITC after different treatments to determine the level of cell apoptosis (n = 3).

[0029] Figure 3 .a) Intravenous injection at different time points 131 Representative SPECT / CT imaging of LLC tumor-bearing mice after i) i.v. injection at different time points 131 Representative SPECT / CT images of LLC tumor-bearing mice after I-Mn / SAE@M. c) Schematic diagram of the in vivo antitumor process. d) Representative images of resected tumors from mice in the corresponding treatment groups after 12 days. e) Tumor weights after different treatments on day 12. fj) Individual (fi) and average (j) tumor growth curves of LLC tumor-bearing mice in the corresponding treatment groups (n=6 per group). k) Body weight change curves of LLC tumor-bearing mice in the corresponding treatment groups (n=6 per group). l) Representative light microscopy images of TUNEL-stained tumor sections from the corresponding treatment groups.

[0030] Figure 4 a,b) Representative flow cytometric profiles and statistical data of DC maturation in spleens from different groups. c,d) Representative flow cytometric profiles and statistical data of DC maturation in tumors from different groups. e,f) Representative flow cytometric profiles and statistical data of CD4+ T cells in tumors from different groups. g,h) Representative flow cytometric profiles and statistical data of CD8+ T cells in tumors from different groups. i,j) Representative flow cytometric profiles and statistical data of Tregs in tumors from different groups.

[0031] Figure 5 a) Representative photographs of primary and distant tumors resected 16 days after treatment in mice of the corresponding treatment groups (n=5 per group). b) Body weight change curves of LLC tumor-bearing mice of the corresponding treatment groups (n=5 per group). cg) Average (c) and individual (dg) tumor growth curves of LLC tumor-bearing mice of the corresponding treatment groups (n=5 per group). (h, i) Flow cytometric analysis of splenic memory T cells in mice of the different treatment groups (n=3 per group). (j, k) Flow cytometric analysis of T cells in distant tumors of mice of the different treatment groups (n=3 per group). l) Serum cytokine expression levels (including TNF-α) in mice of the different treatment groups (n=3 per group). m) Serum cytokine expression levels (including IFN-γ) in mice of the different treatment groups (n=3 per group). n) Schematic diagram of the in vivo anti-tumor process in the bilateral tumor model. DETAILED DESCRIPTION

[0032] To more clearly understand the present invention, the present invention is further described with reference to the following examples and accompanying drawings. The examples are intended to illustrate the present invention only and are not intended to limit the present invention in any way. In the examples, all raw materials and reagents are commercially available. Experimental methods without specific conditions are conventional methods and conditions well known in the art, or according to the conditions recommended by the instrument manufacturer.

[0033] Example 1: 131 Synthesis of I-Mn / SAE@M

[0034] This embodiment provides a 131 I-Mn / SAE@M synthesis method, wherein Mn / SAE@M and 131 The formation diagram of I-Mn / SAE@M is shown in Figure 1 As shown in a, the following steps are included:

[0035] (1) ZIF-8 nanocubes (ZIF-8NCs) were etched with tannic acid (TA) solution to produce hollow structures, and ZIF-8 nanoparticles with hollow mesopores were obtained. Then, Mn 2+ Absorption to obtain Mn-ZIF-8HNC. Specifically, 160 mg of Zn(NO3)2·6H2O and 2 mg of CTAB were dissolved in 2 mL of water, and then added to 14 mL of water containing 0.908 g of 2-methylimidazole (MeIM) under vigorous stirring at room temperature for 2 hours to obtain ZIF-8NC. The resulting precipitate was centrifuged and washed three times with water and vacuum dried at 65°C overnight. Then, 20 mg of ZIF-8NC was dispersed in 3 mL of water under ultrasound at room temperature for 30 minutes. After forming a uniform dispersion, 2 mL of tannic acid aqueous solution (25 mg / mL) was added to the mixture, which was then stirred at room temperature for 2 hours. The obtained ZIF-8 hollow nanocubes (ZIF-8HNC) were centrifuged and vacuum dried at 65°C for 6 hours.

[0036] (2) Mn-ZIF-8HNC was pyrolyzed at 900 °C for 2 h under argon protection to obtain manganese-based single-atom nanozyme (Mn / SAE).

[0037] (3) The non-small cell lung cancer LLC cell membrane was extracted and wrapped on Mn / SAE using the membrane extrusion method to obtain the nanomaterial Mn / SAE@M.

[0038] (4) Using chloramine T labeling method to load nanomaterials Mn / SAE@M 131 I. Obtain radiopharmaceuticals 131 I-Mn / SAE@M.

[0039] 131The synthesis of I-Mn / SAE@M was analyzed as follows:

[0040] 1) The morphology of the synthesized Mn / SAE was characterized by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Figure 1 b and Figure 1 As shown in c, the image shows that the size of Mn / SAE ranges from 100 to 200 nm and presents a hollow nanocube structure.

[0041] 2) The morphology of the synthesized Mn / SAE was characterized by high-angle annular dark field scanning transmission electron microscopy (HAADF-STEM). Figure 1 As shown in d, the scattered individual iron atoms are shown as bright spots (indicated by red circles).

[0042] 3) XRD pattern of Mn / SAE Figure 1 As shown in Figure 5, the X-ray diffraction (XRD) pattern shows that there is a relatively obvious diffraction peak at 25.3°, which can be attributed to the (002) plane of graphitic carbon. The results show that Mn / SAE was successfully synthesized.

[0043] 4) X-ray photoelectron spectroscopy (XPS) of Mn / SAE Figure 1 As shown in g, the XPS spectrum of Mn2p is Figure 1 h. XPS analysis shows that the main components of Mn / SAE are C, N, O, and Mn. X-ray photoelectron spectroscopy revealed that the valence state of manganese was determined by the presence of Mn 2p3 / 2 (642.0 eV) between Mn 4+ (642.5 eV) and Mn 0 (640.9 eV), confirming the ionic Mnδ+ nature of manganese in Mn / SAE (0 < δ < 4). High-resolution analysis of the C 1s spectrum revealed peaks at 284.8 and 285.8 eV, corresponding to C─C and C─N, respectively.

[0044] 5) Energy dispersive spectroscopy (EDS) mapping to characterize the elemental distribution within the single-atom nanozyme, e.g. Figure 1 The results show that manganese (Mn), nitrogen (N), and carbon (C) are uniformly distributed throughout the prepared Mn / SAE, which provides further evidence for the successful synthesis of single-atom nanozymes.

[0045] 6) Based on the poorly immunogenic non-small cell lung cancer LLC, SDS-PAGE was used to identify proteins in Mn / SAE, LLC cell membranes, and Mn / SAE@M. Figure 1 As shown in j, the protein bands of LLC cell membrane and Mn / SAE@M appeared at the same position.

[0046] 7) The morphology of the synthesized Mn / SAE@M was characterized by transmission electron microscopy (TEM). Figure 1 As shown in k, TEM confirmed that the shape of Mn / SAE@M wrapped by tumor cell membrane was consistent with that of the prepared Mn / SAE.

[0047] 8) DLS data showed that the particle size of Mn / SAE@M after tumor cell membrane encapsulation was significantly smaller than that of Mn / SAE, and the Zeta potential was lower.

[0048] 9) 131 The radiolabeled stability analysis of I-Mn / SAE@M after incubation in PBS and FBS (37°C) for 4 h, 12 h, 24 h, and 48 h was as follows: Figure 1 l, loaded with β nuclide 131 I 131 I-Mn / SAE@M has a radiochemical purity of 95% and exhibits excellent radiolabel stability in PBS and FBS at 37°C. Results showed that the residual radiolabel stability reached 80% after 48 hours, which is sufficient for tumor killing.

[0049] Example 2: 131 Catalytic properties of I-Mn / SAE@M

[0050] The following experiments were performed to test the synthesis of Example 1 131 The catalytic properties of I-Mn / SAE@M and the schematic diagram of the multi-enzyme biocatalytic process are shown in Figure 2. Figure 2 As shown:

[0051] 1) Catalase (CAT) can catalyze the decomposition of hydrogen peroxide (H2O2) into water and oxygen. 131 The dissolved oxygen level of I-Mn / SAE@M in H2O2 solution was used to monitor CAT mimic activity. Similar to natural catalase, 131 I-Mn / SAE@M can effectively catalyze the conversion of H2O2 to O2, and the generation of oxygen was observed, confirming that 131 CAT activity of I-Mn / SAE@M.

[0052] 2) Oxidase (OXD) catalyzes the transfer of electrons to O2, thereby generating superoxide radicals. 3,3',5,5'-tetramethylbenzidine (TMB) was used as a colorless substrate to 131 The OXD-like activity of I-Mn / SAE@M was verified. 131 I-Mn / SAE@M can oxidize colorless TMB to generate blue oxide, which can be quantified by measuring the absorbance at 652 nm. 131The OXD-like activity of I-Mn / SAE@M was significantly enhanced with the increase of Mn / SAE concentration. 131 I-Mn / SAE@M catalyzed the massive production of superoxide radicals through OXD-like activity; at the same time, it also assisted in confirming that Mn / SAE produced oxygen through CAT-like activity.

[0053] 3) Peroxidase (POD) can generate superoxide radicals by decomposing H2O2 through catalysis of Fenton-like reaction. 131 The production of ·OH catalyzed by I-Mn / SAE@M. 131 Under the conditions of co-incubation of I-Mn / SAE@M and H2O2, colorless TMB was oxidized to produce blue oxide. The production of TMB oxide increased with the concentration of Mn / SAE, TMB and H2O2. 131 The POD-like activity of I-Mn / SAE@M was also enhanced at weakly acidic pH, indicating that the POD activity of Mn / SAE was more significant in the acidic tumor microenvironment.

[0054] 4) Validation at the cellular level 131 Antitumor effect of I-Mn / SAE@M. ICP-MS showed that the intracellular Mn content of Mn / SAE@M and Mn / SAE increased with time.

[0055] 5) By monitoring the red fluorescent dye Rhodamine B labeled 131 I-Mn / SAE@M was used to measure cellular uptake. Confocal microscopy showed 131 I-Mn / SAE@M can be internalized by cells in a time-dependent manner.

[0056] 6) To verify 131 The ability of I-Mn / SAE@M to generate ROS was detected by flow cytometry (FCM) using dichlorofluorescein diacetate (DCFH-DA) as a probe. After entering the cell, DCFH-DA can be hydrolyzed by esterase to produce DCFH. ROS oxidizes non-fluorescent DCFH to produce fluorescent DCF. The results show 131 I-Mn / SAE@M produced ROS at a level comparable to that of the single 131 I and Mn / SAE were significantly increased, which means that this new radionuclide drug has the strongest ability to induce ROS production through synergistic TRT and CDT strategies.

[0057] 7) Excessive production of ROS in cells leads to redox imbalance, which can induce cell damage and even apoptosis. 131Effect of I-Mn / SAE@M on the proliferation of LLC tumor cells. First, the anti-tumor effect of different concentrations of Mn / SAE was verified. The results showed that under neutral pH conditions, Mn / SAE alone or in combination with H2O2 had little effect on cell viability even at a higher concentration of 250 mg / mL. Under weak acid conditions (pH 6.0), cell proliferation was significantly reduced. At a concentration of 31.25 mg / mL, the cell proliferation under the action of Mn / SAE alone was 45%, and Mn / SAE plus H2O2 was reduced to 27%. This result is consistent with the enzyme assay and confirms 131 The carrier Mn / SAE of I has a higher catalytic effect in the acidic tumor microenvironment.

[0058] 8) For different concentrations 131 I, Mn / SAE and 131 The anti-tumor effect of I-Mn / SAE@M was verified. The results showed that even under neutral pH and H2O2-free conditions, 131 I-Mn / SAE@M exhibited significant anti-tumor effects compared to single 131I and Mn / SAE. Similarly, Calcein-AM and PI co-staining analysis of the cytotoxicity of different treatment groups on tumor cells also confirmed the above results.

[0059] 9) ATP is an energy molecule in cells. Tumor cells require a large amount of energy to support their abnormal proliferation and growth. 131 I, Mn / SAE and 131 After I-Mn / SAE@M treatment, the ATP (adenosine triphosphate) content in tumor cells was found to be 131 The cellular ATP content was significantly reduced after I-Mn / SAE@M treatment, suggesting that it can destroy the metabolic function of tumor cells, leading to reduced ATP synthesis, and then causing tumor cell apoptosis or cessation of proliferation.

[0060] 10) Annexin V-FITC and propidium iodide (PI) co-staining assay was used to distinguish the stages of apoptosis, including early and late apoptosis. 131 I-Mn / SAE@M treatment induced apoptosis in LLC cells mainly in the late stage, and the degree of apoptosis induction was significantly higher than that in other groups.

[0061] Example 3: 131 The potential of integrated diagnosis and treatment of I-Mn / SAE@M

[0062] 1) 131 I-Mn / SAE@M has great potential in in vivo tumor therapy. 131SPECT / CT imaging was used to investigate the tumor targeting ability of I-Mn / SAE@M and determine its peak accumulation time at the tumor site for precise diagnostic imaging and treatment guidance. 131 After I, most of the free 131 I rapidly escaped from the tumor site, indicating low tumor retention. In contrast, i.v. 131 After 24 hours of I-Mn / SAE@M, the drug mainly accumulated in the tumor site and could be maintained for 96 hours. 131 The successful accumulation of I-Mn / SAE@M in tumors and its potential for integrated diagnosis and treatment.

[0063] 2) Before evaluating the effect of tumor treatment, check 131 The biodistribution of I-Mn / SAE@M was revealed 131 The in vivo behavior of I-Mn / SAE@M showed that tumor accumulation increased significantly 24 hours after intravenous injection and remained at a high level 48 hours after injection. This result is consistent with the SPECT / CT results.

[0064] 3) Discussion 131 The anti-tumor effect of I-Mn / SAE@M in vivo. Specifically, we divided the mice into 4 groups and observed the control group, 131 I, Mn / SAE and 131 The therapeutic effect of I-Mn / SAE@M. The tumor growth curves of mice in each group showed that at the given dose, due to its rapid clearance or metabolism, free 131 I also failed to slow tumor growth. In addition, Mn / SAE failed to slow tumor progression. Interestingly, once 131 I combined with Mn / SAE to obtain 131 I-Mn / SAE@M can significantly inhibit tumor growth. Mice were sacrificed 12 days after administration, and their tumor tissues were completely dissected and weighed. It was observed that compared with the control group, 131 The tumor weights of mice treated with I-Mn / SAE@M were much lighter than those of mice in other groups.

[0065] 4) Mouse growth curve shows 131 I, Mn / SAE and 131 Treatment with I-Mn / SAE@M had no effect on the body weight of mice. In addition, hematoxylin and eosin (H&E) staining was performed on the main organs and tumor tissues after administration. No visible damage was observed in normal tissues, indicating that 131 I-Mn / SAE@M has good biocompatibility. Biochemical indicators of mice were also measured. 131 There was no significant difference between the I-Mn / SAE@M treated group and the normal mice. These results indicate that131 Biosafety of I-Mn / SAE@M for in vivo cancer therapy.

[0066] 5) Pathological examinations including hematoxylin and eosin (H&E) staining and terminal deoxynucleotidyl transferase dUTP nickel end labeling (TUNEL) assay were performed to explore cell apoptosis and / or necrosis. The results of H&E showed that the tumor cells in the single therapy (only Mn / SAE or only 131I) group retained a relatively intact structure. 131 In the I-Mn / SAE@M group, the tumors were severely necrotic, in which nuclear condensation and cell shrinkage were clearly identified. These results were further confirmed by TUNEL experiments, in which the combined therapy, i.e. 131 I-Mn / SAE@M led to the most apoptosis. Interestingly, we did immunohistochemical staining of GPX4 (glutathione peroxidase 4) on the tumor tissue and unexpectedly found 131 GPX4 expression in tumor tissues was significantly reduced after I-Mn / SAE@M treatment. GPX4 is a key enzyme that prevents lipid peroxidation and inhibits ferroptosis. Therefore, decreased GPX4 expression suggests that the cell's defense mechanism against ferroptosis is suppressed, potentially enhancing the sensitivity of tumor cells to radiotherapy.

[0067] The above results are as follows Figure 3 shown.

[0068] Example 4: 131 I-Mn / SAE@M enhances anti-tumor immunity

[0069] 1) Evaluation Application 131 Immune response in tumor tissue after I-Mn / SAE@M. After 7 days of treatment, tumor tissue and spleen were dissected for flow cytometry analysis. The results showed that 131 I-Mn / SAE@M can promote the maturation of dendritic cells (DCs) in the spleen.

[0070] 2) Sutra 131 DCs in the tumor microenvironment of I-Mn / SAE@M-treated mice showed higher expression of the DC maturation marker CD86.

[0071] 3) Given that DC maturation can mediate downstream immune responses by regulating T cell proliferation, we further evaluated the content of helper T cells (CD4+ T cells) and cytotoxic T lymphocytes (CD8+ T cells) in tumors. 131 The infiltration of CD4+ T cells and CD8+ T cells was significantly increased in I-Mn / SAE@M-treated tumors, while the expression of regulatory T cells (Tregs) was reduced.

[0072] 4) For further research 131 I-Mn / SAE@M produced immune memory to suppress distant tumors. The primary tumor was injected subcutaneously on the right dorsal side of the mouse, and then the drug was injected intravenously for 7 days. After treatment, the distant tumor was injected subcutaneously on the contralateral side for 3 days. The tumor growth and mouse weight were recorded every two days. After 16 days of treatment, the mouse was sacrificed and the tumor tissue was completely removed. 131 The primary tumors and distant tumors in the I-Mn / SAE@M treatment group were significantly smaller than those in the other groups.

[0073] 5) The body weight of mice increased steadily throughout the treatment period.

[0074] 6) The growth curves of primary tumors and distant tumors show that the control group and 131 The tumors in group I grew rapidly, while those in group Mn / SAE grew relatively slowly, but still less than those in group 131 I-Mn / SAE@M group, which shows 131 I-Mn / SAE@M has good therapeutic effect.

[0075] 7) The spleen was dissected and single cell suspension was prepared to detect the expression of immune memory T cells (TEM: CD3+CD8+CD44+CD62L-). The results showed that 131 The expression of TEM cells in mice treated with I-Mn / SAE@M was significantly increased, indicating the presence of immune memory in the treated mice. Similarly, dissection of distal tumor tissue revealed increased expression of CD8+ T cells in the tumor microenvironment.

[0076] In addition, blood was collected from the mouse orbits and compared with the other groups, 131 Increased expression of TNF-α and IFN-γ was detected in the I-Mn / SAE@M treated group. All these results indicate that 131 The I-Mn / SAE@M treatment group could significantly enhance the anti-tumor immune response of tumor cells.

[0077] The above results are as follows Figure 4 and 5 shown.

[0078] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will readily appreciate that other variations or modifications based on the above descriptions are possible. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A radionuclide-labeled cell membrane-coated nanomaterial, characterized in that: The method is obtained by loading β-nuclides on a manganese-based single-atom nanozyme wrapped in a tumor cell membrane, wherein the manganese-based single-atom nanozyme has hollow mesopores.

2. The radionuclide-labeled cell membrane-coated nanomaterial according to claim 1, characterized in that: The manganese-based single-atom nanozyme has a hollow nanocube structure.

3. The radionuclide-labeled cell membrane-coated nanomaterial according to claim 1, characterized in that: The size of the manganese-based single-atom nanozyme is 100-200 nm.

4. The radionuclide-labeled cell membrane-coated nanomaterial according to claim 1, characterized in that: The tumor cell membrane is a non-small cell lung cancer cell membrane.

5. The radionuclide-labeled cell membrane-coated nanomaterial according to claim 1, characterized in that: The β nuclide is 131 I.

6. A method for preparing the radionuclide-labeled cell membrane-coated nanomaterial according to claim 1, characterized in that: The steps include: (1) ZIF-8 nanocubes were etched with tannic acid solution to produce hollow structures, and ZIF-8 nanoparticles with hollow mesopores were obtained. Then, Mn 2+ absorption to obtain Mn-ZIF-8HNC; (2) Under argon protection, Mn-ZIF-8HNC was pyrolyzed at 900 °C to obtain manganese-based single-atom nanozymes; (3) The tumor cell membrane was wrapped on the manganese-based single-atom nanozyme to obtain the nanomaterial Mn / SAE@M; (4) The chloramine T labeling method is used to load β-nuclides on the nanomaterial Mn / SAE@M, thereby obtaining the nuclide-labeled cell membrane-coated nanomaterial described in claim 1.

7. The preparation method according to claim 6, characterized in that The steps of preparing the Mn-ZIF-8HNC in step (1) include: dissolving Zn(NO3)2·6H2O and CTAB in water, and then adding them to water containing 2-methylimidazole under vigorous stirring at room temperature to obtain ZIF-8 nanocubes; centrifuging the obtained precipitate, washing it with water, and vacuum drying it; then dispersing the ZIF-8 nanocubes in water under ultrasound at room temperature; after forming a uniform dispersion, adding a tannic acid aqueous solution to the mixture, and then stirring it at room temperature to obtain; Preferably, 20 mg of ZIF-8 nanocubes are dispersed in 3 mL of water, the concentration of the tannic acid aqueous solution is 25 mg / mL, the amount of the tannic acid aqueous solution is 3 mL, and the tannic acid aqueous solution is added to the mixture and stirred at room temperature for 2 h.

8. The preparation method according to claim 6, characterized in that In step (2), Mn-ZIF-8HNC was pyrolyzed at 900 °C for 2 h; In step (3), the tumor cell membrane is wrapped in the manganese-based single-atom nanozyme using the membrane extrusion method.

9. Use of the radionuclide-labeled cell membrane-coated nanomaterial according to claim 1 in the preparation of drug targeting carriers or tumor therapeutic drugs.

10. A pharmaceutical composition, characterized in that It comprises the radionuclide-labeled cell membrane-coated nanomaterial according to claim 1.

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