Ultrasonic sensitization nanometer material based on MOFs and preparation method and application of ultrasonic sensitization nanometer material

Abstract

The invention discloses an ultrasonic sensitized nano material based on MOFs (Metal-Organic Frameworks) as well as a preparation method and application thereof, and belongs to the technical field of biological materials. Comprising drug-loaded MOFs; the MOFs is selected from the group consisting of UiO-66-NH2; the UiO-66-NH2 is a particle formed by connecting an amino functional group to UiO-66; an m6A-mRNA (Messenger Ribonucleic Acid) demethylase inhibitor compound is loaded in the medicine carrying MOFs; the m6A-mRNA demethylase inhibitor compound is formed by coordination of a Rhein molecule and a bivalent copper ion; the outer surface of the drug-loaded MOFs is combined with an anti-CD19 antibody; and the outer surface of the drug-loaded MOFs is also modified with mPEG-PO3 (mPEG (Polyethylene Glycol)-PO3). As a replacement therapy of BCL, the MOFs-based ultrasonic sensitized nanomaterial can specifically gather in BCL tissues to play a therapeutic role after being functionalized by mPEG-PO3 and an anti-CD19 antibody.

Description

Technical Field

[0001] The present application relates to an ultrasound-sensitized nanomaterial based on MOFs and a preparation method and application thereof, belonging to the technical field of biomaterials. Background Art

[0002] B-cell lymphoma (BCL) is a highly heterogeneous hematological malignancy that is an aggressive proliferation of mature B cells. Currently, systemic chemotherapy, external beam radiotherapy, and immunotherapy are the main treatments for BCL. Radiotherapy is an effective treatment for patients with aggressive lymphoma, but there is uncertainty as a consolidation therapy after chemotherapy-immunotherapy, especially for advanced patients. In addition, radiotherapy can only target superficial and visible tumor tissues and cannot eliminate lymphocytes that have metastasized or infiltrated deep into the bone marrow. Immunotherapies, including antigen-specific monoclonal antibodies (such as anti-CD20, bispecific antibodies), immune checkpoint inhibitors (such as PD1 / PDL1 blockers), and chimeric antigen receptor (CAR) T cells, have been successfully used to treat BCL in preclinical models or clinical settings and are very effective for specific patients. However, most BCL patients do not benefit from these immunotherapies, and some patients relapse or develop drug resistance, ultimately leading to disease progression. Sonodynamic therapy (SDT) is an emerging non-invasive treatment for solid tumors with the advantages of high specificity, minimal damage to normal cells, deep tissue penetration, high spatial precision, and induction of immune response. SDT utilizes high tissue-penetrating ultrasound (US) to irradiate sonosensitizers, triggering the production of reactive oxygen species (ROS), which then enter deep tumors and induce cell death.

[0003] Although traditional BCL treatments have achieved initial success in clinical trials, most patients eventually develop drug resistance and poor clinical efficacy. Epigenetic dysregulation is a major factor in the pathogenesis of BCL. Given that BCL has characteristics of hematological tumors and solid tumors in terms of programmed death (such as apoptosis, copper mutation, ferroptosis, etc.) and epigenetic regulatory mechanisms, therapies targeting epigenetic pathways are a promising alternative strategy for the treatment of BCL. Summary of the invention

[0004] In order to solve the problems of easy drug resistance and poor clinical efficacy in the existing technology of materials for treating BCL, the present application provides an ultrasound-sensitized nanomaterial based on MOFs and its preparation method and application. 6A-mRNA demethylase inhibitor and copper complexes were encapsulated into UiO-66-NH2, and the Z-type heterostructure of UiO-66-NH2 could efficiently separate electron-hole pairs under ultrasound activation to produce ROS. As an alternative therapy for BCL, when MOFs-based ultrasound-sensitized nanomaterials were functionalized with mPEG-PO3 and anti-CD19 antibodies, the formed CRUPPA19 particles could specifically aggregate in BCL tissues and target lymphoma cells infiltrating the bone marrow. Once internalized, CRUPPA19 could induce intracellular ROS generation and apoptosis under ultrasound. Subsequently, ultrasound stimulation triggered autophagy-mediated release of Cu and Rhein from CRUPPA19, thereby increasing protein lipoylation and global mRNA methylation, leading to copper death and PDL1 transcriptional inhibition, respectively. These cascades synergistically induced immunogenic cell death in tumors and promoted CD8 + The activation of T cells ultimately leads to an anti-lymphoma immune response, allowing CRUPPA19-mediated ultrasound immunotherapy to not only eliminate primary and metastatic lymphomas, but also clear lymphoma cells in the bone marrow, thereby enhancing anti-hematological tumor immunity.

[0005] This application adopts the following technical solutions:

[0006] According to one aspect of the present application, there is provided a MOFs-based ultrasound-sensitized nanomaterial, including drug-loaded MOFs;

[0007] The MOFs are selected from UiO-66-NH2;

[0008] The UiO-66-NH2 is a particle with amino functional groups connected to UiO-66;

[0009] The drug-loaded MOFs are loaded with m 6 A-mRNA demethylase inhibitor complex;

[0010] The m 6 The A-mRNA demethylase inhibitor complex is formed by the coordination of Rhein molecules and divalent copper ions;

[0011] The outer surface of the drug-loaded MOFs is bound to anti-CD19 antibodies;

[0012] The outer surface of the drug-loaded MOFs is also modified with mPEG-PO3.

[0013] Optionally, it is characterized in that the particle size of the drug-loaded MOFs is 200-260 nm.

[0014] Rhein (1,8-dihydroxy-3-carboxyanthraquinone) is an anthraquinone derivative of a natural product that is widely used as an antibacterial and anti-inflammatory drug. Anthraquinone derivatives, such as rhodamine and rhein, are also ideal photosensitizers because they can absorb light from the visible to near-infrared (NIR) region and generate a large amount of ROS. In addition, the photocatalytic activity of anthraquinone derivatives can be enhanced by directly coordinating redox-active metal centers.

[0015] According to another aspect of the present application, a method for preparing the above-mentioned MOFs ultrasound-sensitized nanomaterial is provided, characterized in that it comprises the following steps:

[0016] S1. Add ammonia water to a mixture containing Rhein, a soluble divalent copper salt, and an alcohol until the pH of the mixture reaches 10, then stop adding the mixture, stir, separate, wash, and dry to obtain m 6 A-mRNA demethylase inhibitor complex.

[0017] S2, obtain UiO-66-NH2, and then 6 A mixed solution of the A-mRNA demethylase inhibitor complex and the solvent is stirred, separated, washed, and dried to obtain drug-loaded MOFs;

[0018] S3, obtain mPEG-PO3 and anti-CD19 antibody;

[0019] S4. Add a carboxyl activator to an aqueous suspension containing anti-CD19 antibodies, and after activating the carboxyl groups, add an aqueous suspension containing drug-loaded MOFs to react to obtain an aqueous suspension of drug-loaded MOFs with anti-CD19 antibodies bound to the surface, and then add an aqueous solution containing mPEG-PO3, stir, separate, wash, and dry to obtain the ultrasound-sensitized nanomaterial of the MOFs.

[0020] Optionally, in step S1, the molar ratio of Rhein to soluble divalent copper salt is 1:0.25-0.75.

[0021] Optionally, in step S1, the ratio of Rhein to alcohol is 1 mmol: 70-90 mL.

[0022] Optionally, in step S1, the soluble divalent copper salt is selected from CuCl2.

[0023] Optionally, in step S1, the alcohol is selected from ethanol.

[0024] Optionally, in step S1, the stirring conditions include: stirring at 30-50° C. for 6-18 hours.

[0025] Optionally, in step S2, the method for obtaining UiO-66-NH2 includes: adding a zirconium n-propoxide solution to a mixture containing acetic acid and DMF, placing the mixture in a sealed container, heating the reaction, then adding a linker to the product, stirring, separating, washing, and drying to obtain UiO-66-NH2.

[0026] Optionally, the dosage ratio of the zirconium n-propoxide solution to acetic acid is 1 μg: 53-55 mL.

[0027] Optionally, the dosage ratio of the zirconium n-propoxide solution to DMF is 1 μg: 93-95 mL.

[0028] Optionally, the weight ratio of the zirconium n-propoxide solution to the linker is 1 μg:1.1-1.3 mg.

[0029] Optionally, the linker is selected from at least one of 2-amino-1,4-phthalic acid and 1,4-phthalic acid ester.

[0030] BDC-NH2 can improve the acoustic sensitivity of UiO-66 compared with BDC linker.

[0031] Optionally, the conditions for the heating reaction include: a reaction temperature of 120 to 140° C. and a reaction time of 1 to 3 hours.

[0032] Optionally, in step S2, UiO-66-NH2, m 6 The weight ratio of the A-mRNA demethylase inhibitor complex is 1:0.1-1.5.

[0033] Optionally, in step S2, the solvent is selected from water.

[0034] Optionally, in step S2, the stirring conditions include: ultrasonic treatment for 20 to 40 seconds, and stirring at a rotation speed of 150 to 300 rpm for 16 to 24 hours.

[0035] Optionally, in step S3, the method for obtaining mPEG-PO3 includes:

[0036] Mix a mixture A containing phosphorus oxychloride, dichloromethane and triethylamine with a mixture B containing poly(ethylene glycol) methyl ether and dichloromethane, add water, stir, then remove dichloromethane in vacuo, and dialysis purification to obtain mPEG-PO3.

[0037] Optionally, the content of phosphorus oxychloride in the mixed solution A is 40-60 mmol / L, and the content of triethylamine is 140-160 mmol / L.

[0038] Optionally, the content of poly(ethylene glycol) methyl ether in the mixed solution B is 14-16 mmol / L.

[0039] Optionally, the volume ratio of mixed solution A to mixed solution B is 1:8-10 mL.

[0040] Optionally, in step S4, the carboxyl activator is selected from at least one of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide.

[0041] Optionally, in step S4, the time for activating the carboxyl group is 30 to 60 minutes.

[0042] Optionally, in step S4, the reaction conditions include a reaction temperature of 0 to 5° C. and a reaction time of 6 to 18 h.

[0043] Optionally, in step S4, the content of the carboxyl activator in the aqueous suspension containing the anti-CD19 antibody is 0.01-0.02 mmol / L, and the content of the anti-CD19 antibody is 1-3 mmol / L.

[0044] Optionally, in step S4, the content of drug-loaded MOFs in the aqueous suspension containing drug-loaded MOFs is 1 to 3 mmol / L.

[0045] Optionally, in step S4, the volume ratio of the aqueous suspension containing the anti-CD19 antibody to the aqueous suspension containing the drug-loaded MOFs is 1:0.5-1.5.

[0046] Optionally, in step S4, the content of mPEG-PO3 in the aqueous solution containing mPEG-PO3 is 20 to 30 mg / mL.

[0047] Optionally, in step S4, the volume ratio of the water suspension of drug-loaded MOFs with anti-CD19 antibodies bound to the surface to the aqueous solution containing mPEG-PO3 is 1:2-3.

[0048] Optionally, in step S4, the stirring conditions include: stirring at 0-5° C. for 12-24 hours.

[0049] According to another aspect of the present application, there is also provided an application of the ultrasound-sensitized nanomaterial of MOFs or the ultrasound-sensitized nanomaterial of MOFs obtained according to the above preparation method in the preparation of a drug for ultrasound-driven cascade immunotherapy of B-cell lymphoma.

[0050] SDT can induce immunogenic cell death (ICD), thereby triggering a systemic anti-tumor immune response, a phenomenon known as sono-immunotherapy. Combining sono-immunotherapy with immune checkpoint inhibitors can not only ablate the primary tumor but also inhibit the growth of distant or metastatic tumors through the peritoneal effect.

[0051] The copper (Cu) complex of Rhein, an inhibitor of FTO demethylase, was encapsulated into UiO-66-NH2, which enhanced the generation of singlet oxygen ( 1 O2) and hydroxyl radicals (-OH), thereby increasing the generation of electron-hole pairs. These CuR@UiO66 sonar sensitizers were functionalized with mPEG-PO3 and anti-CD19 antibodies, and the resulting CRUPPA19 particles were highly biocompatible and could specifically target B lymphoma cells via CD19 receptor-mediated endocytosis. Once internalized by lymphocytes, CRUPPA19 triggered intracellular ROS generation and apoptosis under ultrasound. In addition, ultrasound stimulation promoted the autophagy-mediated release of Cu and Rhein from the nanoparticles, which increased protein lipoylation and global mRNA methylation and led to copper death and PDL1 downregulation, respectively. These intracellular cascades promoted immunogenic cell death (ICD) in tumors, which in turn stimulated anti-BCL immune responses by promoting dendritic cell (DC) maturation, enhancing T cell recognition of tumor antigens, and cross-stimulation of CD8+ T cells. CRUPPA19-mediated ultrasound immunotherapy not only suppressed primary and metastatic lymphoma in mouse models, but also eliminated lymphoma cells in the bone marrow.

[0052] The beneficial effects of this application include:

[0053] The MOFs ultrasound-sensitized nanomaterial provided in this application is a new type of MOFs-based "surface nanosonosensitizer" (CRUPPA19). The encapsulated copper and Rhein complex effectively sensitizes the MOFs main body and controls the energy band structure, thereby generating high sonodynamic activity. In the application of BCL therapy, primary lymphoma and infiltrating lymphoma cells are eradicated by sonoimmunotherapy. CRUPPA19 can also effectively accumulate in lymphoma tissues and target CD19+ lymphocytes infiltrating the bone marrow. CRUPPA19 nanoparticles can be internalized by lymphoma cells through CD19 receptors and trigger cascade amplification under ultrasonic stimulation, inducing programmed cell death. CRUPPA19-mediated SDT induces apoptosis by triggering the production of ROS. Subsequently, the activation of autophagy by SDT leads to the disintegration of CRUPPA19, while the Cu and Rhein released from the nanoparticles increase protein lipoylation and global mRNA methylation, leading to copper death and transcriptional downregulation of PDL1. Furthermore, these cascades induced ICD and promoted the maturation of DCs in the tumor microenvironment, thereby enhancing the infiltration of CD8+ T cells and subsequent recognition of tumor cells. Finally, CRUPPA19-mediated ultrasound immunotherapy inhibited the growth of primary tumors, suppressed metastasis, and eliminated lymphoma cells in the bone marrow of A20 lymphoma mice. This study extends the concept of nano-epigenetic therapy for both solid tumors and hematological malignancies, providing another perspective for the treatment of various types of BCLs that can be applied clinically. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 Synthesis and characterization of CuR@UiO66 of the present application: (A) is a schematic diagram of the synthesis of CuRhein; (B) is the UV / visible absorption spectra of CuRhein and Rhein; (C) is the fluorescence spectra of CuRhein and Rhein; (D) is the synthesis of CuR@UiO66; (E) is the TEM image of CuR@UiO66; (F) is the hydrated particle size of UiO-66-NH2 and CuR@UiO66; (G) is the XRD spectrum of UiO-66-NH2 and CuR@UiO66; (H) is the STEM image of CuR@UiO66 and the corresponding elemental spectrum; (I) to (K) are XPS high-resolution spectra of UiO-66-NH2 and CuR@UIO-66, where (I) is the Zr3d peak, (J) is the N1s peak and (K) is the Cu2p peak.

[0055] Figure 2 The mechanism of ROS generation by CuR@UiO66 under ultrasonic treatment in this application: (A) and (B) are the TEMP / 1 (C) is the normalized fluorescence intensity of SOSG, which is used to represent the fluorescence intensity of PBS, CuRhein, UiO-66-NH2 and CuR@UiO66 under ultrasonic treatment (1 W cm -2 , 1MHz, 50% duty cycle) is generated by 1 O2; (D) is the normalized UV-visible absorbance of DPBF, which is used to represent the absorption of PBS, CuRhein, UiO-66-NH2 and CuR@UiO66 under ultrasonic treatment (1 W cm -2 , 1MHz, 50% duty cycle) before and after ultrasonic treatment (1Wcm -2 , 1MHz, 50% duty cycle) is generated by 1 O2; (E) is the MB normalized UV-vis absorbance, which is used to represent the absorption of PBS, CuRhein, UiO-66-NH2 and CuR@UiO66 before and after ultrasonic treatment (1 W cm -2 , 1 MHz, 50% duty cycle), the data are mean ± SD; (F) and (G) are UV-visible diffuse reflectance spectra and band gap spectra of CuRhein, UiO-66-NH2 and CuR@UiO66, respectively; (H) to (J) are Mott-Schottky plots of CuRhein, UiO-66-NH2 and CuR@UiO66, respectively; (K) is the charge density of CuR@UiO66; (L) is a schematic diagram of the Z-type mechanism of ROS generation by CuR@UiO66 under ultrasonic treatment.

[0056] Figure 3In this application, CRUPPA19 can selectively target B lymphoma cells for testing: (A) is a schematic diagram of synthesizing CRUPPA19 by adding anti-CD19 antibody and mPEG-PO3 to CuR@UiO66; (B) is a representative fluorescence image of Cy3-labeled CRUPPA19 (red fluorescence) in A20 cells and normal monocytes (L929) after incubation for 30 minutes, and the cell nuclei are stained with DAPI (blue), scale bar = 20μm; (C) is a fluorescence image of A20 cells treated with CRUPPA19 or CRUPPA19+ultrasound after further culture for 30 minutes and 2 hours, respectively, scale bar = 20μm; (D) is a schematic diagram of establishing a mouse BCL model and evaluating the in vivo distribution of CRUPPA19; (E) is a representative in vivo and in vitro fluorescence image of tumor mice, showing the biodistribution of Cy7-labeled CRUPPA19; (F) is a representative fluorescence image of tumors, thigh bones (TB) and major organs 24 hours after intravenous injection of CRUPPA19. G) Zr content in tumors, bone marrow (BM) and other major organs indicating the biodistribution of nanoparticles; (H) and (I) Flow cytometric analysis of Cy7-labeled CRUPPA19 uptake in lymphoma cells (CD19+) extracted from the bone marrow of A20-bearing mice, respectively. Data are mean ± SD; *P < 0.05, ***P < 0.001, one-way ANOVA and Tukey test.

[0057] Figure 4 The CRUPPA19 cascade amplification induced lymphoma cell death test in this application: (A) 1 Representative fluorescence of A20 cells with different treatments after staining with the O2 probe DCFH-DA, scale bar = 50 μm; (B) Flow cytometry plots showing the percentage of annexin V-FITC / 7-AAD-positive A20 cells after the indicated treatments; (C) GFP / RFP ratio showing autophagic flux in appropriately treated A20 cells after transfection with the GFP-LC3-RFP reporter, an increase in the GFP / RFP ratio indicates a decrease in autophagic flux; (D) Western blot showing the expression of copper death or autophagy-related proteins in A20 cells treated with different doses of CRUPPA19, without / with 1 W cm -2 (E) is the expression of surface PDL1 in A20 cells treated with specified doses of CRUPPA19; (F) is dot blot; (G) is LC-MS / MS detection to determine the expression of surface PDL1 in A20 cells treated with specified doses of CRUPPA19 6 AmRNA levels; (H) is gene-specific m 6 AqPCR showing PDL1 m in A20 cells treated with indicated doses of CRUPPA196 A-mRNA level; (I) PDL1 mRNA stability test, A20 cells were pretreated with CRUPPA19 (80 μg / mL) for 24 h, and then cultured with Act-D for the specified time, the cells were harvested and qPCR was performed; (J) is a graphic illustration of the sonodynamic effect of CRUPPA19 in vitro, the data are mean ± SD; *P < 0.05, **P < 0.01, one-way analysis of variance and Tukey test.

[0058] Figure 5 The ultrasonic immunotherapy effect of CRUPPA19 in the lymphoma mouse model of this application: (A) is a schematic diagram of the establishment of the A20 bilateral tumor model and the CRUPPA19-mediated ultrasonic immunotherapy schedule; (B) is the growth curve of the primary tumor; (C) is the growth curve of the distal tumor; (D) is the TGI ratio of the primary tumor; (E) is the TGI ratio of the distal tumor; (F) is the Kaplan-Meier curve showing the overall survival probability of A20 mice treated with differential treatment at 80 days; (G) is a representative image of the Wright-Giemsa staining of the bone marrow (BM) of the recipient mouse, scale bar = 20μm; (H) is a representative image of the spleen and the corresponding H&E stained tissue sections of the designated group, scale bar = 1cm (spleen image), 100μm (H&E staining); (I) is the CD19 of A20 mice in each group + (J) is the percentage of lymphoma cells; (K) is the relative expression level of HMGB1 in primary tumors of the designated groups; (L) is the percentage of mature DCs (CD80 + CD86 + Gating CD11c + (M) is CD8 + T cells (CD3 + T cells are marked) are the percentages; (N) is the serum IFN-γ level of the specified group; (O) is the expression of PDL1 in the distal tumor of the specified group; (P) is the m 6 AmRNA levels, data are mean ± SD; *P < 0.05, **P < 0.01, ***P < 0.001, one-way ANOVA and Tukey test (n = 6).

[0059] Figure 6 This is the CuRhein theoretical structure under density functional theory for this application.

[0060] Figure 7 Fourier transform infrared spectroscopy (FTIR) of Rhein and CuRhein for this application.

[0061] Figure 8 Photos of UiO-66-NH2, CuRhein and CuR@UiO66 for this application.

[0062] Fig. 9 Pore ​​structure test of UiO-66-NH2 and CuR@UiO66 in this application: (A) is the N2 adsorption-desorption isotherm, and (B) is the corresponding pore size distribution.

[0063] Fig.10 Fourier transform infrared spectra of UiO-66-NH2 and CuR@UiO66 in this application.

[0064] Fig.11 The encapsulation efficiency of CuRhein and the drug loading rate of CuR@UiO66 for this application.

[0065] Fig.12 This is the XPS test of C1s, O1s and N1s in CuR@UiO66 for this application.

[0066] Fig.13 Photoluminescence spectra of UiO-66-NH2 and CuR@UiO66 for this application.

[0067] Fig.14 For this application, the fluorescence intensity of SOSG is calculated according to the specified group 1 The amount of O2 produced.

[0068] Fig.15 The specified group calculated according to the ultraviolet absorption peak intensity of DPBF in this application 1 The generation of O2.

[0069] Fig.16 For the purpose of this application, ·OH is generated in a specified group according to the UV absorption peak intensity of methyl bromide.

[0070] Fig.17 For the present application, the particle size of CRUPPA19 cultured for different time periods in FBS at pH 7.4 or pH 4.5.

[0071] Fig.18 Scanning electron microscopy images of CRUPPA19 cultured with FBS at pH 7.4 or pH 4.5 for different time periods for this application.

[0072] Fig.19 This is the in vitro release curve of Cu from CRUPPA19 in PBS with different pH values ​​in this application. The data are mean ± SD; ***P < 0.001, one-way ANOVA and Tukey test.

[0073] Fig. 20For the present application, the survival rate of normal monocytes from BALB / c mice cultured with CRUPPA19 at different concentrations (0 to 400 μg / mL) for 48 hours was measured.

[0074] Fig.21 Hemolytic effect of CRUPPA19 in this application: (A) is the image of red blood cells cultured with PBS, RBC lysis buffer or different concentrations of CRUPPA19 for 48 h, and (B) is the hemolysis percentage of the designated group.

[0075] Fig. 22 For the present application, the zirconium content in A20 cells or normal monocytes after treatment with different concentrations of CRUPPA19 or CRUPP for 30 min, (A) is CRUPPA19, (B) is CRUPP, the Zr content was measured by ICP-AES, and the data are mean ± SD; **P < 0.01, NS, no significant difference, one-way ANOVA and Tukey test.

[0076] Fig.23 The viability data of A20 cells treated with or without ultrasonic treatment for 48 h in this application are mean ± SD; ***P < 0.001, NS, one-way ANOVA with Tukey test (n = 3).

[0077] Fig.24 For the protein expression level of this application, each sample was normalized with β-Actin, and the data are mean ± SD; *P < 0.05, **P < 0.01, NS, no significant difference, one-way ANOVA and Tukey test (n = 3).

[0078] Fig.25 In vivo therapeutic effects of this application: (A) is a representative IHC image showing the expression of PCNA in tumor tissues of the specified group, and the cell nuclei were stained with DAPI, (B) is the percentage of PCNA+ cells in the specified group, and the data are mean ± SD; **P < 0.001, one-way ANOVA and Tukey test (n = 6).

[0079] Fig.26 The relative expression levels of ICD markers CRT and ATP in the tumor tissues of the specified groups in this application, the data are mean ± SD; **P < 0.001, one-way ANOVA and Tukey test (n = 6).

[0080] Fig. 27 For the present application, the changes in the body weight of mice within 60 days after intravenous injection of PBS or 200 μg / mL CRUPPA19 were shown. The data are mean ± SD (n = 3).

[0081] Fig.28For the present application, the blood biochemical indices of healthy mice (n=3) measured 60 days after intravenous injection of PBS or 200 μg / mL CRUPPA19 (n=3) were as follows. The data are mean ± SD; NS, no significant difference, one-way ANOVA and Tukey test.

[0082] Fig.29 For this application, blood parameters of healthy mice were analyzed 60 days after intravenous injection of PBS or 200 μg / mL CRUPPA19 (n=3), and the data are mean ± SD; NS, no significant difference, one-way ANOVA and Tukey test.

[0083] Fig.30 Representative images of H&E staining of healthy mouse tissues 60 days after intravenous injection of PBS or 200 μg / mL CRUPPA19 (n=3) for the present application, scale bar=100 μm.

[0084] Fig.31 In this application, flow cytometry analysis of CD3 in lymphocytes of A20 mouse model was performed + CD8 + T cells, CD19 + Gating strategy for lymphocytes and PDL1 expression.

[0085] Fig.32 In this application, a MOF-based ultrasound immuno / epigenetic therapy platform is used to treat B-cell lymphoma by cascading induction of apoptosis, autophagy, global mRNA methylation and copper death. DETAILED DESCRIPTION

[0086] The present application is described in detail below with reference to embodiments, but the present application is not limited to these embodiments.

[0087] Unless otherwise specified, the raw materials in the examples of this application were purchased from commercial sources and used without further treatment. Zirconium n-propoxide solution [Zr(OnPr)4], 2-amino-1,4-phthalic acid (BDC-NH2) and acetic acid were purchased from Aladdin. CuCl2-2H2O and Rhein were purchased from Sigma-Aldrich. N,N-dimethylformamide (DMF), anhydrous ethanol and dichloromethane (DCM) were purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0088] Unless otherwise specified, conventional methods were used for testing and instrument settings were those recommended by the manufacturer.

[0089] Material characterization: The morphology of the samples was examined using a JEOL JSM-6700F scanning electron microscope (SEM) and a Tecnai G2S-Twin transmission electron microscope (TEM, Philips-FEI microscope; 200 kV field emission gun). Nitrogen adsorption-desorption isotherms at 77 K were obtained using a Micromeritics 2020 analyzer. Powder X-ray diffraction (PXRD) patterns were obtained by a Rigaku D-Max 2550 diffractometer using CuKα radiation. The wavelength range from 400 to 4000 cm was acquired using a Brunaug IFS66V / S Fourier transform infrared spectrometer and a KBr pellet. -1 Fourier transform infrared spectra of the region. X-ray photoelectron spectroscopy (XPS) was performed using an ESCALAB250 spectrometer. Electron paramagnetic resonance spectra were obtained on a JES-FA200EPR spectrometer. UV-visible diffuse reflectance spectra and UV-visible absorption spectra were obtained using Shimadzu U-4100 and Shimadzu UV3600 spectrometers, respectively. Photoluminescence (PL) spectra were measured using a luminescence spectrophotometer (FLUOROMAX-4). Mott-Schottky plots were obtained by an electrochemical workstation (CHI660E, CHInstrument).

[0090] Computational details: All calculations were performed in the framework of density functional theory (DFT) using the projector-augmented plane wave method and implemented in the Vienna Atomic Bomb Simulation Package. The generalized gradient approximation (GGA) was used in the Perdew-Burke-Ernzerhof (PBE) scheme to describe the exchange-correlation functional. The cutoff energy and energy criterion were set to 450 eV and 10 -4 eV. Until the residual force on the atom drops to less than The structure is relaxed. Due to the huge number of atoms in the full unit cell, a part of the MOFs structure is selected for DFT calculation, and the edges are passivated with hydrogen atoms. The calculation adopts the Monkhorst-Pack scheme, the k-point grid is 1×1×1, and all graphs are drawn using VESTA software.

[0091] Detection 1 O2: To detect using SOSG probe 1For the production of O2, 4 μL of nanoparticles (5 mg / mL) were dissolved in 186 μL PBS and then mixed with 10 μL SOSG (100 mM in methanol). After ultrasonic treatment (1 W cm-2, 1 MHz, 50% duty cycle), unreacted nanoparticles were removed by ultrafiltration (10 KD) to avoid interference with SOSG fluorescence. The filtrate containing SOSG was collected for fluorescence measurement. In order to detect the SOSG fluorescence using TEMP as an indicator by ESR, the TEMP fluorescence was measured by HPLC. 1 To generate O2, 20 μL of nanoparticles (5 mg / mL) were dissolved in 1 mL of PBS. -2 , 1MHz, 50% duty cycle, 5min), 90μL of filtrate was collected by ultrafiltration (10KD) and mixed with 10μL of TEMP (1M). ESR was performed immediately after mixing to measure 1 O2 signal.

[0092] Encapsulation efficiency and drug loading: The loading capacity (LC) and encapsulation efficiency (EE) of CuRhein in CuR@UiO66 were determined by UV-visible spectroscopy. LC and EE were calculated according to the following formula:

[0093]

[0094] Drug release and stability testing of CRUPPA19: To measure in vitro drug release, 2 mL aliquots of CRUPPA19 (5 mg / mL) were placed in disposable dialysis tapes (MWCO: 3.5 kDa, Thermo Scientific) and then placed in 10 mL of PBS at pH 4.5, 5.5, 6.5, or 7.4. Dialysis was performed on a shaker at 37 °C. At different time points, 1 mL of buffer was taken for analysis and then replaced with the same volume of PBS. The amount of copper released into the buffer at each time point was measured by ICP-AES.

[0095] In the experiments in this application, PBS solutions of CRUPPA19 and UPPA19 were prepared at a concentration of 200 μg / mL in the therapeutic cells.

[0096] Cell culture: A20 cells (American Type Culture Collection) were cultured in RPMI1640 medium (Biological Industries) supplemented with 10% fetal bovine serum (FBS) and 1% (v / v) penicillin / streptomycin at 37°C in the presence of 5% carbon dioxide. Normal mononuclear cells were isolated from mouse bone marrow. Bone marrow cells (BMCs) were homogenized into single cells, and then monocytes were isolated using Ficoll-Paque PREMIUM (cytiva) according to our previous report.

[0097] Cellular uptake of nanoparticles: Confocal laser scanning microscopy (CLSM) and flow cytometry (FCM) were used to track the uptake of nanoparticles by cells. 5 The cells were seeded into 24-well plates at a density of 100 cells / well and cultured overnight. The culture medium was discarded and replaced with fresh culture medium containing Cy3-labeled nanoparticles. After 30 min of culture, the cells were harvested and fixed on glass slides using a cell slicer. The cells were rinsed with PBS, fixed with 4% paraformaldehyde (PFA) for 15 min, and counterstained with DAPI, and then the slides were observed using CLSM (Nikon). For flow cytometry, the cells were sliced ​​at 5 × 10 5 The cells were seeded into 6-well plates at a density of 1000 / well and cultured overnight. After culturing with Cy3-labeled nanoparticles for 30 min, the cells were collected at 1000 rpm for 3 min and rinsed three times with PBS. The cells were then resuspended in 500 μL PBS, filtered with a 40 μm nylon mesh filter (Corning), and the single cell suspension was analyzed using BDLSR Fortessa and FlowJo software (FlowJo, LLC, v.10.4). The intracellular uptake of nanoparticles was quantified by measuring the Zr content in the cells using a Perkin-Elmer NEXION300× ICP mass spectrometer.

[0098] Intracellular ROS detection: 1×10 5 The cells were seeded into 24-well plates at a density of 10 cells / well and cultured overnight. The culture medium was discarded and replaced with fresh culture medium containing nanoparticles and the cells were cultured for 30 min. US (1 MHz, 1 W cm -2 After irradiation of cells with 50% duty cycle for 5 min, cells were cultured with FBS-free RPMI1640 medium containing DCFH-DA (10 μM) for 30 min. Confocal laser scanning microscopy (CLSM) was used to capture cell images, and the fluorescence intensity was quantified by flow cytometry using FlowJo software.

[0099] Autophagic flux analysis: Autophagic flux was analyzed using the GFP-LC3-RFP reporter transfection system. Briefly, A20 cells were transfected at 0.3 × 10 6 Cells were seeded in 6-well plates at a density of 10 cells / mL and cultured overnight, and then transfected with 2-10 μg of pCMV-mCherry-GFP-LC3B expression plasmid using Lipofectamine 3000 Reagent (Thermo Fisher Scientific) according to the manufacturer's instructions. Transfected cells were harvested 48 h later, filtered through a 40-μm nylon filter (Corning), and analyzed by flow cytometry.

[0100] Quantitative PCR of methylated RNA transcripts: QPCR of methylated RNA transcripts was performed according to the manufacturer's instructions. Briefly, total cellular RNA was extracted using TRIZOL (Life Technologies) according to the standard manual. Subsequently, RNA was fragmented and stained with anti-m 6 A antibody incubation, and immunoprecipitation of m 6 ARNA. The bound RNA was eluted from the magnetic beads with N6-methyladenosine and then reverse transcribed into cDNA using a cDNA synthesis kit (ABIsystems). Quantitative RT-PCR was performed on an ABI7500 sequence detection system using SYBR green master mix (ABIsystems) and specific primers (sequences are listed in Table 1).

[0101] Table 1

[0102]

[0103] mRNA stability assay: Appropriately treated A20 cells were seeded into 6-well plates and cultured for different periods of time in the presence of actinomycin D (5 μg / mL, Sigma). RNA was extracted at specific time points and subjected to quantitative RT-PCR analysis. The percentage of mRNA remaining after actinomycin D treatment relative to the level before treatment was calculated. The half-life value of mRNA was calculated by linear regression analysis.

[0104] Western blotting: Appropriately treated cells were lysed with cold protein lysis buffer containing 1% phosphatase inhibitor (Cell Signaling Technology), and then centrifuged at 12000 rpm for 10 min to remove debris. The protein content in the supernatant was determined by the bicinchoninic acid (BCA) method. SDS-PAGE separation was performed using Bis-Tris 8-12% gel and then transferred to polyvinylidene fluoride (PVDF) membrane (Millipore, USA). After blocking with 5% skim milk for 1 h at room temperature, the PVDF membrane was incubated with primary antibodies targeting β-actin (sc-47778, Santa Cruz Biotechnology), p62 (23214, Cell Signaling Technology), LIAS (11577-1-AP, Proteintech), DLAT (68303-1-Ig, Proteintech) and FDX1 (12592-1-AP, Proteintech) at 4°C overnight. The blots were washed four times with TTBS and then incubated with horseradish peroxidase-conjugated secondary antibodies. Protein bands were visualized by gel imaging system using an enhanced chemiluminescence (ECL) kit and quantified relative to β-actin using ImageJ software.

[0105] M 6 A point blot detection: m 6 A dot blot assay was performed according to the manufacturer's instructions. Briefly, the GenElute TM mRNA was purified from appropriately treated cells using mRNA Miniprep Kit (Sigma, #MRN70). Denatured in 1x buffer (0.4 M NaOH, 20 mM EDTA) at 100°C for 10 min and then mixed with an equal volume of cold 2 M ammonium acetate (pH 7). Nylon membrane (Hybond-N, Amersham, Buckinghamshire, UK) was moistened with nucleic acid transfer buffer and then assembled on a Bio-Dot instrument (BioRad) connected to a vacuum. The membrane was rehydrated with 500 μl H2O and then loaded with denatured RNA (50 μl). Mouse anti-m 6 RNA spots were incubated with antibody A, and signals were detected with horseradish peroxidase-conjugated secondary antibodies and enhanced chemiluminescence. Membranes were stained with 0.2% methylene blue (Sigma-Aldrich) to verify equal RNA loading. Densitometric quantification was performed using ImageJ (NIH Image, Bethesda, MD).

[0106] Animal experimentation: 6-week-old BALB / c (female) mice were purchased from Charles River Laboratories. All animal experiments were performed in accordance with the Guide for the Care and Use of Animals and approved by the Animal Care and Use Committee of Jilin University. The bilateral tumor model was established according to our previous report62. Briefly, A20 cells (0.5 × 10 per mouse) were injected subcutaneously in the right abdomen of mice. 6 The primary tumor was induced by subcutaneous injection of A20 cells in the left mammary gland of mice 24 hours later, and the distal or metastatic tumor was established. When the tumor volume reached about 250 cubic millimeters, the mice were randomly divided into 9 groups (n=6): PBS group, PBS group + US Group, UPPA19 + US group, Rhein (25 μM) group, CuRhein+US group, anti-PD-L1 antibody (aPDL1) group, anti-CD20 antibody (aCD20) group, CRUPPA19 group, and CRUPPA19 + US group. Mice were injected with 100 μg / mL nanoparticles every other day from day 0 to day 9, and the treatment was repeated every 3 days for a total of 3 cycles (1 cycle = 3 days). In the US irradiation group, the primary tumor was irradiated for 5 min (1 W cm -2 , 1 MHz, 50% duty cycle). The doses of aPDL1 (InVivo Mab anti-mouse PD-L1, BioXcell) and aCD20 (InVivo Mab anti-mouse CD20, BioXcell) were 10 mg / kg each, which is within the range of clinically achievable levels. The dose of CuRhein was 15 μg / mL (25 μM), which is comparable to the dose in CRUPPA19. The tumor volume was calculated as follows: 1 / 2 × (length × width2). Mice were monitored until day 60 after the first injection for survival analysis, and if the tumor volume exceeded 2000 mm 3 , euthanasia was performed. A complete response was defined as a reduction in tumor volume to less than 20 cubic millimeters (lowest detection limit). Mice were sacrificed on day 14, and major organs (heart, liver, spleen, lung, and kidney) and tumors were collected for histological examination. In addition, the tumors were homogenized into single-cell suspensions and stained with fluorochrome antibodies for flow cytometry.

[0107] Hematoxylin and eosin (H&E) staining: After the experiment, the tumor-bearing mice were euthanized, and the tumors and major organs (heart, liver, spleen, lung, and kidney) were harvested and fixed with 4% paraformaldehyde. The fixed organs were embedded in paraffin, cut into 5 μm thick sections, and then H&E staining was performed according to the standard protocol. The stained sections were observed using an optical microscope (Nikon, Japan).

[0108] Immunohistochemistry (IHC): IHC was performed on deparaffinized tumor tissues according to standard protocols. The sections were microwaved in citrate buffer (pH = 6) for 10 min for antigen retrieval and then blocked with 1% bovine serum albumin (BSA) for 30 min at room temperature. They were then incubated with anti-PCNA antibody (1:500, 1% BSA dilution) at room temperature overnight at 4°C and then The 488-conjugated secondary antibody was incubated for 1 h at room temperature. After counterstaining with DAPI, fluorescent images of tumor sections were taken on a Nikon microscope.

[0109] Giemsa staining: Briefly, bone marrow cells were collected, washed, and fixed on a slide using a cell vortexer. The slide was stained with a Giemsa staining kit (BASO) and observed under a bright field microscope.

[0110] Flow cytometry: Fresh tumor tissue was homogenized into a single cell suspension, and red blood cells (RBC) were removed with red blood cell lysis buffer. The cells were filtered through a 40 μm nylon mesh filter (Corning), resuspended in PBS containing 3% FBS, and then incubated with fluorescently labeled antibodies (FITC anti-mouse CD45, BioLegend; PerCP / Cyanine5.5 anti-mouse CD3, BioLegend; Alexa Fluor 500, 100 μg / mL ... 700 anti-mouse CD8, BioLegend; Brilliant Violet 650 TM Anti-mouse PDL1, BioLegend; Brilliant Violet 510 TM Anti-mouse CD19, BioLegend) was incubated at the recommended dilution for 30 min at 4°C. Stained cells were washed, resuspended in PBS, and analyzed on an LSRFortessa instrument (BD). A total of 50,000 to 100,000 cells per sample per mouse were analyzed using FlowJo software, and the gating strategy is shown in Fig.31 .

[0111] In vivo biodistribution: To track the in vivo biodistribution of nanoparticles, 100 μL Cy7-labeled CRUPPA19 (100 μg / mL) was injected intravenously into A20 tumor-bearing mice and anesthetized with 2% isoflurane at 0.5, 2, 12, and 24 h after injection. In vivo fluorescence images were taken at the above time points using the PerkinElmer IVISLuminaIII in vivo imaging system (740 nm excitation filter, 790 nm emission filter). Mice were killed 24 h after injection, and tumor tissues and major organs (heart, liver, spleen, lungs, and kidneys) were taken for fluorescence imaging. Bone marrow was collected and Cy7 was analyzed by flow cytometry. + Lymphoma group (CD19 +The tumor tissues and major organs were then removed and weighed, and then digested in aqua regia at 80°C for 2 h, and the zirconium content therein was analyzed by ICP-MS (Perkin Elmer NEXION300×ICP mass spectrometer).

[0112] Enzyme-linked immunosorbent assay (ELISA): The levels of IL-6 (MultiSciences, Hangzhou, China), TNF-α (MultiSciences, Hangzhou, China), and IFN-γ (MultiSciences, Hangzhou, China) in serum were detected using specific ELISA kits according to the manufacturer’s instructions.

[0113] Hemolysis assay: Hemolysis assay was used to determine the blood compatibility of CRUPPA19. Briefly, 1 mL of whole blood was collected from BALB / c mice and placed in a tube containing heparin. The blood samples were centrifuged at 4000 rpm for 10 min at 4 °C and washed three times with PBS until the supernatant was colorless. The purified RBCs were dispersed in 100 μL PBS, and then 10 μL of RBC solution was mixed with 490 μL of nanoparticles (final concentrations of 10 μg / mL, 20 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL, and 400 μg / mL), PBS (negative control), and RBC lysis buffer (positive control). After incubation at 37 °C for 48 h, the RBC solution was centrifuged (6000 rpm, 10 min) and photographed, and then 100 μL of the supernatant was aliquoted into 96-well plates. The absorbance of the supernatant was measured at a wavelength of 570 nm, and the hemolysis rate was calculated using the following formula:

[0114]

[0115] In vivo biocompatibility assessment: Six-week-old BALB / c mice (n=3) were intravenously injected with 200 μg / mL CRUPPA19 (100 μl) (control group received 100 μl PBS). Blood samples and major organs (liver, spleen, heart, lung and kidney) were collected 60 days after injection. Routine blood tests included red blood cell count (RBC), white blood cell count (WBC), platelets (PLT), mean hemoglobin (MCH), mean hematocrit (MCV), hematocrit (HCT), lymphocytes, mean platelet volume (MPV), Mon (%), Gran (%), hemoglobin (HGB) and mean hemoglobin concentration (MCHC). H&E staining of other tissues was performed as described in the previous section.

[0116] Statistical analysis: All statistical analyses were performed using GraphPad Prism 9 software. Multiple group comparisons were performed using ordinary one-way analysis of variance (ANOVA) and post hoc Tukey HSD test, and two-group comparisons were performed using two-tailed Student's t test. Unless otherwise stated, the results are expressed as the mean ± standard deviation (SD) of at least three biological replicates, and P < 0.05 was considered statistically significant.

[0117] Example 1 Ultrasonic Sensitization Nanomaterials of MOFs

[0118] (1) Preparation of CuRhein

[0119] The synthesis process of CuRhein is as follows Figure 1 As shown in A, Rhein (142 mg, 0.5 mmol) was dissolved in 30 mL of anhydrous ethanol, and CuCl2·2H2O (43 mg, 0.25 mmol) was dissolved in 10 mL of anhydrous ethanol, respectively, and the two vials were filled and ultrasonicated until completely dissolved, and then mixed together. After stirring for 10 min, ammonia water was slowly added thereto until the pH value of the mixed solution reached 10. The mixed solution was stirred at 40°C for 8 h and centrifuged. The precipitate was washed with ethanol, ether and water, and then dried in a vacuum oven at 30°C to obtain CuRhein powder.

[0120] (2) Preparation of UiO-66-NH2:

[0121] 75 μL of zirconium n-propoxide solution was dissolved in 4 mL of acetic acid and 7 mL of DMF, mixed in a scintillation vial with an aluminum block, and heated at 130 °C for 2 h to obtain a mixture, to which 81.6 mg of 2-amino-1,4-benzenedicarboxylic acid (BDC-NH2) linker was added, followed by brief ultrasonication for 30 seconds and stirring at 200 rpm / min at 25 °C for 18 h. The product was then centrifuged at 6000 rpm for 15 min, and the precipitate was washed twice with DMF and ethanol, respectively, and then dried in a vacuum oven at 30 °C to obtain UiO-66-NH2 nanoparticles.

[0122] (3) Preparation of CuR@UiO66

[0123] The schematic diagram of the preparation process is as follows Figure 1 As shown in Figure 4, 20 mg of UiO-66-NH2 nanoparticles and 20 mg of CuRhein were mixed in deionized water, then briefly ultrasonicated for 30 seconds and stirred at 200 rpm / min at 25 °C for 18 h, then centrifuged at 6000 rpm for 15 min, and the precipitate was washed twice with DMF and ethanol, respectively, and then dried in a vacuum oven at 30 °C to obtain CuR@UiO66 nanoparticles.

[0124] (4) Preparation of CRUPPA19

[0125] First, mPEG-PO3 was synthesized by dissolving 50 mM phosphorus oxychloride (POCl3) in 5 mL dichloromethane (DCM), then adding 150 mM triethylamine at 0°C to obtain a mixed solution A, then adding 15 mM poly (ethylene glycol) methyl ether (Mn = 5000) to 45 mL DCM to obtain a solution B, mixing the mixed solution A with the solution B, and stirring at room temperature for 10 h, then adding 20 mL deionized water, stirring for 3 h, and then removing DCM in vacuo, purifying mPEG-PO3 by dialysis (molecular weight cutoff 3500), and then freeze-drying to obtain mPEG-PO3 powder;

[0126] Then, anti-CD19 antibody-conjugated CuR@UiO66 was synthesized: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC, 0.01 mM) and N-hydroxysuccinimide (NHS, 0.01 mM) were mixed with anti-CD19 antibody (2 mM) in 2 mL of water and incubated for 30 min to activate the carboxyl group, and an aqueous solution of CuR@UiO66 nanoparticles (2 mM, 2 mL) was added to the above mixture and incubated at 4 °C for 12 h to conjugate the antibody, thereby obtaining an aqueous suspension of anti-CD19 antibody-conjugated CuR@UiO66 (10 mg / mL);

[0127] Finally, CRUPPA19 was synthesized: an aqueous solution of mPEG-PO3 (25 mg / mL, 5 mL) was added to an aqueous suspension of CuR@UiO66 (10 mg / mL, 2 mL) conjugated to anti-CD19 antibody. After stirring at 4°C for 16 h, the mixture containing the product was centrifuged (6000 g) for 20 min to remove unreacted mPEG-PO3 and then further purified by centrifugal filtration at 8000 g (Amicon ultra-15, Millipore) for 30 min to obtain the product CRUPPA19, which was resuspended in PBS and stored at 4°C.

[0128] Comparative Example 1 Synthesis of UPPA19

[0129] The preparation method of UiO-66-NH2 is the same as that in Example 1;

[0130] The preparation method of UPPA19 is consistent with the preparation of CRUPPA19 in Example 1, except that CuR@UiO66 in the preparation process is replaced by UiO-66-NH2.

[0131] Test Example 1

[0132] The structure of CuRhein based on density functional theory (DFT) is shown in Figure 6Cu(II) forms a square planar geometry and coordinates with the 1-hydroxy-9-keto groups of two Rhein molecules. The Fourier transform infrared spectra of Rhein and CuRhein are shown in Figure 7 As shown, 1692cm -1 and 1266cm -1 The peaks at are the vibration stretching of -C=O and -C-OH groups, respectively. The intensity change and position shift of the peaks prove that there may be interaction between Cu(II) and -C=O and -C-OH groups of Rhein. In addition, compared with Rhein, CuRhein shows stronger absorption intensity, almost extending to 750nm, such as Figure 1 As shown in Figure 2, the maximum absorption band of Rhein in the visible light region is at 428 nm, while that of CuRhein is at 480 nm. This obvious red shift can be attributed to the generation of hydroxyanthraquinone diions and the inductive effect of the Cu(II) center. The greater fluorescence intensity of CuRhein also indicates that a larger complex is generated after coordination with Cu(II). Figure 1 C).

[0133] CuR@UiO66 was synthesized in a two-step process: 1) preparing zirconium clusters as secondary building blocks at high temperature and 2) combining these clusters with BDC-NH2 and CuRhein at room temperature ( Figure 1 D), after CuRein was loaded into UiO-66-NH2, the bright yellow color changed to dark yellow and it was well dispersed in the aqueous solution ( Figure 8 ). Transmission electron microscopy (TEM) showed that CuR@UiO66 nanocrystals have a clear octahedral morphology ( Figure 1 E). In addition, dynamic light scattering (DLS) measurements showed that the particle sizes of UiO-66-NH2 and CuR@UiO66 were 230.7±5.4 nm and 240.9±6.1 nm, respectively ( Figure 1 F).

[0134] The powder X-ray diffraction (PXRD) pattern of CuR@UiO66 nanocrystals is similar to that of UiO-66-NH2, indicating that encapsulating CuRhein does not affect the structural integrity of UiO-66-NH2 ( Figure 1 G) The pore structure of CuR@UiO66 nanostructures Fig. 9 As shown in Figure 2, after CuRhein was encapsulated into UiO-66-NH2, the BET surface area of ​​the nanostructure increased from 792 m 2 / g(0.49cm 3 / g) down to 730m 2 / g(0.44cm 3 / g). The elemental mapping of CuR@UiO66 confirmed the presence of Zr, Cu, O, N and C and the successful loading of CuRhein ( Figure 1 H) Fourier transform infrared spectra of UiO-66-NH2 and CuR@UiO66 are shown in Fig.10 .1570cm -1 and 1575cm -1 The peaks at represent carboxyl and Zr 4+ The synergistic effect between -1 and 1260cm -1 The characteristic vibration band of -NH2 appeared at 1629cm -1 and 1285cm -1 Additional absorption peaks of C=O and CO appeared, corresponding to vibration stretching, which is consistent with the incorporation of CuRhein. The same method as in Example 1 was used to adjust the ratio of CuRhein to CuR@UiO66 ( Fig.11 ), when the CuRhein / CuR@UiO66 mass ratio was 0.1:1, the maximum loading efficiency of CuRhein was 98% and the loading capacity was 9.8%. The reason for the relatively high drug loading efficiency was that UiO-66-NH2 had a porous structure.

[0135] To further evaluate the interaction between CuRhein and MOF, the chemical states of UiO-66-NH2, CuRhein, and CuR@UiO66 were characterized using X-ray photoelectron spectroscopy (XPS). Typical test spectra confirmed the presence of C, N, O, Zr, and Cu elements in CuR@UiO66 ( Fig.12 The Zr3d XPS spectrum of CuR@UiO66 shows two distinct peaks at 186.1 and 183.7 eV, respectively, which are attributed to the 3d 3 / 2 and 3D 5 / 2 Hybrid Tracks Figure 1 I). In addition, compared with UiO-66-NH2, the Zr 3d 3 / 2 and 3D 5 / 2 The peak shifted leftward by 0.5 eV, and the N1s peak shifted leftward by 0.2 eV ( Figure 1 J). On the other hand, compared with pure CuRhein, the Cu 2p 1 / 2 and 2p 2 / 3 The peaks shifted to the right by 0.3 eV, reaching 954.5 and 935.0 eV respectively ( Figure 1K). These results indicate that CuR@UiO66 has more Cu 2p electrons compared to CuRhein, while CuR@UiO66 has fewer Zr 3d electrons compared to UiO-66-NH2, which may be due to the electron transfer from UiO-66-NH2 to CuRhein. These results were further verified by photoluminescence (PL) quenching, where the emission peak intensity of CuR@UiO66 was lower than that of UiO-66-NH2 ( Fig.13 ).

[0136] Test Example 2: Sonodynamic mechanism of CuR@UiO66

[0137] Using 2,2,6,6-tetramethylpiperidine (TEMP) as a trapping agent, the electron spin resonance (ESR) spectroscopy was used to measure 1 The production of O2 was tested to test the sonodynamic performance of CuR@UiO66. No O2 was detected in CuRhein, UiO-66-NH2, and CuR@UiO66 without ultrasonic treatment. 1 The characteristic 1:1:1 triplet of O2 was observed, while in ultrasonic treatment (1 W cm -2 , 1MHz, 50% duty cycle, 5min), weak peaks were observed in CuRhein and UiO-66-NH2, indicating that weak 1 On the other hand, a prominent 1:1:1 triplet peak appeared in the ultrasonically treated CuR@UiO66, indicating that the encapsulation of CuRhein into UiO-66-NH2 enhanced the ultrasound-triggered 1 O2 generation ( Figure 2 A, B). These results were respectively obtained by using Singlet Oxygen Sensor Green (SOSG) ( Figure 2 C and Fig.14 ) and 1,3-diphenylisobenzofuran (DPBF) probes ( Figure 2 D and Fig.15 ) was further confirmed. Interestingly, we detected the generation of hydroxyl radicals (·OH) ( Figure 2 E and S11). The amount of ·OH produced by ultrasonically activated CuR@UiO66 was higher than that of other groups.

[0138] In order to further explore the mechanism of the higher sonodynamic performance of CuR@UiO66 compared with CuRhein or UiO-66-NH2, its optical absorption characteristics and band structures were analyzed: the UV-visible diffuse reflectance spectra (DRS) of UiO-66-NH2, CuRhein and CuR@UiO66 showed that the absorption of CuR@UiO66 was significantly red-shifted, which can be attributed to the synergistic effect between UiO-66-NH2 and CuRhein ( Figure 2 F). Therefore, CuR@UiO66 can be easily excited under ultrasonic stimulation because the acoustic emission in water occurs between 250 and 600 nm. Based on the absorption spectra of UiO-66-NH2, CuRhein, and CuR@UiO66, their band gaps were preliminarily determined by the Tauc plot ( Figure 2 G). Mott-Schottky (MS) measurements show that the flat band positions of UiO-66-NH2, CuRhein, and CuR@UiO66 are -0.60, -0.91, and -0.63 eV, respectively, relative to Ag / AgCl. Figure 2 To further understand the electronic structure of CuR@UiO66, we used density functional theory (DFT) to determine its surface charge density ( Figure 2 K). The differential charge density calculation of CuR@UiO66 shows that the electron density around the C atoms (blue) on the surface of UiO-66-NH2 decreases, while the electron density around the Cu atoms (yellow) of CuRhein increases, indicating that electrons are transferred from UiO-66-NH2 to CuRhein. The Z-type mechanism of CuRhein is shown in Figure 2. Figure 2 L. Under ultrasonic stimulation, both CuRhein and UiO-66-NH2 are excited and generate electron-hole pairs. Since the lowest unoccupied molecular orbital (LUMO) potential (-0.81 V for NHE) is more negative than the O2 / -O2- redox potential (-0.33 V for NHE), CuRhein is more likely to generate 1 O2. On the other hand, the highest occupied molecular orbital (HOMO) potential of UiO-66-NH2 (+2.42 V vs. NHE) is more positive than the H2O / ·OH redox potential (+2.38 V vs. NHE), thus favoring the generation of ·OH. In addition, since the HUMO potential of CuRhein is higher than the H2O / ·OH redox potential, CuRhein cannot produce ·OH products. In summary, during ultrasonic treatment, electrons are transferred from UiO-66-NH2 to CuRhein through an efficient electronic pathway, while holes are confined in the HOMO of UiO-66-NH2, thereby achieving efficient spatial separation of electron-hole pairs.

[0139] Test Example 3 Effect of CuR@UiO66 on selectively targeting B lymphoma cells

[0140] In this application, the anti-CD19 antibody is coupled to the amine of UiO66 through the carboxyl group, and the anti-CD19 antibody is combined with CuR@UiO66 to improve the targeting ability of CuR@UiO66 to B lymphoma cells. mPEG-PO3( Figure 3 A) Improved biocompatibility. The resulting CRUPPA19 was tested for stability, biocompatibility, and B lymphoma cell targeting and controlled drug release: DLS measurements showed minimal size changes of CRUPPA19 nanoparticles when cultured in PBS and fetal bovine serum (FBS) at pH 7.4 ( Fig.17 ), indicating that it has high stability in normal tissues. In contrast, in FBS with a pH of 4.5, which simulates the acidic lysosomal environment, the particle size of CRUPPA19 was significantly reduced, indicating that CRUPPA19 may dissociate in lysosomes during autophagy. Scanning electron microscopy also confirmed that CRUPPA19 disassembled at pH 4.5 (Figure S23). In addition, in FBS solution with a pH value of 4.5, CRUPPA19 rapidly released 95.6 ± 4.2% of copper within 24 h, while only 6.6 ± 4.3% and 8.6 ± 7.2% of copper were released in solutions with pH values ​​of 7.4 and 6.5, respectively, indicating that the autophagolysosomal environment can induce the rapid dissolution of CRUPPA19 (Figure S24. To evaluate the biocompatibility of CRUPPA19, primary mouse monocytes were treated with different concentrations of nanoparticles for 48 h. As shown in Figure S25, even a high dose (400 μg / mL) of CRUPPA19 did not cause any cytotoxicity. In addition, no obvious hemolysis was observed after incubation of erythrocytes with different concentrations of CRUPPA19 at 37 °C for 48 h, which confirmed the biocompatibility of these nanoparticles ( Fig.26 ).

[0141] This application used confocal microscopy to evaluate the uptake of Cy3-labeled CRUPPA19 by A20 cells and primary monocytes ( Figure 3B) To determine whether CRUPPA19 can target B lymphoma cells, A20 cells incubated with Cy3-labeled CRUPPA19 showed stronger red fluorescence compared to normal monocytes. In contrast, no fluorescence signal was detected in A20 cells incubated with CRUPP nanoparticles lacking anti-CD19 antibodies. These results clearly show that CRUPPA19 can be selectively taken up by B lymphoma cells with overexpression of CD19. A20 cells treated with CRUPPA19 did not emit any green fluorescence signal, indicating that Rhein was not released from CRUPPA19 nanoparticles in the absence of ultrasound stimulation. In contrast, A20 cells irradiated with CRUPPA19 showed strong green fluorescence, and the fluorescence intensity increased over time, which is consistent with ultrasound-triggered Rhein release ( Figure 3 C).

[0142] These results were also confirmed by quantification of intracellular Zr content by ICP-AES ( Fig. 22 The biodistribution of CRUPPA19 in A20 tumor-bearing mice was evaluated by in vitro fluorescence imaging and ICP-MS measurement of zirconium content in different tissues ( Figure 3 D) CRUPPA19 nanoparticles were labeled with Cy7 for deeper tissue imaging in vivo. Figure 3 As shown in Figure EG, the accumulation of CRUPPA19 in the liver and spleen was relatively low, which may be due to the rapid clearance of CRUPPA19 by the reticuloendothelial system due to the surface PEG coating. This suggests that CRUPPA19 can not only target lymphoma tissues but also enter the bone marrow microenvironment by targeting infiltrating lymphoma cells. Flow cytometric analysis showed that CD19 isolated from bone marrow + There is a strong Cy7 signal in lymphoma cells ( Figure 3 H, I), further confirming the homing ability of CRUPPA19. In conclusion, the high stability, biocompatibility, and lymphoma targeting ability of CRUPPA19 make it an ideal nanomedicine for the treatment of BCL.

[0143] Test Example 4 Cascade Amplification of CRUPPA19 by Ultrasonication in Inducing Lymphoma Cell Death

[0144] To further explore the intracellular effects of CRUPPA19, the generation of ROS was first detected using 2,7-dichlorofluorescent probe (DCFH-DA). Figure 4 As shown in A, PBS, PBS+US, UPPA19 (UiO-66-NH2 containing mPEG-PO3 and anti-CD19 antibody) and CRUPPA19 (1 W cm -2Cells treated with UPPA19 (1MHz, 50% duty cycle, 5min) showed minimal green fluorescence, while the use of UPPA19 or CuRhein in combination with ultrasonic treatment produced only weak fluorescence. The sonodynamic effect of CuRhein in cells is weak because the formation of copper complexes reduces water solubility and cellular uptake. In contrast, A20 cells treated with CRUPPA19 and irradiated emitted strong fluorescence, indicating that a large amount of ROS was generated. Given that excessive ROS generation usually induces cell apoptosis, apoptotic cells in different treatment groups were detected by co-staining with AnnexinV and 7-AminoactinomycinD (7-AAD). Figure 4 As shown in B, the apoptosis rate of cells in the CRUPPA19+US treatment group was 54% (the sum of early apoptosis and late apoptosis), which was significantly higher than that in the other treatment groups. The survival rate of cells in the CRUPPA19+ultrasound treatment group decreased by 97.4% ( Fig.23 ), which indicates that CRUPPA19-mediated SDT may involve other programmed cell death mechanisms besides apoptosis. Autophagy is a self-metabolic process that also contributes to the dissolution of nanomaterials in cells and the subsequent release of active substances (such as drugs, metal ions, etc.). The CRUPPA19-mediated sonodynamic activity of the present application can trigger autophagy, resulting in the release of Cu and Rhein by CRUPPA19 disintegrated in the acidic lysosomal environment (pH 4.5). Rhein can directly bind to m 6 CuA demethylase FTO binds to and inhibits it, leading to the degradation of gene transcripts such as PDL1 and inhibition of downstream pathways. In addition, in the reducing intracellular environment with high glutathione levels, the released Cu 2+ Can be converted into Cu + .Cu + Ion ratio Cu 2+ Ions are more toxic, and Cu is known to + Excessive accumulation of can trigger a type of programmed cell death called copper death. The autophagic flux in appropriately treated cells was quantified using the pCMV-mCherry-GFP-LC3B plasmid reporter system. LC3B is an autophagic marker that is associated with different stages of autophagic flux. It accumulates in autophagosomes and appears as yellow fluorescent dots due to the simultaneous emission of mCherry (red) and GFP (green fluorescence) signals. After autophagy activation, LC3B is localized to autolysosomes, and the produced LC3 protein only emits red fluorescence because the GFP signal is quenched in the acidic lysosomal lumen. Therefore, a decrease in the GFP / RFP ratio indicates an increase in autophagic flux. CRUPPA19 induced autophagy in a dose-dependent manner under ultrasound treatment, while the higher GFP / RFP ratio in the non-sonicated CRUPPA19 group indicated a lack of autophagy induction ( Figure 4C). Consistent with this, the autophagy-specific substrate p62 was significantly downregulated after CRUPPA19+sonication treatment ( Figure 4 D and Fig.24 ), indicating autophagy induction, whereas there was no significant change in the CRUPPA19 group.

[0145] The expression levels of relevant biomarkers were analyzed to determine the expression of Cu in CRUPPA19-treated cells. + Does overload induce copper death? CRUPPA19-mediated SDT led to oligomerization of lipoylated DLAT in a dose-dependent manner, whereas no significant changes were observed in non-sonicated cells ( Figure 4 D and Fig.24 In addition, the iron-sulfur (Fe-S) cluster protein ferredoxin 1 (FDX1) and lipoyl synthase (LIAS) were down-regulated in CRUPPA19+sonicated A20 cells, which may be due to the Cu + Ions destabilize Fe-S cluster proteins during CUP mutation.

[0146] Through epigenetic modifications (such as 6 Post-transcriptional regulation of RNA by A methylation can control gene expression programs that are critical for cancer cell survival, proliferation, and differentiation, thus playing an important role in cancer progression. The expression of PDL1 in appropriately treated A20 cells was analyzed to determine whether CRUPPA19-mediated SDT regulates PDL1: Figure 4 As shown in E, CRUPPA19+sonication treatment significantly reduced the surface protein level of PDL1, indicating that under the stimulation of ultrasound, Rhein was released from CRUPPA19 and inhibited m 6 A demethylase FTO, in addition, m 6 A dot blot ( Figure 4 F)、LC / MS( Figure 4 G) and gene-specific m 6 AqPCR Figure 4 H) shows that CRUPPA19+ sonication increased the global m 6 ARNA methylation reduces the stability of PDL1 transcripts ( Figure 4 In conclusion, CRUPPA19 triggers a cascade of responses to US irradiation in cells, leading to multimodal programmed cell death ( Figure 4 J)

[0147] Test Example 5 CRUPPA19 showed ultrasound immunotherapy effect in BCL mouse model

[0148] In this application, CRUPPA19-mediated multimodal programmed death can induce a systemic immune response against lymphoma. A mouse bilateral tumor model was established using A20 B lymphoma cells ( Figure 5 A20 cells were transplanted into the right abdomen of each mouse to establish a primary tumor. After 24 hours, the cells were inoculated into the left abdomen to establish a secondary or distant tumor that simulates metastasis. When the tumor grew to about 250 mm 3 The mice were randomly divided into 9 groups (n=6 / group) and treated with PBS, PBS+US, UPPA19+US, Rhein, CuRhein+US, anti-PDL1 antibody, anti-CD20 antibody, CRUPPA19 and CRUPPA19+US. The bilateral tumors of mice in the PBS and PBS+US groups grew rapidly, indicating that ultrasound treatment alone could not inhibit lymphoma cells. All mice in these two groups died within 26 days. On the other hand, UPPA19-mediated SDT had an inhibition rate of only 12.3% on the growth of primary tumors, and had a weak effect on distal tumors, resulting in 100% mortality of mice within 31 days after inoculation. Rhein, due to its FTO demethylase inhibitory activity, had an inhibition rate of 22.2% and 16.1% on the growth of primary and secondary tumors, respectively. CuRhein+US treatment resulted in bilateral tumor growth inhibition rates (TGI) of 28.5% and 24.9%, respectively, and prolonged the survival of mice to 35 days, but the therapeutic effect was not ideal due to poor solubility, weak tumor enrichment, and low drug availability. PD1 / PDL1 or CD20 blockade is effective for patients with relapsed / refractory BCL, but the benefit rate remains low, and a considerable number of patients do not respond to these therapies. In fact, the bilateral TGI rates of lymphoma mice treated with anti-PDL1 or anti-CD20 antibodies were 33.4% and 51.1%, respectively, and the survival was prolonged to 35 days and 42 days, respectively (P<0.05). However, the efficacy of PDL1 / CD20 blockade was limited, which may be due to the fact that these two antibodies are more effective against early and middle stage BCL. In addition, CRUPPA19 had no significant inhibitory effect on tumor growth in the absence of ultrasound treatment. However, the combination of CRUPPA19 and ultrasound treatment achieved almost complete primary tumor ablation and inhibited 95.2% of distal tumor growth, indicating that the CRUPPA19-mediated sonodynamic immune response can exert a powerful shedding effect. All mice in this treatment group survived until day 80 ( Figure 5 BF). Pathological staining of tumor tissue further confirmed the in vivo therapeutic effect of CRUPPA19+US. Fig.25 As shown, CRUPPA19+US significantly reduced the percentage of PCNA-positive cells in bilateral tumor tissues compared with the other groups (by 94.1% and 94.8%, respectively), which is consistent with the anti-proliferative effect of the combined treatment.

[0149] A major pathological feature of BCL is the infiltration of lymphoma cells into the bone marrow. The ultrasound immunotherapy mediated by CRUPPA19 in this application can control the infiltration of lymphocytes into the bone marrow through the shedding effect. CRUPPA19+US can significantly reduce the infiltration of CD19+ lymphocytes in the bone marrow, with a decrease of 91.5% ( Figure 5 G,I). These results suggest that CRUPPA19+US can not only ablate primary lymphoma, but also eliminate lymphoma cells that are free and deep in the bone marrow by activating immune responses. In addition, CRUPPA19+US can reverse splenomegaly in tumor-bearing mice, as evidenced by a decrease in spleen weight and infiltration of tumor cells into the red and white pulp ( Figure 5 H, J). The immunotherapeutic mechanism of CRUPPA19 under ultrasound treatment was evaluated by analyzing ICD induction, DC maturation, and T cell infiltration. The expression levels of HMGB1, CRT, and ATP in primary tumor tissues of mice treated with CRUPPA19 and ultrasound were 5 times higher than those in other groups, confirming that CRUPPA19-mediated SDT triggered ICD in tumors ( Figure 5 K, Fig.26 ). DAMPs released by dying tumor cells lead to the activation and maturation of dendritic cells (DCs) in the tumor environment. Consistent with this, CRUPPA19+US significantly increased CD80 + CD86 + The proportion of mature DCs reached 50.9%, the highest among all groups ( Figure 5 L). DC maturation promotes antigen processing and presentation to T cells, leading to the infiltration of cytotoxic T cells. In fact, CRUPPA19 treatment increased CD8 + The intratumoral infiltration of T cells increased to 26.2% ( Figure 5 M). Consistent with this, serum levels of IFN-γ, a characteristic cytokine released by cytotoxic T cells, were highest in the CRUPPA19+US group ( Figure 5 N). The activation of this group of T cells was also attributed to the epigenetic downregulation of PDL1 in lymphoma cells. Figure 5 As shown in O and 5P, the expression of PDL1 in tumor tissues of the CRUPPA19+US group was the lowest compared with other groups. 6 A-mRNA level was the highest. In summary, CRUPPA19-mediated SDT was mediated by global m 6A-mRNA methylation downregulates PDL1, leading to T cell activation, while SDT-driven immunogenic tumor cell death promotes DC maturation and enhances the tumor recognition ability of CD8+ T cells. Finally, the systemic toxicity of CRUPPA19 was evaluated by injecting healthy mice with higher doses of nanoparticles. There was no significant change in the body weight of mice 60 days after injection of CRUPPA19 ( Fig. 27 There were no significant changes in the blood routine indexes and biochemical indicators of mice treated with CRUPPA19, indicating that there was no inflammation and pathological damage ( Fig.28 and 29 In addition, no obvious histological damage was observed in major organs ( Fig.30 ), indicating that it has good biocompatibility.

[0150] The above are only a few embodiments of the present application and do not constitute any form of limitation to the present application. Although the present application is disclosed as above with preferred embodiments, it is not intended to limit the present application. Any technician familiar with the profession, without departing from the scope of the technical solution of the present application, using the technical content disclosed above to make slight changes or modifications are equivalent to equivalent implementation cases and fall within the scope of the technical solution.

Claims

1. A MOFs-based ultrasound-sensitized nanomaterial, characterized in that: Including drug-loaded MOFs; The MOFs are selected from UiO-66-NH2; The UiO-66-NH2 is a particle with amino functional groups connected to UiO-66; The drug-loaded MOFs are loaded with m 6 A-mRNA demethylase inhibitor complex; The m 6 The A-mRNA demethylase inhibitor complex is formed by the coordination of Rhein molecules and divalent copper ions; The outer surface of the drug-loaded MOFs is bound to anti-CD19 antibodies; The outer surface of the drug-loaded MOFs is also modified with mPEG-PO3.

2. The MOFs-based ultrasound-sensitized nanomaterial according to claim 1, characterized in that: The particle size of the drug-loaded MOFs is 200-260 nm.

3. The method for preparing the MOFs-based ultrasound-sensitized nanomaterial according to claim 1 or 2, characterized in that: The steps include: S1. Add ammonia water to a mixture containing Rhein, a soluble divalent copper salt, and an alcohol until the pH of the mixture reaches 10, then stop adding the mixture, stir, separate, wash, and dry to obtain m 6 A-mRNA demethylase inhibitor complex; S2, obtain UiO-66-NH2, and then 6 A mixed solution of the A-mRNA demethylase inhibitor complex and the solvent is stirred, separated, washed, and dried to obtain drug-loaded MOFs; S3, obtain mPEG-PO3 and anti-CD19 antibody; S4. Add a carboxyl activator to an aqueous suspension containing anti-CD19 antibodies, and after activating the carboxyl groups, add an aqueous suspension containing drug-loaded MOFs to react to obtain an aqueous suspension of drug-loaded MOFs with anti-CD19 antibodies bound to the surface, and then add an aqueous solution containing mPEG-PO3, stir, separate, wash, and dry to obtain the ultrasound-sensitized nanomaterial of the MOFs.

4. The preparation method according to claim 3, characterized in that: In step S1, the molar ratio of Rhein to soluble divalent copper salt is 1:0.25-0.75; Preferably, in step S1, the ratio of Rhein to alcohol is 1 mmol: 70-90 mL; Preferably, in step S1, the soluble divalent copper salt is selected from CuCl2; Preferably, in step S1, the alcohol is selected from ethanol; Preferably, in step S1, the stirring conditions include: stirring at 30-50° C. for 6-18 hours.

5. The preparation method according to claim 4, characterized in that: In step S2, the method for obtaining UiO-66-NH2 includes: adding a zirconium n-propoxide solution to a mixture containing acetic acid and DMF, placing the mixture in a sealed container, heating the mixture for reaction, then adding a linker to the mixture, stirring, separating, washing, and drying the mixture to obtain UiO-66-NH2; Preferably, the dosage ratio of zirconium n-propoxide solution to acetic acid is 1 μg: 53-55 mL; Preferably, the ratio of zirconium n-propoxide solution to DMF is 1 μg: 93-95 mL; Preferably, the weight ratio of zirconium n-propoxide solution to the linker is 1 μg: 1.1-1.3 mg; Preferably, the linker is selected from at least one of 2-amino-1,4-phthalic acid and 1,4-phthalic acid ester; Preferably, the conditions for the heating reaction include: a reaction temperature of 120 to 140° C. and a reaction time of 1 to 3 hours.

6. The preparation method according to claim 3, characterized in that: In step S2, UiO-66-NH2, m 6 The weight ratio of the A-mRNA demethylase inhibitor complex is 1:0.1-1.5; Preferably, in step S2, the solvent is selected from water; Preferably, in step S2, the stirring conditions include: ultrasonic treatment for 20 to 40 seconds, and then stirring at a rotation speed of 150 to 300 rpm for 16 to 24 hours.

7. The preparation method according to claim 3, characterized in that: In step S3, the method for obtaining mPEG-PO3 includes: Mix a mixed solution A containing phosphorus oxychloride, dichloromethane and triethylamine with a mixed solution B containing poly(ethylene glycol) methyl ether and dichloromethane, add water, stir, then remove dichloromethane in vacuo, and dialysis purification to obtain mPEG-PO3; Preferably, the content of phosphorus oxychloride in the mixed solution A is 40-60 mmol / L, and the content of triethylamine is 140-160 mmol / L; Preferably, the content of poly(ethylene glycol) methyl ether in the mixed solution B is 14-16 mmol / L; Preferably, the volume ratio of mixed solution A to mixed solution B is 1:8-10 mL.

8. The preparation method according to claim 3, characterized in that: In step S4, the carboxyl activator is selected from at least one of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide; Preferably, in step S4, the time for activating the carboxyl group is 30 to 60 minutes; Preferably, in step S4, the reaction conditions include a reaction temperature of 0 to 5°C and a reaction time of 6 to 18 hours; Preferably, in step S4, the content of the carboxyl activator in the aqueous suspension containing the anti-CD19 antibody is 0.01-0.02 mmol / L, and the content of the anti-CD19 antibody is 1-3 mmol / L; Preferably, in step S4, the content of drug-loaded MOFs in the aqueous suspension containing drug-loaded MOFs is 1 to 3 mmol / L; Preferably, in step S4, the volume ratio of the aqueous suspension containing the anti-CD19 antibody to the aqueous suspension containing the drug-loaded MOFs is 1:0.5-1.

5.

9. The preparation method according to claim 3, characterized in that: In step S4, the content of mPEG-PO3 in the aqueous solution containing mPEG-PO3 is 20-30 mg / mL; Preferably, in step S4, the volume ratio of the water suspension of drug-loaded MOFs with anti-CD19 antibodies bound to the surface to the aqueous solution containing mPEG-PO3 is 1:2-3; Preferably, in step S4, the stirring conditions include: stirring at 0-5° C. for 12-24 h.

10. Use of the MOFs-based ultrasound-sensitized nanomaterial according to claim 1 or 2 or the MOFs-based ultrasound-sensitized nanomaterial obtained according to the preparation method according to any one of claims 3 to 9 in the preparation of a drug for ultrasound-driven cascade immunotherapy of B-cell lymphoma.

Images