ZIF-8 nano-particles loaded with mitoxantrone, preparation and application of ZIF-8 nano-particles in tumor chemical-immunotherapy

Abstract

The invention relates to the technical field of medicines, and provides mitoxantrone-loaded ZIF-8 nanoparticles, a preparation method thereof and an application of the mitoxantrone-loaded ZIF-8 nanoparticles in tumor chemical-immunotherapy. Aiming at the defects of combined chemotherapy and immunotherapy clinically at the present stage, MIT ZIF-8 synthesized by a normal-temperature full-water-phase one-pot method is used as a novel chemical-immunotherapy scheme, and the capability of inducing cell immunogenic death of a chemotherapeutic drug mitoxantrone is enhanced by utilizing pyroptosis; the anti-tumor immune activation capability is enhanced while tumor cells are killed through chemotherapy, effective chemical-immune therapy is achieved, and the responsiveness of tumors to immune checkpoint inhibitor therapy is enhanced.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, relates to ZIF-8 nanoparticles loaded with mitoxantrone and their application in tumor chemo-immunotherapy, and further relates to their application in enhancing the sensitivity of tumors to antibody therapeutic agents. Background Art

[0002] Immunotherapy represented by immune checkpoint blockade has achieved significant and lasting efficacy in the treatment of solid tumors, but only some tumor patients can benefit from immunotherapy. Some chemotherapeutic drugs can induce immunogenic cell death in tumors, release tumor antigens and damage-associated molecular patterns (DAMPs) including high-mobility group protein B1 (HMGB1) and calreticulin (CRT), thereby promoting the activation of anti-tumor immune responses. The combination of chemotherapeutic drugs and immune checkpoint blockers has been successful in clinical trials of malignant tumors such as urothelial carcinoma and non-small cell lung cancer, making chemo-immunotherapy a promising tumor treatment model. However, in immune "cold" tumors such as prostate cancer, the combination of direct chemotherapy and immune checkpoint inhibitors has failed to prolong the survival of patients. For most tumors, especially immune "cold" tumors, direct chemotherapy is difficult to induce sufficient immunogenic stimulation, and the synergistic effect with immunotherapy is limited. Therefore, it is necessary to develop new chemo-immunotherapy strategies that can effectively enhance anti-tumor immune responses.

[0003] Zeolitic imidazolyl framework-8 (ZIF-8) is a metal organic framework nanoparticle composed of zinc ions and 2-methylimidazole (2-MIm) coordination. It has the advantages of high porosity, adjustable pore size, easy preparation, efficient loading of small molecules to biomacromolecules, good biocompatibility and pH-responsive biodegradation. More importantly, ZIF-8 itself can induce cell pyroptosis after entering tumor cells and degradation. Pyroptosis is a highly inflammatory programmed cell death mode that can activate immune cells in the body and promote systemic immune response. In the classical cell pyroptosis pathway, activated caspase-1 (Caspase-1) cleaves gasdermin-D (GSDMD) protein and releases its N-terminal domain (N-GSDMD). N-GSDMD further destroys the cell membrane, leading to the release of cell contents and cell swelling with large blebs. In this process, DAMPs and tumor-specific antigens are rapidly released, which has been shown to have efficient anti-tumor immune effects in vivo.

[0004] The anthracycline chemotherapy drug mitoxantrone (MIT) is an effective topoisomerase II inhibitor that can inhibit DNA synthesis. It is mainly used to treat malignant lymphoma, breast cancer and acute leukemia, and has certain efficacy on various tumors such as lung cancer, prostate cancer, colorectal cancer, liver cancer, and kidney cancer. Mitoxantrone is also a chemotherapy drug with the potential to induce immunogenic cell death, and is one of the ideal chemotherapy drugs for building a chemo-immunotherapy model. Summary of the invention

[0005] The present invention is conducted to solve the above-mentioned problem, and aims to provide a ZIF-8 nanoparticle loaded with mitoxantrone, a preparation method thereof and application in tumor chemo-immunotherapy.

[0006] The technical scheme of the present invention is as follows: In view of the shortcomings of combined chemotherapy and immunotherapy in clinical practice at this stage, MIT@ZIF-8 is synthesized by a one-pot method in an all-aqueous phase at room temperature as a new chemo-immunotherapy scheme, and cell pyroptosis is used to enhance the ability of the chemotherapy drug mitoxantrone to induce cell immunogenic death, thereby enhancing the ability of anti-tumor immune activation while chemotherapy kills tumor cells, achieving effective chemo-immunotherapy, and enhancing the responsiveness of tumors to treatment with immune checkpoint inhibitors.

[0007] Specifically, the first aspect of the present invention provides a ZIF-8 nanoparticle loaded with mitoxantrone, wherein the mass proportion of mitoxantrone is 4%.

[0008] The preparation method of the nanoparticles is as follows:

[0009] (1) preparing a 290 mg / mL 2-methylimidazole aqueous solution, and then adding a 2 mg / mL mitoxantrone aqueous solution thereto at a volume ratio of 1:1 and stirring the mixture evenly under magnetic stirring;

[0010] (2) preparing a zinc nitrate solution with a concentration of 45 mg / mL, adding it into the mixed solution at a volume ratio of 1:16, and stirring the solution magnetically to make it uniform;

[0011] (3) After the synthesis is completed, the product is separated and impurities are removed to prepare a stable product form.

[0012] Preferably, in step (1) and step (2), the speed of magnetic stirring is 1500 rpm; in step (2), the magnetic stirring time is 30 min; in step (3), after the synthesis is completed, the product is centrifuged at 20000 g, the supernatant is removed, and the product is resuspended and washed twice with deionized water to remove free raw materials, and then resuspended into an aqueous solution or vacuum dried into a powder for later use.

[0013] Scanning electron microscopy showed that the synthesized MIT@ZIF-8 particles were rhombic dodecahedrons with a particle size of about 150 nm. XRD detection showed that MIT@ZIF-8 had the crystal structure of ZIF-8. FTIR detection results showed that a new absorption peak corresponding to the C=O stretching vibration of MIT was found in the detection results of MIT@ZIF-8, which shows that the prepared MIT@ZIF-8 nanoparticles contain MIT. Drug release test results showed that the prepared MIT@ZIF-8 nanoparticles can effectively release the loaded MIT under acidic conditions.

[0014] The results of in vitro cell experiments showed that in colon cancer cells, MIT@ZIF-8 can induce the upregulation of the expression of molecules in the cell pyroptosis signaling pathway, and the induced pyroptosis effect is more obvious; MIT@ZIF-8 can induce the strongest immunogenic cell death in colon cancer cells and prostate cancer cells at the same time, and the synthesized MIT@ZIF-8 nanoparticles can produce a significantly stronger anti-cancer effect than MIT. At the same time, MIT@ZIF-8 treatment makes prostate cancer respond to anti-CTLA-4 antibody treatment.

[0015] Therefore, the third aspect of the present invention provides the use of the above-mentioned ZIF-8 nanoparticles loaded with mitoxantrone in the preparation of tumor chemo-immunotherapy drugs.

[0016] In the specific implementation of the present invention, prostate cancer or colon cancer cells are used for experimental verification. However, the tumor application scope of the present invention is not limited to these two tumors, but also applicable to other immune "cold" tumors, such as ovarian cancer, prostate cancer, pancreatic cancer, glioblastoma, etc.

[0017] In a fourth aspect, the present invention provides a tumor chemo-immunotherapy drug or an enhancer for enhancing the responsiveness of prostate cancer to CTLA-4 antibody treatment, which is composed of the above-mentioned ZIF-8 nanoparticles loaded with mitoxantrone and pharmaceutically acceptable excipients.

[0018] The medicine or pharmaceutical composition of the present invention can be prepared into any dosage form with commonly used pharmaceutical excipients, such as common tablets, capsules, granules, injections, decoctions, powders or pills.

[0019] Functions and Effects of the Invention

[0020] The present invention confirms through in vitro pharmacodynamic experiments that MIT@ZIF-8 can induce upregulation of the expression of molecules in the cell pyroptosis signaling pathway in prostate cancer cells and colon cancer cells, and the induced pyroptosis effect is more obvious; MIT@ZIF-8 can induce the strongest immunogenic cell death in prostate cancer cells and colon cancer cells at the same time, and the synthesized MIT@ZIF-8 nanoparticles can produce significantly stronger anti-cancer effects than MIT. At the same time, MIT@ZIF-8 treatment makes prostate cancer respond to anti-CTLA-4 antibody treatment. Therefore, the present invention utilizes cell pyroptosis to enhance the ability of the chemotherapy drug mitoxantrone to induce cell immunogenic death, enhances the ability of anti-tumor immune activation while killing tumor cells with chemotherapy, realizes effective chemo-immunotherapy, and enhances the responsiveness of tumors to immune checkpoint inhibitor treatment, providing a new research idea for the treatment of immune "cold" tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The transmission electron microscopy results of the prepared ZIF-8 and MIT@ZIF-8 nanoparticles are shown;

[0022] Figure 2 The XRD test results of the prepared ZIF-8 and MIT@ZIF-8 nanoparticles are shown;

[0023] Figure 3 The prepared ZIF-8 and MIT@ZIF-8 nanoparticles and the FTIR detection results of MIT are shown;

[0024] Figure 4 The MIT release profiles of MIT@ZIF-8 nanoparticles in PBS at pH 7.4 and pH 5.0 are shown;

[0025] Figure 5 The western blot results of cell pyroptosis markers are shown;

[0026] Figure 6 Quantitative detection of HMGB1 release in the supernatants of RM-1 and CT26 cells is shown;

[0027] Figure 7 The exposure of CRT on the surface of RM-1 and CT26 cells is shown;

[0028] Figure 8 The in vivo inhibitory effects of ZIF-8, MIT, and MIT@ZIF-8 on CT26 colon cancer cells were shown;

[0029] Fig. 9 The in vivo inhibitory effects of MIT@ZIF-8 and anti-CTLA-4 antibody on RM-1 prostate cancer cells were shown. DETAILED DESCRIPTION

[0030] The present invention is described in detail below in conjunction with the embodiments and the accompanying drawings. However, the following embodiments should not be considered as limiting the scope of the present invention.

[0031] The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturers.

[0032] Example 1. Preparation and characterization of MIT@ZIF-8 and ZIF-8 nanoparticles

[0033] 1. Preparation of MIT@ZIF-8 Nanoparticles

[0034] (1) Dissolve 2-MIm powder (2-methylimidazole, purchased from Aladdin) in deionized water to prepare an aqueous solution of 2-MIm with a concentration of 290 mg / mL;

[0035] (2) Prepare a 10 mg / mL MIT (mitoxantrone, purchased from Solebol) aqueous solution with deionized water, take 0.8 mL of the MIT aqueous solution and 3.2 mL of deionized water, add 4 mL of the 2-MIm aqueous solution in step 1, and mix by magnetic stirring at 1500 rpm;

[0036] (3) Dissolve zinc nitrate hexahydrate powder (purchased from Sigma) in deionized water to prepare a zinc nitrate solution with a concentration of 45 mg / mL. Take 0.5 mL of this solution and add it dropwise to the mixed solution in step 2, and continue to maintain magnetic stirring at 1500 rpm for 30 minutes to synthesize MIT@ZIF-8 nanoparticles;

[0037] (4) After the synthesis is completed, the product is centrifuged at 20,000 g, the supernatant is removed, and the product is resuspended and washed twice with deionized water to remove free raw materials. The product is resuspended into an aqueous solution or vacuum dried into a powder for later use.

[0038] 2. Preparation of ZIF-8

[0039] The difference is in the MIT@ZIF-8 preparation step (2): 0.8 mL of MIT aqueous solution is replaced by an equal volume of deionized water, and the other conditions are the same as the MIT@ZIF-8 preparation step.

[0040] 3. Performance Characterization

[0041] 1. Scanning electron microscope: The synthesized ZIF-8 and MIT@ZIF-8 were tested by scanning electron microscope. The test results are shown in Figure 1 :ZIF-8 and MIT@ZIF-8 have similar appearances, both of which are approximately rhombic dodecahedrons, and their particle sizes are also close, about 150nm. It can be seen that MIT@ZIF-8 nanoparticles are obtained.

[0042] 2. XRD detection: XRD detection was performed on the synthesized ZIF-8 and MIT@ZIF-8. The test results are shown in Figure 2 , it can be seen that MIT@ZIF-8 has the crystal structure of ZIF-8.

[0043] 3. FTIR detection: The synthesized ZIF-8 and MIT@ZIF-8 particles, as well as MIT, were subjected to FTIR detection. The test results are shown in Figure 3 In the detection results of MIT@ZIF-8, a new absorption peak corresponding to the C=O stretching vibration of MIT was found, which shows that the prepared MIT@ZIF-8 nanoparticles ZIF-8 contain MIT.

[0044] 4. Ph-responsive drug release detection: The prepared MIT@ZIF-8 nanoparticles were tested for the release of the chemotherapy drug MIT in an acidic environment of pH 5 and a neutral environment of pH 7.4. The results are shown in Figure 4 The prepared MIT@ZIF-8 nanoparticles can effectively release the loaded MIT under acidic conditions.

[0045] Example 2: Verification of efficacy

[0046] 1: Characterization of MIT@ZIF-8-induced tumor cell pyroptosis in vitro

[0047] Mouse prostate cells RM-1 (purchased from the Cell Bank of the Chinese Academy of Sciences) and mouse colon cancer cells CT26 (purchased from the Cell Bank of the Chinese Academy of Sciences) in the logarithmic growth phase were digested with trypsin to prepare single cell suspension. 6 After the cells adhered to the wall, the cells were inoculated with solvent, MIT (0.6 μg mL -1 )、ZIF-8(15μg·mL -1 ) and MIT@ZIF-8 (15 μg mL -1 ) for 24 hours, the cell proteins of each group were collected and the expressions of activated caspase1 (caspase1 p20) and activated GSDMD (GSDMD-NT) were detected by western blot.

[0048] Test results see Figure 5 Both ZIF-8 and MIT@ZIF-8 can induce the upregulation of the expression of molecules in the cell pyroptosis signaling pathway, but the pyroptosis induced by MIT@ZIF-8 is more obvious.

[0049] 2: Characterization of immunogenic cell death induced by MIT@ZIF-8 in vitro

[0050] Mouse prostate cancer cells RM-1 (purchased from the Cell Bank of the Chinese Academy of Sciences) and mouse colon cancer cells CT26 (purchased from the Cell Bank of the Chinese Academy of Sciences) in the logarithmic growth phase were digested with trypsin to prepare single cell suspensions. 6 After the cells adhered to the wall, the cells were inoculated with solvent, MIT (0.6 μg mL -1 )、ZIF-8(15μg·mL -1 )、MIT@ZIF-8(15μg·mL -1 ) cells. After 24 hours, the cell supernatant was collected and centrifuged at 5000g to remove cell debris. The release of HMGB1 in the cell supernatant was detected by ELISA kit (purchased from Elabscience). The collected treated cells were immunostained with AF488 fluorescently labeled CRT antibody (purchased from Abcam), and then flow cytometry was used to detect the AF488 fluorescence intensity on the cell membrane surface to reflect the exposure of CRT on the cell membrane. The test results are shown in Figure 6 and Figure 7 , thus, it can be seen that MIT@ZIF-8 induces the strongest immunogenic cell death.

[0051] 3: The therapeutic effect of MIT@ZIF-8 in colon cancer model

[0052] 1×10 6 CT26 cells (purchased from the Chinese Academy of Sciences Cell Bank) were injected subcutaneously into Balb / c mice to establish a CT26 mouse tumor model. When the tumor size reached 50 mm on day 7, 3 The mice were randomly divided into 4 groups (5-7 mice in each group): control group, MIT group (20 μg), ZIF-8 group (500 μg) and MIT@ZIF-8 group (500 μg). Intratumoral drug injection was performed on the 7th and 10th days respectively. The tumor volume was monitored and calculated by the formula: length × width 2 / 2. The results are shown in Figure 8 As shown, the ZIF-8 carrier itself is not sufficient to produce significant tumor inhibitory ability, but the synthesized MIT@ZIF-8 nanoparticles can produce significantly stronger tumor inhibitory effects than MIT.

[0053] MIT@ZIF-8 sensitizes prostate cancer to anti-CTLA-4 immunotherapy

[0054] 3×10 5 RM-1 cells (purchased from the Chinese Academy of Sciences Cell Bank) were injected subcutaneously into C57BL / 6J mice to establish the RM-1 mouse tumor model. When the tumor volume reached 50 mm on day 8, 3The mice were further randomly divided into a control group, a MIT@ZIF-8 group, an anti-CTLA-4 group, and a MIT@ZIF-8+anti-CTLA-4 group (5 mice in each group). On days 8 and 11, 100 μL of MIT@ZIF-8 (500 μg / mouse) was injected intratumorally. On days 14, 16, and 18, anti-CTLA-4 antibody (200 μg / mouse) was injected intraperitoneally. The tumor volume was monitored and calculated using the formula: length × width 2 / 2. For experimental results, see Fig. 9 Similar to the clinical trial data, CTLA-4 alone had no significant therapeutic effect on RM-1 prostate cancer, while MIT@ZIF-8 treatment made prostate cancer respond to anti-CTLA-4 antibody treatment.

[0055] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions are only for explaining the principles of the present invention. The present invention may be subject to various changes and improvements without departing from the spirit and scope of the present invention. These changes and improvements fall within the scope of the present invention. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. A ZIF-8 nanoparticle loaded with mitoxantrone, characterized in that: The mass proportion of mitoxantrone is 4%.

2. The mitoxantrone-loaded ZIF-8 nanoparticles according to claim 1, characterized in that: The preparation method is as follows: (1) preparing a 290 mg / mL 2-methylimidazole aqueous solution, and then adding a 2 mg / mL mitoxantrone aqueous solution thereto at a volume ratio of 1:1 and stirring the mixture evenly under magnetic stirring; (2) preparing a zinc nitrate solution with a concentration of 45 mg / mL, adding it into the mixed solution at a volume ratio of 1:16, and stirring the solution magnetically to make it uniform; (3) After the synthesis is completed, the product is separated and impurities are removed to prepare a stable product form.

3. The mitoxantrone-loaded ZIF-8 nanoparticles according to claim 1, characterized in that: in, In step (1) and step (2), the rotation speed of magnetic stirring is 1500 rpm; in step (2), the magnetic stirring time is 30 min; In step (3), after the synthesis is completed, the product is centrifuged at 20,000 g, the supernatant is removed, and the product is resuspended and washed twice with deionized water to remove free raw materials, and then resuspended into an aqueous solution or vacuum dried into a powder for later use.

4. Application of mitoxantrone-loaded ZIF-8 nanoparticles in the preparation of tumor chemo-immunotherapy drugs, characterized in that: The tumor is selected from prostate cancer or colon cancer.

5. The use according to claim 4, characterized in that: in, The mass proportion of mitoxantrone is 4%.

6. The use according to claim 4, characterized in that: in, The preparation method of ZIF-8 nanoparticles loaded with mitoxantrone is as follows: (1) preparing a 290 mg / mL 2-methylimidazole aqueous solution, and then adding a 2 mg / mL mitoxantrone aqueous solution thereto at a volume ratio of 1:1 and stirring the mixture evenly under magnetic stirring; (2) preparing a zinc nitrate solution with a concentration of 45 mg / mL, adding it into the mixed solution at a volume ratio of 1:16, and stirring the solution magnetically to make it uniform; (3) After the synthesis is completed, the product is separated and impurities are removed to prepare a stable product form.

7. The use according to claim 6, characterized in that: in, In step (1) and step (2), the rotation speed of magnetic stirring is 1500 rpm; in step (2), the magnetic stirring time is 30 min; Wherein, in step (3), after the synthesis is completed, the product is centrifuged at 20000g, the supernatant is removed, and then the product is resuspended and washed twice with deionized water to remove free raw materials, and then resuspended into an aqueous solution or vacuum dried into a powder for later use.

8. A tumor chemo-immunotherapy drug, characterized in that: The invention comprises ZIF-8 nanoparticles loaded with mitoxantrone and pharmaceutically acceptable excipients. The ZIF-8 nanoparticles loaded with mitoxantrone are prepared by the method described in any one of claims 5 to 7.

9. An accelerator for improving the responsiveness of prostate cancer to CTLA-4 antibody treatment, characterized in that: The invention comprises ZIF-8 nanoparticles loaded with mitoxantrone and pharmaceutically acceptable excipients. The ZIF-8 nanoparticles loaded with mitoxantrone are prepared by the method described in any one of claims 5 to 7.

10. The sensitizer according to claim 9, characterized in that: in, The prostate cancer is RM-1 prostate cancer.

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