An immune-enhancing targeted hollow manganese dioxide radiotherapy sensitizer and its preparation method and application

Abstract

The present invention discloses an immune-enhancing targeted hollow manganese dioxide radiotherapy sensitizer and its preparation method and application, which belongs to the field of tumor treatment technology. Aiming at the problems of short circulation time and poor targeting of radiotherapy sensitizers, the present invention adopts an engineered extracellular vesicle modification method to improve the circulation time, so that it has tumor targeting; Aiming at the problem of poor improvement effect of hypoxic microenvironment in the prior art, the present invention prepares hollow structure MnO2 by template self-etching method to improve the catalytic efficiency of overexpressed hydrogen peroxide in tumor cells, and uses metformin, a cell respiration inhibitory drug, in synergy to reduce cell oxygen consumption, improve hypoxic microenvironment from two aspects, and enhance radiotherapy sensitization effect; At the same time, the present invention effectively regulates the immunosuppressive microenvironment of tumors by reducing manganese ions generated by sensitizers, and activates the cGAS-STING pathway to enhance the activity of natural immune cells, release immune factors, and inhibit the metastasis and recurrence of tumors after radiotherapy.

Description

Technical Field

[0001] The invention belongs to the technical field of tumor treatment, and in particular relates to an immune-enhancing targeted hollow manganese dioxide radiotherapy sensitizer and a preparation method and application thereof. Background Art

[0002] Cancer has become the number one killer of human health in the 21st century. Currently, most patients with malignant tumors in clinical practice are treated with four methods: surgery, chemotherapy, radiotherapy, and immunotherapy. Among them, radiotherapy has the advantages of wide application range, rapid effect, and non-invasiveness. Therefore, more than 50% of patients with malignant tumors in clinical practice need to receive radiotherapy alone or in combination. However, due to the rapid growth of malignant tumors, the blood oxygen supply in the tumor tissue is relatively insufficient, which leads to the hypoxic microenvironment of the tumor, making it easy for tumor patients to develop radiotherapy resistance after several cycles of radiotherapy. On the other hand, the immunosuppressive microenvironment characteristics of tumor tissue make it easy for residual tumor cells to metastasize and recur after radiotherapy. According to surveys, more than 90% of cancer patients die from metastasis and recurrence after treatment. Therefore, how to improve the hypoxic microenvironment and immunosuppressive microenvironment of the tumor has become the key to improving the effect of radiotherapy.

[0003] The emergence of radiosensitizers has brought hope for improving the hypoxic microenvironment of tumors and solving the problem of radiotherapy resistance. Currently, radiosensitizers can be divided into three categories according to their structure: small molecule radiosensitizers, macromolecule radiosensitizers and nanomaterial radiosensitizers. Among them, nanomaterial sensitizers have shown great potential in tumor radiosensitization due to their high sensitization efficiency, easy functional modification, stable and controllable structure, and the ability to be combined with other cancer therapies for synergistic treatment.

[0004] In recent years, manganese dioxide (MnO2) has attracted widespread attention as a tumor microenvironment-responsive radiosensitizer. Studies have found that MnO2 can catalyze excess hydrogen peroxide in tumor cells to produce oxygen, thereby improving the tumor hypoxic microenvironment and enhancing the effect of radiotherapy; in addition, MnO2 can also be reduced by GSH overexpressed by tumor cells to generate Mn ions, thereby effectively regulating the tumor's immunosuppressive microenvironment, activating the host's own immune system, and inhibiting the metastasis and recurrence of tumors after radiotherapy.

[0005] At present, there are literature reports on the use of MnO2-based nanocomposites for cancer treatment, but they have the following disadvantages: (1) The catalytic efficiency of MnO2 is low and the oxygen produced by unilateral catalysis is easily consumed by the respiration of tumor cells. (2) The circulation time of MnO2 is short and the targeting is poor. (3) The potential anti-metastasis and recurrence ability of MnO2 has not been studied. There has been no report on the preparation of a radiosensitizer with a long circulation time, which can target tumor cells, increase the production of oxygen in tumor cells and reduce oxygen consumption, thereby achieving cancer radiosensitization and anti-tumor metastasis and recurrence. Summary of the invention

[0006] One of the purposes of the present invention is to provide a method for preparing an immune-enhancing targeted hollow manganese dioxide radiotherapy sensitizer, the preparation method comprising the following steps:

[0007] Step 1A) A methanol solution of Zn(NO3)2·6(H2O) (293.4 mg Zn(NO3)2·6(H2O) was poured into a methanol solution of 2-methylimidazole (649 mg 2-methylimidazole was poured into a 20 mL methanol solution), stirred for 1 hour, then centrifuged at 10000× rpm for 5 minutes at room temperature, and washed three times with methanol (centrifuged at 10000× rpm for 5 minutes) to obtain ZIF-8 nanoparticles.

[0008] Step 1B) The ZIF-8 nanoparticles obtained in step 1A) were dispersed in 15 mL of methanol and 20 mL of Tris-HCl (10 mmol mL -1 , pH = 8.5), then dopamine was added under stirring, stirred for 8 hours, centrifuged at room temperature, and washed with methanol to obtain ZIF-8@PDA nanoparticles;

[0009] Step 1C) The ZIF-8@PDA obtained in step 1B) was dispersed in 15 mL of water, and 8.25 mL of KMnO4 aqueous solution was added dropwise under stirring to carry out redox reaction. After stirring for 5 minutes, the mixture was centrifuged at room temperature and washed with water. The mixture was then suspended in ultrapure water under light conditions for 24 hours to obtain hollow MnO2, i.e., HollowMnO2 (HMn) nanoparticles. The obtained HMn solution was centrifuged at room temperature and washed with water, and vacuum dried to obtain HMn powder, which was then resuspended in ultrapure water to prepare 2 mg mL -1 of stock solution.

[0010] Step 2A) Mouse macrophages were maintained in a 37°C, 5% CO2 cell culture incubator. The cells were cultured in DMEM medium containing 10% FBS and 1% penicillin-streptomycin. When the cell density reached about 60-80%, the cells were washed with PBS and incubated with medium without FBS for 12 hours.

[0011] Step 2B) The cell culture medium was collected and centrifuged to remove the precipitate and cells. The collected supernatant was centrifuged at 1000×g for 10 minutes to remove apoptotic bodies and debris, and the supernatant was centrifuged again at 10,000×g for 30 minutes to remove larger microvesicles. The supernatant was passed through a 0.22 μm filter to further remove residual microvesicles, and then the supernatant was collected and centrifuged at 100,000×g for 2 hours to obtain extracellular vesicles (EVs), which were quantified using a nanoparticle tracking analyzer (NTA) and then prepared to a concentration of 1×10 13 mL -1 EVs solution, engineered extracellular vesicles (EVs-RGD) were prepared at 1×10 13 mL -1 EVs and 40-60 μg mL -1 Distearoylphosphatidylethanolamine-polyethylene glycol-arginyl-glycyl-aspartic acid (DSPE-PEG-RGD) was obtained after co-incubation for 24 h at a volume ratio of 2:1;

[0012] Step 3A) The 2 mg mL obtained in step 1C -1 5 mL of HMn nanoparticle aqueous solution was added to 5 mL of metformin aqueous solution (10 mg mL -1 ), stirred at room temperature in the dark for 24 hours, and washed three times with water to obtain a hollow MnO2@Met (HMn@Met) aqueous solution; the HMn@Met aqueous solution was then vacuum dried to obtain HMn@Met powder, which was then resuspended in ultrapure water to prepare 1 mg mL -1 Stock solution;

[0013] Step 3B) Mix the PBS solution of EVs-RGD obtained in step 2B) with the prepared 1 mg / mL -1 The HMn@Met aqueous solution was mixed at a volume ratio of 1:1, incubated for 24 hours, and then extruded through a 0.22 μm membrane to obtain a Hollow MnO2@Met@EVs-RGD (HMnER@Met) solution.

[0014] Preferably, the amount of dopamine added in step 1B) is 100 mg.

[0015] More preferably, it is characterized in that the concentration of KMnO4 aqueous solution in step 1C) is 5 mg / mL -1 .

[0016] More preferably, the concentration of DSPE-PEG-RGD in step 2B) is 50 μg mL -1 .

[0017] More preferably, the particle size of HollowMnO2 (HMn) in step 1C) is 40-60 nm.

[0018] The second object of the present invention is to provide an immune-enhancing targeted hollow manganese dioxide radiotherapy sensitizer prepared by the above preparation method.

[0019] The third object of the present invention is to provide the use of the above-mentioned immune-enhancing targeted hollow manganese dioxide radiosensitizer in the preparation of radiosensitization drugs.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] (1) The HMn of the present invention is loaded with metformin, a drug used to treat diabetes. Metformin can significantly inhibit the respiration process of mitochondria, reduce the intracellular oxygen consumption rate, and synergize with the effect of HMn catalyzing H2O2 to produce O2, thereby greatly improving the hypoxic microenvironment of tumors in a "increasing revenue and reducing expenditure" manner; in addition, metformin blocks the tumor cell cycle at the G2-M phase, and also plays a significant role in improving radiosensitization.

[0022] (2) In the present invention, HMn@Met is coated on the engineered macrophage-derived EVs, giving them the ability of targeting and long circulation.

[0023] (3) In the present invention, the Mn ions generated by the reduction of HMn by GSH can effectively regulate the immunosuppressive microenvironment of the tumor, activate the cGAS-STING pathway, enhance the activity of natural immune cells, release immune factors, and inhibit the metastasis and recurrence of tumors after radiotherapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is the technical roadmap of the present invention.

[0025] Figure 2 TEM characterization diagram of the particle size of each stage of HMnER in Experimental Example 1.

[0026] Figure 3 This is a characterization diagram of the average hydrated particle size of each stage of HMnER in Experimental Example 1.

[0027] Figure 4 This is a characterization diagram of the Zeta potential of each stage of HMnER in Experimental Example 1.

[0028] Figure 5 The stability analysis results of HMnER in Experimental Example 1.

[0029] Figure 6Figure 6 is a graph showing the results of HMnER@Met sensitizing treatment of tumors in Experiment 2, where 6(A) is a diagram showing the treatment plan, 6(B) is a diagram showing the tumor after 8 groups of treatments, 6(C) is a curve showing the changes in mouse weight, and 6(D) is a curve showing the changes in mouse tumor volume.

[0030] Figure 7 The results of the evaluation of the anti-recurrence and lung metastasis efficacy of HMnER@Met in vivo in Experiment 3, wherein 7(A) is a diagram of the anti-recurrence and metastasis scheme, 7(B) is a picture of the induced tumors in the two groups, 7(C) is a comparison of the tumor weights in the two groups, 7(D) is a picture of the lungs in the two groups, and 7(E) is a comparison of the lung weights of the two groups of mice. DETAILED DESCRIPTION

[0031] Example 1

[0032] This embodiment provides a method for preparing an immune-enhancing targeted synergistic radiotherapy sensitizer, the preparation method comprising the following steps:

[0033] Step 1A) A methanol solution of Zn(NO3)2·6(H2O) (293.4 mg Zn(NO3)2·6(H2O) was poured into a methanol solution of 2-methylimidazole (649 mg 2-methylimidazole was poured into a 20 mL methanol solution), stirred for 1 hour, then centrifuged at 10000× rpm for 5 minutes at room temperature, and washed 3 times with methanol (centrifuged at 10000× rpm for 5 minutes) to obtain ZIF-8 nanoparticles;

[0034] Step 1B) The ZIF-8 nanoparticles obtained in step 1A) were dispersed in 15 mL of methanol and 20 mL of Tris-HCl (10 mmol mL -1 , pH = 8.5), then add 100 mg of dopamine under stirring, stir for 8 hours, centrifuge at room temperature, and wash with methanol to obtain ZIF-8@PDA nanoparticles;

[0035] Step 1C) Disperse the ZIF-8@PDA obtained in step 1B) in 15 mL of water, and add 8.25 mL of KMnO4 (5 mg mL -1 ) aqueous solution for redox reaction, stirred for 5 minutes, centrifuged at room temperature, washed with water, and then suspended in ultrapure water under light conditions for 24 hours to obtain hollow MnO2, namely HollowMnO2 (HMn) nanoparticles. The obtained HMn solution was centrifuged at room temperature and washed with water, vacuum dried to obtain HMn powder, and then resuspended in ultrapure water to prepare 1 mg mL -1 of stock solution.

[0036] Step 2A) Mouse macrophages were maintained in a 37°C, 5% CO2 cell culture incubator. The cells were cultured in DMEM medium containing 10% FBS and 1% penicillin-streptomycin. When the cell density reached about 70%, the cells were washed with PBS and incubated with medium without FBS for 12 hours.

[0037] Step 2B) The cell culture medium was collected and centrifuged at 300 × g for 10 minutes to remove the precipitate and cells. The collected supernatant was centrifuged at 1000 × g for 10 minutes to remove apoptotic bodies and debris, and the supernatant was centrifuged again at 10,000 × g for 30 minutes to remove larger microvesicles. The supernatant was passed through a 0.22 μm filter to further remove residual microvesicles, and then the supernatant was collected and centrifuged at 100,000 × g for 2 hours to obtain extracellular vesicles (EVs), which were quantified using a nanoparticle tracking analyzer (NTA) and then prepared to a concentration of 1×10 13 mL -1 EVs solution, engineered extracellular vesicles (EVs-RGD) were prepared at 1×10 13 mL - 1 EVs and 50 μg mL -1 Distearoylphosphatidylethanolamine-polyethylene glycol-arginyl-glycyl-aspartic acid (DSPE-PEG-RGD) was obtained after co-incubation for 24 h at a volume ratio of 2:1. The molecular weight of PEG in DSPE-PEG-RGD was 2000;

[0038] Step 3A) The PBS solution of EVs-RGD obtained in step 2B) was mixed with the prepared HMn aqueous solution (1 mg mL -1 ) were mixed at a volume ratio of 1:1, incubated for 24 hours, and then extruded through a 0.22 μm membrane to obtain Hollow MnO2@EVs-RGD, i.e., HMnER solution.

[0039] Test Example 1

[0040] This test example tests the relevant properties of the HMnER prepared in Example 1:

[0041] 1) The various stages of HMnER prepared in Example 1 were characterized using a transmission electron microscope (TEM). Figure 2 As shown, Figure 2 A, B, C, and D represent ZIF-8, ZIF-8@PDA, HMn, HMnER, and the particle size of HMnER is about 100 nm.

[0042] 2) Dynamic light scattering (DLS) was used to characterize the particle size of each stage of HMnER prepared in Example 1. The results are as follows: Figure 3 As shown, Figure 3 A, B, C, and D represent ZIF-8, ZIF-8@PDA, HMn, HMnER, and the average hydrated particle size of HMnER is 142 nm, respectively.

[0043] 3) The Zeta potential of each phase of the HMnER prepared in Example 1 was characterized using a dynamic light scattering instrument (DLS). The results are as follows: Figure 4 As shown, the Zeta potential of HMnER is -35.3 mV.

[0044] 4) The stability of each stage of HMnER prepared in Example 1 was analyzed. The results are as follows: Figure 5 As indicated, 40 μg mL -1 The particle size of HMnER remained unchanged after being stored at room temperature for 7 days.

[0045] Test Example 2

[0046] This test example evaluates the tumor inhibition effect of radiosensitizer in vivo. The method is as follows: the tumor is inoculated in the right leg of BALB / cA-nu nude mice. Each mouse is inoculated with MCF-7 cells. When the tumor volume reaches 100mm 3 , treatment was started. MCF-7 tumor-bearing mice were randomly divided into 8 groups (n = 3 in each group): Control, Met, HMnER, HMnER@Met, RT (radiotherapy), Met+RT, HMnER+RT, HMnER@Met+RT. The composite material was injected intravenously four times, 200 μL*1 mg mL each time -1 The group receiving combined radiotherapy received radiotherapy (5 Gy) on the second day after injection, and then the tumor volume and mouse body weight were measured every 2 days. Tumor volume was calculated as volume = (tumor length) × (tumor width) 2 / 2. Figure 6 (A) is a treatment plan diagram. Figure 6 (B) is a picture of the tumor after 8 groups of treatment, 6 (C) is a curve of mouse weight change, and 6 (D) is a curve of mouse tumor volume change. The tumor growth of mice treated with the radiotherapy sensitizer combined with radiotherapy of the present invention was significantly inhibited, which was significantly different from the single radiotherapy group, indicating that the composite nanoparticles have a good radiotherapy sensitization effect.

[0047] Test Example 3

[0048] In this test example, the anti-recurrence and lung metastasis efficacy of the radiosensitizer prepared in Example 1 was evaluated in vivo. The method was as follows: First, two groups were set up, a control group and a treatment group, and tumor cells were injected respectively. After induction of tumor formation, the control group was treated with PBS+RT, and the treatment group was treated with HMnER@Met+RT. On the fifth day after the tumors in both groups were completely eliminated, the anti-recurrence and metastasis effects were evaluated. MCF-7 tumor cells (5×10 6 The cells were suspended in 0.1 mL of PBS and injected subcutaneously into the right hind limb of each mouse on the 5th day after the completion of treatment in the control group and the treatment group to induce recurrent tumors. The size of the recurrent tumors was observed on the 21st day. Compared with the control group, the recurrent tumor volume in the treatment group was significantly smaller than that in the control group.

[0049] For the anti-metastasis model, MCF-7 tumor cells (1×10 6 The mice were injected with 100 cells (100 μg / mL) of 50 μg / mL of 10 μg / mL of 2 ... Figure 7 (A) is a diagram of the anti-recurrence and metastasis plan. Figure 7 (B) are pictures of tumors induced in the two groups. Figure 7 (C) is a comparison of tumor weights of the two groups, 7(D) is a picture of the lungs of the two groups, and 7(E) is a comparison of lung weights of the two groups of mice. The radiotherapy sensitizer of the present invention can enhance the immune response in vivo and significantly inhibit the recurrence and metastasis of tumors compared with the control group.

[0050] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for preparing an immune-enhancing targeted hollow manganese dioxide radiotherapy sensitizer, characterized in that: The preparation method comprises the following steps: Step 1A) Pour a methanol solution of Zn(NO3)2·6(H2O) into a methanol solution of 2-methylimidazole, stir for 1 hour, then centrifuge at 10000×rpm for 5 minutes at room temperature, and wash with methanol 3 times to obtain ZIF-8 nanoparticles; the methanol solution of Zn(NO3)2·6(H2O) is prepared by pouring 293.4 mg of Zn(NO3)2·6(H2O) into 20 mL of methanol solution; the methanol solution of 2-methylimidazole is prepared by pouring 649 mg of 2-methylimidazole into 20 mL of methanol solution; Step 1B) Disperse the ZIF-8 nanoparticles obtained in step 1A) in 15 mL of methanol and add 20 mL of 10 mmol / mL -1 , pH = 8.5 Tris-HCl, then dopamine was added under stirring, stirred for 8 hours, centrifuged at room temperature, and washed with methanol to obtain ZIF-8@PDA nanoparticles; Step 1C) The ZIF-8@PDA obtained in step 1B) was dispersed in 15 mL of water, and 8.25 mL of KMnO4 aqueous solution was added dropwise under stirring to carry out redox reaction. After stirring for 5 minutes, the mixture was centrifuged at room temperature and washed with water. The mixture was then suspended in ultrapure water under light for 24 hours to obtain hollow MnO2 (HMn) nanoparticles. The obtained HMn solution was centrifuged at room temperature and washed with water, and vacuum dried to obtain HMn powder, which was then resuspended in ultrapure water to prepare 2 mg mL -1 Stock solution; Step 2A) Maintain mouse macrophages in a 37 °C, 5% CO2 cell culture incubator in DMEM medium containing 10% FBS and 1% penicillin-streptomycin. When the cell density reaches 60-80%, wash the cells with PBS and incubate with medium without FBS for 12 hours. Step 2B) Collect the cell culture medium and centrifuge to remove the precipitate and cells. Centrifuge the collected supernatant at 1000 × g for 10 minutes to remove apoptotic bodies and debris, and centrifuge the supernatant again at 10,000 × g for 30 minutes to remove larger microvesicles. Pass the supernatant through a 0.22 μm filter to further remove residual microvesicles, then collect the supernatant and centrifuge at 100,000 × g for 2 hours to obtain extracellular vesicles EVs at a concentration of 1 × 10 13 mL -1 EVs solution, engineered extracellular vesicles EVs-RGD was present at 1 × 10 13 mL -1 EVs and 40-60 μg mL -1 obtained after DSPE-PEG-RGD was co-incubated at a volume ratio of 2:1 for 24 h; Step 3A) Add 2 mg mL -1 Take 5 mL of HMn nanoparticles aqueous solution and add it to 5 mL of 10 mg mL -1 The mixture was added to the metformin aqueous solution, stirred at room temperature in the dark for 24 h, and washed with water three times to obtain HollowMnO2@Met, i.e., HMn@Met aqueous solution. The HMn@Met aqueous solution was then vacuum dried to obtain HMn@Met powder, which was then resuspended in ultrapure water to prepare 1 mg mL -1 Stock solution; Step 3B) Mix the EVs-RGD PBS solution obtained in step 2B) with the prepared 1 mg mL -1 The HMn@Met aqueous solution was mixed at a volume ratio of 1:1, incubated for 24 hours, and then extruded through a 0.22 μm membrane to obtain Hollow MnO2@Met@EVs-RGD, i.e., HMnER@Met solution.

2. The method for preparing the immune-enhancing targeted hollow manganese dioxide radiotherapy sensitizer according to claim 1, characterized in that: The amount of dopamine added in step 1B) is 100 mg.

3. The method for preparing the immune-enhancing targeted hollow manganese dioxide radiotherapy sensitizer according to claim 2, characterized in that: The concentration of KMnO4 aqueous solution in step 1C) is 5 mg mL -1 .

4. The method for preparing the immune-enhancing targeted hollow manganese dioxide radiotherapy sensitizer according to claim 3, characterized in that: The concentration of DSPE-PEG-RGD in step 2B) was 50 µg mL -1 .

5. The method for preparing the immune-enhancing targeted hollow manganese dioxide radiotherapy sensitizer according to claim 4, characterized in that: The particle size of HMn in step 1C) is 40-60 nm.

6. An immune-enhancing targeted hollow manganese dioxide radiotherapy sensitizer prepared by the preparation method according to any one of claims 1 to 5.

7. Use of the immune-enhancing targeted hollow manganese dioxide radiosensitizer according to claim 6 in the preparation of radiosensitization drugs.

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