Folate-modified organic metal framework-encapsulated trimanganese tetroxide and preparation method and application thereof

Abstract

The application discloses folate modified organic metal framework wrapped trimanganese tetroxide and a preparation method and application thereof. The application is characterized in that: a surfactant is used to modify Mn3O4; then, further reaction is carried out to obtain Mn3O4@ZIF-8; finally, reaction is carried out with a FA-PEG solution to obtain folate modified organic metal framework wrapped trimanganese tetroxide. The substance has good cervical cancer targeting property, can be effectively accumulated to a tumor site, and can be automatically degraded in an acidic microenvironment of the tumor under the action of radiotherapy, releases Mn3O4 in the tumor, oxidizes oxygen ions in excessive hydrogen peroxide in the tumor into oxygen, improves the anoxic environment of the tumor, and achieves ideal radiotherapy synergy and sensitization effect.

Description

Technical Field

[0001] This invention relates to the field of medical materials, and particularly to a folic acid-modified organometallic framework-encapsulated manganese tetroxide, its preparation method, and its application. Background Technology

[0002] Cervical cancer is one of the most common malignant tumors among women worldwide, with approximately 600,000 new cases and 340,000 deaths globally in 2020. Radiation therapy is an important treatment for cervical cancer. The ionizing radiation produced by radiation therapy can directly damage DNA molecules, or indirectly cause irreparable DNA damage such as double-strand breaks in tumor cells by inducing an increase in free radical content, especially reactive oxygen species (ROS), within tumor tissue, leading to tumor cell death. ROS can also cause tumor cell apoptosis by damaging related biomolecules and activating related signaling pathways. The effectiveness of radiation therapy is related to oxygenation; hypoxic tumors are often more tolerant to radiation than well-oxygenated tumors and require higher doses of radiation for treatment. Cells in well-oxygenated conditions are three times more sensitive to X-rays than those in hypoxic conditions. Hypoxia is a key characteristic of solid tumors, and the efficacy of radiation therapy is limited by the hypoxic microenvironment of tumor tissue.

[0003] In recent years, numerous studies have developed nanosystems to enhance the efficacy of radiotherapy. Most existing radiosensitizing nanomaterials are based on high atomic number metals; however, these materials require sufficient oxygen in the environment to function effectively. Therefore, the radiosensitizing effect of metallic nanomaterials is often hindered by the hypoxic microenvironment of tumors. Thus, strategies to overcome tumor hypoxia and enhance radiosensitization are particularly important. Summary of the Invention

[0004] The primary objective of this invention is to overcome the shortcomings and deficiencies of the prior art and provide a method for preparing manganese tetroxide encapsulated in a folic acid-modified organometallic framework.

[0005] Another object of the present invention is to provide manganese tetroxide (MnO) encapsulated in a folic acid-modified organometallic framework obtained by the above preparation method. This folic acid-modified organometallic framework-encapsulated MnO exhibits highly efficient radiosensitizing effects responsive to X-rays.

[0006] Another object of the present invention is to provide the application of the above-mentioned folic acid-modified organometallic framework-encapsulated manganese tetroxide.

[0007] The objective of this invention is achieved through the following technical solution: a method for preparing manganese tetroxide encapsulated in a folic acid-modified organometallic framework, comprising the following steps:

[0008] (1) Modification of Mn3O4

[0009] ① Disperse Mn3O4 with an organic solvent to obtain a Mn3O4 solution;

[0010] ② After dispersing or dissolving the surfactant evenly with an organic solvent, add the Mn3O4 solution obtained in step ① and stir to react; perform solid-liquid separation on the resulting mixture, and disperse the solid evenly with an organic solvent to obtain the surfactant-modified Mn3O4 solution;

[0011] (2) Synthesis of FA-Mn3O4@ZIF-8

[0012] ① Synthesis of FA-PEG: Folic acid and NaHCO3 are dissolved in water to obtain a folic acid solution; NHS, EDC and PEG are dissolved in water to obtain a mixture A; the folic acid solution and mixture A are mixed to obtain a mixture B, and the mixture is stirred to react; after the reaction is completed, the mixture is dialyzed to obtain a FA-PEG solution.

[0013] ② Synthesis of Mn3O4@ZIF-8: 2-methylimidazole was dissolved in an organic solvent to obtain a 2-methylimidazole solution; the 2-methylimidazole solution and the surfactant-modified Mn3O4 solution obtained in step (1) were mixed to obtain a mixture C; zinc nitrate was dissolved in an organic solvent to obtain a zinc nitrate solution; mixture C and zinc nitrate solution were mixed to obtain a mixture D, the mixture was allowed to stand for reaction, solid and liquid were separated, and the obtained solid was washed to obtain solid Mn3O4@ZIF-8;

[0014] ③ Synthesis of FA-Mn3O4@ZIF-8: After dispersing Mn3O4@ZIF-8 evenly in FA-PEG solution, the mixture was stirred and reacted overnight to obtain a mixture E; the mixture E was subjected to solid-liquid separation, and the obtained solid was washed to obtain manganese tetroxide encapsulated by a folic acid-modified organometallic framework.

[0015] The Mn3O4 mentioned in step (1)① is preferably obtained by calcining a manganese source; more preferably, it is prepared by the following steps: dispersing or dissolving a manganese source in solvent A, calcining, cooling, and washing to obtain Mn3O4.

[0016] The manganese source is preferably inorganic manganese; more preferably, it is at least one of manganese acetate, potassium permanganate, and manganese chloride.

[0017] Solvent A is preferably at least one of anhydrous ethanol, water, and methanol.

[0018] The preferred calcination conditions are a reaction at 100–140°C in a muffle furnace for 20–30 hours; more preferably, a reaction at 120°C in a muffle furnace for 24 hours.

[0019] The preferred degree of cooling is cooling to room temperature.

[0020] The room temperature is 10–40°C; more preferably 20–30°C; and most preferably 24–27°C.

[0021] The cleaning process involves washing with anhydrous ethanol and ultrapure water, respectively.

[0022] The cleaning is preferably performed at least once; more preferably three times.

[0023] The organic solvent mentioned in step (1)① is preferably at least one of methanol and anhydrous ethanol.

[0024] The amount of organic solvent used in step (1)① is preferably calculated based on 0.8-1g Mn3O4 mixed with 4mL of organic solvent.

[0025] The organic solvent mentioned in step (1)② is preferably at least one of methanol and anhydrous ethanol.

[0026] The amount of organic solvent used in step (1) ② is preferably calculated as surfactant: organic solvent = 10-11 mg: 1 mL.

[0027] The surfactant mentioned in step (1)② is preferably polyvinylpyrrolidone; more preferably PVP10, PVP15, PVP25 and PVP30, etc.

[0028] The amount of surfactant used in step (1) ② is preferably in the mass ratio of Mn3O4:surfactant = 1 to 1.01:1.

[0029] The stirring reaction time in step (1)② is preferably 10 to 16 hours; more preferably 12 hours.

[0030] The preferred method for solid-liquid separation in step (1)② is centrifugation.

[0031] The preferred centrifugation speed is 10,000 to 12,000 rpm for 10 to 15 minutes.

[0032] The folic acid and NaHCO3 mentioned in step (2)① are preferably mixed in a mass ratio of 5:3.5 to 4; more preferably in a mass ratio of 5:3.8.

[0033] The water mentioned in step (2)① is preferably ultrapure water.

[0034] In step (2)①, NHS, EDC and PEG in the mixture A are preferably mixed in a mass ratio of 1:1:1.4 to 1.8; more preferably in a mass ratio of 1:1:1.6.

[0035] The PEG mentioned in step (2)① is preferably at least one of PEG2000 and PEG5000.

[0036] In step (2)①, folic acid and PEG in the mixture B are preferably mixed in a mass ratio of 1:3 to 5; more preferably in a mass ratio of 1:4.

[0037] The stirring reaction time in step (2)① is preferably 10 to 16 hours; more preferably 12 hours.

[0038] The dialysis described in step (2)① is to remove unreacted reagents.

[0039] The preferred dialysis time in step (2)① is 12 to 36 hours; more preferably 24 hours.

[0040] The concentration of the FA-PEG solution mentioned in step (2)① is preferably 5 mg / mL.

[0041] The organic solvent mentioned in step (2)② is preferably methanol.

[0042] The concentration of the 2-methylimidazole solution mentioned in step (2)② is preferably 2 to 2.1 mg / mL.

[0043] In step (2)②, the mixture D containing 2-methylimidazole, zinc nitrate and Mn3O4 is preferably mixed in a mass ratio of 1 to 1.01:3.5 to 4:1; more preferably in a mass ratio of 1 to 1.01:3.6 to 3.7:1.

[0044] The preferred time for the static reaction in step (2)② is 40 to 80 minutes; more preferably 60 minutes.

[0045] The preferred method for solid-liquid separation in step (2)② is centrifugation.

[0046] The preferred centrifugation speed is 10,000 to 12,000 rpm for 10 to 15 minutes.

[0047] The cleaning described in step (2)② is done with methanol.

[0048] The number of cleaning cycles described in step (2)② is preferably at least once; more preferably three times.

[0049] In step (2)③, the FA-PEG and Mn3O4@ZIF-8 in the mixture E are preferably mixed in a mass ratio of 1 to 5:1; more preferably in a mass ratio of 1:1.

[0050] The dispersion described in step (2) ③ is preferably achieved by ultrasonic dispersion.

[0051] The preferred conditions for ultrasonic dispersion are 120W, 20kHz, and dispersion for 10 minutes.

[0052] The preferred method for solid-liquid separation in step (2)③ is centrifugation.

[0053] The preferred centrifugation speed is 10,000 to 12,000 rpm for 10 to 15 minutes.

[0054] The cleaning described in step (2) ③ is preferably done using ultrapure water.

[0055] The cleaning is preferably performed at least once; more preferably three times.

[0056] A manganese tetroxide encapsulated in a folic acid-modified organometallic framework was prepared by the above method.

[0057] The application of manganese tetroxide encapsulated in a folic acid-modified organometallic framework in the preparation of cancer radiotherapy.

[0058] The cancer mentioned is preferably cervical cancer.

[0059] The folic acid-modified organometallic framework ZIF-8 encapsulates manganese tetroxide, which has good targeting properties for cervical cancer. It can effectively accumulate at the tumor site and automatically degrade in the acidic tumor microenvironment under radiotherapy, releasing Mn3O4 locally in the tumor. This oxidizes the oxygen ions in the excess hydrogen peroxide in the tumor into oxygen, improving the hypoxic environment of the tumor and achieving ideal radiotherapy synergy and sensitization effects.

[0060] The present invention has the following advantages and effects compared with the prior art:

[0061] (1) This invention overcomes the disadvantage that the effect of simple radiotherapy is limited by the hypoxic microenvironment of the tumor. Based on the fact that manganese tetroxide has good H2O2 responsiveness and can generate oxygen to relieve the hypoxic microenvironment of the tumor, manganese tetroxide is selected as a radiosensitizer. In particular, it has a stronger H2O2 responsiveness after being combined with ZIF-8, especially as one of the comprehensive means of cervical cancer treatment.

[0062] (2) Through transmission electron microscopy, we found that the manganese tetroxide encapsulated by the folic acid modified organometallic framework ZIF-8 described in this invention has better response to radiation than ZIF-8 and manganese tetroxide alone.

[0063] (3) Using a portable dissolved oxygen meter, we found that the manganese tetroxide encapsulated by the folic acid-modified organometallic framework ZIF-8 described in this invention has good biological responsiveness to the acidic microenvironment of tumors. At the same time, it can reverse the enrichment of H2O2 in tumors and release oxygen to relieve tumor hypoxia, thereby enhancing the effect of radiotherapy. In addition, the manganese tetroxide encapsulated by the folic acid-modified organometallic framework ZIF-8 described in this invention has a faster and stronger oxygen release capacity than ZIF-8 and manganese tetroxide alone.

[0064] (4) Through cell experiments and subcutaneous tumor formation experiments in nude mice, we found that the manganese tetroxide encapsulated by the folic acid-modified organometallic framework ZIF-8 described in this invention can effectively enhance the inhibitory effect of radiotherapy on cervical cancer. Attached Figure Description

[0065] Figure 1 This is a transmission electron microscope image of manganese tetroxide encapsulated in a folic acid-modified organometallic framework ZIF-8.

[0066] Figure 2 The graph shows the morphological changes of manganese tetroxide encapsulated by the folic acid-modified organometallic framework ZIF-8 under different pH conditions; where A is the environment with pH=5.3; B is the environment with pH=6.8; and C is the environment with pH=7.4.

[0067] Figure 3 This is a diagram showing the morphological changes of a nanosystem and its different components under different doses of radiation.

[0068] Figure 4 The graph shows the oxygen production curves (A) of the nanosystem and its different components in 0.5% H2O2 solution and the bubble generation (B) of the nanoparticles and their different components after reacting with 0.5% H2O2 solution for 20 minutes.

[0069] Figure 5 The figure shows the effect of FA-Mn3O4@ZIF8 (10 mg / L) combined with radiotherapy (4 Gy) on the survival rate of SiHa cells and C33a cells 72 hours later; where * indicates P<0.05.

[0070] Figure 6 This is a graph showing the effect of different nanosystems combined with radiotherapy on SiHa cell clone formation.

[0071] Figure 7This figure shows the apoptosis rate of SiHa cells in different groups after 72 hours of combined radiotherapy with different nanosystems, using Annexin V-FITC / PI double staining kit and flow cytometry. The cell populations shown in the lower right Annexin V-FITC+ / PI- and upper right Annexin V-FITC+ / PI+ quadrants are apoptotic cells.

[0072] Figure 8 The image shows the in vivo antitumor activity results of FA-Mn3O4@ZIF8 combined with radiotherapy. In the image, A represents the tumor weight and picture; B represents the tumor volume growth curve; and C represents the weight curve of the tumor-bearing nude mice.

[0073] Figure 9 The figures show the distribution of FA-ICG@Mn3O4@ZIF8 in nude mice (A) and the distribution of the nanosystems in various organs 72 hours after tail vein injection of FA-ICG@Mn3O4@ZIF8 and ICG@Mn3O4@ZIF8 (B). Detailed Implementation

[0074] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0075] Example 1:

[0076] Preparation of FA-Mn3O4@ZIF-8 nanosystem

[0077] (1) Synthesis and modification of Mn3O4

[0078] Weigh 816.8 mg (3.33 mmol) of MnC4H6O4·4H2O and add it to 40 mL of anhydrous ethanol. Stir until completely dissolved. React in a muffle furnace at 120 °C for 24 hours. After cooling to room temperature, wash three times with anhydrous ethanol, then three times with ultrapure water. Centrifuge at 12000 rpm for 10 minutes each time. Finally, resuspend in 4 mL of methanol to obtain a Mn3O4 methanol solution.

[0079] Weigh 400 mg of polyvinylpyrrolidone (PVP10) and dissolve it in 38 mL of methanol. Add 2 mL of Mn3O4 methanol solution and stir overnight at room temperature. Centrifuge the resulting mixture at 12000 rpm for 10 minutes to remove excess unreacted reagent and resuspend it in 2 mL of methanol to obtain Mn3O4·PVP solution.

[0080] (2) Synthesis of FA-Mn3O4@ZIF-8

[0081] Synthesis of FA-PEG: Weigh 50 mg of folic acid and 38 mg of NaHCO3, mix them in 10 mL of ultrapure water, and stir until completely dissolved to obtain a folic acid solution. Weigh 25 mg of N-hydroxysuccinimide (NHS), 25 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC), and 40 mg of PEG (PEG molecular weight 2000), completely dissolve them in 8 mL of ultrapure water, then add 2 mL of freshly prepared folic acid solution, stir overnight at room temperature, and then dialyze through a dialysis bag with a molecular weight of 3000 for 24 hours to remove unreacted reagents to obtain an FA-PEG solution with a concentration of approximately 5 mg / mL.

[0082] Synthesis of Mn3O4@ZIF-8: 375.6 mg of zinc nitrate was dissolved in 50 mL of methanol to obtain a zinc nitrate methanol solution. 104.7 mg of 2-methylimidazole was dissolved in 50 mL of methanol, and 0.5 mL of Mn3O4·PVP solution (5 mg / mL) was added to the 2-methylimidazole methanol solution. The mixture was ultrasonically mixed to obtain mixed solution 1. The zinc nitrate methanol solution and mixed solution 1 were mixed and allowed to stand for 1 hour. The mixture was then centrifuged at 12000 rpm to remove excess unreacted reagents. The mixture was washed three times with methanol at 12000 rpm each time to obtain Mn3O4@ZIF-8.

[0083] Synthesis of FA-Mn3O4@ZIF-8: The Mn3O4@ZIF-8 precipitate was resuspended in 5 mL of FA-PEG solution, sonicated (120 W, 20 kHz) for 10 minutes, and then stirred overnight at room temperature. The mixture was then centrifuged at 12000 rpm to remove excess unreacted reagents and washed three times with ultrapure water at 12000 rpm each time to obtain FA-Mn3O4@ZIF-8.

[0084] Transmission electron microscopy (TEM) image of FA-Mn3O4@ZIF-8 successfully prepared by in-situ growth method is shown below. Figure 1 As shown.

[0085] Example 2:

[0086] Experimental study on the multiple response capabilities of the FA-Mn3O4@ZIF-8 nanosystem

[0087] (1) Verification of the acid response capability of the FA-Mn3O4@ZIF-8 nanosystem

[0088] PBS buffer (0.01M, pH=7.4) was prepared, and the pH was adjusted to three different values ​​(5.3, 6.8, and 7.4) using HCl and NaHCO3. 100 μL of FA-Mn3O4@ZIF-8 (Mn effective concentration: 45 mg / mL, Zn effective concentration: 100 mg / mL, effective concentrations obtained by ICP-MS) was added to 2 mL of PBS buffer at different pH values. After incubation for 4 hours, the morphological changes of nanoparticles at different pH groups were observed using transmission electron microscopy.

[0089] The results are as follows Figure 2 As shown: In environments with pH = 5.3 and 6.8, FA-Mn3O4@ZIF-8 loses its original regular shape, and ZIF-8 undergoes cleavage, exposing the Mn3O4 within; in an environment with pH = 7.4 ( Figure 2 C) The shape of FA-Mn3O4@ZIF-8 did not change significantly. Therefore, FA-Mn3O4@ZIF-8 exhibits good responsiveness to acidic environments.

[0090] (2) Verification of X-ray response capability of FA-Mn3O4@ZIF-8 nanosystem

[0091] Take 2 mL of FA-Mn3O4@ZIF-8, Mn3O4 and ZIF-8 (effective Mn concentration: 20 mg / mL, effective Zn concentration: 45 mg / mL) and place them on the panel of a biological X-ray irradiator. Simultaneously receive X-ray irradiation of 4 Gy, 16 Gy and 32 Gy. After irradiation, observe the morphological changes of different groups of nanoparticles by TEM.

[0092] The results are as follows Figure 3 As shown, with increasing radiation dose, individual Mn3O4 particles did not exhibit significant morphological changes. For ZIF-8, radiation doses of 4 Gy and 16 Gy did not significantly affect its morphology; however, under 32 Gy radiation, its sharp edges disappeared, its shape became rounded, and surface gaps appeared. For FA-Mn3O4@ZIF-8, under 4 Gy irradiation, the surface of individual particles fractured; under 16 Gy and 32 Gy irradiation, the ZIF-8 structure collapsed, exposing its internal Mn3O4. In summary, compared to individual Mn3O4 and ZIF-8, FA-Mn3O4@ZIF-8 exhibits better responsiveness to radiation, still able to disintegrate and expose Mn3O4 after lower doses of radiation.

[0093] (3) Verification of H2O2 response and oxygen production capacity of Mn3O4@ZIF-8 nanosystem

[0094] Mn3O4@ZIF-8, Mn3O4 and ZIF-8 were prepared into an aqueous dispersion system with the same Mn and Zn concentrations (Mn: 45 mg / mL, Zn: 100 mg / mL). 4 mL of 0.5% v / v H2O2 solution was taken, and the probe of a portable dissolved oxygen meter was inserted below the liquid surface. After the reading stabilized, 0.5 mL of the nano-solution to be tested was added. The change in dissolved oxygen concentration in the solution was monitored in real time using a portable dissolved oxygen meter and recorded by photograph, thereby evaluating the ability of the nanomedicine to respond to H2O2 and generate oxygen.

[0095] The results are as follows Figure 4 As shown: Figure 4 As shown in Table A and Table 1, within 20 minutes of reaction with 0.5% H2O2 solution, the dissolved oxygen in the ZIF-8 group did not change significantly, the dissolved oxygen in the Mn3O4 group increased slightly but at a relatively slow rate, while the dissolved oxygen in the Mn3O4@ZIF-8 group increased rapidly and reached its peak at 15 minutes. After 15 minutes, the upward trend slowed down, and after 20 minutes, the dissolved oxygen in the Mn3O4@ZIF-8 group reached 35.57 mg / L. Figure 4 B clearly shows that after 20 minutes, the Mn3O4@ZIF-8 group produced a large number of large bubbles, while other groups produced only a few small bubbles or no bubbles at all. These results indicate that Mn3O4@ZIF-8 has a good response to H2O2 and can release oxygen to improve the hypoxic microenvironment of tumors. Furthermore, compared to Mn3O4 and ZIF-8 alone, Mn3O4@ZIF-8 has a faster and stronger response to H2O2 and its ability to release oxygen, which is one of the foundations of its radiosensitizing ability.

[0096] Table 1. Dissolved oxygen levels (mg / L) at different times and in different groups

[0097] <![CDATA[Mn3O4@ZIF-8+H2O2]]> <![CDATA[Mn3O4+H2O2]]> <![CDATA[ZIF-8+H2O2]]> <![CDATA[H2O2]]> 0min 5.38 5.68 5.6 7.7 5min 22.37 12.53 7.76 7.06 10min 30.88 12.63 7.78 7.19 15min 35.14 19.36 8.02 7.33 20min 35.57 16.01 8.18 7.43

[0098] Example 3:

[0099] Investigation on the radiosensitizing ability of FA-Mn3O4@ZIF-8 nanosystem in cervical cancer cells

[0100] (1) Effects of FA-Mn3O4@ZIF-8 nanosystem combined with radiotherapy on the viability of cervical cancer cells

[0101] This invention investigates the effect of the FA-Mn3O4@ZIF-8 nanosystem combined with radiotherapy on the viability of cervical cancer cells using an MTT assay: cells in the logarithmic growth phase were used at a concentration of 3 × 10⁻⁶ cells / day. 4Cells were seeded at a density of 100 μL / mL in 96-well plates and cultured for 24 hours until adherence. The cells were then cultured in DMEM medium. FA-Mn3O4@ZIF8 (10 mg / L, DMEM medium, 100 μL / well) was co-incubated with the cells for 6 hours, followed by X-ray irradiation at 4 Gy. After another 72 hours of incubation, 25 μL of freshly prepared MTT solution (5 mg / mL) was added to each well and incubated for 4 hours. After aspirating the supernatant, 150 μL of DMSO was added to each well and the cells were shaken for 15 minutes to dissolve completely. The absorbance (OD) at 570 nm was measured in each well using an automated microplate reader. 570 ), calculate cell viability, cell viability (%) = (OD) 570 Experimental group / OD 570 The experimental group (blank control group) was multiplied by 100%. Control groups were also established, including a radiotherapy-only group and a FA-Mn3O4@ZIF8-only treatment group; and a blank control group was also included. At least three replicates were set up for both the experimental and control groups.

[0102] The results are as follows Figure 5 As shown, cell viability analysis revealed that in SiHa cells (Chinese Academy of Sciences Cell Bank), radiotherapy alone (86.24±7.03%), FA-Mn3O4@ZIF8 alone (65.20±3.64%), and combined therapy (44.53±1.85%) significantly inhibited SiHa cell viability. Furthermore, the cell viability of the combined therapy group was significantly lower than that of the single therapy group. All differences were statistically significant (P<0.05). In C33a cells (ATCC), radiotherapy alone (65.95±18.49%), FA-Mn3O4@ZIF8 alone (55.53±2.60%), and combined therapy (0.41±2.12%) significantly inhibited C33a cell viability. Furthermore, the cell viability of the combined therapy group was significantly lower than that of the single therapy group. All differences were statistically significant (P<0.05).

[0103] In summary, FA-Mn3O4@ZIF8 can significantly enhance the inhibitory effect of radiotherapy on the viability of various cervical cancer cells (including SiHa cells and C33a cells).

[0104] (2) Effects of FA-Mn3O4@ZIF-8 nanosystem combined with radiotherapy on the proliferation of cervical cancer cells

[0105] This invention investigates the effect of the FA-Mn3O4@ZIF-8 nanosystem combined with radiotherapy on the proliferation ability of cervical cancer cells through a clonogenic assay: SiHa cells in logarithmic growth phase were used with 1×10⁻⁶ cells / day of radiotherapy. 3Cells were seeded at a density of 2 mL / mL in 6-well plates and cultured in DMEM medium for 24 hours until adherence. FA-Mn3O4@ZIF8, Mn3O4@ZIF8, and Mn3O4 and ZIF8 were prepared with drugs in DMEM medium at predetermined concentrations (5 mg / L for Mn3O4 and 11 mg / L for Zn) and co-incubated with the cells for 6 hours. Cells were then irradiated with X-rays at predetermined doses and cultured for 10-14 days. The supernatant was then aspirated, and the cells were washed three times with 0.01 M phosphate-buffered saline (pH 7.4). Adherent cells were then fixed with 4% paraformaldehyde at room temperature for 15 minutes. The cells were then washed three times with PBS and stained with 0.5% crystal violet solution for 20 minutes. After washing three times with PBS and air-drying the 6-well plates at room temperature, the cells were observed and photographed.

[0106] The results are as follows Figure 6 As shown, both radiotherapy and FA-Mn3O4@ZIF8 alone can effectively inhibit the formation of cell clonal communities. However, the synergistic effect of FA-Mn3O4@ZIF8 and radiotherapy achieves a better inhibitory effect on cell clonal community formation. These results further demonstrate that FA-Mn3O4@ZIF8 combined with radiotherapy can effectively inhibit the proliferation of cervical cancer cells.

[0107] (3) Effects of FA-Mn3O4@ZIF-8 nanosystem combined with radiotherapy on apoptosis of cervical cancer cells

[0108] This study used Annexin V-FITC / PI double staining assay to analyze apoptosis in cervical cancer SiHa cells: SiHa cells in logarithmic growth phase were sampled at a concentration of 3 × 10⁻⁶ cells / cells. 4 Cells were seeded at a density of 2 mL / mL in 6-well plates and cultured for 24 hours until adherence. FA-Mn3O4@ZIF8, Mn3O4@ZIF8, and Mn3O4 and ZIF8 were co-incubated with cells at predetermined concentrations (Mn3O4 15 mg / L, Zn 33 mg / L) for 6 hours, followed by X-ray irradiation at a predetermined dose (4 Gy). After 48 hours of further culture, the supernatant was collected, cells were digested with EDTA-free trypsin, and washed three times with pre-cooled PBS (4°C). Following the Annexin V-FITC / PI double staining kit, each sample was resuspended in 500 μL of binding buffer and stained with Annexin V-FITC and PI at room temperature for 30 minutes under dark conditions. Finally, the apoptosis rate was analyzed by flow cytometry.

[0109] The results are as follows Figure 7As shown, the apoptosis rate in the radiotherapy-only group was 11.94%. After combined treatment with FA-Mn3O4@ZIF8, the apoptosis rate increased to 85.45%, which was higher than that of the FA-Mn3O4@ZIF8-only group (80.34%) and other treatment groups. Therefore, FA-Mn3O4@ZIF8 can effectively induce apoptosis in cervical cancer cells, thereby enhancing the anti-tumor effect of radiotherapy.

[0110] Example 4:

[0111] Investigation on the radiosensitizing ability of FA-Mn3O4@ZIF-8 nanosystem in a nude mouse subcutaneous xenograft tumor model

[0112] Establishment of a subcutaneous unilateral tumor-bearing model in female Balb / c nude mice (purchased from Guangdong Yaokang Biotechnology Co., Ltd.): Human cervical cancer cells SiHa in the logarithmic growth phase were collected by trypsin / EDTA digestion and resuspended in DMEM to 2×10⁻⁶ cells. 7 Cells / mL, 100 μL of cell suspension was inoculated into the right back of nude mice.

[0113] Study on the sensitizing effect of FA-Mn3O4@ZIF8 in vivo radiotherapy: When the subcutaneous tumor grows to 100-150mm 3 Nude mice bearing tumors were randomly divided into four groups of six each. The following treatments were administered: Group 1 (control group): 100 μL of saline solution was injected intravenously twice weekly; Group 2 (radiotherapy group): 100 μL of saline solution was injected intravenously twice weekly, followed by local radiotherapy to the tumor site 6 hours later (radiation dose: 2 Gy); Group 3 (FA-Mn3O4@ZIF8 treatment group): 100 μL of FA-Mn3O4@ZIF8 (Mn: 5 mg / kg, Zn: 11 mg / kg) was injected intravenously twice weekly; Group 4 (FA-Mn3O4@ZIF8 combined with radiotherapy group): 100 μL of FA-Mn3O4@ZIF8 (Mn: 5 mg / kg, Zn: 11 mg / kg) was injected intravenously twice weekly, followed by local radiotherapy to the tumor site 6 hours later (radiation dose: 2 Gy). The treatment cycle is 24 days. After treatment begins, the length and width of the tumor are measured every day using calipers, and the weight of the tumor-bearing nude mice is also measured. The tumor volume is calculated using the formula: Volume = 1 / 2 × Length × Width. 2 Twenty-four days later, the nude mice were euthanized, and the tumors from each mouse were collected and weighed.

[0114] The results are as follows Figure 8As shown, after 24 days of treatment, the tumor weight in the drug group, radiotherapy group, and drug-plus-radiotherapy group was significantly smaller than that in the control group (P<0.05). Meanwhile, the tumor weight in the control group, radiotherapy group, and drug group was greater than that in the drug-plus-radiotherapy group (P<0.05). These results suggest that FA-Mn3O4@ZIF8 combined with radiotherapy has good in vivo anti-tumor activity.

[0115] Example 5:

[0116] Targeting of FA-Mn3O4@ZIF-8 Nanosystem in a Nude Mouse Subcutaneous Xenograft Model

[0117] First, indocyanine green (ICG)-labeled FA-ICG@Mn3O4@ZIF8 and ICG@Mn3O4@ZIF8 nanosystems were synthesized. The specific steps are as follows: 375.6 mg of zinc nitrate was dissolved in 50 mL of methanol. 104.7 mg of 2-methylimidazole was dissolved in 50 mL of methanol. 0.5 mL of Mn3O4·PVP solution (5 g / L) and 0.5 mL of indocyanine green solution (5 mg / mL) were added to the 2-methylimidazole solution and sonicated until homogeneous. The two solutions were mixed and allowed to stand for 1 hour. The mixture was then centrifuged at 12000 rpm to remove excess unreacted reagents. The mixture was washed three times with methanol at 12000 rpm each time to obtain ICG@Mn3O4@ZIF8. The ICG@Mn3O4@ZIF8 precipitate was resuspended in 5 mL of FA-PEG solution, sonicated for 10 minutes, and then stirred overnight at room temperature in the dark. The mixture was then centrifuged at 12,000 rpm to remove excess unreacted reagents and washed three times with ultrapure water at 12,000 rpm each time to obtain FA-ICG@Mn3O4@ZIF8.

[0118] Following the procedure in Example 4, wait until the subcutaneous tumor in the nude mouse grows to 150 mm. 3 Six tumor-bearing nude mice were randomly divided into two groups of three. One group received a tail vein injection of FA-ICG@Mn3O4@ZIF8, while the other group received a tail vein injection of ICG@Mn3O4@ZIF8 (both groups had concentrations of Mn: 5 mg / kg and Zn: 11 mg / kg). The mice were anesthetized with 4% chloral hydrate (400 mg / kg) at 0, 2, 4, 8, 12, 24, 48, and 72 hours, and the distribution of the drug within the mice was observed using a small animal in vivo imaging system.

[0119] The results are as follows Figure 9 As shown: Figure 9As shown in Figure A, after intravenous injection of FA-ICG@Mn3O4@ZIF8, the nanomaterials were initially distributed throughout the body and liver. At 4 hours, the nanomaterials began to accumulate within the tumor. By 12 hours, significant accumulation was observed at the tumor site. At 72 hours, most of the nanomaterials were metabolized, but a small portion remained distributed and accumulated within the tumor. Figure 9 As shown in Figure B, after 72 hours, the liver and spleen were the organs with the highest accumulation of both nanomaterials after organ ex vivo. Meanwhile, FA-ICG@Mn3O4@ZIF8 still partially accumulated within the tumor, while ICG@Mn3O4@ZIF8 without folic acid modification did not accumulate in the tumor. These results suggest that the folic acid-modified nanosystem has a better tumor-targeting effect in vivo.

[0120] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for preparing manganese tetroxide encapsulated in a folic acid-modified organometallic framework, characterized in that... Includes the following steps: (1) Modification of Mn3O4 ① Disperse Mn3O4 with an organic solvent to obtain a Mn3O4 solution; ② After dispersing or dissolving the surfactant evenly with an organic solvent, add the Mn3O4 solution obtained in step ① and stir to react; perform solid-liquid separation on the resulting mixture, and disperse the solid evenly with an organic solvent to obtain the surfactant-modified Mn3O4 solution; (2) Synthesis of FA-Mn3O4@ZIF-8 ① Synthesis of FA-PEG: Folic acid and NaHCO3 are dissolved in water to obtain a folic acid solution; NHS, EDC and PEG are dissolved in water to obtain a mixture A; the folic acid solution and mixture A are mixed to obtain a mixture B, and the mixture is stirred to react; after the reaction is completed, the mixture is dialyzed to obtain a FA-PEG solution. ② Synthesis of Mn3O4@ZIF-8: 2-methylimidazole was dissolved in an organic solvent to obtain a 2-methylimidazole solution; the 2-methylimidazole solution and the surfactant-modified Mn3O4 solution obtained in step (1) were mixed to obtain a mixture C; zinc nitrate was dissolved in an organic solvent to obtain a zinc nitrate solution; mixture C and zinc nitrate solution were mixed to obtain a mixture D, the mixture was allowed to stand for reaction, solid and liquid were separated, and the obtained solid was washed to obtain solid Mn3O4@ZIF-8; ③ Synthesis of FA-Mn3O4@ZIF-8: After dispersing Mn3O4@ZIF-8 evenly in FA-PEG solution, the mixture was stirred and reacted overnight to obtain mixture E; mixture E was subjected to solid-liquid separation, and the obtained solid was washed to obtain manganese tetroxide encapsulated by a folic acid-modified organometallic framework.

2. The method for preparing manganese tetroxide encapsulated in a folic acid-modified organometallic framework according to claim 1, characterized in that: The Mn3O4 mentioned in step (1)① is prepared by the following steps: dispersing or dissolving the manganese source in solvent A, calcining, cooling, and washing to obtain Mn3O4; The manganese source is inorganic manganese; Solvent A is at least one of anhydrous ethanol, water, and methanol; The calcination conditions are as follows: reaction at 100–140°C in a muffle furnace for 20–30 hours; The cleaning process involves washing with anhydrous ethanol and ultrapure water, respectively.

3. The method for preparing manganese tetroxide encapsulated in a folic acid-modified organometallic framework according to claim 1, characterized in that: The organic solvent mentioned in step (1)① is at least one of methanol and anhydrous ethanol; The organic solvent mentioned in step (1)② is at least one of methanol and anhydrous ethanol; The surfactant mentioned in step (1)② is polyvinylpyrrolidone; The PEG mentioned in step (2)① is at least one of PEG2000 and PEG5000; The organic solvent mentioned in step (2)② is methanol.

4. The method for preparing manganese tetroxide encapsulated in a folic acid-modified organometallic framework according to claim 1, characterized in that: The amount of organic solvent used in step (1)① is calculated based on 0.8-1g Mn3O4 mixed with 4mL of organic solvent; The amount of organic solvent used in step (1) ② is calculated as surfactant: organic solvent = 10-11 mg: 1 mL; The amount of surfactant used in step (1) ② is in the mass ratio of Mn3O4:surfactant = 1 to 1.01:1; The folic acid and NaHCO3 mentioned in step (2)① are mixed in a mass ratio of 5:3.5-4; In step (2)①, NHS, EDC, and PEG are mixed in a mass ratio of 1:1:1.4 to 1.8 in the mixture A. In step (2)①, folic acid and PEG are mixed in a mass ratio of 1:3 to 5 in mixture B. The concentration of the 2-methylimidazole solution mentioned in step (2)② is 2 to 2.1 mg / mL; In step (2)②, the mixture D contains 2-methylimidazolium, zinc nitrate, and Mn3O4 in a mass ratio of 1–1.01:3.5–4:

1. In step (2)③, FA-PEG and Mn3O4@ZIF-8 are mixed in a mass ratio of 1:1 in the mixture E.

5. The method for preparing manganese tetroxide encapsulated in a folic acid-modified organometallic framework according to claim 1, characterized in that: The stirring reaction time described in step (1) ② is 10–16 h; The solid-liquid separation method described in step (1)② is centrifugation; The stirring reaction time mentioned in step (2)① is 10-16 hours; The dialysis time mentioned in step (2)① is 12-36 hours; The static reaction time mentioned in step (2)② is 40-80 min; The solid-liquid separation method described in step (2)② is centrifugation; The cleaning described in step (2)② is done with methanol; The dispersion described in step (2)③ is ultrasonic dispersion; The solid-liquid separation method described in step (2)③ is centrifugation.

6. A manganese tetroxide encapsulated in a folic acid-modified organometallic framework, characterized in that: It is prepared by the method described in any one of claims 1 to 5.

7. The use of manganese tetroxide encapsulated in a folic acid-modified organometallic framework as described in claim 6 in the preparation of cancer radiotherapy.

8. The application according to claim 7, characterized in that: The cancer mentioned is cervical cancer.

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