An artesunate-coordinated metal-organic framework nanoplatform and its preparation method and application

Abstract

The present invention belongs to the technical field of the preparation of medical materials, and particularly relates to an artesunate-loaded coordination metal-organic framework nanoplatform and its preparation method and application. In the present invention, zinc nitrate hexahydrate, 2-methylimidazole, and artesunate are used as precursors, and artesunate is loaded by means of metal coordination to synthesize a hybrid zeolitic imidazolate framework nanomaterial Art-ZIF NPs loaded with artesunate. The size is nanoscale, with good water dispersibility, high loading rate and loading capacity. It has the characteristic of acid-responsive degradation, can be stable under physiological conditions, but disintegrates under acidic conditions (pH < 5.5), thus realizing the intelligent controlled release of drugs. The present invention fully overcomes the deficiency of poor water solubility of artesunate and has a significant inhibitory effect on the growth of tumor cells. The preparation process is simple, the operation is easy, and the reaction conditions are mild; the requirements for instruments are not high, the experimental process is green and environmentally friendly, and it can be applied to large-scale industrial production.

Description

Technical Field

[0001] The present invention belongs to the technical field of medical material preparation, and particularly relates to a coordination metal-organic framework nano preparation loaded with artesunate, a preparation method thereof and an application thereof. Background Art

[0002] Artesunate (Art) is a derivative of artemisinin from traditional Chinese medicine. The addition of a hemisuccinate group to its structure endows it with good anti-cancer effects. Artesunate plays a role in inducing cell cycle arrest and apoptosis, inhibiting cell proliferation and growth, metastasis and angiogenesis during the anti-cancer process through pathways such as down-regulating cyclooxygenase-2 (COX-2), reducing mitochondrial membrane potential, down-regulating Bcl2, up-regulating Bax and activating caspase-3, endoplasmic reticulum stress, iron dependence and ROS-mediated ferroptosis. The combined treatment of artesunate with other therapies also shows good anti-cancer effects in research. However, Art can cause toxic side effects such as a decrease in peripheral blood reticulocytes and teratogenesis, and it has a half-life of only 30 minutes, low bioavailability and poor water solubility. In order to reduce the toxicity of the drug, improve bioavailability and reduce the number of drug administrations, it is necessary to prepare it into biodegradable nanoparticles. Although there is research on loading artemisinin with Zif-8, its physically encapsulated drug loading method is passive, resulting in a low drug loading rate and drug loading amount (only 7%), which affects the therapeutic effect of the drug. Therefore, it is necessary to develop a nanoparticle with a simple preparation method that can achieve a high drug loading amount and drug loading rate. Summary of the Invention

[0003] In view of this, in order to make up for some deficiencies in the prior art, the purpose of the present invention is to provide a coordination metal-organic framework nano preparation loaded with artesunate, a preparation method thereof and an application thereof. The present invention simultaneously performs a coordination reaction on a mixture of zinc ions, artesunate and 2-methylimidazole to successfully load artesunate, thereby synthesizing a coordination metal-organic framework nano preparation loaded with artesunate having a nanoscale size. The reversible coordination bond formed between artesunate and zinc ions enables the prepared nanoparticles to have an intelligent controlled release function while maintaining structural stability and a high drug loading rate.

[0004] In order to achieve the above purpose, the technical solution of the present invention is as follows:

[0005] The present invention provides a preparation method of a coordination metal-organic framework nano preparation loaded with artesunate (Art-ZIF NPs), comprising the following steps:

[0006] (1) Dissolve zinc nitrate hexahydrate in methanol to obtain solution A, dissolve 2-methylimidazole in methanol to obtain solution B, and dissolve artesunate in methanol to obtain solution C;

[0007] (2) Add solution B to solution C at room temperature and stir evenly with a magnetic stirrer to obtain a mixed solution. Dropwise add solution A into the mixed solution and stir thoroughly with a magnetic stirrer until a milky white suspension is formed by the reaction.

[0008] (3) Centrifuge the milky white suspension at high speed. After centrifugation, discard the supernatant to obtain a white precipitate. Rinse it with deionized water 2 - 3 times and then purify it by vacuum freeze - drying to finally obtain the metal - organic framework nano - preparation loaded with artesunate.

[0009] Further, in step (1), the concentration of solution A is 14.88 mg / mL, the concentration of solution B is 32.83 mg / mL, and the concentration of solution C is 0.231 - 23.1 mg / mL.

[0010] In the milky white suspension in step (2), the molar ratio of zinc nitrate, 2 - methylimidazole, and artesunate is 1:8:0.012 - 1.2.

[0011] In step (2), the temperature of the reaction is room temperature and the reaction time is 5 - 10 min.

[0012] In step (3), the conditions for high - speed centrifugation are centrifugation at 8000 - 12000 g for 15 - 25 min.

[0013] The present invention also provides a artesunate - loaded coordination metal - organic framework nano - preparation prepared by the above - mentioned preparation method.

[0014] In the nano - preparation, zinc ions coordinate with 2 - methylimidazole to form zeolitic imidazolate framework material (ZIF - 8), and zinc ions chelate artesunate by metal coordination. The average particle size of the nano - preparation is 70 - 100 nm. The nano - preparation is loaded with artesunate, and the loading amount of artesunate is 48 - 65%.

[0015] The present invention also provides the application of the above - prepared artesunate - loaded coordination metal - organic framework nano - preparation in the preparation of tumor treatment products.

[0016] The tumor is a tumor treated with artesunate as a therapeutic drug.

[0017] Further, the application is to degrade and release artesunate in an acidic environment.

[0018] The present invention uses zinc nitrate hexahydrate, 2-methylimidazole, and artesunate as precursors, and loads artesunate by means of metal coordination to synthesize a hybrid zeolitic imidazolate framework nanomaterial Art-ZIF NPs loaded with artesunate, which is a novel nanoformulation for actively loading artesunate. The prepared Art-ZIF NPs have good water dispersibility and are nanoscale in size; the reversible coordination bonds formed between artesunate and zinc ions, and the metal coordination loading method not only reduces drug leakage, but also greatly improves the drug loading capacity. At the same time, the prepared Art-ZIF NPs have the characteristics of acid-responsive degradation, endowing them with stability under physiological conditions but disintegrating under acidic conditions (pH < 5.5), thus realizing the intelligent controlled release of drugs. The prepared Art-ZIF NPs fully overcome the deficiency of poor water solubility of artesunate and have a significant inhibitory effect on the growth of tumor cells. The preparation process of the present invention is simple, the operation is easy, and the reaction conditions are mild; the instrument requirements are not high, the experimental process is green and environmentally friendly, and it can be applied to large-scale industrial production. Description of the Drawings

[0019] Figure 1 is the schematic diagram of metal coordination for preparing Art-ZIF NPs;

[0020] Figure 2 is the comparison chart of the average hydrodynamic particle size of ZIF-8 NPs and Art-ZIF NPs; in the figure, A is ZIF-8 NPs and B is Art-ZIF NPs;

[0021] Figure 3 is the transmission electron microscope image of Art-ZIF NPs; in the figure, A is the low-resolution image with a scale bar of 0.2 μm, and B is the high-resolution enlarged image with a scale bar of 100 nm;

[0022] Figure 4 is the ultraviolet-visible absorption spectrum of Art-ZIF NPs;

[0023] Figure 5 is the comparison chart of the cumulative drug release curves of artesunate at different pH values;

[0024] Figure 6 is the bar chart of the growth inhibition of Art-ZIF NPs on tumor cells 4T1. Embodiments

[0025] The present invention will be further illustrated below in conjunction with the accompanying drawings. These embodiments are only used to illustrate the present invention and not to limit the scope of the present invention; for the experimental methods without specific conditions noted in the embodiments, they are all carried out according to conventional conditions. The amounts of the kit reagents used in the embodiments are only exemplary, and those skilled in the art can make corresponding adjustments according to the actual situation; the reagents and biological materials, unless otherwise specified, can be obtained from commercial channels. The 4T1 cells were purchased from the Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences; Example

[0026] Dissolve 148.7 mg of zinc nitrate hexahydrate (0.5 mmol) in 10 mL of methanol (analytical grade), dissolve 328.3 mg of 2-methylimidazole (4 mmol) in 10 mL of methanol, and dissolve 23.1 mg of artesunate (0.06 mmol) in 10 mL of methanol. Magnetically stir the prepared 2-methylimidazole methanol solution and artesunate methanol solution at room temperature (200 rpm / min) to fully mix and obtain a mixed solution; then gradually add the prepared 10 mL of zinc nitrate methanol solution dropwise to the mixed solution, continue to stir and mix well, and react for 7 min to form a milky white suspension; the molar ratio of zinc nitrate, 2-methylimidazole, and artesunate in the milky white suspension is 1:8:0.12. Centrifuge at 10000 g for 20 minutes to collect the reaction product, and wash it 3 times by centrifugation with deionized water. Freeze-dry under vacuum to obtain artesunate-loaded hybrid zeolitic imidazolate framework nanoparticles Art-ZIF NPs. It was determined that the loading amount of artesunate in the prepared Art-ZIF NPs was 60%, and the drug loading rate was 87%. Figure 1 It is the schematic diagram of the metal coordination principle for the preparation of Art-ZIF NPs. Example

[0027] Dissolve 148.7 mg of zinc nitrate hexahydrate (0.5 mmol) in 10 mL of methanol (analytical grade), dissolve 328.3 mg of 2-methylimidazole (4 mmol) in 10 mL of methanol, and ultrasonically dissolve 231 mg of artesunate (0.6 mmol) in 10 mL of methanol. Magnetically stir the prepared 2-methylimidazole methanol solution and artesunate methanol solution at room temperature to fully mix and obtain a mixed solution; then gradually add the zinc nitrate methanol solution dropwise to the mixed solution, continue to stir and mix well, and react for 10 min to form a milky white suspension; the molar ratio of zinc nitrate, 2-methylimidazole, and artesunate in the milky white suspension is 1:8:1.2. Centrifuge at 8000 g for 25 minutes to collect the reaction product, and wash it 3 times by centrifugation with deionized water. Freeze-dry under vacuum to obtain artesunate-loaded hybrid zeolitic imidazolate framework nanoparticles. It was determined that the loading amount of artesunate in the prepared Art-ZIF NPs was 65%. Example

[0028] 148.7 mg of zinc nitrate hexahydrate (0.5 mmol) was dissolved in 10 mL of methanol (analytical grade), 328.3 mg of 2-methylimidazole (4 mmol) was dissolved in 10 mL of methanol, and 2.31 mg of artesunate (0.06 mmol) was dissolved in 10 mL of methanol. The prepared 2-methylimidazole methanol solution and artesunate methanol solution were magnetically stirred at room temperature and thoroughly mixed to obtain a mixed solution; then the zinc nitrate methanol solution was added dropwise to the mixed solution, and stirring and mixing were continued. After reacting for 5 min, a milky white suspension was formed; the molar ratio of zinc nitrate, 2-methylimidazole, and artesunate in the milky white suspension was 1:8:0.012. The reaction product was collected by centrifugation at 12000 g for 15 minutes and centrifugally washed 3 times with deionized water. Freeze-drying under vacuum was performed to obtain artesunate-loaded hybrid zeolitic imidazolate framework nanoparticles. It was determined that the loading amount of artesunate in the prepared Art-ZIF NPs was 48%. Example

[0029] Taking the Art-ZIF NPs prepared in Example 1 as an example, the prepared hybrid zeolitic imidazolate framework nanoparticles were physically characterized, and at the same time, a comparison was made with standard ZIF-8 nanoparticles (ZIF-8 NPs). Figure 2 Figure is a comparison chart of the average hydrodynamic particle sizes of ZIF-8 NPs and Art-ZIF NPs; in the figure, A is ZIF-8 NPs and B is Art-ZIF NPs; as Figure 2 shown, the particle sizes of ZIF-8 NPs and Art-ZIF NPs were 73.8 and 88.8 nm respectively, indicating that the Art-ZIF NPs loaded with artesunate had a particle size similar to that of ZIF-8. Figure 3 Figure is a transmission electron micrograph of Art-ZIF NPs; in the figure, A is a low-resolution image with a scale bar of 0.2 μm, and B is a high-resolution magnified image with a scale bar of 100 nm; from Figure 3 it can be further shown that the Art-ZIF NPs had good dispersibility and a relatively uniform particle size distribution. Example

[0030] In this example, the Art-ZIF NPs prepared in Example 1 were analyzed by ultraviolet-visible absorption spectroscopy. For comparison, a pure artesunate solution (0.1 mg / mL) and an artesunate / zinc nitrate mixed solution (0.1 mg / mL) were prepared. Figure 4 Figure is the ultraviolet-visible absorption spectrum of Art-ZIF NPs; from Figure 4As shown, compared with the characteristic peak of pure artesunate (195 nm), the absorption peak shows an obvious red shift (211 nm) after artesunate forms a chelate with zinc ions. It can be seen that artesunate can be chelated into Art-ZIF NPs through coordination, rather than the simple physical encapsulation mediated by traditional MOF materials. The Art-ZIF NPs provided by the present invention integrate artesunate into ZIF nanoparticles through metal coordination rather than simple physical encapsulation, greatly reducing its leakage during in vivo delivery and improving the drug loading efficiency. Example

[0031] In this example, the release of artesunate from Art-ZIF NPs was detected in phosphate buffer solutions with different pH values. Figure 5 It is a comparison chart of the cumulative drug release curves of artesunate at different pH values. As Figure 5 shown, within 24 hours, the release of artesunate is relatively slow in a neutral environment (pH = 7.2), and the maximum release rate is only 23.6%. In a weakly acidic environment with pH = 5.5, there is a burst release (59.5%) within the first hour, and then it gradually approaches a steady state (80.1%). The above results indicate that an acidic environment can induce the rapid release of artesunate from Art-ZIF NPs. This phenomenon may be attributed to protonation induced by low pH values, ultimately leading to the degradation of the ZIF-8-like structure. This characteristic endows Art-ZIF NPs with the ability to target drug delivery to tumors through pH changes. Example

[0032] In this example, the anti-tumor effect of Art-ZIF NPs was studied through in vitro cell experiments. Specifically, triple-negative breast cancer cells (4T1 cells) were co-cultured with different concentrations of Art-ZIF NPs for 24 h, and then the cell viability was measured by the CCK-8 method. Figure 6 It is a bar chart of the growth inhibition of tumor cells 4T1 by Art-ZIF NPs; as Figure 6 shown, the viability of 4T1 cells is significantly negatively correlated with the concentration of Art-ZIF NPs. When the concentration is greater than 12 μg / mL, the viability of 4T1 cells is significantly reduced; when the concentration is greater than 96 μg / mL, the viability of 4T1 cells drops to 15%. The above results preliminarily indicate that Art-ZIF NPs can significantly inhibit cell growth.

[0033] Although the present invention has been described in detail with general descriptions and specific embodiments above, based on the present invention, some modifications or improvements can be made, which are obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection required by the present invention.

Claims

1. A preparation method of an artesunate-loaded coordination metal-organic framework nanoplatform, characterized in that, The following steps are involved: (1) Zinc nitrate hexahydrate is dissolved in methanol to obtain solution A, 2-methylimidazole is dissolved in methanol to obtain solution B, and artesunate is dissolved in methanol to obtain solution C; (2) Add solution B to solution C at room temperature and stir evenly on a magnetic stirrer to obtain a mixed solution; Add solution A dropwise into the mixed solution and stir thoroughly with a magnetic stirrer until a milky white suspension is formed. (3) The milky white suspension was centrifuged at high speed, and the supernatant was discarded after centrifugation to obtain a white precipitate, which was rinsed with deionized water 2-3 times and then purified by vacuum freeze drying to finally obtain a metal organic framework nanoformulation loaded with artesunate; the concentration of the solution A in step (1) was 14.88 mg / mL, the concentration of the solution B was 32.83 mg / mL, and the concentration of the solution C was 0.231 ~ 23.1 mg / mL.

2. The preparation method according to claim 1, characterized in that, The molar ratio of zinc nitrate, 2-methylimidazole and artesunate in the milky white suspension in step (2) is 1:8:0.012~1.

2.

3. The preparation method according to claim 1, characterized in that, The reaction temperature in step (2) is room temperature, and the reaction time is 5 to 10 minutes.

4. The preparation method according to claim 1, wherein The high-speed centrifugation conditions in step (3) are 8000-12000 g for 15-25 min.

5. The artesunate-loaded coordinated metal-organic framework nanoformulation prepared by the preparation method according to any one of claims 1 to 4.

6. The nano - preparation according to claim 5, characterized in that, The zinc ions in the nanoformulation react with 2-methylimidazole to form a zeolite imidazole framework material, and the zinc ions chelate artesunate through metal coordination. The average particle size of the nanoformulation is 70 to 100 nm. The nanoformulation is loaded with artesunate, and the loading amount of artesunate is 48 to 65%.

7. Use of the artesunate-loaded coordinated metal-organic framework nanoformulation prepared by the preparation method according to any one of claims 1 to 4 in the preparation of tumor treatment products.

8. The application according to claim 7, wherein The tumor is a tumor for which artesunate is used as a therapeutic drug.

9. The application according to claim 7, characterized in that, The application is to release artesunate by degradation of the nano preparation in an acidic environment.

Images