Preparation method of nano-metal covalent organic framework material for enhancing cell ferroptosis by consuming glutathione as well as obtained product and application of nano-metal covalent organic framework material

Abstract

The invention belongs to the technical field of medical nano-materials, and particularly relates to a preparation method of a nano-metal covalent organic framework material for enhancing cell ferroptosis by consuming glutathione as well as an obtained product and application of the nano-metal covalent organic framework material. The material is prepared by the following steps: firstly, preparing a metal organic ligand Cu3-PyCA3; the preparation method comprises the following steps: firstly, preparing a metal organic ligand Cu3-PyCA3, then mixing the metal organic ligand Cu3-PyCA3, 2, 5-diethoxybenzene-1, 4-bis (formylhydrazine), mesitylene, 1, 4-dioxane and a trifluoroacetic acid solution, and then reacting to obtain a nano metal covalent organic framework Cu3-MCOF; and finally, uniformly mixing the activated hyaluronic acid solution and the Cu3-MCOF, and intensely stirring to obtain the Cu3-MCOF-coated HA. The nano-metal covalent organic framework provided by the invention can consume glutathione to enhance cell ferroptosis, and the nano-metal covalent organic framework Cu3-MCOF can quickly react with glutathione, so that the framework structure of the Cu3-MCOF is seriously damaged, the crystalline state disappears, a large number of ribbons are further assembled and generated, glutathione is consumed, and the ferroptosis of cells is enhanced. The ferroptosis tumor treatment enhancer has an application prospect.

Description

Technical Field

[0001] This invention belongs to the field of medical nanomaterials technology, specifically relating to a method for preparing a nanomaterial of metal covalent organic framework that consumes glutathione to enhance cellular ferroptosis, as well as the resulting product and its applications. Background Technology

[0002] Malignant tumors have become one of the leading causes of death worldwide, seriously threatening human life and health. To date, the mechanisms of tumor development are not fully understood; various single or combined factors can trigger cancerous changes in the body. Clinically, an effective method to completely cure all types of tumors has yet to be found, and striving to cure patients or prolong their lives has become the primary goal of medical professionals. With the advancement of medical technology and the continuous development of treatment methods, traditional chemotherapy, radiotherapy, or surgery have increased the five-year survival rate for cancer patients to over 50%. However, these three treatment methods all have their own limitations. Therefore, exploring safer and more efficient cancer treatment methods remains a significant challenge. With the rapid development of nanotechnology, nanomedicines have been widely researched and applied in the biomedical field, bringing new hope to cancer patients.

[0003] In recent years, covalent organic frameworks (COFs), as a class of long-range ordered porous crystalline polymers formed by covalent reactions of organic building blocks, have been widely used in various fields such as catalysis, adsorption separation, sensing, and energy storage due to their advantages such as low density, clear structure, large surface area, and tunable pore size. In particular, with the development of nanotechnology, research on the application of nanoscale COFs in the biomedical field has gradually emerged.

[0004] The tumor microenvironment (TME) contains a large amount of reduced glutathione, which can regulate the oxidative stress balance in tumor cells. During ferroptosis therapy, Fe... 2+ The toxic hydroxyl radicals (·OH) generated by the Fenton reaction with intracellular hydrogen peroxide exert a therapeutic effect; however, reduced glutathione reduces the oxidative toxicity of ·OH, thus weakening the therapeutic effect. On the other hand, reduced glutathione can reduce metal ions from high to low valence states, such as reducing Fe. 3+ To Fe 2 + This process consumes glutathione, which makes Fe... 2+ The therapeutic effect of ferroptosis can be greatly enhanced. Therefore, exploring the application of MCOF in tumor treatment is of great significance. Summary of the Invention

[0005] To address the problems existing in the prior art, this invention provides a method for preparing a nano-metal covalent organic framework material that enhances ferroptosis by consuming glutathione. The nano-metal covalent organic framework material prepared by this invention can effectively solve the problems of low Fenton response induction efficiency, poor therapeutic effect, and inability to efficiently exert the therapeutic effect of ferroptosis by single ferroptosis inducers.

[0006] The present invention also provides nano-metal covalent organic framework materials obtained by the above preparation method.

[0007] Another objective of this invention is to provide the application of the above-mentioned nano-metal covalent organic framework material as a drug carrier that consumes glutathione to enhance ferroptosis.

[0008] To overcome the above problems, the present invention adopts the following technical solution: This invention provides a method for preparing a nanomaterial of metal covalent organic framework that enhances ferroptosis by consuming glutathione, comprising the following steps: (1) Cu(NO3)2·3H2O and 1H-pyrazole-4-carboxaldehyde were ultrasonically dissolved in a mixed solution, and the reaction was carried out at high temperature under closed conditions. The product was washed and dried to obtain the organometallic ligand Cu3-PyCA3. (2) The metal-organic ligand Cu3-PyCA3, 2,5-diethoxybenzene-1,4-bis(formylhydrazine), mesitylene, 1,4-dioxane and trifluoroacetic acid solution were ultrasonically dispersed to obtain mixed solution 1. After three cycles of liquid nitrogen freezing-vacuum pumping-thawing, the reaction was carried out at high temperature under sealed conditions. The obtained product was washed and dried to obtain nano-metal covalent organic framework Cu3-MCOF; (3) Activate the hyaluronic acid solution, then add Cu3-MCOF and mix evenly. Stir vigorously and wash to obtain Cu3-MCOF@HA.

[0009] Furthermore, in step (1), the molar ratio of Cu(NO3)2·3H2O to 1H-pyrazole-4-carboxaldehyde is (3~4):5; the mixed solution is composed of N,N-dimethylformamide, pure water and ethanol in a volume ratio of (6~7):5:(6~7).

[0010] Furthermore, in step (1), the concentration of Cu(NO3)2·3H2O in the mixed solution is 0.033~0.044mol / L.

[0011] Furthermore, in step (1), the high-temperature reaction is carried out at a temperature of 100-150°C for 12-24 hours; the washing is carried out using water and acetone respectively; and the drying temperature is 100°C.

[0012] Furthermore, in step (2), the molar ratio of Cu3-PyCA3 and 2,5-diethoxybenzene-1,4-bis(formylhydrazine) is 2:3; the volume ratio of mesitylene, 1,4-dioxane and trifluoroacetic acid is 1:1:0.2.

[0013] Furthermore, in step (2), the concentration of the organometallic ligand Cu3-PyCA3 in mixed solution 1 is 0.02 mol / L; the concentration of 2,5-diethoxybenzene-1,4-bis(formylhydrazine) in mixed solution 1 is 0.03 mol / L; and the concentration of the trifluoroacetic acid solution is 6~9 M.

[0014] Furthermore, in step (2), the high-temperature reaction is carried out at a temperature of 120~130℃ for 3-7 days; the washing is performed using acetone; and the drying temperature is 100℃.

[0015] Further, in step (3), the mass ratio of hyaluronic acid to Cu3-MCOF is 10~20:1-2; the activation is to first add hyaluronic acid to a phosphate buffer solution, then add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and sonicate for 1 hour; then add N-hydroxysuccinimide, and sonicate for 12 hours; the stirring time is 2~6 hours. Furthermore, in step (3), the concentration of hyaluronic acid in phosphate buffer is 10~20 g / L; the mass ratio of Cu3-MCOF, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide is 1~2:4:5.

[0016] The present invention also provides nano-metal covalent organic framework materials prepared by the above preparation method.

[0017] This invention also provides the application of the above-mentioned nano-metal covalent organic framework material as a drug carrier that consumes glutathione to enhance ferroptosis.

[0018] Furthermore, in the preparation method provided by the present invention, the Cu3-PyCA3 prepared in step (1) is a pale yellow needle-like crystal with a microstructure of polygonal nanoplates and uniform size of about 800 nm. The nano-metal covalent organic framework provided by this invention can consume glutathione to enhance ferroptosis. The nano-metal covalent organic framework Cu3-MCOF can react rapidly with glutathione, resulting in severe damage to the framework structure of Cu3-MCOF, loss of crystallization, and further assembly to produce a large number of band-like structures, while consuming glutathione.

[0019] The beneficial results of this invention are as follows: (1) The preparation method provided by the present invention is simple and easy to operate, and the synthesized material has a high specific surface area and low biotoxicity; (2) Based on the nano-metal covalent organic framework, the present invention can consume glutathione to enhance ferroptosis. Cu3-MCOF@HA and three ferroptosis inducing agents (Erastin, Fe3O4 nanoparticles and Cu3-MCOF@Fe-TA coated with Fe-TA) are used together to treat tumor cells. The ferroptosis tumor treatment effect enhanced by consuming glutathione through Cu3-MCOF@HA can be achieved. It is a promising ferroptosis tumor treatment enhancer. Attached Figure Description

[0020] Figure 1 Scanning electron microscope (SEM) image of Cu3-PyCA3. Figure 2 Schematic diagram of Cu3-MCOF synthesis; Figure 3 Transmission electron microscopy (TEM) image of Cu3-MCOF; Figure 4 UV-Vis absorption spectra of Cu3-MCOF-consuming glutathione detected by 5,5'-dithiobis(2-nitrobenzoic acid); Figure 5 Transmission electron microscopy (TEM) images of the products of Cu3-MCOF and glutathione after different reaction times. Figure 6 Cell viability of cancer cells treated with Cu3-MCOF and three ferroptosis-inducing agents; Figure 7 Laser confocal fluorescence imaging of live / dead cancer cells treated with Cu3-MCOF and three ferroptosis-inducing agents. Detailed Implementation

[0021] To more clearly illustrate the present invention, the following description, in conjunction with preferred embodiments and accompanying drawings, further clarifies the invention. Those skilled in the art should understand that the specific description below is illustrative rather than restrictive and should not be construed as limiting the scope of protection of the present invention. Based on this explanation, those skilled in the art can readily conceive of specific embodiments of the present invention without inventive effort, and all related embodiments of the present invention will fall within the scope of protection of the present invention.

[0022] In this invention, unless otherwise specified, the preparation methods are all conventional preparation methods, and the reagents used are all obtained from publicly available commercial sources or according to existing methods unless otherwise specified.

[0023] This invention provides a method for enhancing ferroptosis by consuming glutathione based on a nano-metal covalent organic framework, aiming to solve the problems of low Fenton response induction efficiency, poor therapeutic effect, and inability to effectively exert ferroptosis therapeutic effect by single ferroptosis inducers.

[0024] Example 1 (a) Preparation of organometallic ligand Cu3-PyCA3 (1) In a 25 mL pressure-resistant bottle, 0.20 g Cu(NO3)2·3H2O (0.83 mmol) and 96 mg 1H-pyrazole-4-carboxaldehyde (1.0 mmol) were ultrasonically dissolved in a mixed solution of 7 mL N,N-dimethylformamide, 5 mL pure water and 7 mL ethanol; (2) Seal the pressure-resistant bottle and place it in an oven at 100°C for 12 hours. Wash the product three times with water and acetone respectively, and dry it at 100°C for 2 days to obtain Cu3-PyCA3 organometallic ligand (M = 475.88 g / mol).

[0025] The product Cu3-PyCA3 obtained from the above reaction is a pale yellow needle-like crystal. After grinding, the product becomes a polygonal nanoplate with uniform size and morphology, with a size of about 800 nm (e.g., Figure 1 (As shown).

[0026] (II) Preparation of Cu3-MCOF metal covalent organic framework 19.02 mg of organometallic ligand Cu3-PyCA3 (0.04 mmol), 16.93 mg of 2,5-diethoxybenzene-1,4-bis(formylhydrazine) (0.06 mmol), 0.5 mL of mesitylene, 0.5 mL of 1,4-dioxane, and 0.1 mL of 6 M trifluoroacetic acid were added to a 10 mL glass tube (9 × 6 mm). After ultrasonic dispersion, the mixture was subjected to three cycles of liquid nitrogen freezing-vacuum pump evacuation-thawing, and the glass tube was sealed with an alcohol torch. The glass tube was placed in an oven at 120 °C for 3 days. After opening the tube, the product was washed with acetone and dried at 100 °C for 2 days to obtain Cu3-MCOF (e.g., ...). Figure 2 (As shown).

[0027] The synthesized Cu3-MCOF has a rod-like structure with good dispersibility, a width of approximately 50-80 nm, and a non-uniform length ranging from 250-350 nm (e.g., Figure 3 (As shown).

[0028] (III) Biocompatibility Modification of Cu3-MCOF with Hyaluronic Acid 100 mg of hyaluronic acid was dispersed in 10 mL of phosphate buffer solution at pH 7.4. 40 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride was added, and the mixture was sonicated for 1 hour. Then, 50 mg of N-hydroxysuccinimide was added, and the mixture was sonicated for another 12 hours. 10 mg of Cu3-MCOF was added to the activated hyaluronic acid solution, and the mixture was stirred vigorously (600–1000 rpm) for 4 hours. The product was washed three times with pure water by centrifugation to obtain Cu3-MCOF@HA.

[0029] The tumor microenvironment contains abundant reduced glutathione, which can regulate the oxidative stress balance in tumor cells. During ferroptosis therapy, Fe... 2+ The toxic ·OH produced by the Fenton reaction with intracellular hydrogen peroxide is reduced by glutathione, thereby reducing the oxidative toxicity of ·OH and the therapeutic effect. On the other hand, reduced glutathione can reduce metal ions from a high valence state to a low valence state, reducing Fe... 3+ To Fe 2+ This process consumes glutathione, and the reduced Fe... 2+ The effect is greatly enhanced when ferrode therapy is exerted.

[0030] Example 2 10 mg Cu3-MCOF was dispersed in 10 mL of 10 mM glutathione aqueous solution. Samples were taken at 0, 5, 10, 30, 60 and 120 minutes of reaction for transmission electron microscopy to observe the morphological changes of the reaction product. At the same time, the concentration of glutathione in the solution system was determined by ultraviolet-visible absorption spectroscopy.

[0031] 5,5'-Dithiobis(2-nitrobenzoic acid) reacts with the thiol group of glutathione to form yellow 5-thio-2-nitrobenzoic acid, which has a maximum UV-Vis absorption peak at 412 nm. In contrast, 5,5'-dithiobis(2-nitrobenzoic acid) has almost no absorption peak above 400 nm. Therefore, 5,5'-dithiobis(2-nitrobenzoic acid) is used as a colorimetric indicator for glutathione.

[0032] The glutathione concentration test results showed that within 15 minutes, the absorption intensity at 412 nm decreased with the extension of the interaction time, indicating that the glutathione content in the solution was gradually decreasing. Simultaneously, the color of the reaction solution rapidly changed from yellow to colorless, indicating that Cu3-MCOF consumed glutathione (e.g., ...). Figure 4 (As shown).

[0033] The products of the above reaction at different times were characterized by transmission electron microscopy. The results showed that with the extension of reaction time, Cu3-MCOF underwent significant deformation and aggregation at 5 minutes, and its size had increased significantly. When the reaction reached 10 minutes, obvious long-ribbon fibrous structures with a size greater than 4 μm appeared in the product, but there were still many agglomerated block products. With the extension of time to 30 minutes, the number of long-ribbon fibrous structures increased significantly, while the number of agglomerated blocks decreased significantly. By 120 minutes, it had almost completely transformed into ultra-large ribbon-like structures. This indicates that glutathione plays a role in the reduction of Cu3-MCOF. 2+ Become Cu + During the process, the Cu3-MCOF structure was severely damaged, and further assembly produced a large number of ribbon-like structures (such as...). Figure 5 (As shown).

[0034] Example 3 Cytotoxicity assay: Human alveolar basal epithelial lung cancer cells (A549 cells) were passaged into 96-well plates at a density of 6 × 10⁶ cells per well. 3 After 24 hours of continued growth, 40 µg mL of [amount missing] was added. -1 Cu3-MCOF@HA was cultured for 0, 2, 4, 8, 12, and 24 hours, with three replicates per group, for MTT assays. Additionally, treatments were performed using Erastin, Erastin + Cu3-MCOF@HA, Fe3O4, Fe3O4 + Cu3-MCOF@HA, and Cu3-MCOF@Fe-TA, with the following groupings; the concentration of Cu3-MCOF@HA was 40 µg / mL in all cases. -1 The concentrations of Erastin were 0, 2, 4, 6, 10, and 14 µM, and the concentrations of Fe3O4 were 0, 10, 20, 30, 40, and 50 µg / mL. -1 The concentrations of Cu3-MCOF@Fe-TA were 0, 8, 16, 24, 32, and 40 µg / mL, respectively. -1 Each group has 3 parallel wells, and the cells are cultured for another 24 hours before performing an MTT assay. The result is calculated using the formula: % Cell viability = (ΔC) / (ΔC). samples - △C black ) / (△C control - △C black Cell viability was calculated by multiplying C by 100%. samples C represents the values ​​for drug and cell culture. control For values ​​without drug culture, C black Blank group.

[0035] The results showed that when the concentration of Cu3-MCOF@HA was 40 µg / mL, the activity of tumor cells did not decrease with the extension of culture time, remaining between 70% and 80% from 2 to 24 hours. This indicates that Cu3-MCOF@HA had a certain impact on cell activity, but did not cause complete damage to the cells. This may be because the interaction between Cu3-MCOF@HA and intracellular GSH disrupted the intracellular oxidative stress balance, leading to an accumulation of reactive oxygen species produced by the cells themselves, thus affecting cell viability. Figure 6 a). In the Erastin and Fe3O4NPs groups, cell activity was significantly inhibited. The decrease in activity was even more severe and concentration-dependent upon simultaneous addition of Cu3-MCOF@HA. Figure 6 b and Figure 6 c). Furthermore, the cell viability of the Cu3-MCOF@Fe-TA group also decreased significantly with increasing concentration ( Figure 6 d). These results indicate that Cu3-MCOF@HA enhances the effect of ferroptosis-inducing agents on ferroptosis in tumor cells.

[0036] Example 4 Tumor cell live-death dual-stain fluorescence imaging assay: A549 cells were passaged into laser confocal culture dishes at a density of 4 × 10⁶ cells per well. 5 After 24 hours of continued growth, the cells were treated according to the following groups: PBS, Cu3-MCOF@HA, Erastin, Erastin + Cu3-MCOF@HA, Fe3O4, Fe3O4 + Cu3-MCOF@HA, and Cu3-MCOF@Fe-TA (7 groups in total). The concentration of Cu3-MCOF@HA added was 40 µg / mL. -1 The concentration of Erastin was 4 µM, and the concentration of Fe3O4 was 30 µg / mL. -1 The concentration of Cu3-MCOF@Fe-TA was 32 µg / mL. -1 The cells were further processed for 24 h. Calcein-AM and propidium iodide (PI) were used as fluorescent probes for live and dead cells (Beyotime, Shanghai, China) to detect live / dead cells.

[0037] The above results show that Cu3-MCOF@HA alone did not significantly induce cell death, but adding Cu3-MCOF@HA to the ferroptosis inducing agent group significantly increased the cell death effect, proving that Cu3-MCOF@HA enhanced the effect of the ferroptosis inducing agent on ferroptosis in tumor cells. Figure 7 (As shown).

[0038] The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiments. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing a nanomaterial of metal covalent organic framework that consumes glutathione and enhances ferroptosis, characterized in that, Includes the following steps: (1) Cu(NO3)2·3H2O and 1H-pyrazole-4-carboxaldehyde were ultrasonically dissolved in a mixed solution, and the reaction was carried out at high temperature under closed conditions. The product was washed and dried to obtain the organometallic ligand Cu3-PyCA3. (2) The metal-organic ligand Cu3-PyCA3, 2,5-diethoxybenzene-1,4-bis(formylhydrazine), mesitylene, 1,4-dioxane and trifluoroacetic acid solution were ultrasonically dispersed to obtain mixed solution 1. After three cycles of liquid nitrogen freezing-vacuum pumping-thawing, the reaction was carried out at high temperature under sealed conditions. The obtained product was washed and dried to obtain nano-metal covalent organic framework Cu3-MCOF; (3) Activate the hyaluronic acid solution, then add Cu3-MCOF and mix evenly. Stir vigorously and wash to obtain Cu3-MCOF@HA.

2. The preparation method according to claim 1, characterized in that, In step (1), the molar ratio of Cu(NO3)2·3H2O to 1H-pyrazole-4-carboxaldehyde is (3~4):5; the mixed solution is composed of N,N-dimethylformamide, pure water and ethanol in a volume ratio of (6~7):5:(6~7).

3. The preparation method according to claim 1 or 2, characterized in that, In step (1), the concentration of Cu(NO3)2·3H2O in the mixed solution is 0.033~0.044 mol / L.

4. The preparation method according to any one of claims 1-3, characterized in that, In step (1), the high-temperature reaction is carried out at a temperature of 100-150℃ for 12-24 hours; the washing is carried out with water and acetone respectively; and the drying temperature is 100℃.

5. The preparation method according to claim 1, characterized in that, In step (2), the molar ratio of Cu3-PyCA3 and 2,5-diethoxybenzene-1,4-bis(formylhydrazine) is 2:3; the volume ratio of mesitylene, 1,4-dioxane and trifluoroacetic acid is 1:1:0.

2.

6. The preparation method according to claim 1 or 5, characterized in that, In step (2), the concentration of the organometallic ligand Cu3-PyCA3 in mixed solution 1 is 0.02 mol / L; the concentration of 2,5-diethoxybenzene-1,4-bis(formylhydrazine) in mixed solution 1 is 0.03 mol / L; and the concentration of the trifluoroacetic acid solution is 6~9 M.

7. The preparation method according to claim 1, 4, or 5, characterized in that, In step (2), the high-temperature reaction is carried out at a temperature of 120~130℃ for 3-7 days; the washing is carried out with acetone; and the drying temperature is 100℃.

8. The preparation method according to claim 1, characterized in that, In step (3), the mass ratio of hyaluronic acid to Cu3-MCOF is 10~20:1-2; the activation is to first add hyaluronic acid to phosphate buffer solution, then add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and sonicate for 1 hour; then add N-hydroxysuccinimide, and sonicate for 12 hours; the stirring time is 2~6 hours; the concentration of hyaluronic acid in phosphate buffer solution is 10~20 g / L; the mass ratio of Cu3-MCOF, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide is 1~2:4:

5.

9. A nano-metal covalent organic framework material prepared by the preparation method according to any one of claims 1-8.

10. The use of a nano-metal covalent organic framework material prepared by any one of claims 1-8 or as described in claim 9 as a drug carrier that consumes glutathione to enhance ferroptosis.

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