Hydrogen storage composite metal organic frameworks, methods of making and using the same

Abstract

This invention belongs to the field of pharmaceutical formulation technology, specifically disclosing a hydrogen storage composite metal-organic framework, its preparation method, and its applications. The preparation method of the hydrogen storage composite metal-organic framework includes the following steps: adjusting a gallic acid solution to alkalinity, adding magnesium chloride hexahydrate and reacting, centrifuging the magnesium-gallic acid metal-organic framework material to obtain Mg-GA-MOF; mixing Mg-GA-MOF with anhydrous ethanol, adding anhydrous sodium tetrachloropalladate solution in ethanol, equilibrating the solution, and slowly adding anhydrous sodium tetraborate solution in ethanol to obtain Pd / Mg-GA-MOF; and then loading with hydrogen. This invention utilizes both the porous microstructure of the MOF to physically adsorb hydrogen and the Pd nanoparticles embedded in the framework structure to chemically adsorb hydrogen. The combination of these two methods significantly improves the hydrogen loading efficiency of Pd / Mg-GA-MOF while exhibiting good biocompatibility. Furthermore, cell experiments show that Pd / Mg-GA-MOF can effectively scavenge various ROS and inhibit the inflammatory response of microglia, demonstrating high clinical application potential in stroke repair.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical formulation technology, and particularly relates to hydrogen storage composite metal-organic frameworks, their preparation methods and applications. Background Technology

[0002] After a stroke, the hypoxic lesion site undergoes anaerobic glycolysis, forming an acidic microenvironment. Subsequently, the microenvironment at the lesion site is activated and recruits distal microglia. Ultimately, microglia in situ and those infiltrating the lesion are activated, producing ROS and inflammatory factors, which exacerbate the death of nerve cells.

[0003] Metal-organic frameworks (MOFs), also known as coordination polymers, are a class of crystalline porous materials with a network structure formed by the self-assembly of inorganic metals (metal ions or metal clusters) and organic ligands. These materials differ from both inorganic porous materials and typical organic complexes, possessing both the rigidity of inorganic materials and the flexibility of organic materials. Porous MOFs are used as catalysts, sensors, or ion conductors, primarily in fields such as optics and magnetism. Their porous nature also makes them suitable for the adsorption, separation, and storage of natural gas, air, and inert gases.

[0004] Hydrogen (H2), as an endogenous gas, is not only an important energy source but also exhibits significant physiological and pathological regulatory functions. As early as 1975, hydrogen was discovered to have therapeutic potential for cancer. In 2007, hydrogen was confirmed as a therapeutic antioxidant that selectively reduces cytotoxic oxygen free radicals caused by ischemia-reperfusion and inflammation. Over the past decade, hydrogen has been shown to treat numerous diseases, including cancer, diabetes, stroke, atherosclerosis, Parkinson's disease, Alzheimer's disease, arthritis, dermatitis, colitis, hepatitis, pancreatitis, and myocardial infarction. The selective antioxidant properties of hydrogen are the basis for its therapeutic effects. In addition to its broad biomedical applicability, hydrogen's high biocompatibility has also been well-established. Therefore, hydrogen therapy is receiving increasing attention as clinical trials progress and is considered a promising treatment option.

[0005] Hydrogen possesses several unique properties, including its small molecular size, nonpolarity, low solubility (1.6 ppm) under physiological conditions, and high untargeted biodiffusion. Therefore, traditional drug delivery methods, such as inhalation, oral administration of hydrogen-rich water, and injection of hydrogen-rich saline, cannot precisely deliver hydrogen to the lesion site, resulting in limited therapeutic efficacy. Achieving effective hydrogen storage, targeted delivery, and controlled release is crucial for improving the therapeutic effect of hydrogen therapy, but it remains an unsolved scientific challenge. Pd nanocrystals are excellent hydrogen storage materials and ideal catalysts for hydrogenation reactions. They can chemically bond with hydrogen to form PdH, making Pd a potential candidate component for developing in vivo hydrogen delivery systems. Summary of the Invention

[0006] To address the aforementioned problems, this invention provides hydrogen storage composite metal-organic frameworks, their preparation methods, and applications, primarily to solve the issues of low hydrogen loading capacity and unsatisfactory targeting effects in current hydrogen therapy.

[0007] To solve the above problems, the present invention adopts the following technical solution:

[0008] The first aspect of the present invention relates to Preparation method of hydrogen storage composite metal-organic framework. The term "hydrogen storage composite metal-organic framework" has two different meanings in this section, representing two different substances: one is an organic framework Pd / Mg-GA-MOF for hydrogen storage, and the other is a hydrogen-loaded composite metal-organic framework H2@Pd / Mg-GA-MOF. Furthermore, the high hydrogen loading efficiency of the hydrogen storage composite metal-organic framework is explained by the combined use of physical microporous structure and Pd chemisorption to improve the hydrogen-loading efficiency.

[0009] Regarding Explanation 1, the preparation method of the hydrogen storage composite metal-organic framework Pd / Mg-GA-MOF includes the following steps:

[0010] Preparation of magnesium-gallic acid metal-organic framework:

[0011] The gallic acid (GA) solution was adjusted to a weakly alkaline state (to avoid strong bases damaging the product structure), allowing gallic acid to form gallate anions and exposing the binding sites.

[0012] Add magnesium chloride hexahydrate, and the reaction occurs.

[0013] Magnesium-gallic acid metal-organic frameworks were separated by centrifugation to obtain Mg-GA-MOF;

[0014] In-situ reduction of palladium nanoparticles in Mg-GA-MOF:

[0015] Mg-GA-MOF was mixed with anhydrous ethanol, and anhydrous ethanol solution of sodium tetrachloropalladium was added. The solution was brought to equilibrium (the color of the supernatant after centrifugation no longer changed, and the solution color changed from yellowish-brown to pale yellow or colorless). Anhydrous ethanol solution of sodium tetraborohydride was then slowly added to obtain Pd / Mg-GA-MOF. One purpose of anhydrous ethanol is to protect the reactants and products, preventing denaturation.

[0016] One of these steps, in the preparation of magnesium-gallic acid metal-organic frameworks, can be specifically described as follows for different steps:

[0017] I) Adjust the pH of the gallic acid solution to 7.2–10.0 (generally 8.0–8.5) using NaOH;

[0018] II) After adding magnesium chloride hexahydrate, the reaction is carried out by ultrasonication at 25-35℃. The frequency of the ultrasonication is generally greater than 20 kHz. The short-term heat generated during the generation and rupture of microbubbles during the ultrasonication process promotes the reaction.

[0019] Secondly, in the in-situ reduction step of nanoparticles in Mg-GA-MOF, the specific details for different steps can be as follows:

[0020] I) Mix Mg-GA-MOF with anhydrous ethanol to form an anhydrous ethanol solution of Mg-GA-MOF with a concentration of 8-11 mg / ml;

[0021] II) The concentration of sodium tetrachloropalladium in anhydrous ethanol solution is 2–3 mg / ml;

[0022] III) Sodium tetrahydrogenate in anhydrous ethanol solution is slowly added to Mg-GA-MOF in anhydrous ethanol solution, and the solution color deepens rapidly;

[0023] IV) After adding anhydrous sodium borohydride in ethanol and sealing the reaction, collect Pd / Mg-GA-MOF by centrifugation at room temperature or low temperature (e.g., 4°C).

[0024] Thirdly, the ratio of sodium tetrachloropalladium to Mg-GA-MOF is 5±1%. When a 5% ratio is used, the prepared material is better suited for the treatment of stroke-related central nervous system diseases, exhibiting lower biotoxicity and higher biocompatibility. Of course, this invention does not relinquish the right to other ratios. Appropriate adjustments to the ratio based on this invention to make the material applicable to other diseases should also be considered as application of the preparation method of this invention.

[0025] Fourthly, in processes involving gallic acid, a sealed, oxygen-free reaction is generally employed. The primary purpose is to protect the gallic acid, as oxygen oxidizes it, especially in an aqueous environment, forming two new compounds coordinated with the oxidized gallic acid, leading to material contamination and degradation. When reacting with anhydrous ethanol solution of sodium borohydride, sealing the reaction vessel is not strictly necessary if conditions permit. This is because anhydrous ethanol is used as the solvent in nanoparticle synthesis, preventing material ionization and ensuring stability. However, further sealing of the reaction vessel provides even better protection for the reactants.

[0026] Current research, such as the work published in the journal *Bioactive Materials* by Yue Kang et al., requires high-temperature synthesis of Mg-GA-MOF, and the resulting particles are micrometer-sized, limiting the application of this material in in vivo circulatory systems. The solution of this invention, however, allows for reaction at room temperature and the acquisition of nanometer-sized particles, thereby expanding its in vivo application scenarios.

[0027] Regarding Explanation 2, the preparation method of the hydrogen-loaded composite metal-organic framework H2@Pd / Mg-GA-MOF includes hydrogen loading in addition to the aforementioned preparation steps of the hydrogen storage composite metal-organic framework Pd / Mg-GA-MOF: hydrogen is introduced into a closed reactor containing Pd / Mg-GA-MOF to obtain the hydrogen storage composite metal-organic framework, which is loaded with hydrogen.

[0028] In the hydrogen loading step: Pd / Mg-GA-MOF is dissolved in anhydrous ethanol, and then hydrogen gas is passed into the Pd / Mg-GA-MOF anhydrous ethanol solution. Generally, the gas flow is kept uninterrupted while the material is continuously stirred. If necessary, the reactor can be brought into a high-pressure hydrogen environment.

[0029] The second aspect of the present invention relates to The hydrogen storage composite metal-organic framework Pd / Mg-GA-MOF prepared in the first aspect, or the hydrogen-loaded hydrogen storage composite metal-organic framework H2@Pd / Mg-GA-MOF prepared in the second aspect, are used. Pd / Mg-GA-MOF can better load hydrogen and has a higher loading capacity, and it can increase ROS scavenging ability; when used as H2@Pd / Mg-GA-MOF, it already has H2 loaded on the Pd / Mg-GA-MOF base. Figure 6 As shown, hydrogen gas exists within the lattice of Pd nanoparticles, causing changes in the ultraviolet absorption spectrum.

[0030] The third aspect of the present invention relates to The application of the hydrogen storage composite metal-organic framework obtained in the first aspect above in the preparation of products for the prevention and treatment of stroke.

[0031] When the hydrogen storage composite metal-organic framework is loaded with hydrogen, the framework is H2@Pd / Mg-GA-MOF, and the hydrogen storage composite metal-organic framework has the function of catalytically scavenging ROS and inhibiting microglial inflammatory response.

[0032] When the hydrogen storage composite metal-organic framework is not loaded with hydrogen, Pd / Mg-GA-MOF is used as a carrier particle in the preparation of stroke prevention and treatment products, mainly for loading hydrogen, and its direct anti-inflammatory effect is limited.

[0033] The fourth aspect of the present invention relates to The first aspect above explains the application of the hydrogen storage composite metal-organic framework Pd / Mg-GA-MOF in the preparation of negative hydrogen carriers. It enables efficient hydrogen storage, targeted delivery, and controlled release: the targeted delivery effect is reflected in the acidic environment responsiveness of the framework material. The microenvironment of stroke infarct lesions is weakly acidic, and the prepared framework material will rapidly degrade under this environment, achieving controlled release.

[0034] The beneficial effects of this invention are:

[0035] This invention combines the physical adsorption of hydrogen by a metal-organic framework porous structure with the chemical adsorption of hydrogen by Pd nanoparticles. This improves the hydrogen loading efficiency while ensuring its biocompatibility, and also broadens its ROS catalytic performance and anti-inflammatory properties, greatly enhancing its application potential in stroke treatment. Attached Figure Description

[0036] Figure 1 This is a flowchart of the synthesis path of the present invention;

[0037] Figure 2 SEM results for Mg-GA-MOF (left) and Pd / Mg-GA-MOF (right);

[0038] Figure 3 Results for Pd / Mg-GA-MOFTEM;

[0039] Figure 4 Results of Pd / Mg-GA-MOF and Mg-GA-MOF XRD;

[0040] Figure 5 Results for Pd / Mg-GA-MOF and Mg-GA-MOFFTIR;

[0041] Figure 6 The results are the UV absorption spectra of Pd / Mg-GA-MOF, Mg-GA-MOF and H2@Pd / Mg-GA-MOF.

[0042] Figure 7To obtain the results of ROS probe fluorescence quantification;

[0043] Figure 8 Results of ROS flow cytometry;

[0044] Figure 9 The changes in mRNA levels of inflammatory factors after stimulation by OGD in different groups were investigated.

[0045] Figure 10 The changes in mRNA levels of inflammatory factors after LPS stimulation were inhibited in different groups;

[0046] Figure 11 Changes in biocompatibility at different feed ratios;

[0047] Figure 12 The results show the acid-response degradation of Mg-GA-MOF;

[0048] Figure 13 The results show the acid response hydrogen release of H2@Pd-Mg-GA-MOF. Detailed Implementation

[0049] The invention will now be described in detail with reference to specific research projects.

[0050] The preparation method of hydrogen storage composite metal-organic framework is as follows: :

[0051] Step 1: Magnesium-gallic acid metal-organic framework

[0052] Weigh 3.76g of gallic acid and dissolve it in 50ml of deionized water. Adjust the pH of the solution to 8.0 using NaOH and seal it with a sealing film to ensure airtightness.

[0053] Weigh out 1.02 g of magnesium chloride hexahydrate again and add it to the above system;

[0054] Using parameters of 100W, 40Hz, and 30±5℃, the above reaction flask was placed in an ultrasonic water bath for reaction for 30 minutes.

[0055] Magnesium-gallic acid metal-organic framework Mg-GA-MOF (Mg-MOF can also be expressed in this paper, as shown in the figure) was centrifuged at 6000 rpm, 30 min, and 4 °C.

[0056] The material was then washed a second time with anhydrous ethanol and finally stored in anhydrous ethanol.

[0057] Step 2: In-situ reduction of palladium nanoparticles in Mg-GA-MOF

[0058] Taking a 5% feed ratio of Pd / Mg-GA-MOF (Pd / Mg-MOF can also be expressed in this article, as shown in the figure) as an example, take the Mg-GA-MOF synthesized in the first step, adjust the concentration to 10 mg / ml with anhydrous ethanol, and take 10 ml and put it in a reaction flask.

[0059] Weigh out 2.5 ml of the prepared 2 mg / ml sodium tetrachloropalladium anhydrous ethanol solution and add it to the above reaction flask;

[0060] The above system was sealed with a sealing film and the solution was equilibrated by magnetic stirring at 4°C and 300 rpm for 24 hours.

[0061] Prepare a 2 mg / ml anhydrous ethanol solution of sodium tetraborohydride (prepare fresh for use), and slowly add 2.5 ml to the above reaction flask. At this time, the reaction flask should be magnetically stirred at 300 rpm. After adding sodium tetraborohydride, the color of the material should be observed to darken rapidly.

[0062] The above system was sealed with a sealing film and magnetically stirred at 4°C and 300 rpm for 24 hours to ensure that the reduction reaction was fully carried out;

[0063] Pd / Mg-GA-MOF was collected at 12000 rpm for 30 min at 4 °C and washed twice with anhydrous ethanol to remove excess ions.

[0064] Finally, it is stored in anhydrous ethanol and sealed with sealing film.

[0065] pass Figure 2-4 It can be seen that the corresponding products were successfully prepared using the methods and steps involved in this invention.

[0066] Step 3: Hydrogen Loading

[0067] Taking the chemical reaction to produce hydrogen gas as an example

[0068] A certain amount of zinc granules are reacted with dilute hydrochloric acid to generate hydrogen gas. The hydrogen gas is then passed through water and anhydrous calcium chloride to remove impurities.

[0069] The aforementioned hydrogen gas was introduced into a sealed container containing 10 mg / ml Pd / Mg-GA-MOF, and the gas was introduced into the liquid. According to indirect experimental data, it can be calculated that each milligram of material can load 2.70 μmol of hydrogen gas, which is about 0.089 ml of hydrogen gas at normal atmospheric pressure. This is equivalent to adsorbing 89 ml of hydrogen gas per gram, which is 3 times the hydrogen adsorption level of pure Pd nanoparticles that have been reported so far.

[0070] Seal after 15 minutes and store at 4°C away from light.

[0071] pass Figure 6As shown, hydrogen was successfully loaded onto Pd to form PdH material. The hydrogen will exist in the lattice of Pd nanoparticles, which will cause changes in the ultraviolet absorption spectrum.

[0072] In the preparation of materials such as H2@Mg-GA-MOF (which can also be expressed as H2@Pd / Mg-MOF in this paper, as shown in the attached figure), hydrogen gas is directly introduced into the ethanol solution of Mg-GA-MOF (sealed) to create a high-pressure hydrogen environment. Since the hydrogen loading of a simple MOF is based on its high specific surface area, it's essentially a process of balancing from a high concentration to a low concentration. High-pressure hydrogen storage for a period of time simply replaces the gas in the pores, but this method is extremely unstable and has a limited loading capacity.

[0073] ROS removal via H2@Pd / Mg-GA-MOF:

[0074] ROS probe fluorescence quantification:

[0075] Using the BV-2 cell line, six groups were set up in 12-well plates: no treatment control, PBS positive control, Mg-GA-MOF, Pd / Mg-GA-MOF, H2@Mg-GA-MOF, and H2@Pd / Mg-GA-MOF.

[0076] Gibco's sugar-free DMEM was placed in an anaerobic culture environment overnight to remove oxygen from the culture medium.

[0077] Replace the cell culture medium with sugar-free and oxygen-free DMEM, and add material at a final concentration of 50 μg / ml.

[0078] The above system was treated with OGD in an anaerobic culture environment for 2 hours;

[0079] After processing, cells were collected and diluted 1:6000 with DCFH-DA (Nanjing Jiancheng Biotechnology) probe. Each sample was incubated in 500ml of dilution buffer at 37℃ for 30min.

[0080] After washing twice with PBS, the sample was measured. The excitation light was 488 nm and the emission light was 515 nm.

[0081] The result is as follows Figure 7 As shown, the ROS of the H2@Pd / Mg-GA-MOF group was significantly lower than that of the other groups.

[0082] ROS flow cytometry using H2@Pd / Mg-GA-MOF:

[0083] Using the BV-2 cell line, six groups were set up in 12-well plates: no treatment control, PBS positive control, Mg-GA-MOF, Pd / Mg-GA-MOF, H2@Mg-GA-MOF, and H2@Pd / Mg-GA-MOF.

[0084] Gibco's sugar-free DMEM was placed in an anaerobic culture environment overnight to remove oxygen from the culture medium.

[0085] Replace the cell culture medium with sugar-free and oxygen-free DMEM, and add material at a final concentration of 50 μg / ml.

[0086] The above system was treated with OGD in an anaerobic culture environment for 2 hours;

[0087] After processing, cells were collected and diluted 1:6000 with DCFH-DA (Nanjing Jiancheng Biotechnology) probe. Each sample was incubated in 500ml of dilution buffer at 37℃ for 30min.

[0088] After washing twice with PBS, the sample was resuspended in 200 μL of PBS and then analyzed.

[0089] The result is as follows Figure 8 As shown, H2@Pd / Mg-GA-MOF had the lowest average intracellular ROS level.

[0090] Inhibition of inflammatory factors in an OGD model using H2@Pd / Mg-GA-MOF:

[0091] BV-2 cell line was used, treated with OGD, and material was added to a final concentration of 50 μg / ml for validation. After reaching the specified time, cells were washed with PBS, and RNA was extracted using the Trizol method. cDNA was prepared using a reverse transcription kit (Novitamin), and the expression of relevant mRNA was detected by qRT-PCR using a qPCR kit. The results are as follows: Figure 9 As shown, Figure 9 The two images in the upper middle section show elevated levels of IL10 and TGF-β anti-inflammatory factor mRNA. Figure 9 The two images at the bottom show a decrease in the mRNA levels of pro-inflammatory factors IL6, TNF-α, and IL-1b.

[0092] Inhibition of inflammatory factors in an LPS stimulation model using H2@Pd / Mg-GA-MOF:

[0093] Using the BV-2 cell line, cells were stimulated with lipopolysaccharide (LPS) at a final concentration of 5 μg / ml, with material added at a final concentration of 50 μg / ml for validation. After reaching the specified time, cells were washed with PBS, and RNA was extracted using the Trizol method. cDNA was prepared using a reverse transcription kit (Novozymes), and the expression of relevant mRNAs was detected by qRT-PCR using a qPCR kit. Figure 10 As shown in the three images below the middle, after 3 hours of LPS stimulation, the level of pro-inflammatory factor mRNA decreased; Figure 10 As shown in the upper two figures, after 12 hours of LPS stimulation, the level of anti-inflammatory factor mRNA increased.

[0094] Simulation of in vitro neuron-related studies using the N2a cell line :

[0095] Using the N2a cell line, cells were seeded in 96-well plates with MOF material added at different concentration gradients (PBS, 5, 10, 17.5, 25, 35, 50, 75, 100 μg / ml) at different feed ratios (sodium tetrachloropalladium to Mg-GA-MOF feed ratio). After co-incubation for 24 h, cell viability was measured using a CCK-8 assay kit. The results are as follows: Figure 11 As shown, when the concentration exceeds 25 μg / ml, the material with a 5% feed ratio has better cell compatibility.

[0096] Acid-responsive degradation of Mg-GA-MOF:

[0097] The materials were placed in PBS buffer solutions at pH 6.5, 7.3, and 8.0, and sealed with paraffin oil to avoid additional interference from atmospheric oxygen. The supernatant of the above systems was collected sequentially at specified time intervals, and undegraded materials were removed by centrifugation at 12000 rpm for 30 min at 4°C. The gallic acid content in the supernatant was determined using the Folin-Ciocalteu method to assess the degradation status of the materials. The results are as follows: Figure 12 As shown, the prepared Mg-GA-MOF material exhibits better responsiveness in acidic environments.

[0098] Acid response of H2@Pd-Mg-GA-MOF to hydrogen release:

[0099] The material was placed in PBS buffer solutions at pH 6.5, 7.3, and 8.0, followed by the addition of methylene blue to a final concentration of 300 μg / ml and sealing with paraffin oil. Hydrogen, under palladium catalysis, reduced methylene blue to methylene white, thus altering the absorbance value. The supernatant of the above system was collected sequentially at specified time intervals, and undegraded material was removed by centrifugation at 12000 rpm for 30 min at 4℃. The absorbance of the supernatant was directly measured to calculate the remaining methylene blue content, and then the hydrogen release was calculated. The results are as follows: Figure 12As shown, H2@Pd-Mg-GA-MOF exhibits better acid response and hydrogen release.

[0100] Those skilled in the art will appreciate that various modifications to the above embodiments can be made without departing from the overall spirit and concept of the present invention. All such modifications fall within the protection scope of the present invention. The protection scheme of the present invention is defined by the appended claims.

Claims

1. A method for preparing a hydrogen storage composite metal-organic framework, characterized in that, Includes the following steps Preparation of magnesium-gallic acid framework: The gallic acid solution was adjusted to a weakly alkaline pH of 8.0-8.5 by adding NaOH. Magnesium chloride hexahydrate was added and ultrasonically reacted at 25-35℃. The ultrasonic reaction conditions were 100W and 40Hz. After the reaction, the magnesium-gallic acid metal-organic framework was separated by centrifugation to obtain Mg-GA-MOF. In-situ reduction of palladium nanoparticles in Mg-GA-MOF: Mg-GA-MOF was mixed with anhydrous ethanol to form an anhydrous ethanol solution of Mg-GA-MOF with a concentration of 8~11 mg / ml, and 2~3 mg / ml anhydrous ethanol solution of sodium tetrachloropalladium was added. After equilibration, anhydrous ethanol solution of sodium tetraborohydride was slowly added to obtain Pd / Mg-GA-MOF, wherein the feed ratio of sodium tetrachloropalladium to Mg-GA-MOF was 5%. Hydrogen gas was introduced into a closed reactor containing anhydrous ethanol solution of Pd / Mg-GA-MOF to obtain hydrogen storage composite metal-organic framework H2@Pd / Mg-GA-MOF.

2. The application of the hydrogen storage composite metal-organic framework prepared by the method of claim 1 in the preparation of drugs for the prevention and treatment of stroke, wherein the drug is degraded by H2@Pd / Mg-GA-MOF in the acidic microenvironment of the lesion, releasing hydrogen and gallic acid at the same time, catalyzing the removal of ROS and inhibiting the inflammatory response of microglia.

3. The application according to claim 2, characterized in that, The hydrogen storage composite metal-organic framework can load 2.70 μmol of hydrogen per milligram of material.

4. The application of the hydrogen storage composite metal-organic framework prepared by the method of claim 1 in the preparation of negative hydrogen carriers.

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