A kind of oxidation catalytic nano material and its preparation method and application

Abstract

The present invention relates to a method for preparing an oxidative catalytic nano material, comprising the steps of: S1, dissolving 2-aminoterephthalic acid and ferric chloride hexahydrate in N, N-dimethylformamide, adding acetic acid, and crystallizing in an oil bath; S2, mixing the solid obtained by crystallization, chitosan, and thiamine pyrophosphate, and stirring until the reaction is completed, to obtain the oxidative catalytic nano material. The oxidative catalytic nano material of the present invention enhances the autophagic death of tumor cells through chitosan, and at the same time enhances the autophagic dependence of tumor cells, i.e., ferroptosis, and significantly enhances the therapeutic effect for tumors.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to an oxidation catalytic nano material and a preparation method and application thereof. Background Art

[0002] At present, during nanocatalytic therapy, the rate of metal ion valence state transition is slow and the oxidation catalytic efficiency is low. Generally, compared with normal cells, the unique cell proliferation, energy metabolism and mitochondrial dysfunction in tumor cells will induce excessive accumulation of hydrogen peroxide in tumor tissues. Nanocatalytic medicine utilizes this characteristic of tumor cells and uses Fenton or Fenton-like reactions to induce transition metal ions to catalyze hydrogen peroxide to produce highly cytotoxic reactive oxygen species, such as hydroxyl free radicals, singlet oxygen, and superoxide anions, to kill tumor cells.

[0003] Nanomaterials based on iron ions can undergo Fenton reactions.

[0004] Fe 2+ +H2O2—→Fe 3+ +OH - +OH (i)

[0005] H2O2+Fe 3+ —→Fe 2+ +O2+H + (ii)

[0006] O2+Fe 2+ —→Fe 3+ +O2 - (iii)

[0007] While hydroxyl radicals are being generated, the Fe(II) / Fe(III) cycle of iron ions occurs, but the effectiveness and sustainability of the reaction are often limited by the efficiency of the conversion of iron ions to ferrous ions, which results in a low overall nanocatalytic efficiency.

[0008] Therefore, there is an urgent need for an oxidation catalytic nanomaterial and a preparation method and application thereof. Summary of the invention

[0009] The purpose of the present invention is to provide an oxidation catalytic nano material and a preparation method and application thereof in view of the deficiencies in the prior art.

[0010] To achieve the above object, the technical solution adopted by the present invention is:

[0011] The first aspect of the present invention is to provide a method for preparing an oxidation catalytic nanomaterial, the steps comprising:

[0012] S1, dissolving 2-aminoterephthalic acid and ferric chloride hexahydrate in N,N-dimethylformamide, adding acetic acid, and crystallizing in an oil bath;

[0013] S2. Mix the solid obtained by crystallization, chitosan and thiamine pyrophosphate, and stir until the reaction is completed to obtain the oxidation catalytic nanomaterial.

[0014] Preferably, in step S1, the temperature of the oil bath is 60°C-240°C; and the time of the oil bath is 2h-8h.

[0015] Preferably, the steps further include: after crystallization in an oil bath, cooling to room temperature, separating the crystallized solid by centrifugation, washing the solid, and mixing it with chitosan and thiamine pyrophosphate.

[0016] Preferably, the washing comprises: washing once with N,N-dimethylformamide, and washing twice with ethanol.

[0017] Preferably, in step S2, the mass ratio of the solid obtained by crystallization, chitosan and thiamine pyrophosphate is 1:(0-10):(0-5).

[0018] Preferably, in step S2, the reaction time is overnight.

[0019] Preferably, step S2 further comprises: after the reaction is completed, washing the obtained second metal organic framework.

[0020] Preferably, the washing comprises: washing three times with ethanol.

[0021] The second aspect of the present invention is to provide an oxidation catalytic nanomaterial prepared by the preparation method as described above.

[0022] The third aspect of the present invention is to provide a use of the above-mentioned oxidative catalytic nanomaterial in the preparation of a tumor treatment drug, wherein the tumor treatment drug enhances the autophagic death and / or ferroptosis of tumor cells.

[0023] The present invention adopts the above technical solution, and has the following technical effects compared with the prior art:

[0024] The oxidative catalytic nanomaterial of the present invention enhances the autophagic death of tumor cells through chitosan, and simultaneously enhances the autophagy dependence of tumor cells, namely ferroptosis, thereby significantly enhancing the therapeutic efficacy for tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It is a schematic diagram of the structure of the oxidation catalytic nanomaterial in the present invention;

[0026] Figure 2This is a result diagram of the oxidation catalytic nanomaterial being internalized by cells in the present invention;

[0027] Figure 3 This is a result diagram of cell survival rate after intervention of the oxidative catalytic nanomaterials of the present invention;

[0028] Figure 4 This is a result diagram of the intracellular lipid oxidation level of the oxidation catalytic nanomaterial group of the present invention;

[0029] Figure 5 The result graph is the decrease rate of glutathione peroxidase of the oxidation catalytic nanomaterials;

[0030] Figure 6 The results of the expression of autophagy-related proteins and ferroptosis-key protein GPX4 in oxidative catalytic nanomaterials;

[0031] Figure 7 The results of tumor volume after administration of oxidation catalytic nanomaterials are shown in Figure 2 . DETAILED DESCRIPTION

[0032] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0033] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments may be combined with each other.

[0034] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, but they are not intended to limit the present invention.

[0035] Examples 1-5

[0036] Embodiment 1 provides an oxidation catalytic nanomaterial and a preparation method thereof, the preparation steps comprising:

[0037] S1. Dissolve 0.126 g of 2-aminoterephthalic acid and 0.187 g of ferric chloride hexahydrate in 15 ml of N,N-dimethylformamide, and drop 198 μL of acetic acid, stir and crystallize in an oil bath at 120° C. for 4 hours, cool to room temperature, separate the crystallized metal organic framework by centrifugation, and wash once with N,N-dimethylformamide and twice with ethanol in sequence to remove excess reactants;

[0038] S2. Mix the metal organic framework, chitosan and thiamine pyrophosphate in a mass ratio of 1:0:0, and stir at room temperature overnight. After the reaction is completed, wash three times with ethanol to remove excess reactants to obtain the oxidation catalytic nanomaterial.

[0039] The differences between Example 2-5 and Example 1 are shown in the following table:

[0040] The mixing mass ratio of the metal organic framework, chitosan and thiamine pyrophosphate Example 1 1:0:0 Example 2 1:1:0.5 Example 3 1:2.5:1.25 Example 4 1:5:2.5 Example 5 1:10:5

[0041] Example 1-5 Figure 1 As shown, the first to fifth columns are embodiments 1-5 respectively, the scale bar of the first row is 50 nm, and the scale bar of the second row is 200 nm.

[0042] Application Examples

[0043] CT26 cells (Catalog No. TCM37, identifier CSTR: 19375.09.3101MOUTCM37) were purchased from Shanghai Cell Bank, Chinese Academy of Sciences. Cells were cultured in DMEM high glucose medium (Gibco, USA) containing 10% fetal bovine serum (Gibco, USA) and 1% penicillin / streptomycin (C0222, Beyotime Biotechnology Co., Ltd., China) at 37°C.

[0044] CT26 cells were inoculated into T25 culture flasks and cultured for 24 hours. Cells were treated with NaHS+H2O2, MOF (metal organic framework) + NaHS+H2O2, COS (chitosan) + NaHS+H2O2 or COS@MOF (oxidative catalytic nanomaterial) + NaHS+H2O2 for 24 hours. The cells were scraped with a cell scraper and centrifuged at 3000 rpm for 10 minutes to collect the cells. After adding 2.5% glutaraldehyde fixative precooled at 4°C, store in a 4°C refrigerator. Discard the fixative and rinse three times with 0.1M phosphate buffer (pH 7.0) for 15 minutes each. Fix with 1% osmium acid solution for 1 hour. Carefully remove the osmium acid waste liquid and rinse three times with 0.1M phosphate buffer (pH7.0) for 15 minutes each. Dehydrate with ethanol solutions of gradient concentrations (including 30%, 50%, 70%, 80%, 90% and 95%) for 15 minutes, and then treat with 100% ethanol for 20 minutes. Finally, treat with pure acetone for 20 minutes. Treat the sample with a mixture of embedding agent and acetone (V / V=1 / 1) for 1 hour. Treat the sample with a mixture of embedding agent and acetone (V / V=3 / 1) for 3 hours. Treat with pure embedding agent overnight. After embedding infiltration treatment, the embedded sample was obtained by heating at 70°C overnight. Ultrathin sections with a thickness of 70nm-90nm were obtained by slicing in an ultrathin microtome, stained with lead citrate solution and 50% ethanol saturated solution of uranyl acetate for 5-10 minutes respectively, dried, and observed in a Hitachi H-7650 transmission electron microscope. The results are as shown in Figure 2 As shown, the oxidative catalytic nanomaterials were internalized by cells.

[0045] CT26 cells were seeded into 96-well culture plates and incubated with COS@MOF at different concentrations (0 μg / mL, 12.5 μg / mL, 25 μg / mL, 50 μg / mL, and 100 μg / mL) for 24 hours, and then carefully washed with PBS. After that, cell counting Kit-8 was added to each well to reach a final concentration of 10%, and the plate was incubated for 1 hour at 37°C in an incubator containing 5% CO2. The absorbance value at 450nm was measured using a fluorescence spectrometer. The results are shown in Figure 2. Figure 3 As shown, the cell survival rate was the lowest after intervention with oxidation-catalytic nanomaterials.

[0046] The lipid peroxidation MDA detection kit (S0131S, Bio-Tech Biotech Co., Ltd., China) was used to investigate the oxidative stress level. 6The cells were incubated at an initial density of 10 cells / well for 24 hours. After gently rinsing three times with PBS, different interventions were added to each well. Pure culture medium was used as a control, and the concentration of MOF and COS@MOF was 50 μg / mL, and then NaHS (0.1 mM) and H2O2 (0.1 mM) were added, and the cells were cultured for 24 hours. The cells were washed three times with PBS pre-cooled at 4°C, and then RIPA was added and lysed on ice for 30 minutes. The cells were collected with a scraper and centrifuged at 12000g for 10 minutes. The clear liquid was removed from the upper layer. The reagents in the kit were used to prepare the detection solution, and pure RIPA was used as a control group. After preparing the reaction system, it was heated in a metal bath at 100°C for 15 minutes. It was then cooled to room temperature by a water bath and centrifuged at 1000g for 10 minutes at room temperature. 200 μL of supernatant from each group was added to a 96-well plate, and the absorbance was measured at 532nm using a microplate reader. The results are shown in Figure 4 As shown, the intracellular lipid oxidation level was the highest in the oxidation-catalytic nanomaterial group.

[0047] The residual content of GSH-Px in the cells was detected using a cellular glutathione peroxidase detection kit containing NADPH (S0056, Biotech Biotechnology Co., Ltd., China). CT26 cells were cultured in an incubator for 24 hours. MOF (50 μg / mL), COS (corresponding mass ratio) and COS@MOF (50 μg / mL) were added, followed by NaHS (0.1 mM) and H2O2 (0.1 M). The cells were cultured in a constant temperature incubator at 37°C and 5% CO2 for another 24 hours and then washed with PBS. After hanging with a cell scraper, the cells were lysed by adding the lysate and homogenized with a glass homogenizer in an ice bath. Subsequently, the cells were centrifuged at 12000g for 10 minutes, and the supernatant was taken to determine the enzyme activity. The results are as shown in Figure 5 As shown, the glutathione peroxidase decrease rate of the oxidation catalytic nanomaterials was the highest.

[0048] To detect changes in the expression of NCOA4, GPx4, LC3, and P62, RIPA lysates were added to the cells treated differently. After full lysis on ice for 30 min, proteins were extracted by centrifugation at 12,000 g for 10 min at 4 °C, and the protein concentration of the samples was determined by a BCA protein assay kit. According to the protein concentration of each sample determined, 500 μg of protein was taken, the corresponding amount of 6× loading buffer was added, 1× loading buffer was added to make the volume of each sample equal, and the concentration of each sample was 2.5 mg / mL, and then the samples were heated in a metal bath preheated at 100 °C for 10 min. The extracted proteins were separated by gel electrophoresis and transferred to PVDF membranes and blocked with 5% whey for 1 h at room temperature. Then, the membranes were trimmed and incubated overnight with primary antibodies against GPx4 (1:1000), LC3 (1:10000), p62 (1:1000), and actin (1:1000), respectively. The next day, the membrane was rinsed five times with PBST for 5 minutes each, and then incubated with the corresponding secondary antibodies for 1 hour at room temperature. After incubation, the membrane was washed five times with PBST for 5 minutes each, and then stained with ECL detection reagent for development. Figure 6 As shown, the expression level of autophagy-related proteins in oxidative catalytic nanomaterials increased, and autophagy was enhanced; the expression level of GPX4, a key protein in ferroptosis, decreased, and ferroptosis was activated.

[0049] Balb / c mice (male, 6 weeks old, 20 g-25 g) were purchased from Shanghai Slake Laboratory Animal Co., Ltd. The mice were maintained with standard feed and water under a 12-h dark-light cycle at room temperature (20°C-24°C) and relative humidity (45%-55%). 6 CT26 cells were subcutaneously injected into the right groin of mice to establish a subcutaneous colorectal cancer tumor model. When the tumor grew to about 6 mm × 6 mm, the mice were randomly divided into four groups (PBS group, MOF group, COS group, and COS@MOF group). The weight and tumor volume of the mice were measured every two days, and the tumor volume was calculated by the formula: 1 / 2 × (tumor length) × (tumor width) 2 On day 14, the mice were euthanized and the tumors were removed. Figure 7 As shown, tumor volume was significantly inhibited after administration of oxidative catalytic nanomaterials.

[0050] In summary, the oxidative catalytic nanomaterial of the present invention enhances the autophagic death of tumor cells through chitosan, and at the same time enhances the autophagy dependence of tumor cells, namely ferroptosis, and significantly enhances the therapeutic efficacy for tumors.

[0051] The above description is only a preferred embodiment of the present invention, and does not limit the implementation mode and protection scope of the present invention. For those skilled in the art, it should be aware that all solutions obtained by equivalent substitutions and obvious changes made using the description and illustrations of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing an oxidation catalytic nanomaterial, characterized in that the steps include: S1, dissolving 2-aminoterephthalic acid and ferric chloride hexahydrate in N,N-dimethylformamide, adding acetic acid, and crystallizing in an oil bath; S2, mixing the crystallized solid, chitosan and thiamine pyrophosphate, and stirring until the reaction is complete, thereby obtaining the oxidation catalytic nanomaterial; Wherein, in step S2, the mass ratio of the crystallized solid, chitosan and thiamine pyrophosphate is 1:(1-10):(0.5-5).

2. The preparation method according to claim 1, characterized in that: In step S1, the temperature of the oil bath is 60°C-240°C; the time of the oil bath is 2h-8h.

3. The preparation method according to claim 1, characterized in that: The steps also include: after crystallization in an oil bath, cooling to room temperature, separating the crystallized solid by centrifugation, washing and mixing with chitosan and thiamine pyrophosphate.

4. The preparation method according to claim 3, characterized in that: The washing includes: washing once with N,N-dimethylformamide and washing twice with ethanol.

5. The preparation method according to claim 1, characterized in that: In step S2, the reaction time is overnight.

6. The preparation method according to claim 1, characterized in that: Step S2 also includes: after the reaction is completed, washing the obtained second metal organic framework.

7. The preparation method according to claim 6, characterized in that: The washing comprises: washing three times with ethanol.

8. An oxidation catalytic nanomaterial prepared by the preparation method according to any one of claims 1 to 7.

9. Use of the oxidation catalytic nanomaterial as claimed in claim 8 in the preparation of a drug for treating tumors, characterized in that: The tumor treatment drug enhances the autophagic death and / or ferroptosis of tumor cells.

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