An artificial metabolic enzyme targeting mitochondria and its preparation method and application

Abstract

The invention discloses a kind of artificial metabolic enzyme targeting mitochondria and its preparation method and application, relate to the field of biomedical material technology, the method comprises the following steps:Activated carbon is mixed and stirred with phosphomolybdic acid solution to obtain POM / AC;Mixed with acetylacetonate platinum (II) solution, reduction treatment is carried out to obtain Pt-POM / AC;It is dispersed in ethanol, 3-aminopropyltriethoxysilane is added and stirred, and solid A is obtained by freeze drying;(3-carboxypropyl) triphenylphosphine bromide, dimethyl sulfoxide and 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride are mixed, stirred in dark, N-hydroxysuccinimide is added, activated in dark, and solution B is obtained;Solid A is dissolved, mixed with solution B, stirred in dark, centrifuged, washed and dried, and the artificial metabolic enzyme targeting mitochondria is obtained.The artificial metabolic enzyme can be effectively taken up by tumor cells, and combined radiotherapy anti-tumor effect is better, and there is greater application value.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical materials, and in particular to an artificial metabolic enzyme targeting mitochondria, and a preparation method and application thereof. Background Art

[0002] Data from the Global Cancer Observatory (GCO) indicates that cancer is one of the leading causes of death worldwide. According to 2020 statistics from the International Agency for the Study of Cancer (GLOBOCAN), approximately 130 million new cases of cancer were diagnosed worldwide in 2020. While traditional anti-tumor methods (surgery, radiotherapy, and chemotherapy) have achieved certain results, these methods have significant limitations and are accompanied by incompatible toxic and side effects. These methods not only have low therapeutic efficacy but may even worsen the patient's condition, leading to tumor recurrence and metastasis, and damaging the patient's immune system and function. Therefore, developing more effective anti-tumor methods that simultaneously destroy normal cells is an urgent issue that needs to be addressed.

[0003] In recent years, researchers have discovered that activating or mobilizing the body's immune system to enhance anti-tumor immunity, thereby controlling and killing tumor cells, has attracted widespread attention. Further studies have shown that during tumor proliferation, chromatin fragmentation and micronuclei in the cytoplasm generally increase, correspondingly increasing the probability of cytoplasmic DNA leakage from cancer cells. The immune system recognizes exogenous nucleic acids, particularly through the cGAS-STING pathway. Free dsDNA binds to cGAS, catalyzing the production of cyclic GMP-AMP (cGAMP). Under cGAMP stimulation, STING dimers and translocates from the endoplasmic reticulum to the perinuclear body. STING then recruits and phosphorylates TBK1, activating downstream IRF3, releasing IFN-I, and promoting the maturation and functionalization of DCs, NK cells, and T cells. Activated cGAS-STING pathways can effectively increase the number of CD4+ and CD8+ T cells within tumors and promote their secretion of IFN-γ and IFN-α. The released IFN-I can downregulate the expression of the cystine transporter (SLC7A11) in tumor cells, inhibiting glutathione synthesis and curbing tumor proliferation. Therefore, how to more efficiently activate the innate immune cGAS-STING pathway has become a research focus in tumor immunotherapy.

[0004] To meet their own rapid proliferation needs, tumors need to change their metabolic pathways, of which the accumulation of xanthine is a notable manifestation. Accumulated xanthine can be converted to uric acid by xanthine oxidase. This signaling molecule can stimulate macrophages to secrete pro-inflammatory factors, thereby driving macrophage polarization toward M1 and activating other immune cells. However, xanthine oxidase activity is low in some tumors, and current metabolic regulators have problems such as off-target effects and unstable duration. How to respond to metabolic changes in the tumor microenvironment, convert xanthine to uric acid, activate the polarization of immune cells, and achieve faster tumor immune activation with minimal side effects has become a hot topic in tumor immunology research.

[0005] Radiotherapy (RT), an important clinical treatment for tumors, utilizes high-energy ionizing radiation from X-rays to generate reactive oxygen species (ROS), which induce oxidative stress in tumor cells and damage DNA structure, leading to apoptosis or necrosis. However, the effectiveness of RT is affected by multiple factors in actual treatment. First, RT cannot accurately distinguish between tumor and normal tissue, indiscriminately damaging surrounding normal tissue. Second, the hypoxic tumor microenvironment can easily induce resistance to RT in tumor cells. Third, overexpressed reduced substances such as glutathione in tumor cells can rapidly scavenge ROS, affecting the therapeutic efficacy of RT. Therefore, how to enhance RT's ability to kill tumor tissue while minimizing side effects on normal tissue is a pressing clinical issue that needs to be addressed.

[0006] The rapid development of nanotechnology has brought hope to tumor immunotherapy. Compared with traditional radiosensitizers, personalized nanomaterials are being used to enhance radiation absorption and overcome tumor microenvironment-mediated radioresistance, thereby achieving radiosensitization or mitigating radiotherapy side effects. Therefore, how to use nanomaterials to modulate tumor cell metabolic signaling through autocrine and paracrine pathways to inhibit tumor proliferation and development, while also incorporating external conditions such as light, heat, electricity, and magnetism to respond to significant features of the tumor microenvironment, including hypoxia, mild acidity, and overexpressed glutathione, to achieve more effective anti-tumor effects; how to use nanomaterials to precisely deposit X-rays at the tumor site to transform it from a low-immunogenic "cold tumor" to a high-immunogenic "hot tumor"; how to effectively kill tumors and activate systemic anti-tumor immune responses while reducing radiation dose or reducing the number of radiotherapy sessions to protect normal tissue; and how to improve the biocompatibility and circulation of nanomaterials are all urgent challenges.

[0007] While metal nanomaterials possess excellent anti-tumor potential, they are also associated with potential toxicity issues caused by metal ions, a key factor hindering their widespread application. Therefore, further enhancing the catalytic activity of metal nanomaterials while reducing their inherent toxicity has become a new challenge. In recent years, single-atom nanomaterials have attracted significant attention for their ability to achieve 100% metal atom utilization while maintaining high catalytic activity. Fortunately, optimizing metal nanomaterials at the single-atom scale not only reduces metal content and mitigates biotoxicity issues, but also conserves raw materials and reduces costs. However, as metal particles are shrunk to the single-atom level, their surface energy increases dramatically, leading to particle aggregation and reduced catalytic activity. Therefore, addressing the stability of single atoms and preparing atomically dispersed single-atom materials is crucial. Furthermore, the effective uptake of nanomaterials by tumor cells and their prolonged retention at the tumor site are crucial for precise anti-cancer treatment. Summary of the Invention

[0008] In order to solve the above technical problems, the purpose of the present invention is to provide an artificial metabolic enzyme (Pt-POM / AC@TPP) targeting mitochondria and its preparation method and application. The artificial metabolic enzyme can be effectively taken up by tumor cells, has a good anti-tumor effect when combined with radiotherapy, and has great application value.

[0009] The present invention solves the above technical problems with the following technical solution: a method for preparing an artificial metabolic enzyme targeting mitochondria is provided, comprising the following steps:

[0010] (1) Activated carbon and phosphomolybdic acid solution are mixed, stirred, and then ultrasonically dispersed, centrifuged, and dried to obtain POM / AC;

[0011] (2) mixing the platinum (II) acetylacetonate solution with the POM / AC obtained in step (1), stirring, and then sequentially performing ultrasonic dispersion, centrifugation, and drying, and then heating to 160-180° C. under a reducing gas atmosphere for reduction for 0.5-1.5 h to obtain Pt-POM / AC;

[0012] (3) dispersing the Pt-POM / AC obtained in step (2) in an ethanol solution, then adding 3-aminopropyltriethoxysilane, stirring at room temperature for 10-15 hours, and then freeze-drying to obtain solid A;

[0013] (4) (3-Carboxypropyl)triphenylphosphine bromide, dimethyl sulfoxide, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride were mixed and stirred at room temperature in the dark. Then, N-hydroxysuccinimide was added and the mixture was stirred and activated in the dark for 5-7 hours to obtain solution B.

[0014] (5) The solid A obtained in step (3) is dissolved in ultrapure water to prepare solution A; solution A and solution B are then mixed, stirred in the dark for 10-15 hours, and then centrifuged, washed, and dried in sequence to obtain an artificial metabolic enzyme targeting mitochondria (Pt-POM / AC@TPP).

[0015] Furthermore, in step (1), the mass volume ratio of activated carbon to phosphomolybdic acid solution is 1 g:8-12 mL; and the concentration of phosphomolybdic acid solution is 0.01 g / mL.

[0016] Furthermore, phosphomolybdic acid is phosphomolybdic acid hydrate, and its chemical formula is H3[P(Mo3O 10 )4]·xH2O; the solvent of the phosphomolybdic acid solution is acetone.

[0017] Furthermore, in step (2), the mass volume ratio of the acetylacetonate platinum (II) solution to POM / AC is 4-6 mL:1 g; the concentration of the acetylacetonate platinum (II) solution is 0.0042 g / mL, and the solvent of the acetylacetonate platinum (II) solution is acetone.

[0018] Furthermore, in steps (1) and (2), the dispersion was performed at a frequency of 40 kHz for 10 min; the centrifugation was performed at 10,000 rpm / min for 10 min; and the drying was performed at 70° C. for 30 min.

[0019] Furthermore, in step (2), the reducing gas is 5% H2 / N2. Argon is introduced for 30 minutes before the hydrogen is introduced to exhaust the air in the apparatus.

[0020] Furthermore, in step (3), the mass volume ratio of Pt-POM / AC, ethanol solution and 3-aminopropyltriethoxysilane is 18-22 mg:20 mL:1-3 mg.

[0021] Furthermore, in step (4), the mass volume ratio of (3-carboxypropyl)triphenylphosphine bromide, dimethyl sulfoxide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide is 13-16 mg:5 mL:8-10 mg:4-5 mg.

[0022] Furthermore, in step (5), the mass volume ratio of solid A and ultrapure water is 20 mg:20 mL; the volume ratio of solution A and solution B is 18-22 mL:2 mL.

[0023] Furthermore, in step (5), centrifugation is performed at 15000 rpm / min for 15 min.

[0024] Furthermore, the particle size of Pt-POM / AC is 500-600 nm, and the particle size of the artificial metabolic enzyme targeting mitochondria is 600-800 nm; the electrode potential of Pt-POM / AC is -12.5 mV to -14.3 mV, and the electrode potential of the artificial metabolic enzyme targeting mitochondria is 21.6-22.9 mV.

[0025] Furthermore, the artificial metabolic enzymes targeted to mitochondria appear as amorphous, spherical particle aggregates.

[0026] The present invention also provides an artificial metabolic enzyme targeting mitochondria prepared by the preparation method of the artificial metabolic enzyme targeting mitochondria.

[0027] The artificial metabolic enzyme Pt-POM / AC@TPP, a novel nanomaterial, differs from other nanomaterials in that its core structure, the polyoxometalate, possesses a well-defined molecular structure, facilitating structural control and regulation of the activity and selectivity of single-atom catalysts. Furthermore, the polyoxometalate's abundance of oxygen vacancies provides a suitable carrier for the introduction of single Pt atoms. More importantly, the Pt atoms form a stable electronic structure with the oxygen ligands in the polyoxometalate, enabling efficient electron transfer and improving catalytic efficiency.

[0028] The present method, through modification with (3-carboxypropyl)triphenylphosphonium bromide, not only reverses the surface charge of Pt-POM / AC from negative (-12.5 to -14.3 mV) to positive (21.6 to 22.9 mV), but also enhances tumor cell uptake of the artificial metabolic enzyme. More importantly, the positively charged artificial metabolic enzyme exhibits X-ray deposition and catalytic properties, responding to the characteristics of the tumor microenvironment. This self-catalytic H2O2 reaction generates a large amount of reactive oxygen species, which damage tumor nuclear and mitochondrial DNA, release DNA fragments, activate the cGAS-STING pathway, and promote immunity. Furthermore, the artificial metabolic enzyme catalyzes the conversion of xanthine, abundant in tumors, into uric acid, stimulating macrophage polarization toward M1, further promoting immune activation. Excessive reactive oxygen species also damage and inhibit the expression of the tumor cell cystine transporter (SLC7A11), inhibiting glutathione synthesis and depleting glutathione, weakening the tumor's radiation resistance and further enhancing lipid peroxidation, achieving an ideal anti-tumor effect.

[0029] The present invention also provides the use of the above-mentioned artificial metabolic enzyme targeting mitochondria in the preparation of anti-tumor drugs.

[0030] The present invention has the following beneficial effects:

[0031] 1. First, the artificial metabolic enzyme of the present invention can respond to overexpressed hydrogen peroxide in the tumor microenvironment, catalyzing hydrogen peroxide to produce highly toxic hydroxyl radicals (·OH) through peroxidase, thereby inducing tumor cell apoptosis or necrosis. Second, the artificial metabolic enzyme with a positive surface charge can be more precisely deposited at the tumor site. Under X-ray radiation, the deposition of high atomic number Pt and Mo in the target area generates a large amount of ROS, which cooperates with catalase-like enzymes to overcome hypoxia, thereby further enhancing the product's anti-tumor ability.

[0032] 2. The latent glutathione peroxidase in the artificial metabolic enzyme of this invention oxidizes glutathione, scavenging reducing substances. This effectively ensures that the resulting ROS storm attacks the mitochondria and cell nucleus, leaking DNA and activating the cGAS-STING pathway, thereby activating innate anti-tumor immunity and activating T cells. Activated T cells, on the one hand, transform low-immunogenic "cold tumors" into highly immunogenic "hot tumors," and on the other hand, the released IFN-I can downregulate the expression of cystine transporters in tumor cells, inhibiting glutathione synthesis. This positive feedback regulation leads to glutathione depletion, addressing both the symptoms and the root cause, a two-pronged approach.

[0033] 3. The artificial metabolic enzyme of the present invention has xanthine oxidase activity, which can undergo redox reaction with xanthine, converting xanthine into uric acid, activating macrophages to polarize toward M1, and further activating immunity;

[0034] 4. Compared with natural enzymes, artificial metabolic enzymes make up for the defect of natural enzymes being easily digested by proteases, while improving universality and being able to work at different temperatures and pH levels. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 TEM image of Pt-POM / AC prepared in Example 1;

[0036] Figure 2 This is a transmission electron microscopy image of Pt-POM / AC@TPP prepared in Example 1;

[0037] Figure 3 This is a hydrated particle size analysis diagram of Pt-POM / AC prepared in Example 1;

[0038] Figure 4 This is a hydration particle size analysis diagram of Pt-POM / AC@TPP prepared in Example 1;

[0039] Figure 5 : A comparison of the electrode potentials of Pt-POM / AC and Pt-POM / AC@TPP prepared in Example 1;

[0040] Figure 6The infrared spectra of Pt-POM / AC and Pt-POM / AC@TPP prepared in Example 1;

[0041] Figure 7 This is a graph showing the peroxidase activity evaluation results of Pt-POM / AC@TPP prepared in Example 1;

[0042] Figure 8 This is a graph showing the catalase activity evaluation results of Pt-POM / AC@TPP prepared in Example 1;

[0043] Figure 9 This is a graph showing the evaluation results of glutathione peroxidase activity of Pt-POM / AC@TPP prepared in Example 1;

[0044] Figure 10 This is a graph showing the xanthine oxidase activity evaluation results of Pt-POM / AC@TPP prepared in Example 1;

[0045] Figure 11 This is a diagram showing the cytotoxicity verification results of Pt-POM / AC@TPP prepared in Example 1 on L-929 cells;

[0046] Figure 12 The figure shows the cytotoxicity verification results of Pt-POM / AC@TPP prepared in Example 1 on L-929 and 4T1 cells;

[0047] Figure 13 Figure 2 is the change of tumor volume in mice of each treatment group;

[0048] Figure 14 These are pictures of mouse tumors in each treatment group after the experiment. DETAILED DESCRIPTION

[0049] The principles and features of the present invention are described below. The examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. In the examples, where specific conditions are not specified, conventional conditions or manufacturer-recommended conditions were used. Reagents or instruments used where the manufacturer is not specified are conventional products that can be purchased commercially.

[0050] Example 1

[0051] A method for preparing an artificial metabolic enzyme targeting mitochondria comprises the following steps:

[0052] (1) 1 g of activated carbon was mixed with 10 mL of 0.01 g / mL phosphomolybdic acid solution, stirred for 3 h, and then ultrasonically dispersed, centrifuged, and dried to obtain POM / AC;

[0053] (2) 5 mL of 0.0042 g / mL platinum (II) acetylacetonate solution was mixed with 1 g of the POM / AC obtained in step (1), stirred for 3 h, and then ultrasonically dispersed, centrifuged, and dried in sequence. The mixture was then heated to 170° C. under a reducing gas (5% H 2 / N 2 ) atmosphere for 1 h to obtain Pt-POM / AC;

[0054] (3) 20 mg of the Pt-POM / AC obtained in step (2) was dispersed in 20 mL of ethanol solution, and then 2 mg of 3-aminopropyltriethoxysilane was added. The mixture was stirred at room temperature for 12 h, and then freeze-dried to obtain solid A.

[0055] (4) 14.17 mg of (3-carboxypropyl)triphenylphosphonium bromide, 5 mL of dimethyl sulfoxide, and 9.49 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride were mixed and stirred at room temperature in the dark. Then, 4.56 mg of N-hydroxysuccinimide was added and activated with stirring for 6 h in the dark to obtain Solution B.

[0056] (5) 20 mg of solid A obtained in step (3) was dissolved in 20 mL of ultrapure water to prepare solution A; then 20 mL of solution A and 2 mL of solution B were mixed, stirred in the dark for 12 h, and then centrifuged at 15,000 rpm / min for 15 min. After washing and drying, the artificial metabolic enzyme targeting mitochondria was obtained.

[0057] Example 2

[0058] A method for preparing an artificial metabolic enzyme targeting mitochondria comprises the following steps:

[0059] (1) 1 g of activated carbon was mixed with 8 mL of 0.01 g / mL phosphomolybdic acid solution, stirred for 3 h, and then ultrasonically dispersed, centrifuged, and dried to obtain POM / AC;

[0060] (2) 4 mL of 0.0042 g / mL platinum (II) acetylacetonate solution was mixed with 1 g of the POM / AC obtained in step (1), stirred for 3 h, and then ultrasonically dispersed, centrifuged, and dried in sequence. The mixture was then heated to 160° C. and reduced for 0.5 h under a reducing gas (5% H 2 / N 2 ) atmosphere to obtain Pt-POM / AC;

[0061] (3) 18 mg of the Pt-POM / AC obtained in step (2) was dispersed in 20 mL of ethanol solution, and then 1 mg of 3-aminopropyltriethoxysilane was added. The mixture was stirred at room temperature for 10 h, and then freeze-dried to obtain solid A.

[0062] (4) 13 mg of (3-carboxypropyl)triphenylphosphonium bromide, 5 mL of dimethyl sulfoxide, and 8 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride were mixed and stirred at room temperature in the dark. Then, 4 mg of N-hydroxysuccinimide was added and activated with stirring for 5 h in the dark to obtain solution B.

[0063] (5) 20 mg of solid A obtained in step (3) was dissolved in 20 mL of ultrapure water to prepare solution A; then 18 mL of solution A and 2 mL of solution B were mixed, stirred in the dark for 10 h, and then centrifuged at 15,000 rpm / min for 15 min. After washing and drying, the artificial metabolic enzyme targeting mitochondria was obtained.

[0064] Example 3

[0065] A method for preparing an artificial metabolic enzyme targeting mitochondria comprises the following steps:

[0066] (1) 1 g of activated carbon was mixed with 12 mL of 0.01 g / mL phosphomolybdic acid solution, stirred for 3 h, and then ultrasonically dispersed, centrifuged, and dried to obtain POM / AC;

[0067] (2) 6 mL of 0.0042 g / mL platinum (II) acetylacetonate solution was mixed with 1 g of the POM / AC obtained in step (1), stirred for 3 h, and then ultrasonically dispersed, centrifuged, and dried in sequence. The mixture was then heated to 180° C. under a reducing gas (5% H 2 / N 2 ) atmosphere and reduced for 1.5 h to obtain Pt-POM / AC;

[0068] (3) 22 mg of the Pt-POM / AC obtained in step (2) was dispersed in 20 mL of ethanol solution, and then 3 mg of 3-aminopropyltriethoxysilane was added. The mixture was stirred at room temperature for 15 h, and then freeze-dried to obtain solid A.

[0069] (4) 16 mg of (3-carboxypropyl)triphenylphosphonium bromide, 5 mL of dimethyl sulfoxide, and 10 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride were mixed and stirred at room temperature in the dark. Then, 5 mg of N-hydroxysuccinimide was added and activated with stirring for 7 h in the dark to obtain solution B.

[0070] (5) 20 mg of solid A obtained in step (3) was dissolved in 20 mL of ultrapure water to prepare solution A; then 22 mL of solution A and 2 mL of solution B were mixed, stirred in the dark for 15 h, and then centrifuged at 15,000 rpm / min for 15 min. After washing and drying, the artificial metabolic enzyme targeting mitochondria was obtained.

[0071] Test Example 1

[0072] (1) The polyoxometalate-based platinum single-atom nanomaterial Pt-POM / AC and the mitochondrial-targeted artificial metabolic enzyme (Pt-POM / AC@TPP) prepared in Example 1 were characterized by field emission transmission electron microscopy. The results are shown in Figure 2. Figure 1 and Figure 2 As shown in the figure, it can be seen that the overall morphology of Pt-POM / AC presents an irregular, amorphous spherical polyhedron structure. Figure 2 It can be further observed that there are no obvious particles on the activated carbon support, indicating that the Pt in the material may be in a single atomic state and has not migrated and agglomerated into nanoparticles.

[0073] (2) The Pt-POM / AC and Pt-POM / AC@TPP prepared in Example 1 were characterized by a dynamic light scattering particle size analyzer. The results are as follows: Figure 3-5 It can be seen that the particle size of Pt-POM / AC is 500-600nm, and the particle size of Pt-POM / AC@TPP is 600-800nm. Figure 3 It shows that the electrode potential of Pt-POM / AC is -12.5mV~-14.3mV, and the electrode potential of Pt-POM / AC@TPP is 22.9-23.5mV, indicating that TPP has successfully modified the material and changed the surface charge of the material.

[0074] (3) The Pt-POM / AC and Pt-POM / AC@TPP prepared in Example 1 were characterized by Fourier transform infrared spectrometer. The results are as follows: Figure 6 As shown in the figure, it can be seen that Pt-POM / AC has a -1 and 963cm -1 There are two Keggin-type characteristic peaks belonging to polyoxometalates, which belong to the stretching vibration of PO bond and Mo=O bond respectively. Pt-POM / AC@TPP has a peak at 1734cm -1 The vibration of the amide bond at 1650 cm -1 and 1450cm -1 The characteristic absorption of the benzene ring at the bottom of the PDMS photoelectron spectroscopy indicates that TPP has been successfully modified.

[0075] Test Example 2

[0076] (1) The artificial metabolic enzyme Pt-POM / AC@TPP, hydrogen peroxide and the color developer o-phenylenediamine (OPD) were placed in different groups at room temperature in the dark for 25 minutes, and then their ultraviolet absorbance was measured using an ultraviolet spectrophotometer. The PBS group, X-ray group, Pt-POM / AC@TPP group and Pt-POM / AC@TPP+X-ray group were measured respectively. The concentration of Pt-POM / AC@TPP was 20 μg / mL, the concentration of OPD color developer was 1 mM, the pH of PBS was 4.0, the temperature was 25°C, the concentration of hydrogen peroxide was 1 mM and the X-ray dose was 6 Gy to verify the peroxidase-like activity of the artificial metabolic enzyme. The results are shown in Figure 2. Figure 7 The results showed that there was an obvious absorption peak at around 445 nm, indicating that Pt-POM / AC@TPP had peroxidase activity and could catalyze low concentrations of hydrogen peroxide to generate hydroxyl radicals.

[0077] (2) A portable dissolved oxygen meter was used to verify the change in oxygen content in water within 10 minutes after adding the artificial metabolic enzyme to a 2mM H2O2 solution. The concentration of Pt-POM / AC@TPP, H2O2 concentration, PBS pH, and temperature were 20μg / mL, 2mM, 5.0, and 25°C, respectively. The results are shown in Figure 2. Figure 8 shown.

[0078] Depend on Figure 8 It can be seen that when there is only PBS in the system, the oxygen content in the water gradually decreases; when there is only H2O2 in the system, the oxygen content in the water changes very little over time; when there are artificial metabolic enzymes and H2O2 in the system, the oxygen content in the water increases rapidly over time, indicating that the artificial metabolic enzyme has excellent catalase activity, that is, it can catalyze the decomposition of H2O2 to produce O2.

[0079] (3) Using PBS buffer solution with pH=8.0 and concentration of 0.05M as solvent, glutathione and artificial metabolic enzyme were reacted at room temperature for 3 hours. The PBS group, H2O2 group, and artificial metabolic enzyme were measured by UV spectrophotometer to verify that the artificial metabolic enzyme had glutathione peroxidase activity. The concentration of artificial metabolic enzyme, glutathione concentration, H2O2 concentration, and temperature were: 75μg / mL, 0.2mM, 1mM, and 25℃, respectively. The results are as follows: Figure 9 shown.

[0080] Depend on Figure 9 It can be seen that the artificial metabolic enzyme Pt-POM / AC@TPP can react with glutathione, reduce the GSH content, and reduce the loss of reactive oxygen species.

[0081] (4) Using 0.1M sodium hydroxide solution as solvent, xanthine and artificial metabolic enzyme were reacted at room temperature for 30 minutes. The PBS group and artificial metabolic enzyme were measured by UV spectrophotometer to verify that the artificial metabolic enzyme had xanthine oxidase activity. The concentrations of artificial metabolic enzyme and xanthine were 50μg / mL and 0.2mM, respectively. The results are as follows: Figure 10 The results showed that the artificial metabolic enzyme Pt-POM / AC@TPP can effectively reduce the content of xanthine.

[0082] Test Example 3

[0083] (1) When mouse fibroblasts (L-929) grew to about 80% density, the cells were transferred to a 96-well plate and incubated for 24 hours. The cell density was 5×10 3 After the cells basically covered the entire well, the old culture medium was aspirated and the cells were incubated with fresh culture medium containing different concentrations of Pt-POM / AC and Pt-POM / AC@TPP (0, 6.26μg / mL, 12.5μg / mL, 25μg / mL, 35μg / mL, 50μg / mL and 100μg / mL) for 24 hours. The culture medium was aspirated and the cells were washed three times with PBS. 90μL of DMEM culture medium without FBS was added to each well, followed by 10U of lcck-8 reagent. After incubation for 30 minutes, the 96-well plate was placed under a microplate reader, the absorbance of each well was measured at 450nm, and the survival rate of mouse fibroblasts (L-929) under different concentration conditions was calculated. The results are shown in Figure 2. Figure 11 The results showed that when the concentration was lower than 100 μg / mL, the artificial metabolic enzyme did not produce significant cytotoxicity to L-929 cells.

[0084] (2) The cytotoxicity of artificial metabolic enzymes on L-929 normal cells and 4T1 tumor cells was evaluated. When the cells grew to about 80% density, the cells were transferred to 96-well plates and incubated for 24 hours. The L-929 cell density was 5×10 3 / well, 4T1 cells are 4×10 3 / well. After the cells basically covered the entire well, the old culture medium was aspirated and the cells were incubated with fresh culture medium containing different concentrations of Pt-POM / AC@TPP (0, 6.26μg / mL, 12.5μg / mL, 25μg / mL, 35μg / mL, 50μg / mL, 75μg / mL, 100μg / mL and 200μg / mL) for 24 hours. The culture medium was aspirated and the cells were washed three times with PBS. 90μL of DMEM culture medium without FBS was added to each well, followed by 10U of lcck-8 reagent. After incubation for 30 minutes, the 96-well plate was placed under a microplate reader, and the absorbance of each well was measured at 450nm. The survival rates of mouse fibroblasts (L-929) and mouse breast cancer cells (4T1) under different concentration conditions were calculated. The results are shown in Figure 2. Figure 12 The results showed that when the concentration was lower than 25 μg / mL, the artificial metabolic enzyme did not produce significant cytotoxicity to L-929 and 4T1 cells, and could be further used to evaluate the anti-tumor effect.

[0085] Test Example 4

[0086] (1) Construction of tumor model

[0087] Twelve BALB / c mice (Chengdu Dashuo Experimental Animal Co., Ltd.) were used as experimental animals. 100 μL of the medium containing 1.0×10 cells was implanted subcutaneously in the mice. 6 4T1 cell suspension, 10 days later, when the tumor size of the mouse is about 100mm 3 .

[0088] (2) Evaluate synergistic therapeutic effects in in vivo tumor models

[0089] Mice bearing tumors were randomly divided into four groups: PBS buffer group (PBS), artificial metabolic enzyme alone (Pt-POM / AC@TPP), X-ray alone group (X-ray), and combined treatment group (Pt-POM / AC@TPP+X-ray). Tumor-bearing mice in the PBS group were injected intratumorally with 20 μL of PBS solution; mice in the Pt-POM / AC@TPP group were injected intratumorally with 20 μL of artificial metabolic enzyme aqueous solution; mice in the X-ray group were irradiated with X-ray (6 Gy) for 10 minutes; mice in the Pt-POM / AC@TPP+X-ray group were injected intratumorally with 20 μL of artificial metabolic enzyme and then irradiated with X-ray (6 Gy) for 10 minutes. The changes in mouse tumor volume were measured and recorded using a vernier caliper. The mouse tumor volume is tumor volume V = ab 2 / 2, where a = tumor length, b = tumor width. After the experiment, the tumors of mice in each treatment group were photographed. The results are as follows: Figure 13 and Figure 14As shown, *** indicates significant differences among treatment groups (p<0.01). The results showed that artificial metabolic enzyme Pt-POM / AC@TPP combined with radiotherapy significantly inhibited tumor proliferation in a living tumor model.

[0090] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent replacements or improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for preparing an artificial metabolic enzyme targeting mitochondria, characterized in that: The following steps are involved: (1) Activated carbon and phosphomolybdic acid solution are mixed, stirred, and then ultrasonically dispersed, centrifuged, and dried to obtain POM / AC; (2) mixing the platinum (II) acetylacetonate solution with the POM / AC obtained in step (1), stirring, and then sequentially performing ultrasonic dispersion, centrifugation, and drying, and then heating to 160-180° C. under a reducing gas atmosphere for reduction for 0.5-1.5 h to obtain Pt-POM / AC; (3) dispersing the Pt-POM / AC obtained in step (2) in an ethanol solution, then adding 3-aminopropyltriethoxysilane, stirring at room temperature for 10-15 hours, and then freeze-drying to obtain solid A; (4) (3-Carboxypropyl)triphenylphosphine bromide, dimethyl sulfoxide, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride were mixed and stirred at room temperature in the dark. Then, N-hydroxysuccinimide was added and the mixture was stirred and activated in the dark for 5-7 hours to obtain solution B. (5) dissolving the solid A obtained in step (3) in ultrapure water to prepare solution A; then mixing solution A and solution B, stirring for 10-15 hours in the dark, and then centrifuging, washing and drying in sequence to obtain an artificial metabolic enzyme targeting mitochondria.

2. The method for preparing the mitochondrial-targeting artificial metabolic enzyme according to claim 1, wherein: In step (1), the mass volume ratio of the activated carbon to the phosphomolybdic acid solution is 1 g:8-12 mL; and the concentration of the phosphomolybdic acid solution is 0.01 g / mL.

3. The method for preparing the mitochondrial-targeting artificial metabolic enzyme according to claim 1, wherein: In step (2), the mass volume ratio of the acetylacetonate platinum (II) solution and POM / AC is 4-6 mL:1 g; and the concentration of the acetylacetonate platinum (II) solution is 0.0042 g / mL.

4. The method for preparing the mitochondrial-targeting artificial metabolic enzyme according to claim 1, wherein: In step (3), the mass volume ratio of the Pt-POM / AC, ethanol solution and 3-aminopropyltriethoxysilane is 18-22 mg:20 mL:1-3 mg.

5. The method for preparing the mitochondrial-targeting artificial metabolic enzyme according to claim 1, wherein: In step (4), the mass volume ratio of (3-carboxypropyl)triphenylphosphine bromide, dimethyl sulfoxide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide is 13-16 mg:5 mL:8-10 mg:4-5 mg.

6. The method for preparing the artificial metabolic enzyme targeting mitochondria according to claim 1, wherein: In step (4), stirring is performed at a rotation speed of 500-600 r / min.

7. The method for preparing the artificial metabolic enzyme targeting mitochondria according to claim 1, wherein: In step (5), the mass volume ratio of the solid A and ultrapure water is 20 mg:20 mL; the volume ratio of the solution A and solution B is 18-22 mL:2 mL.

8. The method for preparing the mitochondrial-targeting artificial metabolic enzyme according to claim 1, wherein: In step (5), centrifuge at 15000 rpm / min for 15 min.

9. The artificial metabolic enzyme targeting mitochondria obtained by the method for preparing the artificial metabolic enzyme targeting mitochondria according to any one of claims 1 to 8.

10. Use of the mitochondria-targeted artificial metabolic enzyme according to claim 9 in the preparation of anti-tumor drugs.

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