Artificial metabolic enzyme for targeting mitochondria as well as preparation method and application of artificial metabolic enzyme

Abstract

The invention discloses a mitochondrial targeting artificial metabolic enzyme and a preparation method and application thereof, and relates to the technical field of biomedical materials.The method comprises the following steps that activated carbon and a phosphomolybdic acid solution are mixed and stirred, and POM / AC is obtained; mixing with a platinum acetylacetonate (II) solution, and carrying out reduction treatment to obtain Pt-POM / AC; dispersing in ethanol, adding 3-aminopropyltriethoxysilane, stirring, and freeze-drying to obtain a solid A; mixing (3-carboxypropyl) triphenylphosphonium bromide, dimethyl sulfoxide and 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride, stirring in a dark place, adding N-hydroxysuccinimide, and activating in a dark place to obtain a solution B; dissolving the solid A, mixing with the solution B, stirring in a dark place, centrifuging, washing and drying to obtain the artificial metabolic enzyme of the targeted mitochondria. The artificial metabolic enzyme can be effectively taken by tumor cells, has a good anti-tumor effect in combination with radiotherapy, and has a great 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) show that cancer is one of the leading causes of death worldwide. According to statistics from the International Cancer Organization (GLOBOCAN) in 2020, there will be approximately 130 million new cases of cancer worldwide in 2020. Although traditional anti-tumor methods (surgery, radiotherapy, and chemotherapy) have achieved certain results, these methods have great limitations, and the toxic and side effects that do not match them not only have low therapeutic effects, but may even worsen the patient's condition, easily leading to tumor recurrence and metastasis, and destroying the patient's immune system and function. Therefore, how to develop more efficient anti-tumor methods while destroying normal cells is an urgent problem to be solved.

[0003] In recent years, researchers have found that methods to control and kill tumor cells by activating or mobilizing the body's immune system to enhance its own anti-tumor immunity have attracted widespread attention. Further studies have shown that in the process of tumor proliferation, chromatin fragments and micronuclei in the cytoplasm will generally increase, which will correspondingly increase the probability of cytoplasmic DNA leakage in cancer cells. The immune system recognizes exogenous nucleic acids, especially sensing cytoplasmic DNA through the cGAS-STING pathway. Free dsDNA binds to cGAS and catalyzes the generation of cyclic GMP-AMP (cGAMP). Under the stimulation of cGAMP, STING becomes a dimer and transports from the endoplasmic reticulum to the perinuclear body. Subsequently, STING recruits and phosphorylates TBK1, activates downstream IRF3, releases IFN-I, and promotes the maturation and functionalization of DCs, NK and T cells. The activated cGAS-STING pathway can effectively increase the number of CD4+T and CD8+T cells in tumors and promote their secretion of IFN-γ and IFN-α. The released IFN-I can downregulate the expression of cystine transporter (SLC7A11) in tumor cells, inhibit the synthesis of glutathione, and curb tumor proliferation. Therefore, how to more efficiently activate the innate immune cGAS-STING pathway has become the research focus of tumor immunotherapy.

[0004] In order to meet the needs of their own rapid proliferation, tumors need to change their metabolic pathways, among which the accumulation of xanthine is a significant manifestation. The accumulation of xanthine can be converted into uric acid through xanthine oxidase. Uric acid, a signaling molecule, can stimulate macrophages to secrete proinflammatory factors, thereby promoting the polarization of macrophages to M1 and activating the activation of other immune cells. However, the activity of xanthine oxidase is low in some tumors, and current metabolic regulators have problems such as off-target and unstable duration. How to respond to metabolic changes in the tumor microenvironment, convert xanthine into 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 immunity research.

[0005] Radiotherapy (RT) is one of the important means of treating tumors clinically. It uses high-energy ionizing radiation from X-rays to produce reactive oxygen species (ROS), which induce oxidative stress in tumor cells and damage the DNA structure, thereby causing apoptosis or necrosis of tumor cells. However, in actual treatment, the effect of radiotherapy is affected by many factors. First, radiotherapy cannot accurately distinguish between tumor tissue and normal tissue, and indiscriminately damages surrounding normal tissues; second, the hypoxic tumor microenvironment is prone to cause tumor cells to resist radiotherapy; third, reduced substances such as glutathione overexpressed in tumor cells can quickly clear ROS, affecting the therapeutic effect of radiotherapy. Therefore, how to enhance the killing of tumor tissue by radiotherapy while reducing the side effects on normal tissues is an urgent problem to be solved clinically.

[0006] The rapid development of nanotechnology has brought hope to tumor immunotherapy. Compared with traditional radiosensitizers, personalized nanomaterials are used to enhance radiation absorption and overcome tumor microenvironment-mediated radiotherapy resistance, thereby achieving radiosensitization or reducing the side effects of radiotherapy. Therefore, how to use nanomaterials to regulate tumor cells through autocrine and paracrine metabolic signal transmission to inhibit tumor proliferation and development, and at the same time, combine external conditions such as light, heat, electricity, and magnetism to respond to the significant characteristics of the tumor microenvironment, including hypoxia, microacidity, and overexpressed glutathione, so as to achieve more efficient anti-tumor effects; how to use nanomaterials to accurately deposit X-rays at the tumor to transform the tumor from a low-immunogenic "cold tumor" to a high-immunogenic "hot tumor"; how to better kill the tumor and activate the systemic anti-tumor immune response under the condition of reducing the radiation dose or reducing the number of radiotherapy to protect normal tissues; how to improve the biocompatibility and in vivo circulation of nanomaterials are all urgent problems to be solved.

[0007] While metal nanomaterials have excellent anti-tumor potential, they are also accompanied by potential toxicity problems caused by metal ions, which has become a key factor restricting the widespread application of metal nanomaterials. Therefore, how to further improve the catalytic activity of metal nanomaterials while reducing the toxicity of the material itself has become a new challenge. In recent years, single-atom nanomaterials have attracted attention for their ability to achieve 100% utilization of metal atoms and maintain high catalytic activity. Fortunately, the optimization of metal nanomaterials at the single-atom scale can not only reduce the metal content and weaken the problem of biological toxicity, but also save raw materials and reduce costs. However, as metal particles shrink to the level of single atoms, the surface energy of the metal increases sharply, causing the metal particles to agglomerate and reduce the catalytic activity. Therefore, it is crucial to solve the stability of single atoms and prepare single-atom materials at the atomic dispersion level. In addition, whether nanomaterials can be effectively taken up by tumor cells or prolong their retention effect at the tumor site is also crucial for the precise treatment of anti-tumor. 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 combined with radiotherapy, and has great application value.

[0009] The technical solution of the present invention to solve the above technical problem is as follows: a method for preparing an artificial metabolic enzyme targeting mitochondria is provided, comprising the following steps:

[0010] (1) mixing activated carbon with a phosphomolybdic acid solution, stirring, and sequentially subjecting to ultrasonic dispersion, centrifugation, and drying to obtain POM / AC;

[0011] (2) mixing the acetylacetonate platinum (II) solution with the POM / AC obtained in step (1), stirring, then sequentially performing ultrasonic dispersion, centrifugation and drying, and then heating to 160-180° C. in 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, stirred at room temperature in the dark, and then N-hydroxysuccinimide was added. The mixture was stirred and activated for 5-7 hours in the dark to obtain solution B;

[0014] (5) dissolving the solid A obtained in step (3) in ultrapure water to obtain 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 (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] Further, phosphomolybdic acid is phosphomolybdic acid hydrate, and the chemical formula is H 3 [P(Mo 3 O 10 ) 4 ]·xH 2 O; The solvent of the phosphomolybdic acid solution is acetone.

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

[0018] Furthermore, in steps (1) and (2), dispersion is performed at a frequency of 40 kHz for 10 min; centrifugation is performed at 10000 rpm / min for 10 min; and drying is performed at 70° C. for 30 min.

[0019] Further, in step (2), the reducing gas is 5% H 2 / N 2 Before introducing hydrogen, introduce argon for 30 minutes to exhaust the air in the device.

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

[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 of the present invention is different from other nanomaterials. Its core structure, polyoxometalate, has a clear molecular structure, which is helpful for structural control and regulation of the activity and selectivity of single-atom catalysts. At the same time, polyoxometalate is rich in oxygen vacancies and provides a suitable carrier for the introduction of Pt single atoms. More importantly, Pt single atoms and oxygen ligands in polyoxometalate form a stable electronic structure, which can effectively transmit electrons and improve catalytic efficiency.

[0028] The method of the present invention not only reverses the surface charge of Pt-POM / AC from negative charge (-12.5 to -14.3 mV) to positive charge (21.6 mV to 22.9 mV) by modifying (3-carboxypropyl) triphenylphosphine bromide, but also enhances the uptake of artificial metabolic enzymes by tumor cells. More importantly, the artificial metabolic enzymes with positive charges on the surface have X-ray deposition ability and catalytic performance, can respond to the characteristics of the tumor microenvironment, and achieve H 2 O 2 The self-cascade catalysis produces a large amount of reactive oxygen species, which damages the cell nucleus and mitochondrial DNA of the tumor, releases DNA fragments, activates the cGAS-STING pathway, and promotes immunity; on the other hand, the artificial metabolic enzyme can catalyze the conversion of xanthine, which is abundant in the tumor, into uric acid, stimulates macrophages to polarize to M1, and further promotes immune activation. At the same time, excessive reactive oxygen destroys and inhibits the expression of tumor cell cystine transporter (SLC7A11), inhibits the synthesis of glutathione, depletes glutathione, weakens the tumor's radiation resistance, and further enhances the degree of lipid peroxidation to achieve 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 the overexpressed hydrogen peroxide in the tumor microenvironment, and produce highly toxic hydroxyl radicals (·OH) through peroxidase-catalyzed hydrogen peroxide, thereby inducing apoptosis or necrosis of tumor cells; secondly, the artificial metabolic enzyme with positive charge on the surface can be more accurately deposited at the tumor site under X-ray radiation, and a large amount of ROS is generated through the deposition of high atomic number Pt and Mo in the target area, which cooperates with catalase to overcome hypoxia, thereby further enhancing the anti-tumor ability of the product.

[0032] 2. The potential glutathione peroxidase of the artificial metabolic enzyme of the present invention oxidizes glutathione and removes reducing substances, thereby effectively ensuring that the generated ROS storm attacks the mitochondria and cell nucleus, leaks DNA to activate the cGAS-STING pathway, thereby activating innate anti-tumor immunity and activating T cells. On the one hand, the activated T cells convert the low-immunogenic "cold tumor" into a highly immunogenic "hot tumor", and on the other hand, the released IFN-I can downregulate the expression of tumor cell cystine transporter and inhibit the synthesis of glutathione. The positive feedback regulation realizes the depletion of glutathione, treating the symptoms and the root cause at the same time.

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

[0034] 4. Compared with natural enzymes, artificial metabolic enzymes make up for the defect that natural enzymes are easily digested by proteases, while improving universality and being able to function at different temperatures and pH values. 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 microscope 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 The electrode potential comparison diagram 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 evaluation results of the peroxidase activity of Pt-POM / AC@TPP prepared in Example 1;

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

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

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

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

[0046] Fig.12 The figure is the result of verification of cytotoxicity of Pt-POM / AC@TPP prepared in Example 1 to L-929 and 4T1 cells;

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

[0048] Fig.14 These are the 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, and the examples are only used to explain the present invention and are not used to limit the scope of the present invention. If no specific conditions are specified in the embodiments, they are carried out according to normal conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased commercially.

[0050] Example 1

[0051] An artificial metabolic enzyme targeting mitochondria, the preparation method of which 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 acetylacetonate platinum (II) solution was mixed with 1 g of POM / AC obtained in step (1), stirred for 3 h, and then ultrasonically dispersed, centrifuged and dried in sequence, and then heated in a reducing gas (5% H 2 / N 2 ) atmosphere and heated to 170 °C 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, 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, stirred at room temperature in the dark, and then 4.56 mg of N-hydroxysuccinimide was added. The mixture was stirred and activated for 6 h in the dark to obtain solution B.

[0056] (5) Dissolve 20 mg of the solid A obtained in step (3) in 20 mL of ultrapure water to prepare solution A; then mix 20 mL of solution A and 2 mL of solution B, stir for 12 h in the dark, and then centrifuge at 15,000 rpm / min for 15 min. After washing and drying, an artificial metabolic enzyme targeting mitochondria is obtained.

[0057] Example 2

[0058] An artificial metabolic enzyme targeting mitochondria, the preparation method of which 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 acetylacetonate platinum (II) solution was mixed with 1 g of POM / AC obtained in step (1), stirred for 3 h, and then ultrasonically dispersed, centrifuged and dried in sequence, and then heated in a reducing gas (5% H 2 / N 2 ) atmosphere and heated to 160°C for reduction for 0.5h 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, 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, stirred at room temperature in the dark, and then 4 mg of N-hydroxysuccinimide was added. The mixture was stirred and activated for 5 h in the dark to obtain solution B.

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

[0064] Example 3

[0065] An artificial metabolic enzyme targeting mitochondria, the preparation method of which 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 acetylacetonate platinum (II) solution was mixed with 1 g of POM / AC obtained in step (1), stirred for 3 h, and then ultrasonically dispersed, centrifuged and dried in sequence, and then heated in a reducing gas (5% H 2 / N 2 ) atmosphere and heated to 180 °C 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, 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, stirred at room temperature in the dark, and then 5 mg of N-hydroxysuccinimide was added. The mixture was stirred and activated for 7 h in the dark to obtain solution B.

[0070] (5) Dissolve 20 mg of the solid A obtained in step (3) in 20 mL of ultrapure water to prepare solution A; then mix 22 mL of solution A and 2 mL of solution B, stir for 15 h in the dark, and then centrifuge at 15,000 rpm / min for 15 min. After washing and drying, an artificial metabolic enzyme targeting mitochondria is 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 as follows: 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 carrier, 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 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 ionophore 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 show that there is an obvious absorption peak around 445nm, indicating that Pt-POM / AC@TPP has peroxidase activity and can catalyze low concentrations of hydrogen peroxide to generate hydroxyl radicals.

[0077] (2) The artificial metabolic enzyme was added to 2 mM H 2 O 2 The change of oxygen content in water within 10 minutes was measured after solution was added. The concentration of Pt-POM / AC@TPP, H2O2 concentration, pH of PBS, and temperature were 20 μg / mL, 2 mM, 5.0, and 25 °C, respectively. The results are shown in 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 H 2 O 2 When the system contains artificial metabolic enzymes and H 2 O 2 The oxygen content in the water increased rapidly over time, indicating that the artificial metabolic enzyme had excellent catalase activity, that is, it could catalyze H 2 O 2 Decomposition produces O 2 .

[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 concentrations of PBS group and H 2 O 2 The concentration of artificial metabolic enzyme, glutathione concentration, H 2 O 2 The concentration and temperature were: 75μg / mL, 0.2mM, 1mM and 25℃, and the results were as follows Fig. 9 shown.

[0080] Depend on Fig. 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 ultraviolet 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: Fig.10 The results show 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 / 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 and Pt-POM / AC@TPP (0, 6.26μg / mL, 12.5μg / mL, 25μg / mL, 35μg / mL, 50μg / mL and 100μg / mL) for 24 hours, and then 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, and then 10U of lcck-8 reagent was added. After incubation for 30 minutes, the 96-well plate was placed under an ELISA reader, and the absorbance of each well was measured at 450nm. The survival rate of mouse fibroblasts (L-929) under different concentration conditions was calculated. The results are shown in the figure. Fig.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 a 96-well plate 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, and then the culture medium was aspirated, washed 3 times with PBS, and 90μL of DMEM culture medium without FBS was added to each well, followed by 10U lcck-8 reagent. After incubation for 30 minutes, the 96-well plate was placed under an ELISA 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. Fig.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] The experimental animals were 12 BALB / c mice (Chengdu Dashuo Experimental Animal Co., Ltd.). 100 μL of cells containing 1.0×10 6 4T1 cell suspension. After 10 days, when the tumor size of the mouse is about 100mm 3 .

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

[0089] Mice bearing tumors were randomly divided into 4 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). 20 μL PBS solution was injected into the tumor of the tumor-bearing mice in the PBS group; 20 μL artificial metabolic enzyme aqueous solution was injected into the tumor of the mice in the Pt-POM / AC@TPP group; the mice in the X-ray group were irradiated with X-ray (6Gy) for 10 minutes; and the mice in the Pt-POM / AC@TPP+X-ray group were irradiated with X-ray (6Gy) for 10 minutes after 20 μL artificial metabolic enzyme was injected into the tumor. The changes in the tumor volume of the mice were measured and recorded using a vernier caliper. The tumor volume of the mice 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 shown in the figure. Fig.13 and Fig.14As shown, *** indicates significant differences among the treatment groups (p<0.01). The results showed that artificial metabolic enzyme Pt-POM / AC@TPP combined with radiotherapy significantly inhibited tumor proliferation in the 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 modification, equivalent substitution or improvement made within the spirit and principle of the present invention shall be included in the protection scope 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) mixing activated carbon with a phosphomolybdic acid solution, stirring, and sequentially subjecting to ultrasonic dispersion, centrifugation, and drying to obtain POM / AC; (2) mixing the acetylacetonate platinum (II) solution with the POM / AC obtained in step (1), stirring, then sequentially performing ultrasonic dispersion, centrifugation and drying, and then heating to 160-180° C. in 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, stirred at room temperature in the dark, and then N-hydroxysuccinimide was added. The mixture was stirred and activated for 5-7 hours in the dark to obtain solution B; (5) dissolving the solid A obtained in step (3) in ultrapure water to obtain 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-targeted artificial metabolic enzyme according to claim 1, characterized in that: 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-targeted artificial metabolic enzyme according to claim 1, characterized in that: 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-targeted artificial metabolic enzyme according to claim 1, characterized in that: 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-targeted artificial metabolic enzyme according to claim 1, characterized in that: In step (4), the mass volume ratio of (3-carboxypropyl)triphenylphosphonium 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 mitochondrial-targeted artificial metabolic enzyme according to claim 1, characterized in that: In step (4), stirring is performed at a rotation speed of 500-600 r / min.

7. The method for preparing the mitochondrial-targeted artificial metabolic enzyme according to claim 1, characterized in that: 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-targeted artificial metabolic enzyme according to claim 1, characterized in that: 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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