Preparation and application of rare earth ion doped metal organic framework loaded with lactate oxidase

Abstract

The present invention belongs to the technical field of biomaterials, and specifically relates to a rare earth ion doped metal organic framework loaded with lactate oxidase preparation and application thereof. The metal organic framework loaded with lactate oxidase provided by the present invention comprises a rare earth ion doped metal organic framework and lactate oxidase loaded on the metal organic framework through rare earth ion coordination; the rare earth ion doped metal organic framework is Tb 3+ Doped 5,10,15,20-tetrakis(4-carboxyphenyl)manganese porphyrin. The metal organic framework loaded lactate oxidase provided by the present invention loads lactate oxidase on the metal organic framework through the coordination of rare earth metal ions, and the lactate oxidase is chemically bonded to the metal organic framework carrier, with strong action and good stability, thereby ensuring that more lactate oxidase enters solid tumor cells, effectively increasing the content of H2O2 in the tumor microenvironment, and significantly enhancing the therapeutic effect of PDT and / or CDT.

Description

Technical Field

[0001] The invention belongs to the technical field of biomaterials, and in particular relates to the preparation and application of a rare earth ion-doped metal organic framework loaded with lactate oxidase. Background Art

[0002] Photodynamic therapy (PDT) is an important treatment for subcutaneous tumor tissue and an emerging cancer treatment method. The main principle of photodynamic therapy is that after being irradiated with light of a specific wavelength, the photosensitizer can transfer the excited state energy to the oxygen molecules in the surrounding tissue cells, causing them to produce cytotoxic reactive oxygen species (ROS), thereby inducing apoptosis and necrosis of tumor cells. Compared with other treatment methods, PDT has many advantages: non-invasiveness, low drug resistance, rapid efficacy, no accumulation of toxicity, repeatable medication, and low side effects. Chemodynamic therapy (CDT) is a new in situ treatment method that mainly uses the slightly acidic environment of tumor cells to trigger the Fenton or Fenton-like reaction, catalyzing the conversion of endogenous H2O2 into cytotoxic hydroxyl radicals (·OH), thereby inducing cell apoptosis.

[0003] However, there are still many factors that limit the performance of photodynamic therapy and chemodynamic therapy in solid tumor cells. For example, the rapid growth of tumors leads to severe hypoxia in solid tumors, and the content of H2O2 in the tumor microenvironment is low (0.1-1mM), which cannot achieve the ideal PDT and CDT effects; secondly, most traditional photosensitizers are hydrophobic, which will also lead to energy dissipation induced by photosensitizer aggregation and reduced efficacy.

[0004] In order to solve the above problems, it has been reported that bioenzymes are loaded on MOF to improve the effects of tumor chemodynamics and photodynamic therapy. However, the interaction between the enzyme and the carrier is weak and easy to leak; it cannot effectively improve the tumor treatment effect of PDT / CDT. Summary of the invention

[0005] The purpose of the present invention is to provide a preparation and application of a rare earth ion-doped metal-organic framework-loaded lactate oxidase. The metal-organic framework-loaded lactate oxidase provided by the present invention has good stability, can effectively increase the content of H2O2 in the tumor microenvironment, and significantly enhance the therapeutic effect of PDT and / or CDT.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] The present invention provides a metal organic framework loaded with lactate oxidase, comprising a metal organic framework doped with rare earth ions and lactate oxidase loaded on the metal organic framework through rare earth ion coordination;

[0008] The rare earth ion doped metal organic framework is Tb 3+Doped 5,10,15,20-tetrakis(4-carboxyphenyl)manganese porphyrin.

[0009] Preferably, the particle size of the metal organic framework loaded lactate oxidase is 190 to 210 nm.

[0010] Preferably, the Tb 3+ In the doped 5,10,15,20-tetrakis(4-carboxyphenyl)manganese porphyrin: the mass percentage of Tb element is 0.08-0.125wt%; the mass percentage of Mn element is 0.15-0.2wt%;

[0011] In the metal organic framework loaded lactate oxidase, the mass percentage of lactate oxidase is 10-20wt%.

[0012] The present invention provides a method for preparing the metal organic framework loaded with lactate oxidase described in the above technical solution, comprising the following steps:

[0013] Lactate oxidase solution and Tb 3+ The doped 5,10,15,20-tetrakis(4-carboxyphenyl)manganese porphyrin solution is mixed to carry out coordination reaction to obtain the metal organic framework loaded with lactate oxidase.

[0014] Preferably, Tb 3+ The preparation method of doped 5,10,15,20-tetrakis(4-carboxyphenyl)manganese porphyrin comprises the following steps:

[0015] 5,10,15,20-tetrakis(4-carboxyphenyl)manganese porphyrin, terbium acetate, glacial acetic acid and an organic solvent were mixed to carry out Tb 3+ Doping to obtain Tb 3+ Doped 5,10,15,20-tetrakis(4-carboxyphenyl)manganese porphyrin.

[0016] Preferably, lactate oxidase and Tb 3+ The mass ratio of the doped 5,10,15,20-tetrakis(4-carboxyphenyl)manganese porphyrin is (1-2):1.

[0017] Preferably, the molar ratio of the 5,10,15,20-tetrakis(4-carboxyphenyl)manganese porphyrin to the terbium acetate is 1:(1-8).

[0018] Preferably, the preparation method of 5,10,15,20-tetrakis(4-carboxyphenyl)manganese porphyrin comprises the following steps:

[0019] Mixing manganese acetate, glacial acetic acid and an organic solvent to obtain a manganese acetate solution;

[0020] An organic solution of 5,10,15,20-tetrakis(4-carboxyphenyl)porphyrin is added to the manganese acetate solution to carry out a coordination reaction to obtain the 5,10,15,20-tetrakis(4-carboxyphenyl)manganeseporphyrin.

[0021] The present invention provides a photodynamic and / or chemodynamic therapy reagent, comprising the metal organic framework-loaded lactate oxidase described in the above technical solution or the metal organic framework-loaded lactate oxidase prepared by the preparation method described in the above technical solution.

[0022] The present invention provides the use of the metal organic framework loaded lactate oxidase described in the above technical solution or the metal organic framework loaded lactate oxidase prepared by the preparation method described in the above technical solution in the preparation of anti-tumor drugs.

[0023] The present invention provides a metal organic framework loaded with lactate oxidase, comprising a metal organic framework doped with rare earth ions and lactate oxidase loaded on the metal organic framework through rare earth ion coordination; the metal organic framework doped with rare earth ions is Tb 3+ Doped 5,10,15,20-tetrakis(4-carboxyphenyl)manganese porphyrin. The metal organic framework loaded with lactate oxidase provided by the present invention loads lactate oxidase on the metal organic framework through the coordination of rare earth metal ions. The lactate oxidase and the metal organic framework carrier are chemically bonded, with strong action and good stability, thereby ensuring that more lactate oxidase enters solid tumor cells. After entering the tumor cells: on the one hand, lactate oxidase (LOX) catalyzes lactate to generate pyruvate and H2O2, providing more H2O2 reaction substrates for Fenton-like reactions, and H2O2 participates in Fenton-like reactions, thereby generating more cytotoxic ·OH, inducing tumor cell apoptosis; the generated H2O2 can also react with Mn 3+ The reaction generates oxygen, which produces more cytotoxic ROS after light exposure, thereby enhancing the PDT / CDT treatment effect. 3+ It not only reacts with glutathione to increase the amount of ROS, but also reacts with H2O2 to produce O2, which can also improve the PDT effect. In summary, the metal organic framework loaded with lactate oxidase provided by the present invention has good stability, can effectively increase the content of H2O2 in the tumor microenvironment, and significantly enhance the therapeutic effect of PDT and / or CDT.

[0024] The present invention provides a method for preparing a metal organic framework loaded with lactate oxidase according to the above technical solution, comprising the following steps: mixing a lactate oxidase solution and Tb 3+The doped 5,10,15,20-tetrakis(4-carboxyphenyl)manganese porphyrin solution is mixed for coordination reaction to obtain the metal organic framework loaded lactate oxidase. The preparation method provided by the present invention is simple and suitable for industrial application, through the coordination reaction of lactate oxidase and rare earth metal ions in the solution to obtain the metal organic framework loaded lactate oxidase. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 TEM image of LOX-Mn-TCPP(Tb) prepared in Example 1 of the present invention;

[0026] Figure 2 UV absorption spectra of H2TCPP, Mn-TCPP, Mn-TCPP(Tb), and LOX-Mn-TCPP(Tb);

[0027] Figure 3 The mass fractions of Mn and Tb in Mn-TCPP(Tb) prepared in different embodiments;

[0028] Figure 4 is the fluorescence emission spectra under different conditions;

[0029] Figure 5 After different materials were co-incubated with LO2 cells (left) and MCF-7 cells (right), cell activity was detected using CCK-8. DETAILED DESCRIPTION

[0030] The present invention provides a metal organic framework loaded with lactate oxidase, comprising a metal organic framework doped with rare earth ions and lactate oxidase loaded on the metal organic framework through rare earth ion coordination;

[0031] The rare earth ion doped metal organic framework is Tb 3+ Doped 5,10,15,20-tetrakis(4-carboxyphenyl)manganese porphyrin.

[0032] In the present invention, unless otherwise specified, all preparation raw materials / components are commercially available products well known to those skilled in the art.

[0033] The metal organic framework loaded with lactate oxidase provided by the present invention comprises Tb 3+ Doped 5,10,15,20-tetrakis(4-carboxyphenyl) manganese porphyrin (referred to as Mn-TCPP(Tb) in the present invention). The present invention adopts 5,10,15,20-tetrakis(4-carboxyphenyl) manganese porphyrin (Mn-TCPP) as a MOFs carrier, wherein Mn 3+ It not only reacts with glutathione to increase the amount of ROS, but also reacts with H2O2 to produce O2, which can also improve the PDT effect. 3+Doped Mn-TCPP, Tb 3+ As a connecting group, lactate oxidase can be loaded on the Mn-TCPP molecule in the form of a chemical bond through a coordination reaction, thereby improving the loading stability of lactate oxidase on the Mn-TCPP molecule, avoiding the leakage of lactate oxidase during transportation in the body, and ensuring that lactate oxidase is transported to tumor cells, thereby improving the tumor treatment effect of PDT / CDT.

[0034] In the present invention, the Tb 3+ In the doped 5,10,15,20-tetrakis(4-carboxyphenyl)manganese porphyrin, the mass percentage of Tb element is preferably 0.08-0.125wt%, more preferably 0.1-0.125wt%; the mass percentage of Mn element is preferably 0.15-0.2wt%, more preferably 0.15-0.175wt%.

[0035] The metal organic framework loaded lactate oxidase provided by the present invention includes lactate oxidase loaded on the metal organic framework through rare earth ion coordination. In the present invention, the lactate oxidase and Tb 3+ In the present invention, the lactate oxidase is transported into tumor cells through the Mn-TCPP (Tb) carrier. On the one hand, the lactate oxidase (LOX) catalyzes lactic acid to generate pyruvic acid and H2O2, providing more H2O2 reaction substrates for the Fenton-like reaction. H2O2 participates in the Fenton-like reaction, thereby generating more cytotoxic ·OH, inducing tumor cell apoptosis; the generated H2O2 can also react with Mn 3+ The reaction generates oxygen, which produces more cytotoxic ROS after light exposure, thereby enhancing the PDT / CDT treatment effect at the same time.

[0036] In the present invention, in the metal organic framework loaded lactate oxidase: the mass percentage content (load amount) of lactate oxidase is preferably 10-20wt%, more preferably 12-15wt%, and most preferably 12wt%. The loading amount of lactate oxidase in the metal organic framework loaded lactate oxidase provided by the present invention cannot be too high or too low. When the loading amount of lactate oxidase is too high, the stability of the metal organic framework loaded lactate oxidase deteriorates, and lactate oxidase leakage is easily caused during transportation; when the loading amount of lactate oxidase is too low, the PDT / CDT treatment effect cannot be effectively enhanced.

[0037] In the present invention, the morphology of the metal organic framework loaded with lactate oxidase is spindle-shaped; the particle size is preferably 190 to 210 nm.

[0038] The present invention provides a method for preparing a metal organic framework loaded with lactate oxidase as described in the above technical solution, comprising the following steps:

[0039] Lactate oxidase solution and Tb 3+ The doped 5,10,15,20-tetrakis(4-carboxyphenyl)manganese porphyrin solution is mixed to carry out a coordination reaction (hereinafter referred to as the first coordination reaction) to obtain the metal organic framework loaded with lactate oxidase.

[0040] In the present invention, the solvent in the lactate oxidase solution is preferably N,N'-dimethylformamide (DMF). The mass concentration of the lactate oxidase solution is preferably 2 mg / mL.

[0041] In the present invention, Tb 3+ The solvent used in the doped 5,10,15,20-tetrakis(4-carboxyphenyl)manganese porphyrin solution is preferably DMF. 3+ The mass concentration of the doped 5,10,15,20-tetrakis(4-carboxyphenyl)manganese porphyrin solution is preferably 1 mg / mL.

[0042] In the present invention, the Tb 3+ The preparation method of doped 5,10,15,20-tetrakis(4-carboxyphenyl)manganese porphyrin comprises the following steps:

[0043] 5,10,15,20-tetrakis(4-carboxyphenyl)manganese porphyrin, terbium acetate, glacial acetic acid (hereinafter referred to as the first glacial acetic acid) and an organic solvent (hereinafter referred to as the first organic solvent) are mixed to carry out Tb 3+ Doping to obtain Tb 3+ Doped 5,10,15,20-tetrakis(4-carboxyphenyl)manganese porphyrin.

[0044] In the present invention, the preparation method of 5,10,15,20-tetrakis(4-carboxyphenyl)manganese porphyrin comprises the following steps:

[0045] mixing manganese acetate, glacial acetic acid (hereinafter referred to as the second glacial acetic acid) and an organic solvent (hereinafter referred to as the second organic solvent) to obtain a manganese acetate solution;

[0046] An organic solution of 5,10,15,20-tetrakis(4-carboxyphenyl)porphyrin is added to the manganese acetate solution to carry out a coordination reaction (hereinafter referred to as the second coordination reaction) to obtain the 5,10,15,20-tetrakis(4-carboxyphenyl)manganeseporphyrin.

[0047] The present invention mixes manganese acetate, a second glacial acetic acid and a second organic solvent to obtain a manganese acetate solution. In a specific embodiment of the present invention, the manganese acetate is manganese acetate (III) dihydrate (Mn(OAc)3·2H2O). The second organic solvent is N,N'-dimethylformamide (DMF). In the present invention, the order of mixing the manganese acetate, the second glacial acetic acid and the second organic solvent is preferably: dissolving manganese acetate in part of the second organic solvent to obtain a manganese acetate solution; mixing the second glacial acetic acid and the remaining second organic solvent to obtain a mixed solution; mixing the manganese acetate solution and the mixed solution. The ratio of the mass of the manganese acetate to the volume of part of the second organic solvent is preferably (85-90) mg:10 mL; the ratio of the volume of the second glacial acetic acid to the remaining second organic solvent is preferably 1:4; the ratio of the mass of the manganese acetate to the volume of the mixed solution is preferably (85-90) mg:(25-27) mL.

[0048] After obtaining the manganese acetate solution, the present invention adds an organic solution of 5,10,15,20-tetrakis(4-carboxyphenyl)porphyrin to the manganese acetate solution to carry out a second coordination reaction to obtain the 5,10,15,20-tetrakis(4-carboxyphenyl)manganese porphyrin. In the present invention, the solvent in the organic solution of 5,10,15,20-tetrakis(4-carboxyphenyl)porphyrin is preferably DMF; the mass concentration is preferably 1 mg / mL. The mass ratio of the manganese acetate and the 5,10,15,20-tetrakis(4-carboxyphenyl)porphyrin is preferably (85-90):(10-12). In the present invention, the second coordination reaction is preferably carried out at room temperature, and the reaction time is preferably 12-24 hours. In the present invention, after the second coordination reaction is completed, the present invention preferably separates the obtained second coordination reaction liquid into solid and liquid, and the obtained solid phase product is washed with DMF and ethanol in sequence to obtain the 5,10,15,20-tetrakis(4-carboxyphenyl)manganeseporphyrin. The solid-liquid separation is preferably centrifugation; the number of DMF washing is preferably 3 times, and the number of ethanol washing is preferably 2 times.

[0049] In the present invention, the molar ratio of the 5,10,15,20-tetrakis(4-carboxyphenyl)manganese porphyrin and the terbium acetate is preferably 1:(1-8), more preferably 1:4. The volume ratio of the first glacial acetic acid to the first organic solvent is preferably 1:4. The present invention has no special requirements for the amount of the first organic solvent, as long as the 5,10,15,20-tetrakis(4-carboxyphenyl)manganese porphyrin and the terbium acetate are completely dissolved. In the present invention, the Tb 3+ The doping is preferably carried out at room temperature, and the reaction time is preferably 1 hour. 3+ After the doping is completed, the present invention preferably treats the obtained Tb 3+The doping reaction liquid is solid-liquid separated, and the obtained solid phase product is washed with DMF and dried in sequence to obtain the Mn-TCPP(Tb). The solid-liquid separation is preferably centrifugal; the DMF washing is preferably 3 times, and the drying is preferably oven drying.

[0050] In the present invention, lactate oxidase and Tb 3+ The mass ratio of the doped 5,10,15,20-tetrakis(4-carboxyphenyl)manganese porphyrin is (1-2):1, more preferably 2:1.

[0051] In the present invention, the temperature of the first coordination reaction is preferably 37°C, and the reaction time is preferably 3h. In the present invention, after the first coordination reaction is completed, the present invention preferably separates the first coordination reaction liquid into solid-liquid phase, and the obtained solid phase product is LOX-Mn-TCPP(Tb).

[0052] The present invention provides a photodynamic and / or chemodynamic therapy reagent, comprising the metal organic framework-loaded lactate oxidase described in the above technical solution or the metal organic framework-loaded lactate oxidase prepared by the preparation method described in the above technical solution.

[0053] The present invention provides the use of the metal organic framework loaded lactate oxidase described in the above technical solution or the metal organic framework loaded lactate oxidase prepared by the preparation method described in the above technical solution in the preparation of anti-tumor drugs.

[0054] In the present invention, solid tumors preferably include breast cancer.

[0055] In order to further illustrate the present invention, the technical solutions provided by the present invention are described in detail below in conjunction with embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0056] Explanation of technical terms in this invention: 1. Metal Organic Frameworks (MOFs), which are organic-inorganic hybrid materials with intramolecular pores formed by self-assembly of organic ligands and metal ions or clusters through coordination bonds. 2. Lactate oxidase: LOX; Lactic acid: Lactic acid (LA). 3. 5,10,15,20-tetrakis (4-carboxyphenyl) manganese porphyrin (Mn-TCPP) is a type of metal organic framework material; Tb 3+Doped 5,10,15,20-tetrakis(4-carboxyphenyl) manganese porphyrin: LOX-Mn-TCPP(Tb). 4.2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonate benzene)-2H-tetrazolyl monosodium salt (CCK-8, Cell Counting Kit-8), under the action of electron carrier 1-methoxy-5-methylphenazine dimethyl sulfate (1-MethoxyPMS), is reduced by dehydrogenase in cell mitochondria to a highly water-soluble yellow formazan product (Formazan). The amount of formazan generated is proportional to the number of living cells. Its light absorption value is measured at a wavelength of 450nm using an enzyme marker, which can indirectly reflect the number of living cells. 5. Human breast cancer cells: MCF-7, human normal liver cells: LO2. 6. 2,7-Dichlorofluorescein diacetate, also known as: reactive oxygen species ROS fluorescent probe (DCFH-DA, 4091-99-0): DCFH-DA itself has no fluorescence and can freely pass through the cell membrane. After entering the cell, it can be hydrolyzed by the esterase in the cell to produce DCFH, while DCFH cannot pass through the cell membrane, making it easy for the probe to be labeled into the cell. In the presence of ROS, DCFH is oxidized to produce the green fluorescent substance DCF, and the green fluorescence intensity is proportional to the ROS level.

[0057] Example 1

[0058] Weigh 85 mg of manganese acetate (III) dihydrate (Mn(OAc)3·2H2O) and disperse it in a flat-bottom flask containing 10 mL of N,N'-dimethylformamide (DMF), and stir it evenly by ultrasonic stirring. Add 25 mL of a 1:4 volume ratio of glacial acetic acid (HAc) / DMF mixed solution, mix thoroughly, and continue stirring for 8 minutes. Slowly add 12 mL of a 1 mg / mL 5,10,15,20-tetrakis(4-hydroxyphenyl)porphyrin (H2TCPP) solution (solvent is DMF) to the above solution, react at room temperature for 24 hours, centrifuge the product and wash it three times with DMF and twice with ethanol to obtain Mn-TCPP.

[0059] Under normal temperature conditions, Mn-TCPP and C6H9O6Tb·xH2O with a molar ratio of 1:4 were dissolved in 1 mL of HAc / DMF solution with a volume ratio of 1:4, and reacted for 1 hour at room temperature. After the reaction, the sample was collected by centrifugation, washed three times with DMF, and dried to obtain Mn-TCPP(Tb) for standby use.

[0060] 100 μL of a prepared LOX solution (DMF) with a concentration of 2 mg / mL was fully mixed with 100 μL of a Mn-TCPP(Tb) solution (DMF) with a concentration of 1 mg / mL, and reacted in an oven at 37° C. for 3 hours. After the reaction was completed, the sample was collected by centrifugation to obtain LOX-Mn-TCPP(Tb).

[0061] Test Example 1

[0062] Characterization of LOX-Mn-TCPP(Tb)

[0063] The morphology and size of LOX-Mn-TCPP(Tb) prepared in Example 1 were characterized by transmission electron microscopy (TEM). Figure 1 As shown, the size of LOX-Mn-TCPP(Tb) is 190-210 nm and the morphology is spindle-shaped.

[0064] UV absorption detection

[0065] UV absorption detection was performed on H2TCPP, Mn-TCPP, Mn-TCPP(Tb), and LOX-Mn-TCPP(Tb). Figure 2 As shown in Figure 2, compared with the organic ligand H2TCPP, all the UV absorption peaks of Mn-TCPP are red-shifted, which is due to the increase in the conjugated area of ​​porphyrin, and this also proves the successful synthesis of Mn-TCPP. 3+ After loading LOX, the ultraviolet absorption peak of the material hardly changed, indicating that the crystal structure of Mn-TCPP did not change.

[0066] Embodiments 2 to 4

[0067] The preparation method is basically the same as that in Example 1, except that: in Example 2, n(Mn-TCPP): n(Tb 3+ )=1:1, in Example 3, n(Mn-TCPP):n(Tb 3+ )=1:2, in Example 4, n(Mn-TCPP):n(Tb 3+ )=1:8.

[0068] Test Example 2

[0069] Inductively coupled plasma characterization

[0070] The metal element contents in the Mn-TCPP prepared in Example 1 and the Mn-TCPP(Tb) prepared in Examples 1 to 4 were analyzed by inductively coupled plasma (ICP). Figure 3 As shown, according to the different molar ratios of Mn-TCPP and Tb (n(Mn-TCPP):n(Tb 3+)=1:0, 1:1, 1:2, 1:4, 1:8) to synthesize Mn-TCPP(Tb) with different doping degrees. The ICP results are as follows Figure 3 As shown, with Tb 3+ As the concentration increases, the content of Tb element in Mn-TCPP(Tb) also increases, and the content of Mn element decreases, indicating the successful doping of Tb element.

[0071] Test Example 3

[0072] Reactive oxygen detection

[0073] DCFH was used to further detect the generation of ·OH. First, 0.97 mg DCFH-DA was weighed and dissolved in dimethyl sulfoxide (DMSO) to obtain a 1 mM DCFH-DA solution. Then 0.5 mL of the 1 mM DCFH-DA solution was added to 2 mL of 0.01 M NaOH solution, and the mixed solution was placed in a dark environment to react for 30 minutes. After the reaction was completed, 10 mL of PBS buffer solution with a pH of 4 was added to terminate the reaction to obtain a DCF solution, which was stored at -20°C away from light. The experiment was designed into the following groups: Mn-TCPP(Tb), Mn-TCPP(Tb) + light, Mn-TCPP(Tb) + lactic acid, Mn-TCPP(Tb) + lactic acid + light, LOX-Mn-TCPP(Tb), LOX-Mn-TCPP(Tb) + light, LOX-Mn-TCPP(Tb) + lactic acid, LOX-Mn-TCPP(Tb) + lactic acid + light. Lactic acid was adjusted to pH = 6 using NaOH solution, and the illumination time was 1 minute. Finally, equal amounts of DCF solution were added to the above experimental groups, and the fluorescence intensity of the supernatant was measured under an excitation wavelength of 488nm. The test results are as follows: Figure 4 shown.

[0074] Since 2',7'-dichlorofluorescein diacetate (DCFH-DA) can be oxidized by ROS to generate DCF with green fluorescence, such as Figure 4 As shown, the fluorescence intensity of the LOX-Mn-TCPP(Tb)+Lactic acid+Laser group prepared in Example 1 is the strongest. This is because the H2O2 produced by lactic acid catalyzed by LOX is simultaneously catalyzed by manganese ions and light to produce more ROS.

[0075] Test Example 4

[0076] Cytotoxicity assay

[0077] The present invention studied the cytotoxicity of different materials to human breast cancer cells (MCF-7) and human normal liver cells (LO2) by CCK-8. MCF-7 and LO2 cells were inoculated into 96-well plates at a cell density of 8×104 / well, and cultured in a constant temperature incubator at 37°C for 12 hours. After the cells adhered to the wall, discard the old cell culture medium. Fresh culture medium containing different concentrations of Mn-TCPP (Tb) and LOX-Mn-TCPP (Tb) was added to the 96-well plate and continued to culture. The concentration gradients of Mn-TCPP (Tb) and LOX-Mn-TCPP (Tb) were 0μg / mL, 1μg / mL, 2μg / mL, 4μg / mL, 6μg / mL and 8μg / mL. For the phototoxicity experiment, the Mn-TCPP (Tb) and LOX-Mn-TCPP (Tb) prepared in Example 1 were cultured for 4 hours, irradiated with a 650nm laser for 10 minutes, and continued to incubate for 18 hours. After the incubation, the survival rate of cells in different groups was detected by an enzyme marker. The results are as follows Figure 5 As shown in the figure, when the concentration of Mn-TCPP(Tb) was 8μg / mL, the survival rate of MCF-7 cells was about 75%, while the survival rate of LO2 cells was above 80%, which was attributed to the catalysis of H2O2 overexpressed in tumor cells by Fenton-like materials to generate ROS. After laser irradiation, the survival rate of both cells in the Mn-TCPP(Tb)+L group (where "L" represents light) decreased, which is the result of the synergistic treatment of CDT and PDT. Importantly, compared with Mn-TCPP(Tb), the survival rate of cells in the LOX-Mn-TCPP(Tb) group was lower, because LOX catalyzed lactate to generate pyruvate and H2O2, which improved the therapeutic effect of CDT. Similarly, compared with the LOX-Mn-TCPP(Tb) group, the survival rate of cells in the LOX-Mn-TCPP(Tb)+L (where "L" represents light) group was lower, indicating that the synergistic treatment effect of CDT / PDT was better.

[0078] The nano material LOX-Mn-TCPP (Tb) provided by the present invention is synthesized at room temperature by Mn-TCPP and then doped with Tb 3+ , LOX is loaded by the coordination of rare earth ions to obtain LOX-Mn-TCPP(Tb). After the nanomaterial LOX-Mn-TCPP(Tb) enters the body, LOX catalyzes lactic acid to produce pyruvic acid and H2O2, part of which participates in the Fenton-like reaction to generate ·OH, and part reacts with Mn 3+ The reaction generates oxygen and enhances the PDT / CDT treatment effect. 3+ It not only reacts with glutathione to increase the amount of ROS, but also reacts with H2O2 to produce O2 to improve the PDT effect. The synthesis method of the LOX-Mn-TCPP (Tb) nanomaterial provided by the present invention is simple, has no dangerous operation, and has a stable structure. The loading of LOX can enhance the synergistic treatment effect of chemical dynamics and photodynamics.

[0079] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. Other embodiments can be obtained based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A metal organic framework loaded with lactate oxidase, characterized in that: It includes a rare earth ion doped metal organic framework and lactate oxidase loaded on the metal organic framework through rare earth ion coordination; The rare earth ion doped metal organic framework is Tb 3+ Doped 5,10,15,20-tetrakis(4-carboxyphenyl)manganese porphyrin; The Tb 3+ In the doped 5,10,15,20-tetrakis(4-carboxyphenyl)manganese porphyrin: the mass percentage of Tb element is 0.08-0.125wt%; In the metal organic framework loaded lactate oxidase, the mass percentage of lactate oxidase is 10-20wt%.

2. The metal organic framework loaded lactate oxidase according to claim 1, characterized in that: The particle size of the metal organic framework loaded lactate oxidase is 190-210 nm.

3. The metal organic framework loaded lactate oxidase according to claim 1, characterized in that: The Tb 3+ In the doped 5,10,15,20-tetrakis(4-carboxyphenyl)manganese porphyrin, the mass percentage of Mn element is 0.15-0.2wt%.

4. The method for preparing a metal organic framework loaded with lactate oxidase according to any one of claims 1 to 3, characterized in that: The following steps are involved: Lactate oxidase solution and Tb 3+ The doped 5,10,15,20-tetrakis(4-carboxyphenyl)manganese porphyrin solution is mixed to carry out coordination reaction to obtain the metal organic framework loaded with lactate oxidase.

5. The preparation method according to claim 4, characterized in that: Tb 3+ The preparation method of doped 5,10,15,20-tetrakis(4-carboxyphenyl)manganese porphyrin comprises the following steps: 5,10,15,20-tetrakis(4-carboxyphenyl)manganese porphyrin, terbium acetate, glacial acetic acid and an organic solvent were mixed to carry out Tb 3+ Doping to obtain Tb 3+ Doped 5,10,15,20-tetrakis(4-carboxyphenyl)manganese porphyrin.

6. The preparation method according to claim 4, characterized in that: Lactate oxidase and Tb 3+ The mass ratio of the doped 5,10,15,20-tetrakis(4-carboxyphenyl)manganese porphyrin is (1-2):

1.

7. The preparation method according to claim 5, characterized in that: The molar ratio of the 5,10,15,20-tetrakis(4-carboxyphenyl)manganese porphyrin to the terbium acetate is 1:(1-8).

8. The preparation method according to claim 5, characterized in that: The preparation method of the 5,10,15,20-tetrakis(4-carboxyphenyl)manganese porphyrin comprises the following steps: Mixing manganese acetate, glacial acetic acid and an organic solvent to obtain a manganese acetate solution; An organic solution of 5,10,15,20-tetrakis(4-carboxyphenyl)porphyrin is added to the manganese acetate solution to carry out a coordination reaction to obtain the 5,10,15,20-tetrakis(4-carboxyphenyl)manganeseporphyrin.

9. A photodynamic and / or chemodynamic therapy agent, characterized in that: The invention comprises the metal organic framework-loaded lactate oxidase according to any one of claims 1 to 3 or the metal organic framework-loaded lactate oxidase prepared by the preparation method according to any one of claims 4 to 8.

10. Use of the metal organic framework-loaded lactate oxidase according to any one of claims 1 to 3 or the metal organic framework-loaded lactate oxidase prepared by the preparation method according to any one of claims 4 to 8 in the preparation of anti-tumor drugs.

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