CN108273059B - Preparation method and application of composite nano material for treating tumors by combining photo-thermal treatment and active oxygen treatment - Google Patents
Preparation method and application of composite nano material for treating tumors by combining photo-thermal treatment and active oxygen treatment Download PDFInfo
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Abstract
The invention relates to a preparation method and application of a composite nano material for treating tumors by combining photo-thermal and active oxygen, belonging to the technical field of medicaments for treatment. The method comprises the steps of preparing nano-iron by an ethylene glycol method, oxidizing catechol groups on dopamine molecules in an alkaline solution and under the condition of oxygen to form dihydroxyindole or semiquinone free radicals, carrying out self-polymerization reaction of rearrangement or coupling crosslinking, coating a polydopamine layer outside the nano-iron, realizing covalent connection with glucose oxidase by utilizing an active group of the polydopamine, preparing a nano-iron/polydopamine/glucose oxidase (nano-iron/PDA/GOx) composite nano-material, and constructing a composite material of biomacromolecules and an inorganic nano-material modified by a bionic material. The material prepared by the invention has the advantages of obvious treatment effect, high biocompatibility, no toxicity, no pollution, simple preparation process and lower cost, and is suitable for mass production and clinical medical use.
Description
Technical Field
The invention relates to a preparation method and application of a composite nano material for treating tumors by combining photo-thermal and active oxygen, belonging to the technical field of medicaments for treatment.
Background
In cancer treatment, in order to improve the anti-tumor effect and reduce the toxic and side effects, aiming at the weak points of tumor tissues, compound therapeutic reagents are developed, which is an important strategy for cancer treatment. Tumor cells are very glucose dependent due to their rapid growth and proliferation. Moreover, because of the unique tissue structure and metabolic characteristics of the tumor, the tolerance of the tumor to heat (>42 ℃) and exogenous active oxygen is far lower than that of normal cells; the solid tumor tissue has abundant blood vessels and incomplete tube wall structure, and has obvious retention and accumulation Effects (EPR) on macromolecules and nano materials with certain particle sizes (100-.
Photothermal therapy is a minimally invasive tumor therapy technology developed in recent years, near-infrared laser (700-. Polydopamine (PDA) is a bionic material derived from marine mussel mucin, and has the advantages of high biocompatibility, thermal stability and the like. The poly-dopamine can quickly modify the surface of the nano material, has active groups and can perform secondary grafting reaction. The polydopamine also has high-efficiency photothermal conversion capability, can convert near-infrared light energy into heat, and can be used for photothermal treatment of tumors.
Glucose is an energy source substance necessary for tumors, and the deprivation of glucose can rapidly kill tumor cells. Glucose oxidase (GOx, EC1.1.3.4) is an important class of oxidoreductases in organisms, has the characteristic of substrate specificity, and can oxidize beta-D-glucose into gluconic acid and generate active oxygen hydrogen peroxide (H)2O2). The transformation of macrophages with glucose oxidase-liposome material is believed to have therapeutic effects on chronic granulomatous patients infected with pathogenic microorganisms. Glucose oxidase has been reported to increase active oxygen (H) in vivo2O2) The content of the product can be combined with "Linum Augustin-Linum Augustin" to generate toxic cyanide, and can be used for treating malignant glioma. The nano-iron material can release ferrous ions and catalyze hydrogen peroxide (H) through Fenton reaction2O2) Generate more oxidative hydroxyl radical (. OH) and inhibit early tumor metastasis. Therefore, the glucose oxidase and nano-iron combined material can have the function of generating high-oxidizing hydroxyl radicals.
The composite nano material with photo-thermal conversion and active oxygen generation is constructed to reach a certain particle size, tumor tissue specific accumulation can be realized, and the effect of combined treatment of tumors can be achieved by aiming at different weaknesses of the tumor tissue from multiple angles. At present, no related report exists that nano iron/polydopamine/glucose oxidase is used for constructing a composite material, and photothermal and active oxygen are combined to treat tumors.
Disclosure of Invention
The invention provides a preparation method and application of a composite nano material for treating tumors by combining photo-thermal and active oxygen aiming at the defects of the existing tumor treatment materials. The material is prepared by preparing nano-iron by an ethylene glycol method, a catechol group on dopamine molecules is oxidized under the conditions of alkaline solution and oxygen to form dihydroxyindole or semiquinone free radicals, the self-polymerization reaction of rearrangement or coupling crosslinking is carried out, a polydopamine layer is coated outside the nano-iron, the covalent connection with glucose oxidase is realized by utilizing an active group of the polydopamine, the nano-iron/polydopamine/glucose oxidase (nano-iron/PDA/GOx) composite nano-material is prepared, and the composite material of the biomacromolecule and the inorganic nano-material modified by the bionic material is constructed.
The technical scheme of the invention is as follows:
a preparation method of a composite nano material for treating tumors by combining photo-thermal and active oxygen comprises the following steps:
(1) weighing dopamine hydrochloride and a nano-iron material with the particle size of 10-300nm according to a proportion, adding a Tris-HCl buffer solution with the concentration of 1-50mM, wherein the final concentration of the dopamine hydrochloride is 0.02-6mg/mL, carrying out oscillation reaction at the temperature of 10-60 ℃ for 0.5-16h, and after the reaction is finished, separating and washing to obtain the nano-iron/poly-dopamine composite nano-material with the surface coated with a poly-dopamine layer;
(2) weighing the nano-iron/poly-dopamine composite nano-material prepared in the step (1) and glucose oxidase according to a proportion, adding 1-50mM Tris-HCl buffer solution, wherein the final concentration of the glucose oxidase is 0.04-4mg/mL, carrying out oscillation reaction for 1-36h at the temperature of 0-60 ℃, and after the reaction is finished, carrying out separation, washing and freeze-drying to obtain the nano-iron/poly-dopamine/glucose oxidase (nano-iron/PDA/GOx) composite nano-material.
Preferably, in the step (1), dopamine hydrochloride and the nano-iron material with the particle size of 70-250nm are weighed according to the proportion, a Tris-HCl buffer solution with the concentration of 5-20mM is added, the final concentration of the dopamine hydrochloride is 0.08-4mg/mL, the oscillation reaction is carried out for 1-5h at the temperature of 20-30 ℃, and after the reaction is finished, the nano-iron/poly-dopamine composite nano-material is obtained through separation and washing.
Preferably, in the step (2), the nano-iron/poly-dopamine/glucose oxidase (nano-iron/PDA/GOx) composite nanomaterial prepared in the step (1) is weighed according to a proportion, Tris-HCl buffer solution with the concentration of 5-20mM is added, the final concentration of the glucose oxidase is 0.1-2mg/mL, oscillation reaction is carried out for 8-30h at the temperature of 20-30 ℃, and after the reaction is finished, separation, washing and freeze-drying are carried out to obtain the nano-iron/poly-dopamine/glucose oxidase (nano-iron/PDA/GOx) composite nanomaterial.
According to the present invention, preferably, the preparation method of the nano-iron material in the step (1) is as follows: 1-1.5g of FeCl was weighed3·6H2Dissolving O in 35-45mL of ethylene glycol, and magnetically stirring until FeCl3·6H2Completely dissolving the O; adding 3.5-4.0g of anhydrous sodium acetate, and stirring for 30min until the sodium acetate is completely dissolved; pouring the solution into a high-pressure reaction kettle lined with polytetrafluoroethylene, setting the temperature at 180 ℃ and 220 ℃, carrying out constant-temperature oil bath reaction for 7-9h, taking out the reaction kettle, cooling to room temperature, washing for 5-6 times with methanol, and putting the reaction kettle into a vacuum drying oven for overnight drying to obtain the nano iron material.
According to the invention, the rotation speed of the oscillating reaction in step (1) or (2) is preferably 50-300 rpm.
According to the invention, the separation in the step (1) or (2) is preferably performed by a magnetic separator or a high-speed centrifugal separation at the rotation speed of 1500-.
According to the invention, the pH value of the buffer solution in the step (1) or (2) is 7.0-9.5.
According to the invention, the mass ratio of the nano-iron material to the dopamine hydrochloride in the step (1) is 1: 0.1-10.
According to the present invention, the washing in step (1) or (2) is preferably performed 2 to 5 times with Tris-HCl buffer or sodium phosphate buffer.
Preferably, according to the present invention, the glucose oxidase of step (2) is a system named as β -D-glucose oxidoreductase (EC 1.1.3.4).
According to the invention, the mass ratio of the glucose oxidase to the nano-iron/poly-dopamine composite nano-material in the step (2) is 1: 0.05-5.
The invention relates to application of a nano-iron/polydopamine/glucose oxidase composite nano-material in preparing a targeted drug for treating tumors by combining photo-thermal treatment and active oxygen treatment.
The invention has the beneficial effects that:
1. the invention firstly connects the glucose oxidase to the surface of the nano-iron material through polydopamine, and utilizes the hydrogen peroxide (H) generated by the glucose oxidase2O2) The hydroxyl free radical (OH) with stronger oxidability is generated by the catalysis of ferrous ions released by the nano iron, thereby achieving the purpose of killing tumor cells. Because normal cells have stronger tolerance to heat and active oxygen than tumor cells, the material has much lower damage to the normal cells than the tumor cells, and has strong tumor cell selective killing effect.
2. The invention synthesizes the composite material with the grain diameter of 70-250nm and multiple tumor killing effects, and the material can target the tumor by utilizing the retention and accumulation Effect (EPR) of the tumor on macromolecules.
3. The material prepared by the invention has the advantages of obvious treatment effect, high biocompatibility, no toxicity, no pollution, simple preparation process and lower cost, and is suitable for mass production and clinical medical use.
Drawings
FIG. 1 is a transmission electron microscope image of the nano-iron/PDA/GOx composite nano-material;
FIG. 2 is a graph of the efficiency of nano-iron/PDA/GOx composite nano-material in converting near-infrared light into heat;
FIG. 3 is a bar graph of the survival rate of tumor cells and normal cells after the action of the nano-iron/PDA/GOx composite nanomaterial;
FIG. 4 is a laser confocal diagram of the nano-iron/PDA/GOx composite nano-material for increasing the content of active oxygen in tumor cells;
FIG. 5 shows the tumor-bearing mice treated by the composite nano-material of nano-iron/PDA/GOx.
Detailed Description
The present invention is further illustrated by the following examples, but the scope of the invention is not limited thereto.
The materials and reagents used in the following examples are commercially available biological and chemical laboratory materials unless otherwise specified.
Example 1 preparation of a composite nanomaterial of Nano iron/PDA/GOx
(1) Preparation of nano iron matrix
1.35g of FeCl was weighed3·6H2Dissolving O in 40mL of ethylene glycol, and magnetically stirring until FeCl3·6H2Completely dissolving the O; adding 3.60g of anhydrous sodium acetate, and stirring for 30min until the sodium acetate is completely dissolved; pouring the solution into a high-pressure reaction kettle lined with polytetrafluoroethylene, setting the temperature to be 200 ℃, carrying out constant-temperature oil bath reaction for 8h, taking out the reaction kettle, cooling to room temperature, washing with methanol for 6 times, putting into a vacuum drying oven for overnight drying, and detecting a TEM image as shown in figure 1(a) to obtain the particle size of 200 nm.
(2) Preparation of nano-iron/PDA/GOx composite material
And (2) coating poly dopamine by adopting a self-assembly method, weighing 50mg of nano iron powder prepared in the step (1), meanwhile weighing 50mg of dopamine hydrochloride, dispersing the nano iron powder and the dopamine hydrochloride in 250mL of 10mM Tris-HCl buffer solution with the pH value of 8.0, uniformly oscillating at the room temperature at 150rpm, reacting for 12 hours, separating by using a magnetic separator after the reaction is finished, and washing for 3 times by using the Tris-HCl buffer solution to obtain the nano iron/poly dopamine composite nanomaterial.
And adding the washed nano-iron/polydopamine/glucose oxidase and 50mg of glucose oxidase into 200mL of 10mM Tris-HCl buffer solution with the pH value of 8.0 together, uniformly oscillating at 150rpm at room temperature, reacting for 16h, separating by a magnetic separator after the reaction is finished, washing for 3 times by the Tris-HCl buffer solution, and freeze-drying to obtain the nano-iron/polydopamine/glucose oxidase (nano-iron/PDA/GOx) composite nano-material. The TEM image is detected, and as shown in FIG. 1(b), the composite material is measured to have a diameter of 70 to 250 nm.
Example 2 photo-thermal conversion performance experiment of nano-iron/PDA/GOx composite nano-material
(1) Configuration of reaction System
5mg of the nano-iron/PDA/GOx composite material prepared in example 1 was weighed, dispersed in 5mL of Phosphate (PBS) buffer, and diluted in a gradient manner to obtain suspensions of 10. mu.g/mL, 100. mu.g/mL and 1000. mu.g/mL. The experimental group is that more than 200 mu L of composite material suspension with concentration is respectively added into a single hole of a 96-hole plate; the control group was a 96-well plate with 200. mu.L PBS buffer added to a single well.
(2) Testing of photothermal conversion Performance
The experimental group and the control group are respectively at infrared light of 3.6W/cm2The temperature change was measured and recorded every 30 seconds by an electronic thermometer under the condition of 5 minutes irradiation, and the result is shown in fig. 2, and the higher the concentration of the nano-iron/PDA/GOx composite material is, the larger the amount of heat generated is.
Example 3 specific killing of Breast cancer cells MDA-MB-231 by Nano iron/PDA/GOx composite nanomaterial
The MTT method is adopted to detect the activity of the cells, and the specific steps are as follows:
(1) culturing normal epithelial cell MCF-10A of mammary gland and breast cancer cell MDA-MB-231, culturing the cells to 70% fusion degree, digesting, collecting and counting, and diluting the cell density to 1 × 105Adding 100 mu L of the cell diluent into each well of a 96-well plate, and culturing for 24 hours in a 5% CO2 incubator at 37 ℃;
(2) replacing a new complete culture medium, adding the nano-iron/PDA/GOx composite nano-material to the final concentration of 10 mug/mL, 20 mug/mL, 50 mug/mL, 100 mug/mL, 1000 mug/mL, culturing for 4 hours in a 5% CO2 incubator at 37 ℃;
(3) near infrared light (3.6W/cm) emitted by BST808-5-F laser per hole2) After 3min of irradiation, 5% CO at 37 ℃2Culturing for 12h in an incubator;
(4) the 96-well plate was stained by adding 10. mu.L of MTT (5mg/mL), 37 ℃ and 5% CO per well of the 96-well plate2Culturing for 4h in an incubator, removing the culture medium, adding 100 μ L DMSO into each well, gently mixing for 10min by using a shaking table, dissolving MTT, measuring OD value at λ 490nm, and calculating the inhibition rate.
The inhibition rate of the composite material with different concentrations on breast cancer cell MDA-MB-231 and normal cell MCF-10A is shown in figure 3. The result shows that the nano-iron/PDA/GOx composite nano-material has an inhibition rate on breast cancer cells MDA-MB-231, and the inhibition rate of 50 mug/mL composite nano-material on tumor cells can reach more than 90%. Under the same conditions, the composite nano material can inhibit normal cells by only about 25%, and shows that the material has the characteristic of inhibiting tumor cells selectively.
Example 4 composite nanomaterial of Nano iron/PDA/GOX to increase active oxygen content in Breast cancer cell MDA-MB-231
The intracellular active oxygen content is detected by using a DCFH-DA (2',7' -dichlorofluorescein diacetate) method, and the specific steps are as follows:
(1) breast cancer cells MDA-MB-231 were cultured, cells were cultured to 70% confluence, and digestion counted at 1X 10 per well5Inoculating the cells in a laser confocal dish at 37 deg.C and 5% CO2Culturing for 24h in an incubator;
(2) abandoning the original culture medium, adding 1mL of complete culture medium in each dish, adding the nano-iron/PDA/GOx composite material until the final concentration is 100 mu g/mL, and adding the same amount of PBS buffer solution in a control group. Incubating for 12h at 37 ℃ in a 5% CO2 incubator;
(3) abandoning the original culture medium, adding 1mL of DCFH-DA (final concentration of 10 mu mol/L) into each group, and incubating for 20min in a cell culture box at 37 ℃;
(4) washing the cells with PBS buffer solution for 3 times to fully remove DCFH-DA which does not enter the cells, then adding 1mL of precooled 4% paraformaldehyde into each dish, and fixing for 20min at 4 ℃;
(5) washing with PBS buffer solution for 3 times, each for 3 min; adding 1mL PBS buffer solution containing 0.2% Triton X-100 into each well, and standing for 10min on ice;
(6) washing with PBS buffer solution for 3 times, each for 3 min; adding 500 μ L DAPI (final concentration 10 μ g/mL) staining solution into each well, and staining for 5min at room temperature; washing with PBS buffer solution for 3 times, each for 3 min;
(7) 50 mu L of the anti-fluorescence quenching blocking tablet is dripped into the center, and the content of active oxygen (the wavelength of excitation light is 488nm, and the wavelength of emission light is 525nm) is observed under a laser confocal microscope.
As shown in FIG. 4, after non-fluorescent DCFH-DA entered the cells, it was hydrolyzed by intracellular esterase to generate DCFH that was impermeable to the cell membrane. Intracellular reactive oxygen species can oxidize non-fluorescent DCFH to produce fluorescent DCF. The control group had no intracellular fluorescence generation and the experimental group had significant intracellular fluorescence.
Example 5 Combined photo-thermal and active oxygen treatment of tumors with Nano-iron/PDA/GOx composite nanomaterial
(1) Weighing 1mg of the nano-iron/PDA/GOx composite nano-material prepared in the embodiment 1, dispersing the nano-iron/PDA/GOx composite nano-material in 1mL of PBS buffer solution, preparing 1mg/mL of composite material stock solution for later use, and diluting according to the subsequent experiment requirements;
(2) inoculating a mouse breast cancer cell 4T1 tumor cell on the back of a BALB/c mouse for 6 weeks, constructing a tumor-bearing mouse model, and carrying out a test after keeping the mouse normally fed for one week;
(3) dividing the tumor-bearing mice in the step (2) into three groups, and respectively carrying out intraperitoneal injection of 100 mu L/10g chloral hydrate (5%) for anesthesia; three groups of tumor-bearing mice were subjected to the following procedures: group A is a treatment group, mice are injected with nano-iron/PDA/glucose oxidase composite nano-material with 100 mu L and 200 mu g/mL in tumor, and are injected with 100 mu L/10g chloral hydrate (5%) in abdominal cavity again after 48 hours for anesthesia and the adopted wavelength is 808nm, and the power is 2W/cm2The laser irradiation treatment is carried out for 5min, and the change of the tumor before and after the treatment is recorded; group B is a control group, mice are injected with 100 mu L of normal saline intratumorally, and are injected with 100 mu L/10g chloral hydrate (5%) intraperitoneally again after 48 hours for anesthesia and the adopted wavelength is 808nm and the power is 2W/cm2The laser irradiation treatment was performed for 5min and the changes in the tumor were recorded. Group C was a semi-treatment group, mice used the same injection and materials as group a mice: injecting 100 mu L of nano-iron/PDA/GOx composite nano-material with 200 mu g/mL into the tumor without subsequent laser irradiation, and recording the change of the tumor before and after treatment.
The results are shown in fig. 5, the tumors in the group a treated by the nano-iron/PDA/GOx composite nano-material synthesized in example 1 become red after 5min of laser irradiation treatment, and the tumors become scab in the subsequent raising process until the tumors are completely cured; the group B control group had no change in tumor during laser irradiation treatment; in the C group semi-treatment group, the injection of the medicine is not treated by laser irradiation, the tumor is also inhibited to a certain extent, and the size is reduced compared with that of a control group. The nano-iron/PDA/GOx composite nano-material disclosed by the invention is proved to achieve a treatment effect through a combined mechanism of photo-thermal inhibition and active oxygen inhibition of tumors, and has high-efficiency tumor treatment capability.
Example 6 preparation of a Nano-iron/PDA/GOx composite nanomaterial
(1) Preparation of nano iron matrix
1g of FeCl was weighed3·6H2Dissolving O in 35mL of ethylene glycol, and magnetically stirring until FeCl3·6H2Completely dissolving the O; adding 3.50g of anhydrous sodium acetate, and stirring for 30min until the sodium acetate is completely dissolved; pouring the solution into a high-pressure reaction kettle lined with polytetrafluoroethylene, setting the temperature at 180 ℃, carrying out oil bath reaction at constant temperature for 7h, taking out the reaction kettle, cooling to room temperature, washing with methanol for 5 times, putting into a vacuum drying oven, and drying overnight to obtain a particle size of 100 nm.
(2) Preparation of nano-iron/PDA/GOx composite material
And (2) coating polydopamine on the nano-iron by adopting a self-assembly method, weighing 50mg of nano-iron powder prepared in the step (1), simultaneously weighing 300mg of dopamine hydrochloride, dispersing the dopamine hydrochloride in 75mL of 15mM Tris-HCl buffer solution with pH of 7.8, uniformly oscillating at room temperature at 100rpm, reacting for 5 hours, carrying out high-speed centrifugal separation at 8000rpm after the reaction is finished, and washing for 2 times by using sodium phosphate buffer solution to obtain the nano-iron/polydopamine composite nano-material.
And adding 50mL of 5mM Tris-HCl buffer solution with the pH value of 7.8 into the washed nano iron/polydopamine composite nano material and 100mg of glucose oxidase together, uniformly oscillating at 100rpm at room temperature, reacting for 8h, performing high-speed centrifugal separation at 8000rpm after the reaction is finished, washing for 2 times by using sodium phosphate buffer solution, and freeze-drying to obtain the nano iron/polydopamine/glucose oxidase (nano iron/PDA/GOx) composite nano material.
Example 7 preparation of a Nano-iron/PDA/GOx composite nanomaterial
(1) Preparation of nano iron matrix
1.5g of FeCl were weighed3·6H2Dissolving O in 40mL of ethylene glycol, and magnetically stirring until FeCl3·6H2Completely dissolving the O;adding anhydrous sodium acetate 4.0g, stirring for 30min until the sodium acetate is completely dissolved; pouring the solution into a high-pressure reaction kettle lined with polytetrafluoroethylene, setting the temperature at 220 ℃, carrying out oil bath reaction at constant temperature for 9h, taking out the reaction kettle, cooling to room temperature, washing for 6 times by using methanol, putting the reaction kettle into a vacuum drying oven, and drying overnight to obtain the particle size of 250 nm.
(2) Preparation of nano-iron/PDA/GOx composite material
And (2) coating poly dopamine by adopting a self-assembly method, weighing 50mg of nano iron powder prepared in the step (1), meanwhile weighing 450mg of dopamine hydrochloride, dispersing the nano iron powder and the dopamine hydrochloride in 75mL of 20mM Tris-HCl buffer solution with the pH value of 8.5, uniformly oscillating at 300rpm at room temperature, reacting for 15h, carrying out high-speed centrifugal separation at 5000rpm after the reaction is finished, and washing for 5 times by using the Tris-HCl buffer solution to obtain the nano iron/poly dopamine composite nanomaterial.
And adding 25mL of 20mM Tris-HCl buffer solution with the pH of 8.5 into the washed nano iron/polydopamine composite nano material and 100mg of glucose oxidase together, uniformly oscillating at 300rpm at room temperature, reacting for 30h, carrying out high-speed centrifugal separation at 5000rpm after the reaction is finished, washing with the Tris-HCl buffer solution for 5 times, and freeze-drying to obtain the nano iron/polydopamine/glucose oxidase (nano iron/PDA/GOx) composite nano material.
Experiments prove that the nano-iron/PDA/GOx composite nano-materials prepared in the examples 6 and 7 have higher photo-thermal conversion performance, can effectively improve the active oxygen content in the breast cancer cells MDA-MB-231, and have no substantial difference in the treatment condition of mouse tumors from the composite nano-materials prepared in the example 1.
Claims (6)
1. A preparation method of a composite nano material for treating tumors by combining photo-thermal and active oxygen is characterized by comprising the following steps:
(1) weighing dopamine hydrochloride and a nano-iron material with the particle size of 70-250nm according to a ratio, wherein the mass ratio of the nano-iron material to the dopamine hydrochloride is 1:0.1-10, adding a Tris-HCl buffer solution with the concentration of 5-20mM, the final concentration of the dopamine hydrochloride is 0.08-4mg/mL, carrying out oscillation reaction at the temperature of 20-30 ℃ for 1-5h, and after the reaction is finished, separating and washing to obtain the nano-iron/poly-dopamine composite nano-material with the surface coated with a poly-dopamine layer;
(2) weighing the nano iron/polydopamine composite nano material prepared in the step (1) and glucose oxidase according to a ratio of 1:0.05-5 by mass, adding a Tris-HCl buffer solution with the concentration of 5-20mM, carrying out oscillation reaction for 8-30h at 20-30 ℃ with the final concentration of the glucose oxidase of 0.1-2mg/mL, and after the reaction is finished, separating, washing and freeze-drying to obtain the nano iron/polydopamine/glucose oxidase composite nano material.
2. The method according to claim 1, wherein the nano-iron material in the step (1) is prepared by the following method: 1-1.5g of FeCl was weighed3•6H2Dissolving O in 35-45mL of ethylene glycol, and magnetically stirring until FeCl3•6H2Completely dissolving the O; adding 3.5-4.0g of anhydrous sodium acetate, and stirring for 30min until the sodium acetate is completely dissolved; pouring the solution into a high-pressure reaction kettle lined with polytetrafluoroethylene, setting the temperature at 180 ℃ and 220 ℃, carrying out constant-temperature oil bath reaction for 7-9h, taking out the reaction kettle, cooling to room temperature, washing for 5-6 times with methanol, and putting the reaction kettle into a vacuum drying oven for overnight drying to obtain the nano iron material.
3. The method according to claim 1, wherein the rotation speed of the oscillatory reaction in step (1) or (2) is 50-300rpm, and the separation in step (1) or (2) is magnetic separation or high-speed centrifugation at 1500-8000 rpm.
4. The method according to claim 1, wherein the buffer solution of step (1) or (2) has a pH of 7.0 to 9.5.
5. The method according to claim 1, wherein the washing in step (1) or (2) is performed 2 to 5 times with Tris-HCl buffer or sodium phosphate buffer.
6. The application of the nano-iron/polydopamine/glucose oxidase composite nano-material obtained by the method of claim 1 in preparing a tumor-targeted drug for combined therapy of photo-thermal and active oxygen.
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