Multifunctional cascade nano-enzyme as well as preparation method and application thereof

Abstract

The invention discloses a multifunctional cascade nano-enzyme and a preparation method and application thereof, and belongs to the technical field of medical nano-materials, the multifunctional cascade nano-enzyme is a polydopamine nano-material doped with gold nanoparticles and heme, and the surface of the polydopamine nano-material is modified with a targeting ligand or a stabilizer. According to the nano material, gold ions, heme and dopamine hydrochloride are self-assembled under an alkaline condition to form nano particles, and a targeting ligand or a stabilizer is further modified through a chemical reaction. The multifunctional cascade nano-enzyme disclosed by the invention can convert glucose in a disease microenvironment into hydrogen peroxide in situ and can also convert the glucose into toxic hydroxyl radicals, so that the redox balance of tumor cells and bacteria is destroyed, apoptosis and death of the tumor cells and the bacteria are induced, toxic and side effects on normal cells and tissues can be effectively avoided, and the treatment effect is good. And the polymer has certain microenvironment regulation and control capability, so that the polymer can be used for preparing photo-thermal therapeutic agents, drug therapeutic agents and photo-thermal drug combined therapeutic agents for tumors or infected wounds.

Description

Technical Field

[0001] The present invention belongs to the technical field of medical nanomaterials, and specifically relates to a multifunctional cascade nanozyme based on a disease microenvironment, a preparation method thereof, and an application thereof in the treatment of tumor and diabetic chronic wound infections. Background Art

[0002] The role of microenvironmental biological functions and their reconstruction in the occurrence and development of diseases such as tumors and chronic wound healing is receiving more and more attention and importance, becoming a key and core direction in the diagnosis and treatment of diseases such as tumors, infected wounds, and diabetic ulcers. Catalytic therapy based on enzymatic reactions is an excellent entry point for disease microenvironment intervention. However, natural enzymes have limitations such as poor stability, variability, poor hydrolysis, inability to tolerate harsh reaction conditions, difficulty in purification, and poor reusability, which limit their application in disease treatment. Nanozymes are a type of nanomaterials that contain enzymatic properties. They can catalyze enzyme substrates, produce catalytic reactions similar to natural enzymes, and have characteristics such as enzymatic reaction kinetics. They belong to a new type of mimetic enzyme. Similar to natural enzymes, nanozymes can efficiently catalyze enzyme substrates under mild physiological conditions because of their adjustable catalytic activity, high stability under harsh conditions, flexibility in composition and structural design, and excellent biocompatibility. The development of nanozymes with disease microenvironment intervention functions is of great significance for the treatment of a series of diseases such as autoimmune damage, chronic functional degeneration, tissue repair, and malignant tumors.

[0003] Although the research on nanozymes based on microenvironment regulation has made great progress, the microenvironment is a complex dynamic system, and the regulation of a single factor is difficult to achieve the desired therapeutic effect. As we all know, after millions of years of evolution, chemical transformations and signal transduction in living organisms are characterized by high efficiency and high specificity, and the cascade reaction of multiple enzymes plays a vital role in the life process. Not only that, the cascade reaction in living organisms usually occurs in a confined space, which can not only improve the selectivity of each enzymatic reaction for the substrate, but also effectively prevent the loss of intermediates and improve the reaction efficiency. Inspired by the advantages of these systems, it is of great significance to integrate enzymes on the framework structure and develop cascade nanozymes based on multi-enzyme synergistic catalysis. At present, the patent discloses some cascade nanozymes based on disease microenvironments for the treatment of tumors and chronic wounds. Patent CN202111462047.6 discloses a HABT-C nanomaterial with triple enzyme-like activity, which can cooperate with sonodynamics and cascade enzyme simulation activity, thereby achieving the purpose of killing tumors and inhibiting their recurrence. Patent CN202110582078.9 discloses an antibacterial fiber loaded with metal organic framework nanozymes and glucose. There is a cascade antibacterial property between glucose and metal organic framework nanozymes. It can play an antibacterial role in situ without exogenous supply and has high antibacterial properties against a variety of bacteria. Patent CN202010838122.3 discloses a dual nanozyme antibacterial agent (AuFe3O4@DMSNs), which kills bacteria by generating hydroxyl radicals ·OH through a cascade reaction between the loaded gold nanozymes and ferroferric oxide nanozymes. However, most of the above patent technologies have relatively complex synthesis processes, poor biocompatibility, and poor stability, which to a certain extent limits their biological applications. Summary of the invention

[0004] In view of the deficiencies in the prior art, the present invention aims to provide a multifunctional cascade nanozyme based on biocompatible polydopamine, a preparation method thereof, and its application in the treatment of tumor and diabetic chronic infection wounds. The multifunctional cascade nanozyme provided by the present invention can produce hydrogen peroxide in situ by consuming endogenous glucose in the infection environment of tumor and diabetic chronic wounds, and can also convert it into toxic hydroxyl radicals, destroy the redox balance of tumor cells and bacteria, and induce their apoptosis and death. In addition, the multifunctional cascade nanozyme also has excellent photothermal performance, which can realize photothermal therapy and enhancement of multi-enzyme activity, and achieve the effect of treating tumor and diabetic chronic infection wounds.

[0005] The technical solution of the present invention is as follows: The present invention provides a multifunctional cascade nanoenzyme material, which is a polydopamine nanomaterial doped with gold nanoparticles and heme and modified with different targeting ligands or stabilizers on its surface. The nanomaterial is self-assembled by gold ions, heme and dopamine hydrochloride under alkaline conditions to form nanoparticles, and the targeting ligand or stabilizer is further modified by chemical reaction to enhance its functionality and targeting.

[0006] The method for preparing a multifunctional cascade nanozyme according to the present invention comprises the following steps: Step 1: Preparation of polydopamine nanomaterials doped with gold nanoparticles and heme: Add dopamine hydrochloride solution into tris-HCl buffer solution, stir vigorously to dissolve, slowly add tetrachloroauric acid (HAuCl4) and hemin, stir at room temperature, collect the product by centrifugation after the reaction is completed, and obtain polydopamine nanomaterials doped with gold nanoparticles and hemin.

[0007] Step 2: Modification of targeting ligand or stabilizer: The polydopamine nanomaterial doped with gold nanoparticles and heme prepared in step 1 is reacted with a targeting ligand or stabilizer according to method 1, method 2 or method 3, and dialyzed and freeze-dried to obtain a multifunctional cascade nanozyme modified with a targeting ligand or stabilizer. The targeting ligand or stabilizer includes a peptide containing a carboxyl group, a thiol group or an amino group, a DNA ligand, a polysaccharide, polyethylene glycol (PEG), and folic acid.

[0008] Method 1: Modification of targeting ligands or stabilizers by amide condensation reaction: A targeting ligand or stabilizer containing a carboxyl group, a condensing agent and a base are added to the polydopamine nanomaterial doped with gold nanoparticles and heme prepared in step 1, and a multifunctional cascade nanozyme modified with the targeting ligand or stabilizer is obtained through an amide condensation reaction.

[0009] Method 2: Modification of targeting ligands or stabilizers by thiol coupling: The targeting ligand or stabilizer containing thiol is dissolved in phosphate buffered saline (PBS) containing tri(2-carboxyethyl)phosphine (TCEP), stirred at room temperature for 1h-3h to reduce the thiol, and then the polydopamine nanomaterial doped with gold nanoparticles and heme prepared in step 1 is added, stirred at room temperature, and reacted for 10h-24h. The multifunctional cascade nanozyme modified with the targeting ligand or stabilizer is obtained by coupling the thiol and polydopamine nanomaterials through Michael addition reaction.

[0010] Method 3: Modification of targeting ligands or stabilizers by amino coupling: The targeting ligand or stabilizer containing an amino group is dissolved in phosphate buffered saline (PBS) or water, and then the tris-hydroxymethylaminomethane hydrochloride (Tris-HCl) buffer solution of the polydopamine nanomaterial doped with gold nanoparticles and heme prepared in step 1 is slowly added and stirred at room temperature for 12 h-24 h. The multifunctional cascade nanozyme modified with the targeting ligand or stabilizer is obtained by coupling the amino group with the polydopamine nanomaterial through a Michael addition reaction.

[0011] The above-mentioned method for preparing a multifunctional cascade nanozyme, wherein: Furthermore, in step 1, the concentration of the dopamine hydrochloride solution is 1 mg / mL-4 mg / mL, the concentration of the Tris-HCl buffer solution is 0.01M-0.05M, and the pH is 8.0-9.5.

[0012] Furthermore, in step 1, the HAuCl4 is added dropwise; the HAuCl4 concentration is 0.005%-0.2% (w / v), and the optimal concentration is 0.1% (w / v); the heme concentration is 1 mg / mL-4 mg / mL, and the optimal concentration is 1 mg / mL.

[0013] Furthermore, in step 1, the stirring condition at room temperature is 10°C-40°C for 6h-48h, and the optimal condition is stirring at room temperature for 24h.

[0014] Furthermore, in step 1, the centrifugal speed condition is 8000rpm-15000rpm, and the optimal condition is 10000rpm; the centrifugal time is 5min-35min, and the optimal condition is 10min.

[0015] Furthermore, in step 2, method 1, the concentration of the polydopamine nanomaterial doped with gold nanoparticles and heme is 0.5 g / mL-4 mg / mL; the mass ratio of the targeting ligand or stabilizer containing a carboxyl group to the polydopamine nanomaterial doped with gold nanoparticles and heme is (0.1-2.0):1.0; the molar ratio of the targeting ligand or stabilizer containing a carboxyl group to the condensing agent and the base is 1:(1.0-2.0):(2.0-6.0).

[0016] Further, in the first method of step 2, the condensing agent / base is 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) / N,N-diisopropylethylamine (DIPEA), 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ), 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) / N-hydroxysuccinimide (NHS), N,N'-diisopropylcarbodiimide (DIC) / 4-dimethylaminopyridine (DMAP), 1,3-dicyclohexylcarbodiimide (DCC) / DMAP or 1-hydroxybenzotriazole (HOBT) / DMAP / N,N-diethylethylamine (TEA).

[0017] Furthermore, in step 2, the molar ratio of the targeting ligand or stabilizer containing a thiol group to tri(2-carboxyethyl)phosphine (TCEP) is (0.8-1.2):1; the concentration of the polydopamine nanomaterial doped with gold nanoparticles and heme is 0.5 g / mL-4 mg / mL; the mass ratio of the targeting ligand or stabilizer containing a thiol group to the polydopamine nanomaterial doped with gold nanoparticles and heme is (0.1-2.0):1.0.

[0018] Furthermore, in step 2, method 3, the concentration of the polydopamine nanomaterial doped with gold nanoparticles and heme is 0.5 g / mL-4 mg / mL; the mass ratio of the amino-containing targeting ligand or stabilizer to the polydopamine nanomaterial doped with gold nanoparticles and heme is (0.1-2.0):1.0.

[0019] Furthermore, in step 2 method 3, the concentration of the tris-hydroxymethylaminomethane hydrochloride (Tris-HCl) buffer solution is 0.01M-0.05M, and the pH is 8.0-9.5.

[0020] The multifunctional cascade nanozyme material of the present invention has significant multi-enzyme catalytic activity, including three enzyme activities of glucose oxidase (GOD), peroxidase (POD), and glutathione peroxidase (GPX) ( Figure 2 ). This nanozyme material can produce hydrogen peroxide (H2O2) in situ by consuming endogenous glucose in the context of tumor and diabetic chronic wound infection, and further convert it into toxic hydroxyl radicals. This process destroys the redox balance of tumor cells and bacteria, inducing their apoptosis and death.

[0021] The multifunctional cascade nanozyme of the present invention has no obvious toxicity to cells when the drug concentration is as high as 1000 μg / mL, indicating that it has good biocompatibility.

[0022] The multifunctional cascade nanozyme described in the present invention has a significant photothermal effect and good photothermal stability. Under the irradiation of 808nm near-infrared laser, it can convert light energy into thermal energy. Therefore, it can be used for photothermal therapy of tumors or infected wounds, and photothermal drug combined therapy.

[0023] The multifunctional cascade nanozyme described in the present invention also has peroxidase-like activity and can catalyze the oxidation of glucose into gluconic acid, and thus can be used to prepare a substance for detecting glucose content.

[0024] The multifunctional cascade nanozyme described in the present invention can convert glucose in the disease microenvironment into hydrogen peroxide in situ, and can also convert it into toxic hydroxyl free radicals, thereby destroying the redox balance of tumor cells and bacteria and inducing their apoptosis and death.

[0025] The multifunctional cascade nanozyme described in the present invention can inhibit the proliferation of tumor cells and has a significant inhibitory effect on Staphylococcus aureus and Escherichia coli. The effect is further enhanced after 808nm near-infrared irradiation.

[0026] Compared with the prior art, the present invention has the following significant advantages: (1) Utilizing the self-oxidation property of dopamine under alkaline conditions and its chelation and reduction properties to metals, a nanomaterial with multi-enzyme activity and photothermal properties was constructed by a one-step synthesis method.

[0027] (2) Compared with traditional single enzyme mimics, the multifunctional cascade nanozyme prepared in the present invention has multi-enzyme cascade activities such as glucose oxidase, glutathione peroxidase, and peroxidase.

[0028] (3) The multifunctional cascade nanozyme preparation process disclosed in the present invention is green, simple, low-cost, and easy to prepare in large quantities.

[0029] (4) The multifunctional cascade nanozyme disclosed in the present invention is easy to be modified with a variety of targeting ligands or stabilizers.

[0030] (5) The multifunctional cascade nanozyme disclosed in the present invention can convert glucose in the disease microenvironment into hydrogen peroxide in situ, and can also convert it into toxic hydroxyl radicals, thereby destroying the redox balance of tumor cells and bacteria, inducing their apoptosis and death, and can effectively avoid toxic side effects on normal cells and tissues, and has a certain microenvironment regulation ability. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Schematic diagram of the synthesis of the multifunctional cascade nanozyme of the present invention.

[0032] Figure 2 It is a schematic diagram of the multi-enzyme catalytic activity of the multifunctional cascade nanozyme of the present invention.

[0033] Figure 3 The transmission electron microscope image and particle size distribution diagram of the polydopamine nanomaterial doped with gold nanoparticles and heme prepared in Example 1 of the present invention; wherein a is the transmission electron microscope image; and b is the particle size distribution diagram.

[0034] Figure 4 The photothermal performance analysis of the polydopamine nanomaterial doped with gold nanoparticles and heme prepared in Example 1 of the present invention; a is the temperature rise of 50 μg / mL nanomaterial under different infrared power conditions; b is the temperature rise of nanomaterials with different concentrations under 0.75 W / cm 2 Temperature rise under certain conditions; c is the thermal stability of the nanomaterial.

[0035] Figure 5 It is the multi-enzyme activity of the polydopamine nanomaterial doped with gold nanoparticles and heme prepared in Example 1 of the present invention; wherein a is the peroxidase activity of the nanomaterial; b is the GSH consumption in the solution after treatment with nanomaterials of different concentrations; c is the glucose oxidase activity of the nanomaterial; and d is the cascade catalytic activity of the nanomaterial.

[0036] Figure 6 is the cytotoxicity of the multifunctional cascade nanozyme of the present invention; wherein a is the effect of the multifunctional cascade nanozyme on the proliferation activity of mouse lung fibroblasts (L929); b is the effect of the multifunctional cascade nanozyme on the proliferation activity of mouse breast cancer cells (4T1).

[0037] Figure 7 This is the in vivo anti-tumor effect of the multifunctional cascade nanozyme of the present invention.

[0038] Figure 8 It is the antibacterial effect of the multifunctional cascade nanozyme of the present invention; wherein a is the antibacterial effect of the multifunctional cascade nanozyme on Staphylococcus aureus; b is the antibacterial effect of the multifunctional cascade nanozyme on Escherichia coli.

[0039] Fig. 9 The multifunctional cascade nanozyme of the present invention accelerates the healing of diabetic wounds; wherein a is the use of PBS, PBS+NIR (0.75W / cm 2 ), APH, APH+NIR (0.75W / cm 2 ), APHC, APHC+NIR (0.75W / cm 2 ) The healing process of diabetic wounds after treatment of S. aureus infection; b is the quantification of wound area. DETAILED DESCRIPTION

[0040] The present invention is further described in detail below with specific embodiments, but it should not be understood that the scope of the above subject matter of the present invention is limited to the following embodiments, and all technologies realized based on the content of the present invention belong to the scope of the present invention.

[0041] Unless otherwise specified, all reagents and materials used in the present invention are commercially available.

[0042] Example 1 A method for preparing a multifunctional cascade nanozyme, such as Figure 1 As shown, gold ions, heme and dopamine hydrochloride are self-assembled under alkaline conditions to form nano-ions, and then modified with targeting ligands or stabilizers by amide condensation, thiol coupling, amino coupling, etc. The specific operation steps are as follows: Step 1: Preparation of polydopamine nanomaterials doped with gold nanoparticles and heme: 3.6 mL of dopamine hydrochloride solution (4 mg / mL) was added to 72 mL of tris-HCl buffer solution (0.01 M, pH = 8.5) and stirred vigorously. Then 5.4 mL of tetrachloroauric acid (HAuCl4) ultrapure water (0.1%, w / v) and 1 mL of heme-containing N,N-dimethylformamide (DMF) (1 mg / mL) were added dropwise and stirred at room temperature for 20 h. After the reaction was completed, the mixture was centrifuged at 10,000 rpm for 10 min, the supernatant was discarded, the mixture was washed with ultrapure water and centrifuged twice, and the polydopamine nanomaterial doped with gold nanoparticles and heme was obtained by vacuum drying.

[0043] Characterization and performance: The polydopamine nanomaterials doped with gold nanoparticles and heme obtained in this example were characterized by transmission electron microscopy (Hitachi TEM, Japan). Figure 3 As shown, the nanomaterials present a daisy-shaped ( Figure 3 a), the particle size is normally distributed and the dispersion is good ( Figure 3 b), the particle size is 150.1±16.3nm.

[0044] The polydopamine nanomaterial doped with gold nanoparticles and heme prepared in this embodiment was subjected to photothermal testing. The results are as follows: Figure 4 It has a high efficiency of photothermal conversion, which increases with the material concentration (10, 25, 50, 75, 100µg / mL) and NIR808 power (0.25, 0.50, 0.75W / cm 2 ) increases, the system temperature further increases ( Figure 4 ab, and has good photothermal stability, and does not decay within 5 photothermal cycles ( Figure 4 c).

[0045] The polydopamine nanomaterial doped with gold nanoparticles and heme prepared in this example has multi-enzyme activity. Figure 5Its peroxidase-like activity was determined by the colorimetric reaction of 3,3',5,5'-tetramethylbenzidine (TMB) in the presence of hydrogen peroxide. 100 μL of polydopamine nanomaterials doped with gold nanoparticles and heme (30 μg / mL), 100 μL of TMB (1.2 mM), and 50 μL of H2O2 of different concentrations (6.25, 12.5, 25.0, 50.0, and 100 mM) were sequentially added to 2 mL of NaAc buffer (pH = 5.4, 0.01 M). The absorbance at 652 nm was measured using the time scan mode of an ultraviolet spectrophotometer. The results are shown in FIG. Figure 5 As shown in a, the nanomaterial can effectively catalyze the oxidation of hydrogen peroxide (H2O2) and has a high peroxidase-like activity. The reactive oxygen generated during the reaction is a hydroxyl radical. Using 5,5'-dithiodinitrobenzoic acid (DTNB) as a probe, the consumption capacity of the nanomaterial on GSH was detected. Nanozymes of different concentrations (0, 50, 100, 200, 500 μg / mL) were mixed with GSH (2 mM) solution, incubated at 37 °C for 3 h, and then 10 μL DTNB (5 mM) was added. The mixture was mixed and allowed to react for 3 min. The absorbance change was detected at 412 nm using a UV spectrophotometer. The results are shown in Figure 1. Figure 5 As shown in b, the GSH level gradually decreased with the increase of nanomaterial concentration, which indicates that the nanomaterial can effectively consume GSH through redox reaction, thereby increasing the concentration of ROS in the tumor microenvironment. In addition, the nanomaterial has a significant glucose consumption capacity. The consumption of glucose was determined using the 3,5-dinitrosalicylic acid (DNS) method. Nanozyme solutions of different concentrations (0, 100, 200 μg / mL) were mixed with PBS containing glucose (1 mg / mL). At specific time intervals, 0.5 mL of the above solution was collected and mixed with 1.5 mL of DNS reagent, heated at 100 ° C for 5 minutes, and then quickly transferred to ice water for cooling for 20 minutes. The absorbance at 450 nm was detected using a UV spectrophotometer. The results are shown in Figure 5 As shown in c, the concentration of glucose decreases exponentially, and the increase in the concentration of nanomaterials accelerates the consumption of glucose. The changes in TMB in glucose solutions of different concentrations were quantitatively detected by the TMB method. 100 μg / mL nanomaterial solution was mixed with glucose solutions of different concentrations (0, 0.25, 0.5, 1.0, 2.0 mg / mL), incubated at 37°C for 6 h, and then TMB (2 mM) was added. The absorbance at 652 nm was measured using the time scan mode of the UV spectrophotometer. The results are shown in Figure 5 As shown in FIG. d, the characteristic absorbance of oxTMB oxidized by the increase of glucose concentration and the extension of reaction time can continuously increase, which proves that the nanomaterial prepared by the present invention has significant cascade catalytic activity.

[0046] Step 2: Modification of targeting ligand or stabilizer: (1) Targeted modification of thiol-containing DNA aptamers: 0.2 mg of thiol DNA aptamer Aptamer1 (Apt1) (Wuhan Jinkairui Bioengineering Co., Ltd.) was stirred at room temperature for 1 h in 1 mL of phosphate buffer (PBS) containing 0.1 mM TCEP. Then, phosphate buffer (PBS) (1 mL, 1 mg / mL) of polydopamine nanomaterials doped with gold nanoparticles and heme prepared in step 1 was added, stirred at room temperature, and reacted for 16 h. Then 0.2 mg of Aptamer2 (Apt2) (Wuhan Jinkairui Bioengineering Co., Ltd.) was dissolved in 1 mL of PBS and added to the mixture, and reacted at 60 ° C for 2 h. After cooling to room temperature, stirring was continued for 6 h. The solution was dialyzed in a dialysis bag (Mw 14000) for 48 h and freeze-dried to obtain a multifunctional cascade nanozyme targeted modified by Aptamer DNA aptamer. The particle size distribution diagram is shown in Figure 3 b. The DNA sequence of Apt1 modified with 5′-SH thiol (5′→3′) is shown in SEQ ID NO: 1, and the DNA sequence of Apt2 modified with 5′-FAM (5′-Carboxyfluorescein) green fluorescent probe (5′→3′) is shown in SEQ ID NO: 2.

[0047] (2) Targeted modification of carboxyl-containing folic acid (C-FA): 0.55 μM 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and 0.55 μM N-hydroxysuccinimide (NHS) were added to 0.5 mM folic acid dimethyl sulfoxide (DMSO) solution (1 mL), mixed and stirred, and then slowly added to the aqueous solution of polydopamine nanomaterials doped with gold nanoparticles and heme (1 mL, 1 mg / mL) prepared in step 1, stirred at room temperature, and reacted for 24 hours. The solution was dialyzed in a dialysis bag (Mw 14000) for 48 hours and freeze-dried to obtain a multifunctional cascade nanozyme targeted with folic acid.

[0048] (3) Polyethylene glycol (PEG) modification containing amino groups: Dissolve 1 mg of monoamino polyethylene glycol (PEG) (Mw=5000) in water, slowly add the polydopamine nanomaterial doped with gold nanoparticles and heme (1 mL, 1 mg / mL) prepared in step 1 and tris-HCl buffer solution (0.01 M, pH=8.5), stir at room temperature for 24 h, dialyze the solution in a dialysis bag (Mw 10000) for 48 h, and freeze-dry to obtain the polyethylene glycol (PEG)-modified multifunctional cascade nanozyme.

[0049] Application Example 1 Cytotoxicity test of Aptamer DNA aptamer-targeted multifunctional cascade nanozyme (APHA): The effects of multifunctional cascade nanozymes modified by Aptamer DNA aptamers on the proliferation activity of mouse lung fibroblasts (L929) and mouse breast cancer cells (4T1) were evaluated using the classic methylthiazolyl tetrazolium (MTT) method. After co-incubation with different concentrations of APHA supramolecular nanozyme assemblies for 24 hours, there was no obvious cytotoxicity to normal mouse L929 cells, and even at high concentrations (1000μg / mL), the cell viability remained above 95% ( Figure 6 a), confirming that the nanomaterial APHA has good biocompatibility. In 4T1 tumor cells, the cell viability decreased significantly with the increase of APHA concentration, in a dose-dependent manner, and the inhibition rate of 50μg / mL exceeded 75% ( Figure 6 b). It shows that nanozymes can produce toxic reactive oxygen species through their various enzyme activities, inducing apoptosis and death of tumor cells. The introduction of near-infrared light can further improve enzyme activity and enhance the ability to kill tumor cells during photothermal therapy.

[0050] In vivo anti-tumor effect test of multifunctional cascade nanozymes modified with Aptamer DNA aptamers: 4T1 cells in logarithmic growth phase were digested with trypsin, centrifuged and resuspended in phosphate buffered saline (PBS). After counting the cell suspension, 2×10 6 4T1 cells were subcutaneously inoculated into the right lower limb of female Balb / c mice. The tumor formation and tumor volume were observed every day after inoculation. When the tumor grew to 100 mm 3 At about 14 h, 4T1 tumor-bearing mice were randomly divided into 6 groups (n=5): PBS group, PBS+NIR group (PBS+L group), polydopamine nanomaterials doped with gold nanoparticles and heme group (APH group), Aptamer DNA aptamer cascade nanozyme group (APHA group), polydopamine nanomaterials doped with gold nanoparticles and heme+NIR group (APH+L group), Aptamer DNA aptamer cascade nanozyme+NIR group (APHA+L group). 9 h after intraperitoneal administration, the corresponding NIR laser irradiation groups were given 808 nm laser (0.75 W / cm 2 ) for 5 min. During the treatment period, the tumor volume and body weight were recorded every other day. After the 14-day treatment period, the mice were killed by cervical dislocation. Figure 7 As shown, compared with the control group, the Aptamer DNA aptamer cascade nanozyme group had a significant inhibitory effect on tumors, and the inhibitory effect was further enhanced after 808nm near-infrared irradiation.

[0051] Application Example 2 Antibacterial effect test of PEG-modified multifunctional cascade nanozymes: The plate count method was used to study the synergistic antibacterial effect of polyethylene glycol (PEG)-modified multifunctional cascade nanozymes on Gram-negative bacteria Escherichia coli and Gram-positive bacteria Staphylococcus aureus. Escherichia coli and Staphylococcus aureus were selected and cultured in a fresh LB broth liquid medium to the logarithmic growth phase and diluted to 10 7 The bacteria were added to the culture medium containing glucose, and different concentrations of nanozymes were added with or without near-infrared irradiation. The near-infrared irradiation group was treated with 808nm laser (0.75W / cm 2 ) for 5 min. The phosphate buffered saline (PBS) treatment group served as the control group. The treated bacterial suspension (60 μL) was spread on solid agar medium and incubated at 37°C for 24 h before taking photos and counting colonies. Figure 8 With the increase of the concentration of PEG-modified multifunctional cascade nanozymes, the antibacterial effect was enhanced, and the bacterial killing effect was further enhanced after 808nm near-infrared irradiation. Under the conditions of 1.5mg / mL nanozyme treatment and 808nm near-infrared irradiation, the antibacterial effect on Escherichia coli and Staphylococcus aureus reached 98.2% and 94.2%, respectively.

[0052] Application Example 3 Test on the effect of folic acid (C-FA) targeted modified multifunctional cascade nanozyme on accelerating diabetic wound healing: The diabetic mouse model was established. Five-week-old C57BL / 6 mice (17-20g body weight) were fasted for 12 hours for 3 consecutive days and then intraperitoneally injected with streptozotocin (STZ, 50mg / kg) to establish a type 1 diabetes model. Mice with blood glucose concentrations higher than 16.7mM were observed for 7 days after injection and were considered diabetic mice. The mice were randomly divided into 6 groups, 3 mice in each group, and were named Control group (PBS group), Control+NIR group (Control+L group), polydopamine nanomaterials doped with gold nanoparticles and heme group (APH group), polydopamine nanomaterials doped with gold nanoparticles and heme+NIR group (APH+L group), multifunctional cascade nanozyme modified with folate (APHC group), and multifunctional cascade nanozyme modified with folate + NIR group (APHC+L group). 0.3% sodium pentobarbital (0.2mL / 10g) was injected intraperitoneally for anesthesia, and the back hair was shaved with electric clippers. The shaved area was disinfected with 75% ethanol. Then, a full-thickness wound of equal size was made on the shaved back using a sterile dermal biopsy punch with a diameter of 6 mm. 10 µL of the prepared Staphylococcus aureus suspension (1 × 10 6CFU / mL) was inoculated on the wound surface for 24 hours to form wound infection. From the second day, different nanozymes were applied to the wound surface and photothermal therapy (0.75W / cm 2 , 5min) to treat infected wounds. Optical images of the wound surface were collected on days 0, 3, 7, and 14, and the wound size was recorded. On days 7 and 14, the wound surface and surrounding tissues were collected for histological hematoxylin and eosin (H&E) staining evaluation. The experimental results are shown in Fig. 9 As shown, on day 7, the wound area of ​​the APHC+NIR group was significantly reduced to 13.17%, and the wound healing effect was the best. On day 14, the wounds of the APHC+NIR group were almost healed, with the wound area reduced to 1.52%, while the wounds of the Control group were still not healed, with a wound area of ​​29.17%.

Claims

1. A method for preparing a multifunctional cascade nanozyme, characterized in that: The following steps are involved: Step 1: Preparation of polydopamine nanomaterials doped with gold nanoparticles and heme: Add the dopamine hydrochloride solution to the Tris-HCl buffer solution, stir vigorously to dissolve, slowly add tetrachloroauric acid and heme, stir at room temperature, and collect the product by centrifugation after the reaction is completed to obtain a polydopamine nanomaterial doped with gold nanoparticles and heme; Step 2: Modification of targeting ligand or stabilizer: The polydopamine nanomaterial doped with gold nanoparticles and heme prepared in step 1 is reacted with a targeting ligand or a stabilizer according to method 1, method 2 or method 3, and dialyzed and freeze-dried to obtain a multifunctional cascade nanozyme modified with a targeting ligand or a stabilizer; Method 1: Modification of targeting ligands or stabilizers by amide condensation reaction: Adding a targeting ligand or stabilizer containing a carboxyl group, a condensing agent and a base to the polydopamine nanomaterial doped with gold nanoparticles and heme prepared in step 1, and obtaining a multifunctional cascade nanozyme modified with the targeting ligand or stabilizer through an amide condensation reaction; Method 2: Modification of targeting ligands or stabilizers by thiol coupling: Dissolving a thiol-containing targeting ligand or stabilizer in a phosphate buffer containing tris(2-carboxyethyl)phosphine, reducing the thiol group by stirring at room temperature, then adding the polydopamine nanomaterial doped with gold nanoparticles and heme prepared in step 1, stirring at room temperature, reacting, and coupling the thiol group with the polydopamine nanomaterial through a Michael addition reaction to obtain a multifunctional cascade nanozyme modified with the targeting ligand or stabilizer; Method 3: Modification of targeting ligands or stabilizers by amino coupling: The targeting ligand or stabilizer containing an amino group is dissolved in a phosphate buffer or water, and then the Tris-HCl buffer solution of the polydopamine nanomaterial doped with gold nanoparticles and heme prepared in step 1 is slowly added, stirred at room temperature, and the targeting ligand or stabilizer-modified multifunctional cascade nanozyme is obtained by coupling the amino group with the polydopamine nanomaterial through a Michael addition reaction.

2. The method for preparing a multifunctional cascade nanozyme according to claim 1, characterized in that: In step 1, the concentration of dopamine hydrochloride solution is 1 mg / mL-4 mg / mL, the concentration of Tris-HCl buffer solution is 0.01M-0.05M, and the pH is 8.0-9.

5.

3. The method for preparing a multifunctional cascade nanozyme according to claim 1, characterized in that: In step 1, tetrachloroauric acid is added dropwise; the concentration of tetrachloroauric acid is 0.005%-0.2%, and the concentration of heme is 1 mg / mL-4 mg / mL; the stirring condition at room temperature is 10°C-40°C for 6h-48h; the centrifugal speed condition is 8000rpm-15000rpm, and the centrifugal time is 5min-35min.

4. The method for preparing a multifunctional cascade nanozyme according to claim 1, characterized in that: In step 2, method 1, the concentration of the polydopamine nanomaterial doped with gold nanoparticles and heme is 0.5 g / mL-4 mg / mL; the mass ratio of the targeting ligand or stabilizer containing a carboxyl group to the polydopamine nanomaterial doped with gold nanoparticles and heme is (0.1-2.0):1.0; the molar ratio of the targeting ligand or stabilizer containing a carboxyl group to the condensing agent and the base is 1:(1.0-2.0):(2.0-6.0).

5. The method for preparing a multifunctional cascade nanozyme according to claim 1, characterized in that: In step 2, the molar ratio of the targeting ligand or stabilizer containing a thiol group to tri(2-carboxyethyl)phosphine is (0.8-1.2):1; the concentration of the polydopamine nanomaterial doped with gold nanoparticles and heme is 0.5 g / mL-4 mg / mL; the mass ratio of the targeting ligand or stabilizer containing a thiol group to the polydopamine nanomaterial doped with gold nanoparticles and heme is (0.1-2.0):1.

0.

6. The method for preparing a multifunctional cascade nanozyme according to claim 1, characterized in that: In step 2, method three, the concentration of the polydopamine nanomaterial doped with gold nanoparticles and heme is 0.5 g / mL-4 mg / mL; the mass ratio of the amino-containing targeting ligand or stabilizer to the polydopamine nanomaterial doped with gold nanoparticles and heme is (0.1-2.0):1.0; the concentration of the Tris-HCl buffer solution is 0.01 M-0.05 M, pH 8.0-9.

5.

7. A multifunctional cascade nanozyme, characterized in that: A polydopamine nanomaterial doped with gold nanoparticles and heme, prepared by the preparation method according to any one of claims 1 to 6, and having a targeting ligand or a stabilizer modified on its surface.

8. Use of the multifunctional cascade nanozyme according to claim 7 in the preparation of photothermal therapeutic agents, drug therapeutic agents, and photothermal drug combined therapeutic agents for tumors or infected wounds.

9. Use of the multifunctional cascade nanozyme according to claim 7 in preparing a substance for detecting glucose content.

10. Use of the multifunctional cascade nanozyme according to claim 7 in the preparation of a drug for inhibiting tumor cell proliferation.

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