A multifunctional cascade nanozyme and its preparation method and application

Abstract

The present invention discloses a multifunctional cascade nanozyme and its preparation method and application, belonging to the technical field of medical nanomaterials. It is a polydopamine nanomaterial doped with gold nanoparticles and heme, and a targeting ligand or stabilizer is modified on its surface. The nanomaterial is formed by self-assembly of gold ions, heme and dopamine hydrochloride under alkaline conditions, and the targeting ligand or stabilizer is further modified through a chemical reaction. The multifunctional cascade nanozyme disclosed by the present invention can in-situ convert glucose in the disease microenvironment into hydrogen peroxide, and at the same time can also convert it into toxic hydroxyl radicals, destroy the redox balance of tumor cells and bacteria, and induce their apoptosis and death. It can effectively avoid the toxic and side effects on normal cells and tissues, and has a certain microenvironment regulation ability. Therefore, it can be used to prepare photothermal therapeutic agents, drug therapeutic agents, and combined photothermal drug therapeutic agents for tumors or infected wounds.

Description

Technical Field

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

[0002] The roles of microenvironmental biological functions and their remodeling in the occurrence and development of diseases such as tumors and chronic wound healing are receiving increasing attention and have become a key and core direction in the diagnosis and treatment research 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, poor variability and hydrolysis resistance, inability to tolerate harsh reaction conditions, difficulty in purification, and poor reusability, which limit their application in disease treatment. Nanozymes are a class of nanomaterials that inherently possess enzymatic properties, can catalyze enzyme substrates, produce catalytic reactions similar to those of natural enzymes, and have characteristics such as enzymatic reaction kinetics, belonging to a new type of mimetic enzyme. Similar to natural enzymes, nanozymes can efficiently catalyze enzyme substrates under mild physiological conditions due to their adjustable catalytic activity, high stability under harsh conditions, flexibility in composition and structure design, and excellent biocompatibility. Developing nanozymes with disease microenvironment intervention functions is of great significance for the treatment of a series of diseases such as autoimmune damage, chronic functional degradation, tissue repair, and malignant tumors.

[0003] Although great progress has been made in the research of nanozymes based on microenvironment regulation, the microenvironment is a complex dynamic system, and it is difficult to achieve ideal therapeutic effects by regulating a single factor. As is well known, through millions of years of evolution, chemical transformation and signal transduction in vivo are characterized by high efficiency and high specificity. The cascade reaction of multiple enzymes plays a crucial role in life processes. Moreover, the cascade reaction in vivo usually occurs in a confined space, which can not only improve the selectivity of each enzymatic reaction for substrates, but also effectively prevent the loss of intermediate products 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 cooperative catalysis. At present, some cascade nanozymes based on the disease microenvironment have been disclosed in patents for the treatment of tumors and chronic wounds. Patent CN202111462047.6 discloses a HABT-C nanomaterial, which has triple pseudo-enzyme activities and can synergistically actuate sonodynamic and cascade enzyme mimicking activities to achieve 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, which can act antibacterial in situ without exogenous supply and has high antibacterial performance against a variety of bacteria. Patent CN202010838122.3 discloses a dual nanozyme antibacterial agent (AuFe3O4@DMSNs), which generates hydroxyl radicals ·OH through the cascade reaction between the loaded gold nanoparticles and two kinds of nanozymes of iron tetroxide for sterilization. However, most of the above patent technologies have relatively complex synthesis processes, poor biocompatibility and stability, which to a certain extent limit their biological applications. Summary of the Invention

[0004] Aiming at the deficiencies of the existing technology, the present invention aims to provide a multifunctional cascade nanozyme based on biocompatible polydopamine, its preparation method, and its application in the treatment of tumors and diabetic chronic infected wounds. The multifunctional cascade nanozyme provided by the present invention can in situ generate hydrogen peroxide by consuming endogenous glucose in the tumor and diabetic chronic wound infection environment, and at the same time, it can also convert it into toxic hydroxyl radicals to disrupt the redox balance of tumor cells and bacteria, inducing their apoptosis and death. In addition, the multifunctional cascade nanozyme also has excellent photothermal performance, which can realize photothermal therapy and enhance multi-enzyme activities, achieving the treatment effects of tumors and diabetic chronic infected wounds.

[0005] The technical solution of the present invention is as follows:

[0006] A multifunctional cascaded nanozyme material provided by the present invention is a polydopamine nano material doped with gold nanoparticles and hemin, and different targeting ligands or stabilizers are modified on its surface. The nano material is formed by self-assembly of gold ions, hemin and dopamine hydrochloride under alkaline conditions to form nanoparticles, and further chemically reacts to modify targeting ligands or stabilizers to enhance its functionality and targeting ability.

[0007] A preparation method of a multifunctional cascaded nanozyme described in the present invention includes the following steps:

[0008] Step 1: Preparation of a polydopamine nano material doped with gold nanoparticles and hemin:

[0009] Add the dopamine hydrochloride solution to the tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl) buffer solution, stir vigorously to dissolve, slowly add chloroauric acid (HAuCl4) and hemin, stir at room temperature, and after the reaction is completed, centrifuge to collect the product to obtain a polydopamine nano material doped with gold nanoparticles and hemin.

[0010] Step 2: Modify the targeting ligand or stabilizer:

[0011] React the polydopamine nano material doped with gold nanoparticles and hemin prepared in Step 1 with the targeting ligand or stabilizer according to Method 1, Method 2 or Method 3, and dialyze and freeze-dry to obtain a multifunctional cascaded nanozyme modified with the targeting ligand or stabilizer. The targeting ligand or stabilizer includes peptides containing carboxyl groups or mercapto groups or amino groups, DNA ligands, polysaccharides, polyethylene glycol (PEG), and folic acid.

[0012] Method 1: Modify the targeting ligand or stabilizer through an amide condensation reaction:

[0013] Add the targeting ligand or stabilizer containing a carboxyl group, a condensing agent and a base to the polydopamine nano material doped with gold nanoparticles and hemin prepared in Step 1, and obtain a multifunctional cascaded nanozyme modified with the targeting ligand or stabilizer through an amide condensation reaction.

[0014] Method 2: Modify the targeting ligand or stabilizer through a thiol coupling method:

[0015] Dissolve the targeting ligand or stabilizer containing a mercapto group in a phosphate buffer solution (PBS) containing tris(2-carboxyethyl)phosphine (TCEP), stir at room temperature for 1 h - 3 h to reduce the mercapto group, and then add the polydopamine nano material doped with gold nanoparticles and hemin prepared in Step 1, stir at room temperature, react for 10 h - 24 h, and couple through a Michael addition reaction between the mercapto group and the polydopamine nano material to obtain a multifunctional cascaded nanozyme modified with the targeting ligand or stabilizer.

[0016] Method 3: Modify the targeting ligand or stabilizer through an amino coupling method:

[0017] Dissolve the amino-containing targeting ligand or stabilizer in phosphate buffer (PBS) or water, and then slowly add it to the tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl) buffer solution of the polydopamine nanomaterial doped with gold nanoparticles and heme prepared in step 1. Stir at room temperature for 12 h - 24 h, and couple through the Michael addition reaction between the amino group and the polydopamine nanomaterial to obtain the multifunctional cascade nanozyme modified with the targeting ligand or stabilizer.

[0018] The preparation method of the above-mentioned multifunctional cascade nanozyme, wherein:

[0019] Further, 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 0.01 M - 0.05 M, and the pH is 8.0 - 9.5.

[0020] Further, in step 1, the addition method of HAuCl4 is dropwise addition; the concentration of HAuCl4 is 0.005% - 0.2% (w / v), and the optimal is 0.1% (w / v); the concentration of heme is 1 mg / mL - 4 mg / mL, and the optimal is 1 mg / mL.

[0021] Further, in step 1, the room temperature stirring condition is stirring at 10°C - 40°C for 6 h - 48 h, and the optimal is stirring at room temperature for 24 h.

[0022] Further, in step 1, the centrifugation speed condition is 8000 rpm - 15000 rpm, and the optimal is 10000 rpm; the centrifugation time is 5 min - 35 min, and the optimal is 10 min.

[0023] Further, in method 1 of step 2, 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 carboxyl-containing targeting ligand or stabilizer to the polydopamine nanomaterial doped with gold nanoparticles and heme is (0.1 - 2.0):1.0; the molar ratio of the carboxyl-containing targeting ligand or stabilizer to the condensing agent and the base is 1:(1.0 - 2.0):(2.0 - 6.0).

[0024] Further, in Method 1 of Step 2, the condensing agent / base is 2-(7-azabenzotriazol-1-yl)-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).

[0025] Further, in Method 2 of Step 2, the molar ratio of the thiol-containing targeting ligand or stabilizer to tris(2-carboxyethyl)phosphine (TCEP) is (0.8-1.2):1; the concentration of the doped gold nanoparticles and heme-containing polydopamine nanomaterials is 0.5 g / mL - 4 mg / mL; the mass ratio of the thiol-containing targeting ligand or stabilizer to the doped gold nanoparticles and heme-containing polydopamine nanomaterials is (0.1-2.0):1.0.

[0026] Further, in Method 3 of Step 2, the concentration of the doped gold nanoparticles and heme-containing polydopamine nanomaterials is 0.5 g / mL - 4 mg / mL; the mass ratio of the amino-containing targeting ligand or stabilizer to the doped gold nanoparticles and heme-containing polydopamine nanomaterials is (0.1-2.0):1.0.

[0027] Further, in Method 3 of Step 2, the concentration of the tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl) buffer solution is 0.01 M - 0.05 M, and the pH is 8.0 - 9.5.

[0028] The multifunctional cascade nanozyme material of the present invention has significant multi-enzyme catalytic activities, including three types of enzyme activities: glucose oxidase (GOD), peroxidase (POD), and glutathione peroxidase (GPX) ( Figure 2 ). This nanozyme material can in situ generate hydrogen peroxide (H2O2) by consuming endogenous glucose in the tumor and diabetic chronic wound infection environments, and further convert it into toxic hydroxyl radicals. This process disrupts the redox balance of tumor cells and bacteria, inducing their apoptosis and death.

[0029] The multifunctional cascade nanozyme of the present invention has no obvious toxicity to cells even when the drug concentration is as high as 1000 μg / mL, indicating its good biocompatibility.

[0030] The multifunctional cascade nanozyme described in the present invention has significant photothermal effects and good photothermal stability. Under the irradiation of 808 nm near-infrared laser, it can convert light energy into heat energy, and thus can be used for photothermal therapy and combined photothermal-drug therapy of tumors or infected wounds.

[0031] The multifunctional cascade nanozyme described in the present invention also has peroxidase-like activity and can catalyze the oxidation of glucose to gluconic acid. Therefore, it can be used for the preparation of substances for detecting glucose content.

[0032] The multifunctional cascade nanozyme described in the present invention can in-situ convert glucose in the disease microenvironment into hydrogen peroxide, and at the same time can also convert it into toxic hydroxyl radicals, disrupting the redox balance of tumor cells and bacteria and inducing their apoptosis and death.

[0033] The multifunctional cascade nanozyme described in the present invention can inhibit the proliferation of tumor cells and has obvious inhibitory effects on Staphylococcus aureus and Escherichia coli. The effect is further enhanced after 808 nm near-infrared irradiation.

[0034] Compared with the prior art, the present invention has the following remarkable advantages:

[0035] (1) Utilize the self-oxidation property of dopamine under alkaline conditions, as well as its chelation and reduction properties towards metals, to construct a nanomaterial with multi-enzyme activities and photothermal properties by a one-step synthesis method.

[0036] (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.

[0037] (3) The preparation process of the multifunctional cascade nanozyme disclosed in the present invention is green, simple, low-cost, and easy to prepare in large quantities.

[0038] (4) The multifunctional cascade nanozyme disclosed in the present invention is easy to be modified with various targeting ligands or stabilizers.

[0039] (5) The multifunctional cascade nanozyme disclosed in the present invention can in-situ convert glucose in the disease microenvironment into hydrogen peroxide, and at the same time can also convert it into toxic hydroxyl radicals, disrupting the redox balance of tumor cells and bacteria and inducing their apoptosis and death, which can effectively avoid the toxic and side effects on normal cells and tissues and has a certain ability to regulate the microenvironment. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 It is a schematic diagram of the synthesis of the multifunctional cascade nanozyme of the present invention.

[0041] Figure 2 It is a schematic diagram of the multi-enzyme catalytic activities of the multifunctional cascade nanozyme of the present invention.

[0042] Figure 3 Transmission electron microscopy image and particle size distribution diagram of the poly dopamine nanomaterial doped with gold nanoparticles and hemin prepared in Example 1 of the present invention; wherein a is the transmission electron microscopy image; b is the particle size distribution diagram.

[0043] Figure 4 Photothermal performance analysis of the poly dopamine nanomaterial doped with gold nanoparticles and hemin prepared in Example 1 of the present invention; wherein a is the temperature increase of the 50 μg / mL nanomaterial under different infrared power conditions; b is the temperature increase of the nanomaterials with different concentrations under the condition of 0.75 W / cm 2 ; c is the thermal stability of the nanomaterial.

[0044] Figure 5 Multi-enzyme activity of the poly dopamine nanomaterial doped with gold nanoparticles and hemin prepared in Example 1 of the present invention; wherein a is the peroxidase activity of the nanomaterial; b is the consumption of GSH in the solution after treatment with nanomaterials of different concentrations; c is the glucose oxidase activity of the nanomaterial; d is the cascade catalytic activity of the nanomaterial.

[0045] Figure 6 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).

[0046] Figure 7 In vivo anti-tumor effect of the multifunctional cascade nanozyme of the present invention.

[0047] Figure 8 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.

[0048] Figure 9 Accelerated diabetic wound healing by the multifunctional cascade nanozyme of the present invention; wherein a is the use of PBS, PBS + NIR (0.75 W / cm 2 ), APH, APH + NIR (0.75 W / cm 2 ), APHC, APHC + NIR (0.75 W / cm 2 to treat the healing process of S. aureus-infected diabetic wounds; b is the wound area quantification diagram. Detailed implementation manners

[0049] The following is a further detailed description of the present invention with specific embodiments. However, this should not be construed as limiting the scope of the above-mentioned subject matter of the present invention to the following embodiments. All technologies implemented based on the content of the present invention belong to the scope of the present invention.

[0050] Unless otherwise specified, the reagents and materials used in the present invention are commercially available.

[0051] Example 1

[0052] A preparation method of a multifunctional cascade nanozyme is as Figure 1 shown. Gold ions, heme, and dopamine hydrochloride are self-assembled to form nano-ions under alkaline conditions, and targeting ligands or stabilizers are modified by means of amide condensation, thiol coupling, amino coupling, etc. The specific operation steps are as follows:

[0053] Step 1: Preparation of polydopamine nanomaterials doped with gold nanoparticles and heme:

[0054] 3.6 mL of dopamine hydrochloride solution (4 mg / mL) was added to 72 mL of tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl) buffer solution (0.01 M, pH = 8.5), and stirred vigorously. Subsequently, 5.4 mL of chloroauric 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, centrifuged at 10000 rpm for 10 min, discarded the supernatant, washed and centrifuged twice with ultrapure water, and dried in vacuum to obtain polydopamine nanomaterials doped with gold nanoparticles and heme.

[0055] 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), and the results were as Figure 3 shown. The nanomaterials presented a chrysanthemum shape ( Figure 3 a), the particle size showed a normal distribution, and the dispersion was good ( Figure 3 b), and the particle size was 150.1 ± 16.3 nm.

[0056] The polydopamine nanomaterials doped with gold nanoparticles and heme prepared in this example were subjected to photothermal testing, and the results were as Figure 4 shown. It had high efficient photothermal conversion ability. With the increase of the material concentration (10, 25, 50, 75, 100 μg / mL) and the NIR808 power (0.25, 0.50, 0.75 W / cm 2 ), the temperature of the system further increased ( Figure 4 a-b), and it had good photothermal stability and did not decay within 5 photothermal cycles ( Figure 4 c).

[0057] The prepared poly-dopamine nanomaterial doped with gold nanoparticles and hemin in this example has multi-enzyme activity, and the results are as Figure 5 shown. Its peroxidase-like activity was measured by the color reaction of 3,3',5,5'-tetramethylbenzidine (TMB) in the presence of hydrogen peroxide. 100 μL of the poly-dopamine nanomaterial doped with gold nanoparticles and hemin (30 μg / mL), 100 μL of TMB (1.2 mM), and 50 μL of H2O2 at different concentrations (6.25, 12.5, 25.0, 50.0, and 100 mM) were successively 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 a UV spectrophotometer. The results are as Figure 5 shown in a. This nanomaterial can effectively catalyze the oxidation of hydrogen peroxide (H2O2) and has high peroxidase-like activity. The reactive oxygen species generated during the reaction are hydroxyl radicals. Using 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) as a probe, the ability of the nanomaterial to consume GSH was detected. Different concentrations of nanozyme (0, 50, 100, 200, 500 μg / mL) were mixed with GSH (2 mM) solution. After incubation at 37 °C for 3 h, 10 μL of DTNB (5 mM) was added, and the mixture was incubated for 3 min after mixing and standing still. The change in absorbance was detected at 412 nm using a UV spectrophotometer. The results are as Figure 5 shown in b. As the concentration of the nanomaterial increases, the GSH level gradually decreases, indicating that the nanomaterial can effectively consume GSH through redox reactions, thereby increasing the concentration of ROS in the tumor microenvironment. In addition, this nanomaterial has significant glucose consumption ability. The consumption of glucose was measured using the 3,5-dinitrosalicylic acid (DNS) method. Different concentrations of nanozyme solutions (0, 100, 200 μg / mL) were mixed with PBS containing glucose (1 mg / mL). 0.5 mL of the above solution was collected at specific time intervals and mixed with 1.5 mL of DNS reagent. After heating at 100 °C for 5 min, it was quickly transferred to ice water for cooling for 20 min. The absorbance at 450 nm was detected using a UV spectrophotometer. The results are as Figure 5 shown in c. The concentration of glucose decreased exponentially, and the increase in the concentration of the nanomaterial accelerated the consumption rate of glucose. The change in TMB of different concentrations of glucose solutions was quantitatively detected by the TMB method. 100 μg / mL of the nanomaterial solution was mixed with different concentrations of glucose solutions (0, 0.25, 0.5, 1.0, 2.0 mg / mL). After incubation at 37 °C for 6 h, TMB (2 mM) was added, and the absorbance at 652 nm was measured using the time scan mode of a UV spectrophotometer. The results are as Figure 5As shown in d, both the increase in glucose concentration and the extension of reaction time can continuously increase the characteristic absorbance of the oxidation of oxTMB, confirming that the nanomaterials prepared by the present invention have significant cascade catalytic activity.

[0058] Step 2. Modify the targeting ligand or stabilizer:

[0059] (1) Targeted modification of DNA aptamer containing a thiol group:

[0060] Dissolve 0.2 mg of thiol DNA aptamer Aptamer1 (Apt1) (Wuhan Kingcare Bioengineering Co., Ltd.) in 1 mL of phosphate buffer (PBS) containing 0.1 mM TCEP and stir at room temperature for 1 h. Subsequently, add the phosphate buffer (PBS) (1 mL, 1 mg / mL) of the polydopamine nanomaterial doped with gold nanoparticles and hemin prepared in Step 1, stir at room temperature, and react for 16 h. Then dissolve 0.2 mg of Aptamer2 (Apt2) (Wuhan Kingcare Bioengineering Co., Ltd.) in 1 mL of PBS and add it to the mixture, and react at 60 °C for 2 h. After cooling to room temperature, continue to stir for 6 h. Dialyze the solution in a dialysis bag (Mw 14000) for 48 h and freeze-dry to obtain a multifunctional cascade nanozyme with targeted modification of Aptamer DNA aptamer. The particle size distribution diagram is shown in Figure 3 b. The DNA sequence (5′→3′) of 5′-SH thiol-modified Apt1 is shown in SEQ ID NO: 1, and the DNA sequence (5′→3′) of 5′-FAM (5′-Carboxyfluorescein) green fluorescent probe-modified Apt2 is shown in SEQ ID NO: 2.

[0061] (2) Targeted modification of folic acid (C-FA) containing a carboxyl group:

[0062] Add 0.55 μM 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and 0.55 μM N-hydroxysuccinimide (NHS) to a 0.5 mM folic acid dimethyl sulfoxide (DMSO) solution (1 mL) and stir. Then slowly add it to the aqueous solution (1 mL, 1 mg / mL) of the polydopamine nanomaterial doped with gold nanoparticles and hemin prepared in Step 1, stir at room temperature, and react for 24 h. Dialyze the solution in a dialysis bag (Mw 14000) for 48 h and freeze-dry to obtain a multifunctional cascade nanozyme with targeted modification of folic acid.

[0063] (3) Modification of polyethylene glycol (PEG) containing an amino group:

[0064] Dissolve 1 mg of monoamino polyethylene glycol (PEG) (Mw = 5000) in water, and slowly add dropwise the poly(dopamine) nanomaterial doped with gold nanoparticles and hemin prepared in step 1 (1 mL, 1 mg / mL) to a tris(hydroxymethyl)aminomethane hydrochloride (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 10,000) for 48 h, and freeze-dry to obtain a polyethylene glycol (PEG)-modified multifunctional cascade nanozyme.

[0065] Application Example 1

[0066] Cytotoxicity test of the aptamer DNA aptamer-targeted modified multifunctional cascade nanozyme (APHA):

[0067] The effect of the aptamer DNA aptamer-targeted modified multifunctional cascade nanozyme on the proliferation activity of mouse lung fibroblasts (L929) and mouse breast cancer cells (4T1) was evaluated using the classical 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. After co-incubation with different concentrations of the APHA supramolecular nanozyme assembly for 24 h, there was no obvious cytotoxicity to the normal mouse cells L929. Even at a high concentration (1000 μg / 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 in the concentration of APHA, showing a dose-dependent manner, and the inhibition rate exceeded 75% at 50 μg / mL ( Figure 6 b). It indicates that the nanozyme can generate toxic reactive oxygen species through its various enzyme-like activities, induce apoptosis and death of tumor cells, and the introduction of near-infrared light can further improve the enzyme-like activity during photothermal therapy and enhance the ability to kill tumor cells.

[0068] In vivo anti-tumor effect test of the aptamer DNA aptamer-targeted modified multifunctional cascade nanozyme:

[0069] Digest the 4T1 cells in the logarithmic growth phase with trypsin, centrifuge and resuspend with phosphate buffer solution (PBS). After counting the cell suspension, inoculate 4T1 cells subcutaneously in the right lower limb of Balb / c female mice at a standard of 2×10 6 cells / mouse, and observe the tumor formation situation and measure the tumor volume every day after inoculation. Wait until the tumor grows to 100 mm 3When around, 4T1 tumor-bearing mice were randomly divided into 6 groups (n = 5): PBS group, PBS + NIR group (PBS + L group), polydopamine nanomaterial group doped with gold nanoparticles and heme (APH group), Aptamer DNA aptamer cascade nanozyme group (APHA group), polydopamine nanomaterial 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 group was irradiated with 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 ended, the mice were sacrificed by cervical dislocation. The anti-tumor effect was as Figure 7 shown. Compared with the control group, the Aptamer DNA aptamer cascade nanozyme group had an obvious inhibitory effect on tumors, and the inhibitory effect was further enhanced after 808 nm near-infrared irradiation.

[0070] Application Example 2

[0071] Antibacterial effect test of PEG-modified multifunctional cascade nanozyme:

[0072] The plate counting method was used to study the synergistic antibacterial effect of polyethylene glycol (PEG)-modified multifunctional cascade nanozyme on Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus. Escherichia coli and Staphylococcus aureus were selected and cultured in a fresh liquid LB broth medium until the logarithmic growth phase, and then diluted to 10 7 colony forming units (CFU) / mL. The bacteria were added to the culture medium with glucose, and different concentrations of nanozyme were added with or without near-infrared irradiation. The near-infrared irradiation group was irradiated with 808 nm laser (0.75 W / cm 2 ) for 5 min. The phosphate buffer solution (PBS) treatment group was used as the control group. The treated bacterial suspension (60 μL) was spread on a solid agar medium, and after incubation at 37 °C for 24 h, the colonies were photographed and counted. The results are shown in Figure 8 . As the concentration of PEG-modified multifunctional cascade nanozyme increased, the antibacterial effect enhanced, and the bactericidal effect on bacteria was further strengthened after 808 nm near-infrared irradiation. Under the conditions of 1.5 mg / mL nanozyme treatment and 808 nm near-infrared irradiation, the antibacterial effects on Escherichia coli and Staphylococcus aureus reached 98.2% and 94.2% respectively.

[0073] Application Example 3

[0074] Accelerated diabetic wound healing effect test of folic acid (C-FA)-targeted modified multifunctional cascade nanozyme:

[0075] Establishment of a diabetic mouse model: 5-week-old C57BL / 6 mice (weighing 17 - 20 g) were fasted for 12 h for 3 consecutive days and then intraperitoneally injected with streptozotocin (STZ, 50 mg / kg) to construct a type 1 diabetes model. Mice were observed for 7 days after injection, and mice with blood glucose concentration higher than 16.7 mM were considered diabetic mice. The mice were randomly divided into 6 groups, with 3 mice in each group, named Control group (PBS group), Control + NIR group (Control + L group), polydopamine nanomaterial doped with gold nanoparticles and heme group (APH group), polydopamine nanomaterial doped with gold nanoparticles and heme + NIR group (APH + L group), folic acid-targeted modified multifunctional cascade nanozyme group (APHC group), folic acid-modified multifunctional cascade nanozyme + NIR group (APHC + L group). The mice were anesthetized by intraperitoneal injection of 0.3% sodium pentobarbital (0.2 mL / 10 g), and the back hair was shaved with an electric clipper. The shaved area was disinfected with 75% ethanol. Then, a full-thickness and equal-sized skin wound puncture was made on the shaved back with a sterile dermal biopsy punch with a diameter of 6 mm. 10 µL of the prepared Staphylococcus aureus suspension (1×10 6 CFU / mL) was inoculated on the wound surface for 24 h to form a wound infection. Starting from the next day, different nanozymes were coated on the wound surface every day and photothermal therapy (0.75 W / cm 2 , 5 min) was performed to treat the infected wound. Wound optical images 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 were as Figure 9 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 wound of the APHC + NIR group was almost healed, and the wound area was reduced to 1.52%, while the wound of the Control group was still not healed, and the wound area was 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 dopamine hydrochloride solution to Tris-HCl buffer solution, stir vigorously to dissolve, slowly add tetrachloroauric acid and heme, stir at room temperature for 6h-48h, collect the product by centrifugation after the reaction is completed, and obtain polydopamine nanomaterials doped with gold nanoparticles and heme. The nanomaterials are chrysanthemum-shaped and have a particle size of 150.1±16.3nm; the concentration of dopamine hydrochloride solution is 1mg / mL-4mg / mL, the concentration of Tris-HCl buffer solution is 0.01M-0.05M, and the pH is 8.0-9.5; 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: Add 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 obtain a multifunctional cascade nanozyme modified with the targeting ligand or stabilizer through an amide condensation reaction; 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); the targeting ligand or stabilizer containing a carboxyl group is folic acid; Method 2: Modification of targeting ligands or stabilizers by thiol coupling: The targeting ligand or stabilizer containing a thiol group is dissolved in a phosphate buffer containing tris(2-carboxyethyl)phosphine, and the thiol group is reduced by stirring at room temperature, and then the polydopamine nanomaterial doped with gold nanoparticles and heme prepared in step 1 is added, stirred at room temperature, and reacted to obtain a multifunctional cascade nanozyme modified with the targeting ligand or stabilizer through Michael addition reaction coupling between the thiol group and the polydopamine nanomaterial; the targeting ligand or stabilizer containing a thiol group is thiol DNA aptamer Aptamer1 and Aptamer2, and the DNA sequence is shown in SEQ ID NO: 1~ 2; 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, 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 centrifugal speed condition is 8000rpm-15000rpm, and the centrifugal time is 5min-35min.

3. 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.

4. 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.

5. The method for preparing a multifunctional cascade nanozyme according to claim 1, characterized in that: In step 2, method 1, the carboxyl-containing targeting ligand or stabilizer is folic acid, and a folic acid-targeted modified multifunctional cascade nanozyme is obtained. The folic acid-targeted modified multifunctional cascade nanozyme is used to prepare a drug for accelerating the healing of diabetic wounds.

6. The method for preparing a multifunctional cascade nanozyme according to claim 1, characterized in that: In step 2, the thiol-containing targeting ligand or stabilizer is thiol DNA aptamers Aptamer1 and Aptamer2, and the DNA sequences are shown in SEQ ID NO: 1~ 2, to obtain a multifunctional cascade nanozyme targeted by Aptamer DNA aptamers. The multifunctional cascade nanozyme targeted by Aptamer DNA aptamers is used to prepare a drug for inhibiting tumor cell proliferation.

7. The method for preparing a multifunctional cascade nanozyme according to claim 1, characterized in that: In step 2, method three, the amino-containing targeting ligand or stabilizer is monoamino polyethylene glycol, and a PEG-modified multifunctional cascade nanozyme is obtained. The PEG-modified multifunctional cascade nanozyme is used to prepare anti-inflammatory and antibacterial drugs.

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