Gold nano-enzyme composite material MAuN-coated HA as well as preparation method and application thereof

Abstract

The invention discloses a gold nano-enzyme composite material MAuN-coated HA and a preparation method and application thereof, the preparation method of the MAuN-coated HA comprises the following steps: growing gold nanoparticles on MIL-88 (Fe), specifically, uniformly dispersing the MIL-88 (Fe) in water, adding tetrachloroauric acid trihydrate of which the mass is 0.8-1.2 times of that of the MIL-88 (Fe), uniformly mixing, adding a reducing agent into the system, and reacting for 2-5 hours to obtain MAu; then, norcantharidin is loaded on the MAu, and MAuN is obtained; and modifying hyaluronic acid on the surface of the MAuN to obtain the product. According to the composite material disclosed by the invention, through chemotherapy, chemokinetic therapy, enhancement effect of gold atom nano-enzyme on activity of CAT / POD / OXD-like enzyme and multi-catalytic mechanism of Russell effect, remarkable chemotherapy / CDT synergistic therapy is realized, and the problem of low efficiency of traditional CDT therapy is solved.

Description

Technical Field

[0001] This invention relates to a nanoenzyme composite material, and more particularly to a gold nanoenzyme composite material MAuN@HA, its preparation method and application, belonging to the field of nanobiomedical materials technology. Background Technology

[0002] In recent years, the incidence and mortality rates of malignant tumors have continued to rise. Traditional treatment methods (such as surgery, radiotherapy, and chemotherapy) are difficult to meet clinical needs due to problems such as large toxic side effects, easy recurrence, and drug resistance.

[0003] Nanozymes are a class of artificial enzymes that possess the unique properties of nanomaterials and catalytic functions. Due to their economic efficiency, stability, and durability, they are widely used in medicine, chemical engineering, and environmental fields. With the development of nanotechnology, nanozymes have attracted considerable attention. Compared to natural enzymes, nanozymes offer advantages such as low cost, good stability, and ease of modification. Their catalytic activity can be modulated by controlling their morphology and size, thus allowing them to replace natural enzymes in a wider range of applications. Various artificial enzymes based on nanomaterials have been discovered, including metal oxides, single-metal nanomaterials, carbon-based nanomaterials, and others.

[0004] Chemokinetic therapy (CDT) is a novel therapeutic technique based on the specific activation of the Fenton (or Fenton-like) reaction in the tumor microenvironment, generating hydroxyl radicals (·OH) to selectively kill tumor cells. Its core principle is to trigger a chemical reaction within the tumor using nanocatalytic materials to achieve targeted therapy. Due to its selective killing characteristics dependent on the tumor-specific microenvironment, it has become a research hotspot in recent years. However, this therapy still faces several limitations, including low concentrations of endogenous metal ions in tumor cells and limited catalytic efficiency, low H2O2 concentration, and high glutathione expression leading to limited hydroxyl radical generation.

[0005] To overcome these limitations, nanocatalysis in medicine has been exploring the use of multifunctional materials (such as drug-loaded and enzyme-loaded nanocatalysts) combined with exogenous stimuli (light, ultrasound) to enhance the catalytic efficiency of CDT (tumor-degrading therapy). However, a single catalytic mechanism is still insufficient to achieve radical therapeutic effects. Therefore, designing and preparing nanoenzyme composite materials that combine multiple treatment methods and possess various catalytic effects is of great significance for the treatment of tumors. Summary of the Invention

[0006] The technical problem to be solved by this invention is to provide a gold nanozyme composite material MAuN@HA, its preparation method and application, which aims to solve the problems in tumor treatment, such as the large toxic side effects, easy recurrence, drug resistance and the difficulty in achieving radical efficacy with a single catalytic mechanism.

[0007] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0008] A method for preparing a gold nanozyme composite material MAuN@HA includes the following steps:

[0009] 1) Obtain the MIL-88(Fe) metal-organic framework;

[0010] 2) Growth of gold nanoparticles on MIL-88(Fe) metal-organic framework: MIL-88(Fe) metal-organic framework was uniformly dispersed in water, tetrachloroauric acid trihydrate (HAuCl4·3H2O) was added, and the mixture was stirred. A reducing agent was added to the system under stirring conditions, and the reaction was carried out for 2-5 hours to allow gold nanoparticles to grow uniformly on the surface of MIL-88(Fe) metal-organic framework, thus obtaining the composite material MAu; wherein the mass ratio of MIL-88(Fe) metal-organic framework to tetrachloroauric acid trihydrate was 10:0.8-1.2.

[0011] 3) Norepinephrine (NCTD) was loaded onto MAu to obtain the composite material MAuN;

[0012] 4) Modify the surface of MAuN with hyaluronic acid (HA) to obtain the gold nanozyme composite material MAuN@HA.

[0013] The applicant discovered in its experiments that the mass ratio of MIL-88(Fe) metal-organic framework to tetrachloroauric acid trihydrate in step 2) significantly affected the in vitro antitumor effect of the final gold nanozyme composite material MAuN@HA. The applicant concluded that this was due to the increased particle size of the gold nanoparticles on the MAu composite material and the entire MAu composite material after exceeding the specified ratio in this application. Based on the experimental results, the applicant further optimized the mass ratio of MIL-88(Fe) metal-organic framework to tetrachloroauric acid trihydrate to be 10:1.0~1.2.

[0014] Further, in step 2), a surfactant is added when dispersing the MIL-88(Fe) metal-organic framework in water. The concentration of the surfactant in the water is preferably controlled at 10-15 mg / mL. Preferably, the surfactant is a cationic surfactant, more preferably a quaternary ammonium salt cationic surfactant, and more specifically, it can be hexadecyltrimethylammonium bromide (CTAB) and / or dodecyltrimethylammonium chloride.

[0015] Furthermore, the tetrachloroauric acid trihydrate and reducing agent mentioned in step 2) are added in the form of their aqueous solutions, and their concentrations are not specifically required. Preferably, the reducing agent is NaBH4, and its amount is usually 2 to 3 times the molar amount of tetrachloroauric acid trihydrate.

[0016] Furthermore, the reaction in step 2) is carried out at 23~37°C, and the reaction time is more preferably 2~4h.

[0017] Further, step 3) includes: dissolving norcantharidin in water, and adding the composite material MAu in the presence of EDC (1-ethyl-(3-dimethylaminopropyl)carbodiimide) and NHS (N-hydroxysuccinimide) to carry out a coupling reaction to obtain the composite material MAuN. Preferably, the mass ratio of the composite material MAu to norcantharidin is 1:1.1~1.4, and the amounts of EDC and NHS are preferably 1~1.2 times the molar amount of norcantharidin, respectively; the reaction is carried out under light-protected conditions, preferably at room temperature, and the reaction time is controlled at 20~48h.

[0018] Further, step 4) includes: dissolving hyaluronic acid in water, adding the composite material MAuN to react, thereby obtaining the gold nanozyme composite material MAuN@HA. Preferably, the concentration of hyaluronic acid in water is 0.4~0.8 mg / mL, and the mass ratio of hyaluronic acid to composite material MAuN is 1:1.5~2.5; the reaction is carried out under light-protected conditions, preferably at room temperature, and the reaction time is controlled at 10~24 h.

[0019] In the method described in this invention, the MIL-88(Fe) metal-organic framework can be purchased directly from the market or prepared with reference to existing literature (Acta Pharmaceutica Sinica B (IF 14.6) Pub Date: 2020-08-13, Biyuan Wu, Jintao Fu, Yixian Zhou, Sulan Luo, Yiting Zhao, Guilan Quan, XinPan, Chuanbin Wu.).

[0020] The present invention also includes the gold nanozyme composite material MAuN@HA prepared by the above method.

[0021] This invention further includes the application of the gold nanozyme composite material MAuN@HA prepared by the above method in the preparation of chemokinetic therapy / chemotherapy synergistic tumor drugs.

[0022] Compared with existing technologies, this invention provides a gold nanozyme composite material MAuN@HA for enhancing chemokinetic therapy / chemotherapy synergistic treatment to inhibit tumor cells and its preparation method. In the design of this nanozyme composite material, the MIL-88(Fe) metal-organic framework exhibits excellent drug loading capacity and tumor microenvironment responsiveness, preventing premature drug release. The loaded gold nanozymes enhance the system's multi-enzyme activity, similar to catalase (CAT), peroxidase (POD), and oxidase (OXD), and are generated through a Russell reaction cascade catalysis. 1 O2, ·O2 - The release of NCTD accelerates the generation rate of cytotoxic ROS by adding ·OH; simultaneously, the precise release of NCTD can induce tumor cell apoptosis through chemotherapy. Significant synergistic chemotherapy / chemokinetic therapy is achieved through the multi-catalytic mechanism of chemotherapy, chemokinetics, the enhancement of CAT / POD / OXD enzyme activity by gold atom nanozymes, and the Russell effect. Therefore, the nanozyme composite material described in this invention can solve the problems of high toxicity, easy relapse, drug resistance, and the inability to achieve radical efficacy with a single catalytic mechanism in traditional tumor treatment. Attached Figure Description

[0023] Figure 1 The particle size distribution diagrams are of MIL-88(Fe), MAu, MAuN, and MAuN@HA prepared in Example 1 of this invention.

[0024] Figure 2 The XRD patterns of MIL-88(Fe) and MAuN@HA prepared in Example 1 of this invention are shown.

[0025] Figure 3 This is the NCTD standard curve.

[0026] Figure 4 The oxygen generation curves of MIL-88(Fe) and MAuN@HA in hydrogen peroxide solution are shown.

[0027] Figure 5 To detect ·OH(a), 1 O2(b) and ·O2 - (c) EPR spectrum.

[0028] Figure 6 Bar charts showing cell viability of B16 cells in different groups: (a) represents NCTD, and (b) represents MIL-88(Fe), Mau, MAuN, and MAuN@HA. Data are expressed as mean ± standard deviation (n=3). The groups were set as (1) MIL-88(Fe), (2) MAu, (3) MAuN, and (4) MAuN@HA.

[0029] Figure 7 The bar graph shows the cell viability of B16 cells at the same concentration (50 μg / mL) of MAuN@HA prepared using MIL-88(Fe) metal-organic framework and tetrachloroauric acid trihydrate at different mass ratios.

[0030] Figure 8 The bar graph shows the cell viability of B16 cells at the same concentration (50 μg / mL) of MAuN@HA prepared according to different reaction times after the addition of reducing agent.

[0031] Figure 9 The fluorescence of reactive oxygen species in cells after treatment with different materials. Detailed Implementation

[0032] To better explain the technical solution of the present invention, the present invention will be further described in detail below with reference to the embodiments, but the implementation of the present invention is not limited thereto.

[0033] In the following embodiments, MIL-88(Fe) represents the MIL-88(Fe) metal-organic framework, MAuN represents the composite material MAuN, MAu represents the composite material MAu, and MAuN@HA represents the gold nanozyme composite material MAuN@HA.

[0034] Example 1: Preparation of MAuN@HA

[0035] 1) Synthesis of MIL-88(Fe) metal-organic framework:

[0036] 0.126 g (0.696 mmol) of 2-aminoterephthalic acid (NH2-BDC) and 0.187 g (0.699 mmol) of ferric chloride hexahydrate (FeCl3·6H2O) were dissolved in 15 mL (0.1937 mol) of N,N-dimethylformamide (DMF), and the solution was sonicated for 5 minutes to ensure complete dissolution. 3.45 mmol (197 μL) of acetic acid was added to the solution as a regulator, and the mixture was transferred to an oil bath at 120 °C and reacted at this temperature for 4 hours. After the reaction was complete, heating was stopped, and the mixture was allowed to cool naturally to room temperature with continuous stirring. The product was washed three times each with DMF and anhydrous ethanol until the supernatant was clear. The product was then dried under vacuum (25 °C, 24 h) to obtain MIL-88(Fe) (brownish-gray powder).

[0037] 2) Synthesis of MAu:

[0038] 150 mg (0.042 mmol) of CTAB was dissolved in 10 mL of deionized water, sonicated, and then 100 mg of MIL-88(Fe) was added. Sonication was continued for 10 min to ensure uniform dispersion. 2.54 mL of 10 mM HAuCl4·3H2O (equivalent to 10 mg of HAuCl4·3H2O) was added, and the mixture was vortexed for 5 min to form a homogeneous suspension. Under vigorous stirring (≥800 rpm), 6 mL of 10 mM NaBH4 was rapidly injected, turning the solution amber. The solution was then transferred to a 37°C water bath and stirred continuously for 3 h to ensure uniform growth of gold nanoparticles (Au NPs) on the MIL-88(Fe) surface. After the reaction, the precipitate was collected by high-speed centrifugation (13,000 rpm, 10 min). The precipitate was washed alternately with warm water and anhydrous ethanol to thoroughly remove CTAB and unreacted substances. The precipitate was vacuum dried (25°C, 24 h) to obtain MAu (a gray powder).

[0039] 3) Synthesis of MAuN:

[0040] 0.25 mmol (0.042 g) of NCTD was dissolved in 5 mL of deionized water and sonicated for 10 min. Then, 0.3 mmol (0.575 g) of EDC and 0.25 mmol (0.0288 g) of NHS were added sequentially, and the mixture was stirred at room temperature (25 ± 2 °C) for 4 h to activate the NCTD carboxyl groups. 0.036 g of MAu was dispersed in the above activation solution and reacted under light-protected conditions with magnetic stirring (500 rpm) for 24 h. After the reaction was complete, the precipitate was collected by high-speed centrifugation (12,000 rpm, 15 min), washed multiple times with deionized water to remove unreacted NCTD, EDC / NHS, and vacuum dried (25 °C, 24 h) to obtain the composite material MAuN.

[0041] 4) Synthesis of MAuN@HA:

[0042] Add 5 mg of HA to 10 mL of deionized water and sonicate until completely dissolved to form a homogeneous solution. Add 10 mg of MAuN to the solution and continue sonicating for 5 min to prevent aggregation. React at room temperature (25±2℃) in the dark with magnetic stirring (300 rpm) for 24 h to promote the binding of HA and MAuN surface-active groups. After the reaction is complete, collect the precipitate by low-speed centrifugation, wash repeatedly, and vacuum dry (25℃, 24 h) to obtain the final product MAuN@HA (brown powder).

[0043] Example 2: Characterization of MAuN@HA

[0044] 1. Dynamic Light Scattering (DLS) Test

[0045] Dynamic light scattering tests were performed on the products synthesized in each step of Example 1, and the results are as follows: Figure 1 As shown. By Figure 1 It can be seen that the hydrated particle size of MAuN@HA is 295 nm.

[0046] 2. X-ray powder diffraction (XRD) analysis

[0047] X-ray powder diffraction tests were performed on MIL-88(Fe) synthesized in step 1) of Example 1 and the final product MAuN@HA synthesized in step 4). Figure 2 As shown in the figure. The results further confirm the successful anchoring of Au NPs in the MIL-88(Fe) framework. Figure 2 In the XRD pattern of the composite material MAuN@HA, MIL-88(Fe) exhibits characteristic diffraction peaks consistent with its crystal structure; however, new diffraction peaks appear in the composite material MAuN@HA, directly confirming the formation of Au NPs. Notably, compared to pure MIL-88(Fe), the intensity of the characteristic diffraction peaks of MIL-88(Fe) in MAuN@HA is significantly reduced, indicating that the introduction of Au NPs may have affected the crystallinity of the original framework or led to partial structural reorganization.

[0048] Example 3: Physicochemical properties of MAuN@HA

[0049] 1. Drug loading analysis

[0050] After thoroughly mixing a known concentration of NCTD solution with MAu nanocarrier, the supernatant was obtained by centrifugation, and the peak area of ​​NCTD in the supernatant was determined by high performance liquid chromatography.

[0051] Quantitative analysis based on standard curves showed that ( Figure 3 The loading efficiency of NCTD in the MAu carrier reached 12.7%, which confirms that NCTD was successfully loaded into the nanocarrier system.

[0052] 2. Nanozyme activity detection

[0053] 2. 1 O2 generation assessment

[0054] This application investigated the Russell reaction, catalase, peroxidase, and oxidase-like activities of MAuN@HA. MIL-88(Fe) metal-organic framework and MAuN@HA were added separately to an aqueous solution of H2O2, and the amount of O2 generated was monitored over 600 seconds using a portable dissolved oxygen meter.

[0055] Dissolved oxygen experiment ( Figure 4The results showed that both MAuN@HA and MIL-88(Fe) could degrade H2O2 and catalyze the generation of O2; however, compared with MIL-88(Fe), MAuN@HA induced more significant H2O2 decomposition and O2 release, indicating that its CAT-like enzyme activity was superior to that of MIL-88(Fe).

[0056] 2.2 Detection of reactive oxygen species

[0057] 1 For O2 detection, MIL-88 (Fe) and MAuN@HA were added to an aqueous solution containing glutathione and H2O2, respectively. 2,2,6,6-Tetramethylpiperidine (TEMP) scavenger was added 5 min before the test, and EPR analysis was performed immediately after mixing.

[0058] For OH detection, the same method as for O2 detection is used. After adding the material, incubate at 37°C for 2 h. Add 5,5-dimethyl-1-pyrrolidone N-oxide (DMPO) 5 min before the test and detect directly.

[0059] ·O2 - For the detection, replace the solvent with anhydrous methanol; the remaining steps are the same as for the ·OH detection.

[0060] The results showed that the ·OH ( ) in the MAuN@HA group Figure 5 a) 1 O2 ( Figure 5 b) and O2 - ( Figure 5 c) The ROS signal intensity of MAuN@HA was significantly higher than that of the MIL-88(Fe) group and the MAu group. These EPR results indicate that MAuN@HA possesses excellent nanozyme-like activity. Based on this, we hypothesize that MAuN@HA can catalyze the generation of cytotoxic free radicals from endogenous H2O2 through redox reactions in the tumor microenvironment, disrupting the dynamic balance of free radicals in the system and ultimately leading to tumor cell death.

[0061] Example 4: In vitro antitumor properties

[0062] 1. In vitro cytotoxicity assessment

[0063] Based on the excellent reactive oxygen species generation capacity and enzyme-like activity of MAuN@HA, we further investigated its in vitro antitumor effect. Melanoma cells (B16) were subjected to a 4×10⁻⁶ in vitro culture. 3Cells were seeded at a density of 1:1 in 96-well plates. After cell attachment, experimental groups were set up: MIL-88 group, MAu group, MAuN group, and MAuN@HA group. After incubation at 37°C for 24 hours, 10 μL of MTT solution was added to each well for an additional 4 hours of incubation. The supernatant was carefully aspirated, and 100 μL of DMSO was added to each well. The plates were shaken for 10 minutes, and the absorbance at 490 nm was measured using a multi-plate reader.

[0064] The results are as follows Figure 6 As shown in the figure. The results showed that, with increasing concentration, NCTD alone only inhibited B16 cells by 29.8% at the maximum loading concentration (26 μg / mL). Figure 6 a), while the MAuN@HA group was significantly different from the control group (MIL-88(Fe)). Figure 6 (b) Cell viability decreased significantly. When the concentration reached 200 μg / mL, the inhibition rate of B16 cells in the MAuN@HA group increased sharply to 88.8%, which was mainly attributed to a triple synergistic mechanism, including enhanced oxidative stress induced by ginnerase, the direct antitumor effect of norcantharidin, and the targeting effect mediated by hyaluronic acid.

[0065] 2. In vitro fluorescence imaging detection of ROS

[0066] B16 cells were respectively fed with 1×10 5 Cells were seeded at a density of 1 / well in 6-well plates and cultured at 37°C and 5% CO2 for 24 hours to allow cell adhesion. After removing the old culture medium, fresh culture medium containing 50 μg / mL MIL-88, MAu, MAuN, or MAuN@HA was added to each well, and the cells were cultured for another 24 hours. The culture medium was discarded, and the cells were gently washed three times with PBS. 1 mL of DCFH-DA (20 μM) was added to each well under dark conditions, and the cells were incubated at 37°C for 20 minutes. The cells were washed three times with PBS to remove unbound probes, and images were acquired using a fluorescence microscope at an excitation wavelength of 488 nm.

[0067] The results are as follows Figure 9 As shown. By Figure 9 As can be seen, MIL-88(Fe) and MAu only showed weak green fluorescence, similar to the control group, while the fluorescence signal of the MAuN@HA group was significantly enhanced.

[0068] In summary, this invention prepares a chemokinetic MOF nanocarrier, which is then used as a carrier to load gold atom nanozymes and NCTD, along with their surface-modified hyaluronic acid, to achieve tumor targeting. The MIL-88(Fe) metal-organic framework exhibits excellent drug loading capacity and tumor microenvironment responsiveness, preventing premature drug release. The gold atom nanozyme enhances the CAT / POD / OXD multi-enzyme activity of the system and generates [the desired product] via a Russell reaction cascade.1 O2, ·O2 - The presence of various ROS, including ·OH, significantly improves treatment efficiency. Simultaneously, the precise release of NCTD can induce tumor cell death through chemotherapy. Through chemotherapy, chemokinetics, the enhancing effect of gold atom nanozymes on CAT / POD / OXD-like enzyme activity, and the Russell effect—a multi-catalytic mechanism—significant synergistic chemotherapy / CDT therapy is achieved, systematically addressing the low treatment efficiency of traditional CDT.

[0069] Example 5: Preparation of MAuN@HA

[0070] MAuN@HA was prepared according to the method described in Example 1, except that in step 2), the amount of HAuCl4·3H2O was changed to 8.0 mg (the mass ratio of MIL-88(Fe) to HAuCl4·3H2O was 10:0.8, i.e. 25:2).

[0071] The prepared MAuN@HA was evaluated for in vitro cytotoxicity according to the method described in Example 4, and the results are as follows: Figure 7 As shown. By Figure 7 It can be seen that when the concentration reaches 50 μg / mL, the inhibition rate of B16 cells in the MAuN@HA group is 54.3%.

[0072] Example 6: Preparation of MAuN@HA

[0073] MAuN@HA was prepared according to the method described in Example 1, except that in step 2), the amount of HAuCl4·3H2O was changed to 12.0 mg (the mass ratio of MIL-88(Fe) to HAuCl4·3H2O was 10:1.2, i.e. 25:3).

[0074] The prepared MAuN@HA was evaluated for in vitro cytotoxicity according to the method described in Example 4, and the results are as follows: Figure 7 As shown. By Figure 7 It can be seen that when the concentration reaches 50 μg / mL, the inhibition rate of B16 cells in the MAuN@HA group is 63.5%.

[0075] Example 7: Preparation of MAuN@HA

[0076] MAuN@HA was prepared according to the method described in Example 1, except that in step 2), the reaction time was changed to 2 h and 5 h, respectively, and the resulting composite materials were denoted as MAuN@HA-2 and MAuN@HA-5, respectively.

[0077] The prepared MAuN@HA was evaluated for in vitro cytotoxicity according to the method described in Example 4. The results showed that when the concentration reached 50 μg / mL, the B16 cell inhibition rates of MAuN@HA-2 and MAuN@HA-5 were 50.4% and 52.2%, respectively.

[0078] Comparative Example 1: Preparation of MAuN@HA

[0079] MAuN@HA was prepared according to the method described in Example 1, except that in step 2), the amount of HAuCl4·3H2O was changed to 4.0 mg (the mass ratio of MIL-88(Fe) to HAuCl4·3H2O was 10:0.4, i.e. 25:1).

[0080] The prepared MAuN@HA was evaluated for in vitro cytotoxicity according to the method described in Example 4, and the results are as follows: Figure 7 As shown. By Figure 7 It can be seen that when the concentration reaches 50 μg / mL, the inhibition rate of B16 cells in the MAuN@HA group is 8.6%.

[0081] Comparative Example 2: Preparation of MAuN@HA

[0082] MAuN@HA was prepared according to the method described in Example 1, except that in step 2), the amount of HAuCl4·3H2O was changed to 14.0 mg (the mass ratio of MIL-88(Fe) to HAuCl4·3H2O was 10:1.4, i.e. 50:7).

[0083] The prepared MAuN@HA was evaluated for in vitro cytotoxicity according to the method described in Example 4, and the results are as follows: Figure 7 As shown. By Figure 7 It can be seen that when the concentration reaches 50 μg / mL, the inhibition rate of B16 cells in the MAuN@HA group suddenly increased to 24.4%.

[0084] Comparative Example 3: Preparation of MAuN@HA

[0085] MAuN@HA was prepared according to the method described in Example 1, except that in step 2), the amount of HAuCl4·3H2O was changed to 6.0 mg (the mass ratio of MIL-88(Fe) to HAuCl4·3H2O was 10:0.6, 50:3).

[0086] The prepared MAuN@HA was evaluated for in vitro cytotoxicity according to the method described in Example 4, and the results are as follows: Figure 7 As shown. By Figure 7 It can be seen that when the concentration reaches 50 μg / mL, the inhibition rate of B16 cells in the MAuN@HA group is 28.2%.

[0087] Comparative Example 4: Preparation of MAuN@HA

[0088] MAuN@HA was prepared according to the method described in Example 1, except that in step 2), the reaction time was changed to 1 h, 6 h and 7 h respectively, and the final composite materials were denoted as MAuN@HA-1, MAuN@HA-6 and MAuN@HA-7 respectively.

[0089] The prepared MAuN@HA was evaluated for in vitro cytotoxicity according to the method described in Example 4, and the results are as follows: Figure 8 As shown. By Figure 8 It can be seen that when the concentration reaches 50 μg / mL, the inhibition rates of B16 cells in the MAuN@HA-1, MAuN@HA-6 and MAuN@HA-7 groups are 25.8%, 38.2% and 7.9%, respectively.

[0090] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make several modifications and improvements without departing from the concept of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent.

Claims

1. A method for preparing a gold nanozyme composite material MAuN@HA, comprising the following steps: 1) Obtain the MIL-88(Fe) metal-organic framework; 2) Growth of gold nanoparticles on MIL-88(Fe) metal-organic framework: MIL-88(Fe) metal-organic framework was uniformly dispersed in water, tetrachloroauric acid trihydrate was added, and the mixture was stirred. A reducing agent was added to the system under stirring conditions, and the reaction was carried out for 2-5 hours to allow gold nanoparticles to grow uniformly on the surface of MIL-88(Fe) metal-organic framework, thus obtaining the composite material MAu; wherein, the mass ratio of MIL-88(Fe) metal-organic framework to tetrachloroauric acid trihydrate was 10:0.8-1.2; 3) Loading norepinephrine onto MAu yields the composite material MAuN; 4) Hyaluronic acid is modified on the surface of MAuN to obtain the gold nanozyme composite material MAuN@HA.

2. The preparation method according to claim 1, characterized in that, In step 2), a surfactant is added when dispersing the MIL-88(Fe) metal-organic framework in water.

3. The preparation method according to claim 2, characterized in that, The surfactant is a cationic surfactant.

4. The preparation method according to any one of claims 1 to 3, characterized in that, Step 3) includes: dissolving norcantharidin in water, adding the composite material MAu in the presence of EDC and NHS to carry out a coupling reaction, and obtaining the composite material MAuN.

5. The preparation method according to claim 4, characterized in that, The mass ratio of the composite material MAu to norcantharidin is 1:1.1~1.

4.

6. The preparation method according to any one of claims 1 to 3, characterized in that, Step 4) includes: dissolving hyaluronic acid in water, adding the composite material MAuN to react, thereby obtaining the gold nanozyme composite material MAuN@HA.

7. The preparation method according to claim 6, characterized in that, The concentration of hyaluronic acid in water is 0.4~0.8 mg / mL.

8. The preparation method according to claim 6, characterized in that, The mass ratio of hyaluronic acid to the composite material MAuN is 1:1.5~2.

5.

9. The gold nanozyme composite material MAuN@HA prepared by the method according to any one of claims 1 to 8.

10. The use of the gold nanozyme composite material MAuN@HA according to claim 9 in the preparation of chemokinetic therapy / chemotherapy synergistic tumor drugs.

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