A hollow mesoporous monatomic molybdenum nanoscale enzyme, a preparation method thereof and preparation and application of a nanoscale enzyme reactor based on the same

Abstract

The application discloses a hollow mesoporous single-atom molybdenum nanozyme, a preparation method of the nanozyme and preparation and application of a nanozyme reactor based on the nanozyme, and belongs to the technical field of functional nano material preparation and biological application thereof. The novel hollow mesoporous single-atom molybdenum nanozyme (HMMo-SAzyme) is prepared for the first time, has a large internal space and a high specific surface area, can have a high peroxide enzyme activity and can be used as a carrier for loading natural enzymes. In addition, the large pore size is beneficial to the rapid transmission of a catalytic substrate in the HMMo-SAzyme. Meanwhile, glucose oxidase (GOx) is loaded on the hollow mesoporous single-atom molybdenum nanozyme and is wrapped with hyaluronic acid (HA) to construct a nanozyme reactor (HMMo / GOx@HA) with a broad-spectrum antibacterial effect, which can be used as a carrier for loading natural enzymes and drugs, can cause a cascade catalytic reaction at a bacterial infection site, can effectively inhibit bacterial infection of a wound and can promote wound healing.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of functional nanomaterials preparation and biological application, and particularly relates to a hollow mesoporous single-atom molybdenum nanoscale enzyme, a preparation method thereof, and preparation and application of a nanoscale enzyme reactor based on the same. BACKGROUND

[0002] Diseases caused by bacterial infection seriously threaten human health and cause huge medical and economic burden. Antibiotics are currently the most commonly used drugs against bacterial infection, however, the overuse of antibiotics leads to the emergence and rapid spread of drug-resistant bacteria, so the development of new antibacterial drugs that are not prone to bacterial resistance has important basic research value and social significance.

[0003] Inspired by the fact that the production of reactive oxygen species (ROS) catalyzed by natural enzymes causes irreversible oxidative damage to bacteria, in recent years, antibacterial therapy based on nanoscale enzymes has attracted widespread attention. Nanoscale enzymes are a class of nanomaterials with similar catalytic activity to natural enzymes, and their antibacterial mechanism is to generate hydroxyl radicals (·OH) with higher oxidation by catalyzing hydrogen peroxide (H2O2) through the peroxidase activity of nanoscale enzymes, and then to destroy the cell membrane structure of bacteria, degrade intracellular nucleic acids, and inactivate proteins, so as to achieve high-efficiency antibacterial effect and not prone to bacterial resistance. Although nanoscale enzymes have similar catalytic activity to natural enzymes, the non-uniformity of their structure construction causes the catalytic active sites of nanoscale enzymes to be unable to be effectively controlled, resulting in their catalytic performance being far inferior to that of natural enzymes, which limits their further application in the antibacterial field. The emergence of single-atom nanoscale enzymes effectively solves the deficiency of insufficient activity of nanoscale enzymes. Single-atom nanoscale enzymes are nanoscale enzymes with atomically dispersed metal active sites similar to natural metal enzymes. Compared with the nanoscale enzymes studied in the past, single-atom nanoscale enzymes have a clear electronic and geometric structure, maximum metal atom utilization rate, and unique quantum size effect, so their enzyme-like activity is greatly improved, and therefore they have attracted special attention in the antibacterial field.

[0004] However, studies have shown that single-atom nanoscale enzymes usually have high catalytic activity only under acidic conditions, which seriously limits their application in near-neutral physiological systems. In addition, single-atom nanoscale enzymes often need to use high concentrations of H2O2 to generate ·OH in the antibacterial process, and high concentrations of H2O2 not only cause immunogenicity and inflammation, but also cause damage to normal cells and tissues. These defects seriously limit the application of single-atom nanoscale enzymes in in vivo antibacterial applications.

[0005] Glucose oxidase (GOx) catalyzes the production of large amounts of gluconic acid and H₂O₂ from glucose, providing a substrate and acidic environment for the antibacterial activity of single-atom nanozymes. Combining highly catalytically active single-atom nanozymes with GOx through a cascade reaction holds promise for achieving highly efficient antibacterial effects. However, most currently studied single-atom nanozymes are solid structures, which cannot effectively load GOx. Furthermore, the small pore size of single-atom nanozymes hinders the rapid contact of H₂O₂ with the internal active sites, and its combination with GOx affects the cascade catalytic effect. Therefore, developing a cascade catalytic system with high catalytic efficiency is of great significance for promoting the in vivo antibacterial activity of single-atom nanozymes. Summary of the Invention

[0006] In order to overcome the shortcomings of the prior art, the present invention aims to provide a hollow mesoporous single-atom molybdenum nanozyme, its preparation method, and the preparation and application of a nanozyme reactor based thereon, so as to solve the technical problems of existing single-atom nanozymes having small pore size and mostly being solid structures, which cannot effectively load Gox, resulting in low catalytic efficiency and poor antibacterial effect.

[0007] To achieve the above objectives, the present invention employs the following technical solution:

[0008] This invention discloses a method for preparing hollow mesoporous single-atom molybdenum nanozymes, comprising the following steps:

[0009] 1) Preparation of molybdenum acetylacetone-doped metal-organic framework Mo-ZIF-8;

[0010] 2) Molybdenum acetylacetonate-doped metal-organic framework Mo-ZIF-8 was dispersed in water, tannic acid aqueous solution was added for etching, then stirred, centrifuged to collect the precipitate, washed and dried to obtain HMo-ZIF-8 with a hollow structure.

[0011] 3) Hollow mesoporous single-atom molybdenum nanozyme HMMo-SAzyme was prepared by pyrolysis of HMo-ZIF-8 with a hollow structure.

[0012] Preferably, in step 1), the method for preparing the molybdenum acetylacetonate-doped metal-organic framework Mo-ZIF-8 is as follows: 2-methylimidazole is dissolved in anhydrous methanol and sonicated to obtain a first solution; zinc nitrate hexahydrate and molybdenum acetylacetonate are dissolved in anhydrous methanol and sonicated to obtain a second solution; the second solution is added dropwise to the first solution, stirred, centrifuged to collect the precipitate, washed, and dried to obtain the molybdenum acetylacetonate-doped metal-organic framework (Mo-ZIF-8);

[0013] Further preferably, the mass ratio of the used zinc nitrate hexahydrate and 2-methylimidazole is 1:1-1:3, preferably 1:2.22; the mass ratio of the used molybdenum acetylacetonate and 2-methylimidazole is 1:30-1:50, preferably 1:40; the stirring rate is 1000-1500 rpm, and the stirring time is 8-12 h; the centrifugal rate is 8000-12000 rpm, and the centrifugal time is 5-10 min.

[0014] Preferably, in step 2), the mass ratio of the molybdenum acetylacetonate-doped metal organic framework Mo-ZIF-8 and tannic acid is 1:(2-4), the stirring treatment speed is 500-1000 rpm, and the stirring time is 1-20 min.

[0015] Preferably, in step 3), the pyrolysis treatment is carried out in a nitrogen atmosphere or an argon atmosphere, the pyrolysis temperature is 900-1000℃, the pyrolysis treatment time is 3-5 h, and the heating rate of the pyrolysis treatment is 2-5℃ / min.

[0016] The application further discloses a hollow mesoporous monatomic molybdenum nanozyme prepared by the preparation method.

[0017] Further, the particle size of the hollow mesoporous monatomic molybdenum nanozyme is preferably 120-150 nm, and the pore size is preferably 5-10 nm.

[0018] Further, the hollow mesoporous monatomic molybdenum nanozyme has a large internal space and a high specific surface area; in the application, the hollow mesoporous monatomic molybdenum nanozyme has a high peroxidase activity and can also be used as a carrier for loading natural enzymes.

[0019] The application further discloses a method for preparing a nanozyme reactor based on the hollow mesoporous monatomic molybdenum nanozyme.

[0020] S1: mixing the hollow mesoporous monatomic molybdenum nanozyme HMMo-SAzyme and a glucose oxidase aqueous solution, stirring to assemble, centrifuging to collect the precipitate, and obtaining the monatomic molybdenum nanozyme HMMo / Gox loaded with the glucose oxidase;

[0021] S2: adding the monatomic molybdenum nanozyme HMMo / Gox loaded with the glucose oxidase into a hyaluronic acid aqueous solution, stirring to assemble, centrifuging to collect the precipitate, washing, and freeze-drying to prepare the nanozyme reactor HMMo / GOx@HA.

[0022] Preferably, in S1, the mass ratio of the hollow mesoporous monatomic molybdenum nanoszyme HMMo-SAzyme and glucose oxidase used is 1:(1-3); the stirring assembly temperature is 0-4 DEG C, the stirring rate is 200-1000 rpm, and the stirring time is 8-12 h.

[0023] Preferably, in S2, the mass ratio of the monatomic molybdenum nanoszyme HMMo / Gox loaded with glucose oxidase and hyaluronic acid used is 1:(5-10); the molecular weight of the hyaluronic acid used is 20 kDa-40 kDa; the stirring rate is 500-1000 rpm; and the stirring time is 2-6 h.

[0024] The application further discloses a nanoszyme reactor prepared by the method.

[0025] Further, the particle size of the nanoszyme reactor is preferably 150-200 nm.

[0026] Further, in the application, the nanoszyme reactor can generate hydroxyl radicals by using glucose as a substrate through high-efficiency cascade catalytic reaction.

[0027] The application further discloses application of the nanoszyme reactor as a drug carrier.

[0028] The application further discloses application of the nanoszyme reactor in preparation of an antibacterial drug or an antitumor drug.

[0029] Compared with the prior art, the application has the following beneficial effects:

[0030] The application first prepares a novel hollow mesoporous monatomic molybdenum nanoszyme (HMMo-SAzyme), which has a larger internal space and a higher specific surface area compared with the monatomic nanoszyme reported in the prior art, so that the HMMo-SAzyme has higher peroxidase activity and can be used as a carrier for loading natural enzymes. In addition, the larger pore size is beneficial to rapid transmission of a catalytic substrate in the HMMo-SAzyme.

[0031] Based on the above-mentioned hollow mesoporous single-atom molybdenum nanozyme innovation, the application loads glucose oxidase (GOx) on the hollow mesoporous single-atom molybdenum nanozyme and wraps it with hyaluronic acid (HA) to construct a nanozyme reactor (HMMo / GOx@HA) with broad-spectrum antibacterial effect. The nanozyme reactor (HMMo / GOx@HA) can generate hydroxyl radicals (·OH) by efficient cascade catalytic reaction with glucose as substrate, thereby effectively inhibiting wound bacterial infection and promoting wound healing. The HA shell of the nanozyme reactor (HMMo / GOx@HA) can be degraded by hyaluronidase at the infection site, and the exposed GOx can effectively convert glucose into abundant gluconic acid and H2O2, avoiding the direct use of high-concentration and high-toxicity H2O2 and reducing the damage to normal cells and tissues. At the same time, the generated gluconic acid can reduce the pH value of the bacterial infection site, thereby effectively improving the peroxidase-like activity of HMMo-SAzyme, and H2O2 is further catalyzed to generate ·OH with stronger oxidizing property, thereby destroying the cell membrane and cell wall structure of bacteria, inactivating proteins, and ultimately leading to bacterial death. Therefore, it can be widely used in the preparation of antibacterial drugs and antitumor drugs. BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1 are scanning electron microscope and transmission electron microscope images of HMMo-SAzyme; wherein, a is a scanning electron microscope image; b is a transmission electron microscope image; c is an EDS element mapping image; d is an HAADF-STEM image;

[0033] Figure 2 are nitrogen adsorption isotherm and pore size distribution results of HMMo-SAzyme; wherein, a is a nitrogen adsorption isotherm; b is a pore size distribution;

[0034] Figure 3 are particle size and Zeta potential change results in the preparation process of HMMo / GOx@HA; wherein, a is particle size; b is Zeta potential;

[0035] Figure 4 is a transmission electron microscope image of HMMo / GOx@HA;

[0036] Figure 5 is a cascade catalytic reaction performance of HMMo / GOx@HA;

[0037] Figure 6Fig. 2 is a graph of the survival rate of S. aureus after different treatments; wherein a is the colony photograph of S. aureus after different treatments, and b is the corresponding survival rate column chart; a in which: 1 is PBS, 2 is glucose, 3 is HMMo / GOx@HA, 4 is glucose+HMMo-SAzyme, 5 is glucose+GOx, 6 is glucose+HMMo / GOx@HA;

[0038] Figure 7 Fig. 3 is a graph of the survival rate of E. coli after different treatments; wherein a is the colony photograph of E. coli after different treatments, and b is the corresponding survival rate column chart; a in which: 1 is PBS, 2 is glucose, 3 is HMMo / GOx@HA, 4 is glucose+HMMo-SAzyme, 5 is glucose+GOx, 6 is glucose+HMMo / GOx@HA;

[0039] Figure 8 Fig. 4 is the experimental result of treating wound infection by using the nano-reactor; wherein a is the photograph of the infected wound of the mouse after being treated in different ways at different times; b is the relative wound area of the infected wound of the mouse at different times; c is the bacterial colony photograph of the bacteria formed at the tissue of the wound of the mouse, c in which: 1 is PBS, 2 is glucose, 3 is HMMo / GOx@HA, 4 is glucose+HMMo-SAzyme, 5 is glucose+GOx, 6 is glucose+HMMo / GOx@HA; d is the number of surviving bacteria at the tissue of the wound of the mouse;

[0040] Figure 9 Fig. 5 is the survival rate of 4T1 cells after being treated by the nano-enzyme reactor. DETAILED DESCRIPTION

[0041] In order to enable persons skilled in the art to better understand the present application, the technical solutions in the embodiments of the present application will be described clearly and completely in combination with the drawings in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by persons skilled in the art without creative labor should belong to the protection scope of the present application.

[0042] It should be noted that the terms "first", "second", and the like in the description and in the claims of the present application and above-described accompanying drawings are used to distinguish similar objects, and do not necessarily indicate a specific order or sequence. It should be understood that the data thus used can be interchanged under appropriate circumstances, so that the embodiments of the application described herein can be implemented in an order other than that illustrated or described herein. In addition, the terms "include" and "have" and any variations thereof are intended to cover non-exclusive inclusion, for example, a process, method, system, product or device including a series of steps or units does not necessarily have to be limited to those steps or units clearly listed, but can include other steps or units not clearly listed or inherent to these processes, methods, products or devices.

[0043] The present application will be further described in detail below with reference to the accompanying drawings:

[0044] Example 1

[0045] Preparation of hollow mesoporous monatomic molybdenum nanozyme, comprising the following steps:

[0046] (1) Preparation of Mo-ZIF-8

[0047] Dissolve 90 mmol of 2-methylimidazole in 80 mL of anhydrous methanol, and ultrasonic for 30 min to obtain a first solution. Dissolve 5.6 mmol of zinc nitrate hexahydrate and 130 mg of molybdenum acetylacetonate in 80 mL of anhydrous methanol, and ultrasonic for 30 min to obtain a second solution. Slowly add the second solution to the first solution, then stir the mixed solution at room temperature for 12 h, after standing and precipitation, centrifuge the obtained product at 8500 rpm for 5 min, remove the anhydrous methanol, wash the obtained white product with anhydrous methanol for 3 times, and then freeze-dry for 12 h to obtain a metal organic framework doped with molybdenum acetylacetonate (Mo-ZIF-8).

[0048] (2) Preparation of HMo-ZIF-8

[0049] Weigh 50 mg of Mo-ZIF-8 prepared by the above method and disperse it in 20 mL of ultrapure water, and ultrasonic for 30 min at room temperature to make it disperse into a uniform solution. Dissolve 50 mg of tannic acid in 2 mL of ultrapure water, and ultrasonic for 30 min at room temperature until it is dissolved. Take 0.75 mL of the tannic acid aqueous solution and drop it into the above Mo-ZIF-8 aqueous solution, and slowly stir for 10 min at room temperature. Then centrifuge the obtained product at 12000 rpm for 5 min to remove the supernatant, wash it with ultrapure water for 3 times, and then freeze-dry for 12 h to obtain Mo-ZIF-8 with hollow structure (HMo-ZIF-8).

[0050] (3) Preparation of HMMo-SAzyme

[0051] The HMo-ZIF-8 prepared by the above method was ground into powder, then loaded into a clean corundum magnetic boat, and placed in a tube furnace for high-temperature pyrolysis under the protection of a nitrogen atmosphere, with a heating rate of 5°C / min, and the temperature was raised to 900°C. After high-temperature pyrolysis for 3h, the temperature was naturally cooled to room temperature, and a hollow mesoporous single-atom molybdenum nanoscale enzyme (HMMo-SAzyme) was obtained.

[0052] The preparation of a nanoscale enzyme reactor includes the following steps:

[0053] S1: Preparation of HMMo / GOx

[0054] 4mg of HMMo-SAzyme prepared by the above method was dissolved in 4mL of ultrapure water, and ultrasonic treatment was performed at room temperature for 30min to make it uniformly dispersed. 4mg of glucose oxidase was weighed and dissolved in 4mL of ultrapure water, and ultrasonic treatment was performed at an ice bath for 5min to make it dissolved. 4mL of glucose oxidase solution was added to 4mL of HMMo-SAzyme solution, and slowly stirred at an ice bath for 12h, and then centrifuged and washed with water to obtain a single-atom molybdenum nanoscale enzyme loaded with glucose oxidase (HMMo / GOx).

[0055] S2: Preparation of HMMo / GOx@HA nanoscale reactor

[0056] 32mg of hyaluronic acid was dissolved in 4mL of ultrapure water, and ultrasonic treatment was performed at room temperature for 30min to make it dissolved. Then, the prepared HMMo / GOx was added to 4mL of hyaluronic acid aqueous solution, and stirred at room temperature for 6h, and then centrifuged and washed with water to obtain a nanoscale enzyme reactor (HMMo / GOx@HA).

[0057] Example 2

[0058] Different from example 1, Mo-ZIF-8, HMo-ZIF-8, and HMMo / GOx in example 2 were prepared in different proportions.

[0059] The preparation of a hollow mesoporous single-atom molybdenum nanoscale enzyme includes the following steps:

[0060] (1) Preparation of Mo-ZIF-8

[0061] Molybdenum Acetylacetonate Doped Metal-Organic Framework (Mo-ZIF-8) was prepared by dissolving 45 mmol of 2-methylimidazole in 80 mL of anhydrous methanol under ultrasonic for 30 min to obtain a first solution. Dissolving 5.6 mmol of zinc nitrate hexahydrate and 90 mg of molybdenum acetylacetonate in 40 mL of anhydrous methanol under ultrasonic for 30 min to obtain a second solution. The second solution was slowly added to the first solution, and the mixed solution was stirred at room temperature for 12 h. After standing and precipitation, the obtained product was centrifuged at 8500 rpm for 5 min, the anhydrous methanol was removed, and the obtained white product was washed with anhydrous methanol for 3 times, and then freeze-dried for 12 h to obtain the molybdenum acetylacetonate doped metal-organic framework (Mo-ZIF-8).

[0062] (2) Preparation of HMo-ZIF-8

[0063] The Mo-ZIF-8 prepared by the above method was weighed at 100 mg and dispersed in 20 mL of ultrapure water under ultrasonic for 30 min at room temperature to make it disperse into a uniform solution. 50 mg of tannic acid was dissolved in 2 mL of ultrapure water under ultrasonic for 30 min at room temperature, and after it was dissolved, 1.25 mL of tannic acid aqueous solution was added dropwise to the above Mo-ZIF-8 aqueous solution, and slowly stirred at room temperature for 10 min. Then the obtained product was centrifuged at 12000 rpm for 5 min to remove the supernatant, washed with ultrapure water for 3 times, and then freeze-dried for 12 h to obtain the hollow structure Mo-ZIF-8 (HMo-ZIF-8).

[0064] (3) Preparation of HMMo-SAzyme

[0065] The HMo-ZIF-8 prepared by the above method was ground into powder, then placed in a clean corundum magnetic boat, and placed in a tube furnace for high temperature pyrolysis under the protection of nitrogen atmosphere, and the heating rate was adjusted to 5 ℃ / min, and the temperature was raised to 900 ℃. After high temperature pyrolysis for 3 h, it was naturally cooled to room temperature to obtain the hollow mesoporous single-atom molybdenum nanoscale enzyme (HMMo-SAzyme).

[0066] The preparation of the nanoscale enzyme reactor includes the following steps:

[0067] S1: Preparation of HMMo / GOx

[0068] 4 mg of HMMo-SAzyme prepared by the above method was dissolved in 4 mL of ultrapure water under ultrasonic for 30 min at room temperature to make it disperse uniformly. 8 mg of glucose oxidase was weighed and dissolved in 4 mL of ultrapure water under ultrasonic for 5 min in an ice bath to make it dissolve. 4 mL of glucose oxidase solution was added to 4 mL of HMMo-SAzyme solution, and slowly stirred in an ice bath for 12 h, and then centrifuged and washed with water to obtain the single-atom molybdenum nanoscale enzyme loaded with glucose oxidase (HMMo / GOx).

[0069] S2: Fabrication of HMMo / GOx@HA nanoreactors

[0070] 32 mg of hyaluronic acid was dissolved in 4 mL of ultrapure water and sonicated for 30 min at room temperature until dissolved. Then, the prepared HMMo / GOx was added to 4 mL of hyaluronic acid aqueous solution, stirred at room temperature for 6 h, and then centrifuged and washed with water to obtain the nanoreactor (HMMo / GOx@HA).

[0071] Experimental results: Scanning electron microscopy and transmission electron microscopy images show that the HMMo-SAzyme prepared in Example 2 has a hollow and porous structure. Figure 1 As shown in a and b). Energy dispersive X-ray spectroscopy (EDS) analysis showed that carbon, nitrogen, and molybdenum were uniformly distributed in the HMMo-SAzyme. Figure 1 (As shown in c). Atomically-resolution aberration-corrected high-angle annular dark-field scanning electron microscopy (HAADF-STEM) was used to observe molybdenum in the pyrolysis sample at the atomic scale more directly. The results are as follows... Figure 1 As shown in Figure d, numerous monodisperse bright spots exist in the disordered porous carbon layer of HMMo-SAzyme. Since molybdenum atoms have higher contrast than carbon and nitrogen atoms along the Z-axis, these monodisperse bright spots indicate that molybdenum exists in atomic form within the nitrogen-doped hollow carbon substrate. Nitrogen adsorption isotherms further confirm that HMMo-SAzyme has a hollow mesoporous structure. Figure 2 Its pore size is 7.54 nm, and its total pore volume is 0.33 cm³. 3 / g, with a specific surface area of ​​346.72m². 2 / g. The above results indicate that the hollow mesoporous single-atom molybdenum nanozyme was successfully prepared.

[0072] During the preparation of the HMMo / GOx@HA nanoreactor, the hydration particle size of the product at each step was measured. Figure 3 a) and Zeta potential ( Figure 3 (b) Changes in hydrated particle size and zeta potential support the successful fabrication of the HMMo / GOx@HA nanoreactor. Transmission electron microscopy revealed that the HMMo / GOx surface was coated with a viscous HA layer ( Figure 4 This indicates that HA successfully encapsulated HMMo / GOx.

[0073] This invention presents experimental research on the relevant performance of the prepared hollow mesoporous single-atom molybdenum nanozyme and its nanozyme reactor:

[0074] 1. Study on cascade catalytic reactions in nanozyme reactors

[0075] The cascade catalytic activity of the nanoreactor was detected by 3,3',5,5'-tetramethylbenzidine (TMB). After the HMMo / GOx@HA was treated by 150 U / mL of HAase, it reacted with glucose and TMB in PBS (0.5 mM, pH 7.0) buffer, in which the concentration of HMMo / GOx@HA was 100 μg / mL, the concentration of glucose was 5 mM, the reaction was carried out at 37 °C for 3 h, and the absorbance change of the solution was detected at 652 nm. At the same time, HMMo / GOx+glucose+TMB and HMMo / GOx@HA+glucose+TMB groups were set as controls.

[0076] Experimental results: Under the same glucose concentration, there was no significant change in the absorbance at 652 nm after adding TMB when the HMMo / GOx@HA was not treated by hyaluronidase, while the absorbance change at 652 nm after adding TMB when the HMMo / GOx@HA was treated by hyaluronidase was basically the same as that of HMMo / GOx Figure 5 ). This indicates that the HMMo / GOx@HA can be activated by hyaluronidase to occur cascade catalytic reaction.

[0077] 2. Plate coating method for characterizing the antibacterial performance of nanoscale enzyme reactor

[0078] A sterile inoculation loop was used to pick single colonies of S. aureus from LB agar medium and transfer them to LB liquid medium, which was placed in a 37 °C constant temperature incubator for culture. After culture to the logarithmic growth phase, the OD600 was measured and the bacteria were diluted to 10 7 CFU / mL with LB liquid medium. The bacterial suspension of S. aureus was treated with (1) PBS, (2) glucose, (3) HMMo / GOx@HA, (4) glucose+HMMo-SAzyme, (5) glucose+GOx, (6) glucose+HMMo / GOx@HA, respectively, and the concentrations of glucose and HMMo / GOx@HA were 15 mM and 100 μg / mL, respectively. Each group of bacterial solution was placed in a 37 °C constant temperature incubator (180 rpm) for incubation for 5 h, and then the bacterial solution was diluted 10 4 times, 100 μL of the diluted bacterial suspension was spread on LB agar medium, which was placed in a 37 °C constant temperature incubator for culture for 24 h, and 3 parallel samples were set for each group. Finally, the antibacterial effect of the nanoreactor was evaluated by counting the number of colonies (CFU) on the LB agar plate.

[0079] Experimental results: The results are shown in Figure 6As shown in Figures a and b, compared with the PBS treatment group (1), the bacterial survival rate of the treatment groups (2)-(4) remained basically unchanged, indicating that the antibacterial ability of glucose, HMMo / GOx@HA nanoreactor, and glucose + HMMo-SAzyme alone was negligible. After treatment with glucose + GOx in group (5), the survival rate of Staphylococcus aureus decreased, but it was still as high as 64.77%, indicating that the antibacterial effect of H2O2 produced by GOx catalyzing glucose oxidation was limited. It is worth noting that in group (6), which used both glucose and HMMo / GOx@HA nanoreactor, the survival rate of Staphylococcus aureus decreased significantly, indicating that the HMMo / GOx@HA nanoreactor can undergo a cascade catalytic reaction to catalyze the production of ·OH from glucose, thereby killing bacteria.

[0080] 3. Experiment on the treatment of wound infection using nanoreactors

[0081] Male BALB / c mice aged 6–8 weeks were selected. A 6 mm diameter wound was created on their backs after shaving. 50 μL of 10... 7 A wound infection model was established by injecting a CFU / mL Staphylococcus aureus suspension into the wound. After 24 hours, mice infected with Staphylococcus aureus were randomly divided into 6 groups of 5 mice each. The wounds were treated with different samples every 24 hours: (1) PBS, (2) glucose, (3) HMMo / GOx@HA, (4) glucose + HMMo-SAzyme, (5) glucose + GOx, and (6) glucose + HMMo / GOx@HA, where the glucose concentration was 15 mM and the HMMo / GOx@HA concentration was 100 μg / mL. The infected wounds of each group were photographed and measured every other day. On the 7th day after treatment, wound tissue from the mice was collected, homogenized thoroughly, and placed in 1 mL of sterile saline. The tissue was incubated at 37°C for 24 hours, and then the bacterial samples from the infected site were analyzed using the dilution plate method.

[0082] Experimental results: The results are as follows Figure 8 As shown in a and b, compared with group (1) PBS, the wound healing degree of groups (2)–(5) remained essentially unchanged. However, the wound area of ​​mice treated with group (6) glucose + HMMo / GOx@HA nanoreactor was significantly reduced, and by day 7 of treatment, the wounds were almost completely healed, with new pink skin observed. The number of bacteria on the mouse wounds was determined using the plate count method, and the results are as follows: Figure 8 As shown in c and d, after treatment with glucose and the HMMo / GOx@HA nanoreactor, the bacterial survival rate decreased significantly to only 4.28%, which was significantly lower than that of other treatment groups. This indicates that the ·OH generated by the cascade catalytic reaction of the HMMo / GOx@HA nanoreactor can effectively inhibit bacterial infection and promote wound healing.

[0083] 4. Study on antibacterial performance of nanoreactor against E. coli

[0084] Single colony of E. coli was picked up from LB agar medium with sterile inoculation loop and transferred into LB liquid medium, which was placed in a constant temperature shaking incubator at 37°C for culture. After culture to logarithmic growth phase, OD600 was measured and the bacteria were diluted to 10 7 CFU / mL with LB liquid medium. The bacterial suspension of E. coli was treated with (1) PBS, (2) glucose, (3) HMMo / GOx@HA, (4) glucose + HMMo-SAzyme, (5) glucose + GOx, (6) glucose + HMMo / GOx@HA, respectively. The concentrations of glucose and HMMo / GOx@HA were 15 mM and 100 μg / mL, respectively. Each group of bacterial solution was incubated in a constant temperature shaking incubator (180 rpm) at 37°C for 5 h, and then the bacterial solution was diluted 10 4 times, and 100 μL of the diluted bacterial suspension was spread on LB agar medium and placed in a constant temperature incubator at 37°C for 24 h. Three parallel samples were set for each group. Finally, the antibacterial effect of nanoreactor was evaluated by counting the number of colonies (CFU) on LB agar plates.

[0085] Experimental results: The results are shown in Figure 7 Compared with (1) PBS treatment group, the survival rate of bacteria in (2)-(4) treatment groups was basically unchanged, indicating that the antibacterial ability of glucose, HMMo / GOx@HA nanoreactor and glucose + HMMo-SAzyme alone was negligible. Although the survival rate of E. coli treated with (5) glucose + GOx decreased, it was still as high as 70.26%, indicating that the antibacterial effect of H2O2 produced by GOx catalyzing glucose oxidation was limited. Notably, group (6) used both glucose and HMMo / GOx@HA nanoreactor, and the survival rate of E. coli decreased significantly, indicating that HMMo / GOx@HA nanoreactor could occur cascade catalytic reaction to catalyze glucose to produce ·OH, thereby killing bacteria.

[0086] Example 3

[0087] Killing effect of HMMo / GOx@HA nanoreactor on tumor cells

[0088] Mouse breast cancer cells 4T1 were seeded at 5 x 10 3The cell density of the tumor cells was inoculated on a 96-well plate for a total incubation of 12 hours, and then the culture medium was removed. The HMMo / GOx@HA was dispersed in the culture medium, and was co-incubated with the cells for 12 hours, and the concentration of the HMMo / GOx@HA was 12.5, 25, 50, 100 and 200 mu g / mL respectively. After the co-incubation, the cells were washed with PBS for 3 times, new culture medium was added to each well, and 10% CCK-8 was added to each well for a co-incubation of 2 hours. Finally, the absorbance at 450 nm was detected by an enzyme label instrument, and the survival rate of the cells was calculated.

[0089] The experimental results are shown in Table 1. Figure 9 As shown in Table 1, with the increase of the concentration of the HMMo / GOx@HA, the survival rate of the tumor cells gradually decreased, and when the concentration of the HMMo / GOx@HA reached 100 mu g / mL, the survival rate of the tumor cells was 19.93%, which indicated that the HMMo / GOx@HA nano-reactor had a certain killing effect on the tumor cells.

[0090] In summary, the advantages of the present application are very obvious.

[0091] (1) The present application first prepared a new type of hollow mesoporous single-atom molybdenum nanozyme, which not only has high peroxidase activity, but also can be used as a carrier for loading natural enzymes and drugs.

[0092] (2) The present application provides a nano-reactor, which can occur a cascade catalytic reaction at the site of bacterial infection, effectively inhibit bacterial infection of the wound, and promote wound healing.

[0093] (3) The present application provides a preparation method of the hollow mesoporous single-atom molybdenum nanozyme and the nano-reactor, which has a simple and controllable preparation process, a stable structure of the obtained nano-reactor, and further, the metal material used in the preparation method of the present application is widely available and low in cost.

[0094] The above content only illustrates the technical idea of the present application, and cannot limit the protection scope of the present application. Any modification made according to the technical idea of the present application on the basis of the technical solution falls within the protection scope of the claims of the present application.

Claims

1. A method for preparing a nanozyme reactor based on hollow mesoporous single-atom molybdenum nanozymes, characterized in that, Includes the following steps: S1: The hollow mesoporous single-atom molybdenum nanozyme HMMo-SAzyme and glucose oxidase aqueous solution were mixed, stirred and assembled, and the precipitate was collected by centrifugation to obtain single-atom molybdenum nanozyme HMMo / Gox loaded with glucose oxidase. The hollow mesoporous single-atom molybdenum nanozyme HMMo-SAzyme was prepared according to the following steps: Step 1) Prepare a molybdenum acetylacetone-doped metal-organic framework Mo-ZIF-8; Step 2) Disperse the molybdenum acetylacetonate-doped metal-organic framework Mo-ZIF-8 in water, add tannic acid aqueous solution for etching, then stir, centrifuge to collect the precipitate, wash and dry the precipitate to obtain HMo-ZIF-8 with a hollow structure. Step 3) Pyrolyze HMo-ZIF-8 with a hollow structure to obtain hollow mesoporous single-atom molybdenum nanozyme HMMo-Sazyme; S2: The single-atom molybdenum nanozyme HMMo / Gox loaded with glucose oxidase was added to a hyaluronic acid aqueous solution, stirred and assembled, centrifuged to collect the precipitate, washed and freeze-dried to obtain the nanozyme reactor HMMo / GOx@HA.

2. The method for preparing a nanozyme reactor based on hollow mesoporous single-atom molybdenum nanozymes according to claim 1, characterized in that, In S1, the mass ratio of the hollow mesoporous single-atom molybdenum nanozyme HMMo-SAzyme to glucose oxidase is 1:(1-3); the stirring assembly temperature is 0-4℃, the stirring rate is 200-1000 rpm, and the stirring time is 8-12 h.

3. The method for preparing a nanozyme reactor based on hollow mesoporous single-atom molybdenum nanozymes according to claim 1, characterized in that, In S2, the mass ratio of the single-atom molybdenum nanozyme HMMo / Gox loaded with glucose oxidase to hyaluronic acid is 1:(5-10); the molecular weight of the hyaluronic acid used is 20 kDa-40 kDa; the stirring rate is 500-1000 rpm; and the stirring time is 2-6 h.

4. The method for preparing a nanozyme reactor based on hollow mesoporous single-atom molybdenum nanozymes according to claim 1, characterized in that, In step 2), the mass ratio of molybdenum acetylacetone-doped metal-organic framework Mo-ZIF-8 to tannic acid is 1:(2~4), the stirring speed is 500-1000 rpm, and the stirring time is 1-20 min.

5. The method for preparing a nanozyme reactor based on hollow mesoporous single-atom molybdenum nanozymes according to claim 1, characterized in that, In step 3), the pyrolysis treatment is carried out in a nitrogen atmosphere or an argon atmosphere, the pyrolysis temperature is 900~1000 ℃, the pyrolysis treatment time is 3~5 h, and the heating rate of the pyrolysis treatment is 2-5℃ / min.

6. The method for preparing a nanozyme reactor based on hollow mesoporous single-atom molybdenum nanozymes according to claim 1, characterized in that, The hollow mesoporous single-atom molybdenum nanozyme HMMo-SAzyme has a particle size of 50~200 nm and a pore size of 5~20 nm.

7. A nanozyme reactor prepared by the method according to any one of claims 1 to 6, characterized in that, The nanozyme reactor has a particle size of 100~300 nm.

8. The application of the nanozyme reactor according to claim 7 as a drug carrier, characterized in that, The drug carrier is a drug carrier that encapsulates antibacterial drugs or antitumor drugs.

9. The use of the nanozyme reactor according to claim 7 in the preparation of antibacterial drugs or antitumor drugs.

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