Biomimetic nanomedicines for combating subcutaneous drug-resistant bacterial infections, their preparation methods and applications

Abstract

This invention belongs to the field of pharmaceutical technology, specifically relating to biomimetic nanomedicines for treating subcutaneous drug-resistant bacterial infections, their preparation methods, and applications. In this invention, dopamine hydrochloride is added during the synthesis of a metal-organic framework to prepare a dandelion-shaped carbon nanozyme precursor. This precursor is then carbonized to obtain a carbon nanozyme with targeting, photoresponsiveness, and peroxidase activity. Finally, berberine hydrochloride is loaded onto the carbon nanozyme to obtain the biomimetic nanomedicine. The biomimetic nanomedicine provided by this invention firstly possesses good biocompatibility; secondly, it has a unique dandelion-like morphology, which is beneficial for its targeting effect; and finally, it exhibits strong photoresponsiveness in the near-infrared region, enabling synergistic and efficient bactericidal action through the mechanisms of antibacterial drugs, photothermal therapy, and chemokinetics. It has significant application value in the preparation of drugs for treating subcutaneous drug-resistant bacterial infections.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to biomimetic nanomedicines for combating subcutaneous drug-resistant bacterial infections, their preparation methods, and applications. Background Technology

[0002] The invention and widespread use of antibiotics have provided humanity with a powerful weapon against bacterial infections. However, with the widespread use of antibiotics, bacteria have gradually developed various resistance mechanisms, such as permeability barriers, inactivating enzymes, drug efflux pumps, target modification, and secretory biofilms. This not only reduces the therapeutic effect of antibiotics but also leads to widespread resistance to existing antibiotics. Bacterial biofilms can shield against external damage and prevent drugs from penetrating into the bacterial interior. When the skin barrier is damaged, the permeability of some blood vessels increases, causing nutrients and tissue fluid supplied to normal cells to leak out, creating a moist environment that provides a suitable environment and material basis for bacterial anchoring and proliferation. After anchoring on the wound surface, bacteria multiply rapidly, forming biofilms. The bacteria encased in these biofilms are in a state of slow growth and starvation, resulting in low sensitivity to antibiotics and other chemical drugs. If the bacteria or biofilms remaining after drug-resistant bacterial infection cannot be eradicated, the microenvironment of infection will continue to deteriorate, further leading to high fever and potentially inducing serious complications. Therefore, developing an effective treatment strategy to combat bacterial infections and overcome bacterial resistance is crucial.

[0003] Nanomedicines can reduce the toxic side effects of drugs, increase local tissue uptake, and enable controlled / responsive drug release. Currently reported nanozyme drug delivery systems primarily load Western medicines, antibiotics, and small molecule drugs. However, overuse of antibiotic-loaded nanozymes can lead to the proliferation of drug-resistant bacteria and even the emergence of multidrug-resistant superbugs.

[0004] Berberine, also known as berberine hydrochloride, is a quaternary ammonium alkaloid widely distributed in the plant kingdom. Berberine hydrochloride is the most common form of berberine and is a drug with various medical potentials, possessing antimalarial, antifungal, antioxidant, and anti-inflammatory properties. Berberine hydrochloride can interfere with biofilm formation by inhibiting the formation of extracellular amyloid peptides in bacteria; it can also bind to some proteins on the biofilm, disrupting their structure and further damaging the integrity and permeability of the bacterial biofilm. Given its excellent antibacterial activity, berberine hydrochloride can serve as an ideal alternative to antibiotics; therefore, there is an urgent need to develop a method for preparing a nanoenzyme drug delivery system that uses active ingredients from traditional Chinese medicine to replace antibiotics. Summary of the Invention

[0005] To address the technical problems existing in the prior art, the purpose of this invention is to provide a biomimetic nanomedicine for combating subcutaneous drug-resistant bacterial infections, its preparation method, and its applications. The biomimetic nanomedicine provided by this invention firstly possesses good biocompatibility; secondly, it has a unique dandelion-like morphology, which is beneficial for targeted action; and finally, it exhibits strong photoresponse in the near-infrared region, enabling synergistic and efficient bactericidal action through the mechanisms of antibacterial drugs, photothermal therapy, and chemokinetics. It has significant application value in the preparation of drugs for treating subcutaneous drug-resistant bacterial infections.

[0006] The first aspect of this invention provides a method for preparing a biomimetic nanomedicine for resisting subcutaneous drug-resistant bacterial infections, comprising the following steps:

[0007] 2-Methylimidazole was dissolved in methanol to obtain a mixed methanol solution. A mixed aqueous solution of zinc salt and dopamine hydrochloride was added to the above mixed methanol solution. After centrifugation, a dandelion-shaped carbon nanozyme precursor was obtained.

[0008] Carbon nanozyme precursors were carbonized to obtain dandelion-shaped carbon nanozymes with targeting, photoresponsiveness and peroxidase activity.

[0009] Berberine hydrochloride, which has antibacterial properties, is mixed with carbon nanozymes to load berberine hydrochloride onto the carbon nanozymes. The mixture is then centrifuged and dried to obtain the biomimetic nanomedicine.

[0010] Furthermore, the zinc salt is zinc nitrate, and the Zn in zinc nitrate... 2+ It can coordinate with the imidazole group of dimethylimidazole to form a metal complex. At the same time, zinc nitrate usually only releases nitrate ions in the reaction, which will not interfere with the formation of carbon nanozyme precursors, and it has high water solubility and stability.

[0011] Furthermore, the zinc salt is zinc nitrate hexahydrate.

[0012] Further, the mass ratio of 2-methylimidazole, zinc nitrate hexahydrate and dopamine hydrochloride is 0.32~0.45:1.4375~2.1375:0.095~0.105.

[0013] Furthermore, the mass ratio of berberine hydrochloride to carbon nanozyme is 1~3:1.

[0014] Furthermore, the centrifugation speed is 11000 r / min to 12000 r / min, and the centrifugation time is 8 min to 15 min; under these centrifugation conditions, the precipitate can be effectively separated.

[0015] Furthermore, the drying temperature is 55℃~65℃, and the drying time is 8h~12h; at this drying temperature, the performance of the nanozyme precursor will not be affected, and it can be thoroughly dried overnight.

[0016] Furthermore, the carbonization temperature is 800℃~1000℃ and the time is 2h~4h; under these carbonization conditions, it is possible to avoid the inability to synthesize carbon nanozymes due to excessively low carbonization temperature, and also to avoid the decomposition of zinc ions due to excessively high carbonization temperature.

[0017] A second aspect of the present invention provides a biomimetic nanomedicine prepared using the above-described method for preparing biomimetic nanomedicines against subcutaneous drug-resistant bacterial infections.

[0018] A third aspect of the present invention provides the application of the above-described biomimetic nanomedicine in the preparation of a drug for treating subcutaneous drug-resistant bacterial infections.

[0019] Furthermore, the bacteria is Staphylococcus aureus.

[0020] The fourth aspect of this invention provides the application of the above-described biomimetic nanomedicine in the preparation of chemokinetic therapeutic agents.

[0021] The fifth aspect of this invention provides the application of the above-described biomimetic nanomedicine in the preparation of photothermal therapy reagents.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] This invention discloses for the first time a method for preparing biomimetic nanomedicines against subcutaneous drug-resistant bacterial infections. By adding dopamine hydrochloride during the synthesis of a metal-organic framework, the amino and hydroxyl groups in the dopamine hydrochloride molecule undergo a cross-linking reaction with carbon nanomaterials, enhancing the polymerization effect of the carbon nanozyme precursor. After centrifugation, a dandelion-like carbon nanozyme precursor is obtained. This precursor is then carbonized at 800℃~1000℃ for 2h~4h to obtain a carbon nanozyme with targeting, photoresponsiveness, and peroxidase activity. Finally, berberine hydrochloride, which has antibacterial activity, is loaded onto the carbon nanozyme to obtain the biomimetic nanomedicine. The preparation method provided by this invention is simple and easy to implement, offering new ideas and theoretical basis for developing nanozyme drugs loaded with other effective components of traditional Chinese medicine.

[0024] This invention discloses a biomimetic nanomedicine for combating subcutaneous drug-resistant bacterial infections. This biomimetic nanomedicine uses dandelion-like carbon nanozymes as a carrier, loading berberine hydrochloride, which has antibacterial activity. This invention constructs a synergistic antibacterial platform combining photothermal therapy, chemokinetic therapy, and antibacterial drugs, overcoming the limitations of single antibacterial strategies and the side effects and metabolic burden associated with high-dose antibiotics. The biomimetic nanomedicine exhibits highly efficient targeting. This biomimetic nanomedicine, possessing both three synergistic antibacterial modes and highly efficient targeting capabilities, has significant application value in the preparation of therapeutic drugs for subcutaneous drug-resistant bacterial infections.

[0025] (1) The carbon nanozymes provided by this invention have a higher specific surface area and more efficient targeting compared with conventional metal-organic framework materials ZIF-8. By adding dopamine hydrochloride during the preparation process, the carbon nanozymes are polymerized more effectively, preventing framework collapse and further forming a porous carbon nanozyme material with a dandelion-like structure. In addition, the addition of dopamine hydrochloride does not affect the good photothermal conversion ability of the carbon nanozymes, and the carbon nanozymes also have the catalytic activity and drug loading capacity of peroxidase. Then, the antibacterial substance berberine hydrochloride is loaded to obtain a biomimetic nanomedicine.

[0026] (2) The biomimetic nanomedicine preparation process provided by this invention is simple. In addition, carbon nanoenzymes, as a photothermal agent, can convert light energy into heat energy. They have strong light absorption in the near-infrared region and the local temperature can reach about 50°C. They can effectively kill bacteria without damaging normal tissues due to excessive heat, thus avoiding the generation of drug-resistant bacteria at the source.

[0027] (3) The biomimetic nanomedicine of the present invention has the catalytic activity of peroxidase. Compared with neutral conditions, higher enzyme activity can be observed under acidic conditions with a pH of 3.0 to 6.0, which indicates that it is beneficial to exert good antibacterial performance in the acidic environment of the subcutaneous infection area.

[0028] (4) The biomimetic nanomedicine of the present invention has good biocompatibility. In cell viability staining experiments, carbon nanozymes at concentrations as high as 200 μg / mL showed very limited toxicity to cells, with cell viability > 80%. In vitro hemolysis experiments showed that the hemolytic effect of carbon nanozymes was very low, indicating good blood compatibility. This shows that the effect of carbon nanozymes on cell viability during administration is almost negligible, demonstrating good biocompatibility.

[0029] (5) The biomimetic nanomedicine of the present invention has the ability to target the surface of bacteria. Due to the topological interaction between bacteria and rough surfaces, bacteria can easily attach to the surface of cells, tissues or other biological materials. Compared with conventional spherical carbon nanozymes, carbon nanozymes have a higher specific surface area, which can improve the efficiency of surface-based interactions. Therefore, the drug prepared by using biomimetic nanomedicine has a more efficient ability to treat subcutaneous infections caused by drug-resistant bacteria.

[0030] In summary, the biomimetic nanomedicine for treating subcutaneous drug-resistant bacterial infections provided by this invention has a unique dandelion-like morphology, good photothermal effect, targeting effect, high antibacterial ability and high peroxidase activity, and good biocompatibility. It has important application value in the preparation of drugs for treating subcutaneous drug-resistant bacterial infections. Attached Figure Description

[0031] Figure 1 Characterization results for different ZPDs; Figure 1 Figure A is a scanning electron microscope image of the ZPD prepared in Example 1; Figure 1 Figure B is a scanning electron microscope image of the ZPD prepared in Comparative Example 1; Figure 1 Figure C is a scanning electron microscope image of the ZPD prepared in Comparative Example 2; Figure 1 The D-plot is a particle size distribution diagram of the ZPD prepared in Example 1; Figure 1 Figure E shows the infrared spectrum of ZPD prepared in Example 1. The three curves from top to bottom are the infrared spectra of ZPD, ZPD precursor and dopamine hydrochloride, respectively. Figure 1 Figure F shows the ultraviolet spectrum of the ZPD prepared in Example 1. Curve 1 represents the ultraviolet spectrum of the ZPD precursor, and curve 2 represents the ultraviolet spectrum of the ZPD.

[0032] Figure 2 The results show the peroxidase activity characterization of ZPD. Figure 2 Figure A is a schematic diagram of ZPD enzyme activity. Curve 1 shows the enzyme activity characterization results of ZPD when TMB, hydrogen peroxide and ZPD are present simultaneously. Curve 2 shows the enzyme activity characterization results of ZPD when TMB and hydrogen peroxide are present. Curve 3 shows the enzyme activity characterization results of ZPD when TMB and ZPD are present. Curve 4 shows the enzyme activity characterization results of ZPD when only TMB is present. Figure 2 Figure B is Figure 2 Figure A shows the sample images corresponding to the enzyme activity assay. From left to right, the samples from the four tubes are as follows: Figure 2 The samples corresponding to curves 1, 2, 3 and 4 in Figure A; Figure 2 Figure C is a schematic diagram of ZPD enzyme activity. Curve 1 is a schematic diagram of ZPD enzyme activity when TMB, hydrogen peroxide and ZPD are present simultaneously and under NIR light irradiation. Curve 2 is a schematic diagram of ZPD enzyme activity when TMB, hydrogen peroxide and ZPD are present simultaneously. Curve 3 is a schematic diagram of ZPD enzyme activity when TMB and hydrogen peroxide are present and under NIR light irradiation. Curve 4 is a schematic diagram of ZPD enzyme activity when only TMB and hydrogen peroxide are present. Figure 2 The D diagram is Figure 2 The C-graph shows the sample images corresponding to the enzyme activity determination. From left to right, the samples from the four tubes are as follows: Figure 2 The samples corresponding to curves 1, 2, 3 and 4 in Figure C.

[0033] Figure 3 The standard curve for ZPD loading of berberine hydrochloride is given by the formula y = 0.0531744x + 0.0113125, where x is the concentration of berberine hydrochloride and y is the absorbance value.

[0034] Figure 4Results of photothermal drug release characteristics of ZPD@BBH; Figure 4 Figure A shows the photothermal stability of ZPD@BBH at different concentrations. The four curves from top to bottom are 150 μg / mL ZPD@BBH, 100 μg / mL ZPD@BBH, 50 μg / mL ZPD@BBH and H2O. Figure 4 Figure B shows the effect of different NIR irradiation powers on the photothermal stability of 100 μg / mL ZPD@BBH. The four curves correspond to powers of 1.0 W / cm² from top to bottom. 2 0.8W / cm 2 0.5W / cm 2 0.3W / cm 2 ; Figure 4 Figure C shows the 808 nm and 1.0 W / cm² values ​​of 100 μg / mL ZPD@BBH after three heating / cooling cycles. 2 Repeated experimental results of NIR laser irradiation; Figure 4 The D-plot shows the photothermal drug release characteristics of ZPD@BBH. The curve with the triangle icon represents the drug release rate of ZPD@BBH when the pH value is 5.5 and NIR light is applied. The curve with the square icon represents the drug release rate of ZPD@BBH when the pH value is 5.5. The curve with the circle icon represents the drug release rate of ZPD when the pH value is 7.4.

[0035] Figure 5 The results show the antibacterial properties of ZPD and ZPD@BBH against methicillin-resistant Staphylococcus aureus. Figure 5 Figure A shows the colony images of the untreated control group; Figure 5 Image B shows a colony image after hydrogen peroxide was added and the colony was treated with NIR light. Figure 5 Figure C shows the effect of different treatments on the colonies of methicillin-resistant Staphylococcus aureus. Figure 5 In Figure C, row (a) shows colony images of methicillin-resistant Staphylococcus aureus after treatment with ZPD at concentrations of 20 μg / mL, 40 μg / mL, 60 μg / mL, 80 μg / mL, and 100 μg / mL, from left to right. Figure 5 In Figure C, row (b) shows, from left to right, colony images of methicillin-resistant Staphylococcus aureus after adding 20 μg / mL, 40 μg / mL, 60 μg / mL, 80 μg / mL, and 100 μg / mL ZPD, along with hydrogen peroxide, and then treating with NIR light. Figure 5 In Figure C, row (c) shows colony images of methicillin-resistant Staphylococcus aureus after treatment with ZPD@BBH at concentrations of 20 μg / mL, 40 μg / mL, 60 μg / mL, 80 μg / mL, and 100 μg / mL, respectively, from left to right. Figure 5 In Figure C, row (d) shows, from left to right, colony images of methicillin-resistant Staphylococcus aureus after adding 20 μg / mL, 40 μg / mL, 60 μg / mL, 80 μg / mL, and 100 μg / mL ZPD@BBH, hydrogen peroxide, and NIR light, respectively. Figure 5 Figure D shows the antibacterial rates of ZPD and ZPD@BBH against methicillin-resistant Staphylococcus aureus. Solid bars in the bar chart (columns 1, 3, 5, 7, 9, and 11) represent those treated with NIR light without the addition of hydrogen peroxide. Hollow bars (columns 2, 4, 6, 8, 10, and 12) represent those treated with NIR light after the addition of hydrogen peroxide. The control bars (columns 1 and 2) correspond to... Figure 5 Figure A and Figure 5 The experimental results in Figure B correspond to the results in Figure B; ** indicates the relationship between the two groups. p <0.01.

[0036] Figure 6 The results of the biosafety test for ZPD@BBH; Figure 6 Figure A shows the fluorescence liveness / death staining of L929 cells in the ZPD@BBH treatment group and the blank control group. Figure 6 The top row of images in Figure A is a schematic diagram of the live and dead staining of L929 cells in the blank control group. Figure 6 The bottom row of images in Figure A shows a schematic diagram of the live and dead staining of L929 cells in the 100 μg / mL ZPD@BBH treatment group. Figure 6 In Figure A, each column from left to right represents a schematic diagram of live cell staining, a schematic diagram of dead cell staining, and a schematic diagram of staining a mixture of live and dead cells. Figure 6 Figure B shows the cell viability of L929 cells treated with different concentrations of ZPD@BBH, where ns indicates that there is no significant difference between the two groups. Figure 6 Figure C shows the results of the hemolysis experiment in mice. The standard range for normal hemolysis rate is below 5%. The dashed line indicates that the hemolysis rate of all concentrations of ZPD@BBH is below 2%, which is within the standard range. Figure 6 Figure (a) in Figure C is Figure 6 The actual images corresponding to the bar charts in Figure C. Figure 6 In diagram C(a), the tubes from left to right are connected to... Figure 6 The columns in diagram C correspond from left to right. Detailed Implementation

[0037] The present invention will be described in detail below with reference to specific embodiments, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments can be obtained commercially unless otherwise specified.

[0038] In the embodiments of this invention: carbon nanozymes were labeled as ZPD; berberine hydrochloride was labeled as BBH; biomimetic nanomedicine was labeled as ZPD@BBH; 3,3',5,5'-tetramethylbenzidine was labeled as TMB; NIR labeling was near-infrared light; L929 cells were purchased from Shanghai Chunmai Biotechnology Co., Ltd.

[0039] The overuse of antibiotics has led to widespread bacterial resistance to existing antibiotics. Traditional antibiotic treatments often have limited effectiveness against bacteria in biofilms, resulting in persistent and recurrent infections. Nanomedicines can reduce drug toxicity, increase local tissue uptake, and enable controlled / responsive drug release. Currently reported nanozyme drug delivery systems mostly load Western medicines, antibiotics, and small molecule drugs. Overuse of antibiotic-loaded nanozymes can lead to the proliferation of drug-resistant bacteria and even the emergence of multidrug-resistant superbugs. Berberine hydrochloride possesses excellent antibacterial activity and can serve as an ideal alternative to antibiotics. Therefore, developing a method for preparing a nanozyme drug delivery system that uses active ingredients from traditional Chinese medicine to replace antibiotics is of significant practical importance.

[0040] This invention provides a method for preparing and applying a biomimetic nanomedicine for combating subcutaneous drug-resistant bacterial infections. In this invention, dopamine hydrochloride is added during the synthesis of a metal-organic framework to facilitate the more efficient polymerization of carbon nanozymes and prevent framework collapse. After centrifugation, a dandelion-shaped carbon nanozyme precursor is obtained. This precursor is then carbonized to obtain a carbon nanozyme with targeting, photoresponsiveness, and peroxidase activity. Finally, berberine hydrochloride, which has antibacterial activity, is loaded onto the carbon nanozyme to obtain the biomimetic nanomedicine. The biomimetic nanomedicine provided by this invention firstly exhibits good biocompatibility; secondly, it possesses a unique dandelion-like morphology, which is beneficial for its targeting effect; and finally, it exhibits strong photoresponsiveness in the near-infrared region, enabling efficient bactericidal action through the synergistic effect of antibacterial drugs, photothermal therapy, and chemokinetics. Furthermore, the biomimetic nanomedicine of this invention shows higher enzyme activity under acidic conditions with a pH of 3.0–6.0. ​​Therefore, the biomimetic nanomedicine provided by this invention can exert good antibacterial properties in the acidic environment of subcutaneous infection areas, and has significant application value in the preparation of drugs for treating subcutaneous drug-resistant bacterial infections.

[0041] Example 1

[0042] A biomimetic nanomedicine for combating subcutaneous drug-resistant bacterial infections is prepared using the following steps:

[0043] S1. Preparation of ZPD precursor: 0.32 g of 2-methylimidazole was dissolved in 45 mL of methanol solution and stirred evenly on a magnetic stirrer to obtain a mixed methanol solution; then 1.4375 g of zinc nitrate hexahydrate and 100 mg of dopamine hydrochloride were placed in the same beaker, 21 mL of water was added, and the mixture was stirred at 800 r / min on a magnetic stirrer to fully dissolve the zinc nitrate hexahydrate and dopamine hydrochloride to obtain a mixed aqueous solution; the mixed aqueous solution was poured into the above mixed methanol solution and stirred at 800 r / min for 3 h at room temperature; after stirring, the mixture was centrifuged at 11000 r / min for 10 min, and then the uncarbonized nanozyme precipitate collected by centrifugation was washed with methanol solution. The centrifugation and methanol washing steps were repeated 3 times, and the precipitate was collected by centrifugation at 11000 r / min for 10 min again. The precipitate was placed in a vacuum drying oven and dried at 60 °C for 8 h to obtain a dandelion-shaped ZPD precursor;

[0044] S2. Preparation of ZPD: The ZPD precursor obtained in step S1 is placed in a tube furnace. Argon gas is first purged for 30 minutes to remove the residual air in the tube furnace. Then, in an argon atmosphere, the temperature is increased to 800°C at a rate of 5°C / min and held for 3 hours. After that, it is naturally cooled to 25°C to obtain ZPD.

[0045] S3. Preparation of ZPD@BBH: 10 mg of ZPD obtained in step S2 was ultrasonically dispersed in 10 mL of methanol, and then 10 mg of BBH was added. The mixture was stirred at 800 r / min for 12 h at room temperature on a magnetic stirrer. After stirring, the mixture was centrifuged at 12000 r / min for 10 min, and the precipitate was washed with methanol solution. The centrifugation and methanol washing steps were repeated 3 times. The precipitate was collected by centrifugation at 12000 r / min for 10 min again and placed in a vacuum drying oven. After drying at 60 °C for 8 h, the biomimetic nanomedicine ZPD@BBH was obtained.

[0046] Example 2

[0047] A biomimetic nanomedicine for combating subcutaneous drug-resistant bacterial infections is prepared using the following steps:

[0048] S1. Preparation of ZPD precursor: 0.38 g of 2-methylimidazole was dissolved in 45 mL of methanol solution and stirred evenly on a magnetic stirrer to obtain a mixed methanol solution; then 1.6675 g of zinc nitrate hexahydrate and 95 mg of dopamine hydrochloride were placed in the same beaker, 21 mL of water was added, and the mixture was stirred at 700 r / min on a magnetic stirrer to fully dissolve the zinc nitrate hexahydrate and dopamine hydrochloride to obtain a mixed aqueous solution; the mixed aqueous solution was poured into the above mixed methanol solution and stirred at 700 r / min for 3 h at room temperature; after stirring, the mixture was centrifuged at 11500 r / min for 10 min, and then the uncarbonized nanozyme precipitate collected by centrifugation was washed with methanol solution. The centrifugation and methanol washing steps were repeated 3 times, and the precipitate was collected by centrifugation at 11500 r / min for 10 min again. The precipitate was placed in a vacuum drying oven and dried at 65 °C for 10 h to obtain a dandelion-shaped ZPD precursor;

[0049] S2. Preparation of ZPD: The ZPD precursor obtained in step S1 is placed in a tube furnace. Argon gas is first purged for 30 minutes to remove the air remaining in the tube furnace. Then, the furnace is carbonized and calcined at 900°C in an argon atmosphere at a heating rate of 5°C / min and held for 2 hours. Finally, it is naturally cooled to 25°C to obtain ZPD.

[0050] S3. Preparation of ZPD@BBH: 10 mg of ZPD obtained in step S2 was ultrasonically dispersed in 10 mL of methanol, and then 20 mg of BBH was added. The mixture was stirred at 750 r / min for 10 h at room temperature on a magnetic stirrer. After stirring, the mixture was centrifuged at 11500 r / min for 10 min, and the precipitate was washed with methanol solution. The centrifugation and methanol washing steps were repeated 4 times. The precipitate was collected by centrifugation at 11500 r / min for 10 min again and placed in a vacuum drying oven. After drying at 65 °C for 8 h, the biomimetic nanomedicine ZPD@BBH was obtained.

[0051] Example 3

[0052] A biomimetic nanomedicine for combating subcutaneous drug-resistant bacterial infections is prepared using the following steps:

[0053] S1. Preparation of ZPD precursor: Dissolve 0.45 g of 2-methylimidazole in 45 mL of methanol solution and stir evenly on a magnetic stirrer to obtain a mixed methanol solution; then place 2.1375 g of zinc nitrate hexahydrate and 105 mg of dopamine hydrochloride in the same beaker, add 21 mL of water, and stir on a magnetic stirrer at a rate of 650 r / min to fully dissolve the zinc nitrate hexahydrate and dopamine hydrochloride to obtain a mixed aqueous solution; pour the mixed aqueous solution into the above mixed methanol solution and stir at a rate of 650 r / min for 3 h at room temperature; after stirring, centrifuge at 12000 r / min for 8 min, then wash the uncarbonized nanozyme precipitate collected by centrifugation with methanol solution, repeat the centrifugation and methanol washing steps 3 times, and then centrifuge again at 12000 r / min for 10 min to collect the precipitate, place it in a vacuum drying oven, and dry at 55 °C for 12 h to obtain a dandelion-shaped ZPD precursor;

[0054] S2. Preparation of ZPD: The ZPD precursor obtained in step S1 is placed in a tube furnace. Argon gas is first purged for 30 minutes to remove the air remaining in the tube furnace. Then, the furnace is carbonized and calcined at 1000°C in an argon atmosphere at a heating rate of 5°C / min and held for 4 hours. Finally, it is naturally cooled to 25°C to obtain ZPD.

[0055] S3. Preparation of ZPD@BBH: 10 mg of ZPD obtained in step S2 was ultrasonically dispersed in 10 mL of methanol, and then 30 mg of BBH was added. The mixture was stirred at 650 r / min for 8 h at room temperature on a magnetic stirrer. After stirring, the mixture was centrifuged at 11000 r / min for 15 min, and the precipitate was washed with methanol solution. The centrifugation and methanol washing steps were repeated twice. The precipitate was collected by centrifugation at 11000 r / min for 10 min again and placed in a vacuum drying oven. After drying at 55 °C for 12 h, the biomimetic nanomedicine ZPD@BBH was obtained.

[0056] Comparative Example 1

[0057] A biomimetic nanomedicine for combating subcutaneous drug-resistant bacterial infections is prepared using the following steps:

[0058] S1. Preparation of ZPD precursor: Dissolve 0.15 g of 2-methylimidazole in 45 mL of methanol solution and stir evenly on a magnetic stirrer to obtain a mixed methanol solution; then place 1.4375 g of zinc nitrate hexahydrate and 50 mg of dopamine hydrochloride in the same beaker, add 21 mL of water, and stir on a magnetic stirrer at a rate of 800 r / min to fully dissolve the zinc nitrate hexahydrate and dopamine hydrochloride to obtain a mixed aqueous solution; pour the mixed aqueous solution into the above mixed methanol solution and stir at a rate of 800 r / min for 3 h at room temperature; after stirring, centrifuge at 11000 r / min for 10 min, then wash the uncarbonized nanozyme precipitate collected by centrifugation with methanol solution, repeat the centrifugation and methanol washing steps 3 times, then centrifuge again at 11000 r / min for 10 min to collect the precipitate, and place it in a vacuum drying oven and dry at 60 °C for 8 h to obtain a dandelion-shaped ZPD precursor;

[0059] S2. Preparation of ZPD: The ZPD precursor obtained in step S1 is placed in a tube furnace. Argon gas is first purged for 30 minutes to remove the residual air in the tube furnace. Then, in an argon atmosphere, the temperature is increased to 800°C at a rate of 5°C / min and held for 3 hours. After that, it is naturally cooled to 25°C to obtain ZPD.

[0060] S3. Preparation of ZPD@BBH: 10 mg of ZPD obtained in step S2 was ultrasonically dispersed in 10 mL of methanol, and then 10 mg of BBH was added. The mixture was stirred at 800 r / min for 12 h at room temperature on a magnetic stirrer. After stirring, the mixture was centrifuged at 12000 r / min for 10 min, and the precipitate was washed with methanol solution. The centrifugation and methanol washing steps were repeated 3 times. The precipitate was collected by centrifugation at 12000 r / min for 10 min again and placed in a vacuum drying oven. After drying at 60 °C for 8 h, the biomimetic nanomedicine ZPD@BBH was obtained.

[0061] Comparative Example 2

[0062] A biomimetic nanomedicine for combating subcutaneous drug-resistant bacterial infections is prepared using the following steps:

[0063] S1. Preparation of ZPD precursor: Dissolve 0.65 g of 2-methylimidazole in 45 mL of methanol solution and stir evenly on a magnetic stirrer to obtain a mixed methanol solution; then place 1.4375 g of zinc nitrate hexahydrate and 150 mg of dopamine hydrochloride in the same beaker, add 21 mL of water, and stir on a magnetic stirrer at a rate of 800 r / min to fully dissolve the zinc nitrate hexahydrate and dopamine hydrochloride to obtain a mixed aqueous solution; pour the mixed aqueous solution into the above mixed methanol solution and stir at a rate of 800 r / min for 3 h at room temperature; after stirring, centrifuge at 11000 r / min for 10 min, then wash the uncarbonized nanozyme precipitate collected by centrifugation with methanol solution, repeat the centrifugation and methanol washing steps 3 times, then centrifuge again at 11000 r / min for 10 min to collect the precipitate, and place it in a vacuum drying oven and dry at 60 °C for 8 h to obtain a dandelion-shaped ZPD precursor;

[0064] S2. Preparation of ZPD: The ZPD precursor obtained in step S1 is placed in a tube furnace. Argon gas is first purged for 30 minutes to remove the residual air in the tube furnace. Then, in an argon atmosphere, the temperature is increased to 800°C at a rate of 5°C / min and held for 3 hours. After that, it is naturally cooled to 25°C to obtain ZPD.

[0065] S3. Preparation of ZPD@BBH: 10 mg of ZPD obtained in step S2 was ultrasonically dispersed in 10 mL of methanol, and then 10 mg of BBH was added. The mixture was stirred at 800 r / min for 12 h at room temperature on a magnetic stirrer. After stirring, the mixture was centrifuged at 12000 r / min for 10 min, and the precipitate was washed with methanol solution. The centrifugation and methanol washing steps were repeated 3 times. The precipitate was collected by centrifugation at 12000 r / min for 10 min again and placed in a vacuum drying oven. After drying at 60 °C for 8 h, the biomimetic nanomedicine ZPD@BBH was obtained.

[0066] Examples 1 to 3 have similar effects. For the convenience of subsequent discussion and reference, the experimental results of Example 1 shall be used as the standard for the following experiments.

[0067] Experimental Example 1: Physical Characterization of Different ZPDs

[0068] The inventors first characterized the ZPDs prepared in Example 1, Comparative Example 1, and Comparative Example 2 using a scanning electron microscope, and the results are as follows: Figure 1 As shown, Figure 1 Figure A is a scanning electron microscope image of the ZPD prepared in Example 1. Figure 1 Figure B is a scanning electron microscope image of the ZPD prepared in Comparative Example 1. Figure 1 Figure C is a scanning electron microscope image of the ZPD prepared in Comparative Example 2.

[0069] Observation of scanning electron microscopy results revealed that the ZPD prepared in Example 1 had a rough surface, a special dandelion-like morphology, and a large specific surface area, which could improve the efficiency of topological interaction between bacteria and the rough surface, thus making it more conducive to targeting the bacterial surface and playing a more efficient antibacterial role. However, the ZPD prepared in Comparative Example 1 could not effectively polymerize and was difficult to form carbon nanozyme materials. The ZPD prepared in Comparative Example 2 could not form carbon nanozymes with the special dandelion-like morphology, could not conduct topological interactions with bacteria to achieve the purpose of targeting, and the formed nanoparticles were stuck together and disordered.

[0070] Therefore, the ZPD prepared in Example 1 was subsequently used for further experimental investigation in this invention.

[0071] The inventors characterized the ZPD prepared in Example 1 by measuring its particle size, infrared spectrum, and ultraviolet spectrum. Figure 1 The D-plot is the particle size distribution diagram of ZPD. Figure 1 The E-graph is the infrared spectrum of the ZPD. Figure 1 The F-plot is the ultraviolet spectrum of the ZPD.

[0072] like Figure 1 As shown in the D-plot, the diameter of the ZPD is approximately 500 nm. Figure 1 As shown in Figure E, ZPD, its precursor, and dopamine hydrochloride were characterized using Fourier transform infrared spectroscopy. Overlapping peaks were observed between the ZPD precursor and dopamine hydrochloride, indicating that dopamine hydrochloride could be successfully loaded onto the ZPD precursor. Figure 1 As shown in Figure F, the full spectrum of ZPD and ZPD precursor was scanned by a UV spectrophotometer. It can be seen that the ZPD precursor has absorbance due to the presence of dopamine hydrochloride; while ZPD is a carbonized nanozyme, so it also has absorbance at 808 nm.

[0073] Experimental Example 2: Enzyme Activity Characterization of ZPD

[0074] This invention utilizes a colorimetric reaction of TMB oxidation assisted by hydrogen peroxide to determine the peroxidase catalytic performance of ZPD prepared in Example 1 in 10 mM PBS at pH 4.0. All reactions were carried out at 37°C for 5 min, and the UV-Vis absorption spectra were recorded. The concentrations were: hydrogen peroxide 25 mM, TMB 1 mM, ZPD 40 μg / mL, and the total reaction volume 400 μL. For peroxidase activity under NIR laser irradiation, the reaction system was irradiated with an NIR laser for 5 min under the same conditions, and the UV absorption spectra were recorded.

[0075] Figure 2The results of enzyme activity characterization of ZPD peroxidase were obtained from... Figure 2 Figure A and Figure 2 As shown in Figure B, ZPD exhibits good peroxidase activity, with the highest activity observed when ZPD, hydrogen peroxide, and TMB are present together. Figure 2 The C diagram and Figure 2 As shown in the D diagram, ZPD exhibits the highest enzyme activity when ZPD, hydrogen peroxide, and TMB are present together and irradiated with an NIR laser.

[0076] Experimental Example 3: Detection of the photothermal drug release capacity of ZPD@BBH

[0077] This invention first constructs a standard curve of ZPD on the loading of berberine hydrochloride, such as... Figure 3 As shown; through linear regression analysis, the standard curve equation was obtained as y = 0.0531744x + 0.0113125, where x represents the concentration of berberine hydrochloride and y is the absorbance value. Based on this standard curve, the loading of berberine hydrochloride on ZPD in the ZPD@BBH prepared in Example 1 was calculated to be 18.55% ± 1.8%. Furthermore, the drug release capacity of ZPD@BBH was further determined in this invention.

[0078] Depend on Figure 4 It can be seen that when the pH value is 5.5, i.e. under acidic conditions, the cumulative drug release rate of ZPD@BBH in 48h is 55.46%±3.25%, while when the pH value is 7.4, i.e. under neutral conditions, the cumulative drug release rate of ZPD@BBH in 48h is 37.37%±0.69%. Therefore, the drug release capacity of ZPD@BBH under acidic conditions is higher than that under neutral conditions, indicating that the biomimetic nanomedicine provided by the present invention has better drug release characteristics in the microenvironment of subcutaneous drug-resistant bacterial infection area.

[0079] To evaluate the photothermal stability of ZPD@BBH, different concentrations of ZPD@BBH were subjected to 808nm NIR irradiation with different powers for three heating / cooling cycles. The results are as follows: Figure 4 As shown.

[0080] At 808nm, 1.0W / cm 2 Under NIR laser irradiation, the photothermal properties of ZPD@BBH exhibited a concentration-dependent effect, with the maximum temperature increasing with increasing concentration. At a fixed concentration of 100 μg / mL, ZPD@BBH also showed a power gradient-related photothermal effect, with higher power resulting in higher temperatures, and the local tissue temperature reaching approximately 50 °C. Furthermore, no significant difference was observed in the temperature of ZPD@BBH after three cycles of experiments, indicating that ZPD@BBH possesses good photothermal stability.

[0081] Because carbon nanozymes possess excellent photothermal properties, this invention also measured the photoresponsive drug release characteristics of ZPD@BBH, such as... Figure 4 The ZPD@BBH sample was obtained from three replicates at 1.0 W / cm². 2 Under near-infrared laser irradiation, the cumulative drug release rate of ZPD@BBH after 48 hours was 68.19% ± 0.87%, significantly higher than that of the control group that did not undergo near-infrared laser irradiation. Therefore, this demonstrates that ZPD@BBH can enhance drug release through near-infrared laser irradiation, proving that ZPD@BBH possesses photoresponsive drug release characteristics.

[0082] Experimental Example 4: Antibacterial Performance Testing of ZPD and ZPD@BBH

[0083] Methicillin-resistant Staphylococcus aureus (MRSA) is a common and challenging bacterium in clinical practice that can cause subcutaneous infections. This invention systematically evaluated the in vitro antibacterial activity of ZPD and ZPD@BBH prepared in Example 1 using the agar plate method, and statistically analyzed the antibacterial rates of ZPD and ZPD@BBH against MRSA. The antibacterial rate was calculated using the following formula:

[0084] ;

[0085] In the formula for calculating the antibacterial rate: C represents the antibacterial rate, A represents the number of methicillin-resistant Staphylococcus aureus colonies on the untreated control group agar plate, and B represents the number of methicillin-resistant Staphylococcus aureus colonies on the agar plates of other groups besides the untreated control group.

[0086] The experiment was divided into the following groups: (a) untreated control group; (b) untreated control group + hydrogen peroxide + NIR light irradiation group; (c) ZPD treatment group, with ZPD concentrations of 20 μg / mL, 40 μg / mL, 60 μg / mL, 80 μg / mL and 100 μg / mL, respectively; (d) ZPD + hydrogen peroxide + NIR light irradiation treatment group, with ZPD concentrations of 20 μg / mL, 40 μg / mL, 60 μg / mL and 80 μg / mL, respectively. (e) ZPD@BBH treatment group, ZPD concentrations in ZPD@BBH were 20 μg / mL, 40 μg / mL, 60 μg / mL, 80 μg / mL and 100 μg / mL respectively; (f) ZPD@BBH + hydrogen peroxide + NIR light irradiation treatment group, ZPD concentrations in ZPD@BBH were 20 μg / mL, 40 μg / mL, 60 μg / mL, 80 μg / mL and 100 μg / mL respectively. Methicillin-resistant Staphylococcus aureus was cultured for 10 h, hydrogen peroxide concentration was 100 μM, and NIR light intensity was 1 W / cm². 2 The NIR illumination duration was 10 minutes.

[0087] Depend on Figure 5 The results show that when the concentration of ZPD is 100 μg / mL, combined with 100 μM hydrogen peroxide and 1 W / cm², the effect is significant. 2 When combined with low-intensity near-infrared light irradiation for treatment, the antibacterial rate can reach over 90%; when using ZPD@BBH, only a ZPD@BBH concentration of 80 μg / mL is needed, combined with 100 μM hydrogen peroxide and 1 W / cm². 2 Treatment with low-intensity near-infrared light can achieve an antibacterial rate of over 90%, and when the concentration of ZPD@BBH is 100μg / mL, the bacteria are almost completely killed.

[0088] comprehensive Figure 5 The results show that ZPD@BBH can achieve synergistic treatment of enzyme catalysis and photoresponse in vitro at low concentrations, and has a strong antibacterial effect. This is because ZPD@BBH has multiple antibacterial modes: (1) the local heat generated by near-infrared laser irradiation can kill some bacteria; (2) the heat generated by near-infrared laser irradiation increases the catalytic activity of ZPD peroxidase, and more effectively converts hydrogen peroxide into toxic hydroxyl radicals. The strong oxidizing properties of hydroxyl radicals can quickly destroy bacterial cell membranes and intracellular substances, leading to bacterial death; (3) ZPD has a dandelion-like surface, which makes it easier to target the bacterial surface. The effective ingredient of traditional Chinese medicine, berberine hydrochloride, has a high-efficiency antibacterial ability and the photothermal killing method is unlikely to cause the generation of drug-resistant bacteria. This synergistic bactericidal system of photothermal therapy, chemokinetic therapy, and antibacterial drugs reduces the amount of drugs and the intensity of near-infrared laser light, achieving the effect of high efficiency with less drugs, greatly reducing the toxic side effects on the body, and achieving a satisfactory antibacterial effect.

[0089] Experimental Example 5: Biosafety Testing of ZPD@BBH

[0090] Mouse epithelial-like fibroblast L929 cells were selected as the research subject. The in vitro safety of ZPD@BBH was verified by the MTT assay. The specific steps were as follows: the cells were packed at a density of 1×10⁶ cells in a 96-well plate. 4 L929 cells were added to complete medium containing DMEM, and then ZPD@BBH at concentrations of 0 μg / mL, 12.5 μg / mL, 25 μg / mL, 50 μg / mL, 100 μg / mL, and 200 μg / mL were co-incubated with the cells. The control group was treated with PBS only. After 24 h of co-incubation, the medium was aspirated, the cells were washed once with PBS, and then MTT complete medium was added to each well. The cells were incubated in a cell culture incubator for 4 h, after which MTT was aspirated, and finally 100 μL of DMSO was added to each well. Cell viability was detected at 490 nm using a microplate reader. The results are shown below. Figure 6 As shown.

[0091] from Figure 6 Figure A shows that the toxicity of 100 μg / mL ZPD@BBH to L929 cells is negligible. Cells treated with ZPD@BBH exhibited normal morphology and no abnormal cell concentration. Figure 6 As shown in Figure B, ZPD@BBH at concentrations as high as 200 μg / mL had very limited toxicity to L929 cells, with a cell survival rate >80%. The highest concentration of ZPD@BBH in the experimental group was 100 μg / mL. These results indicate that ZPD@BBH has good cell compatibility.

[0092] Subsequently, this invention tested the hemolysis rate of ZPD@BBH, such as... Figure 6 As shown in Figure C, the hemolysis rate after treatment with ZPD@BBH at concentrations of 200 μg / mL and below was less than 5%. Blood cells treated with hydrogen peroxide served as the positive control group, and the supernatant was bright red. However, after treatment with physiological saline, 12.5 μg / mL ZPD@BBH, 25 μg / mL ZPD@BBH, 50 μg / mL ZPD@BBH, 100 μg / mL ZPD@BBH, and 200 μg / mL ZPD@BBH, the supernatant of each group was colorless and transparent, indicating that the hemolytic effect of ZPD@BBH is very low and its blood compatibility is good.

[0093] It should be noted that when numerical ranges are involved in this invention, it should be understood that both endpoints of each numerical range and any value between the two endpoints can be selected. Since the steps and methods used are the same as in the embodiments, preferred embodiments are described here to avoid redundancy. Although preferred embodiments of the invention have been described, those skilled in the art, once they understand the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of this invention.

[0094] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A biomimetic nanodrug against subcutaneous drug-resistant bacterial infection, characterized in that, The preparation method of the biomimetic nanodrug has the following steps: S1, preparation of ZPD precursor: 0.32g of 2-methyl imidazole is dissolved in 45mL of methanol solution, placed on a magnetic stirrer to stir uniformly to obtain a mixed methanol solution; then 1.4375g of zinc nitrate hexahydrate and 100mg of dopamine hydrochloride are placed in the same beaker, 21mL of water is added, and the magnetic stirrer is stirred at a speed of 800r / min to make the zinc nitrate hexahydrate and dopamine hydrochloride fully dissolved to obtain a mixed aqueous solution; the mixed aqueous solution is poured into the above mixed methanol solution, and stirred at a speed of 800r / min at room temperature for 3h; after stirring, centrifugation is performed at 11000r / min for 10min, then the non-carbonized nanometer enzyme precipitate collected by centrifugation is washed with methanol solution, the centrifugation and methanol washing steps are repeated for 3 times, then the precipitate is collected by centrifugation at 11000r / min for 10min, and placed in a vacuum drying oven, dried at 60℃ for 8h, to obtain a ZPD precursor with dandelion shape; S2, preparation of ZPD: the ZPD precursor obtained in step S1 is placed in a tube furnace, first purged with argon for 30min to remove the air remaining in the tube furnace, then carbonized and calcined at 800℃ at a temperature rising rate of 5℃ / min in an argon atmosphere for 3h, and then naturally cooled to 25℃ to obtain ZPD; S3, preparation of ZPD@BBH: 10mg of ZPD obtained in step S2 is ultrasonically dispersed in 10mL of methanol, then 10mg of berberine hydrochloride BBH is added, and stirred at a speed of 800r / min at room temperature on a magnetic stirrer for 12h; after stirring, centrifugation is performed at 12000r / min for 10min, the centrifugal precipitate is washed with methanol solution, the centrifugation and methanol washing steps are repeated for 3 times, then the precipitate is collected by centrifugation at 12000r / min for 10min, and placed in a vacuum drying oven, dried at 60℃ for 8h to obtain a biomimetic nanodrug ZPD@BBH.

2. A biomimetic nanodrug against subcutaneous drug resistant bacterial infection, characterized in that, The preparation method of the biomimetic nanodrug has the following steps: S1, preparation of ZPD precursor: 0.32g of 2-methyl imidazole is dissolved in 45mL of methanol solution, placed on a magnetic stirrer to stir uniformly to obtain a mixed methanol solution; then 1.4375g of zinc nitrate hexahydrate and 100mg of dopamine hydrochloride are placed in the same beaker, 21mL of water is added, and the magnetic stirrer is stirred at a speed of 800r / min to make the zinc nitrate hexahydrate and dopamine hydrochloride fully dissolved to obtain a mixed aqueous solution; the mixed aqueous solution is poured into the above mixed methanol solution, and stirred at a speed of 800r / min at room temperature for 3h; after stirring, centrifugation is performed at 11000r / min for 10min, then the non-carbonized nanometer enzyme precipitate collected by centrifugation is washed with methanol solution, the centrifugation and methanol washing steps are repeated for 3 times, then the precipitate is collected by centrifugation at 11000r / min for 10min, and placed in a vacuum drying oven, dried at 60℃ for 8h, to obtain a ZPD precursor with dandelion shape; S2, preparation of ZPD: the ZPD precursor obtained in step S1 is placed in a tube furnace, first purged with argon for 30min to remove the air remaining in the tube furnace, then carbonized and calcined at 800℃ at a temperature rising rate of 5℃ / min in an argon atmosphere for 3h, and then naturally cooled to 25℃ to obtain ZPD; S2, preparing ZPD: the ZPD precursor obtained in step S1 was placed in a tube furnace, first purged with argon for 30 min to remove the residual air in the tube furnace, then carbonized and calcined at a temperature of 900 DEG C at a heating rate of 5 DEG C / min under argon atmosphere for 2 h, and then naturally cooled to 25 DEG C to obtain ZPD; S3, preparing ZPD@BBH: 10 mg of ZPD obtained in step S2 was ultrasonically dispersed in 10 mL of methanol, then 20 mg of BBH was added, and the mixture was stirred at a speed of 750 r / min on a magnetic stirrer at room temperature for 10 h; after stirring, the mixture was centrifuged at a speed of 11500 r / min for 10 min, the precipitate was washed with methanol solution, the centrifugation and methanol washing steps were repeated 4 times, then the precipitate was collected by centrifugation at a speed of 11500 r / min for 10 min, and placed in a vacuum drying oven for drying at 65 DEG C for 8 h to obtain the biomimetic nanodrug ZPD@BBH.

3. A biomimetic nanodrug against subcutaneous drug resistant bacterial infection, characterized in that, The preparation method of the biomimetic nanodrug has the following steps: S1, preparing ZPD precursor: 0.45 g of 2-methylimidazole was dissolved in 45 mL of methanol solution, and the mixture was stirred on a magnetic stirrer to obtain a mixed methanol solution; then 2.1375 g of zinc nitrate hexahydrate and 105 mg of dopamine hydrochloride were placed in the same beaker, 21 mL of water was added, and the mixture was stirred on a magnetic stirrer at a speed of 650 r / min to dissolve the zinc nitrate hexahydrate and dopamine hydrochloride to obtain a mixed aqueous solution; the mixed aqueous solution was poured into the above mixed methanol solution, and the mixture was stirred at a speed of 650 r / min at room temperature for 3 h; after stirring, the mixture was centrifuged at a speed of 12000 r / min for 8 min, then the uncarbonized nanoscale enzyme precipitate collected by centrifugation was washed with methanol solution, the centrifugation and methanol washing steps were repeated 3 times, then the precipitate was collected by centrifugation at a speed of 12000 r / min for 10 min, and placed in a vacuum drying oven for drying at 55 DEG C for 12 h to obtain a dandelion-shaped ZPD precursor; S2, preparing ZPD: the ZPD precursor obtained in step S1 was placed in a tube furnace, first purged with argon for 30 min to remove the residual air in the tube furnace, then carbonized and calcined at a temperature of 900 DEG C at a heating rate of 5 DEG C / min under argon atmosphere for 2 h, and then naturally cooled to 25 DEG C to obtain ZPD; S3, preparing ZPD@BBH: 10 mg of ZPD obtained in step S2 was ultrasonically dispersed in 10 mL of methanol, then 20 mg of BBH was added, and the mixture was stirred at a speed of 750 r / min on a magnetic stirrer at room temperature for 10 h; after stirring, the mixture was centrifuged at a speed of 11500 r / min for 10 min, the precipitate was washed with methanol solution, the centrifugation and methanol washing steps were repeated 4 times, then the precipitate was collected by centrifugation at a speed of 11500 r / min for 10 min, and placed in a vacuum drying oven for drying at 65 DEG C for 8 h to obtain the biomimetic nanodrug ZPD@BBH.

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