Bionic nano-drug for resisting subcutaneous drug-resistant bacterial infection as well as preparation method and application of bionic nano-drug

Abstract

The invention belongs to the technical field of medicines, and particularly relates to a bionic nano-drug for resisting subcutaneous drug-resistant bacterial infection as well as a preparation method and application thereof. Dopamine hydrochloride is added in the synthesis process of a metal-organic framework to prepare a dandelion-shaped carbon nano-enzyme precursor, then the nano-enzyme precursor is carbonized to obtain a carbon nano-enzyme with targeting property, photoresponse and peroxidase activity, and finally berberine hydrochloride is loaded on the carbon nano-enzyme to obtain the bionic nano-drug. The bionic nano-drug provided by the invention firstly has good biocompatibility, secondly has a unique dandelion-shaped form, is beneficial to playing a targeting role, and finally has relatively strong light response capability in a near-infrared region, and can synergistically and efficiently sterilize through action mechanisms of antibacterial drugs, photothermal therapy and chemical kinetics, so that the bionic nano-drug has a good application prospect. The compound has an important application value in preparation of drugs for treating subcutaneous drug-resistant bacterium infection.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and specifically relates to a bionic nano drug for resisting subcutaneous drug-resistant bacterial infection, a preparation method and an application thereof. Background Art

[0002] The invention and widespread use of antibiotics have provided humans with a powerful weapon to fight bacterial infections. However, with the widespread use of antibiotics, bacteria have gradually developed a variety of resistance mechanisms such as permeability barriers, inactivation enzymes, drug efflux pumps, target modifications, and secretion biofilms to resist antibiotics, which not only reduces the therapeutic effect of antibiotics, but also leads to widespread resistance of bacteria to existing antibiotics. The bacterial biofilm can shield external damage and prevent drugs from penetrating into the interior of the bacteria. When the skin barrier is damaged, the permeability of some blood vessels increases, causing the nutrients and tissue fluid supplied to normal cells to overflow, forming a humid environment, providing a suitable environment and material basis for the anchoring and proliferation of bacteria; after anchoring on the wound surface, bacteria multiply in large numbers to form biofilms, and the bacteria wrapped in biofilms are in a state of slow growth and hunger, so they are very sensitive to chemical drugs such as antibiotics. If the bacteria or biofilms that remain after resistant bacterial infection cannot be eradicated, the microenvironment of the infection will continue to deteriorate, further leading to high fever and may even induce serious complications. Therefore, it is crucial to develop an effective treatment strategy to combat bacterial infection and overcome bacterial resistance.

[0003] Nanomedicine can reduce drug toxicity and side effects, increase local tissue uptake, and perform controlled / responsive drug release. Most of the drugs currently reported in nanozyme drug delivery systems are Western medicines, antibiotics, small molecule drugs, etc. However, excessive use of nanozymes loaded with antibiotics can lead to the proliferation of drug-resistant bacteria and even the mutation of multi-drug resistant super bacteria.

[0004] Berberine, also known as berberine, is a quaternary ammonium alkaloid that is widely distributed in the plant kingdom. Berberine hydrochloride is the most common form of berberine. Berberine hydrochloride is a drug with multiple medical potentials, including antimalarial, antifungal, antioxidant, and anti-inflammatory properties. Berberine hydrochloride can interfere with the formation of biofilms by inhibiting the formation of extracellular amyloid peptides in bacteria; it can also bind to some proteins on the biofilm, destroying the structure of these proteins, and further destroying the integrity and permeability of the bacterial biofilm. In view of its excellent antibacterial activity, berberine hydrochloride can be used as an ideal substitute for antibiotics. Therefore, it is urgent to develop a method for preparing a nanoenzyme drug delivery system that uses active ingredients of traditional Chinese medicine to replace antibiotics. Summary of the invention

[0005] In view of the technical problems existing in the above-mentioned prior art, the purpose of the present invention is to provide a bionic nanomedicine for resisting subcutaneous drug-resistant bacterial infection, a preparation method and an application thereof. The bionic nanomedicine provided by the present invention first has good biocompatibility, secondly has a unique dandelion-like morphology, which is conducive to exerting a targeting effect, and finally, has a strong light response ability in the near-infrared region, and can synergistically and efficiently kill bacteria through the mechanism of action of antibacterial drugs, photothermal therapy and chemical kinetics, and has important application value in the preparation of drugs for treating subcutaneous drug-resistant bacterial infections.

[0006] The first aspect of the present invention provides a method for preparing a bionic nanomedicine for resisting subcutaneous drug-resistant bacterial infection, comprising the following steps: Dissolving 2-methylimidazole in methanol to obtain a mixed methanol solution, adding a mixed aqueous solution of zinc salt and dopamine hydrochloride to the mixed methanol solution, and centrifuging to obtain a dandelion-shaped carbon nanozyme precursor; Carbonizing the carbon nanozyme precursor to obtain a dandelion-shaped carbon nanozyme with targeting, light response and peroxidase activity; Berberine hydrochloride with antibacterial effect is mixed with carbon nanozyme to load berberine hydrochloride on the carbon nanozyme, and then the mixture is centrifuged and dried to obtain the bionic nanomedicine.

[0007] Furthermore, the zinc salt is zinc nitrate, and the Zn in the zinc nitrate is 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 during the reaction, which will not interfere with the formation of carbon nanozyme precursors, and has high water solubility and stability.

[0008] Furthermore, the zinc salt is zinc nitrate hexahydrate.

[0009] Furthermore, the mass ratio of the 2-methylimidazole, zinc nitrate hexahydrate and dopamine hydrochloride is 0.32-0.45:1.4375-2.1375:0.095-0.105.

[0010] Furthermore, the mixing mass ratio of berberine hydrochloride to carbon nanozyme is 1~3:1.

[0011] Furthermore, the centrifugal speed is 11000r / min~12000r / min, and the centrifugal time is 8min~15min; under this centrifugal condition, the precipitated material can be effectively precipitated.

[0012] Furthermore, the drying temperature is 55°C to 65°C, and the drying time is 8h to 12h. At this drying temperature, the performance of the nanozyme precursor will not be affected and the nanozyme precursor can be completely dried overnight.

[0013] Furthermore, the carbonization temperature is 800°C~1000°C, and the time is 2h~4h; under this carbonization condition, it can avoid the inability to synthesize carbon nanozymes due to the carbonization temperature being too low, and it can also avoid the digestion of zinc ions due to the carbonization temperature being too high.

[0014] The second aspect of the present invention provides a bionic nano drug prepared by the above-mentioned method for preparing the bionic nano drug for resisting subcutaneous drug-resistant bacterial infection.

[0015] The third aspect of the present invention provides a use of the above-mentioned bionic nanomedicine in the preparation of a drug for treating subcutaneous drug-resistant bacterial infection.

[0016] Furthermore, the bacteria is Staphylococcus aureus.

[0017] The fourth aspect of the present invention provides a use of the above-mentioned bionic nanomedicine in the preparation of chemodynamic therapy agents.

[0018] A fifth aspect of the present invention provides an application of the above-mentioned bionic nanomedicine in the preparation of photothermal therapy therapeutic agents.

[0019] Compared with the prior art, the present invention has the following beneficial effects: The present invention discloses for the first time a method for preparing a bionic nano drug for resisting subcutaneous drug-resistant bacterial infection. Dopamine hydrochloride is added during the synthesis of a metal-organic framework, and the amino and hydroxyl groups in the dopamine hydrochloride molecules undergo cross-linking reactions with carbon nanomaterials to enhance the polymerization effect of a carbon nano enzyme precursor. A dandelion-shaped carbon nano enzyme precursor is prepared after centrifugation, and then the nano enzyme precursor is carbonized at 800°C to 1000°C for 2h to 4h to obtain a carbon nano enzyme with targeting, light response and peroxidase activity. Finally, berberine hydrochloride with antibacterial effect is loaded on the carbon nano enzyme to obtain a bionic nano drug. The preparation method provided by the present invention is simple and easy to implement, and provides a new idea and theoretical basis for the development of nano enzyme drugs loaded with other effective ingredients of traditional Chinese medicine.

[0020] The present invention discloses a bionic nanomedicine for resisting subcutaneous drug-resistant bacterial infection. The bionic nanomedicine uses a dandelion-shaped carbon nanoenzyme as a carrier and is loaded with berberine hydrochloride with antibacterial effect. The present invention constructs a three-mode synergistic antibacterial platform of photothermal therapy-chemodynamic therapy-antibacterial drug, which overcomes the problems of limited antibacterial effect of a single antibacterial strategy or side effects and metabolic burden caused by the use of large doses of antibiotics. The bionic nanomedicine has efficient targeting. The bionic nanomedicine with three synergistic antibacterial modes and efficient targeting ability has important application value in the preparation of therapeutic drugs for subcutaneous drug-resistant bacterial infection.

[0021] (1) Compared with the conventional metal organic framework material ZIF-8, the carbon nanozyme provided by the present invention has a higher specific surface area and has efficient targeting. By adding dopamine hydrochloride during the preparation process, the carbon nanozyme can be polymerized more effectively, the skeleton collapse can be prevented, and a multi-pore and dandelion-shaped carbon nanozyme material can be further formed. In addition, the addition of dopamine hydrochloride does not affect the good photothermal conversion ability of the carbon nanozyme, and the carbon nanozyme also has the catalytic activity and drug loading capacity of peroxidase, and then the antibacterial substance berberine hydrochloride is loaded to obtain a bionic nanomedicine.

[0022] (2) The bionic nanomedicine preparation process provided by the present invention is simple. In addition, the nanomaterial carbon nanozyme, as a photothermal agent, can convert light energy into heat energy and has strong light absorption in the near-infrared region. The local temperature can reach about 50°C, which can effectively kill bacteria without damaging normal tissues due to excessively high temperatures, thus avoiding the generation of drug-resistant bacteria at the source.

[0023] (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 value of 3.0 to 6.0, which indicates that it is beneficial to exert good antibacterial properties in the acidic environment of the subcutaneous infection area.

[0024] (4) The biomimetic nanomedicine of the present invention has good biocompatibility. In the cell live-dead staining experiment, the toxicity of carbon nanozymes at a concentration of up to 200 μg / mL to cells is very limited, and the cell survival rate is >80%. The in vitro hemolysis experiment shows that the hemolytic effect of carbon nanozymes is very low and the blood compatibility is good. This shows that the effect of carbon nanozymes on cell viability during administration is almost negligible and has good biocompatibility.

[0025] (5) The bionic nanomedicine of the present invention has the ability to target the bacterial surface. Due to the topological interaction between bacteria and rough surfaces, bacteria can easily adhere to the surface of cells, tissues or other biomaterials. Compared with conventional spherical particle carbon nanozymes, carbon nanozymes have a higher specific surface area, which can improve the efficiency of surface-based interactions. Therefore, drugs prepared using bionic nanomedicines have the ability to more effectively treat subcutaneous infections caused by drug-resistant bacteria.

[0026] In summary, the bionic nanomedicine for treating subcutaneous drug-resistant bacterial infection provided by the present invention has a special dandelion-like morphology, good photothermal effect, targeting effect, efficient antibacterial ability and efficient peroxidase activity, and has good biocompatibility, and has important application value in the preparation of drugs for treating subcutaneous drug-resistant bacterial infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 is the characterization result of different ZPD; 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 Figure D is a particle size distribution diagram of ZPD prepared in Example 1; Figure 1 Figure E is an infrared spectrum of ZPD prepared in Example 1, and the three curves from top to bottom are infrared spectra of ZPD, ZPD precursor and dopamine hydrochloride respectively; Figure 1 Figure F is the ultraviolet spectrum of ZPD prepared in Example 1, curve 1 represents the ultraviolet spectrum of ZPD precursor, and curve 2 represents the ultraviolet spectrum of ZPD.

[0028] Figure 2 The results are for characterization of the peroxidase activity of ZPD; Figure 2 Figure A is a schematic diagram of ZPD enzyme activity, curve 1 is the characterization result of ZPD enzyme activity when TMB, hydrogen peroxide and ZPD exist simultaneously, curve 2 is the characterization result of ZPD enzyme activity when TMB and hydrogen peroxide exist, curve 3 is the characterization result of ZPD enzyme activity when TMB and ZPD exist, and curve 4 is the characterization result of ZPD enzyme activity when only TMB exists; Figure 2 Figure B is Figure 2 The corresponding sample pictures when the enzyme activity is measured in Figure A. From left to right, the samples in the four tubes are Figure 2 The samples corresponding to curve 1, curve 2, curve 3 and curve 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 at the same time and NIR light is applied, curve 2 is a schematic diagram of ZPD enzyme activity when TMB, hydrogen peroxide and ZPD are present at the same time, curve 3 is a schematic diagram of ZPD enzyme activity when TMB and hydrogen peroxide are present and NIR light is applied, and 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 corresponding sample pictures when measuring enzyme activity in Figure C. The samples in the 4 tubes from left to right are Figure 2 The samples corresponding to curve 1, curve 2, curve 3 and curve 4 in Figure C.

[0029] Figure 3 This is the standard curve of ZPD versus berberine hydrochloride loading, and the standard curve formula is y=0.0531744x+0.0113125, where x is the berberine hydrochloride concentration and y is the absorbance value.

[0030] Figure 4 The 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 power corresponding to the four curves from top to bottom is 1.0 W / cm 2 , 0.8W / cm 2 , 0.5W / cm 2 , 0.3W / cm 2 ; Figure 4 Figure C shows the 808 nm, 1.0 W / cm 2 Repeated experimental results of NIR laser irradiation; Figure 4 Figure D shows the photothermal drug release characteristics of ZPD@BBH. The curve with a triangle icon is a schematic diagram of the drug release rate of ZPD@BBH when the pH value is 5.5 and treated with NIR light. The curve with a square icon is a schematic diagram of the drug release rate of ZPD@BBH when the pH value is 5.5. The curve with a circle icon is a schematic diagram of the drug release rate of ZPD when the pH value is 7.4.

[0031] Figure 5 The antibacterial performance test results of ZPD and ZPD@BBH against methicillin-resistant Staphylococcus aureus; Figure 5 Figure A is a picture of the colonies in the untreated control group; Figure 5 Figure B shows the colony after adding hydrogen peroxide and being treated with NIR light; Figure 5 Figure C shows the effects of different treatments on the colonies of methicillin-resistant Staphylococcus aureus. Figure 5 The (a) row in Figure C shows, from left to right, the colony images of methicillin-resistant Staphylococcus aureus after treatment with 20 μg / mL, 40 μg / mL, 60 μg / mL, 80 μg / mL, and 100 μg / mL of ZPD. Figure 5 The (b) row in Figure C shows, from left to right, the 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 and hydrogen peroxide respectively and then treated with NIR light. Figure 5 The (c) row in Figure C shows, from left to right, the colony images of methicillin-resistant Staphylococcus aureus after treatment with 20 μg / mL, 40 μg / mL, 60 μg / mL, 80 μg / mL, and 100 μg / mL of ZPD@BBH, respectively; Figure 5The (d) row in Figure C shows, from left to right, the 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 and hydrogen peroxide and then irradiated with NIR light. Figure 5 Figure D is a statistical chart of the antibacterial rate of ZPD and ZPD@BBH against methicillin-resistant Staphylococcus aureus. The solid columns in the bar graph, i.e., the 1st column, the 3rd column, the 5th column, the 7th column, the 9th column, and the 11th column, all indicate that no hydrogen peroxide was added and the samples were treated with NIR light. The hollow columns in the bar graph, i.e., the 2nd column, the 4th column, the 6th column, the 8th column, the 10th column, and the 12th column, all indicate that hydrogen peroxide was added and the samples were treated with NIR light. The 1st column and the 2nd column corresponding to Control are respectively Figure 5 Figure A and Figure 5 The experimental results of Figure B correspond to the results of Figure B; ** indicates the difference between the two groups. p <0.01.

[0032] Figure 6 The biosafety test results of ZPD@BBH; Figure 6 Figure A shows the fluorescent live-dead staining of L929 cells in the ZPD@BBH treatment group and the blank control group. Figure 6 The upper row of pictures in Figure A are schematic diagrams of live-dead staining of L929 cells in the blank control group. Figure 6 The lower row of pictures in Figure A are schematic diagrams of live-dead staining of L929 cells in the 100 μg / mL ZPD@BBH treatment group. Figure 6 In Figure A, from left to right, each column is a live cell staining schematic diagram, a dead cell staining schematic diagram, and a mixed live and dead cell staining schematic diagram; Figure 6 Figure B shows the cell viability of L929 cells treated with different concentrations of ZPD@BBH. ns indicates that there is no significant difference between the two groups. Figure 6 Figure C shows the hemolysis test results of mice. The standard range of normal hemolysis rate is below 5%. The dotted line indicates that the hemolysis rate of all concentrations of ZPD@BBH is less than 2%, which is within the standard range. Figure 6 Figure (a) in Figure C is Figure 6 The actual picture corresponding to the bar chart in Figure C, Figure 6 The tubes in (a) of Figure C are connected from left to right in sequence with Figure 6 The columns from left to right in Figure C correspond to . DETAILED DESCRIPTION

[0033] The present invention is described in detail below in conjunction with specific examples, but it should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0034] In the embodiments of the present invention: the carbon nanozyme is labeled as ZPD; berberine hydrochloride is labeled as BBH; the biomimetic nanomedicine is labeled as ZPD@BBH; 3,3',5,5'-tetramethylbenzidine is labeled as TMB; NIR is labeled as near infrared light; L929 cells were purchased from Shanghai Chunmai Biotechnology Co., Ltd.

[0035] Due to the abuse of antibiotics, bacteria have developed widespread resistance to existing antibiotics. Traditional antibiotic treatments often have limited effects on bacteria in biofilms, leading to persistent and recurrent infections. Nanomedicines can reduce drug toxicity and side effects, increase local tissue uptake, and perform controlled / responsive drug release. Most of the drugs loaded by the currently reported nanozyme drug delivery systems are Western medicines, antibiotics, small molecule drugs, etc. Excessive use of nanozyme therapy loaded with antibiotics will cause drug-resistant bacteria to proliferate and even mutate into multi-drug resistant super bacteria. Berberine hydrochloride has excellent antibacterial activity and can be used as an ideal substitute for antibiotics. Therefore, it is of great practical significance to develop a method for preparing a nanozyme drug delivery system that uses active ingredients of traditional Chinese medicine to replace antibiotics.

[0036] The present invention provides a preparation method and application of a bionic nano drug for resisting subcutaneous drug-resistant bacterial infection. In the synthesis process of the metal-organic framework, dopamine hydrochloride is added to make the carbon nano enzyme polymerize more effectively, prevent the collapse of the skeleton, obtain a dandelion-shaped carbon nano enzyme precursor after centrifugation, and then carbonize the nano enzyme precursor to obtain a carbon nano enzyme with targeting, light response and peroxidase activity, and finally load berberine hydrochloride with antibacterial effect on the carbon nano enzyme to obtain a bionic nano drug; the bionic nano drug provided by the present invention first has good biocompatibility, and secondly has a unique dandelion-like morphology, which is conducive to exerting a targeting effect, and finally, it has a strong light response ability in the near-infrared region, and can be efficiently sterilized through the synergistic effect of antibacterial drugs, photothermal therapy and chemical kinetics, and the bionic nano drug of the present invention is observed to have higher enzyme activity under acidic conditions with a pH value of 3.0 to 6.0, so the bionic nano drug provided by the present invention can play a good antibacterial performance in the acidic environment of the subcutaneous infection area, and has important application value in the preparation of drugs for treating subcutaneous drug-resistant bacterial infection.

[0037] Example 1 A bionic nanomedicine for resisting subcutaneous drug-resistant bacterial infection, the preparation method of which comprises the following steps: 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 a rate of 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-mentioned mixed methanol solution, and stirred at a rate of 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 a methanol solution, and the centrifugation and methanol washing steps were repeated 3 times, and the precipitate was collected by centrifugation at 11000 r / min for 10 min, and placed in a vacuum drying oven, and dried at 60°C for 8 h to obtain a dandelion-shaped ZPD precursor; S2. Preparation of ZPD: The ZPD precursor obtained in step S1 is placed in a tube furnace, and ventilated with argon for 30 minutes to expel the air remaining in the tube furnace, and then carbonized and calcined at a heating rate of 5°C / min to 800°C in an argon atmosphere and kept at this temperature for 3 hours, and then naturally cooled to 25°C to obtain ZPD; S3. Preparation of ZPD@BBH: ultrasonically disperse 10 mg of ZPD obtained in step S2 in 10 mL of methanol, then add 10 mg of BBH, and stir on a magnetic stirrer at 800 r / min for 12 h at room temperature; after stirring, centrifuge at 12000 r / min for 10 min, wash the centrifugal precipitate with methanol solution, repeat the centrifugation and methanol washing steps 3 times, centrifuge again at 12000 r / min for 10 min to collect the precipitate, and place it in a vacuum drying oven, and dry it at 60°C for 8 h to obtain the bionic nanomedicine ZPD@BBH.

[0038] Example 2 A bionic nanomedicine for resisting subcutaneous drug-resistant bacterial infection, the preparation method of which comprises the following steps: S1. Preparation of ZPD precursor: Dissolve 0.38 g of 2-methylimidazole in 45 mL of methanol solution, stir evenly on a magnetic stirrer to obtain a mixed methanol solution; then place 1.6675 g of zinc nitrate hexahydrate and 95 mg of dopamine hydrochloride in the same beaker, add 21 mL of water, stir on a magnetic stirrer at a rate of 700 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-mentioned mixed methanol solution, and stir at a rate of 700 r / min for 3 h at room temperature; after stirring, centrifuge at 11500 r / min for 10 min, and then wash the uncarbonized nanozyme precipitate collected by centrifugation with methanol solution, repeat the centrifugation and methanol washing steps 3 times, and then centrifuge at 11500 r / min for 10 min to collect the precipitate, and place it in a vacuum drying oven, and dry it at 65°C for 10 h to obtain a ZPD precursor with a dandelion shape; S2, preparation of ZPD: the ZPD precursor obtained in step S1 is placed in a tube furnace, ventilated with argon for 30 minutes to expel the air remaining in the tube furnace, and then carbonized and calcined at a heating rate of 5°C / min to 900°C in an argon atmosphere and kept at this temperature for 2 hours, and then naturally cooled to 25°C to obtain ZPD; S3. Preparation of ZPD@BBH: ultrasonically disperse 10 mg of ZPD obtained in step S2 in 10 mL of methanol, then add 20 mg of BBH, and stir at 750 r / min on a magnetic stirrer at room temperature for 10 h; after stirring, centrifuge at 11500 r / min for 10 min, wash the centrifugal precipitate with methanol solution, repeat the centrifugation and methanol washing steps 4 times, centrifuge at 11500 r / min for 10 min again to collect the precipitate, and place it in a vacuum drying oven, and dry it at 65°C for 8 h to obtain the bionic nanomedicine ZPD@BBH.

[0039] Example 3 A bionic nanomedicine for resisting subcutaneous drug-resistant bacterial infection, the preparation method of which comprises the following steps: S1. Preparation of ZPD precursor: 0.45 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 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 at a rate of 650 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-mentioned mixed methanol solution, and stirred at a rate of 650 r / min for 3 h at room temperature; after stirring, the mixture was centrifuged at 12000 r / min for 8 min, and then the uncarbonized nanozyme precipitate collected by centrifugation was washed with a methanol solution, and the centrifugation and methanol washing steps were repeated 3 times, and the precipitate was collected by centrifugation at 12000 r / min for 10 min, and placed in a vacuum drying oven, and dried at 55°C for 12 h to obtain a dandelion-shaped ZPD precursor; S2, preparation of ZPD: the ZPD precursor obtained in step S1 is placed in a tube furnace, ventilated with argon for 30 minutes to expel the air remaining in the tube furnace, and then carbonized and calcined at a heating rate of 5°C / min to 1000°C in an argon atmosphere and kept at this temperature for 4 hours, and then naturally cooled to 25°C to obtain ZPD; S3. Preparation of ZPD@BBH: ultrasonically disperse 10 mg of ZPD obtained in step S2 in 10 mL of methanol, then add 30 mg of BBH, and stir on a magnetic stirrer at 650 r / min for 8 h at room temperature; after stirring, centrifuge at 11000 r / min for 15 min, wash the centrifugal precipitate with methanol solution, repeat the centrifugation and methanol washing steps twice, centrifuge again at 11000 r / min for 10 min to collect the precipitate, and place it in a vacuum drying oven, and dry it at 55°C for 12 h to obtain the bionic nanomedicine ZPD@BBH.

[0040] Comparative Example 1 A bionic nanomedicine for resisting subcutaneous drug-resistant bacterial infection, the preparation method of which comprises the following steps: S1. Preparation of ZPD precursor: Dissolve 0.15 g of 2-methylimidazole in 45 mL of methanol solution, 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, 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-mentioned 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, and then wash the uncarbonized nanozyme precipitate collected by centrifugation with methanol solution, repeat the centrifugation and methanol washing steps 3 times, and then centrifuge at 11000 r / min for 10 min to collect the precipitate, and place it in a vacuum drying oven, and dry it at 60°C for 8 h to obtain a dandelion-shaped ZPD precursor; S2. Preparation of ZPD: The ZPD precursor obtained in step S1 is placed in a tube furnace, and ventilated with argon for 30 minutes to expel the air remaining in the tube furnace, and then carbonized and calcined at a heating rate of 5°C / min to 800°C in an argon atmosphere and kept at this temperature for 3 hours, and then naturally cooled to 25°C to obtain ZPD; S3. Preparation of ZPD@BBH: ultrasonically disperse 10 mg of ZPD obtained in step S2 in 10 mL of methanol, then add 10 mg of BBH, and stir on a magnetic stirrer at 800 r / min for 12 h at room temperature; after stirring, centrifuge at 12000 r / min for 10 min, wash the centrifugal precipitate with methanol solution, repeat the centrifugation and methanol washing steps 3 times, centrifuge again at 12000 r / min for 10 min to collect the precipitate, and place it in a vacuum drying oven, and dry it at 60°C for 8 h to obtain the bionic nanomedicine ZPD@BBH.

[0041] Comparative Example 2 A bionic nanomedicine for resisting subcutaneous drug-resistant bacterial infection, the preparation method of which comprises the following steps: S1. Preparation of ZPD precursor: Dissolve 0.65 g of 2-methylimidazole in 45 mL of methanol solution, 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, 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-mentioned mixed methanol solution, and stir at a rate of 800 r / min for 3 hours at room temperature; after stirring, centrifuge at 11000 r / min for 10 minutes, and then wash the uncarbonized nanozyme precipitate collected by centrifugation with methanol solution, repeat the centrifugation and methanol washing steps 3 times, and then centrifuge at 11000 r / min for 10 minutes to collect the precipitate, and place it in a vacuum drying oven, and dry it at 60°C for 8 hours to obtain a dandelion-shaped ZPD precursor; S2. Preparation of ZPD: The ZPD precursor obtained in step S1 is placed in a tube furnace, and ventilated with argon for 30 minutes to expel the air remaining in the tube furnace, and then carbonized and calcined at a heating rate of 5°C / min to 800°C in an argon atmosphere and kept at this temperature for 3 hours, and then naturally cooled to 25°C to obtain ZPD; S3. Preparation of ZPD@BBH: ultrasonically disperse 10 mg of ZPD obtained in step S2 in 10 mL of methanol, then add 10 mg of BBH, and stir on a magnetic stirrer at 800 r / min for 12 h at room temperature; after stirring, centrifuge at 12000 r / min for 10 min, wash the centrifugal precipitate with methanol solution, repeat the centrifugation and methanol washing steps 3 times, centrifuge again at 12000 r / min for 10 min to collect the precipitate, and place it in a vacuum drying oven, and dry it at 60°C for 8 h to obtain the bionic nanomedicine ZPD@BBH.

[0042] Examples 1 to 3 have similar effects. For the convenience of subsequent discussion and reference, the following experiments are conducted based on the test results of Example 1.

[0043] Experimental Example 1: Physical Characterization of Different ZPDs The inventors first used a scanning electron microscope to characterize the ZPD prepared in Example 1, Comparative Example 1 and Comparative Example 2. 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.

[0044] By observing the scanning electron microscopy results, it can be found that the ZPD prepared in Example 1 has a rough surface, a special dandelion-like morphology and a large specific surface area, which can improve the efficiency of topological interaction between bacteria and rough surfaces, thereby being more conducive to targeting the bacterial surface and more efficiently playing an antibacterial role; however, the ZPD prepared in Comparative Example 1 cannot be effectively polymerized and it is difficult to form a carbon nanozyme material; the ZPD prepared in Comparative Example 2 cannot form a carbon nanozyme with a special dandelion-like morphology, cannot interact topologically with bacteria to achieve the purpose of targeting, and the formed nanoparticles stick together and are disorderly.

[0045] Therefore, the present invention subsequently selected the ZPD prepared in Example 1 for further experimental exploration.

[0046] The inventors characterized the ZPD prepared in Example 1 by particle size, infrared spectrum and ultraviolet spectrum. Figure 1 Figure D is the particle size distribution diagram of ZPD. Figure 1 Figure E is the infrared spectrum of ZPD. Figure 1 Figure F is the UV spectrum of ZPD.

[0047] like Figure 1 As shown in Figure D, the diameter of ZPD is about 500nm. Figure 1 As shown in Figure E, ZPD, ZPD precursor and dopamine hydrochloride were characterized by Fourier transform infrared spectrometer, and it can be found that ZPD precursor and dopamine hydrochloride have intersection peaks, indicating that dopamine hydrochloride can be successfully loaded onto ZPD precursor. Figure 1 As shown in Figure F, the full spectrum of ZPD and ZPD precursor was scanned with a UV spectrophotometer. It can be seen that the ZPD precursor has absorbance due to the presence of dopamine hydrochloride; and ZPD is a carbonized nanozyme, so it also has absorbance at 808nm.

[0048] Experimental Example 2: Characterization of ZPD enzyme activity The present invention utilizes the color development reaction of TMB oxidation assisted by hydrogen peroxide to measure the catalytic performance of the peroxidase of ZPD prepared in Example 1 in PBS with pH=4.0 and 10mM. All reactions are carried out at 37°C for 5 minutes, and the UV-visible absorption spectrum is recorded. Among them, the concentration of hydrogen peroxide is 25mM, the concentration of TMB is 1mM, the concentration of ZPD is 40μg / mL, and the total reaction system is 400μL. For the peroxidase activity under NIR laser irradiation, under the same conditions, the reaction system is irradiated with NIR laser for 5 minutes and the UV absorption spectrum is recorded.

[0049] Figure 2 The characterization results of the peroxidase activity of ZPD were as follows: Figure 2 Figure A and Figure 2As shown in Figure B, ZPD has good peroxidase activity. When ZPD, hydrogen peroxide and TMB coexist, the enzyme activity of ZPD is the highest. Figure 2 Figure C and Figure 2 As shown in Figure D, the enzyme activity of ZPD is the highest when ZPD, hydrogen peroxide and TMB co-exist and are irradiated by NIR laser.

[0050] Experimental Example 3: Photothermal drug release ability test of ZPD@BBH The present invention firstly constructs a standard curve of ZPD versus berberine hydrochloride loading, such as Figure 3 As shown; through linear regression analysis, the standard curve equation is y=0.0531744x+0.0113125, where x represents the concentration of berberine hydrochloride and y is the absorbance value. Based on the standard curve, it is calculated that the loading amount of berberine hydrochloride on ZPD in ZPD@BBH prepared in Example 1 is 18.55%±1.8%. On this basis, the present invention further determines the drug release capacity of ZPD@BBH.

[0051] Depend on Figure 4 It can be seen that when the pH value is 5.5, that is, under acidic conditions, the cumulative drug release rate of ZPD@BBH in 48 hours is 55.46%±3.25%, and when the pH value is 7.4, that is, under neutral conditions, the cumulative drug release rate of ZPD@BBH in 48 hours is 37.37%±0.69%. Therefore, the drug release ability of ZPD@BBH under acidic conditions is higher than that under neutral conditions, indicating that the bionic nanomedicine provided by the present invention has better drug release characteristics in the microenvironment of the subcutaneous drug-resistant bacteria infection area.

[0052] To evaluate the photothermal stability of ZPD@BBH, ZPD@BBH with different concentrations were subjected to three heating / cooling cycles of 808 nm NIR irradiation with different powers. Figure 4 shown.

[0053] At 808nm, 1.0W / cm 2 Under NIR laser irradiation, the photothermal properties of ZPD@BBH showed concentration dependence, and the maximum temperature increased with increasing concentration; ZPD@BBH also exhibited power gradient-related photothermal properties at a fixed concentration of 100 μg / mL. The higher the power, the higher the temperature, and the local temperature of the tissue could reach about 50°C; and in three cycle experiments, no obvious difference was found in the temperature of ZPD@BBH, indicating that ZPD@BBH has good photothermal stability.

[0054] Since carbon nanozymes have good photothermal properties, the present invention also measured the photoresponsive drug release properties of ZPD@BBH, such as Figure 4The ZPD@BBH was shown in Figure 1 after 3 repetitions of 1.0 W / cm 2 Under near-infrared laser irradiation, the cumulative drug release rate of ZPD@BBH in 48 hours was 68.19%±0.87%, which was significantly higher than that of the control group without near-infrared laser irradiation. Therefore, it can be shown that ZPD@BBH can enhance the drug release rate through near-infrared laser irradiation, proving that ZPD@BBH has light-responsive drug release characteristics.

[0055] Experimental Example 4: Antibacterial performance test of ZPD and ZPD@BBH Methicillin-resistant Staphylococcus aureus is a common and difficult bacterium that can cause subcutaneous infection in clinical practice. The present invention uses an agar plate method to systematically evaluate the in vitro antibacterial ability of ZPD and ZPD@BBH prepared in Example 1, and statistically calculates the antibacterial rate of ZPD and ZPD@BBH against methicillin-resistant Staphylococcus aureus. The antibacterial rate calculation formula is as follows: ; In the antibacterial rate calculation formula: C represents the antibacterial rate, A represents the colony count of methicillin-resistant Staphylococcus aureus on the agar plate of the untreated control group, and B represents the colony count of methicillin-resistant Staphylococcus aureus on the agar plates of other groups except the untreated control group.

[0056] The experiment was divided into the following groups: (a) no treatment control group; (b) no treatment control group + hydrogen peroxide + NIR light group; (c) ZPD treatment group, the concentration of ZPD was 20μg / mL, 40μg / mL, 60μg / mL, 80μg / mL and 100μg / mL; (d) ZPD + hydrogen peroxide + NIR light treatment group, the concentration of ZPD was 20μg / mL, 40μg / mL, 60μg / mL, 80μg / mL and 100μg / mL; (e) ZPD@BBH treatment group, the ZPD concentrations in ZPD@BBH were 20μg / mL, 40μg / mL, 60μg / mL, 80μg / mL and 100μg / mL; (f) ZPD@BBH+hydrogen peroxide+NIR light treatment group, the ZPD concentrations in ZPD@BBH were 20μg / mL, 40μg / mL, 60μg / mL, 80μg / mL and 100μg / mL. The MRSA culture time was 10h, the hydrogen peroxide concentration was 100μM, and the NIR light intensity was 1W / cm 2 , the NIR illumination duration is 10 minutes.

[0057] 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 / cm 2When combined with low-intensity near-infrared light, the antibacterial rate can reach more than 90%; when using ZPD@BBH, only 80 μg / mL of ZPD@BBH is required, combined with 100 μM hydrogen peroxide and 1W / cm 2 The antibacterial rate can reach more than 90% by co-treatment with low-intensity near-infrared light, and when the concentration of ZPD@BBH is 100 μg / mL, the bacteria are almost completely killed.

[0058] comprehensive Figure 5 The results show that ZPD@BBH can achieve enzyme catalysis-photoresponse synergistic treatment in vitro at a low concentration and has a strong antibacterial effect. The reason is that ZPD@BBH has multiple antibacterial modes: (1) The local warmth generated under near-infrared laser irradiation can kill some bacteria; (2) The heat generated under near-infrared laser irradiation increases the catalytic activity of ZPD's peroxidase, more effectively converting hydrogen peroxide into toxic hydroxyl radicals. The strong oxidizing property of hydroxyl radicals can quickly destroy bacterial cell membranes and intracellular substances, leading to bacterial death; (3) ZPD has a dandelion-like surface, which is easier to target the bacterial surface; berberine hydrochloride, an effective ingredient in traditional Chinese medicine, has high antibacterial ability and the photothermal killing method of bacteria is not easy to cause the production of drug-resistant bacteria. This three-way synergistic bactericidal system of photothermal therapy-chemodynamic therapy-antibacterial drugs reduces the amount of drugs and the intensity of near-infrared lasers, achieving the effect of less drugs and high efficiency, greatly reducing the toxic side effects to the body, and achieving a satisfactory antibacterial effect.

[0059] Experimental Example 5: Biosafety Testing of ZPD@BBH Mouse epithelial fibroblast L929 cells were selected as the research object, and the in vitro safety of ZPD@BBH was verified by MTT colorimetry. The specific steps were as follows: the cells were plated in a 96-well plate at a density of 1×10 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, and only PBS was used in the control group; after 24 hours of co-incubation, the medium was aspirated, and the cells were washed once with PBS. Then, complete medium containing MTT was added to each well, and the cells were placed in a cell culture incubator for 4 hours, and then MTT was aspirated. Finally, 100 μL of DMSO was added to each well, and the cell viability was detected at 490 nm using an enzyme marker. The test results are shown in Figure 2. Figure 6 shown.

[0060] from Figure 6As shown in Figure A, the toxicity of 100 μg / mL ZPD@BBH to L929 cells is negligible. The cells treated with ZPD@BBH have normal morphology and no abnormal cell concentration. Figure 6 As shown in Figure B, when the concentration of ZPD@BBH is as high as 200 μg / mL, its toxicity to L929 cells is very limited, and the survival rate of L929 cells is >80%. The maximum concentration of ZPD@BBH in the experimental group is 100 μg / mL. These results indicate that ZPD@BBH has good cell compatibility.

[0061] Then the present invention tested the hemolysis rate of ZPD@BBH. Figure 6 As shown in Figure C, the hemolysis rate after treatment with ZPD@BBH at a concentration of 200 μg / mL and below was less than 5%. The blood cells treated with hydrogen peroxide served as the positive control group, and the supernatant was bright red. After treatment with normal 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 was very low and the blood compatibility was good.

[0062] It should be noted that when the present invention involves a numerical range, it should be understood that the two 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 those in the embodiments, in order to avoid redundancy, the present invention describes a preferred embodiment. Although the preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they know the basic creative concept. Therefore, the attached claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0063] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A method for preparing a bionic nanomedicine for resisting subcutaneous drug-resistant bacterial infection, characterized in that: The following steps are involved: Dissolving 2-methylimidazole in methanol to obtain a mixed methanol solution, adding a mixed aqueous solution of zinc salt and dopamine hydrochloride to the mixed methanol solution, and centrifuging to obtain a dandelion-shaped carbon nanozyme precursor; carbonizing the carbon nanozyme precursor to obtain the carbon nanozyme; Berberine hydrochloride is mixed with carbon nanozyme to load berberine hydrochloride on the carbon nanozyme, and then the mixture is centrifuged and dried to obtain the bionic nanomedicine.

2. The method for preparing the bionic nanomedicine for resisting subcutaneous drug-resistant bacterial infection according to claim 1, characterized in that: The zinc salt is zinc nitrate.

3. The method for preparing the bionic nanomedicine for resisting subcutaneous drug-resistant bacterial infection according to claim 1, characterized in that: The zinc salt is zinc nitrate hexahydrate; the mass ratio of the 2-methylimidazole, zinc nitrate hexahydrate and dopamine hydrochloride is 0.32-0.45:1.4375-2.1375:0.095-0.

105.

4. The method for preparing the bionic nanomedicine for resisting subcutaneous drug-resistant bacterial infection according to claim 1, characterized in that: The mixing mass ratio of the berberine hydrochloride to the carbon nanozyme is 1-3:

1.

5. The method for preparing the bionic nanomedicine for resisting subcutaneous drug-resistant bacterial infection according to claim 1, characterized in that: The centrifugal speed is 11000r / min~12000r / min, and the centrifugal time is 8min~15min; the drying temperature is 55℃~65℃, and the drying time is 8h~12h.

6. The method for preparing the bionic nanomedicine for resisting subcutaneous drug-resistant bacterial infection according to claim 1, characterized in that: The carbonization temperature is 800° C. to 1000° C., and the time is 2 h to 4 h.

7. A bionic nanomedicine prepared by the method for preparing a bionic nanomedicine for resisting subcutaneous drug-resistant bacterial infection according to any one of claims 1 to 6.

8. Use of the bionic nanomedicine according to claim 7 in the preparation of a drug for treating subcutaneous drug-resistant bacterial infection, characterized in that: The bacteria is Staphylococcus aureus.

9. Use of the bionic nano drug according to claim 7 in preparing chemodynamic therapeutic agents.

10. Use of the bionic nanomedicine according to claim 7 in preparing a photothermal therapy agent.

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