Nano-antibacterial agent for treating biofilm infection by using Prussian blue as well as preparation method and application of nano-antibacterial agent

Abstract

The invention discloses a nano-antibacterial agent for treating biofilm infection through Prussian blue and a preparation method and application thereof.According to the nano-antibacterial agent, hollow Prussian blue (HPB) serves as a carrier to load a substance with anti-inflammatory and anti-oxidation effects, a metal polyphenol network structure is formed through chelating of tannic acid (TA) modified by hyaluronic acid (HA) and high-valence metal ions, and the nano-antibacterial agent is used for treating biofilm infection through Prussian blue. And the MPNs are packaged to obtain the product. All the components of the nano antibacterial agent have a synergistic effect, MPNs formed by chelating TA modified by hyaluronic acid and high-valence metal ions in the antibacterial agent have good photo-thermal performance, and light energy can be converted into heat energy for photo-thermal therapy. According to the invention, the nanoparticles for photo-thermal antisepsis and anti-inflammation are constructed, so that comprehensive treatment of anti-biofilm and anti-inflammatory effect is realized, and healing of infected parts is promoted. And the antibacterial agent has good biocompatibility and high biological safety, and is very suitable for replacing antibiotics to treat biological membrane infection.

Description

Technical Field

[0001] The present invention belongs to the technical field of antibacterial materials, and more specifically, a nano antibacterial agent for treating biofilm infections with Prussian blue, its preparation method and application. Background Art

[0002] In recent years, bacterial infections have seriously endangered human health and become one of the main causes of human death. Research shows that about 65% - 80% of human bacterial infections are related to bacterial biofilms, and multi-species biofilms play an important role in persistent infections. Bacterial biofilms are three-dimensional microbial communities formed by extracellular polymeric matrices secreted by bacteria, which wrap the bacteria and the bacteria themselves. Compared with planktonic bacteria that do not form biofilms, biofilm bacteria have 10 - 1000 times higher tolerance to antibiotics and stronger resistance to complex environments. Since the discovery of antibiotics, due to their high efficiency, broad-spectrum antibacterial and other characteristics, they have been widely used to treat bacterial infections in humans and animals. However, with the overuse of antibiotics, the bacterial drug resistance has increased, leading to global problems of drug-resistant bacterial infections, posing a major threat to human health. Antibiotic resistance has become one of the greatest challenges facing global public health. Therefore, there is an urgent need to explore new and efficient means of anti-bacterial biofilms.

[0003] Photothermal Antibacterial Therapy (PTAT), as a non - antibiotic - dependent treatment strategy, has the advantages of being rapid, controllable, non - invasive, non - resistant, and low - toxic. It utilizes photothermal agents to convert light energy into local heat energy under near - infrared light irradiation, disrupting the permeability of bacterial cell membranes and denaturing nucleic acids and proteins inside bacteria, thereby killing bacteria. Many nanomaterials with photothermal properties have been applied in photothermal antibacterial applications. Among them, metal - phenolic networks (MPNs) are rapidly self - assembled in aqueous solution by polyphenolic substances and metal ions through coordination chelation. The coordination of metal ions with polyphenols in MPNs results in a relatively broad absorption band in the near - infrared region. Therefore, MPNs have a high photothermal conversion efficiency and good photothermal stability, and can be used as photothermal conversion materials for photothermal therapy. In addition, MPNs have pH responsiveness. When the pH value is higher than 7, each metal ion in the MPN structure tightly binds to three parts of polyphenols in a stable network structure. However, when the pH value is lower than 6, most phenolic hydroxyl groups on polyphenols are protonated under acidic conditions, weakening their chelation with metal ions and leading to the decomposition of the metal - polyphenol network. Therefore, MPNs will decompose in the acidic microenvironment of bacterial biofilms. In addition, the synthesis process of MPNs is simple, with good biocompatibility and biodegradability, and strong adhesion to the surfaces of various carriers. Therefore, MPNs can be used as a gating for drug release to control the release of drugs at the site of bacterial biofilms.

[0004] Prussian Blue (PB) is a metal - organic framework composed of alternating iron and ferrous ions, which coordinates with cyanide. PB nanoparticles can be synthesized under mild conditions, with hollow mesopores, a large specific surface area, effective iron ion active sites, a high affinity for cyanide ions, and a chemically stable structure, making it an ideal candidate for applications in the biomedical field. PB nanoparticles have been proven to be safe and biocompatible and have been approved by the US Food and Drug Administration (FDA) for clinical use. Recent theoretical studies have shown that PB nanoparticles have gained great popularity in PTT due to their strong near - infrared radiation absorption ability, high photothermal efficiency, excellent chemical stability, outstanding biocompatibility, and low cost. In addition, PB nanoparticles, also known as Prussian blue nanozymes, have a good affinity for hydroxyl radicals, enabling them to act as ROS scavengers. Due to their natural catalytic activity and enzyme specificity, PB nanoparticles are very suitable for functioning in physiological solutions. In addition, PB nanoparticles can be further etched into a hollow structure to become hollow mesoporous Prussian blue nanoparticles, with retained photothermal properties and a greatly increased specific surface area, thus enabling overloading of drugs. Summary of the Invention

[0005] The object of the present invention is to provide a nano-antibacterial agent for treating biofilm infections with Prussian blue and its preparation method and application. The technical problem to be solved by the present invention is to synergistically exert anti-inflammatory and antioxidant effects by loading drugs with anti-inflammatory and antioxidant effects on hollow Prussian blue (HPB). In addition, HPB and metal polyphenol networks (MPNs) synergistically exert photothermal antibacterial effects, effectively solving the problem of the non-persistent photothermal effect when HPB and MPNs are used alone, and providing a nanoparticle for photothermal antibacterial and anti-inflammatory treatment to achieve comprehensive treatment of anti-biofilm and anti-inflammatory effects and promote the healing of the infected site.

[0006] The nano-antibacterial agent is prepared by loading drugs with anti-inflammatory and antioxidant effects using HPB as a carrier and encapsulating it with MPNs formed by chelating tannic acid (TA) modified with hyaluronic acid (HA) and high-valent metal ions. Each component of the nano-antibacterial agent acts synergistically. Among them, MPNs formed by chelating TA modified with HA and high-valent metal ions have good photothermal properties and can convert light energy into heat energy for photothermal treatment. In addition, MPNs have pH responsiveness, and in the acidic microenvironment of bacterial biofilms, the structure of MPNs will decompose and release drugs with anti-inflammatory and antioxidant effects to exert anti-inflammatory and antioxidant effects. HPB can not only act as a photothermal agent to synergistically exert photothermal antibacterial effects with MPNs, but also act as a nanozyme to synergistically exert anti-inflammatory and antioxidant effects with drugs with anti-inflammatory and antioxidant effects. The present invention constructs a nanoparticle for photothermal antibacterial and anti-inflammatory treatment to achieve comprehensive treatment of anti-biofilm and anti-inflammatory effects and promote the healing of the infected site. Moreover, the antibacterial agent has good biocompatibility and high biosafety and is very suitable for replacing antibiotics to treat biofilm infections.

[0007] The object of the present invention can be achieved by the following technical solutions: A nano-antibacterial agent for treating biofilm infections with Prussian blue, wherein the nano-antibacterial agent is prepared by loading drugs with anti-inflammatory and antioxidant effects using hollow Prussian blue (HPB) as a carrier and encapsulating it with metal polyphenol networks (MPNs) formed by chelating tannic acid (TA) modified with hyaluronic acid (HA) and metal ions.

[0008] As a further solution of the present invention: the anti-inflammatory and antioxidant drugs include epigallocatechin gallate (EGCG), tanshinol (TAN), resveratrol (RES), berberine (BBR), baicalin (BI), etc.

[0009] As a further solution of the present invention: the high-valence metal ions include metal ions such as iron, calcium, aluminum, vanadium, chromium, manganese, copper, and zinc.

[0010] A preparation method of a nano antibacterial agent for treating biofilm infection with Prussian blue specifically includes the following steps: S1. First, synthesize Boc group-protected TA: Dissolve TA (20.0 g, 11.8 mmol) in 50 mL of dimethylformamide (DMF), then add 3-(Boc-amino)-propyl bromide (11.7 g, 49.0 mmoL) and potassium carbonate (K2CO3, 20.3 g, 147.0 mmoL). Under argon protection, heat to 60 °C and stir for 6 hours. After cooling to room temperature, add 100 mL of deionized water, acidify the solution with HCI (6M), extract three times with 100 mL of ethyl acetate (EA). The separated EA layer is washed twice with 100 mL of deionized water, dried with an appropriate amount of anhydrous sodium sulfate, then remove EA with a rotary evaporator, redissolve in 50 mL of absolute ethanol, dialyze with a 3500 Da dialysis bag for two days, and then remove absolute ethanol with a rotary evaporator, and dry by suction to obtain a white solid TA-Boc-NH2; Subsequently, remove the Boc group and expose the amino group: Weigh TA-Boc-NH2 (10 g) and dissolve it in 100 mL of absolute ethanol (EtOH). Pass the HCI vapor generated by the reaction of concentrated sulfuric acid and sodium chloride (NaCI) into the above solution for Boc group removal treatment for 6 h, continue to stir at room temperature for 24 h, remove absolute ethanol with a rotary evaporator, redissolve the remaining solid in 50 mL of deionized water, dialyze with a 3500 Da dialysis bag for two days, and freeze-dry to obtain a light yellow solid TAA; S2. Weigh 30 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 19 mg of N-hydroxysuccinimide (NHS) and dissolve them in 20 mL of phosphate buffer solution (PBS) with pH 5.0 for activation for 30 min. Then add 50 mg of HA and stir until completely dissolved. Weigh 96 mg of TAA, dissolve it in a small amount of dimethyl sulfoxide (DMSO, 2 - 3 mL), and add it to the above solution. Stir overnight under nitrogen protection, then dialyze with a DMSO:H2O (1:10) mixed solution using a 3500 Da dialysis bag, and then dialyze with deionized water. Freeze-dry to obtain a TA-HA solid; S3. Synthesize solid Prussian blue (PB) with a size of 110 - 190 nm by hydrothermal method. First, prepare a hydrochloric acid solution (HCl, 40 mL, 0.01 M), add 3 g of polyvinylpyrrolidone (PVP) to the solution until it dissolves, then add 131.7 mg of potassium ferricyanide (K3[Fe(CN)6]) and stir for 30 min until the solution becomes clear. Transfer the above solution into a vial and place it in an 80°C oven for reaction for 20 - 24 h. After the reaction, centrifuge at 10600 rpm / min for 10 min, wash the precipitate in the solvent order of "water ethanol ethanol water", and dry at 60°C to obtain solid Prussian blue nanoparticles; Weigh 100 mg of PVP and dissolve it in 4 mL of deionized water, weigh 20 mg of PB and dissolve it in 6 mL of deionized water, and dissolve 1.72 mL of HCl in 10 mL of deionized water. Then add the three solutions to the reaction kettle in sequence, stir at room temperature for 30 min, and then carry out hydrothermal reaction at 140°C for 4 h. After the reaction, cool it and centrifuge at 8000 - 10000 rpm / min for 10 min. Finally, wash the precipitate in the solvent order of "water ethanol water" to obtain hollow Prussian blue nanoparticles (HPB); S4. Weigh 20 mg of HPB and dissolve it in 10 mL of deionized water, and ultrasonically disperse it evenly. Then weigh 10 mg of the drug with anti - inflammatory and antioxidant effects and dissolve it in 10 mL of deionized water to prepare a 1 mg / mL solution. After dissolution, add the solution of the drug with anti - inflammatory and antioxidant effects dropwise to the HPB solution, stir the reaction in the dark for 24 h, centrifuge at 8000 rpm for 10 min, and wash it with deionized water 2 - 3 times. Collect the supernatant to obtain drug - loaded Prussian blue; S5. Redisperse the drug - loaded Prussian blue in 10 mL of deionized water, stir it magnetically, add 2 mL of TA - HA solution (5 mg / mL prepared with deionized water) and stir for 20 min. Then use a syringe pump to uniformly add 12 mL of high - valence metal ions (1 mg / mL) within 10 min. Finally, adjust the pH of the solution to alkaline with Tris buffer solution (pH 8.5) and react at room temperature for 30 min to prepare the nano - antibacterial agent, and store it at 4°C.

[0011] Application of the nano - antibacterial agent of Prussian blue for treating biofilm infections, wherein the nano - antibacterial agent has a synergistic killing effect on bacterial biofilms and has an anti - inflammatory effect.

[0012] Advantages of the present invention: Each component of the nano-antibacterial agent acts synergistically. Among them, the MPNs formed by the chelation of TA modified with hyaluronic acid (HA) and high-valent metal ions in the antibacterial agent have good photothermal properties and can convert light energy into heat energy for photothermal therapy. In addition, MPNs have pH responsiveness. In the acidic microenvironment of bacterial biofilms, the MPNs structure will decompose and release substances with anti-inflammatory and antioxidant effects to exert anti-inflammatory and antioxidant effects. And HPB can not only act as a photothermal agent to synergistically enhance the photothermal antibacterial effect of MPNs, but also act as a nanozyme to synergistically enhance the anti-inflammatory and antioxidant effects of substances with anti-inflammatory and antioxidant effects. The present invention constructs nanoparticles for photothermal antibacterial and anti-inflammatory treatment to achieve comprehensive treatment of anti-biofilm and anti-inflammatory effects and promote the healing of the infected area. Moreover, the antibacterial agent has good biocompatibility and high biosafety and is very suitable for replacing antibiotics to treat biofilm infections. Brief Description of the Drawings

[0013] For the convenience of those skilled in the art to understand, the present invention will be further described below with reference to the accompanying drawings.

[0014] Figure 1 It is a schematic diagram of the preparation process and antibacterial effect of a nano-antibacterial agent for treating biofilm infections with Prussian blue according to the present invention.

[0015] Figure 2 It is a transmission electron microscope image of the HPB (A) and EGCG@HPB@MPN-HA (EHM) nano-antibacterial agents (B) prepared in Example 1 of the present invention.

[0016] Figure 3 It is a photothermal heating curve graph and its photothermal conversion efficiency graph of different concentrations of the EHM nano-antibacterial agent prepared in Example 1 of the present invention under 808 nm near-infrared light irradiation.

[0017] Figure 4 It is the test of the reactive oxygen species scavenging ability of the EHM nano-antibacterial agent prepared in Example 1 of the present invention.

[0018] Figure 5 It is the plate coating and survival rate column graph of the inhibitory effect of the EHM nano-antibacterial agent prepared in Example 1 of the present invention on Staphylococcus aureus and Escherichia coli biofilms under light and non-light conditions.

[0019] Figure 6 It is the scanning electron microscope (SEM) image of the EHM nano-antibacterial agent prepared in Example 1 of the present invention against Staphylococcus aureus and Escherichia coli biofilms under light and non-light conditions.

[0020] Figure 7 It is the performance study of the EHM nano-antibacterial agent prepared in Example 1 of the present invention for in vivo anti-bacterial biofilm infection.

[0021] Figure 8 This is the hemolysis experiment graph of the EHH nano-antibacterial agent prepared in Example 1 of the present invention at different concentrations. Detailed implementation manners

[0022] The technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0023] As Figure 1-3 shown, a nano-antibacterial agent for treating biofilm infections with Prussian blue, the nano-antibacterial agent is prepared by loading a drug with anti-inflammatory and antioxidant effects using hollow Prussian blue (HPB) as a carrier and encapsulating it with a metal polyphenol network (MPNs) formed by chelating tannic acid (TA) modified with hyaluronic acid (HA) and metal ions; the anti-inflammatory and antioxidant drugs include epigallocatechin gallate (EGCG), tanshinol (TAN), resveratrol (RES), berberine (BBR), baicalin (BI), etc.; the high-valent metal ions include metal ions such as iron, calcium, aluminum, vanadium, chromium, manganese, copper, and zinc.

[0024] A preparation method of a nano-antibacterial agent for treating biofilm infections with Prussian blue specifically includes the following steps: S1. First, synthesize TA protected by Boc group: Take TA (20.0 g, 11.8 mmol) and dissolve it in 50 mL of dimethylformamide (DMF), then add 3-(Boc-amino)-propyl bromide (11.7 g, 49.0 mmoL) and potassium carbonate (K2CO3, 20.3 g, 147.0 mmoL). Under argon protection, heat to 60 °C and stir for 6 hours. After cooling to room temperature, add 100 mL of deionized water, acidify the solution with HCI (6M), extract it three times with 100 mL of ethyl acetate (EA). The separated EA layer is washed twice with 100 mL of deionized water, dried with an appropriate amount of anhydrous sodium sulfate, then remove EA with a rotary evaporator, redissolve it in 50 mL of absolute ethanol, dialyze it for two days with a 3500 Da dialysis bag, and then remove absolute ethanol with a rotary evaporator, and dry it to obtain a white solid TA-Boc-NH2; Subsequently, the Boc group was removed to expose the amino group: 10 g of TA-Boc-NH2 was weighed and dissolved in 100 mL of absolute ethanol (EtOH). HCl vapor generated by the reaction of concentrated sulfuric acid and sodium chloride (NaCl) was introduced into the above solution for Boc group removal treatment for 6 h, and stirring was continued at room temperature for 24 h. Absolute ethanol was removed using a rotary evaporator. The remaining solid was redissolved in 50 mL of deionized water and dialyzed using a 3500 Da dialysis bag for two days. After freeze-drying, a pale yellow solid TAA was obtained; S2: 30 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 19 mg of N-hydroxysuccinimide (NHS) were weighed and dissolved in 20 mL of phosphate buffered saline (PBS) solution with pH 5.0 for activation for 30 min. Subsequently, 50 mg of HA was added and stirred until completely dissolved. 96 mg of TAA was weighed, dissolved in a small amount of dimethyl sulfoxide (DMSO, 2 - 3 mL), and added to the above solution. Stirring was carried out overnight under nitrogen protection. Subsequently, dialysis was performed using a DMSO:H2O (1:10) mixed solution with a 3500 Da dialysis bag, and then dialysis was carried out with deionized water. Freeze-drying yielded a TA-HA solid; S3: Solid Prussian blue (PB) with a size of 110 - 190 nm was synthesized by a hydrothermal method. First, a hydrochloric acid solution (HCl, 40 mL, 0.01 M) was prepared. 3 g of polyvinylpyrrolidone (PVP) was added to the solution until dissolved, and then 131.7 mg of potassium ferricyanide (K3[Fe(CN)6]) was added and stirred for 30 min until the solution became clear. The above solution was placed in a vial and reacted in an 80°C oven for 20 - 24 h. After the reaction ended, centrifugation was carried out at 10600 rpm / min for 10 min. The precipitate was washed in the solvent order of "water ethanol ethanol water", and dried at 60°C to obtain solid Prussian blue nanoparticles; 100 mg of PVP was weighed and dissolved in 4 mL of deionized water, 20 mg of PB was weighed and dissolved in 6 mL of deionized water, and 1.72 mL of HCl was dissolved in 10 mL of deionized water. The three solutions were successively added to the reaction kettle and stirred at room temperature for 30 min. Subsequently, hydrothermal reaction was carried out at 140°C for 4 h. After the reaction ended, it was cooled and centrifuged at a speed of 8000 - 10000 rpm / min for 10 min. Finally, the precipitate was washed in the solvent order of "water ethanol water" to obtain hollow Prussian blue nanoparticles (HPB); S4. Weigh 20 mg of HPB and dissolve it in 10 mL of deionized water, then ultrasonically disperse it evenly. Weigh another 10 mg of the drug with anti-inflammatory and antioxidant effects and dissolve it in 10 mL of deionized water to prepare a 1 mg / mL solution. After dissolution, add the solution of the drug with anti-inflammatory and antioxidant effects dropwise to the HPB solution, stir the reaction in the dark for 24 h, centrifuge at 8000 rpm for 10 min, and wash it with deionized water 2 - 3 times. Collect the supernatant to obtain the drug-loaded Prussian blue. S5. Redisperse the drug-loaded Prussian blue in 10 mL of deionized water, stir it magnetically to disperse, add 2 mL of TA-HA solution (5 mg / mL prepared with deionized water), stir for 20 min, then use a syringe pump to uniformly add 12 mL of high-valent metal ions (1 mg / mL) within 10 min. Finally, adjust the pH of the solution to alkaline with Tris buffer (pH 8.5), react at room temperature for 30 min to obtain the nano-antibacterial agent, and store it at 4°C. Application of the nano-antibacterial agent for treating biofilm infections with Prussian blue, wherein the nano-antibacterial agent has a synergistic killing effect on bacterial biofilms and has an anti-inflammatory effect.

[0025] Example 1: A nano-antibacterial agent for treating biofilm infections with Prussian blue (EGCG@HPB@MPN-HA, EHM) The preparation of the nano-antibacterial agent for treating biofilm infections with Prussian blue (EGCG@HPB@MPN-HA, EHM) specifically includes the following steps: S1. First, synthesize Boc-group protected TA: Dissolve TA (20.0 g, 11.8 mmol) in 50 mL of DMF, then add 3-(Boc-amino)-propyl bromide (11.7 g, 49.0 mmoL) and K2CO3 (20.3 g, 147.0 mmoL). Under argon protection, heat to 60°C and stir for 6 hours. After cooling to room temperature, add 100 mL of deionized water, acidify the solution with HCl (6M), extract it three times with 100 mL of EA. Wash the separated EA layer twice with 100 mL of deionized water, dry it with an appropriate amount of anhydrous sodium sulfate, then remove EA with a rotary evaporator, redissolve it in 50 mL of absolute ethanol, dialyze it with a 3500 Da dialysis bag for two days, and then remove absolute ethanol with a rotary evaporator and dry it to obtain a white solid TA-Boc-NH2. Subsequently, the Boc group was removed to expose the amino group: 10 g of TA-Boc-NH2 was weighed and dissolved in 100 mL of EtOH. HCl vapor generated by the reaction of concentrated sulfuric acid and NaCl was introduced into the above solution for Boc group removal treatment for 6 h, and stirring was continued at room temperature for 24 h. Absolute ethanol was removed using a rotary evaporator. The remaining solid was redissolved in 50 mL of deionized water and dialyzed using a 3500 Da dialysis bag for two days. After freeze-drying, a pale yellow solid TAA was obtained. S2: 30 mg of EDC and 19 mg of NHS were weighed and dissolved in 20 mL of PBS solution with pH 5.0 for activation for 30 min. Subsequently, 50 mg of HA was added and stirred until completely dissolved. 96 mg of TAA was weighed, dissolved in a small amount of DMSO (2 - 3 mL), and added to the above solution. Stirring was carried out overnight under nitrogen protection. Subsequently, dialysis was carried out using a 3500 Da dialysis bag with a DMSO:H2O (1:10) mixed solution, and then dialysis was carried out with deionized water. After freeze-drying, a TA-HA solid was obtained. S3: Solid Prussian blue with a size of 110 - 190 nm was synthesized by the hydrothermal method. First, a hydrochloric acid solution (HCl, 40 mL, 0.01 M) was prepared. 3 g of polyvinylpyrrolidone (PVP) was added to the solution until dissolved, and then 131.7 mg of potassium ferricyanide (K3[Fe(CN)6]) was added and stirred for 30 min until the solution became clear. The above solution was placed in a vial and reacted in an 80 °C oven for 20 - 24 h. After the reaction, centrifugation was carried out at 10600 rpm / min for 10 min, and the precipitate was washed in the solvent order of "water ethanol ethanol water", and dried at 60 °C to obtain solid Prussian blue nanoparticles. 100 mg of PVP was weighed and dissolved in 4 mL of deionized water, 20 mg of PB was weighed and dissolved in 6 mL of deionized water, and 1.72 mL of HCl was in 10 mL of deionized water. The three solutions were successively added to the reaction kettle and stirred at room temperature for 30 min. Subsequently, hydrothermal reaction was carried out at 140 °C for 4 h. After the reaction, it was cooled and centrifuged at 8000 - 10000 rpm / min for 10 min. Finally, the precipitate was washed in the solvent order of "water ethanol water" to obtain hollow Prussian blue nanoparticles (HPB). S4: 20 mg of HPB was weighed and dissolved in 10 mL of deionized water and ultrasonically dispersed evenly. Then, 10 mg of EGCG was weighed and dissolved in 10 mL of deionized water to prepare a 1 mg / mL solution. After dissolution, the EGCG solution was added dropwise to the HPB solution, and stirring reaction was carried out in the dark for 24 h. Centrifugation was carried out at 8000 rpm for 10 min, and washed with deionized water 2 - 3 times. The supernatant was collected to obtain drug-loaded Prussian blue. S5. Redisperse the drug-loaded Prussian blue in 10 mL of deionized water, stir magnetically for dispersion, add 2 mL of TA-HA solution (5 mg / mL prepared with deionized water), stir for 20 min, then use a syringe pump to uniformly drip 12 mL of FeCl3·6H2O aqueous solution (1 mg / mL) within 10 min. Finally, adjust the pH of the solution to alkaline with Tris buffer (pH 8.5), react at room temperature for 30 min to obtain the nano-antibacterial agent, and store it at 4°C.

[0026] Example 2: A nano-antibacterial agent (TAN@HPB@MPN-HA) for treating biofilm infections with Prussian blue The preparation of the nano-antibacterial agent (TAN@HPB@MPN-HA, EHM) for treating biofilm infections with Prussian blue specifically includes the following steps: S1. First, synthesize TA protected by Boc group: Dissolve TA (20.0 g, 11.8 mmol) in 50 mL of DMF, then add 3-(Boc-amino)-propyl bromide (11.7 g, 49.0 mmol) and K2CO3 (20.3 g, 147.0 mmol). Under argon protection, heat to 60°C and stir for 6 hours. After cooling to room temperature, add 100 mL of deionized water, acidify the solution with HCl (6M), extract three times with 100 mL of EA. The separated EA layer is washed twice with 100 mL of deionized water, dried with an appropriate amount of anhydrous sodium sulfate, then remove EA with a rotary evaporator, redissolve in 50 mL of absolute ethanol, dialyze with a 3500 Da dialysis bag for two days, and then remove absolute ethanol with a rotary evaporator, and vacuum-dry to obtain a white solid TA-Boc-NH2. Subsequently, remove the Boc group and expose the amino group: Weigh TA-Boc-NH2 (10 g) and dissolve it in 100 mL of EtOH. Pass the HCl vapor generated by the reaction of concentrated sulfuric acid and NaCl into the above solution for Boc group removal treatment for 6 h, continue to stir at room temperature for 24 h, remove absolute ethanol with a rotary evaporator. The remaining solid is redissolved in 50 mL of deionized water and dialyzed with a 3500 Da dialysis bag for two days, and then freeze-dried to obtain a light yellow solid TAA. S2. Weigh 30 mg of EDC and 19 mg of NHS and dissolve them in 20 mL of PBS solution with pH 5.0 for activation for 30 min. Then add 50 mg of HA and stir until completely dissolved. Weigh 96 mg of TAA, dissolve it in a small amount of DMSO (2 - 3 mL), and add it to the above solution. Stir overnight under nitrogen protection, then dialyze with a DMSO:H2O (1:10) mixed solution using a 3500 Da dialysis bag, and then dialyze with deionized water, and freeze-dry to obtain a TA-HA solid. S3. Synthesize solid Prussian blue with a size of 110 - 190 nm by hydrothermal method. First, prepare a hydrochloric acid solution (HCl, 40 mL, 0.01 M), add 3 g of polyvinylpyrrolidone (PVP) into the solution until it dissolves, then add 131.7 mg of potassium ferricyanide (K3[Fe(CN)6]) and stir for 30 min until the solution becomes clear. Transfer the above solution into a vial and place it in an 80 °C oven for reaction for 20 - 24 h. After the reaction, centrifuge at 10600 rpm / min for 10 min, wash the precipitate in the solvent order of "water ethanol ethanol water", and dry it at 60 °C to obtain solid Prussian blue nanoparticles. Weigh 100 mg of PVP and dissolve it in 4 mL of deionized water, weigh 20 mg of PB and dissolve it in 6 mL of deionized water, and dissolve 1.72 mL of HCl in 10 mL of deionized water. Then add the three solutions into the reaction kettle in sequence, stir at room temperature for 30 min, and then carry out hydrothermal reaction at 140 °C for 4 h. After the reaction, cool it down and centrifuge at 8000 - 10000 rpm / min for 10 min. Finally, wash the precipitate in the solvent order of "water ethanol water" to obtain hollow Prussian blue nanoparticles (HPB). S4. Weigh 20 mg of HPB and dissolve it in 10 mL of deionized water, and disperse it evenly by ultrasonic wave. Then weigh 10 mg of TAN and dissolve it in 10 mL of deionized water to prepare a 1 mg / mL solution. After dissolution, add the TAN solution dropwise to the HPB solution, stir the reaction in the dark for 24 h, centrifuge at 8000 rpm for 10 min, and wash it with deionized water 2 - 3 times. Collect the supernatant to obtain drug-loaded Prussian blue. S5. Redisperse the drug-loaded Prussian blue in 10 mL of deionized water, stir it magnetically to disperse, add 2 mL of TA-HA solution (5 mg / mL prepared with deionized water) and stir for 20 min. Then use a syringe pump to uniformly drip 12 mL of FeCl3·6H2O aqueous solution (1 mg / mL) within 10 min. Finally, adjust the pH of the solution to alkaline with Tris buffer solution (pH 8.5), and react at room temperature for 30 min to prepare the nano-antibacterial agent, and store it at 4 °C.

[0027] Example 3: A nano-antibacterial agent (RES@HPB@MPN-HA) for treating biofilm infection with Prussian blue The preparation of the nano-antibacterial agent (RES@HPB@MPN-HA, EHM) for treating biofilm infection with Prussian blue specifically includes the following steps: S1. First, synthesize Boc-group protected TA: Dissolve TA (20.0 g, 11.8 mmol) in 50 mL of DMF, then add 3-(Boc-amino)-propyl bromide (11.7 g, 49.0 mmoL) and K2CO3 (20.3 g, 147.0 mmoL). Under argon protection, heat to 60 °C and stir for 6 hours. After cooling to room temperature, add 100 mL of deionized water, acidify the solution with HCI (6M), extract three times with 100 mL of EA. The separated EA layer is washed twice with 100 mL of deionized water, dried with an appropriate amount of anhydrous sodium sulfate, and then EA is removed using a rotary evaporator. Add 50 mL of absolute ethanol to redissolve, dialyze with a 3500 Da dialysis bag for two days, and then remove absolute ethanol using a rotary evaporator. After drying, a white solid TA-Boc-NH2 is obtained. Subsequently, remove the Boc group and expose the amino group: Weigh TA-Boc-NH2 (10 g) and dissolve it in 100 mL of EtOH. Pass the HCI vapor generated by the reaction of concentrated sulfuric acid and NaCI into the above solution for Boc group removal treatment for 6 h, continue to stir at room temperature for 24 h, remove absolute ethanol using a rotary evaporator. The remaining solid is redissolved in 50 mL of deionized water and dialyzed with a 3500 Da dialysis bag for two days. After freeze-drying, a light yellow solid TAA is obtained. S2. Weigh 30 mg of EDC and 19 mg of NHS and dissolve them in 20 mL of PBS solution with pH 5.0 for activation for 30 min. Then add 50 mg of HA and stir until completely dissolved. Weigh 96 mg of TAA, dissolve it in a small amount of DMSO (2 - 3 mL), and add it to the above solution. Stir overnight under nitrogen protection, then dialyze with a DMSO:H2O (1:10) mixed solution using a 3500 Da dialysis bag, and then dialyze with deionized water. After freeze-drying, a TA-HA solid is obtained. S3. Synthesize solid Prussian blue with a size of 110 - 190 nm by hydrothermal method. First, prepare a hydrochloric acid solution (HCl, 40 mL, 0.01 M), add 3 g of polyvinylpyrrolidone (PVP) to the solution until dissolved, then add 131.7 mg of potassium ferricyanide (K3[Fe(CN)6]) and stir for 30 min until the solution is clear. Pour the above solution into a vial and place it in an 80 °C oven for reaction for 20 - 24 h. After the reaction, centrifuge at 10600 rpm / min for 10 min, wash the precipitate in the solvent order of "water ethanol ethanol water", and dry at 60 °C to obtain solid Prussian blue nanoparticles. Weigh 100 mg of PVP and dissolve it in 4 mL of deionized water. Weigh 20 mg of PB and dissolve it in 6 mL of deionized water. Dissolve 1.72 mL of HCl in 10 mL of deionized water. Then, add the three solutions to the reaction kettle in sequence, stir at room temperature for 30 min, and then carry out a hydrothermal reaction at 140 °C for 4 h. After the reaction, cool it and centrifuge for 10 min at a speed of 8000 - 10000 rpm / min. Finally, wash the precipitate in the solvent order of "water - ethanol - water" to obtain hollow Prussian blue nanoparticles (HPB). S4: Weigh 20 mg of HPB and dissolve it in 10 mL of deionized water, and disperse it evenly by ultrasonic wave. Then, weigh 10 mg of RES and dissolve it in 10 mL of deionized water to prepare a 1 mg / mL solution. After dissolution, add the RES solution dropwise to the HPB solution, stir the reaction in the dark for 24 h, centrifuge at 8000 rpm for 10 min, and wash it with deionized water 2 - 3 times. Collect the supernatant to obtain drug - loaded Prussian blue. S5: Redisperse the drug - loaded Prussian blue in 10 mL of deionized water, stir it magnetically to disperse, add 2 mL of TA - HA solution (5 mg / mL prepared with deionized water), stir for 20 min, then use a syringe pump to uniformly drip 12 mL of CaCl2 (1 mg / mL) within 10 min. Finally, adjust the pH of the solution to alkaline with Tris buffer (pH 8.5), and react at room temperature for 30 min to prepare the nano - antibacterial agent, and store it at 4 °C.

[0028] Comparative Example 1: A nano - antibacterial agent (HPB@MPN - HA, HM) for treating biofilm infections with Prussian blue The preparation of the nano - antibacterial agent (HPB@MPN - HA, HM) specifically includes the following steps: S1: First, synthesize TA protected by Boc group: Take TA (20.0 g, 11.8 mmol) and dissolve it in 50 mL of DMF. Then, add 3 - (Boc - amino) - propyl bromide (11.7 g, 49.0 mmoL) and K2CO3 (20.3 g, 147.0 mmoL). Under the protection of argon, heat it to 60 °C and stir for 6 hours. After cooling to room temperature, add 100 mL of deionized water, acidify the solution with HCl (6M), extract it three times with 100 mL of EA. The separated EA layer is washed twice with 100 mL of deionized water, dried with an appropriate amount of anhydrous sodium sulfate, then remove EA with a rotary evaporator, redissolve it in 50 mL of absolute ethanol, dialyze it with a 3500 Da dialysis bag for two days, and then remove absolute ethanol with a rotary evaporator and dry it to obtain a white solid TA - Boc - NH2. Subsequently, the Boc group was removed to expose the amino group: Weigh 10 g of TA-Boc-NH2 and dissolve it in 100 mL of EtOH. Introduce the HCl vapor generated from the reaction of concentrated sulfuric acid and NaCl into the above solution for Boc group removal treatment for 6 h, and continue stirring at room temperature for 24 h. Use a rotary evaporator to remove absolute ethanol. The remaining solid is redissolved in 50 mL of deionized water and dialyzed with a 3500 Da dialysis bag for two days. After freeze-drying, a pale yellow solid TAA is obtained. S2: Weigh 30 mg of EDC and 19 mg of NHS and dissolve them in 20 mL of PBS solution with pH 5.0 for activation for 30 min. Subsequently, add 50 mg of HA and stir until completely dissolved. Weigh 96 mg of TAA, dissolve it in a small amount of DMSO (2 - 3 mL), and add it to the above solution. Stir overnight under nitrogen protection. Then, dialyze with a DMSO:H2O (1:10) mixed solution using a 3500 Da dialysis bag, and then dialyze with deionized water. After freeze-drying, a TA-HA solid is obtained. S3: Solid Prussian blue with a size of 110 - 190 nm was synthesized by a hydrothermal method. First, prepare a hydrochloric acid solution (HCl, 40 mL, 0.01 M). Add 3 g of polyvinylpyrrolidone (PVP) to the solution until dissolved, and then add 131.7 mg of potassium ferricyanide (K3[Fe(CN)6]) and stir for 30 min until the solution becomes clear. Load the above solution into a vial and place it in an 80°C oven for reaction for 20 - 24 h. After the reaction, centrifuge at 10600 rpm / min for 10 min, and wash the precipitate in the solvent order of "water ethanol ethanol water", and dry at 60°C to obtain solid Prussian blue nanoparticles. Weigh 100 mg of PVP and dissolve it in 4 mL of deionized water, weigh 20 mg of PB and dissolve it in 6 mL of deionized water, and 1.72 mL of HCl in 10 mL of deionized water. Add the three solutions to the reaction kettle in sequence, stir at room temperature for 30 min, and then carry out a hydrothermal reaction at 140°C for 4 h. After the reaction, cool and centrifuge at a speed of 8000 - 10000 rpm / min for 10 min. Finally, wash the precipitate in the solvent order of "water ethanol water" to obtain hollow Prussian blue nanoparticles (HPB). S4: Redisperse the obtained HPB in 10 mL of deionized water, stir magnetically for dispersion, add 2 mL of TA-HA solution (5 mg / mL prepared with deionized water) and stir for 20 min. Then, use a syringe pump to uniformly add 12 mL of FeCl3·6H2O aqueous solution (1 mg / mL) within 10 min. Finally, adjust the pH of the solution to alkaline with Tris buffer solution (pH 8.5), and react at room temperature for 30 min to prepare the nano-antibacterial agent, which is stored at 4°C.

[0029] Figure 2 Transmission electron microscopy (TEM) images of the HPB(A) and EGCG@HPB@MPN-HA (EHM) nano-antibacterial agents (B) prepared in Example 1 of the present invention. It can be seen from the figure that the average particle sizes of HPB and EHM are ~120 and ~150 nm, respectively. Figure 3 Photothermal heating curve and photothermal conversion efficiency diagram of different concentrations of EHM nano-antibacterial agents prepared in Example 1 of the present invention under 808 nm near-infrared light irradiation. EHM suspensions with different concentrations (2.5, 5.0, 10, 20 μg / mL) were irradiated with an 808 nm laser with a power of 1.0 W / cm2. As shown in the figure, the temperature of each concentration of nano-antibacterial agent gradually increased with the increase of irradiation time, and the higher the concentration, the faster the temperature increased. When the concentration of the EHM nano-antibacterial agent was 20 μg / mL and irradiated for 5 min, its temperature could rise to 55 °C. When the concentration of the EHM nano-antibacterial agent was 20 μg / mL, the photothermal conversion efficiency could reach 48.3%, demonstrating the excellent photothermal conversion performance of EHM. Figure 4 Test of the reactive oxygen species scavenging ability of the EHM nano-antibacterial agent prepared in Example 1 of the present invention. To prove the ability of the EHM nano-antibacterial agent to scavenge ROS, we selected DPPH•, PTIO• and •OH as model free radicals to react with aqueous solutions of HPB@MPN (HM) or EHM with different concentrations (2.5, 5, 10, 20 μg / mL) (1 mL each). It can be seen from the figure that EHM has a concentration- and time-dependent ROS scavenging ability. With the increase of the EHM concentration, the ultraviolet-visible absorption peaks of DPPH•, PTIO• and •OH at 517, 557 and 652 nm decreased significantly, while the treatment with HM only led to a slight decrease. This indicates that the main reason for the good free radical scavenging ability of EHM is the encapsulation of EGCG, rather than HPB or the outer membrane HM. These experimental results suggest that EHM may be a promising antioxidant that can eliminate ROS during anti-inflammatory treatment. Figure 5Bar chart of the inhibitory effect of the EHM nano-antibacterial agent prepared in Example 1 of the present invention on Staphylococcus aureus and Escherichia coli biofilms under light and non-light conditions, and the survival rate. The inhibitory effect of the EHM nano-antibacterial agent on Staphylococcus aureus and Escherichia coli biofilms under light and non-light conditions was verified by the method of counting by spreading bacteria on a plate. Different concentrations of the EHM nano-antibacterial agent (2.5, 5, 10, 20 μg / mL) were added to the well plates containing mature mixed biofilms of Staphylococcus aureus and Escherichia coli for incubation, and physiological saline was used as the control group. Then, the well plates were irradiated with 808 nm laser at a power of 1.0 W / cm2 for 5 min. The culture medium and materials were aspirated, 1 mL of physiological saline was added and ultrasonicated for 1 min to obtain a bacterial suspension. Subsequently, the bacterial suspension was diluted and spread on the plate, and each plate was photographed and recorded. As shown in the figure, under non-light conditions, when the concentration of the EHM nano-antibacterial agent was 20 μg / mL, the bacterial survival rates of Staphylococcus aureus and Escherichia coli were as high as 55.1% and 50.8% respectively; while under light conditions, as the concentration of the EHM nano-antibacterial agent group increased, the number of colonies decreased significantly. When the concentration of the EHM nano-antibacterial agent was 20 μg / mL, the bacterial survival rates of Staphylococcus aureus and Escherichia coli were only 8.4% and 3.7% respectively, indicating that the EHM nano-antibacterial agent under light conditions had an obvious inhibitory effect on biofilms. Figure 6 Scanning electron microscopy (SEM) images of the EHM nano-antibacterial agent prepared in Example 1 of the present invention against Staphylococcus aureus and Escherichia coli biofilms under light and non-light conditions. The following eight images are magnifications of the above ones. We observed the effects of the EHM nano-antibacterial agent at a concentration of 20 μg / mL on the bacterial morphology of Staphylococcus aureus and Escherichia coli biofilms after treatment under light and non-light conditions by SEM. As shown in the figure, the bacteria in the EHM group under dark conditions all had complete bacterial structures, and the bacterial cell membranes were not damaged. It was possible to observe that the bacteria were dividing and proliferating. However, under light, it led to bacterial collapse and protein denaturation, and serious damage to the bacterial structure was observed, with cytoplasmic leakage. This strongly demonstrated that EHM had strong antibacterial ability under light. Figure 7 Performance study of the EHM nano-antibacterial agent prepared in Example 1 of the present invention against bacterial biofilm infection in vivo. We used a mouse model infected with Staphylococcus aureus to evaluate the anti-biofilm effect of EHM ( Figure 7 a), The infected Balb / c mice were randomly divided into 6 treatment groups (n = 6): PBS, PBS+NIR, HM, HM+NIR, EHM, and EHM+NIR. On day 0, 50 μL of PBS, HM, and EHM (1 mg / mL) were injected intravenously. The mice in the light treatment group received near-infrared irradiation (808 nm, 1 W / cm2) for 5 min, and photothermal imaging was used to ensure that the temperature did not exceed 47°C.Figure 7 b), Record the body weight and biofilm healing of each group of mice every day. Figure 7 The photos in c depict the changes in the infected sites of the six groups during the entire treatment period. By the 10th day, the abscess size in the EHM+NIR group significantly decreased, indicating that EHM has a strong therapeutic effect in vivo. To further quantify the anti-biofilm activity of EHM against biofilm infections in vivo, on the 5th day, the direct plate count method was used. The infected tissues of each group were collected, homogenized in physiological saline, and analyzed ( Figure 7 d and e). The results showed that the antibacterial rates of the HM+NIR and EHM+NIR groups exceeded 85%, significantly higher than those of the HM (18.5%) and EHM (34.68%) groups. This indicates that EHM has an effective PTT-mediated bactericidal effect on bacterial biofilms. In addition, Giemsa and Gram staining were performed 5 days after treatment to evaluate the residual bacteria in the wound ( Figure 7 f and g). The Giemsa staining images showed a large number of bacteria (black arrows) in the tissues of the PBS, PBS+NIR, HM, and EHM groups, indicating severe bacterial invasion of the surrounding tissues ( Figure 5 f). In contrast, the number of bacteria in the HM+NIR and EHM+NIR groups significantly decreased, and due to the excellent bactericidal effect of PTT, the bacteria were almost completely eliminated. Similarly, Gram staining ( Figure 7 g) showed a high level of Staphylococcus aureus (red arrows) in the PBS, PBS+NIR, HM, and EHM groups, while only a small amount of bacterial residue was observed in the HM+NIR and EHM+NIR groups. These findings emphasize that when EHM is used in combination with near-infrared irradiation, EHM has good anti-biofilm activity against bacterial biofilm infections. During the entire treatment period, there were no significant changes in the body weights of all groups, indicating that EHM has good biocompatibility in vivo ( Figure 7 h). Figure 8 This is the hemolysis experiment diagram of the EHM nano-antibacterial agent prepared in Example 1 of the present invention at different concentrations. As can be seen from the figure, when the mass concentration is not higher than 20 μg / mL, the hemolysis rate of the EHM nano-antibacterial agent is lower than the international standard requirement of 5%. This further proves that the EHM nano-antibacterial agent has good biocompatibility and safety when the mass concentration is not higher than 20 μg / mL.

[0030] The preferred embodiments of the present invention disclosed above are only used to help illustrate the present invention. The preferred embodiments do not describe all the details in detail, nor do they limit the invention to the specific embodiments described. Obviously, many modifications and variations can be made according to the content of this specification. These embodiments are selected and specifically described in this specification in order to better explain the principles and practical applications of the present invention, so that those skilled in the art can well understand and utilize the present invention. The present invention is only limited by the claims and their full scope and equivalents.

Claims

1. A nano antibacterial agent for treating biofilm infection with Prussian blue, characterized in that: The nano antibacterial agent uses hollow Prussian blue (HPB) as a carrier to load drugs with anti-inflammatory and antioxidant effects, and is encapsulated by metal polyphenol networks (MPNs) formed by tannic acid (TA) modified by hyaluronic acid (HA) and chelating metal ions.

2. The nano antibacterial agent for treating biofilm infection with Prussian blue according to claim 1, characterized in that: The anti-inflammatory and antioxidant drugs include epigallocatechin gallate (EGCG), tanshinol (TAN), resveratrol (RES), berberine (BBR), baicalin (BI), etc.

3. The nano antibacterial agent for treating biofilm infection with Prussian blue according to claim 1, characterized in that: The high-valent metal ions include metal ions such as iron, calcium, aluminum, vanadium, chromium, manganese, copper, and zinc.

4. The method for preparing the nano antibacterial agent for treating biofilm infection with Prussian blue according to claim 1, characterized in that: The specific steps include: S1. First, synthesize TA protected by Boc group: take TA (20.0 g, 11.8 mmol) and dissolve it in 50 mL of dimethylformamide (DMF), then add 3-(Boc-amino)-propyl bromide (11.7 g, 49.0 mmol) and potassium carbonate (K2CO3, 20.3 g, 147.0 mmol), heat to 60 ° C and stir for 6 hours under argon protection, cool to room temperature, add 100 mL of deionized water, acidify the solution with HCI (6M), extract with 100 mL of ethyl acetate (EA) three times, add 100 mL of deionized water to the separated EA layer, wash it twice, dry it with an appropriate amount of anhydrous sodium sulfate, then remove EA with a rotary evaporator, add 50 mL of anhydrous ethanol to dissolve it again, dialyze it with a 3500 Da dialysis bag for two days, remove anhydrous ethanol with a rotary evaporator, and dry it to obtain a white solid TA-Boc-NH2; Then, the Boc group was removed and the amino group was exposed: TA-Boc-NH2 (10 g) was weighed and dissolved in 100 mL of anhydrous ethanol (EtOH), and the HCI vapor produced by the reaction of concentrated sulfuric acid and sodium chloride (NaCl) was introduced into the above solution to remove the Boc group for 6 h. Stirring was continued at room temperature for 24 h, and anhydrous ethanol was removed by rotary evaporator. The remaining solid was re-dissolved in 50 mL of deionized water and dialyzed with a 3500 Da dialysis bag for two days. After freeze-drying, pale yellow solid TAA was obtained; S2. Weigh 30 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 19 mg of N-hydroxysuccinimide (NHS) and dissolve them in 20 mL of pH 5.0 phosphate buffered saline (PBS) solution for activation for 30 min, then add 50 mg of HA and stir until completely dissolved, weigh 96 mg of TAA and add a small amount of dimethyl sulfoxide (DMSO, 2-3 mL) to dissolve and add to the above solution, stir overnight under nitrogen protection, then dialyze with a 3500 Da dialysis bag with a mixed solution of DMSO:H2O (1:10), and then dialyze with deionized water. Freeze-dry to obtain TA-HA solid; S3. Solid Prussian blue (PB) with a size of 110-190 nm was synthesized by a hydrothermal method. First, a hydrochloric acid solution (HCl, 40 mL, 0.01 M) was prepared, 3 g of polyvinyl pyrrolidone (PVP) was added to the solution until dissolved, and then 131.7 mg of potassium ferrocyanide (K3[Fe(CN)6]) was added and stirred for 30 min until the solution was clear. The above solution was placed in a vial and placed in an 80°C oven for reaction for 20-24 h. After the reaction was completed, centrifuged at 10600 rpm / min for 10 min, and the precipitate was washed in the solvent sequence of "water, ethanol, ethanol, water", and dried at 60°C to obtain solid Prussian blue nanoparticles. Weigh 100 mg PVP and dissolve it in 4 mL deionized water, weigh 20 mg PB and dissolve it in 6 mL deionized water, and 1.72 mL HCl and dissolve it in 10 mL deionized water. Add the three solutions into the reactor in turn, stir at room temperature for 30 min, and then hydrothermally react at 140 °C for 4 h. After the reaction, cool and centrifuge at 8000-10000 rpm / min for 10 min. Finally, wash the precipitate in the order of "water, ethanol, water" to obtain hollow Prussian blue nanoparticles (HPB). S4, weigh 20 mg HPB and dissolve it in 10 mL deionized water for uniform ultrasonic dispersion, then weigh 10 mg of the drug with anti-inflammatory and antioxidant effects and dissolve it in 10 mL deionized water to prepare a 1 mg / mL solution, after dissolution, add the solution of the drug with anti-inflammatory and antioxidant effects dropwise into the HPB solution, stir and react for 24 h in a dark place, centrifuge at 8000 rpm for 10 min, wash with deionized water 2-3 times, collect the supernatant to obtain the drug-loaded Prussian blue; S5. Re-disperse the drug-loaded Prussian blue in 10 mL of deionized water, disperse it with magnetic stirring, add 2 mL of TA-HA solution (5 mg / mL prepared in deionized water) and stir for 20 min, then use a syringe pump to uniformly add 12 mL of high-valent metal ions (1 mg / mL) within 10 min, and finally adjust the pH of the solution to alkaline with Tris buffer (pH 8.5). React at room temperature for 30 min to obtain the nano antibacterial agent, which is stored at 4°C.

5. The use of the nano antibacterial agent Prussian blue for treating biofilm infection according to claim 1, characterized in that: The nano antibacterial agent has a synergistic killing effect on bacterial biofilm and has an anti-inflammatory effect.

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