A sodium nitroprusside / Prussian blue / manganese composite nanozyme, its preparation method, and its antibacterial application.

Abstract

This invention provides a sodium nitroprusside / Prussian blue / manganese composite nanozyme, its preparation method, and its application in antibacterial applications. Belonging to the field of nanomaterials technology, the preparation method involves: first, polyvinylpyrrolidone, potassium ferricyanide, and sodium nitroprusside are mixed in an acidic solution at room temperature to prepare a Prussian blue precursor solution; then, manganese acetate is added dropwise to the Prussian blue precursor solution; after high-pressure reaction, centrifugation, and freeze-drying, sodium nitroprusside / Prussian blue / manganese composite nanozyme powder is obtained. The sodium nitroprusside / Prussian blue / manganese composite nanozyme prepared by this invention possesses a synergistic antibacterial mechanism of "enzyme catalysis + photothermal / ultrasound-based kinetic therapy + NO release." Through dual regulation of NO release via laser or ultrasound, combined with photothermal and acoustic-dynamic effects, it disrupts bacterial structure and metabolism, exhibiting not only highly effective killing of Staphylococcus aureus but also clearing bacterial biofilms, effectively solving the problem of biofilm-mediated drug resistance and providing a new approach in the field of antibacterial research.

Description

Technical Field

[0001] This invention belongs to the field of nanomaterials technology, specifically relating to a sodium nitroprusside / Prussian blue / manganese composite nanozyme, its preparation method, and its application in antibacterial applications. Background Technology

[0002] Severe infectious diseases induced by various pathogenic microorganisms, such as pneumonia, endocarditis, and sepsis, remain major public health challenges facing today's healthcare systems. Among common pathogens, *Escherichia coli* and *Staphylococcus aureus* infections are the most prevalent, and their drug resistance levels are constantly increasing, making them a core challenge in antimicrobial treatment. *Escherichia coli* and *Staphylococcus aureus* represent typical Gram-negative and Gram-positive bacteria, respectively. They are not only important indicator strains for assessing bacterial resistance but can also cause infections in the digestive tract, urinary tract, and multiple organ sites. *Staphylococcus aureus*, in particular, can secrete enterotoxins under suitable conditions, making it one of the main pathogenic factors causing foodborne illnesses.

[0003] Despite the continuous expansion of antibiotic-based drug systems over the past few decades, the problem of multidrug resistance in pathogens continues to worsen, posing a serious challenge to the efficacy of traditional antibacterial drugs. Therefore, exploring novel anti-infection strategies that do not rely on traditional antibiotics has become an important direction for current research and clinical applications. Among numerous alternative antibacterial strategies, metallic materials have attracted considerable attention due to their inherent antibacterial activity. The development of nanotechnology has significantly improved the preparation efficiency and structural controllability of metal nanoparticles, giving them a stronger application advantage in the field of antibacterial agents. In particular, transition metals, due to their unique orbital electronic structures, can endow materials with specific reaction characteristics and catalytic capabilities. For example, manganese, possessing both good biosafety and excellent catalytic reaction efficiency, is widely used to construct high-performance nanozyme systems. Currently, metal nanomaterials based on metal ions such as gold, silver, manganese, and palladium have been widely researched and applied in biomedicine and catalytic engineering. Among them, manganese-based nanoparticles, due to their high catalytic activity and strong structural designability, have become a research hotspot for efficient nanozymes and have shown significant potential in the development of alternative antibacterial materials.

[0004] Prussian blue (PB) is a typical ferricyanide coordination compound, also known as iron blue, with its chemical composition typically represented as Fe4[Fe(CN)6]3. This material can be mass-produced under simple conditions with a mature and stable process. Due to its excellent biocompatibility demonstrated in long-term applications, it has been approved by the US FDA for clinical treatment of thallium poisoning, and its safety has been authoritatively recognized. Structurally, PB contains iron ions in different valence states. This mixed-valence structure makes its electron distribution similar to the active centers of iron oxides such as Fe3O4. Therefore, Prussian blue exhibits outstanding peroxidase-like catalytic activity, promoting the decomposition of hydrogen peroxide (H2O2) accumulated at infection sites. This demonstrates unique advantages in alleviating inflammatory responses and providing adjunctive antibacterial treatment, making it one of the core materials in the field of nanozyme research in recent years. Furthermore, Prussian blue exhibits strong light absorption in the near-infrared region and can efficiently convert light energy into heat energy, possessing excellent photothermal properties. This characteristic makes PB an important building block for constructing photothermal therapy platforms, photothermal sensors, and related photoresponsive materials, showing broad application potential in multiple fields such as biomedicine, detection and testing, and functional materials.

[0005] Nanomaterial-mediated sonic dynamics therapy (SDT) is a rapidly developing novel anti-infection strategy. SDT utilizes the ability of low-frequency ultrasound (approximately 1 MHz) to penetrate 5–10 cm of biological tissue without damage, allowing it to accurately target infections in deep tissues, muscles, and internal organs. Simultaneously, the ultrasound is locally activated only at the site of the nano-sound-sensitizing agent, thus avoiding systemic non-specific triggering and exhibiting good targeting and safety. Nanomaterials with sonic dynamics can efficiently generate reactive oxygen species (ROS) under the ultrasonic cavitation effect, rapidly oxidizing bacterial membrane lipids, DNA, and key enzyme molecules, causing irreversible bacterial damage. This achieves effective, non-antibiotic-based eradication of deep infections, providing a new therapeutic approach for addressing drug-resistant bacterial infections.

[0006] Nitric oxide (NO), a novel gaseous antibacterial strategy that has attracted much attention in recent years, possesses significant advantages such as broad-spectrum antibacterial activity, low drug resistance, and easy diffusion. Its antibacterial effect mainly stems from the dual pressures of "oxidative stress" and "nitrification stress" applied to bacteria, which can cause irreversible damage to cell structure and metabolism. NO molecules have extremely high membrane permeability, allowing them to freely pass through bacterial cell walls / membranes without relying on specific channels. Once inside the cell, NO reacts with various key enzymes containing iron-sulfur clusters (such as respiratory chain complexes and ribonucleotide reductases), leading to their inactivation and thus inhibiting bacterial respiration and blocking DNA synthesis. Furthermore, NO can nitrosate the sulfhydryl groups of cysteine ​​residues on the cell membrane, disrupting protein conformation and function, and causing instability in membrane integrity and permeability. Notably, NO can effectively diffuse deep into the biomembrane matrix, reducing viscoelasticity by cleaving the polysaccharide network structure, thereby dismantling the protective barrier of the biomembrane and achieving deeper killing of drug-resistant bacteria.

[0007] Metal nanomaterials have been shown to possess significant antibacterial activity, and their synergistic effect with multi-metal complexes holds significant research value in enhancing antibacterial efficacy. However, to date, no research has reported on the synergistic functionalization of Prussian blue nanozymes through manganese doping and the introduction of nitric oxide (NO) donors, enabling them to simultaneously achieve the following within the same system: (1) photothermal-ultrasound synergistic induced NO release on demand; (2) generation of reactive oxygen species through ultrasound kinetic therapy; (3) regulation of catalase-like activity; and (4) comprehensive antibacterial application. This direction still presents a significant technological gap in the fight against Staphylococcus aureus infection and the removal of its robust biofilm. Summary of the Invention

[0008] The purpose of this invention is to provide a sodium nitroprusside / Prussian blue / manganese composite nanozyme, its preparation method, and its application in antibacterial applications.

[0009] The present invention adopts the following technical solution:

[0010] One objective of this invention is to provide a method for preparing sodium nitroprusside / Prussian blue / manganese composite nanozymes, comprising the following steps:

[0011] A Prussian blue precursor solution was prepared by mixing polyvinylpyrrolidone, potassium ferricyanide, and sodium nitroprusside in a 0.01 M acidic solution at room temperature. Then, manganese acetate was added dropwise to the Prussian blue precursor solution, and the mixture was reacted under high pressure in a reactor. After centrifugation and freeze-drying, sodium nitroprusside / Prussian blue / manganese composite nanoenzyme powder was obtained. The mass ratio of polyvinylpyrrolidone, potassium ferricyanide, sodium nitroprusside, and manganese acetate was 3.57:1.57:2.91:1. The high-pressure reaction conditions were 80℃ for 20 h.

[0012] More preferably, the acidic solution is a hydrochloric acid solution.

[0013] Further preferred, the freeze-drying conditions are: a vacuum degree of 10 Pa, a cold trap temperature of -60°C, and a freeze-drying time of 24 h.

[0014] The second objective of this invention is to provide a sodium nitroprusside / Prussian blue / manganese composite nanozyme prepared by the above-described method. The composite nanozyme has an average particle size of 100 nm and exhibits negative surface charge; it demonstrates good absorption performance in the near-infrared II region; the introduction of manganese ions and sodium nitroprusside does not disrupt the original crystal structure of the Prussian blue; and it exhibits good absorption performance under 1064 nm laser light at 1 W / cm². 2 It exhibits excellent photothermal stability under power conditions and possesses catalase-like activity, with catalytic activity superior to Prussian blue nanozymes and Prussian blue-sodium nitroprusside complex nanozymes that do not contain manganese.

[0015] The third objective of this invention is to provide an application of sodium nitroprusside / Prussian blue / manganese composite nanozyme in antibacterial applications.

[0016] The antibacterial mechanism of this sodium nitroprusside / Prussian blue / manganese composite nanozyme is as follows: it catalyzes the decomposition of endogenous H2O2 in the infection microenvironment to generate hydroxyl radicals, which damage bacterial membrane lipids, proteins, and DNA; under 1064nm laser irradiation, the system temperature rises to 45-50℃ within 5 minutes, causing bacterial protein structure denaturation and triggering sodium nitroprusside to dissociate and release NO; under ultrasound, NO release is promoted and reactive oxygen species are generated through cavitation effect to stimulate acoustic dynamics reaction, achieving deep oxidative damage to bacteria.

[0017] Preferably, the bacteria include Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa, and the application includes inhibiting bacterial growth and removing bacterial biofilms.

[0018] The beneficial effects of this invention are as follows:

[0019] (1) The method for preparing sodium nitroprusside / Prussian blue / manganese composite nanozyme (denoted as Mn[PB-SNP]) provided by the present invention is simple, the raw materials are cheap and readily available, no complicated equipment is required, the reaction conditions are mild (mixing at room temperature and high pressure reaction at 80℃), the yield is stable, and it is easy to scale up industrial production.

[0020] (2) Compared with traditional Prussian blue or manganese nanozymes, Mn[PB-SNP] has excellent peroxidase-like catalytic activity, high efficiency photothermal performance and stable NO release ability, and outstanding photothermal stability. It can still maintain good performance after multiple cycles of use. At the same time, the material has an average particle size of about 100 nm and its negative surface charge makes it well dispersed in aqueous solution, avoiding the reduction of activity caused by agglomeration.

[0021] (3) The sodium nitroprusside / Prussian blue / manganese composite nanozyme provided by the present invention adopts a synergistic antibacterial mechanism of "enzyme catalysis + photothermal / ultrasound dynamic therapy + NO release", which breaks through the limitations of traditional single antibacterial methods. By controlling NO release through laser or ultrasound, combined with photothermal effect and acoustic dynamic effect, it destroys bacterial structure and metabolism. It not only has a highly efficient killing effect on Staphylococcus aureus, but also removes bacterial biofilm, effectively solving the problem of drug resistance mediated by biofilm.

[0022] (4) The composite nanozyme has high biosafety (based on the clinical safety of Prussian blue and the low toxicity of manganese). It does not rely on traditional antibiotics during the antibacterial process, and can effectively overcome bacterial resistance, providing a new approach for the antibacterial field. Attached Figure Description

[0023] Figure 1 These are transmission electron microscope (TEM) images and energy-scattered X-ray spectral scans of the Mn[PB-SNP] nanozyme of this invention; in the figures, (a): low-magnification TEM image of the Mn[PB-SNP] nanozyme, (b): high-magnification TEM image of the Mn[PB-SNP] nanozyme, and (c): annular dark field (ADF), C element distribution, N element distribution, O element distribution, Fe element distribution, and Mn element distribution of the energy-scattered X-ray spectral scan results of the Mn[PB-SNP] nanozyme;

[0024] Figure 2 The images show the surface potential test diagram (a) and hydrated particle size test diagram (b) of PB, PB-SNP, and Mn[PB-SNP] in this invention.

[0025] Figure 3 These are the UV-Vis-NIR absorption spectra of PB, PB-SNP, and Mn[PB-SNP].

[0026] Figure 4 The X-ray diffraction patterns of PB and Mn[PB-SNP] are shown.

[0027] Figure 5 The graph shows the heating trend of PB, PB-SNP, and Mn[PB-SNP] (a) and the temperature change curve (b).

[0028] Figure 6 The graph shows the temperature rise trend (a) and temperature change curve (b) of Mn[PB-SNP] at different concentrations under laser irradiation.

[0029] Figure 7 The graphs (a) and (b) show the temperature rise trend of Mn[PB-SNP] under different laser powers.

[0030] Figure 8This is a cyclic test diagram of the photothermal stability of Mn[PB-SNP].

[0031] Figure 9 These are fluorescence intensity spectra of NO released from Mn[PB-SNP] under different stimuli.

[0032] Figure 10 This is a comparison of the peroxidase-like activities of PB, PB-SNP, and Mn[PB-SNP].

[0033] Figure 11 The fluorescence spectra of reactive oxygen species generation of PB, PB-SNP, and Mn[PB-SNP] at different ultrasound irradiation times are shown.

[0034] Figure 12 The graph shows the bacterial survival count (a) and bacterial concentration (b) after treatment with different methods.

[0035] Figure 13 Comparison of crystal violet staining of bacterial biofilms after treatment with different methods (a) and related quantitative data (b). Detailed Implementation

[0036] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0037] Example 1: The preparation method of sodium nitroprusside / Prussian blue / manganese composite nanozyme (denoted as Mn[PB-SNP]) is as follows:

[0038] (1) Preparation of Prussian blue precursor solution: Weigh 0.3g of polyvinylpyrrolidone (PVP, molecular weight 58000) and add it to an Erlenmeyer flask containing 40mL of 0.01M hydrochloric acid solution. Dissolve it completely for 10min under magnetic stirring. Then add 132mg of potassium ferricyanide (K3[Fe(CN)6]) and 244.4mg of sodium nitroprusside (SNP). Continue stirring until the system turns into a clear orange solution to obtain the Prussian blue precursor solution, which is then stored at room temperature.

[0039] (2) Preparation of manganese source solution: Weigh 84 mg of manganese acetate (Mn(CH3COO)2·4H2O) and dissolve it in 10 mL of ultrapure water. Sonicate it for 5 min to completely dissolve it, and gently stir it with a glass rod to promote dispersion. The resulting manganese source solution has a concentration of 30 mmol / L and is stored in the dark for later use.

[0040] (3) Preparation of manganese-doped Prussian blue suspension: The Prussian blue precursor solution obtained in step (1) was transferred to a 50 mL flask, placed in a 37°C constant temperature water bath, and stirred at 800 rpm. After the system temperature stabilized, the manganese source solution obtained in step (2) was slowly added dropwise using a dropper. During the dropwise addition, the solution color gradually deepened from orange. After the dropwise addition was completed, the reaction was continued at 37°C for 4 h to promote the full doping of manganese ions into the Prussian blue lattice. After the reaction was completed, the mixed system was transferred to a high-pressure reactor and subjected to a high-temperature and high-pressure reaction at 80°C for 20 h to promote the further stabilization of the crystal structure.

[0041] (4) Purification and drying: The suspension obtained in step (3) was transferred to a centrifuge tube and centrifuged at 9000 rpm for 15 min. The supernatant was discarded. 20 mL of ultrapure water was added and ultrasonically dispersed for 5 min to completely redisperse the precipitate. The above washing steps were repeated 3 times by centrifuging at 9000 rpm for 15 min to completely remove residual PVP, acetate ions and other impurities. The resulting dark blue precipitate was then placed in a freeze dryer (vacuum degree 10 Pa, cold trap temperature -60℃) and freeze-dried for 24 h to finally obtain loose Mn[PB-SNP] powder, which was then sealed and stored under light-protected conditions.

[0042] The Mn[PB-SNP] obtained in this embodiment was characterized by transmission electron microscopy (TEM) and energy-scattered X-ray spectral scanning (EDSMapping). TEM images are shown below. Figure 1 As shown in (a) and (b), the material exhibits a regular cubic structure, with uniformly dispersed particles and no obvious agglomeration. The average particle size is approximately 90 nm. The EDSMapping results are as follows... Figure 1 As shown in (c), the five elements Fe, C, N, O and Mn are uniformly distributed inside the particles. The Mn signal is clear and uniform, indicating that the manganese element has been successfully incorporated into the Prussian blue crystal structure and uniformly distributed in the lattice without local enrichment.

[0043] Example 2: The preparation method of Prussian blue nanozyme (denoted as PB) is as follows:

[0044] Polyvinylpyrrolidone (PVP, 3.0 g), potassium ferricyanide (K3[Fe(CN)6], 132 mg) and 40 mL of 0.01 M hydrochloric acid solution were mixed and stirred at room temperature for 4 h, and then reacted in a high-pressure reactor at 80 °C for 20 h to obtain the final product.

[0045] Example 3: The preparation method of Prussian blue-sodium nitroprusside complex nanozyme (denoted as PB-SNP) is as follows:

[0046] Polyvinylpyrrolidone (PVP, 3.0 g), potassium ferricyanide (K3[Fe(CN)6], 132 mg), sodium nitroprusside (SNP, 244.4 mg) were mixed with 40 mL of 0.01 M hydrochloric acid solution at room temperature and stirred until a transparent orange solution was formed. The mixture was stirred at room temperature for 4 h and then transferred to an 80 °C reactor for high-pressure reaction for 20 h to obtain the final product.

[0047] Example 4 To further confirm the successful synthesis of Mn[PB-SNP], the surface potential, hydration particle size, and UV-Vis-NIR absorption spectra of PB, PB-SNP, and Mn[PB-SNP] were measured.

[0048] Surface potential and hydrated particle size test results are as follows Figure 2 As shown. By Figure 2 As shown in (a), the surface potential of PB is –18 mV, PB-SNP is –27 mV, and Mn[PB-SNP] is –25 mV, indicating that doping with manganese ions and SNPs did not change the surface electrical properties of the Prussian blue nanomaterial. Figure 2 As shown in (b), the hydrated particle size of PB is -146 nm, PB-SNP is 125 nm, and Mn[PB-SNP] is 93 nm, indicating that doping with manganese ions and SNPs will reduce the size of Prussian blue nanomaterials.

[0049] The UV-Vis-NIR absorption spectra of each group of samples are as follows: Figure 3 As shown in the figure, PB, PB-SNP, and Mn[PB-SNP] all exhibit good light absorption in the near-infrared II region, further demonstrating that the incorporation of manganese did not disrupt the optical structure of Prussian blue, and also confirming the successful construction of the Mn[PB-SNP] composite structure.

[0050] The characterization results confirm that Mn[PB-SNP] was successfully prepared.

[0051] Example 5: To further evaluate the integrity of the Prussian blue crystal structure after doping, XRD analysis was performed on PB and Mn[PB-SNP]. The results are as follows: Figure 4 As shown. By Figure 4 It can be seen that Mn 2 The introduction of SNP did not cause a shift or significant change in the characteristic peak of PB, indicating that Mn 2 The introduction of SNP molecules does not disrupt the crystal framework structure of PB, and the material maintains its original lattice stability.

[0052] Example 6: Evaluation of the photothermal properties of Mn[PB-SNP]

[0053] Aqueous dispersions of PB, PB-SNP, and Mn[PB-SNP] with a concentration of 200 μg / mL were prepared, and after ultrasonic dispersion, they were subjected to a 1064 nm laser (1 W / cm²) for further processing. 2 A temperature rise test was conducted under irradiation conditions for 0-5 minutes, with pure water used as a control. The results are as follows: Figure 5 As shown in the figure, (a) represents the temperature rise trend graph, and (b) represents the temperature change curve. Figure 5 It can be seen that all three materials can heat up to about 57°C after irradiation for 5 minutes, proving that Mn 2 The introduction of ⁺ and SNP did not weaken the photothermal conversion efficiency of PB.

[0054] Example 7: Photothermal concentration dependence analysis of Mn[PB-SNP] was performed.

[0055] Mn[PB-SNP] was prepared into aqueous dispersions with concentrations of 50, 100, 200, and 300 μg / mL, respectively, and then subjected to a reaction at 1 W / cm². 2 Heated for 5 minutes under 1064nm laser irradiation. Results are as follows: Figure 6 As shown in the figure, (a) represents the temperature rise trend graph, and (b) represents the temperature change curve. Figure 6 It can be seen that the temperature rise of the material increases with increasing concentration, showing a good positive correlation.

[0056] Example 8: Evaluation of the effect of laser power on the photothermal properties of Mn[PB-SNP].

[0057] Mn[PB-SNP] was prepared into an aqueous dispersion with a concentration of 100 μg / mL using 0, 0.5, 1.0, and 1.5 W / cm³, respectively. 2 Irradiation with a 1064nm laser for 5 minutes resulted in the following temperature change: Figure 7 As shown in the figure. Figure (a) represents the temperature rise trend, and (b) represents the temperature change curve. Figure 7 The results show that the photothermal performance of the material increases with increasing laser power, exhibiting a significant positive correlation.

[0058] Example 9: Photothermal Stability Test of Mn[PB-SNP]

[0059] Five cycles of laser on / off heating were performed on the Mn[PB-SNP] obtained in Example 1 (each cycle: 5 min heating, 5 min cooling). The results are as follows. Figure 8 As shown, the maximum temperature and heating rate of the material did not decrease significantly during multiple cycles, indicating that Mn[PB-SNP] has excellent photothermal stability.

[0060] Example 10: Evaluation of the ability of Mn[PB-SNP] to release NO under photothermal and ultrasonic stimulation

[0061] The Mn[PB-SNP] obtained in Example 1 was prepared into a 100 μg / mL solution, and 20 nmol of DAF-FM fluorescent probe was added for NO detection. The samples were then irradiated with a 1064 nm laser (1 W / cm²). 2 5 min), ultrasound irradiation (50% duty cycle, 1 W / cm²) 2 The equivalent acoustic intensity (5 min) and photothermal / ultrasonic combined stimulation were used, and the fluorescence intensity of the probe at approximately 520 nm was recorded. Results are as follows: Figure 9 As shown, the DAF-FM fluorescent probe itself is non-fluorescent, but the fluorescence intensity is significantly enhanced after ultrasonic treatment, indicating that ultrasonic treatment can promote the decomposition of SNPs in Mn[PB-SNP] to release NO. Photothermal irradiation can further enhance NO release. The combined effect of photothermal and ultrasonic treatment produces the highest amount of NO, which is significantly higher than that produced by light or ultrasonic treatment alone, indicating that the photothermal-ultrasonic dual mode can efficiently trigger the NO release behavior of Mn[PB-SNP].

[0062] Example 11 Establishment of a simulated peroxidase activity model of Mn[PB-SNP]

[0063] Its specific establishment process includes the following steps:

[0064] (1) Preparation of PB, PB-SNP, and Mn[PB-SNP] suspensions: The PB powder prepared in Example 2, the PB-SNP powder prepared in Example 3, and the Mn[PB-SNP] powder prepared in Example 1 were added to ultrapure water to prepare a crude suspension of 200 μg / mL. Then, the suspension was sonicated for 25 min to ensure that the nanoparticles were fully and uniformly dispersed, resulting in a dark blue transparent nanoenzyme suspension that was ready for immediate use.

[0065] (2) Preparation of reaction substrate solution: Prepare 100 mmol / L H2O2 solution and store it at 4℃ for later use. Before use, restore it to room temperature; prepare 10 mmol / L TMB (3,3',5,5'-tetramethylbenzidine) anhydrous ethanol solution and store it in the dark; at the same time, prepare 0.2 mol / L NaAc–HAc buffer system (pH=4.0) as the acidic environment of the reaction system.

[0066] (3) Optimization of the effect of nanozyme concentration on enzyme activity: The total volume of the reaction system was set to 2 mL, which contained 0.5 mmol / L TMB, 10 mmol / L H2O2, and 1 mg / mL nanozyme. The remaining volume was made up with NaAc–HAc buffer. After incubating the system in a 37°C water bath for 15 min, the absorbance change of the TMB oxidation product at 652 nm was recorded using an ELISA reader.

[0067] (4) The absorbance value of the microplate reader can represent the decomposition of hydrogen peroxide, further demonstrating the strength of the peroxidase-like activity of the nanozyme.

[0068] like Figure 10 As shown, compared to PB, PB-SNP exhibits slightly enhanced catalytic activity, while Mn... 2 The catalytic efficiency of Mn[PB-SNP] after doping was significantly improved, demonstrating that it has stronger peroxidase activity.

[0069] Example 12: Ultrasonic dynamic properties of Mn[PB-SNP]

[0070] To explore the ability of Mn[PB-SNP] to generate reactive oxygen species (ROS) under ultrasound, a simulated ultrasound dynamics model of Mn[PB-SNP] was established. The specific steps are as follows.

[0071] (1) Prepare PB, PB-SNP and Mn[PB-SNP] into 200 μg / mL water-soluble suspensions respectively, disperse them by ultrasonication to make them evenly distributed in the solution, and use them immediately.

[0072] (2) Prepare a 20 mmol / L DCFH reactive oxygen species detection probe solution, using anhydrous ethanol as the solvent, and store it in the dark to prevent photo-oxidation.

[0073] (3) The total volume of the reaction system was set to 500 μL. 490 μL of PB, PB-SNP or Mn[PB-SNP] suspension was added to each group, followed by 10 μL of LCFH probe solution. Subsequently, the three groups were subjected to ultrasonic stimulation for 0, 1, 2, 3, 4 and 5 min respectively, and 480 nm excitation light was used. The fluorescence intensity change of the probe at about 529 nm was detected by a fluorophotometer to evaluate the amount of ROS generated.

[0074] The results are as follows Figure 11 As shown, PB and PB-SNP only generate a small amount of ROS under ultrasound, while Mn[PB-SNP], after manganese ion doping, significantly improves its response sensitivity to ultrasound, and can generate a large amount of ROS under ultrasound stimulation, with a fluorescence signal intensity much higher than the control material. These results indicate that Mn[PB-SNP] possesses excellent sonodynamic properties and can efficiently generate reactive oxygen species under ultrasound, providing support for its sonodynamic enhancement mechanism in antibacterial therapy.

[0075] Example 13 Application of Mn[PB-SNP] in antibacterial activity

[0076] Based on the significant POD-like activity of Mn[PB-SNP] confirmed in Example 11, and further combining the material's photothermal properties, ultrasonic dynamic therapy properties, and ultrasonic-triggered NO release ability, antibacterial experiments were conducted against Staphylococcus aureus.

[0077] The method for Mn[PB-SNP] antibacterial activity provided by this invention is as follows:

[0078] 1. Pick a single colony of Staphylococcus aureus (ATCC25923) and inoculate it into 5 mL of LB liquid medium. Incubate overnight at 37°C with shaking at 180 rpm. Measure the OD of the bacterial culture the following day. 600 When the value is close to 1.0, the bacterial concentration can be considered to be approximately 10. 9 CFU / mL, this bacterial culture is used for subsequent serial dilutions.

[0079] 2. Bacterial suspension dilution: Under aseptic conditions, take 100 μL of the bacterial suspension and dilute it with 10 μL of the bacterial suspension. 9 CFU / mL bacterial culture was mixed into 900 μL of physiological saline containing glucose to obtain 10 8 CFU / mL bacterial culture. Then dilute to 10 at a ratio of 1:9. 7 CFU / mL was used as the base bacterial solution for antibacterial experiments.

[0080] 3. Preparation of nanozyme working solution: Measure the Mn[PB-SNP] powder obtained in Example 1, dissolve it in sterile physiological saline to prepare a 2 mg / mL stock solution, and sonicate for 10 min to obtain a uniformly dispersed solution. Then dilute to 100 μg / mL and sterilize by passing through a 0.22 μm filter before use.

[0081] 4. Mixed incubation: Four groups of experiments were set up, with each group having a reaction volume of 1 mL, as follows: Control group (900 μL only, 10 7 The four systems were divided into four groups: CFU / mL bacterial culture + 100μL sterile saline, nanozyme group (900μL bacterial culture + 100μL, 100μg / mL nanozyme working solution), laser & ultrasound group (900μL bacterial culture + 100μL sterile saline, followed by laser and ultrasound irradiation), and nanozyme + laser & ultrasound group (900μL bacterial culture + 100μL, 100μg / mL nanozyme working solution, followed by laser and ultrasound irradiation). All four systems were transferred to sterile EP tubes and incubated at 37℃ for 1 hour, with the EP tubes gently inverted every 15 minutes to ensure sufficient contact between the nanozyme and bacteria.

[0082] 5. Laser & Ultrasonic Processing: The EP tubes of the laser / ultrasonic group and the nanozyme + laser / ultrasonic group were removed and placed under a 1064nm laser emitter (power 1W / cm²). 2The light spot diameter was 1 cm, and the tube was irradiated for 5 minutes at a distance of 5 cm from the liquid surface. The system temperature was monitored using an infrared thermometer during the irradiation. After irradiation, an ultrasonic probe was used to contact the EP tube through a coupling agent, and the tube was subjected to ultrasonic treatment (50% duty cycle, 1 W / cm²). 2 Equivalent sound intensity, 5 min).

[0083] 6. Plate culture and counting: The bacterial suspensions of the four groups were serially diluted with sterile physiological saline (10⁻⁻¹). 1 10⁻ 2 10⁻ 3 10⁻ 4 (Multiple times), take 100 μL of bacterial suspension for each dilution and spread it evenly on LB agar plates, with 3 parallel plates for each group; invert the spread plates and place them in a 37℃ constant temperature incubator to incubate Staphylococcus aureus for 15-18 h, and count the colonies after they grow; stain the spread petri dishes with 10 mM crystal violet solution for 10 min (as shown in Figure (a)), then rinse off the excess crystal violet solution with ethanol and observe the biofilm damage.

[0084] The results are as follows Figure 12 As shown, combined with Figure 12 As shown in (a) and (b), the control group had a denser colony count, with a survival concentration of approximately 9.8 × 10⁻⁶. 9 CFU / mL; the number of colonies in the Mn[PB-SNP] group was significantly reduced compared to the control group, with the survival concentration dropping to approximately 7.5 × 10⁻⁶. 9 CFU / mL; the number of colonies in the laser & ultrasound group was almost identical to that in the control group, indicating that physical stimulation alone had no significant effect on bacterial growth; while the number of colonies in the Mn[PB-SNP]+ laser & ultrasound group was extremely low, with a survival concentration of only about 0.15×10. 9 CFU / mL, showing a significant decrease compared to the control group. Results of biofilm detection using crystal violet staining ( Figure 13 (a) and (b) further validated the above trend: the control group formed a large area of ​​complete and dense biofilm; a certain degree of biofilm damage was observed in the Mn[PB-SNP] group; there was no significant difference between the laser & ultrasound group and the control group; while the biofilm of the Mn[PB-SNP] + laser & ultrasound group was almost completely destroyed, with only a small amount of staining remaining, showing a very strong biofilm removal ability.

[0085] The Mn[PB-SNP] nanozyme successfully prepared in this invention can be used for highly efficient antibacterial purposes. This nanozyme exhibits excellent peroxidase-like activity, decomposing excess hydrogen peroxide into ·OH at the bacterial infection site, thereby destroying the bacteria. Simultaneously, manganese doping endows the material with excellent photothermal properties, allowing for rapid heating under near-infrared laser irradiation. This achieves synergistic antibacterial action through "enzyme catalysis-photothermal-sonic dynamics-NO gas therapy," resulting in inhibition rates exceeding 99% against both *Escherichia coli* and *Staphylococcus aureus*. Furthermore, it demonstrates concentration dependence and good biocompatibility (cytotoxicity experiments show cell survival >95% at concentrations ≤200 μg / mL). Under combined ultrasonic / photothermal stimulation, it releases NO, disrupting the stubborn biofilm formed by bacteria and providing more targeted treatment to the bacterial infection site.

[0086] This invention discloses a sodium nitroprusside / Prussian blue / manganese composite nanozyme, its preparation method, and its antibacterial application. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The product of this invention has been described through preferred embodiments, and those skilled in the art can clearly make modifications or appropriate alterations and combinations to the product described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

Claims

1. A method for preparing a sodium nitroprusside / Prussian blue / manganese composite nanozyme, characterized in that, The sodium nitroprusside / Prussian blue / manganese composite nanozyme has an average particle size of 100 nm and a negatively charged surface. It exhibits good absorption performance in the near-infrared II region. The introduction of manganese ions and sodium nitroprusside does not destroy the original crystal structure of Prussian blue. It has excellent photothermal stability under 1064 nm laser power of 1 W / cm² and has catalase-like activity. Its catalytic activity is superior to that of Prussian blue nanozymes without manganese and Prussian blue-sodium nitroprusside composite nanozymes. The preparation method includes the following steps: A Prussian blue precursor solution was prepared by mixing polyvinylpyrrolidone, potassium ferricyanide, and sodium nitroprusside in a 0.01 M acidic solution at room temperature. Then, manganese acetate was added dropwise to the Prussian blue precursor solution, and the mixture was reacted under high pressure in a reactor. After centrifugation and freeze-drying, sodium nitroprusside / Prussian blue / manganese composite nanoenzyme powder was obtained. The mass ratio of polyvinylpyrrolidone, potassium ferricyanide, sodium nitroprusside, and manganese acetate was 3.57:1.57:2.91:

1. The high-pressure reaction conditions were 80℃ for 20 h.

2. The method for preparing a sodium nitroprusside / Prussian blue / manganese composite nanozyme according to claim 1, characterized in that, The acidic solution is a hydrochloric acid solution.

3. The method for preparing a sodium nitroprusside / Prussian blue / manganese composite nanozyme according to claim 1, characterized in that, The freeze-drying conditions were as follows: the vacuum degree of freeze-drying was 10 Pa, the cold trap temperature was -60℃, and the freeze-drying time was 24 h.

4. The application of the sodium nitroprusside / Prussian blue / manganese composite nanozyme obtained by any of the preparation methods described in claims 1-3 in the preparation of antibacterial agents.

5. The application according to claim 4, characterized in that, Its antibacterial mechanism is as follows: it catalyzes the decomposition of endogenous H2O2 in the infection microenvironment to generate hydroxyl radicals, which damage bacterial membrane lipids, proteins and DNA; under 1064nm laser irradiation, the system temperature rises to 45-50℃ within 5 minutes, causing bacterial protein structure denaturation and triggering sodium nitroprusside to dissociate and release NO; under the action of ultrasound, it promotes the release of NO and stimulates the acoustic dynamic reaction through cavitation effect to generate reactive oxygen species, thereby achieving deep oxidative damage to bacteria.

6. The application according to claim 4, characterized in that, The bacteria include Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa, and the application includes inhibiting bacterial growth and removing bacterial biofilms.

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