A polydopamine manganese-based nanoscale enzyme with mild photo-thermal enhancement enzyme-like activity and a preparation method and application thereof

Abstract

This invention belongs to the field of pharmaceutical preparation and relates to a polydopamine manganese-based nanozyme with mildly photothermally enhanced enzyme-like activity, its preparation method, and its application. For the first time, a simple and biocompatible PDA@MnOOH nanozyme is prepared using a one-pot method. First, MOOH with enzyme-like activity is prepared in the system. Dopamine (DA) is added without post-treatment. DA modifies the surface of MOOH through self-polymerization and polyphenol metal chelation, forming a polydopamine manganese-based nanozyme with thermo-enhanced enzyme-like activity. Under light irradiation, PDA exhibits photothermal activity and GSH-consuming function, increasing the ROS concentration at the wound site and enhancing the bactericidal effect. CAT enzyme activity can continuously catalyze H2O2 to O2 at the wound site, providing a reaction substrate for OXD activity and improving the bactericidal effect. Furthermore, during the wound recovery period, it eliminates inflammatory responses caused by oxidative stress, promotes angiogenesis, and accelerates the healing of infected wounds.

Description

Technical Field

[0001] This invention belongs to the field of drug preparation and relates to a manganese-based nanozyme. Background Technology

[0002] As the body's largest barrier organ, the skin is easily damaged by factors such as trauma, surgery, and chronic diseases, leading to wounds. Bacterial infection is a core cause of hindered wound healing, resulting in prolonged wound development and even systemic infection. The discovery of antibiotics greatly improved the treatment prognosis of wound infections, but the overuse and misuse of antibiotics accelerated the spread of antimicrobial resistance (AMR). The emergence of multidrug-resistant bacteria and even "superbugs" has rendered once-effective antibiotics increasingly ineffective, leaving traditional treatments in a predicament where "bacterial mutation rates far outpace new drug development." Therefore, the development of non-antibiotic drugs is of great significance for the treatment of infected wounds.

[0003] The rapid development of nanotechnology has brought new breakthroughs to anti-infective therapy. Among these breakthroughs, nanozymes, a key concept first proposed by Chinese scientists, have rapidly become a research hotspot in the biomedical field since the discovery of peroxidase (POD)-like activity in Fe3O4 nanoparticles in 2007. Nanozymes are a class of nanomaterials with enzyme-like catalytic activity, combining the catalytic function of natural enzymes with the unique physicochemical properties of nanomaterials. Their core innovation lies in breaking through the limitations of traditional antibiotics' "single-target killing" and constructing a new "multi-dimensional, physicochemical antibacterial" model, fundamentally reducing the risk of bacterial resistance. Nanozymes possess dual functions of antibacterial and tissue repair. Some nanozymes can regulate local oxidative stress levels in wounds, reduce inflammatory responses, and promote angiogenesis, accelerating wound healing while eliminating bacteria, thus overcoming the shortcomings of traditional antibiotics that can only kill bacteria and cannot assist in tissue repair.

[0004] Among them, Fe, Cu, Mn, Ce, and Co-based nanozymes have been widely studied due to their core advantages such as low preparation cost, strong chemical stability, and good controllability. For example, application CN121731487A discloses a copper-based nanozyme, its preparation method, and its application for eliminating drug-resistant bacteria in the bladder; among inorganic metal nano-oxidases, manganese-based nanozymes benefit from the rich valence state changes of manganese (Mn... 2+ / Mn 3+ / Mn 4+ In recent years, inorganic metal nanozymes have become a research hotspot in the field due to their high biocompatibility and good biosafety. However, standalone inorganic material nanozymes are unstable, prone to aggregation, and still pose a certain risk of neurotoxicity at high doses. Therefore, to enable the widespread application of inorganic metal nanozymes in biomedicine, their surface needs to be modified for biocompatibility and stability.

[0005] Dopamine (DA) is an endogenous neurotransmitter in the human body, and at low concentrations, it is almost non-toxic to cells. Its metabolites (such as homovanillic acid) can also be processed normally by the human body without triggering strong immune rejection. Its safety in the human body has been fully verified in pharmaceutical applications (such as the treatment of shock). Under alkaline conditions, it can polymerize to form polydopamine (PDA), which also has good biocompatibility. PDA contains abundant amino groups and hydroxyl groups, which can chelate with metals to form biocompatible organic-inorganic nanocomposites widely used in biomedicine. Simultaneously, PDA has photothermal effects; suitable photothermal temperatures can catalyze the activity of nanozymes, enhancing their therapeutic effects. Currently, patent application CN121490078A describes a complex nanozyme prepared by combining dopamine, tea polyphenols, and copper manganese oxide, primarily for the inhibition of Escherichia coli and Staphylococcus aureus, but no in vivo studies have been conducted.

[0006] In summary, improving the stability, biocompatibility, and enzyme-like activity of manganese-based nanozymes in vivo are key issues that urgently need to be addressed for their efficient antibacterial therapeutic effects in vivo. Summary of the Invention

[0007] To address the aforementioned technical problems, this invention proposes a polydopamine manganese-based nanozyme with mildly photothermal-enhanced enzyme activity, its preparation method, and its applications.

[0008] The technical solution of this invention is implemented as follows:

[0009] On the one hand, the present invention provides a method for preparing polydopamine manganese-based nanozymes with mild photothermal enhanced enzyme activity. The simple PDA@MnOOH nanozymes were successfully prepared by a one-pot method. First, MnOOH nanozymes were prepared by improving the literature method. DA was added without treatment, causing it to polymerize on the MnOOH surface to form sheet-like PDA@MnOOH nanozymes.

[0010] The preparation steps are as follows:

[0011] (1) Dissolve MnCl2·4H2O and sodium ascorbate in deionized water, then add NaOH solution and let the reaction stand to obtain solution I;

[0012] (2) Dissolve F127 in a mixed solvent of anhydrous ethanol and water and stir at room temperature. Then add 1,3,5,-trimethylbenzene and continue stirring to obtain solution II.

[0013] (3) After mixing solution I and solution II, add tris(hydroxymethyl)aminomethane and dopamine, and continue the reaction until complete. After centrifugation and washing, PDA@MnOOH nanosheets, i.e. polydopamine manganese-based nanozymes, are obtained.

[0014] This system exhibits photothermal, OXD-like and CAT-like nanozyme activities, GSH-consuming ability, and antioxidant properties. The OXD-like activity catalyzes the generation of superoxide anions from O2, killing bacteria.

[0015] Furthermore, in step (1) above, the molar ratio of MnCl2·4H2O, sodium ascorbate, and NaOH is 0.07-0.08:0.040-0.05:4-6; the concentration of the NaOH solution is 1 M; and the volume ratio of deionized water to NaOH solution is 1:1. In step (1) above, the temperature for the static reaction is 36-38 ℃, and the time is 3.5-4.5 h. In step (2) above, the volume ratio of anhydrous ethanol to water in the mixed solvent is 1:2; the concentration of F127 in the mixed solvent is 3.1 mg / mL; and the volume ratio of the mixed solvent to 1,3,5,-trimethylbenzene is 46-48:0.19-0.21.

[0016] Furthermore, in step (2) above, the stirring time at room temperature is 25-35 min; the stirring time continues for 2.5-3.5 h. In step (3) above, the mass ratio of 1,3,5,-trimethylbenzene, tris(hydroxymethyl)aminomethane, and dopamine is 173-174:36-38:11-13. The reaction continues for 23-25 ​​h.

[0017] Secondly, the present invention provides a polydopamine manganese-based nanozyme with mildly photothermally enhanced enzyme activity prepared using the above-described method. The polydopamine manganese-based nanozyme can reach a temperature of 44-45°C after 5 minutes of NIR laser irradiation.

[0018] Thirdly, this invention provides the application of the aforementioned polydopamine manganese-based nanozyme in the preparation of drugs with antibacterial, anti-inflammatory, and wound-healing properties. PDA@MnOOH has a GSH-consuming effect; the reduction of GSH at the infected wound site increases the ROS concentration, enhancing the bactericidal effect. Its CAT-like activity can catalyze H2O2 at the infected site into O2. The generation of O2 provides reactant substrates for OXD-like activities; furthermore, the continuous generation of O2 greatly alleviates the hypoxic microenvironment of bacterial infection, which is beneficial for alleviating excessive inflammatory responses, promoting angiogenesis, and accelerating wound healing.

[0019] The present invention has the following beneficial effects:

[0020] 1. This invention utilizes MnOOH, which possesses OXD-like and CAT enzyme activities and GSH-consuming functions, as a nanozyme. Dopamine (DA), with good biocompatibility and metal chelating properties, is used as an organic modification material. Dopamine is added simultaneously with the preparation of MnOOH, leveraging its good biocompatibility, adhesion, excellent metal ion chelating ability, and self-polymerization to form a photothermal PDA film on its surface. A simple and multifunctional PDA@MnOOH is prepared via a one-pot method. PDA@MnOOH exhibits OXD-like activity, catalyzing the generation of superoxide anions from O2 to achieve highly efficient bactericidal effects. Simultaneously, PDA, as a photothermal agent, can increase the local temperature; under NIR laser irradiation, the temperature can rise to 44-45 °C within 5 minutes, enhancing OXD-like activity, increasing the catalytic rate of the nanozyme, and thus accelerating ROS generation.

[0021] 2. The PDA@MnOOH prepared in this invention consumes GSH, and the reduction of GSH increases the concentration of ROS at the infected wound site, resulting in a more efficient bactericidal effect. PDA@MnOOH also possesses CAT activity, which can catalyze H2O2 into O2. The generation of O2 provides reactant substrates for OXD-like activities and, moreover, the continuously generated O2 greatly alleviates the hypoxic microenvironment of the infected wound. This activity alleviates the inflammatory response of the wound and promotes angiogenesis. Furthermore, the preparation process of PDA@MnOOH is simple, the raw materials are readily available, and the constructed manganese-based nanomaterials exhibit excellent biocompatibility and can be directly used for the treatment of infected wounds. This provides an efficient, safe, and intelligent treatment solution for the clinical treatment of drug-resistant bacterial infections, and has broad prospects for translational applications. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 SEM image of PDA@MnOOH prepared in Example 1.

[0024] Figure 2 TEM image of PDA@MnOOH prepared in Example 1.

[0025] Figure 3 The mapping image of PDA@MnOOH prepared in Example 1.

[0026] Figure 4 EDS spectrum image of PDA@MnOOH prepared in Example 1.

[0027] Figure 5 Fourier transform infrared (FTIR) image of PDA@MnOOH prepared in Example 1.

[0028] Figure 6 XPS image of PDA@MnOOH prepared in Example 1.

[0029] Figure 7 The results of temperature changes for PDA@MnOOH prepared in Example 1 with different contents.

[0030] Figure 8 Temperature variation results for PDA@MnOOH prepared in Example 1 with different power levels.

[0031] Figure 9 Oxygen generation results of PDA@MnOOH prepared for different pH values ​​in Example 1.

[0032] Figure 10 Results of reactive oxygen generation for PDA@MnOOH prepared in Example 1 with different contents.

[0033] Figure 11 The results show the type of reactive oxygen species generated by the PDA@MnOOH prepared in Example 1.

[0034] Figure 12 The enzyme cascade results of PDA@MnOOH prepared in Example 1.

[0035] Figure 13 The photothermal ROS-promoted results of PDA@MnOOH prepared in Example 1.

[0036] Figure 14 The GSH consumption results of PDA@MnOOH prepared in Example 1 with different contents.

[0037] Figure 15 The in vitro biocompatibility results of PDA@MnOOH prepared in Example 1 are shown.

[0038] Figure 16 The diagram shows the ROS production in bacteria of PDA@MnOOH prepared in Example 1.

[0039] Figure 17 The graph shows the in vitro antibacterial properties of PDA@MnOOH prepared in Example 1.

[0040] Figure 18 The image shows the in vivo antibacterial effect of PDA@MnOOH prepared in Example 1. Detailed Implementation

[0041] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. 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 of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0042] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0043] The compound MnOOH was synthesized using the method described in the reference (Zhu P, Pu Y, Wang M, et al. MnOOH-catalyzed autoxidation of glutathione for reactive oxygen species production and nanocatalytic tumorinnate immunotherapy[J]. Journal of the American Chemical Society, 2023, 145(10): 5803–5815.).

[0044] Example 1

[0045] The preparation method of manganese-based nanozyme PDA@MnOOH in this embodiment includes the following specific steps:

[0046] 14.4 mg of MnCl2·4H2O and 8.8 mg of AsA were weighed and dissolved in 5 mL of deionized water. Then, 5 mL of NaOH (1 M) solution was slowly added, and the mixture was allowed to stand at 37 °C for 4 h. 0.147 g of F127 was dissolved in a mixed solvent of 47 mL of anhydrous ethanol and water and stirred at room temperature for 30 min. 200 μL of 1,3,5,-trimethylbenzene was added to the solution, and stirring was continued at room temperature for 3 h. The two solutions were then mixed. 37 mg of tris(hydroxymethyl)aminomethane was weighed and dissolved in 4 mL of distilled water, and 12 mg of DA was added to the mixed solution. The reaction was continued for 24 h. The reaction solution was centrifuged and washed twice with ethanol and acetone, respectively, to obtain PDA@MnOOH nanosheets.

[0047] The prepared PDA@MnOOH dispersion was dropped onto a copper sheet and allowed to dry naturally at room temperature. The morphology of the PDA@MnOOH was observed using a field emission scanning electron microscope (SEM). The results are as follows: Figure 1As shown in the figure. Simultaneously, a dispersion of PDA@MnOOH was dropped onto a copper grid, allowed to dry naturally at room temperature, and its morphology was characterized using transmission electron microscopy (TEM). The results are shown in the figure. Figure 2 As shown. Next, the elemental characterization of PDA@MnOOH was performed using elemental scanning spectroscopy (EDS), and the results are as follows. Figure 3 and 4 The image shows that the prepared nanosheets contain O, N, and Mn elements, further confirming the successful preparation of PDA@MnOOH. The structure was characterized using infrared spectroscopy, and the results are as follows: Figure 5 As shown, by comparing with PDA, PDA@MnOOH at 3438 cm⁻¹ -1 The characteristic absorption peak of -NH is present at 1502, 1265 and 1092 cm⁻¹. −1 The characteristic peaks are attributed to the NH bending, CN stretching, and aromatic ring vibrations of the PDA structure. The spectra of MnOOH and PDA@MnOOH were observed to have peaks at 570 and 2900 cm⁻¹. -1 There is a peak at each location, which is attributed to the presence of the Mn-O characteristic peak and the stretching vibration peak of -OH. Infrared spectroscopy results indicate the successful preparation of PDA@MnOOH. X-ray photoelectron spectroscopy (XPS) of PDA@MnOOH was also performed, and the results are as follows... Figure 6 As shown, the results indicate that PDA@MnOOH contains four chemical elements: C, N, O, and Mn, which is consistent with the results in the EDS table.

[0048] Example 2

[0049] The preparation method of manganese-based nanozyme PDA@MnOOH in this embodiment includes the following specific steps:

[0050] 13.85 mg of MnCl2·4H2O and 9.0 mg of AsA were weighed and dissolved in 5 mL of deionized water. Then, 4 mL of NaOH (1 M) solution was slowly added, and the mixture was allowed to stand at 37 °C for 4 h. 0.143 g of F127 was dissolved in a mixed solvent of 46 mL of anhydrous ethanol and water and stirred at room temperature for 25 min. 190 μL of 1,3,5,-trimethylbenzene was added to the solution, and stirring was continued at room temperature for 2.5 h. The two solutions were then mixed. 36 mg of tris(hydroxymethyl)aminomethane was weighed and dissolved in 4 mL of distilled water, and 11 mg of dopamine (DA) was added to the mixed solution. The reaction was continued for 23 h. The reaction solution was centrifuged and washed twice with ethanol and acetone, respectively, to obtain PDA@MnOOH nanosheets.

[0051] Example 3

[0052] The preparation method of manganese-based nanozyme PDA@MnOOH in this embodiment includes the following specific steps:

[0053] 15.80 mg of MnCl2·4H2O and 9.9 mg of AsA were weighed and dissolved in 5 mL of deionized water. Then, 6 mL of NaOH (1 M) solution was slowly added, and the mixture was allowed to stand at 37 °C for 4 h. 0.149 g of F127 was dissolved in a mixed solvent of 48 mL of anhydrous ethanol and water and stirred at room temperature for 35 min. 210 μL of 1,3,5,-trimethylbenzene was added to the solution, and stirring was continued at room temperature for 3.5 h. The two solutions were then mixed. 38 mg of tris(hydroxymethyl)aminomethane was weighed and dissolved in 4 mL of distilled water, and 13 mg of dopamine (DA) was added to the mixed solution. The reaction was continued for 25 h. The reaction solution was centrifuged and washed twice with ethanol and acetone, respectively, to obtain PDA@MnOOH nanosheets.

[0054] Implementation Results Example

[0055] Experimental Procedure: First, the OXD activity of PDA@MnOOH prepared in Example 1 was evaluated using 3,3',5,5'-tetramethylbenzidine (TMB) as a probe. Briefly, mixed solutions containing different concentrations (0, 25, 50, 100, 150, 200 μg / mL) of PDA@MnOOH and TMB (50 μg / mL) were co-incubated in PBS buffer for 10 min, and then the absorbance at 652 nm was measured using a UV-spectrum spectrophotometer.

[0056] We also investigated the types of ROS generated. Electron spin resonance (ESR) technology was used to analyze the free radical signals to determine the types of ROS. The specific steps were as follows: PDA@MnOOH was prepared into a 2 mg / mL dispersion with methanol; for each measurement, 500 μL of this dispersion was mixed with 5 μL of the scavenging agent 5,5-dimethyl-1-pyrrolline-N-oxide (DMPO), allowed to stand for 5 min, and then analyzed.

[0057] Next, the generation of ROS by the CAT and OXD cascade was evaluated. DPBF was used as a probe to investigate the ROS generation. Specifically, two PDA@MnOO H solutions of the same concentration (100 μL, 100 μg / mL) were prepared. One of these solutions was incubated in buffer with a mixture of DPBF (100 μL) and H2O2 (100 μL, 0.75 mM). The absorbance at 420 nm was measured every 2 min using a UV-Vis spectrophotometer.

[0058] Nanozyme activity is temperature-dependent, so we used a TMB probe to verify the effect of temperature on enzyme activity. Specifically, a mixed solution containing PDA@MnOOH (100 μL, 100 μg / mL) and TMB (50 μg / mL) was co-incubated in PBS (5.0) buffer. During incubation, the reaction system was irradiated with an 808 nm laser. The temperature was varied by changing the irradiation time (0, 1, 2, 3, 5 min). Finally, the absorbance at 652 nm was measured using a UV-Vis spectrophotometer.

[0059] Results analysis:

[0060] I. The photothermal properties of the manganese-based nanozyme PDA@MnOOH prepared in Example 1 were tested, and the results are as follows:

[0061] The photothermal properties of PDA@MnOOH were investigated using a photothermal analyzer. The effects of sample concentration, laser irradiation intensity, and irradiation time on the photothermal properties were studied. The results are as follows: Figure 7 As shown, we found that different concentrations (0, 50, 100, 150, 200 μg / mL) of PDA@MnOOH under specific laser conditions (1 W / cm²). 2 After irradiation for 10 minutes, the solution temperature increased with increasing concentration. After 10 minutes of irradiation, the temperatures of samples with different concentrations reached 31.5 ℃, 43.1 ℃, 48.7 ℃, 59.6 ℃, and 68.7 ℃, respectively, demonstrating that higher sample concentrations result in stronger photothermal conversion capabilities. Samples of specific concentrations were irradiated with lasers of specific power for different durations, and the sample temperature increased with prolonged irradiation time. The results are as follows... Figure 8 The figure shows the results using different power levels (0, 0.5, 1.0, 1.5, 2.0 W / cm). 2 The results showed that after irradiating a PDA@MnOOH solution of a specific concentration with an 808 nm laser for 10 min, the solution temperature increased from 26.5 ℃ to 38.3 ℃, 48.7 ℃, 58.8 ℃, and 70 ℃, respectively. All results demonstrate that the PDA@MnOOH solution is affected by sample concentration, power intensity, and irradiation time.

[0062] II. The oxygen generation performance of the manganese-based nanozyme PDA@MnOOH prepared in Example 1 was tested, and the results are as follows:

[0063] Because bacterial infection wounds have low oxygen levels, and oxygen is a key factor in wound healing, we evaluated the O2 production capacity of PDA@MnOOH. To investigate the CAT activity of PDA@MnOOH, O2 production was monitored in real-time using a dissolved oxygen analyzer. O2 production under different pH conditions was studied. Figure 9As shown, oxygen content was measured using a dissolved oxygen meter at different pH values ​​(7.4, 6.5, 5.0). The results showed that PDA@MnOOH was more efficient at catalyzing O2 production under acidic conditions than under neutral conditions. This is because manganese ions are released more effectively in an acidic environment. Therefore, PDA@MnOOH is more likely to produce more O2 in the slightly acidic environment of bacterial infection sites.

[0064] III. The ROS generation performance of the manganese-based nanozyme PDA@MnOOH prepared in Example 1 was tested, and the results are as follows:

[0065] ROS are crucial for killing bacteria. Therefore, we used the TMB probe to evaluate the ability of PDA@MnOOH to generate ROS. TMB can be oxidized by ROS to generate the blue product oxTMB, which has a maximum absorption wavelength of 652 nm. The ROS generation of PDA@MnOOH at different concentrations (0, 25, 50, 100, 150, 200 μg / mL) was investigated. The results are as follows: Figure 10 As shown, the amount of ROS generated increases with increasing PDA@MnOOH concentration, and the blue product from TMB oxidation becomes increasingly darker. Therefore, ROS generation by PDA@MnOOH is positively correlated with its concentration. To determine the type of ROS generated by PDA@MnOOH, DMPO was used as a trapping agent, and ESR was used to study the ROS types. The results are as follows. Figure 11 As shown, the characteristic quartet (1:1:1:1) of DMPO confirms the presence of O2. – Therefore, it can be concluded that PDA@MnOOH catalyzes the production of O2 using O2 as a reaction substrate. – This ROS further leads to the conclusion that PDA@MnOOH possesses OXD-like activity.

[0066] IV. The enzyme cascade performance of the manganese-based nanozyme PDA@MnOOH prepared in Example 1 was tested, and the results are as follows:

[0067] Experiments verified that PDA@MnOOH can generate more O2 under acidic conditions, which can alleviate hypoxia at the wound site. Furthermore, O2 generation can further increase the activity of OXD-like enzymes and enhance ROS generation. To investigate the cascade activities of the CAT and OXD-like enzymes, the effect of H2O2 on the cascade activities of the two enzymes in PDA@MnOOH was studied using a DPBF probe. The results are as follows: Figure 12 As shown, the absorbance of DPBF at 420 nm decreased more significantly after the addition of H2O2, which verifies that PDA@MnOOH generates O2 in the presence of H2O2, providing sufficient substrate for OXD-like structures and thus generating more ROS.

[0068] V. The photothermal-promoted ROS performance of the manganese-based nanozyme PDA@MnOOH prepared in Example 1 was tested, and the results are as follows:

[0069] The catalytic activity of nanozymes is temperature-dependent. Since PDA@MnOOH exhibits good photothermal performance at 808 nm, the ROS formation of PDA@MnOOH under 808 nm illumination was evaluated. The results are as follows: Figure 13 As shown, the results indicate that ROS production increases with prolonged irradiation time. Previous studies have shown that the sample temperature also increases with prolonged irradiation time. Therefore, photothermal activity can catalyze nanozyme activity.

[0070] VI. The GSH consumption performance of the manganese-based nanozyme PDA@MnOOH prepared in Example 1 was tested, and the results are as follows:

[0071] PDA@MnOOH has the ability to consume GSH at the wound site, indirectly increasing the ROS concentration at the wound site and effectively killing bacteria. Using DTNB as an indicator, it reacts with GSH to generate a stable yellow 5-thio-2-nitrobenzoate ion (TNB). - The characteristic absorption peak of PDA@MnOOH was observed at 412 nm, while that of DTNB was also observed at 325 nm. Different concentrations (0, 50, 100, 150, 200, and 400 μg / mL) of PDA@MnOOH and GSH (10 mM) were investigated after incubation at 37 ℃ for 40 min. The results are as follows: Figure 14 As shown, the characteristic absorption peak of DTNB at 325 nm increases with the increase of PDA@MnOOH concentration, indicating that the DTNB content increases, proving that the GSH reacting with it is consumed. The above study demonstrates that PDA@MnOOH has the effect of consuming GSH, thereby enhancing the bactericidal effect of ROS.

[0072] VII. The in vitro biocompatibility of the manganese-based nanozyme PDA@MnOOH prepared in Example 1 was tested, and the results are as follows:

[0073] The biosafety of antimicrobial drugs is crucial in biopharmaceutical therapy; therefore, it is necessary to evaluate the biosafety of PDA@MnOOH. We co-incubated mouse erythrocytes with both PDA@MnOOH and PDA@MnOOH+NIR groups and calculated the hemolysis rate. The results are as follows: Figure 15 As shown in (A), hemolysis occurred in the positive control group (deionized water), while the experimental and negative groups (physiological saline groups) showed no hemolysis, even at a material concentration as high as 200 μg / mL, with a hemolysis rate of less than 5%. This demonstrates that PDA@MnOOH has excellent blood compatibility. Furthermore, we also investigated the toxicity of PDA@MnOOH to 3T3 cells. The results are as follows... Figure 15 (B) The results showed that when the concentration of PDA@MnOOH was as high as 200 μg / mL, the cells in the laser-irradiated group still retained 89% viability. This indicates that PDA@MnOOH has good biocompatibility under NIR irradiation.

[0074] 8. The ROS levels in bacteria were tested on the manganese-based nanozyme PDA@MnOOH prepared in Example 1, and the results are as follows:

[0075] The ROS content in bacteria treated with PDA@MnOOH was detected using the DCFH-DA fluorescent probe. Results are as follows: Figure 16 The ROS fluorescence intensity in the control group and PDA group was relatively low, and the ROS expression level did not change significantly even under NIR irradiation. However, the MnOOH group and the MnOOH+NIR group showed obvious green fluorescence, which is due to the O2 generated by the MnOOH-mediated OXD-like nanozyme activity. – The PDA@MnOOH group also exhibited significant green fluorescence. Notably, the PDA@MnOOH+NIR group showed the strongest green fluorescence intensity. This is likely due to the increased temperature under NIR laser irradiation, which enhances OXD-like activity and generates more ROS, thus enabling more efficient bacterial killing.

[0076] IX. The in vitro antibacterial properties of the manganese-based nanozyme PDA@MnOOH prepared in Example 1 were tested, and the results are as follows:

[0077] The OXD-like activity of PDA@MnOOH allows it to generate sufficient ROS to achieve a bactericidal effect. Simultaneously, irradiation of the PDA@MnOOH solution with an 808 nm laser for 5 min raises the temperature to approximately 45℃, providing gentle photothermal stimulation of enzyme activity. *S. aureus*, *E. coli*, and MRSA were used as model bacteria to test the antibacterial activity of PDA@MnOOH. All three bacteria were treated with PBS, PBS+NIR, PDA (10 μg / mL), PDA+NIR (10 μg / mL), MnOOH (90 μg / mL), MnOOH+NIR (90 μg / mL), PDA@MnOOH (100 μg / mL), and PDA@MnOOH+NIR (100 μg / mL). Colonies were observed and counted. Results are as follows: Figure 17(AD), PBS, and PBS+NIR groups showed no significant reduction in colonies. PDA (10 μg / mL) and PDA+NIR (10 μg / mL) treatments showed some colony reduction, indicating certain antibacterial activity. MnOOH (90 μg / mL) and MnOOH+NIR (90 μg / mL) treatments showed a more significant reduction in colonies, demonstrating strong bactericidal effects. The survival rates of MRSA treated with these methods were 63.73% and 58.13%, respectively, due to their OXD-like activity, which generates a certain amount of O2. – Killing bacteria. The PDA@MnOOH (100 μg / mL) treatment group showed stronger bactericidal activity against all three bacteria compared to other groups. This is due to the combined antibacterial effect of CAT cascade OXD enzyme activity and PDA. The PDA@MnOOH+NIR (100 μg / mL) treatment group showed the strongest antibacterial effect against all three bacteria, with almost all of them being killed. This was significantly different from other treatment groups. This is due to the antibacterial effect of PDA combined with mild photothermal activation of the OXD cascade CAT, demonstrating that the prepared nanomedicine PDA@MnOOH has a strong broad-spectrum bactericidal ability.

[0078] To further evaluate the antibacterial effect of the composite nanomaterial PDA@MnOOH, bacteria treated under different conditions were collected and their microscopic morphological changes were observed and analyzed using SEM. Figure 17 (E) The control group of E. coli maintained its characteristic rod-shaped form with an intact surface, while S. aureus and MRSA exhibited typical spherical structures, and the cell walls of all three bacteria remained intact. In contrast, the bacterial morphology of all treatment groups showed varying degrees of damage and disruption, with wrinkled bacterial surfaces and even cell wall rupture. Furthermore, the PDA@MnOOH+NIR group showed the most severe morphological damage, consistent with the results of bacterial plate colony determination. Simultaneously, we performed live / dead staining assays on the bacteria in different treatment groups. Bacteria were stained with SYTO9 green fluorescent nucleic acid dye and PI; live bacteria stained green fluorescence, and dead bacteria stained red fluorescence. Figure 17 (F) The control group showed almost complete green fluorescence, indicating that almost all bacteria were viable. In the PDA, PDA+NIR, MnOOH, MnOOH+NIR, and PDA@MnOOH treatment groups, partial red fluorescence was observed, demonstrating that these groups possessed certain antibacterial capabilities. The PDA@MnOOH+NIR group showed almost complete red fluorescence, consistent with the results of bacterial plate colony determination, reflecting the strongest bactericidal effect of synergistic treatment. This further proves that PDA@MnOOH is a highly promising antibacterial nanomedicine.

[0079] 10. The in vivo antibacterial effect of the manganese-based nanozyme PDA@MnOOH prepared in Example 1 was tested, and the results are as follows:

[0080] We established an in vivo antibacterial model by infecting mouse wounds with MRSA bacteria, and compared the in vivo antibacterial activity of PDA@MnOOH by treating different sample groups. The results are as follows: Figure 18 As shown in (AB), compared with other treatment groups, the PDA@MnOOH+NIR treatment group exhibited faster wound healing, with almost complete healing within 12 days. We also simulated the wound healing process of different treatment groups using ImageJ. The results showed that the PDA@MnOOH+NIR treatment group healed faster and with better results. This is attributed to the combined antibacterial and wound-healing effects of the OXD-like and CAT cascade activities of the prepared composite nanomaterials, as well as the mild photothermal promotion of OXD-like activity. Simultaneously, to ensure effective low-temperature enzymatic activity and avoid destructive tissue damage caused by excessively high wound temperatures, we used a near-infrared thermal imager to monitor wound temperature changes in real time. Figure 18 (C) The results showed that the temperature in the blank group did not change significantly under laser irradiation, while the temperature at the wound site in the PDA@MnOOH treatment group rose rapidly under NIR laser irradiation, reaching 44.6℃ in 5 minutes. Therefore, 5 minutes was selected as the phototherapy time in subsequent treatments to ensure that the wound temperature was around 45℃. ImageJ was used to quantitatively analyze the wound area (…). Figure 18 (D) MnOOH or PDA@MnOOH wounds healed faster than the PBS group, showing a clear healing trend on days 4 and 8. This is attributed to the excellent OCD and CAT enzyme cascade activities of MnOOH, which generate ROS to kill bacteria while the consumption of GSH further increases the concentration of ROS at the wound site, effectively killing infected bacteria. Furthermore, the CAT activity of MnOOH generates O2 to alleviate wound hypoxia and promote angiogenesis at the wound site, thereby promoting healing. In addition, it was observed that the wound healing rate was faster in the PDA@MnOOH+NIR group. The wound size in the PDA@MnOOH+NIR treatment group reached 6% of the original wound area on day 12, while the wound size in the PDA@MnOOH group remained at 17.43% of the original wound area. This is attributed to the photothermal effect leading to increased temperature at the wound site, further enhancing OXD-like activity for better bactericidal action and promoting rapid wound healing. In summary, PDA@MnOOH can consume GSH and generate ROS in the bacterial infection microenvironment to effectively kill infected bacteria, while CAT-like activity generates O2 at the wound site. – It provides substrates for OXD-like cells to generate more ROS, while alleviating the hypoxic environment at the wound site, promoting angiogenesis and accelerating wound healing. At the same time, the temperature at the wound site increases under NIR laser irradiation, further promoting nanozyme activity to generate more ROS and kill bacteria.

[0081] To further verify the antibacterial activity of PDA@MnOOH, wound tissue was collected 12 days after treatment. The tissue was aseptically lysed, and the culture medium was collected and spread onto LB agar plates for culture. Bacterial growth was observed, and the bacterial count in each group was statistically analyzed. Figure 18 (EF) The results showed that the number of bacteria in the PDA@MnOOH group was significantly reduced, thanks to the OXD-like activity of MnOOH generating ROS that destroys bacteria. Furthermore, the PDA@MnOOH+NIR treatment group showed the fewest bacteria in the smear results. This result is attributed to the increased temperature at the wound site after NIR irradiation, which promotes the activity of nanozymes, generating a large amount of ROS to kill bacteria, thus achieving optimal therapeutic effect. This result is consistent with the results of wound imaging in mice. In addition, we monitored the weight of mice during the treatment process. Figure 18 (G) shows that during the treatment of mice with PDA@MnOOH, there was no significant decrease in mouse body weight, indicating its excellent biocompatibility. These results demonstrate that PDA@MnOOH is a promising antibacterial and wound-healing composite nanomedicine.

[0082] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing a polydopamine manganese-based nanozyme with mildly photothermally enhanced enzyme activity, characterized in that, The steps are as follows: (1) Dissolve MnCl2·4H2O and sodium ascorbate in deionized water, then add NaOH solution and let the reaction stand to obtain solution I; (2) Dissolve F127 in a mixed solvent of anhydrous ethanol and water and stir at room temperature. Then add 1,3,5,-trimethylbenzene and continue stirring to obtain solution II. (3) After mixing solution I and solution II, add tris(hydroxymethyl)aminomethane and dopamine, and continue the reaction until complete. After centrifugation and washing, PDA@MnOOH nanosheets, i.e. polydopamine manganese-based nanozymes, are obtained.

2. The method for preparing polydopamine manganese-based nanozymes with mildly photothermally enhanced enzyme activity according to claim 1, characterized in that: In step (1), the molar ratio of MnCl2·4H2O, sodium ascorbate and NaOH is 0.07-0.08:0.040-0.05:4-6; the concentration of NaOH solution is 1 M; and the volume ratio of deionized water to NaOH solution is 1:

1.

3. The method for preparing polydopamine manganese-based nanozymes with mildly photothermally enhanced enzyme activity according to claim 2, characterized in that: In step (1), the temperature for static reaction is 36-38 ℃ and the time is 3.5-4.5 h.

4. The method for preparing polydopamine manganese-based nanozymes with mildly photothermally enhanced enzyme activity according to claim 1, characterized in that: In step (2), the volume ratio of anhydrous ethanol to water in the mixed solvent is 1:2; the concentration of F127 in the mixed solvent is 3.1 mg / mL; and the volume ratio of the mixed solvent to 1,3,5,-trimethylbenzene is 46-48:0.19-0.

21.

5. The method for preparing polydopamine manganese-based nanozymes with mildly photothermally enhanced enzyme activity according to claim 4, characterized in that: In step (2), the stirring time at room temperature is 25-35 min; the stirring time is 2.5-3.5 h.

6. The method for preparing polydopamine manganese-based nanozymes with mildly photothermally enhanced enzyme activity according to claim 5, characterized in that: In step (3), the mass ratio of 1,3,5,-trimethylbenzene, tris(hydroxymethyl)aminomethane and dopamine is 163-181:36-38:11-13.

7. The method for preparing polydopamine manganese-based nanozymes with mildly photothermally enhanced enzyme activity according to claim 6, characterized in that: The reaction continues for 23-25 ​​hours.

8. A polydopamine manganese-based nanozyme with mildly photothermal-enhanced enzyme activity prepared by the method according to any one of claims 1-7.

9. The polydopamine manganese-based nanozyme according to claim 8, characterized in that: The polydopamine manganese-based nanozyme can reach a temperature of 44-45℃ after 5 minutes of NIR laser irradiation.

10. The use of the polydopamine manganese-based nanozyme of claim 8 in the preparation of a drug with antibacterial, anti-inflammatory, and wound-healing properties.

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