Preparation method and application of metal aerogel with photo-thermal and multi-enzyme performance

Abstract

The invention relates to the technical field of antibacterial drugs, and discloses a preparation method of metal aerogel with photo-thermal and multi-enzyme performance, the preparation method comprises the following steps: S1, adding an AuCl3 solution and CuCl2 into ethanol, mixing to obtain a faint yellow clear solution, then quickly adding a reducing agent solution, and reacting until the color becomes black; the invention also provides an application of the metal aerogel with photo-thermal and multi-enzyme properties, and an application of the metal aerogel with photo-thermal and multi-enzyme properties, which is prepared according to any one of the preparation methods in claims 2-3, in an anti-drug-resistant bacterium treatment reagent. The photosensitizer provided by the invention has excellent photothermal conversion capability, meets the requirements of photothermal therapy, has good active oxygen generation capability, and can destroy drug-resistant bacteria under laser irradiation induction, thereby improving the bacterial infection treatment efficiency.

Description

Technical Field

[0001] The present invention relates to the technical field of antibacterial drugs, and specifically to a preparation method and application of a metal aerogel with both photothermal and multiple enzyme properties. Background Art

[0002] Bacterial infections are regarded as a serious threat to global public health. The traditional treatment method for bacterial infections is antibiotics, which have significantly improved living standards and saved countless lives. However, the abuse and misuse of antibiotics have led to multi-drug resistance (MDR), increased morbidity and mortality, and produced serious side effects such as low immunity, hematopoietic dysfunction, and intestinal flora imbalance. Methicillin-resistant Staphylococcus aureus (MRSA) has been recognized by the World Health Organization (WHO) as an important pathogen for community- and hospital-acquired infections because it is resistant to antibiotics and can produce a biofilm composed of extracellular polymeric substances (EPS). After bacteria form a biofilm, the challenges faced in clinically controlling bacterial infections become more severe because the barrier effect of the biofilm may hinder the penetration of antibiotic molecules and promote the growth of persistent bacterial cells. Therefore, it is crucial to design effective and safe antibiotic alternatives for antibacterial treatment to eliminate MRSA.

[0003] Due to its strong structural stability and adjustable catalytic activity, metal aerogels have become a research topic in the biological field, providing opportunities for the development of innovative and effective antibacterial agents. Metal aerogels with peroxidase (POD) mimic or oxidase (OD) mimic activity can convert hydrogen peroxide (H2O2) or oxygen (O2) into highly toxic hydroxyl radicals (·OH) or superoxide anion radicals (·O 2- )). The generated free radicals can penetrate cells and damage the membranes, DNA, and proteins of bacteria, thereby overcoming the resistance of bacteria to traditional antibiotics. In addition, metal aerogels accelerate the decomposition of various biofilm matrix components, including polysaccharides, lipids, proteins, and extracellular DNA. However, the moderate activity of metal aerogels hinders their bactericidal effect. In addition, overexpressed glutathione (GSH) in the infectious microenvironment can scavenge reactive oxygen species (ROS), resulting in a decrease in the bactericidal effect. To improve the treatment effect, it is necessary to simultaneously increase the activity of metal aerogels and regulate the microenvironment at the infection site.

[0004] In recent years, metal aerogels based on MgO have been widely used as a new type of porous nanomaterials due to their extraordinary physicochemical properties. They not only inherit the high catalytic activity of original metal nanoparticles, but also maintain the large specific characteristic surface area of ​​aerogels, rich multi-scale pores, and release huge potential in the field of catalysis. They show significant advantages, including large surface area for efficient electron transfer, rich active sites, and independent architecture for enhanced stability. Metal aerogels based on MgO usually have high near-infrared (NIR) photothermal conversion efficiency, providing promising, economical and non-invasive nanotherapy for light-induced therapy. The energy greatly increases the temperature for treating local bacterial infections and enhances enzyme-like catalytic reactions to increase ROS generation efficiency, thereby improving the overall antibacterial efficacy through synergistic effects. Therefore, it may be possible to construct multifunctional metal aerogels to achieve effective in vivo antibacterial therapy. Summary of the invention

[0005] 1. Technical issues to be resolved

[0006] In view of the shortcomings of the prior art, the present invention provides a preparation method and application of a metal aerogel with both photothermal and multi-enzyme properties. AuCu@Pd metal aerogel is prepared by a one-pot reduction method in ethanol phase for photothermal enhanced chemodynamic antibacterial therapy. AuCu@Pd metal aerogel exhibits similar activity to POD and OD, producing a certain amount of ·OH and ·O 2- , destroying the integrity of the cell membrane, thereby killing bacteria. In addition, glutathione peroxidase (GPX)-like activity can deplete glutathione to regulate the infection microenvironment, which is used to amplify the microenvironment of ROS generation, thereby enhancing the bactericidal effect against E. coli (Escherichia coli, Gram-negative) and MRSA (Gram-positive). In addition, AuCu@Pd metal aerogel has excellent photothermal conversion ability, which can generate high heat in AuCu@Pd metal aerogel to ablate bacteria in a short time, and greatly improve the intrinsic multi-enzyme-like activity to produce abundant ROS to eliminate bacteria, enhance in vivo anti-resistant bacteria treatment and accelerate wound healing.

[0007] (II) Technical solution

[0008] To achieve the above object, the present invention provides the following technical solutions:

[0009] A method for preparing a metal aerogel having both photothermal and multi-enzyme properties, the preparation method comprising the following steps:

[0010] S1: Add AuCl3 solution and CuCl2 into ethanol and mix to obtain a light yellow clear solution, then quickly add the reducing agent solution to react, and the color changes to black.

[0011] S2: Under stirring conditions, add the NaPbCl4 solution to the black solution in S1, stir the AuCu@Pd metal aerogel, then add the reducing agent solution for reaction. After that, place the reaction solution in a water bath and let it stand. The AuCu@Pd metal aerogel is formed at the bottom. Wash it with water and ethanol 3 - 5 times respectively, and then convert it into an aerogel by freeze-drying technology.

[0012] Further, the reducing agent in S1 is NaBH4.

[0013] On the basis of the above scheme, the temperature for the coupling reaction of the reaction solution in S2 in the water bath is 40 - 50 °C, and the time is 0.5 - 1 h. The reducing agent in S2 is ascorbic acid.

[0014] The present invention also proposes an application of a metal aerogel with both photothermal and multiple enzyme properties. The application of the metal aerogel with both photothermal and multiple enzyme properties prepared by the preparation method according to any one of claims 2 - 3 in anti-drug-resistant bacteria treatment reagents.

[0015] (III) Beneficial effects

[0016] Compared with the prior art, the present invention provides a preparation method and an application of a metal aerogel with both photothermal and multiple enzyme properties, having the following beneficial effects:

[0017] 1. In the present invention, Au, Cu, and Pd metals are combined to obtain a metal aerogel with both photothermal and multiple enzyme properties. This nanoparticle has photothermal conversion performance, peroxidase (POD)-like activity, and glutathione peroxidase (GPX)-like activity, and can catalyze low-concentration H2O2 to simultaneously perform photothermal / chemical dynamics for anti-drug-resistant bacteria treatment.

[0018] 2. The photothermal conversion efficiency (η) of the photosensitizer AuCu@Pd metal aerogel provided by the present invention is 61%, which has excellent photothermal conversion ability and meets the requirements of photothermal therapy for anti-drug-resistant bacteria. The AuCu@Pd metal aerogel provided by the present invention has good reactive oxygen species (ROS) generation ability. Under laser irradiation induction, the AuCu@Pd metal aerogel has the combined anti-drug-resistant bacteria effect of PTT / CDT, can destroy bacteria, and thus improve the anti-drug-resistant bacteria treatment efficiency. Experimental results show that the AuCu@Pd metal aerogel provided by the present invention can effectively inhibit the growth of bacteria and accelerate wound healing. Description of the drawings

[0019] Figure 1 It is a schematic diagram of the synthesis of the AuCu@Pd metal aerogel;

[0020] Figure 2 It is the synthesis characterization of the AuCu@Pd metal aerogel;

[0021] Figure 3 is the absorption spectrum of AuCu@Pd metal aerogel in aqueous solution;

[0022] Figure 4 is the temperature change curve of AuCu@Pd metal aerogel under 808nm laser irradiation with different concentrations and powers;

[0023] Figure 5 is the photothermal stability and photothermal conversion efficiency of AuCu@Pd metal aerogel;

[0024] Figure 6 is the catalytic activity of AuCu@Pd metal aerogel for H2O2 detected by steady-state kinetics;

[0025] Figure 7 is the MB fading experiment of AuCu@Pd metal aerogel;

[0026] Figure 8 is the GPX activity detection of AuCu@Pd metal aerogel;

[0027] Figure 9 is the in vitro antibacterial experiment of AuCu@Pd metal aerogel and the Michaelis constant detection of AuCu@Pd metal aerogel;

[0028] Figure 10 is the survival rate of E.coli and MRSA bacteria after different treatments;

[0029] Figure 11 is the confocal microscope images of live / dead staining of E.coli and MRSA bacteria after different treatments;

[0030] Figure 12 is the ROS confocal fluorescence images of E.coli and MRSA bacteria induced after different treatments;

[0031] Figure 13 is the flow chart of in vivo anti-drug-resistant bacteria infection and wound healing monitoring of AuCu@Pd metal aerogel;

[0032] Figure 14 is the thermal imaging map of MRSA model mice after different treatments under laser irradiation;

[0033] Figure 15 is the wound bacteria plating, wound tissue H&E and

[0034] Masson staining map of MRSA model mice after the treatment ends;

[0035] Figure 16 is the body weight fluctuation curve of MRSA model mice after different treatments;

[0036] Figure 17 Hemolysis experiment of AuCu@Pd metal aerogel;

[0037] Figure 18 H&E staining of important organs after different treatments of MRSA model mice;

[0038] Figure 19 Liver and kidney indexes of MRSA model mice after treatment;

[0039] Figure 20 Schematic diagram of the structure of AuCu@Pd metal aerogel;

[0040] Figure 21 Schematic diagram of wound healing of mice after different treatments. Specific implementation manners

[0041] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0042] Example 1

[0043] Referring to Figures 1 - 21 , a preparation method of a metal aerogel with both photothermal and multiple enzyme properties, the preparation method includes the following steps:

[0044] S1: Add AuCl3 solution and CuCl2 to ethanol and mix to obtain a light yellow clear solution, and then quickly add a reducing agent solution for reaction, and the color changes to black.

[0045] S2: Under the condition of stirring, add NaPbCl4 solution to the black solution in S1, stir the AuCu@Pd metal aerogel and then add a reducing agent solution for reaction, and then place the reaction solution in a water bath and let it stand. The AuCu@Pd metal aerogel is formed at the bottom. After washing with water and ethanol 5 times respectively, it is then converted into an aerogel by freeze-drying technology. In the present invention, Au, Cu and Pd metals are combined to obtain a metal aerogel with both photothermal and multiple enzyme properties. The nanoparticles have photothermal conversion performance, peroxidase-like (POD) activity and glutathione peroxidase-like (GPX) activity, and can catalyze low-concentration H2O2 to simultaneously perform photothermal / chemical dynamics for anti-drug-resistant bacteria treatment.

[0046] In the present invention, the reducing agent in S1 is NaBH4, the temperature for the coupling reaction of placing the reaction solution in a water bath in S2 is 50 °C, the time is 1 h, and the reducing agent in S2 is ascorbic acid.

[0047] The present invention also provides an application of a metal aerogel with both photothermal and multiple enzyme properties, which is an application of the metal aerogel with both photothermal and multiple enzyme properties prepared by the preparation method according to any one of claims 2 to 3 in an anti-drug-resistant bacteria therapeutic reagent.

[0048] The present invention provides an application of the photosensitizer with both photothermal and chemical kinetic effects in the preparation of antibacterial drugs.

[0049] In the present invention, the bacteria killed by the antibacterial drug are preferably methicillin-resistant Staphylococcus aureus.

[0050] The present invention provides an application of the photosensitizer with both photothermal and chemical kinetic effects in the preparation of anti-drug-resistant bacteria reagents.

[0051] Example 2

[0052] Referring to Figures 1 - 21 , a preparation method of a metal aerogel with both photothermal and multiple enzyme properties, the preparation method includes the following steps:

[0053] S1: Mix the AuCl3 solution and CuCl2 in ethanol to obtain a light yellow clear solution, and then quickly add a reducing agent solution for reaction, and the color changes to black.

[0054] S2: Under stirring conditions, add the NaPbCl4 solution to the black solution in S1, stir the AuCu@Pd metal aerogel and then add a reducing agent solution for reaction, and then place the reaction solution in a water bath and let it stand. The AuCu@Pd metal aerogel is formed at the bottom. After washing with water and ethanol 4 times respectively, and then converted into an aerogel by freeze-drying technology. The present invention combines Au, Cu and Pd metals to obtain a metal aerogel with both photothermal and multiple enzyme properties. The nanoparticles have photothermal conversion performance, peroxidase (POD)-like activity and glutathione peroxidase (GPX)-like activity, and can catalyze low-concentration H2O2 and simultaneously perform photothermal / chemical kinetics for anti-drug-resistant bacteria treatment.

[0055] In the present invention, the reducing agent in S1 is NaBH4, the temperature for the coupling reaction of placing the reaction solution in a water bath in S2 is 45 °C, the time is 0.7 h, and the reducing agent in S2 is ascorbic acid.

[0056] The present invention also provides an application of a metal aerogel with both photothermal and multiple enzyme properties, which is an application of the metal aerogel with both photothermal and multiple enzyme properties prepared by the preparation method according to any one of claims 2 to 3 in an anti-drug-resistant bacteria therapeutic reagent.

[0057] The present invention provides an application of the photosensitizer with both photothermal and chemical kinetic effects in the preparation of antibacterial drugs.

[0058] In the present invention, the bacteria killed by the antibacterial drug are preferably methicillin-resistant Staphylococcus aureus.

[0059] The present invention provides the application of the above-mentioned photosensitizer with both photothermal and chemodynamic effects in the preparation of anti-drug-resistant bacteria reagents.

[0060] Example 3

[0061] Refer to Figures 1 - 21 , a preparation method of a metal aerogel with both photothermal and multi-class enzyme properties, the preparation method includes the following steps:

[0062] Add 1 mL of chloroauric acid (HAuCl4) solution and 3.5 mg of copper chloride (CuCl2) (the molar ratio of Au:Cu is 1:1) to 20 mL of ethanol to obtain a light yellow clear solution, and then quickly add freshly prepared sodium borohydride (NaBH4) aqueous solution (2 mL, 0.05 M). After adding NaBH4, the solution quickly changes from clear to black, and then 0.5 mL of 0.05 M sodium tetrachloropalladate (Na2PdCl4) solution is added under stirring conditions. After stirring the AuCu@Pd metal aerogel, 0.5 mL of ascorbic acid solution (0.25 M) is added, and then the reaction liquid is placed in a 40 °C water bath and left standing. After 0.5 h, AuCu@Pd metal aerogel is formed at the bottom of the reaction flask, and after washing with water and ethanol three times respectively, the AuCu@Pd metal gel is converted into an aerogel by freeze-drying technology. The synthesis process is as Figure 1 shown.

[0063] (1) Structural characterization of AuCu@Pd aerogel

[0064] The morphology of the AuCu@Pd metal aerogel was observed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). As Figure 2 shown in A, the SEM image shows that the AuCu@Pd metal aerogel has a sponge-like structure with three-dimensional (3D) porosity. The TEM image shows the interconnected metal nanowire structure of the AuCu@Pd metal aerogel ( Figure 2 B). The high-resolution TEM (HRTEM) image shows the lattice row spacing distribution corresponding to the (111) plane of face-centered cubic phase Au, Cu, and Pd ( Figure 2 C). In addition, four clear diffraction rings can be found in the SAED pattern ( Figure 2 D), indicating the polycrystalline structure of the AuCu@Pd metal aerogel. High-angle annular dark-field scanning transmission electron microscopy (HAADF) is as Figure 2 shown in E. The elemental mapping images ( Figure 2 F-H) show the distribution of Au, Cu, and Pd elements on the uniform AuCu@Pd metal aerogel, indicating the successful synthesis of the AuCu@Pd metal aerogel.

[0065] (2) Ultraviolet-Visible-Near Infrared Absorption Spectrum

[0066] The absorption spectrum was measured at 200 nm by an ultraviolet-visible spectrophotometer (MAPADA UA-3200S). The absorption spectrum of AuCu@Pd metal aerogel in aqueous solution is as Figure 3 shown, and AuCu@Pd metal aerogel has obvious absorption in the whole wavelength range.

[0067] (3) In Vitro Photothermal Performance of AuCu@Pd Metal Aerogel

[0068] To evaluate the in vitro photothermal (PTT) performance, an infrared thermal imager was used to record the temperature of AuCu@Pd metal aerogel. An aqueous solution of AuCu@Pd metal aerogel (200 μg / ml, 1.0 mL) was irradiated with an 808 nm laser at different laser intensities (0.25, 0.5, 0.75, and 1 W·cm -2 -2). In addition, an aqueous solution of 1.0 mL AuCu@Pd metal aerogel with different concentrations (0, 50, 100, 150, and 200 μg / ml) was irradiated with an 808 nm laser at a power of 1.0 W·cm 2 -2. The photothermal conversion efficiency (η) of AuCu@Pd metal aerogel was calculated by recording the relationship between the temperature change and time of the aqueous solution after irradiating AuCu@Pd metal aerogel (200 μg / ml) with an 808 nm laser (1 W·cm -2 -2) and cooling it to the ambient temperature. The photothermal conversion efficiency (η) was calculated according to formulas (1) - (5).

[0069] η = (hs(T max -T surr ) - Q0) / (I(1 - 10 -A808 -A)) (1)

[0070] hs can be calculated by the following formula.

[0071] hs = m / τ s (2)

[0072] τ s = t / (-lnθ) (3)

[0073] θ = (T - T surr ) / (T max -T surr ) (4)

[0074] Q0 = hs(T max -T surr ) (5)

[0075] where h represents the heat transfer coefficient, S represents the surface area of the sample container, Tmax represents the highest steady-state temperature, T surr represents the ambient normal temperature, T represents the instantaneous temperature during the cooling process, t represents the time of the cooling stage, θ represents the specific heat capacity of water, m represents the mass of the solution (g), and Q0 represents the energy input of the same solvent without the material after laser irradiation under the same conditions.

[0076] First, the photothermal properties of the aqueous solution of AuCu@Pd metal aerogel under 808 nm laser irradiation were investigated. The temperature change curve of AuCu@Pd metal aerogel under 808 nm laser irradiation is as Figure 4 shown in A. After laser irradiation of AuCu@Pd metal aerogel, at a laser power density of 1 W·cm -2 , the temperature of the 200 μg / ml solution concentration increased by 38 °C. At the same time, the photothermal properties of AuCu@Pd metal aerogel with concentrations ranging from 0 μg / ml to 200 μg / ml were further evaluated ( Figure 4 B). The results show that as the concentration increases, the temperature of AuCu@Pd metal aerogel also increases significantly. In addition, the thermal cycle experiment clearly demonstrates that AuCu@Pd metal aerogel has excellent photothermal stability ( Figure 5 A). By calculation, the photothermal conversion efficiency of AuCu@Pd metal aerogel is 61% ( Figure 5 B-C). These results together confirm the strong photothermal efficacy of AuCu@Pd metal aerogel under 808 nm laser irradiation.

[0077] (4) Detection of peroxidase-like (POD) activity of AuCu@Pd metal aerogel

[0078] The peroxidase-like activity of AuCu@Pd metal aerogel was studied using a standard substrate color reaction. First, TMB (10 mM, 50 μL) and H2O2 (0.1 M, 50 μL) were mixed into HAc-NaAc buffer (0.2 M, pH 6.0, 850 μL). Then, AuCu@Pd metal aerogel (200 μg / mL, 50 μL) was added to the above solution. After AuCu@Pd metal aerogel, the absorption spectrum was collected using a UV-visible spectrophotometer, as Figure 6 shown in A. At the same time, the peroxidase-like activity was detected at different pH values ( Figure 6 B), temperatures ( Figure 6 C), and reaction times ( Figure 6 D). The results show that AuCu@Pd metal aerogel has peroxidase-like activity, and the optimal reaction conditions are pH 6 and 40 °C.

[0079] (5) Steady-state kinetic experiments of AuCu@Pd metal aerogel

[0080] By varying the concentration of H2O2 and determining the reaction steady-state kinetic parameters with TMB fixed, the catalytic activity of AuCu@Pd metal aerogel towards H2O2 was further analyzed ( Figure 7 ). The maximum initial velocity (V max = 8.79×10 -8 M s -1 ) and Michaelis constant (Km = 2.86 mM) of the reaction were calculated from the Lineweaver-Burk plot. The Km value of AuCu@Pd metal aerogel was much smaller than that of HRP (Km = 3.7 mM), indicating that AuCu@Pd metal aerogel has excellent affinity for H2O2.

[0081] (6) MB decolorization experiment of AuCu@Pd metal aerogel

[0082] The POD-like activity was detected again using the MB probe. The 2 mg / mL AuCu@Pd aerogel, 200 μM H2O2 and MB (0.5 mM) were reacted at room temperature for different times (0, 10, 20, 30, 40, 50, 60, 90 and 120 min), and the UV-visible spectrum of the mixed solution was measured with a UV-visible spectrophotometer. The results showed that the fluorescence of MB decreased significantly with the increase of reaction time ( Figure 8 ), confirming that a large amount of ·OH was generated in this reaction system, showing strong POD-like enzyme activity.

[0083] (7) Detection of glutathione peroxidase-like (GPX) activity of AuCu@Pd metal aerogel

[0084] The DTNB probe was used to detect the GPX-like activity. The 5 mL AuCu@Pd aerogel with different concentrations (0.1, 0.2 and 0.4 mg / mL) was reacted with GSH (1 M, 5 μL) at room temperature. Then, 100 μL of the culture solution at different times was mixed with 900 μL of PBS (pH 7.4) respectively. Then 10 μL of DTNB (10 mM) was added to the mixed solution, and the UV-visible spectrum of the mixed solution was measured with a UV-visible spectrophotometer. The results showed that the fluorescence of DTNB decreased significantly with the increase of the concentration of AuCu@Pd aerogel, confirming that AuCu@Pd aerogel could consume a large amount of GSH ( Figure 9 ), and could reshape the bacterial infection microenvironment, thus showing great antibacterial application potential.

[0085] (8) In vitro antibacterial test of AuCu@Pd metal aerogel

[0086] Using Escherichia coli (E. coli) and methicillin-resistant Staphylococcus aureus (MRSA) as models, the antibacterial effects of AuCu@Pd metal aerogels were evaluated with Gram-negative and Gram-positive bacterial strains, respectively. A single E. coli or MRSA colony was inoculated into 8 mL of LB liquid medium at a temperature of 37 °C. In the standard antibacterial test, Escherichia coli or methicillin-resistant Staphylococcus aureus was divided into eight categories: I: control, II: control + NIR, III: H2O2, IV: H2O2 + NIR, V: AuCu@Pd, VI: AuCu@Pd + NIR, VII: AuCu@Pd + H2O2, VIII: AuCu@Pd + H2O2 + NIR. After culturing the bacterial suspensions of each group with AuCu@Pd metal aerogel at 37 °C, they were irradiated with an 808 nm laser (1 W·cm -2 ) or treated in the dark. The final concentrations of AuCu@Pd, H2O2, and bacteria were 200 μg / mL, 200 μM, and 1×10 7 CFU / mL, respectively. Take 100 μL of the diluted (30,000-fold) bacterial suspension after treatment and spread it evenly on an agar plate, and place it in a bacterial incubator at 37 °C for 18 h. Bacterial survival rate (%) = C t / C0 × 100% (N = 3), where C0 is the number of colonies in the control group and C t is the number of colonies in the experimental group. As Figure 10 shown, in the solid medium, a large number of bacterial colonies were found in both the H2O2 group and the NIR + H2O2 group, indicating that irradiation with NIR laser alone or low-concentration H2O2 could not limit bacterial proliferation. It is worth noting that the AuCu@Pd + H2O2 group had a certain antibacterial effect on MRSA and E. coli compared with the AuCu@Pd group and the H2O2 group, confirming that AuCu@Pd could induce the generation of ·OH by low-concentration H2O2 leading to bacterial death. After laser treatment, the antibacterial rates of the AuCu@Pd + H2O2 + NIR group against MRSA and E. coli reached more than 99%, significantly higher than those of the AuCu@Pd + NIR group, indicating that the AuCu@Pd + NIR group could not provide the necessary antibacterial effect against bacterial infection, and the antibacterial rate increased significantly when reacting with low-concentration H2O2, indicating that the combination of the high photothermal effect and multi-enzyme-like action of AuCu@Pd greatly improved the antibacterial effect.

[0087] (9) Anti-bacterial live / dead staining experiment of AuCu@Pd metal aerogel

[0088] The disruption of bacterial membranes was detected using a Live / Dead kit containing N01 (labeling live bacteria) and PI (labeling dead bacteria). Briefly, E. coli and MRSA (10 8Staining was carried out at (CFU / mL). After incubating the AuCu@Pd metal aerogel in the dark, the stained images of live bacteria (green) and dead bacteria (red) were recorded using a laser scanning confocal microscope as Figure 11 shown, and the results showed consistent results with the above in vitro antibacterial experiments.

[0089] (10) Determination of intracellular ROS in bacteria by AuCu@Pd metal aerogel

[0090] To determine the chemodynamic effect of the AuCu@Pd metal aerogel, 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) solution is a typical ROS probe that can be oxidized to fluorescent DCF in the presence of ROS.

[0091] The method for determining reactive oxygen species (ROS) is as follows:

[0092] E. coli or MRSA was divided into 8 groups for different treatments: I: PBS, II: PBS + NIR, III: H2O2, IV: H2O2 + NIR, V: AuCu@Pd, VI: AuCu@Pd + NIR, VII: AuCu@Pd + H2O2, VIII: AuCu@Pd + H2O2 + NIR.

[0093] First, 1 μL of DCFH-DA solution (20 μM) was incubated with different groups of bacterial suspensions with the AuCu@Pd metal aerogel at 37 °C. Then, the bacterial suspensions were irradiated with an 808 nm laser or placed in the dark with the AuCu@Pd metal aerogel. They were washed 3 times with sterile PBS and then redispersed in 1 mL of PBS. Then, the ROS levels of each group were detected using a laser confocal scanning microscope. In Figure 12 it, when the AuCu@Pd metal aerogel (200 μg / ml) was irradiated with an 808 nm laser (1.0 W·cm -2 ), it could exhibit POD-like performance and catalyze H2O2 to generate ·OH. These results indicate that the AuCu@Pd metal aerogel has good potential for CDT treatment.

[0094] (11) Establishment of MRSA model mice and antibacterial treatment

[0095] All animal experiments in this study were carried out in accordance with the guidelines of the Animal Care and Use Committee of Xiangnan University. A wound with a diameter of 8 mm was created on the back of anesthetized mice, and 50 μL of MRSA suspension (1×10 8CFU / mL). Taking the AuCu@Pd + H2O2 + NIR group as an example, 200 μL of AuCu@Pd metal aerogel (final concentration 200 μg / mL), 200 μL of H2O2 (final concentration 200 μM), and 600 μL of PBS were mixed, with a final volume of 1 mL. After 1 day, 100 μL of the therapeutic agent was dropped onto the infected wound in each group, and irradiated with an 808 nm laser (1.0 W·cm -2 ) or treated in the dark. Representative thermal images of the mice were recorded using a thermal imager. The body weight and wound diameter of the mice in each group were recorded every two days. The wound healing effect was as Figure 13 shown in A. After 10 days of treatment, the wound area of the AuCu@Pd + H2O2 + NIR group was significantly smaller than that of the other groups, indicating that the combination therapy could effectively prevent wound infection and promote wound healing ( Figure 13 B - C).

[0096] On the 10th day after treatment, the mice were euthanized, and their skin tissues were taken for hematoxylin and eosin (H&E) and Masson staining. The blood of the mice was collected for routine biochemical examinations of blood and serum. To study the biosafety of the AuCu@Pd aerogel, important organs (heart, liver, spleen, lung, and kidney) were extracted from the mice. At the end of the treatment, the mice were sacrificed and whole wound and surrounding skin tissue samples were collected from each group of mice to study the bacterial colonies in the wound skin tissue of the mice. Subsequently, these samples were separately placed into 2 mL of sterile PBS and ultrasonicated. Next, the ultrasonicated PBS of each group was diluted 1000 - fold. Then, 100 μL of the suspension was evenly spread on an LB solid agar plate, and the agar plate was placed in a 37 °C bacterial incubator for 14 h. Finally, the degree of wound infection in the mice was judged by the number of colonies on the solid agar plate.

[0097] (12) In vivo thermal imaging of AuCu@Pd metal aerogel

[0098] The temperature changes of the NIR laser irradiation group were recorded using a thermal imager, Figure 14 showing the temperature changes of the wound during the treatment. When the AuCu@Pd metal aerogel was irradiated with the laser, the wound temperature rapidly increased, indicating that the AuCu@Pd metal aerogel still had excellent photothermal performance in vivo.

[0099] (13) Evaluation of AuCu@Pd metal aerogel in promoting wound repair

[0100] After the treatment ended, the wound tissues of the mice were collected for a plate count experiment. Figure 15The photograph in A shows that the number of bacterial colonies in the AuCu@Pd + H2O2 + NIR group is significantly lower than that in other groups, which explains why the wound in the combination treatment group heals the fastest. Histological changes in the wounds of mice after different treatments were studied using hematoxylin-eosin (H&E) and Masson staining to further evaluate wound healing. The H&E results showed that there were no signs of inflammation in the wounds of the AuCu@Pd + H2O2 + NIR group, and a more complete epidermis was formed ( Figure 15 B), and the collagen fibers grew denser than in other groups ( Figure 15 C). The AuCu@Pd aerogel combined with PTT and multi-enzyme-like catalytic treatment can achieve a good bactericidal effect, thus promoting wound healing.

[0101] (14) Biosafety evaluation of AuCu@Pd metal aerogel

[0102] During the anti-infection treatment process, the body weight changes of mice in each group were monitored, and they were not significant, indicating that the treatments in different groups had little effect on the growth of mice ( Figure 16 ). Even at a high dose of 400 μg / mL, no obvious hemolysis was found, indicating that the AuCu@Pd aerogel has excellent blood compatibility ( Figure 17 ).

[0103] After the treatment, the main organs (heart, liver, spleen, lung, kidney) of the mice were taken for H&E staining, and no obvious pathological damage and inflammatory reaction were found in the PBS group and the combination treatment group ( Figure 18 ). The blood of the mice was collected for routine and biochemical analysis, and the results showed that the routine and biochemical indexes of the PBS group and the AuCu@Pd metal aerogel + H2O2 + NIR group were normal, and there was no obvious difference between the two groups ( Figure 19 ). The above results prove that the AuCu@Pd metal aerogel has good biosafety in photothermal catalytic antibacterial treatment.

[0104] Although the embodiments of the present invention have been shown and described, those of ordinary skill in the art can understand that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A preparation method of a metal aerogel with both photothermal and multiple enzyme properties, characterized in that, The preparation method includes the following steps: S1: Add AuCl3 solution and CuCl2 to ethanol and mix to obtain a light yellow clear solution, then quickly add a reducing agent solution for reaction, and the color changes to black. S2: Under stirring conditions, add NaPbCl4 solution to the black solution in S1, stir the AuCu@Pd metal aerogel and then add a reducing agent solution for reaction. Then, let the reaction solution stand in a water bath, and AuCu@Pd metal aerogel is formed at the bottom. Wash it with water and ethanol 3 - 5 times respectively, and then convert it into an aerogel by freeze-drying technology.

2. The preparation method of a metal aerogel with both photothermal and multiple enzyme properties according to claim 1, characterized in that The reducing agent in S1 is NaBH4.

3. The preparation method of a metal aerogel with both photothermal and multiple enzyme properties according to claim 1, characterized in that In S2, the temperature for the coupling reaction of the reaction solution in the water bath is 40 - 50 °C, and the time is 0.5 - 1 h. The reducing agent in S2 is ascorbic acid.

4. Application of a metal aerogel with both photothermal and multiple enzyme properties, characterized in that, Application of the metal aerogel with both photothermal and multi-class enzyme properties prepared by the preparation method according to any one of claims 2 to 3 in anti-drug-resistant bacteria treatment reagents.

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