A composite material containing platinum nanozyme, its preparation method and application

Abstract

The present invention belongs to the technical field of nanomaterials, and particularly relates to a composite material containing platinum nanozyme, a preparation method thereof and an application. The composite material forms a hydrophobic layer with phase change melting on the surface of amorphous calcium carbonate nanoparticles through electrostatic interaction and coordination with fatty acids. On the surface of the hydrophobic layer, a photothermal effect layer with a photothermal effect is formed with a self-oxidizing polymer through self-oxidizing polymerization. After fixing platinum ions on the surface of the photothermal effect layer, in-situ reduction is carried out to obtain the composite material; the self-oxidizing polymer is dopamine. The composite material of the present invention can change the pH of the wound microenvironment to regulate the POD and CAT activities of the nanozyme, so as to meet the needs of different stages of infectious wound treatment.

Description

Technical Field

[0001] The present invention belongs to the technical field of nanomaterials, and particularly relates to a platinum-containing nanozyme composite material, a preparation method thereof, and an application thereof. Background Art

[0002] After skin injury, it is vulnerable to bacterial infection. Bacterial infection can lead to delayed wound healing, exacerbate the inflammatory response, and may cause systemic infection, sepsis, organ failure, and even death. Therefore, bacterial infection is one of the most severe challenges during the wound healing process. The toxins secreted by bacteria not only cause the degradation of wound collagen, hinder wound repair, and further lead to a persistent inflammatory response in the wound. The acidic microenvironment formed by the persistent inflammation will stimulate inflammatory cells to secrete inflammatory cytokines and chemokines, further enhance the inflammatory response, induce the further recruitment of inflammatory cells such as neutrophils, and delay the apoptosis of neutrophils and the like, thus exacerbating the inflammatory response and slowing down the resolution of inflammation. At the same time, vascular damage at the infected wound site will cause local tissue hypoxia, severely hinder the process of wound tissue remodeling, and lead to delayed wound healing. Therefore, sterilization, anti-inflammation, regulating the inflammatory acidic microenvironment, and relieving tissue hypoxia to promote wound repair are the keys to treating infectious wounds.

[0003] In recent years, nanozymes have shown unique advantages in treating bacterial-infected wounds. As a class of nanomaterials with mimetic enzyme activity, compared with natural enzymes, nanozymes have multiple enzyme activities such as peroxidase (POD), superoxide dismutase (SOD), and catalase (CAT) activities, and have characteristics such as high catalytic activity and stable performance; moreover, the activities of some of its enzymes, such as POD and CAT, are related to the pH of the environment. In an acidic environment, it mainly exhibits POD activity, which can catalyze H2O2 to produce -OH and is commonly used in antibacterial, anti-tumor and other research; while in a neutral environment, it mainly exhibits CAT activity, which can catalyze H2O2 to produce O2, produce oxygen while scavenging ROS, can relieve tissue oxidative stress and hypoxia, and is commonly used in the research of inflammatory diseases, acute kidney injury, promoting wound healing, etc. Utilizing the multiple enzyme activities of nanozymes to simultaneously exert bactericidal, anti-inflammatory, and relieve tissue hypoxia is a very efficient means for the treatment of bacterial-infected wounds. However, the pH of the wound microenvironment during the treatment process of existing treatment materials cannot be quickly changed from weakly acidic to neutral after the bactericidal effect of the nanozyme is completed, thus affecting the subsequent exertion of the anti-inflammatory and hypoxia-relieving effects brought about by the conversion of enzyme activity. Summary of the Invention

[0004] To solve the above problems, the present invention provides a platinum-containing nanozyme composite material, a preparation method thereof, and an application thereof.

[0005] A composite material containing platinum nanozyme. The composite material forms a hydrophobic layer with phase change melting on the surface of amorphous calcium carbonate nanoparticles through electrostatic interaction and coordination with fatty acids. On the surface of the hydrophobic layer, a photothermal effect layer with photothermal effect is formed through self-oxidative polymerization with a self-oxidative polymer. After fixing platinum ions on the surface of the photothermal effect layer, in-situ reduction is carried out to obtain the composite material;

[0006] The self-oxidative polymer is dopamine.

[0007] Preferably, the fatty acid is at least one of long-chain fatty acids, medium-chain fatty acids and short-chain fatty acids.

[0008] Preferably, the long-chain fatty acid is palmitic acid or oleic acid.

[0009] Preferably, the medium-chain fatty acid is lauric acid.

[0010] The preparation method of the composite material includes the following steps:

[0011] The amorphous calcium carbonate nanoparticles react with the fatty acid, so that the electrostatic interaction and coordination between the fatty acid and the calcium ions on the surface of the amorphous calcium carbonate nanoparticles form the hydrophobic layer to obtain a first intermediate product;

[0012] The first intermediate product reacts with the self-oxidative polymer, and the self-oxidative polymerization of the self-oxidative polymer forms the photothermal effect layer to obtain a second intermediate product;

[0013] After fixing platinum ions on the surface of the second intermediate product, the composite material is obtained by in-situ reduction method.

[0014] Preferably, the mass ratio of the amorphous calcium carbonate nanoparticles to the fatty acid is 1:1 to 20;

[0015] The mass ratio of the first intermediate product to the self-oxidative polymer is 1:2 to 15;

[0016] The mass ratio of the second intermediate product to the platinum ions is 1:1 to 5.

[0017] The application of the platinum nanozyme material in the preparation of drugs for treating wound infections.

[0018] Preferably, the infection refers to bacterial infection.

[0019] Preferably, the drug promotes wound healing.

[0020] The application of the platinum nanozyme material in the preparation of materials for combined treatment of wound infections by photothermal and the wound infection drugs.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0022] The present invention designs a composite material containing platinum nanozyme (ACC@LPDAPtNPs), which can change the pH of the wound microenvironment, regulate the POD and CAT activities of platinum nanozyme, and meet the needs of different stages of infectious wound treatment. The dissolution of ACC caused by photothermal action of ACC@LPDAPtNPs can change the pH of the environment, and then regulate the POD and CAT activities of platinum nanozyme, showing effects such as sterilization, anti-inflammatory and alleviating tissue hypoxia. After being applied to skin bacterial infection wounds, it can effectively promote the healing of infected wounds. Based on the regulation of enzyme activity conversion, ACC@LPDAPtNPs show excellent bactericidal, anti-inflammatory and wound healing promotion effects in a mouse bacterial infection wound model and have good biocompatibility.

[0023] ACC in ACC@LPDAPt NPs can regulate the pH of the wound, thereby improving the treatment effect on bacterial infection wounds. In vitro experiments show that ACC@LPDAPtNPs have good POD, SOD and CAT activities, can effectively increase the pH of the system, and regulate the POD and CAT activities of ACC@LPDAPtNPs by changing the pH of the system. In vivo experiments confirm that ACC@LPDAPtNPs have the effects of sterilization, anti-inflammatory and alleviating tissue hypoxia, and then promoting the healing of skin bacterial infection wounds. The present invention promotes the healing of infectious wounds by actively regulating the conversion of multiple enzyme activities of PtNPs, providing a new strategy for the application of nanozymes with multiple enzyme activities in the treatment of bacterial infection wounds. Description of the Drawings

[0024] Figure 1 For the characterization of ACC@LPDAPtNPs, (a-d) TEM images of ACC NPs, ACC@LANPs, ACC@LPDANPs and ACC@LPDAPtNPs, (e) hydrodynamic diameters of ACC NPs, ACC@LA NPs, ACC@LPDANPs and ACC@LPDAPtNPs, (f) Zeta potentials of ACC NPs, ACC@LANPs, ACC@LPDANPs and ACC@LPDAPtNPs, (g) XPS spectrum of ACC@LPDAPtNPs, (h) platinum 4f XPS spectrum of ACC@LPDAPtNPs, (i) XRD spectra of ACC NPs, ACC@LANPs, ACC@LPDANPs and ACC@LPDAPtNPs and Pt JCPDS PDF#65-2868. (j) DSC curves of LA and ACC@LPDAPtNP.

[0025] Figure 2For the photothermal properties of ACC@LPDAPtNPs, infrared thermal images (a) and temperature change curves (b) of ACC@LPDAPtNPs at different concentrations (50, 100, and 200 μg / mL) after being irradiated with 808 nm near-infrared light at 1.5 W / cm 2 for 10 minutes in vitro, infrared thermal images (c) and temperature change curves (d) of 100 μg / mL ACC@LPDAPtNPs after being irradiated with different powers (0, 1.0, 1.5, and 2.0 W / cm 2 ) for 10 minutes in vitro, n = 3, mean ± SD.

[0026] Figure 3 For the effect of ACC@LPDAPtNPs on the solution pH value, the ability of ACC NPs (a), ACC@LA NPs (b), and ACC@LPDAPtNPs (c) to adjust the pH value in 1 mL of 0.001 mol / L pH 5.5 PBS at 25 °C, and the ability of ACC NPs (d), ACC@LA NPs (e), and ACC@LPDAPtNPs (f) to adjust the pH value in 1 mL of 0.001 mol / L pH 5.5 PBS when heated to 45 °C by irradiation with 808 nm near-infrared light at 1.5 W / cm 2 in vitro, n = 3, mean ± SD.

[0027] Figure 4 For the initial and final pH value measurement results, (a) the initial and final pH value measurement results in the experiment, the effect of the pH change in the photothermal-triggered ACC dissolution solution system on the pod-like activity of ACC@LPDAPtNPs, (b) the initial and final pH value measurement results in the experiment, the effect of the pH change in the photothermal-triggered ACC dissolution solution system on the cat-like activity of ACC@LPDAPtNPs, n = 3, mean ± SD.

[0028] Figure 5 For the study on the enzyme-mimicking activity of ACC@LPDAPtNPs, (a) the peroxidase-like and catalase-like activity mechanisms of ACC@LPDAPtNPs, (b) the visible absorption spectra and photos of ACC@LPDAPtNPs before and after reacting with TMB and H2O2, (c) the visible absorption spectra of the mixture at different pH values, (d) the change in the visible absorption rate of ACC@LPDAPtNPs and PDAPtNPs at a wavelength of 652 nm in PBS with a pH value of 5.5 over time before and after using near-infrared light (at the 30th minute, irradiated with 1.5 W / cm 2Irradiate the mixed solution with 808-nm near-infrared light for 5 minutes, and use TMB as a chromogenic substrate to detect -OH. (e) O₂ generated after ACC@LPDAPtNPs react with H₂O₂ in PBS at different pH values (5.5, 6.5, and 7.4) for 10 minutes. (f) Use [Ru(dpp)₃]Cl₂ as a fluorescence probe to detect the generation of oxygen, and detect the change in fluorescence intensity of ACC@LPDAPtNPs and PDAPtNPs in PBS at pH 5.5 before and after using near-infrared (at the 30th minute, with 1.5 W / cm 2 The change in fluorescence intensity of the 808-nm near-infrared light irradiated mixed solution for 5 minutes), n = 3, mean ± SD.

[0029] Figure 6 shows the antibacterial rate of ACC@LPDAPtNPs at different times. (a) Photograph of the agar plate and (c) After ACC@LPDAPt NPs are incubated with Staphylococcus aureus in PBS at pH 5.5 for different times, at 1.5 W / cm 2 The antibacterial rate after heating to 45 °C under 808-nm near-infrared light irradiation. (b) Photograph of the agar plate and (d) After ACC@LPDAPtNPs are incubated with Escherichia coli in PBS at pH 5.5 for different times, at 1.5 W / cm 2 The antibacterial rate after heating to 45 °C under 808-nm near-infrared light irradiation, n = 3, mean ± SD.

[0030] Figure 7 shows the in vitro bactericidal effect of ACC@LPDAPtNPs. Colony pictures of Staphylococcus aureus (a) and Escherichia coli (c) on agar plates after different treatments, corresponding statistical data of the number of CFU colonies per milliliter of Staphylococcus aureus (b) and Escherichia coli (d), representative fluorescence staining images of Staphylococcus aureus (e) and Escherichia coli (f) after different treatments (scale bar: 10 μm), live bacteria are labeled green by N01, dead bacteria are labeled red by PI, scanning electron microscope images of Staphylococcus aureus (g) and Escherichia coli (h) after different treatments (scale bar: 1 μm), n = 3, mean ± SD; ***P < 0.001.

[0031] Figure 8 shows the in vitro biosafety evaluation of ACC@LPDANPs, PDAPtNPs, and ACC@LPDAPtNPs. The survival of HUVECs (a) and L929s (b) after incubation with different concentrations of ACC@LPDANPs, PDAPtNPs, and ACC@LPDAPtNPs for 24 h, n = 3, mean ± SD.

[0032] Figure 9For the regulation of cell culture medium pH by ACC@LPDANPs, PDAPtNPs and ACC@LPDAPtNPs, photos of different culture media and corresponding pH values after culturing HUVEC (a, b) and L929 (c, d) cells. Group 1 was the untreated culture medium, Group 2 was the culture medium treated with PBS for 24 h, Group 3 was the culture medium treated with ACC@LPDANPs for 24 h, Group 4 was the culture medium treated with PDAPtNPs for 24 h, Group 5 was the culture medium treated with ACC@LPDAPtNPs for 24 h. All the above culture media were added with fetal bovine serum (10%, v / v), n = 3, mean ± SD.

[0033] Figure 10 For the ability of ACC@LPDAPtNPs to promote cell migration and angiogenesis, (a) Photos of HUVECs and (b) L929s after being treated with PBS, ACC@LPDANPs, PDAPt NPs and ACC@LPDAPt NPs for 0, 12 and 24 hours; (c) Images of tubules formed by HUVECs after being treated with PBS, ACC@LPDANPs, PDAPt NPs and ACC@LPDAPtNPs for 6 hours; (d) Migration rates of HUVECs and (e) L929s after different treatments, (f) Detection of the total length of the capillary network formation, n = 3, mean ± SD; ***p < 0.001.

[0034] Figure 11 For the hemolysis analysis of ACC@LPDAPtNPs (using Triton-X solution as the positive control and ultrapure water as the negative control), n = 3, mean ± SD; ***p < 0.001.

[0035] Figure 12 For the role of ACC@LPDAPtNPs in promoting the healing of skin wound-infected mice, (a) Procedures for modeling and treating infected wounds, (b) Representative real-time thermal images after irradiation with near-infrared light with a wavelength of 808 nm and a power of 1.5 W / cm 2 for 2 minutes, (c) Representative photos of skin wound tissues in different experimental groups on days 0, 3, 7, 10 and 14, (d) Temperature change curves of the wounds in the treatment group with NIR during the treatment process, (e) Quantitative evaluation of the infected wound area, (f) Changes in the skin wound area over time in different experimental groups, (g) Images of Staphylococcus aureus colonies on agar plates on days 1, 3, 7 and 14 after different treatments, (h) Quantitative results of bacterial counts in the wounds of mice in each group on the first day of modeling and (i) the 7th day, n = 3, mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001.

[0036] Figure 13H&E staining and Masson's trichrome staining of mouse wound tissues, (a) H&E staining of newly regenerated skin in each group on days 3, 7, and 14, and (b) Masson staining, scale bar = 100 μm.

[0037] Figure 14 Immunohistochemical detection of mouse wound sites, (a) Immunohistochemical photographs and relative intensities of TNF-α (b), IL-1β (c), IL-10 (d), and TGF-β1 (e) in wound tissues of the control group, ACC@LPDA group, PDAPt group, and ACC@LPDAPtNPs treatment group on day 7, n = 3, mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001.

[0038] Figure 15 Immunofluorescence detection of mouse wound sites, (a) Double immunofluorescence staining photographs and relative intensities of CD31 (red, b) and α-SMA (green, c) in wound tissues of the control group, ACC@LPDA group, PDAPt group, and ACC@LPDAPtNPs treatment group on day 7, Immunofluorescence detection of mouse wound sites, (d) Immunofluorescence photographs and (f) relative intensities of HIF-1α (red) in wound tissues of the control group, ACC@LPDA group, PDAPt group, and ACC@LPDAPtNPs treatment group on day 7, (e) Immunofluorescence photographs and (g) relative intensities of Col-I (red) in wound tissues of the control group, ACC@LPDA, PDAPt, and ACC@LPDAPtNPs treatment groups on day 14, n = 3, mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001.

[0039] Figure 16 H&E staining of the heart, liver, spleen, lungs, and kidneys on day 14 after treatment with ACC@LPDAPtNPs, scale bar = 100 μm.

[0040] Figure 17 Weight changes after different treatments, n = 9, mean ± SD. Detailed Description of the Invention

[0041] The following is a detailed description of the specific embodiments of the present invention. However, it should be understood that the protection scope of the present invention is not limited by the specific embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts belong to the protection scope of the present invention. The experimental methods described in the embodiments of the present invention are conventional methods unless otherwise specified.

[0042] The inventive concept of the present invention is as follows: Fatty acids form a hydrophobic layer wrapped by fatty acids on the surface of amorphous calcium carbonate nanoparticles (ACC NPs). On the surface of the hydrophobic layer, a photothermal effect layer with a photothermal effect is formed through autoxidative polymerization with an autoxidative polymer. After fixing platinum ions and then in-situ reduction, a composite material containing platinum nanozymes is obtained. The present invention triggers the dissolution of ACC NPs in water by the phase change melting of fatty acids caused by the photothermal effect of the photothermal effect layer, thereby changing the pH of the environment, and further regulating the POD and CAT activities of platinum nanozymes, demonstrating effects such as sterilization, anti-inflammatory, and alleviating tissue hypoxia. The principle of the present invention is specifically illustrated below using lauric acid and dopamine as examples:

[0043] The preparation process of ACC@LPDAPtNPs is as follows: First, ACC NPs were prepared by the gas diffusion method. Secondly, to improve the stability of ACC NPs in water, the carboxyl group of LA (lauric acid) was used to form a hydrophobic layer wrapped by LA on the surface of ACC through the electrostatic interaction and coordination interaction between Ca on the surface of ACC 2+ to prevent its dissolution in water during the subsequent preparation process. Therefore, an ethanol solution of ACC NPs was added to an ethanol solution of LA to prepare ACCNPs with a surface wrapped by LA, that is, ACC@LANPs. Among them, based on the characteristic that LA undergoes phase change melting at 45 °C, the dissolution of ACCNPs in water can be triggered by photothermal action. For this purpose, ACC@LANPs and hydrochloric acid dopamine were added to Tris-HCl buffer solution, and a PDA (polydopamine) layer was formed on the surface of ACC@LANPs through the autoxidative polymerization of dopamine to obtain ACC@LPDANPs, so as to trigger the dissolution of ACCNPs in water by the phase change melting of LA caused by the photothermal effect of PDA. Next, H2PtCl6 was added, and Pt was fixed by the coordination action of amino groups and phenolic hydroxyl groups on the surface of PDA 4+ , and then NaBH4 was added to generate PtNPs on the surface of ACC@LPDANPs by in-situ reduction method to obtain ACC@LPDAPtNPs (a polydopamine-wrapped amorphous calcium carbonate material loaded with platinum nanozymes).

[0044] Examples

[0045] Preparation of ACC@LPDAPtNPs

[0046] Synthesis of ACC NPs: Weigh exactly 200 mg of CaCl2 and dissolve it in 400 μL of deionized water to obtain a calcium chloride solution. While stirring, slowly add the calcium chloride solution dropwise to 100 mL of absolute ethanol to obtain Solution A. Transfer Solution A to a round-bottom flask, cover it with a sealing film, and pierce several small holes on the surface of the sealing film. Then, place the round-bottom flask together with two glass bottles containing ammonium carbonate in a desiccator and carry out a gas diffusion reaction at 30 °C. After 24 hours, obtain Solution B. Centrifuge the Solution B in the round-bottom flask at 8000 rpm for 10 minutes to collect the white precipitate, obtaining ACC NPs. Wash it three times with absolute ethanol and disperse it in absolute ethanol for storage for later use to obtain an ACC NPs ethanol solution.

[0047] While stirring at 500 rpm, slowly add 1 mL of an ACC NPs ethanol solution with a concentration of 1 mg / mL dropwise to 10 mL of an LA ethanol solution with a concentration of 1 mg / mL, and continue stirring for 5 hours. Then, centrifuge at 11000 rpm for 10 minutes to collect the white precipitate, obtaining ACC@LANPs. Wash it three times with absolute ethanol and dry it under vacuum at 30 °C for later use.

[0048] While stirring at 700 rpm, add 5 mg of ACC@LANPs and 25 mg of dopamine hydrochloride to 10 mL of a pH 8.5 Tris-HCl buffer solution (10 mmol / L) respectively, and stir in the dark for 4 hours until the reaction solution turns black to generate ACC@LPDANPs. Then, while under 400 W ultrasonic irradiation, slowly add 10 mg of H2PtCl6 liquid dropwise to the above reaction solution and continue ultrasonic irradiation for 10 min. Subsequently, add 20 mg of sodium borohydride powder and continue ultrasonic irradiation for 20 min. Finally, centrifuge the reaction solution at 11000 rpm for 10 min to collect the black precipitate, obtaining ACC@LPDAPtNPs. Wash it 3 times with deionized water and dry it under vacuum at 30 °C for later use.

[0049] Material Characterization and Performance Testing

[0050] 1. Characterization

[0051] The particle size, polydispersity index (PDI), and zeta potential of ACCNPs, ACC@LANPs, ACC@LPDANPs, and ACC@LPDAPtNPs were measured by dynamic light scattering (DLS) using a Zetasizer Nano ZS90 (Malvern Instruments, UK). The morphology of ACC NPs, ACC@LANPs, ACC@LPDANPs, and ACC@LPDAPtNPs was characterized by transmission electron microscopy (TEM, Thermo Fisher, USA). The crystal morphology of ACC NPs, ACC@LANPs, ACC@LPDANPs, and ACC@LPDAPtNPs was determined by X-ray diffraction (XRD, D8 ADVANCE, Bruker, Germany). The endothermic curves of LA and ACC@LPDAPtNPs were measured using a differential scanning calorimeter (DSC, DSC250, TA Instruments, USA). The elemental composition of ACC@LPDAPtNPs was analyzed using an X-ray photoelectron spectrometer (XPS, Thermo Scientific K-Alpha+, Thermo Fisher, USA). The concentrations of Ca and Pt in ACC NPs, ACC@LANPs, ACC@LPDANPs, PDAPtNPs, and ACC@LPDAPtNPs were detected by inductively coupled plasma mass spectrometry (ICP-MS).

[0052] 2. Photothermal property study

[0053] At room temperature, 1 mL of ACC@LPDAPtNP solutions with different concentrations (0, 50, 100, and 200 μg / mL) were added into 24-well plates respectively, and irradiated with 808 nm near-infrared light from a near-infrared light source (Beijing Honglan Optoelectronic Technology Co., Ltd.) at 1.5 W / cm 2 The temperature changes of the solutions before irradiation and within 10 min after irradiation were measured using an infrared thermal imager (Uni-trend, China), once every 2 min.

[0054] 3. pH regulation ability study

[0055] ACC NPs, ACC@LANPs, or ACC@LPDAPtNP solutions with different concentrations (0.00, 0.05, 0.10, and 0.20 mg / mL) were prepared using 0.001 mol / L PBS at pH 5.5 as the solvent. At room temperature, 1 mL of the above solutions were added into test tubes respectively, at 1.5 W / cm 2Under the conditions of 808 nm near-infrared irradiation or non-irradiation, the pH values of the solutions in each test tube were measured with a pH meter (PB-10, Sartorius instruments, Germany) within 10 min, once every 1 min, to investigate the ability of ACC@LPDAPtNPs, etc. to regulate the pH of the solution system under photothermal or non-photothermal conditions.

[0056] 4. Determination of POD-like activity

[0057] The peroxidase (POD)-like activity of ACC@LPDAPtNPs was determined by the 3,3',5,5'-tetramethylbenzidine (TMB) method. First, 100 μL of TMB (10 mg / mL), 100 μL of ACC@LPDAPtNPs (1 mg / mL) solution were added to 700 μL of 0.001 mol / L PBS solution at pH 5.5, pH 6.5 and pH 7.4, and then 100 μL of H2O2 (100 mmol / L) was added and mixed well. Then, the solution was irradiated with 808 nm near-infrared light at 1.5 W / cm 2 to raise the temperature to 45 °C or not irradiated, and the absorbance of the solution was measured at a wavelength of 652 nm. Secondly, 100 μL of TMB (10 mg / mL), 700 μL of 0.001 mol / L PBS at pH 5.5, 100 μL of H2O2 (100 mmol / L) and 100 μL of ACC@LPDAPtNPs (1 mg / mL), PDAPtNPs (1 mg / mL) or PBS solution were mixed well, and the change in absorbance of the solution within 30 min was measured at 652 nm, once every 5 min. Then, the solutions in each group were irradiated with 808 nm near-infrared light at 1.5 W / cm 2 to raise the temperature of each group of solutions to 45 °C. The change in absorbance of the solution within 30 min was measured at 652 nm, once every 5 min, and the final pH value of each group of solutions was measured.

[0058] 5. Determination of CAT-like activity

[0059] The catalase (CAT)-like activity of ACC@LPDAPtNPs was investigated by the amount of oxygen produced by the reaction with H2O2. First, 100 μL of H2O2 (100 mmol / L) and 100 μL of ACC@LPDAPtNPs (1 mg / mL) solution were successively added to 800 μL of 0.001 mol / L PBS at pH 5.5, pH 6.5 and pH 7.4 and mixed well. At 1.5 W / cm 2Under the condition of heating to 45 °C by 808 nm near-infrared irradiation or without irradiation, the change in the amount of oxygen generated within 10 minutes was measured using a dissolved oxygen meter (JPB-607A, Shanghai Leichi Biotechnology Co., Ltd., China), and the measurement was taken once every 1 minute. Secondly, tris(4,7-diphenyl-1,10-phenanthroline) ruthenium(II) dichloride ([Ru(dpp)3]Cl2) was used as a fluorescent probe to detect the amount of oxygen produced, and further investigate the effect of the change in the pH of the solution system caused by the photothermal-triggered dissolution of ACC on the CAT-like activity of ACC@LPDAPtNPs. 800 μL of 0.001 mol / L PBS at pH 5.5, 0.2 mL of H2O2 (100 mmol / L), and 1 mL of [Ru(dpp)3]Cl2 (10 mg / L) solution were added to a cuvette, and then 0.2 mL of ACC@LPDAPtNPs (1 mg / mL), PDAPtNPs (1 mg / mL), or PBS solution was added and mixed well. After standing at room temperature for 15 minutes, the change in the fluorescence intensity of each group of solutions within 30 minutes was recorded using a fluorescence spectrophotometer (λex = 488 nm, λem = 620 nm), and the measurement was taken once every 5 minutes. Then, each group of solutions was heated to 45 °C using 808 nm near-infrared irradiation with a power of 1.5 W / cm 2 The fluorescence intensity change of the solution within 30 minutes was recorded, and the measurement was taken once every 5 minutes; and the final pH value of each group of solutions was measured.

[0060] 6. Evaluation of in vitro bactericidal activity

[0061] The in vitro bactericidal activity of ACC@LPDAPtNPs was evaluated using Escherichia coli (E. coli, ATCC12228) and Staphylococcus aureus (S. aureus, ATCC8739). The revived strains were inoculated in Luria Bertani (LB) medium and incubated overnight at 37 °C; the absorbance of the bacterial solution at 600 nm was measured to determine the bacterial concentration. First, ACC@LPDAPt NPs and H2O2 were mixed with Staphylococcus aureus or Escherichia coli in PBS at pH 5.5, and samples were taken and placed in a 96-well plate after culturing at 37 °C for 30 min, 60 min, 90 min, and 120 min, respectively. Using 1.5 W / cm 2Irradiate with 808 nm near-infrared light to raise the temperature to 45 °C or not, then take 20 μL of the bacterial suspension and evenly spread it on the LB agar plate, culture overnight at 37 °C, count the number of colonies formed to evaluate the bactericidal effect, and determine the timing of near-infrared light irradiation after drug administration accordingly. Secondly, investigate the bactericidal effects of ACC@LPDANPs, PDAPt, and ACC@LPDAPtNPs. The grouping is as follows: (1) bacteria + H2O2; (2) bacteria + H2O2 + ACC@LPDANPs; (3) bacteria + H2O2 + PDAPt NPs; (4) bacteria + H2O2 + ACC@LPDAPtNPs; (5) bacteria + H2O2 + near-infrared; (6) bacteria + H2O2 + ACC@LPDANPs + near-infrared; (7) bacteria + H2O2 + PDAPt NPs + near-infrared; (8) bacteria + H2O2 + ACC@LPDAPtNPs + near-infrared. Among them, the concentration of H2O2 is 1 mmol / L, and the bacterial concentration is 1×10 6 CFU / mL. The components of each group are mixed in PBS at pH 5.5, and after culturing at 37 °C for 30 min, groups (6)-(10) are irradiated with 808 nm near-infrared light at 1.5 W / cm 2 to raise the temperature to 45 °C. Then, take 20 μL of the bacterial suspension from each group and evenly spread it on the LB agar plate, culture overnight at 37 °C, and count the number of colony formations; take another part of the bacterial suspension and stain it with N01 / PI, and observe the viability of the bacteria under an inverted fluorescence microscope (AxioVert.A1, Zeiss, Germany); observe the bacterial morphology of the remaining bacterial suspension with a scanning electron microscope (SEM, HITACHI, Japan).

[0062] 7. Cytotoxicity test

[0063] Seed HUVECs or L929s into 96-well plates at a density of 5×10 3 cells per well. After culturing for 24 h, discard the culture medium, and then add serum-free medium containing different concentrations of ACC@LPDANPs, PDAPt NPs, or ACC@LPDAPtNPs. After culturing at 37 °C for 30 min, irradiate with 808 nm near-infrared light at 1.5 W / cm 2 to raise the temperature to 45 °C or not, and continue to culture for 24 h. Then, replace the original medium with 200 μL of fresh medium containing 0.5 mg / mL MTT and continue to culture at 37 °C for 4 h. Then add dimethyl sulfoxide to replace the medium containing MTT to lyse the cells, and measure the absorbance at 450 nm to estimate the cell viability of each group.

[0064] 8. Cell migration assay

[0065] Seed HUVECs or L929s (2 mL, 2×104 Cells / mL) were seeded into 6-well plates. After culturing until the cells were completely attached, a straight line was scratched with a sterile pipette tip. Then the cells were rinsed with PBS to remove cell debris, and serum-free medium containing ACC@LPDANPs, PDAPtNPs, or ACC@LPDAPtNPs, or PBS was added for culture. After culturing at 37 °C for 30 min, the cells were irradiated with 808 nm near-infrared light at 1.5 W / cm 2 to raise the temperature to 45 °C or not irradiated, and the culture was continued for 24 h. The closure of the scratch was observed with an optical microscope at 0 h, 12 h, and 24 h of culture, and photographs were taken. ImageJ software was used to calculate the migration rate. Migration rate (%) = (A0 - At) / A0, where A0 is the initial scratch area and At represents the scratch area at different time points. In addition, after culturing for 24 h, the pH of the medium in different administration groups was measured with a pH meter.

[0066] 9. Angiogenesis ability test

[0067] A 96-well plate was placed in an ice bath. After adding 10 μL of Matrigel to each well, it was incubated at 37 °C for 30 min. Then, HUVEC cells were seeded at a density of 2×104 cells / mL, and ACC@LPDANPs, PDAPtNPs, or ACC@LPDAPtNPs, or PBS were added. After culturing at 37 °C for 30 min, the cells were irradiated with 808 nm near-infrared light at 1.5 W / cm 2 to raise the temperature to 45 °C or not irradiated, and then the cells were cultured for another 12 h. The angiogenesis was observed under an optical microscope and photographed, and ImageJ software was used to quantify the total length of the blood vessels.

[0068] 10. Hemolysis test

[0069] Fresh whole blood of rats was taken, and red blood cells were collected by centrifugation at 1500 rpm at 4 °C. After washing with physiological saline, a 5% red blood cell suspension was prepared. 1 mL of the red blood cell suspension was mixed with 1 mL of ACC@LPDAPt NPs solution at different concentrations. After incubation at 37 °C for 30 min, the cells were irradiated with 808 nm near-infrared light at 1.5 W / cm 2 to raise the temperature to 45 °C. Then, after incubating for another 2 h, the supernatant was taken by centrifugation and the absorbance was measured at 540 nm. Physiological saline and 1% Triton solution were used as negative and positive controls, respectively. The hemolysis rate (HR) was calculated as follows: Hemolysis rate (%) = (Asample - Asaline) / (ATriton - Asaline) × 100%, where Asample, ATriton, and Asaline are the absorbances of the experimental sample, Triton, and physiological saline supernatants, respectively.

[0070] 11. Role in promoting the healing of skin wounds infected in mice

[0071] Take healthy male ICR mice, depilate and disinfect their backs. Under isoflurane anesthesia, establish a circular skin wound with a diameter of about 1 cm on the back of the mice, and then inoculate 100 μL of Staphylococcus aureus at 2×106 CFU / mL to establish a mouse infected wound model. On the second day, the mice are randomly divided into 8 groups: (1) control group; (2) ACC@LPDA NPs; (3) PDAPtNPs; (4) ACC@LPDAPtNPs; (5) control group + NIR; (6) ACC@LPDANPs + NIR; (7) PDAPtNPs + NIR; (8) ACC@LPDAPtNPs + NIR. Among them, 30 minutes after topical administration to the wound, groups (5) - (8) are irradiated with 808 nm near-infrared light at 1.5 W / cm 2 to raise the temperature of the mouse wound to 45 °C. On the day of model establishment and on the 3rd, 7th, and 14th days of treatment, measure the size of the wound and take images; use a cotton swab moistened with normal saline to sample from the infected wound, and use the LB agar plate dilution method to evaluate the bacterial load. At the same time, record the weight changes and survival of each group of mice during the treatment period. On the 14th day, the mice are sacrificed under anesthesia, the skin tissue of the wound is taken, fixed with 4.0% paraformaldehyde, embedded in paraffin, the section thickness is 4 μm, and H&E staining or Masson's trichrome staining is used to observe the healing of the wound tissue. Immunohistochemistry is used to detect the expression of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin 10 (IL-10), and transforming growth factor-β1 (TGF-β1) in the wound tissue; immunofluorescence staining is used to detect the expression of platelet endothelial cell adhesion molecule (CD31), α-smooth muscle actin (α-SMA), hypoxia-inducible factor (HIF-1α), and type I collagen (Col-I) in the wound tissue; and the ImageJ software is used to analyze the images. In addition, after sacrificing the mice on the 14th day, the heart, liver, spleen, lungs, and kidneys of the mice in the ACC@LPDAPtNPs + NIR group are dissected, fixed with 4% paraformaldehyde solution, embedded in paraffin, the section thickness is 4 μm, and H&E staining is used to observe the histopathological changes of the main organs of the mice at the treatment dose of ACC@LPDAPtNPs.

[0072] Results

[0073] 1. Characterization

[0074] The morphologies of ACC NPs, ACC@LANPs, ACC@LPDANPs, and ACC@LPDAPtNPs are observed by transmission electron microscopy (TEM). As Figure 1 shown in a - d, the above-mentioned nanoparticles all present spherical shapes. The measurement results of dynamic light scattering (DLS) are shown in Figure 1 e andFigure 1 f. The hydrodynamic size of the ACC NPs was 98.25 ± 12.27 nm (PDI = 0.13), and the zeta potential was +30.85 ± 0.96 mV; after coating with the LA layer, the particle size of the ACC@LANPs increased to 161.03 ± 17.23 nm (PDI = 0.20), and the zeta potential decreased to +5.93 ± 0.19 mV; when the surface of the ACC@LANPs was further coated with the PDA layer and PtNPs were in-situ reduced, the particle size of the ACC@LPDAPtNPs increased to 350.50 ± 35.64 nm (PDI = 0.43), and the zeta potential further decreased to -24.55 ± 0.59 mV.

[0075] Since the enzyme activity of metal nanozymes is closely related to their valence states, the elemental composition and the valence state of elemental platinum of the ACC@LPDAPtNPs were determined by XPS. Figure 1 g shows that the ACC@LPDAPtNPs contain C, N, O, Ca, and Pt elements. As Figure 1 shown in h, the high-resolution spectra of platinum 4f at 78.48, 73.58, and 70.28 eV show three main peaks originating from platinum 4f5 / 2 and platinum 4f7 / 2; the peaks at 78.48 and 73.58 eV can be attributed to Pt 4+ and Pt 2+ , while the peak at 70.28 eV can be attributed to Pt 2+ and Pt 0 . According to the calculation results of the peak areas, Pt 4+ , Pt 2+ and Pt 0Accounting for 29.80%, 43.67% and 26.53% respectively, the presence of multivalent platinum ions lays the foundation for the multiple enzyme activities exhibited by ACC@LPDAPtNPs. In addition, X-ray diffraction (XRD) pattern analysis shows that both ACC NPs and ACC@LANPs exist in an amorphous form, indicating the successful synthesis of ACC NPs and that the method of improving their stability in water by forming a LA hydrophobic layer on their surface does not affect their amorphous form. Moreover, no diffraction peaks of crystalline calcium carbonate are detected in ACC@LPDANPs and ACC@LPDAPtNPs, which indicates that no crystal form transformation of ACC occurs during the preparation of ACC@LPDAPtNPs and it remains in an amorphous state, which also ensures its easy solubility in water. At the same time, three diffraction peaks can be observed in the XRD pattern of ACC@LPDAPtNPs. By comparing with the standard card of platinum, the typical peaks at 2θ = 39.74°, 47.04° and 67.72° are located on the (111), (200) and (220) planes of cubic platinum crystal (JCPDS PDF#65-2868) 52, 53 respectively. These above results indicate the successful synthesis of ACC@LPDAPtNPs( Figure 1 i). Next, the differential scanning calorimetry curve (DSC) shows that both ACC@LPDAPtNPs and LA have similar endothermic peaks at about 45 °C, which should be caused by the endothermic melting of LA. This provides the possibility for ACC@LPDAPtNPs to trigger the dissolution of ACC NPs in water by photothermal heating to initiate the phase change melting of LA and thus regulate the pH value of the microenvironment( Figure 1 j).

[0076] 2. Photothermal property determination

[0077] The photothermal performance of ACC@LPDAPtNPs is crucial for the exertion of its function. Irradiate the ACC@LPDAPtNPs solution with 808-nm near-infrared light and record its temperature change with an infrared thermal imager to investigate its photothermal conversion ability. First, the effect of the concentration of the ACC@LPDAPtNPs solution on its photothermal conversion ability was studied. As Figure 2 shown in Figs. 2a and 2b, with a power density of 1.5 W / cm 2Irradiate with 808 nm near-infrared light for 10 min. When the concentration of ACC@LPDAPtNPs is 50 μg / mL, the solution temperature rises from 23 °C to 36 °C; as the concentration of the ACC@LPDAPtNPs solution increases, when the concentration reaches 200 μg / mL, the solution temperature after irradiation rises to nearly 60 °C; under the same irradiation conditions, the temperature of the PBS solution hardly increases. The photothermal effect of ACC@LPDAPtNPs mainly stems from PDA in its composition, and the photothermal effect increases with the increase in concentration, which facilitates controlling the temperature of the photothermal effect by dose selection when applying ACC@LPDAPtNPs for treatment. Secondly, the effect of power density on the photothermal conversion ability of ACC@LPDAPtNPs was further studied. As shown in Figure 2 Figures 2c and 2d, irradiate the ACC@LPDAPtNPs solution with a concentration of 100 μg / mL with near-infrared light with a wavelength of 808 nm. When the power is 0.5 W / cm 2 , the solution temperature rises from 23 °C to about 37 °C, while when the power is 1.5 W / cm 2 , the solution temperature rises to about 45 °C. Continuing to increase the power to 2.0 W / cm 2 , the solution temperature rises to nearly 50 °C. This indicates that the photothermal effect of ACC@LPDAPt NPs increases with the increase in the irradiation power density. The above results show that ACC@LPDAPtNPs have good photothermal conversion ability. Given that low-temperature photothermal therapy at 45 °C is a recommended sterilization method, which can produce good sterilization effects and protect healthy tissues from damage caused by high temperatures. At the same time, at 45 °C, LA in ACC@LPDAPtNPs can undergo phase change melting to trigger the dissolution of ACC NPs in water, thereby regulating the pH of the microenvironment. Therefore, for subsequent experiments on the related enzyme-like activity and pH regulation of ACC@LPDAPtNPs, the selected irradiation conditions are to irradiate with 808 nm near-infrared light at 1.5 W / cm 2 to raise the temperature to 45 °C.

[0078] 3. Determination of pH regulation ability

[0079] First, the ability of ACC@LPDAPtNPs to regulate the pH value at room temperature was investigated. As shown in Figure 3As shown in a, the ability of ACC NPs, which is the pH regulating component in ACC@LPDAPtNPs, to regulate the pH value of the solution increases with the increase of mass. 0.05 mg can adjust the pH value of the PBS solution from 5.5 to 7.2, and 0.2 mg can adjust the pH value of the PBS solution to 7.7; while ACC@LANPs and ACC@LPDAPtNPs containing 0.2 mg ACC can only adjust the pH value of the pH 5.5 PBS solution to 6.8 and 6.1. This is because ACC is water-soluble and weakly alkaline, and can increase the pH value of the solution after dissolution; and after the LA layer is coated on its surface, the hydrophobicity of LA reduces the soluble amount of ACC, and its ability to regulate the pH value of the solution is reduced; ACC@LPDAPtNPs further coat the surface with PDA, which greatly reduces the soluble amount of ACC, so the pH value of the solution increases less ( Figure 3 b and 3c). Secondly, in order to verify that the photothermal effect of PDA induces the melting phase transition of LA in ACC@LPDAPtNPs to trigger the dissolution of ACC and play a role in regulating the pH value of the solution, the present invention measured the photothermal effects of ACC NPs, ACC@LANPs and ACC@LPDAPtNPs at 1.5W / cm 2 The ability to adjust the pH value of the solution under 808nm near-infrared light irradiation. Figure 3 As shown in a and 3d, there is almost no difference in the ability of ACC to adjust the pH value of the solution with or without near-infrared light irradiation; similarly, after near-infrared light irradiation, the ability of ACC@LANPs to adjust the pH value of the solution did not increase significantly, and the solution temperature did not change ( Figure 3 b and 3e); ACC@LPDAPtNPs showed good ability to increase the pH value of the solution when the solution was heated to 45°C under near-infrared light irradiation, and the pH value of the solution could be increased to 7.7 (0.2 mg Figure 3 f), which is close to the regulation ability of the same amount of ACC at room temperature. This improvement in pH regulation mainly comes from the fact that under near-infrared light irradiation, the photothermal effect of PDA triggers the melting phase transition of LA, triggering the dissolution of ACC, thereby increasing the pH value of the solution. The above results show that the regulation of the pH value of the solution by ACC@LPDAPtNPs can be triggered by photothermal effect, laying the foundation for accelerating wound healing by regulating the pH value of the wound microenvironment.

[0080] 4. Enzyme activity determination

[0081] Previous studies have shown that the POD-like activity and CAT-like activity of Pt nanozymes change with the pH of their microenvironment. In a weakly acidic environment, PtNPs mainly exhibit POD-like activity, which can catalyze H2O2 to generate ·OH, playing roles such as sterilization and anti-tumor; while in a neutral or weakly alkaline environment, PtNPs exhibit CAT-like activity, which can scavenge H2O2 and produce O2, playing roles such as promoting tissue healing. In the present invention, ACC@LPDAPtNPs are constructed to actively regulate the pH of the wound microenvironment, and then regulate the transformation of Pt nanozymes from sterilization to promoting healing effects, in order to meet the treatment needs of different stages of infected wounds. For this purpose, the present invention further studied the changes in the POD-like activity and CAT-like activity of ACC@LPDAPtNPs with the environmental pH. Based on the fact that POD enzyme catalyzes H2O2 to produce -OH, and -OH can oxidize colorless TMB into blue, which has the maximum absorption at 652 nm ( Figure 5 a and 5b), to measure the POD-like activity of ACC@LPDAPtNPs. First, the POD-like activity of ACC@LPDAPtNPs under near-infrared light irradiation or without irradiation was investigated at pH 5.5, pH 6.5, and pH 7.4. As Figure 5 shown in c, when the pH value of the reaction system was 5.5, its absorbance value was the highest, about 1.1; as the pH of the reaction system increased, the absorbance of the solution decreased, and the absorbance was about 0.1 at pH 7.4, indicating that its POD-like enzyme activity decreased with the increase of the reaction system pH. In addition, when near-infrared irradiation was superimposed to increase the temperature, the absorbance of the same reaction system increased, such as the absorbance increased to 1.3 at pH 5.5, probably because the increase in the reaction system temperature accelerated the enzymatic reaction rate and produced more -OH. And this effect is also more helpful to enhance the sterilization effect of ACC@LPDAPtNPs.

[0082] Secondly, the present invention investigated the change in the POD-like activity of ACC@LPDAPtNPs with the change of the reaction system pH. As Figure 5 shown in d, the absorbance of the reaction system first increased rapidly and then remained at about 1.2. At 30 min, near-infrared light was irradiated to raise the solution temperature to 45 °C, and the absorbance of the solution first increased and then continued to decrease. This may be because the increase in the reaction system temperature increased the enzymatic reaction rate of the POD-like enzyme of ACC@LPDAPtNPs, so the absorbance of the solution first increased. Thereafter, the increase in the reaction system temperature triggered the melting phase change of LA and triggered the dissolution of ACC, and the increase in the pH of the solution caused the POD activity of ACC@LPDAPtNPs to immediately decrease, and it could be observed that the absorbance of the solution continued to decrease. Since PDAPtNPs cannot regulate the pH value of the solution, therefore, the absorbance of its solution remained basically unchanged after a slight increase under near-infrared light irradiation. The measurement results of the initial and final pH values of the reaction are shown in Figure 4a. Next, an oxygen meter was used to measure the oxygen production of ACC@LPDAPt NPs in the presence of H2O2 to investigate their CAT-like activity. As Figure 5 shown in e, as the pH of the reaction system increased, the oxygen content in the solution increased, indicating that the CAT-like activity of ACC@LPDAPt NPs increased with the increase in the pH of the reaction system. In addition, when near-infrared light irradiation was superimposed to increase the temperature, the oxygen content in the solution under each pH condition was twice that without near-infrared irradiation. This may be the result of the photothermal effect increasing the temperature of the reaction system and the pH of the solution, greatly enhancing the CAT activity of ACC@LPDAPt NPs. Finally, [Ru(dpp)3]Cl2 was used as a fluorescence probe to detect the production of oxygen, further demonstrating the effect of changes in the pH value of the reaction system on the CAT-like activity of ACC@LPDAPt NPs. As Figure 5 shown in f, in a weakly acidic system, the CAT-like activity of ACC@LPDAPt NPs was weak, and the solution maintained a certain fluorescence intensity. When the temperature was increased to 45 °C by near-infrared light irradiation at 30 min, the fluorescence intensity of the solution continued to decrease. This is because the increase in the temperature of the reaction system triggered the dissolution of ACC, and the increase in the pH of the solution increased the CAT-like activity of ACC@LPDAPt NPs, generating a large amount of O2 that interacted with [Ru(dpp)3]Cl2, causing its fluorescence intensity to continue to decrease. However, since PDAPt NPs could not adjust the pH value of the solution, the fluorescence intensity of its solution did not change significantly before and after photothermal treatment. The measurement results of the initial and final pH values are shown in Figure 4 b.

[0083] The above results indicate that by utilizing the characteristics that the POD enzyme activity and CAT enzyme activity of Pt nanozymes change with the different pH of the microenvironment, triggering the dissolution of ACC by photothermal effect to change the pH of the environment, and regulating the POD-like activity and CAT-like activity of ACC@LPDAPt NPs, the design strategy of satisfying sterilization and enhancing wound oxygenation to promote the healing of infected wounds is feasible.

[0084] 5. In vitro bactericidal activity

[0085] Bacterial infection is an important reason for the failure of wounds to heal in a timely manner. Therefore, an ideal wound treatment drug should have good bactericidal properties. Considering common bacteria in wound infections such as Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, and Enterococcus faecalis, etc., in the present invention, Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli were used to investigate the bactericidal properties of ACC@LPDAPt NPs. Based on the design strategy of ACC@LPDAPt NPs, in order to fully exert its bactericidal efficiency, the present invention first determined the timing of near-infrared light irradiation after administration. As Figure 6 shown, after ACC@LPDAPt NPs were cultured with bacteria for 30 min, 1.5 W / cm2 When the temperature is raised to 45 °C by irradiating with 808 nm near-infrared light, the bactericidal rate of ACC@LPDAPtNPs against Staphylococcus aureus reaches 99.21%, and the bactericidal rate against Escherichia coli reaches 99.94%. However, as the culture time prolongs, the bactericidal rate decreases. After culturing for 120 min and then irradiating with near-infrared light again, the bactericidal rates of ACC@LPDAPtNPs against Staphylococcus aureus and Escherichia coli decrease to 96.49% and 95.67% respectively. The reason for this result is related to the bactericidal mechanism of Pt nanozyme. The ·OH generated by Pt nanozyme catalyzing H2O2 plays a bactericidal role, and when there is insufficient H2O2 at the action site, its bactericidal effect weakens. Therefore, when applying it for sterilization, the bactericidal effect is often enhanced by increasing the concentration of H2O2 at the action site or combining with photothermal and other means. In this invention, when irradiating with near-infrared light 30 min after administration, in addition to the pod enzyme activity of Pt nanozyme superimposing the photothermal effect, the photothermal temperature rise generates a high concentration of ·OH to enhance the pod enzyme activity of Pt nanozyme, thus enhancing the bactericidal effect. When administered for 120 min, the concentration of H2O2 in the system decreases. At this time, the enhancement of pod enzyme activity brought about by photothermal temperature rise cannot generate a high concentration of ·OH, so the bactericidal effect decreases. In addition, under the condition of no near-infrared light irradiation, the bactericidal effects of ACC@LPDAPtNPs against Staphylococcus aureus and Escherichia coli are both lower than those when near-infrared light irradiation is superimposed. This further illustrates the importance of the timing selection in combination and treatment. In subsequent animal experiments, near-infrared light irradiation is applied 30 min after administration to achieve the best treatment effect.

[0086] Secondly, under the condition of simulating the microenvironment of an infected wound (pH 5.5, 1 mmol / L H2O2), the bactericidal effects of ACC@LPDAPtNPs etc. were investigated. As Figure 7 shown, in the absence of near-infrared light, the bactericidal effects of the ACC@LPDANPs group against Staphylococcus aureus and Escherichia coli are weak, while both the PDAPt NPs group and the ACC@LPDAPtNPs group show stronger bactericidal effects. This is mainly because PDAPtNPs and ACC@LPDAPtNPs contain Pt nanozyme, which shows POD activity in the simulated weakly acidic inflammatory environment and catalyzes H2O2 to generate -OH to produce a bactericidal effect. After near-infrared light irradiation is given, due to the good photothermal properties of ACC@LPDANPs, its bactericidal effect is significantly enhanced; while the PDAPt NPs group and the ACC@LPDAPtNPs group show a more significant bactericidal effect. The significant enhancement of its bactericidal effect is due to not only the superimposition of its pod-like activity and photothermal effect, but also the fact that the photothermal temperature rise generates a high concentration of ·OH to enhance the pod-like activity of Pt nanozyme, which is also one of the reasons for enhancing the bactericidal effect.

[0087] Next, the excellent bactericidal effect of ACC@LPDAPtNPs was further verified by N01 / PI double fluorescence staining. As Figure 7 shown in Figures 6e and 7f, almost all stained bacteria in the control group emitted green fluorescence, indicating good bacterial growth. After drug administration, red fluorescence appeared in the bacteria of each group after staining. Especially in the ACC@LPDAPtNPs + NIR group, it was almost red fluorescence, demonstrating good bactericidal effect. In addition, scanning electron microscopy was used to observe the morphology of bacteria after different treatments. As Figure 7 shown in Figures 6g and 7h, the Staphylococcus aureus and Escherichia coli in the control group had regular shapes and intact biofilm structures. After drug administration, perforations appeared in the bacteria of each group, and the biofilm ruptured, resulting in the leakage of bacterial contents. This was particularly obvious in the bacteria of the ACC@LPDAPt NPs + NIR group. The above results further demonstrated the excellent bactericidal effect of ACC@LPDAPtNPs under photothermal conditions.

[0088] 6. Cytotoxicity

[0089] Since nanomaterials containing metal elements have potential toxicity problems, cytotoxicity studies are required before they can be used as therapeutic agents. As Figure 8 shown, in the absence or presence of near-infrared light irradiation to raise the temperature to 45 °C, after co-incubation of different concentrations of ACC@LPDANPs, PDAPt NPs, and ACC@LPDAPt NPs with HUVECs or L929, the survival rate of cells in each group was greater than 80%. This indicated that ACC@LPDANPs, PDAPtNPs, and ACC@LPDAPt NPs had no obvious cytotoxicity at the experimental doses, and at the same time, it also indicated that raising the temperature to 45 °C did not cause obvious damage to the cells.

[0090] 7. Cell migration and tube formation assay

[0091] The proliferative phase of wound healing is characterized by angiogenesis, granulation formation, and the beginning of epithelialization. The migration of vascular endothelial cells is the basis of angiogenesis, and fibroblasts are the main functional cells in the wound healing process. Their migration is crucial for wound healing and tissue remodeling. Therefore, in this invention, the scratch test was used to evaluate the effects of ACC@LPDANPs, PDAPtNPs, and ACC@LPDAPtNPs on the migration of HUVEC and L929 cells. As Figure 10As shown in Figs. a and 10b, in the absence of near-infrared light irradiation, no obvious cell migration was observed in HUVEC or L929 cells of each group until 24 h. When the near-infrared light irradiation increased the temperature to 45 °C and then cultured, there was no difference in the migration rate of ACC@LPDANPs, PDAPtNPs, and ACC@LPDAPt NPs within the first 12 hours, while at the 24th hour, the migration rate of the ACC@LPDAPt NPs group was significantly higher than that of other groups ( Figure 10 Figs. c and d). At the same time, it was found that in the presence of near-infrared light irradiation, the cell migration rates of the ACC@LPDANPs and ACC@LPDAPt NPs groups were higher than that of the PDAPt NPs group, which may be related to the fact that both contain ACC. The photothermal effect of near-infrared light irradiation triggers the dissolution of ACC, releasing Ca 2+ , and it has been found that Ca 2+ promotes the migration of HUVECs and L929s. Finally, by measuring the pH values of the culture media of different groups 24 h after administration ( Figure 9 ), it was found that the pH values of the culture media of the ACC@LPDANPs group and the ACC@LPDAPt NPs group under near-infrared light irradiation were higher than those of the corresponding groups without near-infrared irradiation. In contrast, the pH values of the culture media of the PBS group and the PDAPt NPs group showed little difference whether under near-infrared light irradiation or not. This indicates that under photothermal conditions, the ACC@LPDANPs and ACC@LPDAPt NPs groups can regulate the pH value of the culture medium through the dissolution of ACC. The above results show that in addition to regulating the pH of the system, the ACC in ACC@LPDAPt NPs, the released Ca 2+ also promotes the migration of HUVECs and L929s, contributing to wound healing.

[0092] New blood vessels can ensure the sufficient supply of oxygen and nutrients to the wound, providing the possibility for the growth of granulation tissue and wound healing. Therefore, in this invention, an endothelial tube formation assay was used to evaluate the in vitro angiogenesis-promoting ability of ACC@LPDAPt NPs. As Figure 10 shown in Fig. c, under the condition of no near-infrared light irradiation, there was no obvious difference in the total length of blood vessel formation between each administration group and the control group. However, under the condition of applying near-infrared light irradiation, the total lengths of blood vessel formation of the ACC@LPDANPs, PDAPtNPs, and ACC@LPDAPt NPs groups increased by 78.9%, 54.8%, and 94.8% respectively compared with those without near-infrared light irradiation at the same dose, and the number of new blood vessels in the ACC@LPDANPs and ACC@LPDAPt NPs groups was more; while there was no obvious change in the total length of blood vessel formation in the control group.

[0093] In addition to the photothermal effect itself, the increased influx of Ca2+ into endothelial cells caused by photothermal action may be the main reason for the promotion of angiogenesis by ACC@LPDAPtNPs and others. According to research reports, photothermal action can lead to an increase in the influx of Ca2+ into endothelial cells, and the elevation of cytoplasmic Ca2+ levels increases the number of activated calmodulin (CaM), thereby promoting the activation of Ca2+ / CaM-dependent protein kinase II, further activating the PI3K / Akt and MAPK signaling pathways, triggering cell cycle changes and proliferation, and thus promoting angiogenesis66. After semi-quantitative analysis by ImageJ, the ACC@LPDAPtNPs group generated the largest number of tubes ( Figure 10 f). The above results indicate that ACC@LPDAPtNPs can significantly promote the in vitro migration of HUVECs and L929s and the angiogenesis of HUVECs. Therefore, it has the potential to promote wound angiogenesis and re-endothelialization.

[0094] 8. Hemolysis test

[0095] The hemolysis of different concentrations of ACC@LPDAPtNPs is as Figure 11 shown. The hemolysis rate of ACC@LPDAPt NPs at a concentration of 150 μg / mL is less than 5%, indicating that ACC@LPDAPt NPs have good blood compatibility within this dose range.

[0096] 9. Effect of promoting the healing of mouse skin wound infections

[0097] Inspired by the in vitro bactericidal and cell experiment results, the present invention established a mouse skin infection wound model using Staphylococcus aureus and further studied the effect of ACC@LPDAPtNPs on promoting the healing of skin infection wounds. The experimental protocol is as Figure 12 shown in a. 30 minutes after drug administration to the wound, near-infrared light with a wavelength of 808 nm and a power density of 1.5 W / cm 2 was irradiated for about 2 minutes. The wound temperatures of the mice in the ACC@LPDANPs, PDAPtNPs, and ACC@LPDAPtNPs groups could rapidly increase to 45 °C ( Figure 12 b and d). This temperature can produce a bactericidal effect and avoid overheating damage to tissues; at the same time, it can cause the phase change melting of LA to trigger the dissolution of ACC, thereby raising the pH of the wound microenvironment to near neutral. The rapid restoration of the pH of the wound microenvironment to neutral can form a microenvironment conducive to wound healing, enhance the CAT-like activity of ACC@LPDAPtNPs, relieve the wound hypoxia state by scavenging ROS and generating oxygen, and thus accelerate the healing process. The wound photos, dynamic changes in wound area, and percentage of wound area of each group of mice at the time of model establishment and on the 3rd, 7th, and 14th days of treatment are respectively as Figure 12as shown in c, 12e, and 12f. Observation of the wound area and Staphylococcus aureus infection in each group of mice after modeling showed that an infected wound model was successfully constructed ( Figure 12 g, 12h).

[0098] On the 3rd day after drug administration, under the conditions of with or without near-infrared light irradiation, compared with the control group, the wound areas of each drug administration group of mice were significantly reduced; moreover, the wound areas of each drug administration group under near-infrared light irradiation were smaller than those of the corresponding groups without near-infrared light irradiation. The difference in the wound healing rate of each group may be related to the magnitude of its bactericidal effect. According to research reports, the self-healing ability of wounds due to bacterial infection is very poor. Due to the continuous inflammatory response in the wound caused by bacterial infection, the accumulation of inflammatory factors at the wound site will lead to the degradation of collagen fibers, thus resulting in the obstruction of wound healing. The results of the bacterial count in the mouse wounds showed that compared with the control group, the bacterial counts in each drug administration group were significantly decreased, and the bacterial residues in each drug administration group under near-infrared light irradiation were less than those of the corresponding groups without near-infrared light irradiation. This result is consistent with the results of the wound healing area of each group of mice, and further indicates that the good bactericidal effect of the drug for treating bacterial-infected wounds is the key to promoting wound healing. On the 7th day, the wound areas of each treatment group of mice continued to decrease significantly; among them, the relative wound areas of the PDAPtNPs and ACC@LPDAPtNPs groups under near-infrared light irradiation were 14.6% and 13.8% respectively; while the relative wound areas of the corresponding PDAPtNPs and ACC@LPDAPtNPs groups without near-infrared light irradiation were 30.1% and 21.1% respectively. In addition, the relative infected wound area of the ACC@LPDANPs+NIR group was 40.7%. At the same time, as Figure 12 shown in i, the results of the bacterial residues in the mouse wounds were also consistent with the results of the wound healing area of each group. On the 14th day, the wounds of each group of mice further recovered, but the relative area of the control group wound was still 21.8%, while the wounds of the ACC@LPDAPtNPs+NIR group of mice were almost completely healed, and the relative wound area was only 2.7%, showing a good wound-healing promoting effect. At this time, the results of the bacterial count in the mouse wounds showed that there were still a large number of bacteria in the wounds of the control group mice, while almost no bacteria were detected in the ACC@LPDAPtNPs+NIR group. The above results indicate that ACC@LPDAPtNPs has a good effect on promoting the healing of bacterial-infected wounds; its wound-healing ability may be related to the synergistic bactericidal effect produced by its photothermal and POD-like activities, as well as the effects of improving the acidic microenvironment of the wound and the release of calcium ions.

[0099] In addition, histological analysis was also performed on the mouse wound tissues on the 3rd, 7th, and 14th days to observe the infiltration of inflammatory cells in the wounds of each group of mice. As Figure 13As shown in a, on the 3rd day, the infiltration of inflammatory cells such as neutrophils in the wounds of mice in each group was relatively obvious, indicating that the wounds infected with bacteria were in the inflammatory phase. On the 7th day of treatment, there were still a large number of neutrophils infiltrating in the wound tissues of mice in the control group and each drug administration group without near-infrared light irradiation; while in the ACC@LPDANPs+NIR, PDAPtNPs+NIR, and ACC@LPDAPt NPs+NIR groups, the infiltration of neutrophils in the wound tissues of mice was significantly reduced, which was due to their good bactericidal effect alleviating the inflammatory reaction of the wounds. On the 14th day, compared with the control group and the group without near-infrared light irradiation, there were very few neutrophils in the wound tissues of mice in the ACC@LPDANPs+NIR, PDAPtNPs+NIR, and ACC@LPDAPt NPs+NIR groups, indicating that the inflammation of the wounds of mice in these groups had basically subsided at this time, and the wound healing had entered the proliferation and remodeling phase. This also coincided with the results of the wound healing area of the mice.

[0100] Collagen is the core component of the extracellular matrix (ECM), the framework of the extracellular matrix, and has the properties of promoting tissue regeneration and repair. In the proliferation phase of wound healing, fibroblasts proliferate in large numbers and synthesize extracellular matrix such as collagen. Collagen fills the wound defect and increases the tensile strength of the wound. As collagen accumulates, the wound gradually shrinks, which is crucial for wound healing. Therefore, in this invention, the Masson trichrome staining method was used to evaluate the collagen deposition in the wound healing area. As Figure 13 shown in b, on the 3rd day, due to the destruction of the normal function of fibroblasts by bacterial infection and the resulting inflammatory reaction, no collagen was observed in the wounds of mice in each group. On the 7th day, some collagen tissues stained blue appeared in the wound tissues of mice in each group, indicating that the number of fibroblasts in the wound area increased at this time and began to secrete collagen for tissue repair, but there was no obvious difference between the control group and the treatment group. On the 14th day, due to relatively severe bacterial infection in the control group, the collagenase secreted by bacteria destroyed the structure of collagen, and no obvious collagen deposition was observed in the wound. Compared with the control group, whether there was near-infrared light irradiation or not, collagen deposition could be observed in the skin wound tissues of mice treated with ACC@LPDANPs, PDAPt NPs, and ACC@LPDAPt NPs, and the distribution of collagen in the ACC@LPDAPt NPs+NIR group was more uniform and orderly, which may be related to the good bactericidal effect of ACC@LPDAPt NPs+NIR treatment, as well as the role of improving the acidic microenvironment of the wound, scavenging excessive ROS in the wound, and generating O2 to relieve wound hypoxia at the same time.

[0101] The persistent inflammatory response in the wound hinders normal wound healing, and the change in the level of inflammatory factors can reflect the process of wound healing. The expression levels of pro-inflammatory factors IL-1β and TNF-α, as well as anti-inflammatory factors IL-10 and TGF-β1, in the wound tissues of mice on the 7th day of treatment were detected by immunohistochemistry to evaluate the inhibitory effect on inflammation during the wound healing process in each administration group. As Figure 14 shown, compared with the control group and the corresponding group without near-infrared light irradiation, the expressions of pro-inflammatory factors TNF-α and IL-1β in the wound tissues of mice in the ACC@LPDA NPs+NIR group, PDAPt NPs+NIR group, and ACC@LPDAPt NPs+NIR group were lower, and the expressions of anti-inflammatory factors IL-10 and TGF-β1 were higher. Among them, compared with the control group, there were significant differences in the expressions of pro-inflammatory and anti-inflammatory factors after treatment in the ACC@LPDAPt NPs+NIR group, indicating that it had the optimal anti-inflammatory effect and could effectively relieve the inflammatory response of infected wounds.

[0102] Platelet-endothelial cell adhesion molecule (CD31) is a transmembrane protein expressed during early angiogenesis and plays a crucial role in promoting wound healing. Smooth muscle actin (α-SMA) is a heterodimer of vascular smooth muscle actin, mainly expressed in vascular smooth muscle cells, and is a key indicator of angiogenesis in wound tissues. To evaluate the formation of new blood vessels in the wound tissues of mice after treatment, the expression levels of CD31 and α-SMA in the wound tissues of mice on the 7th day after administration were examined by immunofluorescence staining. As Figure 15 shown in a, 15b, and 15c, there was almost no expression of CD31 (red) and α-SMA (green) in the wound tissues of mice in the control group and the group without near-infrared light irradiation, indicating that angiogenesis in the wound tissues was severely blocked at this time. However, there was a certain expression of CD31 and α-SMA in the wound tissues of mice in the ACC@LPDANPs+NIR and PDAPtNPs+NIR groups; moreover, the expression levels of CD31 and α-SMA in the skin wound tissues of mice in the ACC@LPDAPt+NIR group were higher than those in other groups, indicating that it had a better ability to promote wound angiogenesis.

[0103] Hypoxia-inducible factor 1-α (HIF-1α) reflects the oxygenation level of wound tissues, and its increased expression in wound tissues will hinder angiogenesis and wound healing. As Figure 15As shown in Figures d and f, on the 7th day of treatment, immunofluorescence staining of the wound tissues of the control group mice showed a relatively high expression level of HIF-1α, indicating that the wound tissues were in a hypoxic state, which was not conducive to wound healing. However, immunofluorescence staining of the wound tissues of the mice in the ACC@LPDA NPs+NIR group, PDAPt NPs+NIR group, and ACC@LPDAPt NPs+NIR group showed that the expression of HIF-1α was significantly lower than that of the control group and the corresponding group without near-infrared light irradiation; among them, the expression of HIF-1α in the wound tissues of the mice in the ACC@LPDAPt NPs+NIR group was the least. This effect may be due to the fact that the photothermal-triggered dissolution of ACC raises the pH value of the wound microenvironment to neutral, and the CAT-like activity of ACC@LPDAPt NPs can scavenge H2O2 in the wound microenvironment and generate oxygen, relieve tissue hypoxia, reduce the expression of HIF-1α, and promote wound healing.

[0104] Next, the immunofluorescence staining method was used to detect the expression of the main collagen type I (ColI) in the wound tissues of the mice in each group after 14 days of treatment. As Figure 15 shown in Figures e and 15g, compared with the control group and the corresponding group without near-infrared light irradiation, immunofluorescence staining of the wound tissues of the mice in the ACC@LPDA NPs+NIR, PDAPt NPs+NIR, and ACC@LPDAPt NPs+NIR groups showed more type I collagen (ColI) in the wounds; among them, the expression level of ColI in the ACC@LPDAPt NPs+NIR group was higher, and was significantly higher than that in the ACC@LPDA NPs+NIR and PDAPt NPs+NIR groups. This further indicates that ACC@LPDAPt NPs have a good effect on promoting the healing of bacteria-infected wounds.

[0105] In addition, as Figure 16 shown, after 14 days of treatment, no obvious bleeding, necrosis, inflammatory lesions, or tissue damage were observed in the main organs such as the heart, liver, spleen, lungs, and kidneys of the mice in the ACC@LDAPt NPs+near-infrared group. As Figure 17 shown, during the treatment process, the body weights of the mice in each group showed a natural growth trend, and there were no significant differences. These results indicate that ACC@LPDAPt NPs have good biosafety at the treatment dose, which lays a foundation for its application in clinical treatment.

[0106] It should be noted that when the claims of the present invention involve numerical ranges, it should be understood that any value between the two endpoints of each numerical range and the two endpoints can be selected. To avoid repetition, the preferred embodiments of the present invention are described.

[0107] Although the preferred embodiments of the present invention have been described, additional changes and modifications can be made to these embodiments by those skilled in the art once they learn of the basic creative concept. Therefore, the appended claims are intended to be construed to include the preferred embodiments as well as all changes and modifications that fall within the scope of the present invention.

[0108] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention is also intended to include these modifications and variations.

Claims

1. A composite material containing platinum nanozyme, characterized in that, The composite material is formed by electrostatic interaction and coordination between the surface of amorphous calcium carbonate nanoparticles and fatty acids to form a hydrophobic layer with phase change melting. On the surface of the hydrophobic layer, a photothermal effect layer with a photothermal effect is formed by self-oxidative polymerization with a self-oxidative polymer. After fixing platinum ions on the surface of the photothermal effect layer, the composite material is obtained by in-situ reduction; The self-oxidative polymer is dopamine.

2. The composite material according to claim 1, characterized in that, The fatty acid is at least one of long-chain fatty acids, medium-chain fatty acids, and short-chain fatty acids.

3. The composite material according to claim 2, characterized in that, The long-chain fatty acid is palmitic acid or oleic acid.

4. The composite material according to claim 2, characterized in that, The medium-chain fatty acid is lauric acid.

5. The preparation method of the composite material according to claim 1, characterized in that, It includes the following steps: The amorphous calcium carbonate nanoparticles react with the fatty acid, so that the electrostatic interaction and coordination between the fatty acid and the calcium ions on the surface of the amorphous calcium carbonate nanoparticles form the hydrophobic layer, and a first intermediate product is obtained; The first intermediate product reacts with the self-oxidative polymer, and the photothermal effect layer is formed by the self-oxidative polymerization of the self-oxidative polymer to obtain a second intermediate product; After fixing platinum ions on the surface of the second intermediate product, the composite material is obtained by an in-situ reduction method.

6. The preparation method according to claim 5, wherein The mass ratio of the amorphous calcium carbonate nanoparticles to the fatty acid is 1:1 to 20; The mass ratio of the first intermediate product to the self-oxidative polymer is 1:2 to 15; The mass ratio of the second intermediate product to the platinum ions is 1:1 to 5.

7. Use of the composite material containing platinum nanozyme according to claim 1 in the preparation of a drug for treating wound infection.

8. The application according to claim 7, wherein The infection refers to bacterial infection.

9. The application according to claim 7, characterized in that, The drug promotes wound healing.

10. Use of the composite material containing platinum nanozyme according to claim 1 in the preparation of a material for photothermal treatment of wound infection.

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