Photothermal antibacterial anti-inflammatory antioxidant amino acid cerium particles, and preparation method and application thereof

Abstract

The present application relates to the technical field of biomimetic nanocatalyst, in particular to a photo-thermal antibacterial anti-inflammatory antioxidant amino acid cerium particle and a preparation method and application thereof.The preparation method comprises the following steps: preparation of phenylalanine cerium cluster: adding cerium ammonium nitrate into a phenylalanine anhydrous methanol solution, stirring at room temperature, and obtaining a phenylalanine cerium cluster; preparation of amino acid cerium particle: dissolving indocyanine green in water; dissolving the phenylalanine cerium cluster in water; under ultrasonic, alternately adding the water solution of indocyanine green and the water solution of the phenylalanine cerium cluster into deionized water.The prepared amino acid cerium particle can effectively scavenge reactive oxygen species, promote the formation of anti-inflammatory M2 type macrophages, and effectively kill bacteria under photo-thermal conditions, and has good biological safety, solving the technical problems that the existing biomimetic nanocatalyst has poor biocompatibility, and cannot simultaneously treat ROS excess, excessive inflammation and bacterial infection.

Description

Technical Field

[0001] This invention relates to the field of biomimetic nanocatalyst technology, and in particular to photothermal antibacterial, anti-inflammatory and antioxidant amino acid cerium particles, their preparation methods and applications. Background Technology

[0002] Diabetic wounds (such as diabetic foot ulcers, DFU) are a common and serious complication in diabetic patients. Their main causes include the accumulation of glycation end products (GEP), increased oxidative stress, microvascular complications, nerve damage, and abnormal immune function (macrophage polarization disorder). The core reason for the difficulty in healing diabetic wounds lies in the metabolic disorder driven by excessive production of reactive oxygen species (ROS): ROS can directly damage cell membranes, proteins, DNA, and lipids, exacerbate the inflammatory response by activating pro-inflammatory signaling pathways, inhibit growth factor activity to hinder cell proliferation, and induce vascular endothelial damage leading to thrombosis. Glycation end products, ROS, and hyperglycemia themselves continuously activate innate immune cells (neutrophils, macrophages), promoting the excessive release of pro-inflammatory factors (TNF-α, IL-1β, IL-6), causing persistent damage to local tissues. The transformation of macrophages from pro-inflammatory M1 to anti-inflammatory M2 is hindered, and the function of M2 macrophages is impaired, making them unable to effectively clear apoptotic cells and tissue debris. A hyperglycemic environment provides ideal conditions for the growth of bacteria (especially Staphylococcus aureus and Streptococcus) and fungi, while immune cell deficiencies (decreased phagocytic and bactericidal capabilities) weaken the host's defense function, and vascular lesions make it difficult for antibiotics and immune cells to effectively reach the site of infection. Infection further exacerbates inflammation, tissue damage, and ischemia, creating a vicious cycle. Therefore, there is an urgent need to develop novel treatment strategies that can simultaneously address the problems of ROS excess, excessive inflammation, and bacterial infection.

[0003] Currently, research on biomimetic nanocatalysts with highly efficient catalytic properties similar to natural enzymes has attracted widespread attention. Compared with natural enzymes, they have multiple advantages such as excellent stability, adjustable catalytic performance, and large-scale industrial synthesis. Biomimetic nanocatalysts are usually composed of inorganic materials (such as Fe3O4, CeO2, carbon materials, etc.), which have extremely high stability and the following advantages: (1) Long-term storage: They are not easily degraded and have a shelf life much longer than natural enzymes; (2) Simple preparation: Many biomimetic nanocatalysts can be produced on a large scale and repeatedly through simple chemical synthesis methods (such as hydrothermal method and coprecipitation method); (3) Low raw material cost: Their raw materials (such as iron salts and carbon sources) are much cheaper than the extraction and purification of natural enzymes (which require complex biological fermentation and purification processes); (4) Multifunctional Catalytic activity: A biomimetic nanocatalyst can often mimic the activity of multiple natural enzymes. For example, Fe3O4 nanoparticles simultaneously possess multiple activities such as peroxidase (POD), catalase (CAT), and superoxide dismutase (SOD). This intrinsic multi-enzyme activity is not possessed by natural enzymes, allowing for synergistic treatment or detection in complex systems; (5) Designable catalytic properties: By precisely controlling the size, shape, surface modification, and crystal face exposure of biomimetic nanocatalysts through nanotechnology, their catalytic activity and selectivity can be adjusted. However, a serious challenge for biomimetic nanocatalysts lies in biosafety issues, such as immunogenicity. Although they do not easily cause strong immune responses like some foreign proteins, biomimetic nanocatalysts may still be recognized and cleared by the immune system, affecting their efficacy and bringing potential risks. How to develop a biomimetic nanocatalyst based on a synergistic treatment mechanism to simultaneously regulate uncontrolled oxidative stress, inflammatory response, and bacterial infection in diabetic wounds is the core technical challenge currently faced. Summary of the Invention

[0004] In response to the problems raised in the background technology, the purpose of this invention is to propose a method for preparing photothermal antibacterial, anti-inflammatory, and antioxidant amino acid cerium particles. The prepared amino acid cerium particles can effectively scavenge reactive oxygen species, promote the formation of anti-inflammatory M2 macrophages, effectively kill bacteria under photothermal conditions, and have good biocompatibility. This solves the technical problems of poor biocompatibility of existing biomimetic nanocatalysts and their inability to simultaneously treat excessive ROS, excessive inflammation, and bacterial infection.

[0005] Another objective of this invention is to propose a photothermal antibacterial, anti-inflammatory, and antioxidant amino acid cerium particle, which has good photothermal conversion characteristics and photothermal stability, can effectively scavenge reactive oxygen species, promote the formation of anti-inflammatory M2 macrophages, effectively kill bacteria under photothermal conditions, inhibit infection, and has good biocompatibility.

[0006] Another objective of this invention is to propose the application of the aforementioned photothermal antibacterial, anti-inflammatory, and antioxidant amino acid cerium particles in the preparation of a treatment for infectious diabetic wounds. Using these photothermal antibacterial, anti-inflammatory, and antioxidant amino acid cerium particles in the preparation of a treatment for infectious diabetic wounds can significantly alleviate wound hypoxia, promote the formation of anti-inflammatory M2 macrophages, significantly enhance cell proliferation and migration capabilities, and effectively eliminate wound bacteria through the photothermal effect, thereby accelerating wound healing with good biocompatibility.

[0007] The above-mentioned technical objective of the present invention is achieved through the following technical solution:

[0008] A method for preparing photothermal antibacterial, anti-inflammatory, and antioxidant amino acid cerium particles includes the following steps:

[0009] Step S1, Preparation of phenylalanine cerium clusters: Cerium ammonium nitrate was added to an anhydrous methanol solution of phenylalanine and stirred at room temperature to obtain phenylalanine cerium clusters; Step S2, Preparation of amino acid cerium particles: Indocyanine green was dissolved in water to obtain an aqueous solution of indocyanine green; phenylalanine cerium clusters were dissolved in water to obtain an aqueous solution of phenylalanine cerium clusters; the aqueous solution of indocyanine green and the aqueous solution of phenylalanine cerium clusters were alternately added dropwise to deionized water under ultrasonication, and after ultrasonic treatment, photothermal antibacterial, anti-inflammatory and antioxidant amino acid cerium particles were obtained.

[0010] Optionally, in step S1, the molar ratio of phenylalanine to cerium ammonium nitrate is 2:1. Optionally, in step S1, the mixture is stirred at room temperature for 24 h.

[0011] Optionally, in step S1, after adding cerium ammonium nitrate to the anhydrous methanol solution of phenylalanine, triethylamine is added to adjust the pH of the solution to 6-7.

[0012] Optionally, in step S2, the concentration of the aqueous solution of indocyanine green is 1 mg / mL, the concentration of the aqueous solution of cerium phenylalanine cluster is 1 mg / mL, and the mass ratio of indocyanine green to cerium phenylalanine cluster is 1:1.

[0013] Optionally, in step S2, under ice bath and light-protected conditions, an aqueous solution of indocyanine green and an aqueous solution of cerium phenylalanine clusters are alternately added dropwise to deionized water under ultrasonication, followed by ultrasonic treatment for 15 min.

[0014] Optionally, after ultrasonic treatment for 15 min, the mixture is filtered, and after filtration, it is allowed to stand at 4 ℃ for 12–24 h. After ultrafiltration and centrifugation, amino acid cerium particles with photothermal antibacterial, anti-inflammatory, and antioxidant properties are obtained.

[0015] Optionally, the molecular cutoff of the ultrafiltration centrifugation is 100 kDa, the rotation speed is 2000-2500 rpm, and the time is 8-10 min.

[0016] A photothermal antibacterial, anti-inflammatory, and antioxidant amino acid cerium particle is prepared using the aforementioned method for preparing photothermal antibacterial, anti-inflammatory, and antioxidant amino acid cerium particles.

[0017] The application of the aforementioned photothermal antibacterial, anti-inflammatory, and antioxidant amino acid cerium particles in the preparation of drugs for the treatment of infectious diabetic wounds.

[0018] Compared with the prior art, the embodiments of the present invention have the following beneficial effects:

[0019] This invention relates to Phe-Ce@ICG amino acid cerium particles, which possess excellent photothermal conversion properties and stability. They can effectively scavenge reactive oxygen species, alleviate the hypoxic environment at diabetic wound sites, promote macrophage differentiation into M2 type, secrete anti-inflammatory cytokines, promote angiogenesis, and accelerate wound healing. Furthermore, they can effectively kill bacteria and inhibit infection under photothermal conditions. In addition, the Phe-Ce@ICG nanoparticles exhibit good biocompatibility, solving the technical problems of poor biocompatibility and inability to simultaneously handle excessive ROS, excessive inflammation, and bacterial infection in existing biomimetic nanocatalysts. Attached Figure Description

[0020] Figure 1 This is a diagram illustrating the preparation and physicochemical characterization of photothermal antibacterial, anti-inflammatory, and antioxidant amino acid cerium particles according to Example 1 of the present invention. Figure 1 (A) in the diagram is the synthesis flowchart of Phe-Ce@ICG; Figure 1 (B) in the image is a transmission electron microscope (TEM) image of Phe-Ce@ICG (scale bar: 200 nm). Figure 1 (C) in the image is a scanning transmission electron microscope (STEM) image of Phe-Ce@ICG (scale bar: 50 nm). Figure 1 (D), (E) and (F) are the energy dispersive spectroscopy (EDS) elemental distribution maps of N, O and Ce elements in Phe-Ce@ICG, respectively (scale bar: 50 nm). Figure 1 (G) in the figure is the particle size distribution of Phe-Ce@ICG; Figure 1 (H) in the diagram is the Zeta potential map of Phe-Ce@ICG; Figure 1 (I) in the figure is the ultraviolet absorption spectrum of ICG and Phe-Ce@ICG; Figure 1 (J) in the image is the Fourier transform infrared spectrum of Phe-Ce@ICG; Figure 1(K) in the image is a high-resolution X-ray photoelectron spectroscopy (XPS) fine spectrum of the C1s, N1s, O1s and Ce 3d peaks of Phe-Ce@ICG; Figure 1 (L) in the image is the XPS full spectrum of Phe-Ce@ICG.

[0021] Figure 2 This is the EDS spectrum of Phe-Ce@ICG from Example 1.

[0022] Figure 3 This is the X-ray diffraction (XRD) pattern of Phe-Ce in Example 1.

[0023] Figure 4 This is a thermal stability test diagram of Phe-Ce in Example 1.

[0024] Figure 5 This is a graph showing the ability of Phe-Ce@ICG to scavenge reactive oxygen species (ROS) in Example 1; Figure 5 (A) in the diagram is a schematic diagram of the antioxidant mechanism of Phe-Ce@ICG; Figure 5 (B) in the diagram is a schematic diagram of the mechanism by which Phe-Ce@ICG scavenges reactive oxygen species (ROS); Figure 5 (C) in the figure is a graph showing the scavenging ability of different concentrations of Phe-Ce@ICG for H2O2; Figure 5 (D) in the figure is a test graph showing the scavenging ability of different concentrations of Phe-Ce@ICG against •OH; Figure 5 (E) represents the effect of different concentrations of Phe-Ce@ICG on superoxide anions (E). The diagram shows the cleaning ability test results. Figure 5 (F) in the figure is a graph of superoxide dismutase (SOD) activity assay of Phe-Ce@ICG at different concentrations; Figure 5 (G) in the figure represents the total antioxidant capacity of different concentrations of Phe-Ce@ICG.

[0025] Figure 6 This is a diagram of the biosafety assessment experiment for Phe-Ce@ICG. Figure 6 (A) in the diagram is a graph from a HUVEC cytotoxicity assay. Figure 6 (B) in the figure is a diagram of the Raw264.7 cytotoxicity assay.

[0026] Figure 7 This is a graph showing the in vitro ROS scavenging capacity of Phe-Ce@ICG. Figure 7 (A) in the figure is an experimental diagram of quantifying intracellular ROS levels in HUVEC cells using DCFH-DA as a specific probe (n = 3, where n is the number of independent replicates). Figure 7(B) is an experimental diagram showing the quantitative O2 levels of 4T1 cells under normoxic and hypoxic conditions using RDPP as a specific probe (scale bar: 200 μm). Figure 7 (C) in the graph represents the average fluorescence intensity of DCFH-DA; Figure 7 (D) in the figure represents the average fluorescence intensity of RDPP.

[0027] Figure 8 This is a Western blot analysis of HIF-1α expression levels after different sample treatments.

[0028] Figure 9 This is a diagram of a cell scratch assay. Figure 9 (A) in the diagram is a schematic diagram of the cell scratch test process; Figure 9 (B) in the image is a representative scratch experiment image of HUVEC cells after treatment with 300 μM H2O2 and intervention with different samples (scale bar: 200 μm). Figure 7 (C) and Figure 7 (D) in the figure represents the scratch healing rate test at 12 hours and 24 hours, respectively.

[0029] Figure 10 This is a diagram from an in vitro photothermal antibacterial performance test. Figure 10 (A) in the figure is a temperature change curve of different concentrations of Phe-Ce@ICG under near-infrared light irradiation; Figure 10 (B) in the figure is the repeated irradiation-cooling curve of Phe-Ce@ICG and ICG; Figure 10 (C) in the image is an infrared thermal image of Phe-Ce@ICG, ICG, and H2O under near-infrared illumination; Figure 10 (D) in the diagram is a schematic diagram of the in vitro antibacterial experiment of Phe-Ce@ICG; Figure 10 (E) in the figure are scanning electron microscope images (scale bar: 1 μm) of bacteria from different treatment groups and corresponding agar plate colony morphology diagrams; Figure 10 (F) represents Escherichia coli treated with different concentrations of Phe-Ce@ICG. E. coli ) and Staphylococcus aureus ( S.aureus A graph showing the number of survivors; Figure 10 (G) in the middle is Figure 10 Statistical analysis of the survival rate of (F) in the figure (n=3, n is the number of independent repeated trials); Figure 10 (H) in the middle is Figure 10 The colony count of Escherichia coli (F) in the figure (n=3, where n is the number of independent replicates). Figure 10 (I) in the middle is Figure 10 The colony count of Staphylococcus aureus in (F) (n=3, n is the number of independent replicates).

[0030] Figure 11 This is a diagram from an in-situ spraying healing efficacy test in a diabetic mouse model. Figure 11 (A) in the diagram is a schematic diagram of the in vivo antibacterial experiment process; Figure 11 (B) in the image is a real-time infrared thermal imaging image of a mouse; Figure 11 (C) in the diagram is a schematic diagram of the temperature change curves of the wounds in each group as the irradiation time increases.

[0031] Figure 12 These are representative photographs of each group of wounds and images showing the wound healing and closure at specific time points (scale bar: 5mm).

[0032] Figure 13 This is a diagram from an in-situ spraying healing efficacy test in a diabetic mouse model. Figure 13 (A) in the figure is a graph showing the change in relative wound area of ​​mice after different treatments; Figure 13 (B) in the figure is a graph showing the changes in body weight of mice after different treatments.

[0033] Figure 14 These are hematoxylin-eosin staining images of wound sections from different treatment groups.

[0034] Figure 15 These are images of the wound's immunohistochemical and immunofluorescence quantitative analysis results. Figure 10 (A) in the figure shows the results of CD31 immunohistochemical (IHC) staining and VEGF and HIF-1α immunofluorescence (IF) staining of wound tissue after different treatments (scale bar: 50 μm). Figure 15 (B), (C), and (D) in the figure are quantitative analysis diagrams of CD31, VEGF, and HIF-1α, respectively.

[0035] Figure 16 This is a diagram illustrating the ability to regulate macrophage polarization. Figure 16 (A) in the diagram is a schematic diagram of Phe-Ce@ICG regulating macrophage polarization in vitro; Figure 16 (B) in the figure shows the concentration of TNF-α in the supernatant of macrophages after different treatment groups; Figure 16 (C) in the figure shows the concentration of TGF-β in the supernatant of macrophages after different treatment groups; Figure 16 (D) in the figure shows the flow cytometry results of M2 macrophages (F4 / 80 and CD206+) after different treatment groups.

[0036] Figure 17 This is a diagram of an in vivo anti-inflammatory effect test. Figure 17 (A) shows the macrophage polarization under different treatments on day 11—immunofluorescence staining of CD206 (green) and CD86 (red) (scale bar: 50 μm). Figure 17 In the diagram, (B) and (C) are the average fluorescence intensity graphs of CD206 (green) and CD86 (red), respectively. Figure 17 (D) and (E) in the figure are the mean fluorescence intensity diagrams of TNF-α and IL-10 in the wound tissue, respectively; Figure 17 (F) in the image is an immunofluorescence staining image of IL-10 (red) and TNF-α (red) on day 11.

[0037] Figure 18 This is a biosafety assessment of Phe-Ce@ICG: photos of blood hemolysis experiments and hemolysis rate analysis graphs.

[0038] Figure 19 This is a biosafety assessment of Phe-Ce@ICG: hematoxylin-eosin (H&E) staining results of major organs after different treatment groups.

[0039] in, Figure 12 , Figure 13 , Figure 14 , Figure 15 and Figure 19 In this context, "PBS+" means "PBS + laser", "ICG+" means "ICG + laser", and "Phe-Ce@ICG+" means "Phe-Ce@ICG + laser". Detailed Implementation

[0040] A method for preparing photothermal antibacterial, anti-inflammatory, and antioxidant amino acid cerium particles includes the following steps:

[0041] Step S1, Preparation of phenylalanine cerium cluster compound (Phe-Ce): Cerium ammonium nitrate was added to an anhydrous methanol solution of phenylalanine and stirred at room temperature to obtain phenylalanine cerium cluster compound;

[0042] Step S2, Preparation of amino acid cerium particles (Phe-Ce@ICG): Indocyanine green (ICG) was dissolved in water to obtain an aqueous solution of indocyanine green; phenylalanine cerium cluster compound was dissolved in water to obtain an aqueous solution of phenylalanine cerium cluster compound; the aqueous solution of indocyanine green and the aqueous solution of phenylalanine cerium cluster compound were alternately added dropwise to deionized water under ultrasonic treatment, and after ultrasonic treatment, amino acid cerium particles with photothermal antibacterial, anti-inflammatory and antioxidant properties were obtained.

[0043] CeO2 nanoparticles consist of cerium atoms (in Ce) 3+ and Ce 4+ (In its existing state) it is formed by oxygen atoms. In redox reactions, oxygen and cerium undergo reversible bonding to achieve Ce. 4+ With Ce 3+Interconversion between oxidation states. Based on this characteristic, cerium-based nanocatalysts possess a variety of enzyme-like activities, such as catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD). The reversible redox conversion between Ce(III) and Ce(IV) enables cerium-based nanoparticles to mimic antioxidant enzymes and selectively scavenge ROS under physiological conditions.

[0044] However, cerium dioxide has poor water solubility. This invention selects cerium ammonium nitrate as a raw material, which has the advantage of low cost. It also uses amino acids (phenylalanine), a basic building block of living organisms, as ligands to form Phe-Ce. The resulting Phe-Ce molecules have excellent water solubility and uniform dispersion. Furthermore, through a one-step synthesis method, ICG is introduced. Under ultrasonic conditions, Phe-Ce and ICG self-assemble to form highly water-soluble, uniformly sized, positively charged nanoparticles, Phe-Ce@ICG, effectively solving the problems of water dispersibility and biocompatibility. This invention constructs a cerium-based nanocatalyst, Phe-Ce@ICG, with antibacterial, antioxidant, and macrophage polarization regulation functions through self-assembly. In the Phe-Ce system, phenylalanine is used to surface-functionalize cerium, effectively solving the water solubility problem. Amino acids are naturally occurring molecules in biological systems, allowing a hydrophilic protective layer to form on the catalyst surface, which not only enhances water solubility but also significantly reduces toxicity and improves biocompatibility. In addition, it prevents nanoparticle aggregation, thereby enhancing colloidal stability. More importantly, this invention introduces the biocompatible material ICG, encapsulating ICG within nanoparticles to form a water-soluble nanocomposite material. This integrates phototherapy technology into a nanocatalytic system, achieving highly efficient photothermal conversion. Under near-infrared (NIR) laser irradiation, the excellent photothermal properties of ICG can effectively kill bacteria and reduce bacterial infection at the wound site. Thus, in photothermal therapy (PTT), Phe-Ce@ICG generates local heat through laser excitation, effectively and selectively killing bacteria.

[0045] The removal of ROS at the wound site is mainly achieved by cerium nanoparticles. The large specific surface area and abundant oxygen vacancies of Phe-Ce@ICG enable cerium ions to be effectively eliminated by Ce. 3+ With Ce 4+ A redox cycle occurs between them. In cerium-based biomimetic nanocatalysts, the three core reactions catalyzed by SOD-like and CAT-like enzymes are: + Ce 4+ →O2+ Ce 3+ , + Ce 3+ + 2H + →H2O2+ Ce 4+The reaction 2H₂O₂ → 2H₂O + O₂. The clearance of ROS and the release of oxygen significantly reduced the expression of hypoxia-inducible factor 1α (HIF-1α), promoted the expression of pro-angiogenic factors (VEGF and CD31) in endothelial cells, and enhanced cell proliferation and migration, thereby alleviating wound hypoxia and promoting angiogenesis. Furthermore, Phe-Ce@ICG promoted the generation of anti-inflammatory M2 macrophages and inhibited the production of M1 macrophages, accompanied by the secretion of the anti-inflammatory cytokine IL-10, significantly accelerating the wound healing process.

[0046] This invention relates to Phe-Ce@ICG amino acid cerium particles, which possess excellent photothermal conversion properties and photothermal stability. They can effectively scavenge reactive oxygen species, alleviate the hypoxic environment at diabetic wound sites, promote macrophage differentiation into M2 type, secrete anti-inflammatory cytokines (promoting the formation of anti-inflammatory M2 macrophages), promote angiogenesis, and accelerate wound healing. Furthermore, they can effectively kill bacteria and inhibit infection under photothermal conditions. In addition, the Phe-Ce@ICG nanoparticles exhibit good biocompatibility, solving the technical problems of poor biocompatibility and inability to simultaneously handle excessive ROS, excessive inflammation, and bacterial infection in existing biomimetic nanocatalysts.

[0047] To further clarify, in step S1, the molar ratio of phenylalanine to cerium ammonium nitrate is 2:1.

[0048] To further explain, in step S1, the mixture is stirred at room temperature for 24 hours.

[0049] To further explain, in step S1, after adding cerium ammonium nitrate to the anhydrous methanol solution of phenylalanine, triethylamine is added to adjust the pH of the solution to 6-7.

[0050] To further clarify, in step S2, the concentration of the aqueous solution of indocyanine green is 1 mg / mL, the concentration of the aqueous solution of cerium phenylalanine cluster is 1 mg / mL, and the mass ratio of indocyanine green to cerium phenylalanine cluster is 1:1.

[0051] To further explain, in step S2, under ice bath and light-protected conditions, the aqueous solution of indocyanine green and the aqueous solution of cerium phenylalanine clusters are alternately added dropwise to deionized water under ultrasonication, and then ultrasonic treatment is performed for 15 min.

[0052] Phe-Ce@ICG nanoparticles are nanoparticles assembled through electrostatic interactions driven by the negative charge of the sulfonate group in ICG and the positive charge of the amino group in Phe-Ce. Higher temperatures intensify Brownian motion, leading to an overly rapid and uncontrollable assembly process, easily resulting in large particles with uneven size, loose structure, and agglomeration. Using an ice bath allows for temperature control of the reaction system, making the assembly process more manageable and yielding nanoparticles with good size uniformity and structural stability. Furthermore, reacting under light-protected conditions (such as in darkness) avoids light-induced damage to the conjugated structure of ICG, preventing degradation and inactivation during synthesis, and ensuring the effectiveness of Phe-Ce@ICG nanoparticles in subsequent photothermal therapy.

[0053] To further explain, after ultrasonic treatment for 15 minutes, the mixture was filtered and then allowed to stand at 4 ℃ for 12–24 hours. After ultrafiltration and centrifugation, cerium amino acid particles with photothermal antibacterial, anti-inflammatory, and antioxidant properties were obtained.

[0054] Preferably, after ultrasonic treatment for 15 min, the solution is filtered with filter paper. After filtration, the filtrate is allowed to stand at 4 °C for 24 h. After ultrafiltration and centrifugation, amino acid cerium particles with photothermal antibacterial, anti-inflammatory and antioxidant properties are obtained.

[0055] To further explain, the molecular cutoff of the ultrafiltration centrifugation is 100 kDa, the rotation speed is 2000-2500 rpm, and the time is 8-10 min, in order to remove free Phe-Ce and ICG.

[0056] Preferably, the molecular cutoff of the ultrafiltration centrifugation is 100 kDa, the rotation speed is 2000 rpm, and the time is 8 min.

[0057] A photothermal antibacterial, anti-inflammatory, and antioxidant amino acid cerium particle is prepared using the aforementioned method. The photothermal antibacterial, anti-inflammatory, and antioxidant amino acid cerium particle prepared by the above method exhibits excellent photothermal conversion characteristics and photothermal stability. It can effectively scavenge reactive oxygen species, promote the formation of anti-inflammatory M2 macrophages, effectively kill bacteria and inhibit infection under photothermal conditions, and possesses good biocompatibility.

[0058] The application of the aforementioned photothermal antibacterial, anti-inflammatory, and antioxidant amino acid cerium particles in the preparation of a treatment for infectious diabetic wounds. These photothermal antibacterial, anti-inflammatory, and antioxidant amino acid cerium particles can perform multiple functions, including photothermal therapy, antibacterial activity, oxygen supply, ROS scavenging, and immunomodulation, demonstrating excellent potential in the treatment of diabetic wounds. (Laser (808nm, 0.5 W / cm²)) 2Under irradiation, ICG, with its highly efficient photothermal conversion, can mediate sterilization and disinfection of the wound surface. Phe-Ce can remove excess ROS, generate oxygen, effectively solve the problem of hypoxia at the wound site, enhance cell proliferation and migration, and thus promote angiogenesis. Phe-Ce effectively polarizes and guides pro-inflammatory M1 macrophages to anti-inflammatory M2 macrophages, and in synergy with the photothermal antibacterial effect of ICG, it can effectively promote the healing of infectious diabetic wounds.

[0059] The photothermal antibacterial, anti-inflammatory, and antioxidant amino acid cerium particles of this invention, as a nanocatalyst, can be conveniently administered via a spray formulation, offering advantages such as ease of operation and portability. When sprayed in situ onto the wound, it can both remove excess ROS and simultaneously generate oxygen, exhibiting excellent enzymatic activity similar to catalase (CAT) and superoxide dismutase (SOD), thereby significantly alleviating wound hypoxia. Furthermore, through light intensity modulation, Phe-Ce@ICG exhibits extremely high photothermal conversion efficiency; under near-infrared light irradiation, its photothermal effect can effectively eliminate bacteria on the wound surface. Phe-Ce@ICG can significantly enhance cell proliferation and migration capabilities, thereby promoting wound healing. Phe-Ce@ICG can effectively promote the formation of anti-inflammatory M2 macrophages while inhibiting the generation of pro-inflammatory M1 macrophages. The biomimetic nanocatalyst Phe-Ce@ICG of this invention can accelerate wound healing with good biocompatibility, showing significant potential in the clinical treatment of diabetic wounds.

[0060] To facilitate understanding of the present invention, a more comprehensive description is provided below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a more thorough and complete understanding of the disclosure of the present invention. Where specific techniques or conditions are not specified in the embodiments, they are performed in accordance with techniques or conditions described in the literature in the art or according to product instructions. Reagents or instruments used, unless otherwise specified, are all conventional products that can be obtained commercially.

[0061] Raw materials: Cerium ammonium nitrate and phenylalanine were purchased from Sigma-Aldrich. Phenylalanine is a mixed amino acid of D and L (DL-phenylalanine), CAS number 150-30-1, product number 147966.

[0062] Experimental Instruments: Laser Particle Size / Zeta Potential Analyzer: Malvern Zetasizer Nano As (UK); X-ray Photoelectron Spectrometer (XPS): Thermo Fisher Scientific K-Alpha (USA); Field Emission High-Resolution Scanning / Transmission Electron Microscope: Thermo Fisher Scientific Talos F200i S / TEM (USA); Inductively Coupled Plasma Mass Spectrometer (ICP-MS): Agilent Technologies 7700s (USA); Fourier Transform Infrared Spectrometer (FT-IR): Bruker TENSOR27 (Germany); Ultraviolet-Vis Absorption Spectrometer (UV-Vis): Shimadzu UV-2501PC (Japan); Laser Confocal Microscope: Leica TCS SP5-Ⅱ (Germany); Flow Cytometer: Beckman Coulter (USA).

[0063] Concentration determination of Phe-Ce clusters and Phe-Ce@ICG nanoparticles: The concentration of Phe-Ce clusters was determined by measuring the concentration of cerium using inductively coupled plasma mass spectrometry (ICP-MS) and calculating the concentration of the Phe-Ce clusters. The concentration of ICG in Phe-Ce@ICG nanoparticles was obtained by combining the total mass of the Phe-Ce@ICG nanoparticles with the concentration of the Phe-Ce clusters.

[0064] Statistical analysis: Statistical calculations were performed using software such as Origin 9 and GraphPad Prism 8. Measurement results are expressed as mean ± standard deviation (SD). Differences between groups were statistically analyzed using t-tests (and non-parametric tests).

[0065] Example 1

[0066] like Figure 1 As shown in (A), a method for preparing photothermal antibacterial, anti-inflammatory, and antioxidant amino acid cerium particles includes the following steps:

[0067] Step S1, Preparation of phenylalanine cerium cluster compound (Phe-Ce): 15 mL of anhydrous methanol was added to a single-necked flask, 1 mmol of phenylalanine was added to the anhydrous methanol to obtain an anhydrous methanol solution of phenylalanine, 0.5 mmol of cerium ammonium nitrate was added to the anhydrous methanol solution of phenylalanine, 2-3 drops of triethylamine were added to adjust the pH to 6-7, the reaction was stirred at room temperature for 24 h, the solution was filtered and allowed to stand to obtain phenylalanine cerium cluster compound;

[0068] Step S2, Preparation of amino acid cerium particles (Phe-Ce@ICG): Weigh 1 mg of indocyanine green (ICG) and dissolve it in 1 mL of deionized water to obtain an aqueous solution of indocyanine green; weigh 1 mg of phenylalanine cerium cluster compound (Phe-Ce) and dissolve it in 1 mL of deionized water to obtain an aqueous solution of phenylalanine cerium cluster compound; under ice bath and light-protected conditions, alternately add the aqueous solution of indocyanine green and the aqueous solution of phenylalanine cerium cluster compound to 5 mL of deionized water under ultrasonication. After ultrasonic treatment for 15 min, filter with filter paper, and then place the filtrate in a refrigerator at 4 ℃ for 24 h. Finally, perform ultrafiltration centrifugation (molecular cutoff of 100 kDa, rotation speed of 2000 rpm, time of 8 min) to remove free Phe-Ce and ICG, and obtain photothermal antibacterial, anti-inflammatory and antioxidant amino acid cerium particles Phe-Ce@ICG (stored in a refrigerator at 4 ℃).

[0069] The physicochemical properties of the photothermal antibacterial, anti-inflammatory, and antioxidant amino acid cerium particles prepared in Example 1 were characterized, their ability to scavenge reactive oxygen species (ROS) was determined, their in vitro photothermal antibacterial performance was determined, their in situ spraying healing efficacy in a diabetic mouse model was tested, their wound immunohistochemical and immunofluorescence quantitative analysis was performed, their ability to regulate macrophage polarization was tested, and their in vivo anti-inflammatory effect was tested. The experimental procedures and results are as follows:

[0070] 1. Physicochemical characterization.

[0071] (1) Morphology and size analysis:

[0072] Analysis was performed using transmission electron microscopy (TEM) and dynamic light scattering (DLS), such as... Figure 1 As shown in (B) and (C), Phe-Ce@ICG particles are spherical, uniform in size, and well-dispersed, with an average size of approximately 50 nm. Figure 1 As shown in (G), dynamic light scattering (DLS) measured its hydrodynamic peak size to be approximately 56.82 nm, slightly larger than the TEM result, which may be due to its dispersion state in water. Figure 1 As shown in (H), the Phe-Ce@ICG surface carries a charge of approximately +7.4 mV, a property that enhances its binding ability to bacteria.

[0073] (2) Chemical composition and elemental distribution analysis:

[0074] Analysis was performed using energy-dispersive spectroscopy (EDS), such as... Figure 1 (D), (E), (F) and Figure 2 As shown, Phe-Ce@ICG is composed of C, N, O and Ce elements.

[0075] (3) Crystal structure and thermal stability analysis:

[0076] Crystal structure and thermal stability were analyzed using X-ray diffraction (XRD) and thermogravimetric analysis (TGA), such as... Figure 3 As shown, the characteristic diffraction peaks of Phe-Ce@ICG are in high agreement with the single-crystal structure data predictions, confirming the high purity and well-defined crystal structure of Phe-Ce. Figure 4 Thermogravimetric analysis (Nuclear atmosphere, heating rate 10 °C / min, from room temperature to 800 °C) showed that coordinated water molecules and nitrate ions experienced mass loss between room temperature and 200 °C, while the core structure of the Phe-Ce complex only began to decompose above 240 °C, indicating that it has high thermal stability.

[0077] (4) Verification of chemical structure and functional groups:

[0078] Fourier transform infrared spectrometer (FT-IR) was used (wavenumber range 4000-500 cm⁻¹). -1 Analysis was performed as follows: ICG, Phe-Ce clusters, and Phe-Ce@ICG nanoparticles were accurately weighed and mixed thoroughly with spectral-grade potassium bromide in an agate mortar at a mass ratio of 1:100. The mixture was then dried under reduced pressure and subjected to Fourier transform infrared spectroscopy analysis to collect characteristic absorption band spectra. Figure 1 As shown in (J), Phe-Ce@ICG at 1180 cm⁻¹ -1 The S=O stretching vibration peak of the ICG appears at 1407 cm⁻¹; compared with the ICG spectrum, the peak is at 1407 cm⁻¹. -1 and 1557 cm -1 The peak at that point is the stretching vibration peak of the Ce-O bond, confirming the successful coordination between phenylalanine and cerium.

[0079] (5) Verification of optical properties:

[0080] The synthesis of Phe-Ce@ICG was further verified using ultraviolet-visible absorption spectroscopy (UV-Vis), such as... Figure 1 As shown in (I), free ICG has characteristic absorption peaks at 715 nm and 780 nm, while the absorption peak of Phe-Ce@ICG is red-shifted, indicating the formation of ICG oligomers, which may be due to the interaction between Phe-Ce and ICG.

[0081] (6) Analysis of elemental valence state and surface chemical state:

[0082] like Figure 1As shown in (K), the valence state and surface chemical composition of Phe-Ce@ICG were analyzed by X-ray photoelectron spectroscopy (XPS). Characteristic peaks appeared at 900 eV (Ce), 533 eV (O), 400 eV (N) and 285 eV (C), indicating that Phe-Ce@ICG is mainly composed of C, N, O and Ce. Figure 1 The (L) in the figure specifically shows that Ce has different valence states: Ce(IV) and The binding energies are 882.13 eV, 889.67 eV, and 916.72 eV, respectively; Ce(III) and The binding energies are 855.67 eV and 903.6 eV, respectively, and the ratio of Ce(III) to Ce(IV) is 1.3:1.

[0083] 2. Determination of the ability to scavenge reactive oxygen species (ROS).

[0084] like Figure 5 (A) and Figure 5 As shown in (B), the key reason why wounds in diabetic patients are difficult to heal is the excessive production of reactive oxygen species (ROS), including H2O2, Cerium (Ce) exists primarily in two oxidation states (Ce and •OH). 4+ and Ce 3+ The two states exist and can interconvert. This variable valence state characteristic enables it to participate in redox reactions, exhibiting various enzyme-like activities, thereby catalyzing the scavenging of various reactive oxygen species. Based on this, the scavenging capacity of hydrogen peroxide, hydroxyl radical (•OH) and superoxide anion (…) can be measured. The ROS scavenging performance of Phe-Ce@ICG was comprehensively evaluated by measuring its scavenging capacity, superoxide dismutase (SOD) activity, and total antioxidant capacity.

[0085] Hydrogen peroxide scavenging capacity determination: Based on the reaction of hydrogen peroxide with titanium sulfate to form a yellow titanium peroxide complex precipitate, the yellow intensity of this precipitate after dissolving in strong acid shows a linear relationship with the H2O2 concentration within a certain range. Different concentrations of Phe-Ce@ICG (converted to 10, 20, 50, 100, and 200 μg / mL according to cerium content) were reacted with 10 mM H2O2 solution in ultrapure water for 4 hours. The absorbance at 412 nm was measured using a microplate reader to detect the residual H2O2 content. Figure 5 As shown in (C), the results indicate that Phe-Ce@ICG can effectively remove H2O2. When the cerium concentration increases from 10 μg / mL to 100 μg / mL, its H2O2 removal capacity increases from 10.6% to 51.1%.

[0086] Hydroxyl radical (•OH) scavenging ability determination: Based on the property that hydroxyl radicals (•OH) can bleach methylene blue, the scavenging ability of Phe-Ce@ICG for •OH was evaluated by measuring the absorbance of the remaining methylene blue. First, according to the Fenton reaction principle, a •OH working solution was prepared by mixing 600 μL FeCl2·4H2O and 400 μL H2O2. After 5 minutes, different concentrations of Phe-Ce@ICG were added, and methylene blue was added after 4 hours of reaction. The final concentrations of methylene blue, FeCl2·4H2O, and H2O2 in the system were 15 μM, 1.5 mM, and 1.0 mM, respectively. Finally, the absorbance of the methylene blue solution was measured at a wavelength of 664 nm to evaluate the •OH scavenging effect of Phe-Ce@ICG. Figure 5 As shown in (D), at a cerium concentration of 200 μg / mL, Phe-Ce@ICG can remove 88.7% of •OH; even at a low concentration of 10 μg / mL, it can still achieve a removal rate of 38.7%.

[0087] Superoxide anion ( Scavenging capacity assay: The superoxide anion scavenging assay kit from Nanjing Jiancheng Bioengineering Institute was used for detection. This method simulates the system of superoxide anion free radical generation by xanthine / xanthine oxidase in vivo. The system turns purple-red by adding electron transporters and chromogenic agents, and the absorbance is measured at 550 nm. Different concentrations of Phe-Ce@ICG were mixed with the reagents in the kit, vortexed, and incubated at 37 ℃ for 40 minutes. The chromogenic agent was added and mixed well, and the mixture was centrifuged at 3500 rpm for 10 minutes. The supernatant was collected, and double-distilled water was used as a blank control. The absorbance at 550 nm was measured under a 1 cm optical path. Figure 5 As shown in (E), the scavenging rate of superoxide anions gradually increases with increasing cerium concentration.

[0088] Superoxide dismutase (SOD) activity assay: The SOD activity of Phe-Ce@ICG was evaluated using the Tongren Chemical SOD activity assay kit. The principle is that the highly water-soluble tetrazolium salt WST®-1 reacts with superoxide anions to generate a water-soluble dye. The degree of this reduction reaction is linearly correlated with xanthine oxidase activity and can be inhibited by SOD. The specific steps are as follows: 20 μL of Phe-Ce@ICG solution of different concentrations was added to sample wells and blank well 2; 20 μL of ultrapure water was added to blank wells 1 and 3; 200 μL of WST® working solution was added to each well and mixed well; 20 μL of dilution buffer was added to blank wells 2 and 3 respectively; 20 μL of enzyme working solution was added to sample wells and blank well 1 respectively; after thorough mixing, the mixture was incubated at 37 ℃ for 20 minutes, and the absorbance was measured at 450 nm. Similarly, in the dismutation reaction assay, the simulated SOD activity of Phe-Ce@ICG showed a concentration-dependent increase, such as... Figure 5 As shown in (F), the clearance rate increased from 3.9% at 10 μg / mL to 56.9% at 200 μg / mL.

[0089] Total antioxidant capacity determination of Phe-Ce@ICG: Total antioxidant capacity was determined using the ABTS method. ABTS can be oxidized to green ABTS under the action of appropriate oxidants. •+ Antioxidants inhibit ABTS •+ The formation of ABTS was determined by measuring the concentration at 734 nm. •+ The total antioxidant capacity of a sample can be calculated from its absorbance. The specific steps are as follows: a. Add 20 μL of peroxidase working solution to each well of a 96-well plate; b. Add 10 μL of distilled water or PBS buffer to the blank control wells, 10 μL of Trolox standard solutions of different concentrations to the standard curve wells, and 10 μL of Phe-Ce@ICG solutions of different concentrations to the sample detection wells, and mix gently; c. Add 170 μL of ABTS working solution to each well and mix gently; d. After reacting at room temperature for 6 minutes, measure the absorbance at a wavelength of 734 nm. Figure 5 As shown in (G), the total antioxidant capacity of Phe-Ce@ICG increases with increasing cerium concentration, reaching a scavenging rate of 51.4% at 200 μg / mL. These results demonstrate that Phe-Ce@ICG can effectively scavenge H₂O₂, •OH, and... It can eliminate various ROS in diabetic wounds to promote healing.

[0090] Further investigation into the ability of Phe-Ce@ICG to scavenge intracellular reactive oxygen species (ROS):

[0091] Before cell experiments, the cytotoxicity of different concentrations of Phe-Ce@ICG on cells was assessed using the CCK-8 assay. HUVEC and Raw 264.7 cells were seeded in 96-well plates and cultured for 12 hours. Then, different concentrations of Phe-Ce@ICG (based on ICG concentration) were added to each well, and incubation continued for another 12 hours. After adding CCK-8 solution, the cells were co-incubated for another 2 hours. Finally, the absorbance at 490 nm was measured using a standard procedure. Cell viability was calculated using the CCK-8 assay. Figure 6 As shown, the results indicate that even when the concentration is increased to 80 μg / mL, the cell viability of HUVEC cells and Raw264.7 cells remains close to 100%, fully demonstrating that Phe-Ce@ICG has good biosafety.

[0092] Intracellular reactive oxygen species (ROS) scavenging capacity was detected using the 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) probe. Confocal culture dishes containing cultured cells (human umbilical vein endothelial cells, HUVECs) were treated with PBS, H2O2+PBS, H2O2+ICG, H2O2+Phe-Ce, and H2O2+Phe-Ce@ICG for 12 hours, respectively. After incubation with the DCFH-DA probe for another 20 minutes, the green fluorescence signal of DCFH-DA was detected using a laser confocal microscope. Figure 7 (A) and Figure 7 As shown in (C), both the Phe-Ce group and the Phe-Ce@ICG group showed significant ROS scavenging activity (weakened green fluorescence) compared to the PBS group.

[0093] Oxygen production capacity assay: [Ru(dpp)3]Cl2 (RDPP) was used as a fluorescent probe to monitor intracellular oxygen production in different treatment groups under hypoxic conditions. 4T1 cells were seeded in 6-well plates. After adhesion, the cells were treated with 100 μM H2O2 for 4 hours. After removing the H2O2, the cells were co-incubated with the substrate for 12 hours according to their respective groups. The substrate was then removed and the cells were washed three times with PBS. RDPP was added to a final concentration of 20 μM, and the cells were incubated for another 4 hours. After washing with PBS, the cells were imaged using a confocal laser scanning microscope. Figure 7 (B) and Figure 7 As shown in (D), both the Phe-Ce group and the Phe-Ce@ICG group exhibited significant oxygen generation capacity. As the oxygen concentration increased, the red fluorescence signal of RDPP weakened accordingly. This effect is attributed to the ROS scavenging capacity conferred by cerium ions in Phe-Ce and Phe-Ce@ICG.

[0094] Excessive ROS can induce increased oxidative stress and cell damage, thereby delaying wound healing. Effective ROS clearance and restoration of homeostasis are crucial for normal tissue repair. To further verify the ability of Phe-Ce@ICG to alleviate hypoxia, materials from different treatment groups were added to 4T1 cells treated with H2O2. After incubation in a hypoxic incubator for 12 hours, cells were collected and proteins were extracted. HIF-1α expression levels in each group were detected by Western blotting. Figure 8 As shown, the expression of HIF-1α in the Phe-Ce group and the Phe-Ce@ICG group was significantly reduced, further confirming the property of Phe-Ce to scavenge reactive oxygen species and generate oxygen to alleviate hypoxia.

[0095] like Figure 9 As shown in (A), the effect of Phe-Ce@ICG nanoparticles on promoting cell migration was investigated using a scratch assay: 1×10 5 HUVEC cells were seeded in 12-well plates and incubated for 24 hours before a scratch test was performed. Using a 100 μL pipette tip, a straight line of uniform width was drawn in the center of each well, and cells within the scratched area were removed. Cells were then placed in different treatment environments and divided into five groups: PBS group, H2O2+PBS group, H2O2+ICG group, H2O2+Phe-Ce group, and H2O2+Phe-Ce@ICG group (H2O2 is one of the main sources of ROS, which decomposes within cells to produce hydroxyl radicals, causing oxidative stress. Oxidative stress damages cells, and high levels of ROS impair cell migration; H2O2 was added to establish a cell model of oxidative stress damage). Cell migration was recorded at 0, 12, and 24 hours.

[0096] like Figure 9 As shown in (B) of the diagram, the scratches on the Phe-Ce@ICG group essentially disappeared after 24 hours. Quantitative analysis showed that, as Figure 9 (C) and Figure 9 As shown in (D), the scratch healing rate of human umbilical vein endothelial cells (HUVECs) in the H2O2+Phe-Ce@ICG group was approximately 75.5%, which was nearly 8 times that of the control group (H2O2+PBS group, approximately 9.04%). This confirms that Phe-Ce@ICG treatment can effectively remove ROS, thereby restoring HUVEC activity and promoting cell migration.

[0097] 3. In vitro photothermal antibacterial performance test.

[0098] Effective infection suppression is key to promoting tissue repair in wounds treated with drug-resistant bacteria. Unlike antibiotic therapies that easily induce resistance, photothermal therapy (PTT) kills pathogens through its physical heat ablation mechanism, thus preventing the evolution of drug resistance. The Phe-Ce@ICG nanoparticles described in this invention successfully integrate the antioxidant properties of Phe-Ce with the photothermal conversion properties of ICG. The synergistic effect of these two components endows Phe-Ce@ICG nanoparticles with both highly efficient reactive oxygen species scavenging capabilities and significant photothermal effects.

[0099] Photothermal performance testing of Phe-Ce@ICG: Phe-Ce@ICG solutions of different concentrations (1 mL) were tested under an 808 nm laser (0.5 W / cm²). 2 Irradiation was performed under the irradiation conditions, and the temperature changes of the solution were recorded at 30-second intervals using an infrared thermal imager.

[0100] like Figure 10 As shown in (A), under 808 nm laser irradiation (1.0 W / cm²), 2 At this temperature, the solution temperature gradually increases with increasing Phe-Ce@ICG concentration, indicating its good photothermal properties. Figure 10 As shown in (B), after 5 laser switching cycles, the highest temperature that Phe-Ce@ICG can reach did not change significantly, while the highest temperature of the pure ICG solution gradually decreased, proving that Phe-Ce@ICG nanoparticles have excellent photothermal stability.

[0101] In addition, when using the same amount of ICG, Figure 1 (F) shows that the Phe-Ce@ICG nanoparticles exhibit a redshift in ultraviolet absorption due to aggregation, which may contribute to improving the stability and photothermal properties of the nanomaterial. Figure 10 As shown in (C), the photothermal imaging image clearly shows that the temperature of Phe-Ce@ICG nanoparticles increases significantly with increasing concentration and irradiation time, indicating that they can be used as an excellent photothermal agent for photothermal therapy.

[0102] Based on the excellent photothermal properties of Phe-Ce@ICG, experiments were conducted to specifically target its antibacterial activity. This method avoids the problems associated with traditional antibacterial strategies (such as antibiotic resistance resulting from long-term use). To verify its broad-spectrum antibacterial activity, Gram-positive Staphylococcus aureus was selected. S. aureus ) and Gram-negative Escherichia coli ( E. coli ( ) Perform photothermal treatment. For example Figure 10 As shown in (D), the antibacterial activity of Phe-Ce@ICG was evaluated using the plate count method. Different concentrations of Phe-Ce@ICG were mixed with 1 mL of Escherichia coli or Staphylococcus aureus bacterial suspension (1×10⁻⁶). 7After co-incubation at 37 °C for 12 hours, the cells were irradiated with an 808 nm laser (1.0 W / cm²). 2 After 10 minutes, dilute the bacterial solution and spread it on LB plates. Continue to incubate at 37 °C for 12 hours. The antibacterial performance is evaluated by the difference in colony count.

[0103] To further verify the antibacterial effect, morphological changes of bacteria in different treatment groups were observed using scanning electron microscopy. Bacteria were collected by centrifugation (5000 rpm, 5 minutes) and purified 2-3 times with PBS (pH=7.4). The bacterial samples were then fixed in 2.5% glutaraldehyde solution at 4 °C for 1 hour. The fixed samples were then subjected to a gradient dehydration using ethanol solutions of increasing concentrations (10%, 30%, 50%, 70%, and 90%), with each concentration treated for 10 minutes. Finally, the samples were dispersed in anhydrous ethanol. After sample preparation, morphological changes of the bacteria were observed using scanning electron microscopy.

[0104] like Figure 10 As shown in (E), scanning electron microscopy (SEM) and colony plate results indicate that, compared with the control group, the bacteria in the light-treated group showed significantly damaged morphology and inhibited growth, proving that Phe-Ce@ICG can effectively kill bacteria under light conditions. Figure 10 As shown in (F) and (G), the survival rate of both bacteria gradually decreased as the concentration of Phe-Ce@ICG increased.

[0105] like Figure 10 (H) and Figure 8 As shown in (I), when the concentration reaches 40 μg / mL, the inhibition rates against Staphylococcus aureus and Escherichia coli are 85% and 95%, respectively, indicating that Phe-Ce@ICG has good broad-spectrum antibacterial activity.

[0106] 4. In situ spraying healing efficacy test in a diabetic mouse model.

[0107] To investigate the promoting effect of Phe-Ce@ICG nanoparticles on wound healing, a full-thickness skin defect repair experiment was conducted in a diabetic mouse model. Six-week-old female BALB / c mice (weighing 14-16 grams) provided by Guangdong Yaokang were used. All animals were acclimatized in a specific pathogen-free (SPF) environment for two weeks with free access to food and water. During the experiment, animals were housed in a standardized environment (temperature 23-26 ℃, humidity 40-60%, 12-hour light-dark cycle, 6 mice per cage). Mice were randomly divided into 6 groups (5 mice per group): PBS group, PBS+laser group, Phe-Ce group, Phe-Ce@ICG group, ICG+laser group, and Phe-Ce@ICG+laser group, with the PBS group serving as a control. A 10 mm diameter full-thickness skin wound was created on the back of the mice and inoculated with Staphylococcus aureus to establish an infection model. Treatment was administered 24 hours after infection. The laser treatment group received an 808 nm laser (intensity 0.5 W / cm²). 2 The wound was irradiated for 5 minutes to sterilize it through photothermal conversion, and the healing process was continuously monitored. Wound photographs were taken on days 1, 3, 5, 7, 9, and 11. On day 11, mice were euthanized by cervical dislocation after carbon dioxide inhalation, and wound skin tissue was collected for hematoxylin-eosin (HE) staining and immunofluorescence staining. The indicators detected included CD31, VEGF, HIF-1α, TNF-α, IL-10, and CD86 / CD206. Figure 11 (A) in the diagram shows a schematic of the experiment.

[0108] like Figure 11 As shown in (B), the ICG+ laser group and the Phe-Ce@ICG+ laser group received an 808 nm laser (1.0 W / cm²) after material treatment. 2 Irradiation was performed for 10 minutes, and photothermal images were recorded using an infrared thermal imager for 0-10 minutes. It was observed that the wound temperature increased over time, and... Figure 11 As shown in (C), the wound temperature of the Phe-Ce@ICG+ laser group was higher than that of the ICG+ laser group, and... Figure 8 The experimental results in (C) are consistent.

[0109] like Figure 12 As shown, on day 11, the Phe-Ce@ICG+laser group showed the best healing effect (complete wound closure), while the wounds in the PBS group and the PBS+laser group did not close completely. Figure 13 As shown in (A), the wound healing rate of the Phe-Ce@ICG+laser group was significantly faster than that of the other groups. Figure 13 (B) shows that the mouse weight was not significantly affected during the treatment, further demonstrating the safety of the treatment method.

[0110] To assess the healing efficiency of different groups, the wound skin was stained with hematoxylin and eosin (H&E) on day 11. Figure 14 As shown, the wound in the PBS group was not completely closed and showed extensive inflammatory cell infiltration; while the Phe-Ce@ICG+laser group showed an intact epidermal layer, exhibiting high re-epithelialization and a significant reduction in inflammatory cells. These results indicate that the prepared Phe-Ce@ICG nanoparticles possess excellent wound healing promoting capabilities.

[0111] 5. Immunohistochemical and immunofluorescence quantitative analysis of the wound.

[0112] To further investigate the ability and potential mechanism of Phe-Ce@ICG in scavenging ROS and accelerating wound healing in vivo, immunohistochemical and immunofluorescence staining analyses were performed on wound tissue. CD31 (platelet-endothelial cell adhesion molecule) and VEGF (vascular endothelial growth factor) play key synergistic roles in wound healing and angiogenesis promotion, and are often used as indicators for monitoring the healing process: VEGF is the most important angiogenic factor and a core signaling molecule driving new blood vessel formation during wound healing; CD31 mediates endothelial cell adhesion, which helps form and maintain vascular integrity and reduce leakage. The expression level of HIF-1α is positively correlated with the degree of hypoxia, and therefore can also be used as a biomarker for wound healing.

[0113] The inventors discovered through research that, for example Figure 15 As shown in (A), compared with the control group (PBS + laser group), the expression levels of CD31 and VEGF in the wound tissue of the Phe-Ce@ICG + laser group were significantly increased, while the expression of HIF-1α was significantly inhibited. Figure 15 As shown in (B), (C), and (D), the quantitative fluorescence results of the wound site further confirm that Phe-Ce@ICG nanoparticles can alleviate hypoxia, promote angiogenesis, and thus accelerate wound healing.

[0114] 6. Experiment on the ability to regulate macrophage polarization.

[0115] Reactive oxygen species (ROS) play a crucial role in macrophage polarization: excessive ROS promotes macrophage polarization towards the M1 type. M1 macrophages secrete pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α), thereby exacerbating the inflammatory response; while M2 macrophages secrete anti-inflammatory cytokines such as interleukin-10 (IL-10) or transforming growth factor-β (TGF-β), thereby alleviating the inflammatory response and promoting wound healing. CD206 (mannose receptor) is often used as a marker for recognizing M2 macrophages. We analyzed macrophages from different treatment groups using flow cytometry, detecting the expression of CD206 on their cell surface by fluorescence signal intensity.

[0116] To verify the polarization ability of Phe-Ce@ICG on macrophages, such as Figure 16 As shown in (A), M0 type RAW 264.7 macrophages were first pretreated with 10 μg / mL LPS (lipopolysaccharide) and IFN-γ (interferon-γ) to induce their differentiation into M1 type, followed by treatment with different materials. Cells and cell supernatants were collected separately for flow cytometry and cytokine analysis. Figure 16 As shown in (B) and (C), enzyme-linked immunosorbent assay (ELISA) revealed that macrophages treated with Phe-Ce@ICG secreted a large amount of the anti-inflammatory cytokine TGF-β, while the expression of the pro-inflammatory cytokine TNF-α was significantly reduced. Meanwhile, as... Figure 16 As shown in (D), compared with the PBS group, the proportion of M2 macrophages in the Phe-Ce@ICG treatment group increased from 1.9% to 24.6%. Treatment of macrophages with free Phe-Ce alone also significantly affected these cytokines, indicating that both Phe-Ce and Phe-Ce@ICG have anti-inflammatory effects. In vitro experiments demonstrated that Phe-Ce@ICG nanoparticles exhibit excellent effects in remodeling macrophage polarization and inhibiting inflammation.

[0117] 7. In vivo anti-inflammatory effect test.

[0118] To further evaluate the in vivo anti-inflammatory effect of Phe-Ce@ICG, wound tissue was collected from mice on day 11 post-treatment. Immunofluorescence staining was performed on sections to analyze the ratio of M1 / M2 macrophages and the corresponding cytokine levels at the wound site. CD86 and CD206 were selected as biomarkers for detecting M1 and M2 macrophages, respectively.

[0119] like Figure 17 As shown in (A), (B), and (C), the experimental results indicate that the CD206 green fluorescence of M2 macrophages in the Phe-Ce@ICG group was significantly enhanced, while the CD86 red fluorescence of M1 macrophages producing inflammation was significantly weakened. Furthermore, as shown in (A), (B), and (C), the experimental results show that the CD206 green fluorescence of M2 macrophages in the Phe-Ce@ICG group was significantly enhanced, while the CD86 red fluorescence of M1 macrophages producing inflammation was significantly weakened. Figure 17 As shown in (D), (E), and (F), compared with the PBS and ICG groups, the Phe-Ce@ICG group exhibited weaker red fluorescence of the pro-inflammatory factor TNF-α and stronger red fluorescence of the anti-inflammatory factor IL-10, indicating that this group has a good anti-inflammatory effect. Combined with the above experimental results, this further verifies that Phe-Ce@ICG has excellent anti-inflammatory effects both in vitro and in vivo.

[0120] To conduct biosafety testing, a hemolysis test was performed: Fresh mouse blood was placed in an anticoagulant tube, centrifuged (3000 rpm, 4 ℃, 5 minutes), and the supernatant was discarded. The blood was resuspended and washed with PBS, and the above operation was repeated until the supernatant was colorless. Red blood cells from the bottom were collected and diluted with PBS to 9 mL. The resulting red blood cell suspension was stored at 4 ℃ for later use. The above red blood cell suspension was then mixed with Phe-Ce@ICG nanoparticle solutions at concentrations of 5 μg / mL, 10 μg / mL, 20 μg / mL, 40 μg / mL, and 80 μg / mL (calculated according to ICG concentration), respectively. Pure water was used as a positive control, and PBS as a negative control. 0.4 mL of the red blood cell suspension was added to 1 mL of each concentration solution in a 1.5 mL centrifuge tube, gently mixed, and incubated at 4 ℃ for 2 hours. After standing, centrifuge (3000 r / min, 4 ℃, 5 minutes), take 1 mL of supernatant and dilute to 3 mL. Calculate the hemolysis rate using the UV absorption spectrum of the test solution at 541 nm. The calculation formula is: Hemolysis rate (%) = (Experimental group absorbance - Negative control group absorbance) / (Positive control group absorbance - Negative control group absorbance) × 100%. Figure 18 As shown, the hemolysis rate of Phe-Ce@ICG nanoparticles was less than 5% as determined by the hemolysis test, confirming that they have good blood compatibility.

[0121] To conduct biosafety testing, a major organ sampling experiment was performed: 11 days after treatment, one mouse was randomly selected from each group, and its heart, liver, spleen, lungs, and kidneys were collected and preserved in 4% paraformaldehyde solution for hematoxylin-eosin (H&E) staining. Figure 19 As shown, H&E staining of the major organs of the experimental mice revealed no pathological lesions in any tissue, indicating that Phe-Ce@ICG nanoparticles have good biocompatibility in vivo.

[0122] In summary, the photothermal antibacterial, anti-inflammatory, and antioxidant amino acid cerium particles of this invention achieve scarless wound repair through the synergistic effects of photothermal effect, antibacterial, anti-inflammatory, and antioxidant properties. This invention synthesizes a water-soluble cerium-based biomimetic nanocatalyst, Phe-Ce@ICG, using a green method. This catalyst not only possesses the ability to scavenge ROS and promote macrophage polarization towards the anti-inflammatory M2 phenotype, but also exhibits photothermal antibacterial function. Under light irradiation, it achieves specific bactericidal effects both in vitro and in vivo, and demonstrates excellent biocompatibility.

[0123] Addressing the pathological characteristics of chronic diabetic wounds, including severe hypoxia, excessive ROS, a complex inflammatory microenvironment, and bacterial infection, this invention utilizes the antioxidant, anti-inflammatory, and antibacterial properties of Phe-Ce@ICG for wound repair. It effectively removes excess ROS and generates oxygen, thereby improving local hypoxia, enhancing cell proliferation and migration, and promoting angiogenesis. Simultaneously, it polarizes pro-inflammatory M1 macrophages into an anti-inflammatory M2 phenotype. This effect synergizes with the photothermal antibacterial activity of ICG, ultimately accelerating the healing of diabetic wounds in mice. The Phe-Ce@ICG of this invention can be formulated as a spray-type treatment for infectious diabetic wounds. The spray formulation allows for convenient administration, offering advantages such as ease of use and portability, providing an efficient and convenient solution for the clinical treatment of diabetic wounds.

Claims

1. A method for preparing photo-thermal antibacterial anti-inflammatory antioxidant amino acid cerium particles, characterized in that, The method comprises the following steps: Step S1, preparation of phenylalanine cerium cluster: adding cerium ammonium nitrate into a phenylalanine anhydrous methanol solution, stirring at room temperature to obtain a phenylalanine cerium cluster; Step S2, preparation of amino acid cerium particles: dissolving indocyanine green in water to obtain an indocyanine green aqueous solution; dissolving the phenylalanine cerium cluster in water to obtain a phenylalanine cerium cluster aqueous solution; under ultrasonic, the indocyanine green aqueous solution and the phenylalanine cerium cluster aqueous solution are alternately added into deionized water, and after ultrasonic treatment, the photo-thermal antibacterial anti-inflammatory antioxidant amino acid cerium particles are obtained.

2. The process for the preparation of photo-thermal, antibacterial, anti-inflammatory and antioxidant amino acid cerium particles as claimed in claim 1, wherein, In the step S1, the molar ratio of phenylalanine to cerium ammonium nitrate is 2:

1.

3. The process for the preparation of photo-thermal, antibacterial, anti-inflammatory and antioxidant amino acid cerium particles as claimed in claim 1, wherein, In the step S1, the stirring is performed at room temperature for 24 h.

4. The process for the preparation of photo-thermal, antibacterial, anti-inflammatory and antioxidant amino acid cerium particles as claimed in claim 1, wherein, In the step S1, after adding the cerium ammonium nitrate into the phenylalanine anhydrous methanol solution, triethylamine is added to adjust the pH of the solution to 6-7.

5. The process for the preparation of photo-thermal, anti-bacterial, anti-inflammatory and anti-oxidant amino acid cerium particles as claimed in claim 1, wherein, In the step S2, the concentration of the indocyanine green aqueous solution is 1 mg / mL, the concentration of the phenylalanine cerium cluster aqueous solution is 1 mg / mL, and the mass ratio of indocyanine green to phenylalanine cerium cluster is 1:

1.

6. The process for the preparation of photo-thermal, anti-bacterial, anti-inflammatory and anti-oxidant amino acid cerium particles as claimed in claim 1, wherein, In the step S2, under ice bath and light shielding conditions, the indocyanine green aqueous solution and the phenylalanine cerium cluster aqueous solution are alternately added into deionized water under ultrasonic, and then ultrasonic treatment is performed for 15 min.

7. The process for the preparation of photo-thermal, antibacterial, anti-inflammatory and antioxidant amino acid cerium particles as claimed in claim 6, wherein, After the ultrasonic treatment for 15 min, filtration is performed, and after the filtration, the photo-thermal antibacterial anti-inflammatory antioxidant amino acid cerium particles are obtained after standing at 4 ℃ for 12-24 h and after ultrafiltration centrifugation.

8. The process for the preparation of photo-thermal, antibacterial, anti-inflammatory and antioxidant amino acid cerium particles as claimed in claim 7, wherein, The molecular cut-off of the ultrafiltration centrifugation is 100 kDa, the rotation speed is 2000-2500 rpm, and the time is 8-10 min.

9. A photo-thermal antibacterial anti-inflammatory antioxidant amino acid cerium particle, characterized in that, The photo-thermal antibacterial anti-inflammatory antioxidant amino acid cerium particles are prepared by using the preparation method of the photo-thermal antibacterial anti-inflammatory antioxidant amino acid cerium particles according to any one of claims 1-8.

10. The photo-thermal antibacterial anti-inflammatory antioxidant amino acid cerium particles according to claim 9 are used in the preparation of an infectious diabetic wound treatment drug.

Images