A method for synthesizing photothermal antibacterial nanoparticles

Abstract

The application provides a synthesis method of photothermal antibacterial nanoparticles, and belongs to the technical field of nanopharmaceutical preparations. Silk fibroin nanogold clusters are added drop by drop into an acetone solution, stirred until the acetone is completely volatilized, and in the process of volatilization of the acetone, the silk fibroin nanogold clusters self-assemble to form nanoparticles; under weak reduction conditions, a silver solution is added to the nanoparticles, in-situ growth is performed to obtain silk fibroin / gold / silver nanoparticles; the silk fibroin / gold / silver nanoparticles are dispersed in deionized water, a buffer solution, a tyrosine aqueous solution and tyrosinase are added, and in-situ growth is performed to obtain melanin-like coated photothermal antibacterial nanoparticles. The photothermal antibacterial nanoparticles constructed by the method have high efficient near-infrared region photothermal conversion performance, excellent free radical scavenging capacity, synergistic antibacterial property and good biocompatibility.

Description

Technical Field

[0001] This application relates to a method for synthesizing photothermal antibacterial nanoparticles, belonging to the field of nanomedicine formulation technology. Background Technology

[0002] Photothermal therapy (PTT) has gained increasing attention due to its advantages such as spatiotemporal controllability, low invasiveness, and low likelihood of inducing drug resistance. This therapy converts light energy, such as near-infrared light with deep tissue penetration, into heat energy, using localized high temperatures to cause irreversible damage to bacteria. However, existing photothermal antibacterial systems still face several key challenges: First, most photothermal conversion agents (such as gold nanorods, carbon-based materials, and near-infrared dyes) suffer from poor photostability, insufficient biocompatibility, or a tendency to aggregate. Second, single photothermal antibacterial strategies often neglect the regulation of the infection microenvironment while killing bacteria. Bacterial infections are often accompanied by excessive inflammatory responses; the accumulation of large amounts of reactive oxygen species (ROS) not only exacerbates tissue damage but also inhibits immune cell function and delays wound healing. Therefore, ideal antibacterial materials should not only effectively kill bacteria but also possess the ability to scavenge excess free radicals and alleviate oxidative stress, thereby achieving synergistic "bactericidal-anti-inflammatory" treatment. However, there are currently few reports on functional materials that meet these conditions.

[0003] Silk fibroin, as a natural polymer material, possesses excellent biocompatibility, biodegradability, and abundant functional group modification sites, making it an ideal matrix for constructing functional biomedical nanomaterials. In recent years, functional nanomedicines with photothermal effects, constructed using silk fibroin as a carrier, have been widely reported, demonstrating broad application potential in antibacterial, antitumor, and tissue engineering fields. However, many of these silk fibroin-based nanomedicines, especially photothermal nanomedicines, suffer from problems such as single function, low photothermal activity efficiency, and poor stability due to simple composite construction, making it difficult to effectively meet complex clinical application needs. For anti-infective and anti-inflammatory applications, silk fibroin nanomaterials with both highly efficient synergistic antibacterial and excellent free radical scavenging functions remain to be developed. Summary of the Invention

[0004] In view of this, this application provides a method for synthesizing photothermal antibacterial nanoparticles, which endow the constructed photothermal antibacterial nanoparticles (SAAM) with efficient near-infrared photothermal conversion performance, excellent free radical scavenging ability, synergistic antibacterial properties and good biocompatibility.

[0005] Specifically, this application is implemented through the following scheme: A method for synthesizing photothermal antibacterial nanoparticles, comprising the following steps: Step 1: Silk fibroin gold nanoclusters (SF-AuNCs) are added dropwise to an acetone solution and stirred until the acetone is completely evaporated. During the evaporation of acetone, the silk fibroin gold nanoclusters self-assemble into nanoparticles. The nanoparticles are collected, washed, and dispersed under ice bath conditions to obtain a nanoparticle solution (SA).

[0006] Step 2: Under weak reducing conditions, silver solution is added to the nanoparticle solution (SA). After in-situ growth, the precipitate is collected by centrifugation, the supernatant is discarded, and the precipitate is resuspended in ultrapure water to obtain silk fibroin / gold / silver nanoparticles (SAA).

[0007] Step 3: Disperse silk fibroin / gold / silver nanoparticles (SAA) in deionized water, add buffer solution, tyrosine aqueous solution and tyrosinase, grow in situ, collect the precipitate, discard the supernatant, resuspend the precipitate with ultrapure water to obtain melanin-like photothermal antibacterial nanoparticles (SAAM).

[0008] Furthermore, as a preferred option: In step one, The preparation method of the silk fibroin gold nanoclusters is as follows: a silk fibroin solution is mixed with a glutathione aqueous solution to obtain a GSH / SF mixed solution; an HAuCl4 aqueous solution is added, and the silk fibroin gold nanoclusters are prepared in a one-pot process at 70-80°C. More preferably, in the GSH / SF mixed solution, the concentration of silk fibroin is 1-20 mg / mL (preferably 10-15 mg / mL); the concentration of glutathione is 0.4-1.6 mmol / L (preferably 1.0-1.2 mmol / L); and the concentration of the HAuCl4 aqueous solution is 0.25-5 mmol / L (preferably 0.8-1.4 mmol / L). As a preferred embodiment, in the GSH / SF mixed solution, the concentration of silk fibroin is 10 mg / mL, the concentration of glutathione is 1.2 mmol / L, and the concentration of the HAuCl4 aqueous solution is 1 mM. The silk fibroin gold nanoclusters prepared by this method have a spherical structure, and their performance is comparable to that of fluorescent proteins, exceeding that of most molecular dyes and fluorescent proteins by approximately three orders of magnitude. Compared with classic glutathione-templated gold nanoclusters (GSH-AuNCs), its stability is significantly improved.

[0009] The volume ratio of the silk fibroin gold nanoclusters to the acetone solution is 1:3 to 10, with 1:3 to 5 being preferred.

[0010] In step two, The weak reducing conditions are achieved by adding any one of the following reagents: sodium citrate, ascorbic acid, dopamine, and hydroxylamine hydrochloride. Under the condition that the concentration of the nanoparticle solution is 1.0 mg / mL, the concentration of the added reagent is 0.5–5 mM, with 1–2 mM being preferred.

[0011] The silver solution is any one of silver nitrate solution, silver acetate, or silver chlorate. Under the condition that the concentration of the nanoparticle solution is 1.0 mg / mL, the concentration of the silver solution is 0.1–0.5 mM, preferably 0.2–0.25 mM.

[0012] The in-situ growth temperature is 80–100℃.

[0013] In step three, The buffer solution is any one of Tris-HCl buffer solution (concentration 50-200 mM, pH=6.8-8.8), phosphate buffer solution (concentration 10-200 mM, pH=6.5-7.5), or HEPES buffer solution (concentration 10-50 mM, pH=7.0-7.5).

[0014] When the concentration of the silk fibroin / gold / silver nanoparticles dispersed in deionized water is 1.0 mg / mL, the concentration of the tyrosine aqueous solution is 0.5–2 mg / mL (preferably 1–2 mg / mL), and the concentration of the tyrosinase is 200–500 U / mL (preferably 250–350 U / mL).

[0015] The above scheme uses silk fibroin as a carrier and completes the stepwise construction of nanoparticles through self-assembly and gradient in-situ growth strategies: First, silk fibroin gold nanoclusters (SF-AuNCs) are synthesized in situ. Then, the intermolecular interactions of silk fibroin allow the gold nanoclusters to form self-assembled nanoparticles (SA). Simultaneously, silver is grown in situ on the self-assembled nanoparticles using the gold nanoclusters as anchors to obtain silk fibroin / gold / antibacterial nanoparticles (SAA). Finally, melanin-like pigments are synthesized in situ on the nanoparticle surface using an enzymatic method with tyrosine-rich silk fibroin as anchors to obtain melanin-like pigment-coated photothermal antibacterial nanoparticles (SAAM), thus completing the in-situ growth of gold and silver nanoparticles and the synthesis of melanin-like pigments. The polyphenolic structure of melanin-like pigments endows the nanomedicine with excellent antioxidant activity. The synergistic effect of gold and silver nanoparticles and melanin-like pigments forms a highly efficient near-infrared photothermal conversion efficiency. The photothermal effect and Ag synergistically achieve highly efficient antibacterial activity. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of this application, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application.

[0017] Figure 1 The figure shows the physicochemical properties of SAAM. Part (a) is the particle size distribution and part (b) is the TEM image.

[0018] Figure 2 This is an elemental surface scan of SAAM's X-ray energy spectrum.

[0019] Figure 3 The figure shows the XPS spectrum of SAAM. Parts (a) to (f) are the full XPS spectrum, C 1s, O 1s, N 1s, Au4f and Ag 3d spectra, respectively.

[0020] Figure 4 The figure shows the antioxidant properties of SAAM. Part (a) represents the ABTS free radical scavenging ability, and part (b) represents the DPPH free radical scavenging ability.

[0021] Figure 5 This is the UV-Vis absorption spectrum of SAAM.

[0022] Figure 6 To illustrate the photothermal properties of SAAM, part (a) shows the temperature rise curves of SAAM at different concentrations; part (b) shows the temperature rise curves of SAAM at different near-infrared light power densities; and part (c) shows the temperature rise curves of SAAM at 1.0 W / cm². 2 The temperature change curve after 4 switching cycles under 808 nm laser irradiation; (d) part is the linear time data of the cooling period and the linear fit of -lnθ.

[0023] Figure 7 This is due to the cytotoxicity of SAAM.

[0024] Figure 8 To demonstrate the hemolytic properties of SAAM, part (a) of the figure shows a representative image of freshly isolated red blood cells after incubation in SAAM at a concentration of 100–500 μg / mL for 1 hour, and part (b) shows the hemolysis rate.

[0025] Figure 9 To demonstrate the photothermal antibacterial properties of SAAM, part (a) of the figure shows photographs of Escherichia coli and Staphylococcus aureus LB culture plates after treatment with PBS, PBS+near-infrared light, SAAM, and SAAM+near-infrared light; part (b) shows the bacterial survival rates of Escherichia coli and Staphylococcus aureus. Detailed Implementation

[0026] To make the technical problems, technical solutions, and beneficial effects to be solved by this application clearer, the technical solutions in the embodiments of this application will be further described in detail below with reference to the accompanying drawings. It should be understood that the specific embodiments described herein are only used to explain this application and are not intended to limit the technical solutions of this application. All other embodiments obtained by those skilled in the art based on the embodiments in this application without creative effort are within the scope of protection of this application.

[0027] The reagent information used in this embodiment is as follows: L-tyrosine (99%) and sodium citrate (99.0%) were purchased from Shanghai Maclean Biochemical Technology Co., Ltd. Tyrosinase was purchased from Shanghai E. En Chemical Technology Co., Ltd. Tris-HCl buffer (0.5 M, pH 6.8) and acetone were purchased from Shanghai Sangon Biotech Co., Ltd. Tris-HCl buffer (1.5 M, pH 8.8) was purchased from Shanghai Beyotime Biotechnology Co., Ltd. Tetrachloroauric acid (HAuCl4·4H2O) was purchased from Shanghai Haoyuan Pharmaceutical (Leyan). Silver nitrate was purchased from Sigma-Aldrich.

[0028] All chemical reagents used are reagent grade and are used directly without any additional treatment.

[0029] All other unlisted reagents are commercially available and can be used directly without additional processing.

[0030] Deionized water was used throughout the entire experiment.

[0031] This embodiment provides a method for synthesizing photothermal antibacterial nanoparticles, the steps of which are as follows: S1, Synthesis of Silk Fibroin Nanogold Cluster Self-Assembled Nanoparticles (SA) A solution of silk fibroin gold nanoclusters (SF-AuNCs) was slowly added dropwise to an acetone solution (SF-AuNCs / acetone = 1:5, v / v). The mixture was magnetically stirred at room temperature in a fume hood until the acetone was completely evaporated. During the acetone evaporation process, SF-AuNCs gradually self-assembled into nanoparticles (SA). The self-assembled nanoparticles were then collected by centrifugation at 12000 rpm / min for 10 min, washed three times with ultrapure water, and then ultrasonically dispersed at 195 W for 10 min in an ice bath. Finally, the solution concentration was determined by weighing, and the synthesized SA was stored at 4 ℃ for later use.

[0032] The silk fibroin gold nanoclusters (SF-AuNCs) were prepared by the following method: a silk fibroin (SF) solution (10 mg / mL) was mixed with a glutathione (GSH) aqueous solution (1.2 mM) to obtain a GSH / SF mixed solution. Subsequently, an aqueous solution of HAuCl4 (1.0 mM) was added to the GSH / SF mixed solution, and the reaction was carried out at 80 °C for 6 hours. After the reaction, the solution turned light yellow, indicating that the silk fibroin gold nanoclusters were successfully synthesized. The average diameter was about 1.8 nm. Its UV-Vis absorption spectrum showed an absorption peak of SF at 275 nm and a characteristic weak peak of AuNCs near 410 nm. The fluorescence emission peak was located near 600 nm, and its excitation peak was located near 400 nm. The Stokes shift was 200 nm, the quantum yield was 5.42%, and the fluorescence lifetime was near 3.47 μs.

[0033] S2, Synthesis of Silk Fibroin / Gold / Silver Nanoparticles (SAA) SAA was prepared by in-situ growth of silver nanoparticles using silk fibroin gold nanoclusters (SA) as a template under weak reducing conditions. SA solution (1.0 mg / mL), sodium citrate (1 mM), silver nitrate (0.25 mM), and deionized water were mixed and reacted in a 90 ℃ water bath for 45 min. The precipitate (SAA) was collected by centrifugation (12000 rpm, 10 min), the supernatant was discarded, and the precipitate was resuspended in ultrapure water. This process was repeated three times by centrifugation. Finally, the purified SAA was dispersed in deionized water and stored at 4 ℃ in the dark for later use.

[0034] Synthesis of S3, melanin-like coated SAA nanoparticles (SAAM) SAAM was prepared by in-situ enzymatic growth of melanin on the surface of SAA. Silk fibroin / gold / silver nanoparticle aqueous solution (SAA, 1.0 mg / mL), tyrosine aqueous solution (1.0 mg / mL), and tyrosinase (250 U / mL) were added sequentially to Tris-HCl buffer (100 mM, pH 7.8). The mixture was magnetically stirred for 1 hour in a 37 ℃ water bath. The black precipitate (SAAM) was collected by centrifugation (12000 rpm, 10 min), the supernatant was discarded, and the precipitate was resuspended in ultrapure water. The centrifugation and washing were repeated three times. Finally, the purified SAAM was dispersed in deionized water and stored at 4 ℃ in the dark for later use.

[0035] The preparation process for the Tris-HCl buffer solution (100 mM, pH 7.8) is as follows: 1. Stock solutions: Two commercially available Tris-HCl stock solutions were used: Tris-HCl buffer (0.5 M, pH 6.8) and Tris-HCl buffer (1.5 M, pH 8.8).

[0036] 2. Dilution: Dilute both stock solutions with ultrapure water to prepare 100 mM single-component buffer solutions. ① 0.5 M (pH 6.8) stock solution: Take 1 part of stock solution + 4 parts of ultrapure water and make up to volume to obtain Tris-HCl (100mM, pH 6.8). ② 1.5 M (pH 8.8) stock solution: Take 1 volume of stock solution and dilute it with water to 15 times the volume to obtain Tris-HCl (100 mM, pH 8.8). Dilution follows the law of dilution (C1V1=C2V2) (a general formula in physicochemical science).

[0037] 3. Preparation of working solution: Mix equal volumes of Tris-HCl (100mM, pH 6.8) and Tris-HCl (100mM, pH 8.8) in a 1:1 ratio. The final concentration after mixing is 100 mM and pH=7.8, which is the Tris-HCl buffer solution (100mM, pH 7.8).

[0038] The product synthesized by the above method was characterized, and the results are as follows: 1. Physicochemical characterization of SAAM The synthesized SAAM aqueous dispersion was grayish-black in appearance, with no obvious precipitation, exhibiting good colloidal dispersion behavior. Dynamic light scattering (DLS) results. Figure 1 As shown: The average hydrodynamic diameter of this nanoparticle is approximately 270 nm (see...). Figure 1 The polydispersity index (PDI) of part (a) is 0.286, indicating a relatively narrow particle size distribution and good dispersion uniformity. Transmission electron microscopy (TEM) observation shows (see...) Figure 1 (Part (b)) shows that the nanoparticles are spherical or near-spherical with an average diameter of approximately 100 nm. The hydrodynamic particle size is larger than the electron microscopic observation particle size, which can be attributed to the presence of a hydration layer on the surface of the nanoparticles and their slight aggregation in the aqueous dispersion system. In addition, the zeta potential of the SAAM aqueous dispersion is −44.3 mV, indicating excellent colloidal stability.

[0039] The UV-Vis absorption spectra were measured in the wavelength range of 200 nm to 1000 nm using a multifunctional microplate reader (Shanghai Flash Spectro Biotechnology Co., Ltd., SuperMax 3200). The average diameter and zeta potential of the nanoparticles were determined using a nanoparticle analyzer (HORIBA Scientific, nanoPartica SZ-100V2). Transmission electron microscopy (TEM) images and elemental mapping images were acquired using a transmission electron microscope (Hitachi High Technology Co., Ltd., Japan) at an accelerating voltage of 200 kV. X-ray photoelectron spectroscopy (XPS) analysis was performed using an ESCALAB 250Xi electron spectrometer (Thermo Fisher Scientific, USA).

[0040] X-ray energy dispersive spectroscopy elemental surface scan results are as follows: Figure 2As shown, SAAM is composed of elements such as C, N, O, Au, and Ag. The C, N, and O elements are distributed continuously and uniformly on the surface, completely covering the nanoparticle region and together forming the organic framework of silk fibroin-like melanin, providing support for the metal nanoparticles. The Au element is distributed in a discrete point-like form, which highly overlaps with the distribution area of ​​the Ag element. Both are distributed inside the nanoparticles, indicating that the Au and Ag nanoparticles achieve in-situ loading and uniform dispersion in the organic matrix.

[0041] X-ray photoelectron spectroscopy (XPS) results further confirmed the surface elemental composition and chemical valence state of SAAM. Full-spectrum scanning revealed characteristic peaks for C 1s, O 1s, N 1s, Au 4f, and Ag 3d on the SAAM surface (see...). Figure 3 The results were consistent with those obtained from EDS elemental surface scanning. The binding energies of Au 4f were measured to be 84.4 eV and 88.2 eV, respectively. The binding energies of Ag 3d were 368.3 eV and 374.3 eV, corresponding to Ag 3d5 / 2 and Ag 3d3 / 2 orbitals, respectively. Notably, the XPS high-resolution O 1s spectrum showed a characteristic peak at 533.2 eV, attributed to the phenolic hydroxyl group (C-OH) in the catechol structure. Simultaneously, the UV-Vis absorption spectrum showed a distinct characteristic absorption peak at 280 nm, further confirming the presence of a large number of catechol groups in the SAAM molecular structure. This structural feature lays the structural foundation for its subsequent antioxidant properties.

[0042] 2. Evaluation of SAAM's antioxidant properties To clarify the in vitro antioxidant activity of SAAM, this invention employs two methods: the 2,2-diphenyl-1-picrylhydrazine (DPPH) free radical scavenging assay and the total antioxidant capacity of 2'-azobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), to detect its free radical scavenging capacity and total antioxidant level, respectively.

[0043] The DPPH free radical scavenging experiment was performed as follows: First, a 50 µM DPPH ethanol solution was prepared. Then, 10 μL of SAAM samples at different concentrations (25-400 μg / mL) were added to a 96-well plate and thoroughly mixed with the DPPH ethanol solution. The reaction mixture was incubated at room temperature in the dark for 30 min, and the absorbance of each well at 517 nm was measured using a microplate reader. The DPPH scavenging rate (%) was calculated using the following formula: .

[0044] Among them: A S For the sample group, the absorbance value of the mixture of the test solution and DPPH ethanol solution; A bFor the blank group, the absorbance value of the mixture of the test solution and anhydrous ethanol solution; A c The absorbance value of the mixture of DPPH ethanol solution and ethanol solution serves as a control group.

[0045] The determination of total antioxidant capacity of ABTS was strictly performed according to the instructions of the Total Antioxidant Capacity Assay Kit (ABTS method, S0119, Beyotime). The specific steps were as follows: 200 μL of ABTS working solution was added to each well of a 96-well plate. 10 μL of PBS solution was added to the blank control wells, and 10 μL of SAAM at different concentrations (final concentrations of 25, 50, 100, 200, and 400 μg / mL) was added to the sample wells. After gentle mixing, the plates were incubated at room temperature in the dark for 6 min. The absorbance at 734 nm was measured using a microplate reader, and the ABTS scavenging rate (%) was calculated using the following formula: .

[0046] Among them: A S A represents the absorbance value of the sample group. b The absorbance value is for the blank control group.

[0047] SAAM's antioxidant properties, such as Figure 4 As shown, SAAM is related to ABTS. + • The scavenging ability of SAAM exhibits a clear concentration-dependent effect. Within 5 minutes, the scavenging rates of SAAM solutions at concentrations of 200 and 400 μg / mL reached over 85.4% ± 0.31% and 97.8% ± 0.20%, respectively (see...). Figure 4 (See part (a)). This effect is mainly due to the large number of catechol groups in the SAAM molecular structure. These groups can effectively scavenge free radicals in the system by donating hydrogen atoms or electrons, thus endowing SAAM with excellent antioxidant activity. In the DPPH free radical scavenging experiment, the scavenging rate also gradually increased with the increase of SAAM concentration. The scavenging rates of different concentration groups were 20.7%±0.34%, 32.1%±0.47%, 46.8%±1.60%, 69.3%±3.23%, and 83.4%±3.55%, respectively (see [reference]). Figure 4 (part (b) of the text).

[0048] This demonstrates that SAAM possesses excellent antioxidant activity, making it a promising candidate for use as a reactive oxygen species scavenger in alleviating oxidative stress.

[0049] 3. Photothermal characterization To systematically investigate the photothermal conversion performance of SAAM, experiments were conducted from three dimensions: solution concentration, laser power density, and photothermal cycling stability. SAAM aqueous dispersions with concentrations of 0, 250, 500, 750, and 1000 μg / mL were prepared, and a power density of 1.0 W / cm² was used. 2 An 808 nm laser was used to irradiate the solution for 10 min. A SAAM aqueous dispersion with a concentration of 500 μg / mL was then used, and the solutions were irradiated with lasers at power densities of 0.5, 0.75, 1.0, 1.25, and 1.5 W / cm². 2 Irradiate with an 808 nm laser for 10 min. A 500 μg / mL SAAM aqueous dispersion is then prepared using a 1.0 W / cm² laser. 2 Four photothermal cycles were conducted using an 808nm laser (one cycle consisted of 5 minutes of heating followed by 5 minutes of cooling). Throughout the experiment, a FLIR E76 infrared thermal imager was used to monitor and record the corresponding temperature changes in real time.

[0050] UV-Vis absorption spectra, such as Figure 5 As shown, SAAM exhibits a broad and strong absorption band in the near-infrared (NIR) region (700–1000 nm), which indicates a correlation between its near-infrared absorption capacity and photothermal activity.

[0051] Based on this, the photothermal conversion efficiency of SAAM under near-infrared illumination was evaluated using an infrared thermal imaging system, and the results are as follows: Figure 6 As shown. First, we recorded different concentrations (e.g., 0, 0.25, 0.5, 0.75, 1.0 mg / mL) of SAAM at a power density of 1.0 W / cm². 2 The temperature rise under 808 nm laser irradiation was investigated. Results showed that in in vitro experiments, a 0.5 mg / mL SAAM solution increased in temperature by 26.5 °C within 10 min, while the temperature of the water-based control group increased by only 4.9 °C (see [link to study]). Figure 6 (See part (a)). Furthermore, the temperature of the SAAM solution is also affected by the laser power, with laser powers of 0.5, 0.75, 1.0, 1.25, and 1.5 W / cm² respectively. 2 For example, when using a lower power density (0.5 W / cm²) 2 After 10 minutes of irradiation, the temperature of the SAAM solution only increased by 18.6 °C; however, when the laser power density was increased to 1.5 W / cm², the temperature of the solution increased by 18.6 °C. 2 At that time, the temperature suddenly rose by 43.5 ℃ (see...) Figure 6 (See section (b)). Even after four laser switching cycles, SAAM still exhibits excellent photothermal stability, with no significant change in its maximum transition temperature (see section (b)). Figure 6(See section (c)). Furthermore, test results show that the photothermal conversion efficiency η of SAAM is as high as 44.55% (see section (c)). Figure 6 (d) of the text has excellent photothermal conversion potential, which provides important performance support for its subsequent practical applications such as photothermal antibacterial.

[0052] 4. SAAM biocompatibility Good biocompatibility is the most basic property that biomaterials must possess. To comprehensively evaluate the biocompatibility of SAAM, L929 cytotoxicity and hemolysis experiments were conducted.

[0053] 1) Cytotoxicity test Cells were cultured at 37°C in an incubator containing 5% CO2. DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin was used for cell culture, and the medium was changed every 2 days. L929 (mouse fibroblast cell line) cells were used to evaluate SAAM cytotoxicity. First, L929 cells (10⁴ cells per well) were seeded in 96-well plates and cultured overnight, then co-incubated with different concentrations (0, 25, 50, 100, 200, and 400 μg / mL) of SAAM. After 12 hours of incubation, the medium was removed and replaced with phenol-free DMEM. CCK-8 solution was added to each well, and the cells were incubated for 4 hours. Finally, the absorbance of each well was measured at 490 nm using a microplate reader.

[0054] The results of the L929 cytotoxicity assay are as follows: Figure 7 As shown, at test concentrations of 25–400 μg / mL, the survival rate of L929 cells remained above 90%, meeting the criteria for non-cytotoxicity of medical materials, thus confirming that SAAM has good cell compatibility.

[0055] 2) Hemolysis test: Hemolysis is a physiological and pathological process in which the red blood cell membrane ruptures or is damaged, leading to the release of hemoglobin and other cellular contents into the surrounding environment. Its evaluation result is one of the key indicators for assessing the blood compatibility of materials. To verify the blood compatibility of SAAM, blood samples from each group were centrifuged at 3000 rpm for 10 minutes after incubation. Hemolysis was observed visually to preliminarily determine the blood safety of the material.

[0056] This embodiment uses fresh sheep blood samples treated with EDTA anticoagulation. First, whole blood was placed in a centrifuge tube and centrifuged at 3000 rpm for 5 min. The supernatant plasma and white cell layer were discarded, and red blood cells were obtained. The red blood cells were washed three times with PBS until the supernatant was clear after centrifugation. The washed red blood cells were then prepared into a 4% red blood cell suspension using PBS. SAAM sample working solution, a positive control group, and a negative control group (Ctrl) were set up. The concentrations of the SAAM sample working solution were set at 100, 200, 300, 400, and 500 μg / mL; the positive control group used a 2% Triton-X-100 solution; and the negative control group used PBS buffer. Each group of solutions was mixed with an equal volume of 4% erythrocyte suspension and incubated at 37 ℃ for 1 hour. After incubation, all samples were centrifuged at 3000 rpm for 10 min, and the supernatant was visually inspected for hemolysis. Then, 100 μL of the supernatant was transferred to a 96-well plate, and the absorbance (OD) at 542 nm was measured using a microplate reader. 542nm Quantitative analysis of hemolysis was performed using absorbance values. The hemolysis rate (%) was calculated using the following formula: .

[0057] Experimental results showed that the positive control group (with Triton-X-100 added) solution was a clear red color with no residual red blood cells at the bottom, clearly indicating that the red blood cells ruptured and there was significant hemolysis. In contrast, after centrifugation, the red blood cells in the SAAM sample test group all settled as a whole, and the supernatant remained clear and colorless. No hemoglobin release was observed, indicating that the red blood cell structure in the sample test group was intact and no hemolysis occurred (see...). Figure 8 (See part (a)). The above comparative experiments verified the effectiveness and accuracy of this hemolysis test system. To further investigate the effect of SAAM concentration on blood compatibility, SAAM was diluted with PBS buffer (10 mM, pH=7.2) to gradient concentrations of 100, 200, 300, 400 and 500 μg / mL, and then co-incubated with blood samples. The results showed that even with a nanoparticle concentration as high as 500 μg / mL, SAAM only caused very slight or even no hemolytic reaction (see Part (a)). Figure 8 In part (b) of the study, the hemolysis rate was less than 3%, which indicates that SAAM has good blood compatibility.

[0058] 5. SAAM photothermal antibacterial effect To quantitatively evaluate the photothermal antibacterial activity of SAAM, *Staphylococcus aureus* (Gram-positive) and *Escherichia coli* (Gram-negative) were selected as test strains. Single colonies of both strains were inoculated into 3 mL of LB medium and incubated overnight at 37 °C and 220 rpm in a shaker to obtain activated bacterial solutions. The solutions were then diluted to 1 × 10⁻⁶ with LB medium. 7 CFU / mL. PBS or SAAM (500 μg / mL) was mixed with the diluted bacterial suspension (labeled Control, indicating the group where PBS was mixed with the diluted bacterial suspension). Samples irradiated with near-infrared light were treated with a power density of 1.25 W / cm². 2 The bacterial culture was irradiated with an 808 nm laser for 10 min, and the group incubated in the dark under the same conditions for 10 min. The mixed bacterial cultures of the different treatment groups were further serially diluted 10 times with PBS. 3 The solution was then spread in 100 µL onto LB agar plates using a disposable spreader. The plates were incubated upside down in a 37 °C incubator for 12–16 h. After incubation, the plates were removed and the agar plates were counted.

[0059] The antibacterial properties of SAAM were evaluated using the plate coating method, and the results are as follows: Figure 9 ( Figure 9 Part (a) of the text "near-infrared light" 808nm This column ("-" indicates light avoidance, "+" indicates laser irradiation) shows that under light-avoidance conditions, the colony count and cell viability of the SAAM group were not significantly different from the control group. After 10 minutes of irradiation with an 808 nm near-infrared laser, the SAAM group achieved 100% bactericidal activity against both *Escherichia coli* and *Staphylococcus aureus*, with no visible colonies on the agar plates. 808 nm laser irradiation does not damage the viability of normal cells, and local heating to 50–60 °C for a short period will not damage normal human tissues. Normal tissues have better heat resistance and regeneration capacity than bacteria, and can tolerate a certain degree of temperature increase for a short time without causing significant pathological changes.

[0060] The above results further confirm that SAAM's excellent photothermal properties can achieve rapid and efficient killing of bacteria, and it has good application prospects in the field of photothermal antibacterial.

Claims

1. A method for synthesizing photothermal antibacterial nanoparticles, characterized in that, The steps are as follows: Step 1: Add silk fibroin gold nanoclusters dropwise to an acetone solution and stir until the acetone is completely evaporated. During the evaporation of acetone, the silk fibroin gold nanoclusters self-assemble into nanoparticles. Collect the nanoparticles, wash them, and disperse them under ice bath conditions to obtain a nanoparticle solution. Step 2: Under weak reducing conditions, silver solution is added to the nanoparticle solution. After in-situ growth, the precipitate is collected by centrifugation, the supernatant is discarded, and the precipitate is resuspended in ultrapure water to obtain silk fibroin / gold / silver nanoparticles. Step 3: Disperse silk fibroin / gold / silver nanoparticles in deionized water, add buffer solution, tyrosine aqueous solution, and tyrosinase, grow in situ, collect the precipitate, discard the supernatant, resuspend the precipitate in ultrapure water, and obtain melanin-like photothermal antibacterial nanoparticles.

2. The method for synthesizing photothermal antibacterial nanoparticles according to claim 1, characterized in that, The preparation method of the silk fibroin gold nanoclusters is as follows: a silk fibroin solution is mixed with a glutathione aqueous solution to obtain a GSH / SF mixed solution. In the GSH / SF mixed solution, the concentration of silk fibroin is 1~20 mg / mL and the concentration of glutathione is 0.4~1.6 mmol / L. An aqueous solution of HAuCl4 with a concentration of 0.25~5 mmol / L is added, and the silk fibroin gold nanoclusters are prepared in a one-pot method at a temperature of 70~80℃.

3. The method for synthesizing photothermal antibacterial nanoparticles according to claim 1, characterized in that: In step one, the volume ratio of the silk fibroin gold nanoclusters to the acetone solution is 1:3 to 10.

4. The method for synthesizing photothermal antibacterial nanoparticles according to claim 1, characterized in that: The volume ratio of the silk fibroin gold nanoclusters to the acetone solution is 1:3 to 5.

5. The method for synthesizing photothermal antibacterial nanoparticles according to claim 1, characterized in that: In step two, the weak reducing conditions are achieved by adding any one of sodium citrate, ascorbic acid, dopamine, or hydroxylamine hydrochloride at a concentration of 0.5–5 mM.

6. The method for synthesizing photothermal antibacterial nanoparticles according to claim 1, characterized in that: In step two, the silver solution is any one of silver nitrate solution, silver acetate, or silver chlorate, and the concentration of the silver solution is 0.1–0.5 mM.

7. The method for synthesizing photothermal antibacterial nanoparticles according to claim 1, characterized in that: In step two, the in-situ growth temperature is 80–100℃.

8. The method for synthesizing photothermal antibacterial nanoparticles according to claim 1, characterized in that: In step three, the buffer solution is any one of Tris-HCl buffer solution, phosphate buffer solution, and HEPES buffer solution.

9. The method for synthesizing photothermal antibacterial nanoparticles according to claim 1, characterized in that: In step three, the concentration of the tyrosine aqueous solution is 0.5–2 mg / mL, and the concentration of tyrosinase is 200–500 U / mL.

10. The method for synthesizing photothermal antibacterial nanoparticles according to claim 1, characterized in that: In step three, the concentration of the tyrosine aqueous solution is 1–2 mg / mL, and the concentration of tyrosinase is 250–350 U / mL.

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