Photo-thermal tanshinol-iron nano composition as well as preparation method and application thereof

Abstract

The invention relates to the technical field of medicines, in particular to a photo-thermal tanshinol-iron nano composition as well as a preparation method and application thereof. The preparation method of the tanshinol-iron nano composition comprises the following steps: preparing a tanshinol solution and a ferric trichloride solution; slowly dropwise adding the ferric trichloride solution into the tanshinol solution to obtain a mixed solution; and reacting the mixed solution under the dark and stirring conditions, and freeze-drying the obtained reaction solution. The tanshinol-iron nano composition prepared by the method disclosed by the invention can effectively improve the inherent defect that tanshinol is easy to oxidize, obviously improve the antioxidant activity and antibacterial activity of the tanshinol, promote proliferation and migration of vascular endothelial cells and formation of a lumen-like structure, and efficiently induce angiogenesis. Meanwhile, the nanometer composition can synergistically exert the biological effects of tanshinol and iron ions, and the photo-thermal synergistic effect is achieved. The invention provides a technical support for developing novel and efficient drugs for promoting healing of diabetic wounds.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of medicine, in particular to a photothermal Danshensu-iron nano composition, a preparation method and application thereof. BACKGROUND

[0002] Diabetes is a serious chronic metabolic disease. In recent years, the population with the disease has continued to expand, and the prevalence rate has been rising, which has become a global health challenge. Among various complications caused by diabetes, wound damage is relatively common. Compared with normal people, the inflammation stage of the wound healing process of diabetic patients is longer, which delays wound healing and at the same time promotes oxidative stress and aggravates inflammation, ultimately leading to aggravated tissue damage and delayed healing. Among diabetic patients, about 30% are plagued by chronic and non-healing wounds, and some patients even need to undergo amputation treatment, which seriously affects the quality of life of patients.

[0003] Diabetic wounds are a kind of chronic wounds, which become a favorable place for bacteria to breed due to their difficulty in healing. Bacterial infections can be treated with antibiotics, but the abuse of antibiotics can easily lead to bacterial drug resistance, making antibiotic treatment ineffective. At the same time, traditional wound repair is mostly based on a single pathological mechanism, and the treatment method usually adopts simple anti-infection treatment, which is difficult to achieve complex synergistic regulation of diabetic wounds. Therefore, it is urgent to develop new and efficient drugs for the treatment of diabetic wounds.

[0004] Photothermal therapy mainly uses heat to kill bacteria by destroying bacterial membranes and / or denaturing bacterial proteins. Photothermal therapy has the advantages of avoiding drug resistance and non-invasive treatment, and has become a research focus in recent years. The photothermal effect under near-infrared light irradiation has high photothermal conversion efficiency and good penetration ability into the body of mammals, and causes less damage to normal tissues, so it is widely used in clinical practice.

[0005] Danshensu is a water-soluble component in Danshen. Studies have found that Danshensu has clear pro-angiogenic activity and anti-inflammatory and antioxidant properties, which can provide sufficient blood supply and nutritional support for wound repair. However, as a phenolic acid compound, Danshensu has the disadvantages of easy oxidation, short half-life, low bioavailability, poor stability, and rapid decomposition in aqueous media, which limits its clinical application. Therefore, it is of great significance to develop a safe and efficient drug delivery system to improve the drugability of Danshensu and improve its bioavailability.

[0006] Iron is one of the important trace elements in life activities and has certain antibacterial effect. Iron nano materials have good photothermal conversion performance and can convert light energy into heat energy under near-infrared laser irradiation, forming a local warm environment, which can further enhance the antibacterial effect and promote local blood circulation, thereby accelerating the resolution of inflammation and creating favorable conditions for cell proliferation and tissue regeneration.

[0007] Based on this, the application designs a multifunctional Danshensu-iron nano composition with Danshensu and iron as raw materials, aims to overcome the inherent defect that Danshensu is easy to be oxidized, fully retain the antioxidant activity and antibacterial activity of Danshensu, and synergistically exert the biological effects of Danshensu and iron, and provide technical support for developing a new type of efficient drug for promoting wound healing of diabetes. SUMMARY

[0008] The application aims to provide a Danshensu-iron nano composition with photothermal effect and a preparation method thereof.

[0009] Another object of the application is to provide application of the above-mentioned Danshensu-iron nano composition with photothermal effect in preparation of a drug for promoting repair of infected wounds of diabetes.

[0010] To achieve the above object, the technical scheme adopted by the application is as follows: The preparation method of the Danshensu-iron nano composition with photothermal effect comprises the following steps: S1, Danshensu and trichloride iron hexahydrate are weighed respectively, and are added into ultrapure water to be fully dissolved to prepare Danshensu solution and trichloride iron solution; S2, the prepared trichloride iron solution is slowly added dropwise into the Danshensu solution, and is mixed to obtain a mixed solution; S3, the mixed solution is placed on a magnetic stirrer to react in the dark, and after the reaction is completed, the reaction solution is freeze-dried.

[0011] Preferably, in the preparation method of the Danshensu-iron nano composition with photothermal effect, the concentration of the Danshensu solution and the trichloride iron solution in step S1 is 0.1 mol / L.

[0012] Preferably, in the preparation method of the Danshensu-iron nano composition with photothermal effect, the volume ratio of the Danshensu solution and the trichloride iron solution in step S2 is 1:1.

[0013] Preferably, in the preparation method of the Danshensu-iron nano composition with photothermal effect, the time of the light-avoiding reaction in step S3 is 10-15 h.

[0014] Further preferably, in the preparation method of the Danshensu-iron nano composition with photothermal effect, the time of the light-avoiding reaction in step S3 is 12 h.

[0015] The Danshensu-iron nano composition with photothermal effect is prepared by the above preparation method.

[0016] The application of the Danshensu-iron nano composition with photothermal effect in the preparation of a drug for promoting the repair of diabetic infected wounds.

[0017] Preferably, in the application of the Danshensu-iron nano composition with photothermal effect in the preparation of a drug for promoting the repair of diabetic infected wounds, the drug is prepared by taking the Danshensu-iron nano composition as the main active ingredient and adding a pharmaceutically acceptable excipient to prepare a pharmaceutically acceptable preparation.

[0018] Further preferably, in the application of the Danshensu-iron nano composition with photothermal effect in the preparation of a drug for promoting the repair of diabetic infected wounds, the preparation is a hydrogel, and the preparation method is as follows: Danshensu-iron nano composition is weighed and completely dissolved in ultrapure water to obtain a Danshensu-iron aqueous solution; poloxamer 407 powder is weighed and added to the Danshensu-iron aqueous solution to obtain the hydrogel.

[0019] More preferably, in the application of the Danshensu-iron nano composition with photothermal effect in the preparation of a drug for promoting the repair of diabetic infected wounds, in the preparation method of the hydrogel: the concentration of the Danshensu-iron aqueous solution is 6 mg / mL; and the amount of poloxamer 407 added is 3 g per 10 mL of the Danshensu-iron aqueous solution.

[0020] The application has the following beneficial effects: 1. The Danshensu-iron nano composition provided by the application has better stability and sustained-release performance than Danshensu. Meanwhile, the Danshensu-iron nano composition provided by the application exhibits good in-vitro photothermal effect under near-infrared light (808 nm), can quickly and effectively convert laser energy into heat, and achieves a photothermal antibacterial effect.

[0021] 2. It is found through in-vitro antibacterial activity determination experiments that, under no NIR irradiation, the quantitative inhibition zones of SA-Fe with concentrations of 400 μg / mL and 800 μg / mL on Staphylococcus aureus are 22.18±2.47 mm and 24.97±2.14 mm respectively, and the quantitative inhibition zones of SA-Fe with concentrations of 400 μg / mL and 800 μg / mL on Escherichia coli are 16.25±2.64 mm and 19.37±0.34 mm respectively; under NIR irradiation, the quantitative inhibition zones of SA-Fe with concentrations of 400 μg / mL and 800 μg / mL on Staphylococcus aureus are 27.26±1.24 mm and 31.27±1.04 mm respectively, and the quantitative inhibition zones of SA-Fe with concentrations of 400 μg / mL and 800 μg / mL on Escherichia coli are 22.25±1.17 mm and 24.98±1.46 mm respectively. The above indicates that the Danshensu-iron nano composition provided by the application has a significant antibacterial effect on Staphylococcus aureus and Escherichia coli and can achieve photothermal synergistic antibacterial effect.

[0022] 3. The Danshensu-iron nano-composition has good blood compatibility, cell compatibility and tissue compatibility, and does not have adverse effects on the daily behavior of mice and important organs of the body within the experimental dose range. The above shows that the Danshensu-iron nano-composition provided by the application has good in-vivo biological safety.

[0023] 4. By evaluating the effects of the Danshensu-iron nano-composition on the proliferation, migration and angiogenesis of HUVECs cells and L929 cells, it is found that the Danshensu-iron nano-composition has better effects on promoting the proliferation, migration and angiogenesis of HUVECs cells than free Danshensu, and shows stronger promoting effects under NIR irradiation; the Danshensu-iron nano-composition can significantly promote the migration of L929 cells. The above shows that the Danshensu-iron nano-composition provided by the application overcomes the defect of easy oxidation of free Danshensu, significantly improves the biological activity, and can realize the photothermal synergistic effect.

[0024] 5. HUVECs cells and L929 cells have good uptake of the Danshensu-iron nano-composition. Meanwhile, the Danshensu-iron nano-composition has excellent antioxidant effect, and the clearance rate of SA-Fe with a concentration of 250 μg / mL on ABTS reaches 92.77±0.29%, and the clearance rate of SA-Fe with a concentration of 100 μg / mL on DPPH reaches 96.45±0.69%.

[0025] 6. The Danshensu-iron hydrogel provided by the application is used for in-vivo research on wound healing of diabetic rats, and it is found that on the 11th day, the wound healing rate of the Danshensu-iron hydrogel+NIR group is 94.56±0.94%, which is significantly higher than that of the Danshensu-iron hydrogel group (87.21±2.65%), the Danshensu hydrogel group (82.63±4.04%) and the control group (75.18±2.32%). The above shows that the Danshensu-iron hydrogel provided by the application has stronger effects on promoting the healing of diabetic wounds than the Danshensu hydrogel, and can realize the photothermal synergistic effect.

[0026] 7. The Danshensu-iron hydrogel provided by the application can reduce local inflammatory response by significantly down-regulating the expression of pro-inflammatory factors IL-1β and TNF-α, up-regulating the expression of anti-inflammatory factors IL-10 and TGF-β, promoting the expression of blood vessel-related factors VEGF and CD31, effectively regulating the transformation of M1 macrophages on the wound surface to M2 type, realizing the regulation of inflammatory factors and angiogenesis factors, and achieving the photothermal synergistic effect of antibacterial, anti-inflammatory and pro-angiogenesis. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1Characterization of SA-Fe (Figures: A is the optical photograph of SA-Fe in water medium, the inset is the freeze-dried picture of SA-Fe; B is the average hydrodynamic size of SA-Fe; C is the Zeta potential of SA and SA-Fe in water medium; D is the TEM observation of SA-Fe; E is the full wavelength scan of SA-Fe, ferric chloride trihydrate and SA; F is the FTIR spectrum of SA-Fe and SA; G is the XRD pattern of SA-Fe and SA; H and I are wide-range XPS spectra); Figure 2 Photothermal properties of SA-Fe under near-infrared light (Figures: A is the temperature change diagram of SA-Fe aqueous solution after irradiation with different power near-infrared light for 5 min; B is the infrared thermal image of SA-Fe aqueous solution after irradiation with different power near-infrared light for 5 min; C is the temperature change diagram of SA-Fe aqueous solution after irradiation with near-infrared light for 5 min for 3 cycles; D is the temperature change diagram of SA-Fe aqueous solution after irradiation with different power near-infrared light for 5 min; E is the infrared thermal image of SA-Fe aqueous solution after irradiation with different power near-infrared light for 5 min; F is the drug release curve of SA-Fe and SA); Figure 3 Experimental results of synergistic antibacterial effect of photothermal therapy (PTT) (Figures: A is the observation diagram of inhibition zone after different treatments for 18 h; B is the live / dead bacteria fluorescence image after different treatments for 18 h; C is the SEM image of bacteria after different treatments for 18 h; D is the inhibition zone quantification of Staphylococcus aureus after different treatments for 18 h; E is the inhibition zone quantification of Escherichia coli after different treatments for 18 h; F is the live / dead bacteria fluorescence quantification after different treatments for 18 h); Figure 4 Biocompatibility evaluation results of SA-Fe (Figures: A is the live / dead fluorescence image of L929 cells after co-incubation with different treatment groups for 24 h; B is the hemolysis analysis picture and hemolysis rate after co-incubation with different concentrations of SA-Fe and red blood cell suspension; C is the cell viability of L929 cells and HUVECs cells after co-incubation with different concentrations of SA-Fe for 24 h; D is the live / dead fluorescence image of HUVECs cells after co-incubation with different treatment groups for 24 h; E is the fluorescence quantification of L929 cells and HUVECs cells; F is the pathological section H&E staining image of main organs of control group and experimental group after injection of SA-Fe solution for 48 h; G is the absorbance of HUVECs cells after co-incubation with different treatment groups for 1, 2 and 5 days); Figure 5Results of HUVECs cell proliferation, migration and angiogenesis and L929 cell migration experiments (in the figure: A is the image of L929 cell migration after different treatments for 12h and 24h; B is the quantitative analysis of blood vessel network-node number; C is the quantitative analysis of blood vessel network-branch number; E is the quantitative analysis of blood vessel network-total length; D is the optical image of blood vessel formation in HUVECs cells after different treatments; F is the image of HUVECs cell migration after different treatments for 12h and 24h; G is the migration rate of L929 cells and HUVECs cells after different treatments for 12h and 24h); Figure 6 Results of SA-Fe antioxidant capacity and time-dependent uptake (in the figure: A is the image of L929 cell uptake of SA-Fe at different times; B is the image of HUVECs cell uptake of SA-Fe at different times; C is the quantitative results of L929 cells and HUVECs cells for SA-Fe at different times; D is the clearance rate of ABTS and DPPH; E is the ABTS reagent color development image; F is the DPPH reagent color development image); Figure 7 Results of in vivo study of wound healing effect in diabetic rats (in the figure: A is the method of inducing the establishment of diabetic rats and the scheme of dynamic changes of healing; B is the representative image of the wound at 0, 1, 3, 7, 11d; C is the wound healing trajectory of different treatment groups; D is the representative image of bacterial growth on agar plate at the wound site at 5d; E is the statistical data of wound healing rate at the set time point; F is the corresponding bacterial activity of different treatment groups); Figure 8 Results of histological analysis (in the figure: A is the H&E and Masson staining image of representative tissues of different treatment groups at 5d; B is the H&E and Masson staining image of representative tissues of different treatment groups at 11d; C is the collagen deposition rate of different treatment groups at 5d and 11d; D is the scar width of different treatment groups at 11d); Figure 9 Results of immunohistochemical and immunofluorescence analysis of different samples (in the figure: A is the immunohistochemical staining image under different conditions at 11d; B is the CD206 and CD86 immunofluorescence image and its fusion image; C-I is the quantitative analysis of the corresponding markers; J is the quantitative analysis of the average fluorescence intensity). DETAILED DESCRIPTION

[0028] The technical solutions of the present application are described in detail below in combination with specific examples. The following examples are only used for explanation and illustration, and do not constitute a limitation on the technical solutions of the present application.

[0029] Example 1 A Danshensu-iron nano composition with photothermal effect, the preparation method of which is as follows: (1) Take Danshensu and ferric chloride hexahydrate respectively, add ultrapure water to dissolve thoroughly, and prepare Danshensu solution and ferric chloride solution with a concentration of 0.1 mol / L.

[0030] (2) Slowly add the ferric chloride solution drop by drop into the Danshensu solution and mix (volume ratio 1:1), and ensure that the two solutions are fully contacted to obtain a mixed solution.

[0031] (3) Place the mixed solution on a magnetic stirrer and react in the dark for 12 h. After the reaction is completed, transfer the reaction solution to a beaker, pre-freeze in a -80℃ ultra-low temperature refrigerator for 2 h, and then place it in a freeze-drying machine until the sample is completely dried.

[0032] Example 2 A Danshensu-iron nano composition with a photothermal effect is prepared by the following method: (1) Take Danshensu and ferric chloride hexahydrate respectively, add ultrapure water to dissolve thoroughly, and prepare Danshensu solution and ferric chloride solution with a concentration of 0.1 mol / L.

[0033] (2) Slowly add the ferric chloride solution drop by drop into the Danshensu solution and mix (volume ratio 1:1), and ensure that the two solutions are fully contacted to obtain a mixed solution.

[0034] (3) Place the mixed solution on a magnetic stirrer and react in the dark for 10 h. After the reaction is completed, transfer the reaction solution to a beaker, pre-freeze in a -80℃ ultra-low temperature refrigerator for 2 h, and then place it in a freeze-drying machine until the sample is completely dried.

[0035] Example 3 A Danshensu-iron nano composition with a photothermal effect is prepared by the following method: (1) Take Danshensu and ferric chloride hexahydrate respectively, add ultrapure water to dissolve thoroughly, and prepare Danshensu solution and ferric chloride solution with a concentration of 0.1 mol / L.

[0036] (2) Slowly add the ferric chloride solution drop by drop into the Danshensu solution and mix (volume ratio 1:1), and ensure that the two solutions are fully contacted to obtain a mixed solution.

[0037] (3) Place the mixed solution on a magnetic stirrer and react in the dark for 15 h. After the reaction is completed, transfer the reaction solution to a beaker, pre-freeze in a -80℃ ultra-low temperature refrigerator for 2 h, and then place it in a freeze-drying machine until the sample is completely dried.

[0038] Example 4 The Danshensu-iron nano composition prepared in Examples 1-3 is prepared into a hydrogel preparation for promoting the repair of diabetic wounds by the following method: The prepared danshensu-iron nano-composition was weighed, and ultrapure water was added to prepare a danshensu-iron aqueous solution with a concentration of 6 mg / mL. 3 g of poloxamer 407 powder was weighed into a 50 mL centrifuge tube, and 10 mL of the above-mentioned danshensu-iron aqueous solution was added to prepare a danshensu-iron hydrogel containing 30% poloxamer.

[0039] In order to further verify the reliability of the present application and screen out the best scheme, the inventors carried out a series of tests, as follows: 1. Main reagents and materials Danshensu (item number S31458, Shanghai Yuanye Biotechnology Co., Ltd.); iron (III) chloride hexahydrate (item number 31232, Sigma-Aldrich (Shanghai) Trading Co., Ltd.); Matrigel (item number 356234 BD Biocoat); 1,1-diphenyl-2-picrylhydrazyl radical (item number D273092), 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (item number A109612), 50% glutaraldehyde (item number G105905), and dimethyl sulfoxide (item number D103272) were all purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; streptozotocin (STZ, item number S8050), Luria-Bertani (LB) nutrient agar (item number L8290), poloxamer 407 (item number S7071), dialysis bag MD34 (item number YA1077), thiazolyl blue (item number M8180), and PBS phosphate buffer dry powder (item number P1003) were purchased from Beijing Solabio Technology Co., Ltd. Kunming mice (KM mice, 4-6 weeks) were purchased from the Animal Institute of Guizhou University of Chinese Medicine, and Sprague-Dawley rats (body weight 280±20 g) were purchased from Zhiqinshuo (Chongqing) Biological Technology Co., Ltd. The experimental animals were raised in a sterile laboratory and had free access to feed and water. All animal care and experimental operations were approved by the Animal Experiment Ethics Committee of Guizhou University of Chinese Medicine (approval numbers: 20250827005, 20250827004).

[0040] 2. Experimental methods 2.1 Preparation of SA-Fe Take 19.8 mg (0.1 mmol) of Danshensu (SA), add 1 mL of ultrapure water to dissolve thoroughly, and prepare a Danshensu solution; take 27.0 mg (0.1 mmol) of iron trichloride hexahydrate (FeCl3·6H2O), add 1 mL of ultrapure water to dissolve thoroughly, and prepare an iron trichloride solution. Slowly add the above-mentioned iron trichloride solution drop by drop into the Danshensu solution, mix well, and ensure that the two solutions are in sufficient contact. Then place the mixed solution on a magnetic stirrer and react for 12 h in the dark. After the reaction is completed, remove the mixed solution and transfer it to a beaker, pre-freeze it in a -80℃ ultra-low temperature refrigerator for 2 h, and then place it in a freeze dryer until the sample is completely dried, thus obtaining a SA-Fe complex lyophilisate, which is collected and stored at 4℃ in the dark.

[0041] 2.2 Characterization of SA-Fe The particle size distribution and polydispersity index (PDI) of SA-Fe were determined by dynamic light scattering (DLS), and the zeta potential value was analyzed. The absorbance curves of Danshensu, iron trichloride, and SA-Fe aqueous solutions were determined in the wavelength range of 240-250 nm by ultraviolet-visible spectrophotometry (UV-Vis). The functional group analysis of the samples was performed by Fourier transform infrared spectroscopy (FT-IR). The structure and particle size of SA-Fe were observed by transmission electron microscopy (TEM). The characteristic diffraction peaks of SA-Fe were analyzed by X-ray diffraction (XRD), and the information such as the elemental composition, chemical state, and molecular structure of the sample surface was obtained by X-ray photoelectron spectroscopy (XPS).

[0042] Drug release behavior: 1 mL of SA solution and SA-Fe solution was taken and placed in a dialysis bag (MWCO 3.5kD), the dialysis bag was immersed in 10 mL of PBS (pH 7.4) and placed in a 37℃ environment, 1 mL of dialysis PBS solution was collected at 0.5, 1, 2, 4, 8, 12, 24, and 36 h, and fresh PBS was supplemented. The concentration of SA was determined by high performance liquid chromatography, and the cumulative release amount (Qn) and cumulative percentage release amount (Q) were calculated according to the following formula, with 3 replicates for each group: . Wherein: Ct is the measured concentration of SA at t, Ct-1 is the measured concentration of SA before t, V is the total amount of medium released, and V is the volume of medium removed each time, C0 is the total amount of SA in the drug pool at the beginning.

[0043] 2.3 In vitro photothermal effect determination Different concentrations (0-800 μg / mL) of SA-Fe were used to irradiate with a 1 W / cm 2808 nm laser irradiation and different power (0.2-1.5 W / cm 2 The temperature changes of SA-Fe after 5 min were recorded in real time by an infrared thermal imager to study the photothermal stability. For easy observation, the temperature changes were recorded every 60 s by a handheld temperature imaging instrument.

[0044] 2.4 Photothermal therapy (PTT) synergistic antibacterial effect In vitro antibacterial activity test: The antibacterial ability of SA-Fe was evaluated by gram-positive Staphylococcus aureus and gram-negative Escherichia coli. The bacteria were activated by “Z” line culture, and the round and moist single colonies on the plate were picked up with a gun head and punched into a 15 mL centrifuge tube containing 4 mL LB broth medium, which was placed in a constant temperature incubator (37°C, 220 rpm) for 18 h to obtain the bacterial suspension.

[0045] Oxford cup diffusion method was used to determine the antibacterial ability of SA-Fe: 200 μL of bacterial suspension (108 CFU / mL) was uniformly coated on LB agar plates with an L-type coating rod. The inner diameter of the Oxford cup was 6 mm, and 20 μL of liquid sample was added to each Oxford cup hole. After incubation at 37°C for 18 h, the diameter of the inhibition zone was observed and measured, and the experiment was repeated three times. The experimental groups were as follows: SA-Fe concentrations of 400 μg / mL and 800 μg / mL were experimental groups; normal saline was the blank control group; gentamicin sulfate (50 μg / mL) was the positive control group. The antibacterial activity of SA-Fe was evaluated by 808 nm laser irradiation at a power of 1 W / cm 2

[0046] Live / dead bacterial staining: N,N-dimethyl aniline N-oxide / propyl iodide (DMAO / PI) kit was used for detection. First, the bacteria treated with different SA-Fe were cultured in liquid medium until they reached the logarithmic growth phase. Then, the bacterial suspension was centrifuged at room temperature for 5 min. The supernatant was discarded, and the bacteria were washed twice with normal saline to completely remove the residual liquid medium. The bacterial concentration was adjusted to 108 CFU / mL, and the bacteria were stained with DMAO / PI working solution and incubated at 37°C in the dark for 30 min. The stained samples were placed on a sterile cover, and finally the staining was observed under a fluorescence inverted microscope.

[0047] ​​​Bacterial morphology observation: The morphology of Staphylococcus aureus and Escherichia coli after co-culturing with SA-Fe was observed using scanning electron microscopy. First, bacterial samples were treated with different amounts of SA-Fe, washed twice with PBS, and then resuspended in 3% glutaraldehyde for fixation at 4°C for 4 h to preserve their morphology and structure. After fixation, the samples were washed twice with PBS, and then eluted sequentially with 50%, 70%, 90%, and 100% ethanol. The samples were then dried, sputter-coated with gold, and observed using SEM.

[0048] 2.5 Biocompatibility evaluation of SA-Fe Fresh whole blood from rats was collected and washed with physiological saline at 2000 rpm for 15 min until the supernatant was clear. The erythrocyte suspension was then diluted to 2% (v / v) with physiological saline. SA-Fe was diluted with physiological saline to obtain a series of solutions at concentrations of 12.5, 25, 50, 100, and 200 μg / mL. 0.5 mL of each of these concentrations was added to a 2 mL EP tube containing 0.5 mL of erythrocyte suspension to obtain samples. The samples were incubated at 37℃ and 100 rpm for 30 min, followed by centrifugation for 15 min. The samples were then removed, photographed, and the supernatant of each group was collected. Each group was replicated in triplicate. The absorbance of the supernatant at 540 nm was measured using a microplate reader. Physiological saline was used as the negative control group, and deionized water as the positive control group. The hemolysis rate (HR) was calculated using the following formula: ; In the formula: The absorbance values ​​of the sample group; The absorbance value is for the saline group; The absorbance value is for the deionized water group.

[0049] The cytotoxicity of SA-Fe material was evaluated using the MTT assay and live / dead cell staining. L929 cells and HUVECs were subjected to... Cells were seeded at a density of cells / well in 96-well plates and incubated overnight in a cell culture incubator. Two cell lines were then treated with different concentrations of SA-Fe and control material, with three replicates for each concentration. After 24 h of incubation, 20 μL of freshly prepared MTT solution was added to each well, and incubation continued for 4 h. The supernatant was removed, and 100 μL of DMSO was added to each well. After thorough mixing, the absorbance at 490 nm was measured using a microplate reader. Cell viability (CV) was calculated using the following formula: ; In the formula: The absorbance values ​​of the sample group; Blank group absorbance value; Control group absorbance value.

[0050] Calcein-AM / PI double staining method was used to evaluate the cell compatibility of SA-Fe. L929 cells and HUVECs cells were inoculated in 96-well plates at a density of 1 000 cells / well, and after adhering for 12 h, the culture medium was replaced with culture medium containing SA and SA-Fe, and the control material was used to treat the cells for 24 h. The culture solution was aspirated, washed twice with PBS, and then stained with Calcein-AM / PI staining solution, followed by fluorescence detection of the sample under a fluorescence microscope. The 4-6 week old Kunming mice were randomly divided into a blank group (normal saline) and an SA-Fe group (3 mg / mL). The blank group was injected with 200 μL of normal saline through the tail vein, and the SA-Fe group was injected with 200 μL of the dispersion liquid. After 48 h, the mice were euthanized, dissected, and the heart, liver, spleen, lung, and kidney tissue samples were collected, washed with normal saline, and then fixed in a 4% paraformaldehyde solution. Xylene and ethanol were used for gradient dehydration and transparency, followed by paraffin embedding, sectioning, and staining. The morphology of the cells in each organ and the infiltration of inflammatory cells in the tissue were observed under an optical microscope.

[0051] 2.6 Cell proliferation, migration, and angiogenesis

[0052] HUVECs cell proliferation: MTT method was used to evaluate the effect of SA-Fe on HUVECs cell proliferation. HUVECs cells were inoculated in 96-well plates at a density of 1 000 cells / well, and after incubation overnight, the culture medium was replaced with culture medium containing SA and SA-Fe. On the 1st, 2nd, and 5th days of culture, 20 μL of freshly prepared MTT solution was added to each well, and incubation was continued for 4 h. The supernatant was removed, 100 μL of DMSO was added to each well, and after thorough mixing, the absorbance value at 490 nm was detected using an enzyme-labeled instrument. HUVECs cell migration: logarithmically growing HUVECs cells were inoculated in 24-well plates at a density of 1 000 cells / well, and after the cell confluence reached 90%, a 10 μL pipette tip was used to make a vertical scratch on the plate. The old culture medium was discarded, and the cells were removed by gently washing twice with PBS. Then, the culture medium was replaced with culture medium containing SA and SA-Fe, and serum-free DMEM medium was used as a control. The migration area at 0, 12, and 24 h was observed under an inverted microscope. Software was used to quantify the cell migration rate, and the cell migration rate (CMR) was calculated according to the following formula:

[0053] HUVECs cell migration: logarithmically growing HUVECs cells were inoculated in 24-well plates at a density of 1 000 cells / well, and after the cell confluence reached 90%, a 10 μL pipette tip was used to make a vertical scratch on the plate. The old culture medium was discarded, and the cells were removed by gently washing twice with PBS. Then, the culture medium was replaced with culture medium containing SA and SA-Fe, and serum-free DMEM medium was used as a control. The migration area at 0, 12, and 24 h was observed under an inverted microscope. Software was used to quantify the cell migration rate, and the cell migration rate (CMR) was calculated according to the following formula: ​​ ; wherein: A0is the initial scratch area; A is the scratch area at the corresponding time point.

[0054] Angiogenesis assay: 20 μL Matrigel was evenly spread on 24-well plates, and incubated at 4°C overnight. After the Matrigel was spread, the plates were incubated at 37°C for 1 h. The HUVECs cells in the logarithmic growth phase were starved for 24 h, and then seeded into 24-well plates at a density of 2.5 x 104cells / well. SA-Fe-containing medium was added, and blank medium was used as a control. After 6 h of incubation, the images of the tube formation were taken by microscopy, and the total tube length, node number, and branch number were quantified by software.

[0055] L929 cell migration: L929 cells in the logarithmic growth phase were obtained, and seeded into 24-well plates at a density of 2.5 x 104cells / well, and incubated overnight. After the cell confluence reached 90%, a 10 μL pipette tip was used to make a vertical scratch on the plate. The old medium was discarded, and the cells were washed twice with PBS to remove the ex vivo cells. Then, the medium was replaced with SA-Fe-containing medium, and serum-free medium was used as a control. The migration area at 0, 12, and 24 h was observed under an inverted microscope. The migration rate was quantified by software, and the cell migration rate was calculated (formula same as "HUVECs cell migration").

[0056] 2.7 Antioxidant capacity and in-time uptake Antioxidant capacity: The antioxidant capacity of SA-Fe was evaluated by ABTS and DPPH methods.

[0057] ABTS solution at 7 mmol / L and potassium persulfate solution at 2.45 mmol / L were prepared, mixed at 1:1 (v / v), and darkened at room temperature for 12-16 h. Before use, the solution was diluted to an absorbance value of about 0.7 to obtain the ABTS working solution. 0.9 mL of the ABTS working solution was added to different centrifuge tubes, and then 0.1 mL of SA-Fe at different concentrations was added. After mixing in the dark for 10 min, the absorbance value was measured at a wavelength of 734 nm . The above operation was repeated using an equal amount of distilled water instead of the sample solution, and the absorbance value was recorded as . Each sample was repeated three times, and the radical scavenging rate was calculated according to the following formula: ; wherein: A is the sample absorbance value; B is the blank absorbance value.

[0058] ​​Take 0.0040 g DPPH, dissolve in anhydrous ethanol and dilute to 50 mL to prepare a DPPH-ethanol solution with a concentration of 0.2 mmol / L. Take 0.5 mL of sample solution with different concentrations and 0.5 mL of DPPH-ethanol solution in a 2 mL centrifuge tube, shake well, and incubate at 37°C in the dark for 30 min. Take the supernatant and measure the absorbance value at 517 nm wavelength, denoted as A1. Under the same reaction conditions, replace the above DPPH-ethanol solution with anhydrous ethanol to measure the absorbance value, denoted as A2. Replace the sample with pure water to measure the absorbance value, denoted as A3. Each sample is repeated three times, and the free radical scavenging rate is calculated according to the following formula: In the formula: A1 is the absorbance value of the sample+DPPH group; A2 is the absorbance value of the sample+ethanol group; A3 is the absorbance value of the pure water+DPPH group.

[0059] Time-dependent uptake: L929 cells and HUVECs cells in the logarithmic growth phase were seeded in a 96-well plate at a density of 5×103 cells / well and incubated overnight. Coumarin 6 (C6)-labeled SA-Fe was administered at a time gradient (12, 8, 4, and 1 h), and after administration, the cells were washed twice with PBS to remove the interference of residual serum and free drugs. After adding 100 μL of PBS to the culture plate, the cells were observed under an inverted fluorescence microscope and photographed.

[0060] 2.8 Preparation of SA-Fe hydrogel Weigh SA-Fe and add it to 10 mL ultrapure water to completely dissolve it, preparing SA-Fe aqueous solutions with concentrations of 2, 4, 6, 8, and 10 mg / mL. Weigh 3 g of poloxamer 407 powder and place it in a 50 mL centrifuge tube. Add 10 mL of the above SA-Fe aqueous solution with different concentrations to prepare SA-Fe hydrogel containing 30% poloxamer. Place the mixture in a 4°C refrigerator until the poloxamer is completely dissolved, forming a transparent and uniform solution (no visible particles with the naked eye), which is SA-Fe hydrogel.

[0061] To study the photothermal properties of the prepared SA-Fe hydrogel, the above hydrogels with different concentrations were irradiated by near-infrared radiation (808 nm, 1.0 W / cm 2 , 5 min), and the temperature change was measured using an infrared thermal imager. To further study the in vivo photothermal effect, SA-Fe hydrogel was applied to the injured back of rats, and the temperature change was measured under near-infrared radiation (808 nm, 1.0 W / cm​​2 The temperature change was measured by an infrared thermal imager (Flir Systems, USA) under the condition of 5 min irradiation.

[0062] 2.9 In vivo study of wound healing effect in diabetic rats Male Sprague-Dawley rats weighing 280 ± 20 g were selected. After fasting for 12 h, 1% STZ (60 mg / kg) was injected intraperitoneally to induce diabetes. On the 3rd day after injection, the blood glucose level of the rats was monitored through the tail vein. If the blood glucose value remained above 16.7 mmol / L for 3 consecutive days, it indicated that the diabetes model was successful. Then the rats were anesthetized, the dorsal hair was removed, and the skin was disinfected. Four full-thickness circular skin wounds with a diameter of 10 mm were constructed on the dorsal skin of each diabetic rat, and 200 μL of S. aureus (1.0 × 108CFU / mL) was injected into each wound. Subsequently, the experimental animals were randomly divided into 4 groups: blank hydrogel group, SA hydrogel group, SA-Fe hydrogel group, and SA-Fe+NIR hydrogel group. Each wound was coated with 200 μL of hydrogel material. Near-infrared laser irradiation (808 nm, 1.0 W / cm 2 ) was performed at 0 d, 1 d, 3 d, 5 d, and 7 d, each for 5 min. The wound healing process was evaluated by recording the wound size at 0 d, 1 d, 3 d, 7 d, and 11 d, and the wound healing rate was calculated according to the following formula: ; In the formula: A0is the initial wound area; Anis the wound area on the nth day (n = 1, 3, 7, 11).

[0063] On the 5th day after different treatment regimens, the survival rate of S. aureus in the wounds of diabetic rats was evaluated. At the predetermined time point of 11 d, the rats were euthanized and dissected to obtain the regenerated dorsal skin tissue for pathological evaluation. The obtained tissue samples were immediately immersed in a 4% paraformaldehyde solution for fixation. After fixation, tissue sections were prepared and subjected to histological analysis using various staining methods. Hematoxylin-eosin (H&E) staining was used to evaluate the histological features of the tissue, and Masson staining was used to observe collagen deposition. Immunohistochemical staining was used to detect platelet endothelial cell adhesion molecule (CD31), alpha-smooth muscle actin (a-SMA), and vascular endothelial growth factor (VEGF), transforming growth factor-β (TGF-β), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), interleukin-10 (IL-10), and p-ERK1 / 2. CD86 (M1 macrophage marker) and CD206 (M2 macrophage marker) staining were used to characterize the polarization state of macrophages.

[0064] Statistical software and one-way ANOVA were used to analyze the experimental data. All data were expressed as mean ± SD. The significant levels were expressed as ns: not significant, p < 0.05, p < 0.01, p < 0.001.

[0065] 3. Results and discussion 3.1 Preparation and characterization of SA-Fe The properties of SA-Fe were characterized as shown in Figure 1 The SA-Fe powder was dispersed in pure water, and the laser pen was used to irradiate, and the Tyndall phenomenon was observed Figure 1 A), indicating that the SA-Fe suspension was in a colloidal dispersion state, and the upper left corner was the property after freeze-drying. Dynamic light scattering supplemented this observation Figure 1 B), and the average size of the nanoparticles was 119.6 ± 0.5 nm. In addition, the Zeta potential value of SA-Fe nanoparticles was -18.98 ± 0.68 mV Figure 1 C), while the Zeta potential value of SA was -10.42 ± 0.40 mV, and the potential increased after the formation of nanoparticles, which may be related to the polyphenol groups on the surface of SA-Fe, indicating that the synthesized SA-Fe material had excellent stability. Transmission electron microscopy (TEM) images Figure 1 D) showed that the SA-Fe nanoparticles were uniform in shape and were different nanospheres, without obvious aggregation. The full wavelength scan Figure 1 E) results showed that the maximum absorption peak of SA-Fe appeared red shift compared with SA, which may be related to the formation of coordination bond between SA and Fe ions. FT-IR Figure 1 F) showed that the phenolic hydroxyl group of SA had two small absorption bands at 3601 and 3478 cm -1 , compared with Danshensu, the combined SA-Fe shifted to 3392 cm -1 to form a wide absorption band, and the Fourier infrared spectrum of SA-Fe showed a wide and obvious infrared absorption peak in the range of 490 to 690 cm -1 , indicating that SA-Fe formed Fe-O bond. The X-ray diffraction pattern Figure 1 G) showed that SA had sharp diffraction peaks, indicating high crystallinity, and SA-Fe had no obvious sharp diffraction peaks, showing a diffuse diffraction characteristic. Compared with SA, SA-Fe may be due to the Fe 3+The introduction of [a substance] disrupted the original crystal structure, resulting in a significant decrease in crystallinity. X-ray photoelectron spectroscopy (XPS) was used to determine the surface elemental composition of the aforementioned SA-Fe. Figure 1 The Fe 2p characteristic peak of SA-Fe is located between 709 eV and 725 eV, indicating the presence of Fe in SA-Fe. The elemental contents on the surface of SA-Fe are Fe 2p (1.55%), C 1s (59.20%), and O 1s (39.24%).

[0066] Drug release behavior: We determined the cumulative release of SA and SA-Fe. The results showed that ( Figure 2 In the initial stage (0-12 h), SA release rate was high, with a cumulative release of 89.08±2.84% at 12 h. Simultaneously, in SA-Fe, SA release exhibited sustained-release characteristics, with a cumulative release of 53.13±3.66% at 12 h. Subsequently, the release rates of both components tended to balance, ultimately reaching 95.77±2.67% for SA and 56.80±3.66% for SA-Fe at 36 h. These results indicate that SA-Fe possesses good sustained-release properties. Rapid release in the initial stage reaches therapeutic concentrations, while sustained release in the later stages maintains long-term efficacy.

[0067] 3.2 Determination of in vitro photothermal effect The photothermal properties of SA-Fe under near-infrared light are as follows: Figure 2 As shown. The results show that the temperature increases with increasing irradiation power (as shown). Figure 2 A), with a power of 1.5 W / cm². 2 The temperature change was 35.9℃, while the power was 0.2 W / cm². 2 The temperature change was only 7°C, of ​​which, Figure 2 The corresponding thermal image in B and Figure 2 The results in A are consistent; the temperature increases significantly with different concentrations of SA-Fe. Figure 2 (D) Compared to ddH2O, the temperatures after 5 min of irradiation at concentrations of 400 μg / mL and 800 μg / mL were 56.7℃ and 58.0℃, respectively, with very little difference between the two. Figure 2 The thermal imaging results of E were consistent; the temperature change was monitored by 3 cycles of near-infrared irradiation of SA-Fe aqueous solution for 5 min ( Figure 2 (C) The results show that SA-Fe has good photothermal stability. SA-Fe exhibits a significant photothermal effect under near-infrared (808 nm) irradiation, a property derived from Fe 3+can effectively absorb near-infrared light, making it a promising candidate for photothermal therapy (PTT). These results show that SA-Fe can quickly and effectively convert laser energy into heat, and the large amount of heat generated can destroy bacterial membranes and denature bacterial proteins, achieving an antibacterial effect. These findings highlight the outstanding photothermal effect of SA-Fe nanoparticles, indicating their great potential in photothermal antibacterial applications.

[0068] 3.3 Synergistic antibacterial effect of photothermal therapy (PTT) The experimental results of the synergistic antibacterial effect of photothermal therapy (PTT) are shown in Figure 3 . In this study, the antibacterial activity of SA-Fe was determined using the Oxford cup diffusion method combined with PTT. The inhibition zone images of different groups after 18 h of treatment ( Figure 3 A) show that SA-Fe has good antibacterial effect on both Staphylococcus aureus and Escherichia coli compared to the control group; without NIR irradiation, the quantitative inhibition zone of SA-Fe at concentrations of 400 μg / mL and 800 μg / mL against Staphylococcus aureus was 22.18±2.47 mm and 24.97±2.14 mm, respectively, and against Escherichia coli was 16.25±2.64 mm and 19.37±0.34 mm, respectively; with NIR irradiation, the quantitative inhibition zone of SA-Fe at concentrations of 400 μg / mL and 800 μg / mL against Staphylococcus aureus was 27.26±1.24 mm and 31.27±1.04 mm, respectively, and against Escherichia coli was 22.25±1.17 mm and 24.98±1.46 mm, respectively. The results show that the heat generated after NIR irradiation can inhibit the growth of drug-resistant bacteria, significantly improving the antibacterial efficiency of SA-Fe, indicating that PTT synergistic antibacterial effect is effective.

[0069] The live / dead bacteria fluorescence images after 18 h of different treatments ( Figure 3 B) confirm that SA-Fe has excellent antibacterial effect under PTT: the inhibition rates of Staphylococcus aureus and Escherichia coli in the normal group were 1.10±0.04% and 0.97±0.41%, respectively; without NIR irradiation, the inhibition rates of SA-Fe against Staphylococcus aureus and Escherichia coli were 10.05±0.57% and 21.54±5.67%, respectively; with NIR irradiation, the inhibition rates of SA-Fe against Staphylococcus aureus and Escherichia coli were 20.59±8.70% and 39.49±3.6%, respectively. The above results show that the antibacterial effect of SA-Fe is significantly increased after NIR irradiation.

[0070] To study the mechanism of SA-Fe destroying Staphylococcus aureus and Escherichia coli, scanning electron microscopy (SEM) was used for observation, and the results show that Figure 3C): Under no NIR irradiation, the control group exhibited typical normal morphological characteristics, with smooth and flat cell membrane surfaces, intact and undamaged cell wall structures, and no obvious structural abnormalities. However, bacteria treated with SA-Fe showed some morphological changes; the cell membrane surface was no longer uniformly smooth, exhibiting localized irregular protrusions or depressions. More importantly, microscopic cavities were observed in the cell wall regions of some bacteria, accompanied by slight bacterial shrinkage, suggesting that SA-Fe had already had a preliminary impact on bacterial structure in the absence of light. When NIR irradiation was added, the morphological damage to bacteria in the SA-Fe treatment group was significantly more severe than in the unirradiated state, with more pronounced and widespread bacterial shrinkage. This was particularly evident in Staphylococcus aureus, which showed the most severe structural damage under SA-Fe combined with NIR irradiation: severe disruption of cell wall integrity, with large-area ruptures and defects, breakage of the originally continuous cell wall structure, and drastic shrinkage of the cell membrane, resulting in disordered spatial relationships with the cell wall and overall distorted bacterial morphology. Escherichia coli showed relatively milder structural damage under these treatment conditions. Iron can damage bacterial biofilms, and this damaging effect on biofilms is the key reason why it helps enhance the antibacterial activity of traditional antibiotics.

[0071] 3.4 Biocompatibility evaluation of SA-Fe To assess the biocompatibility of SA-Fe, we evaluated its blood compatibility, cell compatibility, and tissue compatibility. The biocompatibility evaluation results of SA-Fe are as follows: Figure 4 As shown.

[0072] First, the hemolysis rate was evaluated to assess the toxicity of the biomaterials. The results showed (4B): the positive control group using distilled water was bright red, indicating that the rupture of the red blood cell membrane led to the release of hemoglobin; in contrast, the colors of the negative control group (physiological saline) and the SA-Fe group (12.5-200 μg / mL) were similar; the hemolysis rate of all SA-Fe concentrations was less than 5%, showing good blood compatibility.

[0073] Subsequently, the cytotoxicity of SA-Fe on L929 cells and HUVECs cells was quantitatively evaluated using the MTT assay. The results showed (4C): after co-culturing with SA-Fe at concentrations of 5-400 μg / mL for 24 h, the viability values ​​of both L929 cells and HUVECs cells were higher than 100%, indicating good cell activity. SA-Fe did not significantly affect the proliferation of L929 cells, with cell viability around 110%. At a SA-Fe concentration of 100 μg / mL, HUVECs cells exhibited the highest viability, at 135.18±1.92%, indicating good cell compatibility at this concentration. The cytocompatibility of SA-Fe was further evaluated using live / dead cell staining technology. L929 cells (4A) and HUVECs (4D) were co-cultured with SA-Fe for 24 h, and then stained with Calcein-AM / PI (live / dead) staining solution. Live cells showed green fluorescence due to calcein AM, while dead cells showed red fluorescence due to propidium iodide (PI) staining. The figure shows that live cells exhibited the largest proportion of green fluorescence, while only a few dead cells showed red fluorescence in each group; live cells were the most abundant. These results indicate that co-culturing SA-Fe with cells did not significantly decrease cell viability, demonstrating good cytocompatibility.

[0074] We further observed the daily behavior of mice injected with SA-Fe via the tail vein for 48 hours. The results showed that the behavior of the mice after administration was highly consistent with that of normal mice, such as eating and drinking rhythms, nesting behavior, and diurnal activity cycles. No behavioral abnormalities caused by the drug were observed, indicating that the behavioral state of the mice after administration was similar to that of normal mice. Pathological H&E staining analysis of major organs (heart, liver, spleen, lung, and kidney) was performed to verify whether SA-Fe caused systemic pathological toxicity. The results (4F) showed that compared with the control group, the experimental group animals treated with SA-Fe had intact tissue structures and normal cell morphology in all organs, without obvious pathological changes such as inflammatory cell infiltration, cell degeneration and necrosis, tissue fibrosis, or pathological hyperplasia. The ratio and structure of organ parenchyma and interstitium also maintained a normal physiological state. The results of this series of histopathological observations corroborate the conclusion in the previous cytotoxicity experiments that SA-Fe caused no damage to L929 and HUVECs cells, fully demonstrating that the synthesized SA-Fe will not cause adverse effects on vital organs within the experimental dosage range and has good in vivo biosafety, providing key safety evidence for its subsequent biomedical applications.

[0075] 3.5 Cell proliferation, migration, and angiogenesis Results of experiments on HUVECs cell proliferation, migration, angiogenesis, and L929 cell migration are as follows: Figure 5As shown. The proliferation capacity of HUVECs was assessed using the MTT assay. Figure 4 (G) HUVECs treated with SA-Fe showed significantly enhanced proliferation activity compared to the control group after 1, 2, and 5 days of culture. A key observation was observed: on day 2 of culture, the absorbance value of cells in the tanshinone-treated group was lower than that of the blank control group, which differed from the expected proliferation-promoting effect. Considering the chemical properties of tanshinone, it was speculated that this phenomenon might be related to the possible oxidative degradation of tanshinone to generate oxidation products with unknown structures. These oxidation products lost their original activity in promoting HUVEC proliferation. To further verify this hypothesis, the culture medium containing fresh tanshinone was replaced every 24 hours after the fifth day of proliferation. The results showed no significant difference in absorbance between the free tanshinone group and the blank group. Even when fresh tanshinone was added to increase the absorbance to some extent, it failed to promote cell proliferation. However, the prepared SA-Fe-treated group showed a significant increase in absorbance, indicating that SA-Fe not only successfully overcame the defect of easy oxidation of free tanshinone and maintained its biological activity, but also achieved effective promotion of HUVEC proliferation through the synergistic effect of tanshinone and iron.

[0076] To investigate the regulatory effect of SA-Fe on cell migration, the scratch assay was used to assess its influence on the migration behavior of HUVECs. HUVECs were cultured under different conditions, and their migration process was dynamically monitored using a microscope. The results showed that the migration ability of HUVECs in the SA-Fe treatment group was significantly enhanced compared with the control group. Quantitative analysis indicated that after 24 h of co-culture, the migration rates of HUVECs in the SA-Fe group under no-light and NIR light irradiation were 94.03±0.69% and 95.78±1.13%, respectively, significantly higher than the 43.90±0.81% of the control group. The SA-Fe group exhibited the most significant migration-promoting effect under NIR light irradiation.

[0077] To comprehensively evaluate the angiogenesis capabilities of different treatment groups, this study quantitatively analyzed the core parameters of the vascular network (number of nodes, number of branches, and total length) through in vitro angiogenesis experiments. The results (5B, 5C, 5E) showed that, compared with the untreated control group, both the SA-treated and SA-Fe-treated groups significantly promoted the extension of vascular branches and formed a stable three-dimensional vascular network structure, demonstrating that SA-Fe has the ability to activate angiogenesis-related signaling pathways and enhance angiogenesis activity. Under near-infrared (NIR) light stimulation, the SA-Fe-treated group exhibited better angiogenesis capabilities. SA-Fe may promote the release of tanshinone and iron through NIR light triggering, synergistically enhancing cell proliferation, migration, and angiogenesis, thus establishing an optimal microenvironment for tissue regeneration and wound healing. In diabetic wound repair, it can accelerate the tissue repair process in multiple dimensions, providing important theoretical and experimental support for wound healing and the functional optimization of tissue engineering scaffolds.

[0078] L929 cells were co-cultured under different conditions, and their migration process was dynamically monitored using a microscope. The results showed that the migration ability of L929 cells in the SA-Fe treatment group was significantly enhanced compared to the control group. Quantitative analysis indicated that after 24 h of co-culture, the migration rates of L929 cells in the SA-Fe group under no-light and NIR light irradiation were 18.97±2.81% and 24.50±2.96%, respectively, significantly higher than the 7.71±0.76% in the control group. The SA-Fe group showed the most significant migration-promoting effect under NIR light irradiation.

[0079] 3.6 Antioxidant capacity and intake over time The antioxidant capacity and time-dependent uptake of SA-Fe are as follows: Figure 6 As shown in Figure 6A, Figure 7B, and Figure 8C, the uptake of SA-Fe prepared in L929 and HUVECs cells was investigated. Fluorescence quantitative analysis was performed using SA-Fe labeled with the fluorescent reagent coumarin-6. The results (6A, 6B, 6C) showed that both L929 and HUVECs cells exhibited good uptake of SA-Fe, but HUVECs cells showed better uptake. Coumarin-6-labeled SA-Fe could enter the cytoplasm, and this phagocytosis provides a biological basis for the potential functional impact of SA-Fe on L929 and HUVECs cells.

[0080] The antioxidant capacity of different concentrations of SA-Fe was evaluated using the ABTS and DPPH methods. The results showed that ( Figure 6 D): SA-Fe exhibits excellent antioxidant effects. A concentration of 250 μg / mL of SA-Fe showed a ABTS scavenging rate of 92.77±0.29%, and a concentration of 100 μg / mL of SA-Fe showed a DPPH scavenging rate of 96.45±0.69%. From... Figure 6 As can be seen from E, the blue-green color of ABTS turned clear, indicating that ABTS was reduced and acted upon as an antioxidant. Figure 7 The purple color in F gradually faded, changing to a light yellow, reflecting an enhanced free radical scavenging ability. This color change indicates that SA-Fe has a more significant effect in scavenging DPPH. This suggests that the SA-Fe obtained in this study can be used as an antioxidant in many applications, such as promoting wound healing in diabetes and anti-inflammation.

[0081] 3.7 Preparation of SA-Fe hydrogel To facilitate drug administration during wound healing in diabetic rats, a non-functional hydrogel, poloxamer 407, was introduced as a carrier. Experiments showed that the temperature increased with increasing concentration; at a concentration of 6 mg / mL, the temperature reached 52.5℃, which was therefore selected as the drug administration concentration. To investigate the in vivo photothermal properties, SA-Fe hydrogel (10 mm in diameter) was applied to rat wounds and subjected to laser irradiation (1 W / cm²). 2 Infrared thermal imaging was performed, and the temperature reached 49.3℃ after 5 minutes of irradiation, confirming its excellent in vivo properties. In summary, these findings highlight the unique combination of hydrogels in terms of tunable photothermal response, strong thermal stability, and physiological temperature regulation. Its ability to achieve bactericidal temperatures (≥45℃) while avoiding tissue damage makes it an advanced platform for near-infrared controlled photothermal antibacterial therapy, particularly suitable for the dynamic microenvironment of diabetic wounds.

[0082] 3.8 In vivo study on wound healing effect in diabetic rats In vivo study results on wound healing effect in diabetic rats are as follows: Figure 7 As shown. This study used a diabetic full-thickness skin defect model to evaluate its treatment effect ( Figure 7 A). Wound images at different time intervals ( Figure 7 B) indicates that the SA-Fe+NIR hydrogel group exhibited the fastest healing process throughout the 14-day experiment, followed by the SA-Fe hydrogel group, the SA hydrogel group, and the blank control group. Furthermore, quantitative analysis of wound healing rates confirmed a similar trend, such as... Figure 7 As shown in Figure E, on day 11, the wound healing rate in the SA-Fe+NIR hydrogel group was 94.56±0.94%, significantly higher than that in the SA-Fe hydrogel group (87.21±2.65%) and the SA hydrogel group (82.63±4.04%). The healing rate in the blank control group was 75.18±2.32%. This may be attributed to the excellent antioxidant and anti-inflammatory properties of tanshinone, which can effectively alleviate oxidative stress and harmful microenvironment in the early stage of diabetic wound healing. These results indicate that although the effect of tanshinone alone is limited, the combination of the anti-inflammatory and antioxidant capabilities of SA-Fe shows a synergistic effect, especially after adding NIR, the effect is more obvious, and the promoting effect on diabetic wound healing is the most significant.

[0083] To evaluate the effect of SA-Fe hydrogel on wound healing, histological analysis was performed, including H&E staining and Masson staining. The results are as follows: Figure 8 As shown. H&E staining revealed that, compared with the control group and the SA group, the SA-Fe+NIR group had a stronger ability to promote chronic wound healing; on day 5, the SA-Fe+NIR treatment group already showed substantial granulation tissue formation.Figure 8 A, 8C); Over time, the SA-Fe+NIR group showed reduced inflammatory penetration, increased granulation tissue thickness, and enhanced epithelialization. Masson staining also confirmed that the collagen deposition rate in the SA-Fe+NIR group was significantly higher than that in the control group. In H&E staining on day 11, the tissue in this group was nearly continuous with well-organized cells. Masson staining showed that the collagen regions were denser, and the collagen deposition rate in this group was the highest among all groups. Figure 8 B, 8C). From Figure 8 As shown in Figure D, on day 11, the wound width in the SA-Fe+NIR group was the smallest compared to the other groups. Overall, SA-Fe+NIR treatment not only promotes collagen production in the early stages but also improves healing quality and reduces scarring in the later stages, demonstrating excellent ability to promote the healing of chronic wounds.

[0084] Immunohistochemical and immunofluorescence analysis results of different samples are as follows: Figure 9 As shown in the results, the expression of CD31 and VEGF in the SA-Fe+NIR group on day 11 was significantly higher than that in other groups. Figure 9 (A, 9C, 9E) verified the ability of SA-Fe+NIR to promote angiogenesis in chronic wounds, mainly because SA can activate the VEGF pathway, thereby increasing VEGF expression; the expression of TGF-β and α-SMA in the SA-Fe+NIR group on day 11 was significantly higher than that in other groups. Figure 9 (D, 9G) indicates that SA-Fe+NIR has a better effect on promoting fibroblast and endothelial cell migration and collagen cell deposition; compared with other groups, the expression of anti-inflammatory factor (IL-10) in the SA-Fe+NIR group was significantly upregulated on day 11. Figure 9 F) The expression of pro-inflammatory factors (TNF-α, IL-1β) was significantly downregulated. Figure 9 H, 9I), suggesting that the addition of NIR to the hydrogel as a wound dressing can modulate macrophages, reduce inflammatory response, exert a good anti-inflammatory effect, and promote wound healing; fluorescence staining and quantitative results ( Figure 9 (B, 9J) showed that, compared with other groups, the fluorescence intensity of the inflammatory macrophage marker (CD86) in the SA-Fe+NIR group was significantly reduced, while the fluorescence intensity of the anti-inflammatory macrophage marker (CD206) was significantly increased, confirming that SA-Fe+NIR retained the significant ability of tanshinone to induce macrophage polarization. In summary, SA-Fe+NIR exerts good tissue repair and anti-inflammatory effects by promoting angiogenesis, promoting fibrosis, and regulating inflammatory factors and macrophage phenotype.

[0085] Although the present invention has been described in detail above with general descriptions, specific embodiments, and experiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A method for preparing a Danshensu-iron nano-composition with photothermal effect, characterized in that, The preparation method steps are as follows: S1, respectively, take Danshensu and iron trichloride hexahydrate, add ultrapure water to dissolve thoroughly, prepare Danshensu solution and iron trichloride solution; S2, slowly drop the prepared iron trichloride solution into the Danshensu solution, mix well, get the mixed solution; S3, the mixed solution is placed on the magnetic stirrer to avoid light reaction, after the reaction is completed, the reaction solution is freeze-dried, and the Danshensu-iron nano composition with photothermal effect is obtained.

2. The method of claim 1, wherein the preparation of the Danshensu-iron nano-composition with photothermal effect is characterized in that, The concentration of Danshensu solution and iron trichloride solution in step S1 is 0.1 mol / L.

3. The method of claim 1, wherein the preparation of the Danshensu-iron nano-composition with photothermal effect is characterized by, The volume ratio of Danshensu solution and iron trichloride solution in step S2 is 1:

1.

4. The method of claim 1, wherein the preparation of the Danshensu-iron nano-composition with photothermal effect is characterized by, The time of light-avoiding reaction in step S3 is 10-15 h.

5. The method of claim 4, wherein the preparation of the Danshensu-iron nano-composition with photothermal effect is characterized in that, The time of light-avoiding reaction in step S3 is 12 h.

6. A Danshensu-iron nano composition with photothermal effect prepared by the preparation method of any one of claims 1-5.

7. The use of the Danshensu-iron nano composition with photothermal effect of claim 6 in the preparation of a drug for promoting the repair of diabetic infected wounds.

8. Use according to claim 7, characterized in that, The drug for promoting the repair of diabetic infected wounds takes Danshensu-iron nano composition as the main active ingredient, adds pharmaceutically acceptable adjuvants, and is prepared into a pharmaceutically acceptable preparation.

9. Use according to claim 8, characterized in that, The preparation is a hydrogel, and its preparation method is: taking Danshensu-iron nano composition, adding ultrapure water to completely dissolve, preparing Danshensu-iron aqueous solution; taking poloxamer 407 powder, adding it into the Danshensu-iron aqueous solution, and obtaining the hydrogel.

10. Use according to claim 9, characterized in that, In the preparation method of the hydrogel: the concentration of Danshensu-iron aqueous solution is 6 mg / mL; the amount of poloxamer 407 added is 3 g per 10 mL of Danshensu-iron aqueous solution.

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