IR780 (95%) and Tanshinone IIA (98%) were sourced from Shanghai Macklin Biochemical Co., Ltd., while Bovine Serum Albumin (98%) was acquired from Shanghai Aladdin Reagent Co., Ltd. Dithiothreitol (DTT) was provided by Nanjing Wanqing Chemical Glassware Instrument Co. Ltd. (China). The cell counting kit-8 (CCK-8) was obtained from Dojindo Laboratories (Japan). Unless specified otherwise, all additional reagents were purchased from Nanjing Wanqing Chemical Glassware Instrument Co. Ltd. and were used without further modification.

Using the “molecular switch” method, Tanshinone IIA (TSIIA) and IR780 (BSA-TSIIA-IR780 NPs) dual-loaded albumin nanoparticles were prepared by a two-step method. Initially, 100 mg of Bovine Serum Albumin (BSA) was completely dissolved in 50 mL of phosphate-buffered saline (PBS). Subsequently, 100 μL of dithiothreitol (DTT) solution (10 mg/mL) was added to the BSA solution at 45°C. During the reaction, DTT effectively opened the hydrophobic spaces within BSA. Then, while continuously stirring, 2 mL of ethanol solution containing Tanshinone IIA (10 mg/mL) was gradually added to the BSA solution, resulting in the formation of BSA-TSIIA nanoparticles (BSA-TSIIA NPs).

In the second step, 1 mL of ethanol solution containing IR780 (2 mg/mL) was added to the aforementioned solution. The interaction of intermolecular electrostatic forces and hydrophobic interactions facilitated the formation of BSA-TSIIA-IR780 nanoparticles. Subsequently, the nanoparticle solution was ultrafiltered three times to remove free IR780, TSIIA, and ethanol. The final concentrated solution was about 3 mL, suitable for subsequent experiments.

The content of IR780 in the BSA-TSIIA-IR780 nanoparticles was analyzed using a spectrophotometric method. Briefly, the BSA-TSIIA-IR780 nanoparticles were degraded by acetonitrile (volume ratio 1:1). Afterwards, the resulting mixture was diluted 25-fold in chloroform and sonicated for 10 min to ensure complete extraction of IR780. The concentration of IR780 was determined by measuring the absorbance at 785 nm using a UV-vis-NIR spectrophotometer (UV-2450, Shimadzu, Japan) and calculating it from the standard curve of IR780 in chloroform. The content of TSIIA was calculated using high-performance liquid chromatography (HPLC) with reference to the standard curve. TSIIA detection was conducted at a wavelength of 270 nm, using a reverse phase C18 column (5 μm, 4.6 mm × 250 mm, Agilent, United States) at 25°C. The flow rate was maintained at 1.0 mL/min. The elution solvents were phase A (methanol) and phase B (water), with a ratio of 85% A to 15% B (Meng et al., 2015). The BSA concentration was determined using the Coomassie Brilliant Blue method.

The particle size of the BSA-TSIIA-IR780 nanoparticles was determined by Dynamic Light Scattering (DLS) using a Zeta Plus (Brookhaven Instruments Corporation, United States). Before and after irradiation with an 808 nm laser (power density of 1w/cm², for 5 min), the nanoparticles were negatively stained with phosphotungstic acid, and their morphology was assessed using a Transmission Electron Microscope (TEM, Hitachi H-600, Japan).

BSA-TSIIA-IR780 nanoparticles were diluted to 0.05 mg/mL (concentration of IR780) and exposed to an 808 nm near-infrared laser at a power density of 1.0 W/cm² for a continuous duration of 3 min. A thermometer was used to measure the temperature every 30 s. BSA-IR780 nanoparticles, free IR780, and PBS were used as controls.

To study the release curve of TSIIA from nanoparticles, 1 mL of BSA-TSIIA-IR780 nanoparticles and BSA-TSIIA nanoparticles were separately placed into dialysis bags (molecular weight cutoff of 3.5 kDa) and immersed in 15 mL of release medium (PBS containing 1% Tween 80, pH 7.4). The release behavior of BSA-TSIIA-IR780 nanoparticles was investigated with and without irradiation by an 808 nm laser (power of 1w/cm² for 5 min). Samples (release solution) were collected at predetermined time points (0–72 h) and the same volume of release medium was replenished. The content of TSIIA was measured by HPLC (as previously described), and the drug release curve was obtained based on the standard curve.

The model bacterium used in the antibacterial experiment was methicillin-resistant S. aureus (MRSA, ATCC 43300). MRSA cells were cultured in lysogeny broth (LB) medium at 37°C under aerobic conditions until they reached mid-exponential growth phase (Liu et al., 2022). To assess the in vitro antibacterial activity of various concentrations of BSA-TSIIA-IR780 nanoparticles, a suspension of MRSA (100 μL, 1 × 10⁵ CFU/mL) was added to a 96-well plate, along with a range of concentrations of BSA-TSIIA-IR780 nanoparticles (100 μL; 0.5, 1.0, 2.0, and 5 μg/mL IR780; 5.0, 10.0, 20.0, and 50.0 μg/mL TSIIA), BSA-TSIIA nanoparticles (5.0, 10.0, 20.0, and 50.0 μg/mL TSIIA), and BSA-IR780 nanoparticles (100 μL; 0.5, 1.0, 2.0, and 5 μg/mL IR780). LB solution was used as a control. In the photothermal treatment group, the 96-well plate was irradiated with a near-infrared laser (808 nm, 1.0 W/cm²) for 5 min. After incubation for 120 min at 37°C, CCK-8 solution (10 µL/well) was added to the 96-well plate. After incubating for 30 min in the dark at room temperature, the plate was placed in an enzyme-linked immunosorbent assay reader to measure the absorbance at 450 nm, which reflects the number of live bacteria in each well. Each group included five replicate wells. Cell viability was expressed as the mean absorbance ±standard deviation (SD) of the five wells per group. The experiment was repeated three times (Xu et al., 2021).

All animal experiments were conducted in compliance with the Animal Care and Use Committee of Nantong University. For the wound healing model, this experiment used 5 week-old male ICR mice. Prior to wound creation, the mice were given an intraperitoneal injection of an anesthetic mixture (ketamine/xylazine), and then the hair on their dorsal surface was shaved. A skin wound measuring 1 × 1 cm² was created on the dorsal surface of the mice using a 28-gauge needle. Five minutes after creating the wound, each scratch area was inoculated with 40 µL of a suspension containing 1 × 10⁸ CFU/mL MRSA, dispersed in PBS. Twenty-4 h after the wounds were infected with the MRSA suspension, the mice were randomly assigned to different treatment groups (each group, n = 4) as follows: control group (100 µL PBS), BSA-TSIIA NPs (100 μL, 200 μg/mL TSIIA), BSA-TSIIA-IR780 NPs without laser (100 μL, 200 μg/mL TSIIA and 20 μg/mL IR780), BSA-IR780 NPs (100 μL, 20 μg/mL IR780) plus laser, and BSA-TSIIA-IR780 NPs (100 μL, 200 μg/mL TSIIA and 20 μg/mL IR780) plus laser. A near-infrared laser (808 nm, 1.0 W/cm²) was used to irradiate the treatment area for 5 min, 24 h after nanoparticle application to the mice. Wound size was measured using a digital caliper and photographed on days 0, 2, 4, 8, and 16. Wound healing rate was calculated using the following equation: Relative wound area  % = W △   /   W 0 × 100 %

Where W_△ is the wound area on a specific day, and W₀ is the wound area on day 0.

Comparison between two groups was performed using Student’s t-test. Comparisons among more than two groups were conducted using two-way Analysis of Variance (ANOVA), followed by Tukey post-hoc analysis to compare the means of two groups. ^* p < 0.05, ^** p < 0.01, ^# p < 0.05, ^## p < 0.01, data are presented as mean ± standard deviation.

This experiment employed two model molecules, including the hydrophobic Tanshinone IIA and the near-infrared small molecule IR780. As shown in Figure 2A, Tanshinone IIA belongs to the diterpene class of compounds, featuring a typical diterpene ketone skeleton that includes multiple rings and functional groups. The molecule of Tanshinone IIA contains a ketone group, which is one of the key functional groups contributing to its activity. Tanshinone IIA also features a complex side-chain structure, which may influence its pharmacological activity and bioavailability (Jiang et al., 2019; Huang et al., 2022). The structure of the compound accounts for its multiple biological activities, including anti-inflammatory, antioxidant, antitumor, and antimicrobial properties. IR780 (Figure 2B) is a cyanine dye compound with near-infrared light absorption properties, commonly used in photothermal therapy and biological imaging. The indocyanine group in IR780 molecules is one of the main reasons for its absorption capability in the near-infrared region. Additionally, the presence of an amino group endows IR780 with a certain positive charge, offering unique advantages during the nanoparticle assembly process (Zhang et al., 2014). Figure 2C shows the ultraviolet characteristic absorption peak of Tanshinone IIA at 270 nm, while IR780 has a characteristic absorption peak at 785 nm, with no interference between them, which can be utilized to separate them in quantitative experiments using the differences in ultraviolet characteristic absorption.

According to the literature and experimental procedures, we used UV-vis-NIR spectroscopy to determine IR780, and high-performance liquid chromatography (HPLC) for Tanshinone IIA. As shown in Supplementary Figure S1A, there is a very good linear relationship within a certain concentration range (0.0625–2.0 μg/mL) in organic solvents (R ² = 1). From Supplementary Figure S1B, it can be observed that Tanshinone IIA is well separated in the HPLC, with the elution time at 5.5 min and a regular peak shape without significant tailing, which can serve as a quantitative method for Tanshinone IIA.

DTT (dithiothreitol) used in nanoparticle preparation reduces the disulfide bonds in albumin, forming free thiol groups and opening hydrophobic regions. Tanshinone IIA is then added to form initial nanoparticles. IR780, as a hydrophobic charged small molecule, further promotes the assembly of albumin nanoparticles, resulting in drug-loaded nanoparticles with an average diameter of about 185 nm (Figure 3A). Under laser irradiation, IR780 degrades, leading to the “disassembly” of the nanoparticles, which enables the release of Tanshinone IIA. From Figure 3B, it can also be seen that the nanoparticle distribution is broad, with smaller nanoparticles (42 nm) and larger nanoparticles (285 nm), indicating significant changes in the nanoparticles under laser irradiation. The process of IR780 acting as a photosensitizer to generate heat under laser exposure can be explained by the photothermal conversion mechanism. In the presence of a photosensitizer, the laser energy is absorbed and excites the electrons of the photosensitizer to an excited state, forming excited-state photosensitizer molecules. These excited-state molecules, possessing higher energy, release some energy during non-radiative decay, a process known as internal conversion. The released energy is then absorbed by the internal vibrations and rotations of the molecules, causing an increase in internal temperature. This leads to the degradation of the nanoparticles from within, reflected in changes in size and in transmission electron microscopy images (Figures 3C, D). In Figure 3C transmission electron microscopy image, we observed that the albumin nanoparticles were generally uniform and spherical in structure, contributing to their stability in solution. The nanoparticles demonstrated excellent stability in phosphate-buffered saline (PBS) over a period of 7 days, with minimal aggregation or changes in particle size (Narayanan and El-Sayed, 2005). However, after laser irradiation, this regular structure was disrupted, resulting in the appearance of irregular and larger particles. It was demonstrated that after laser irradiation, the structure of the albumin nanoparticles was altered, making the drug more easily released, thereby achieving enhanced therapeutic effects.

In addition to leveraging the anti-inflammatory, antioxidant, antitumor, and antimicrobial biological activities of Tanshinone IIA itself, this project primarily utilizes IR780 as a near-infrared molecule for its heat-producing characteristics. Through photothermal action, it aims to kill bacteria, thus accelerating the wound healing process. Photothermal therapy uses the heat effect produced after a photosensitizer absorbs light to destroy the structure and function of bacterial cells, achieving an antibacterial purpose. Bacteria are sensitive to high temperatures; localized heat effects can cause disruption of bacterial cell membranes, denaturation of proteins, and DNA damage (Xu et al., 2019). Furthermore, high temperatures can also impact bacterial growth and metabolic processes, thereby effectively killing bacteria. Therefore, we evaluated the heat production characteristics of BSA-TSIIA-IR780 NPs in solution (Figure 4). Under conditions of 50 μg/mL IR780 concentration, within 30 s of laser irradiation, the temperature of the BSA-TSIIA-IR780 NPs solution increased from 24°C to 37°C, reached 46.8°C at 60 s, and a maximum of 58.9°C, indicating that under laser irradiation, the photosensitizer IR780 can rapidly heat up and thus kill bacteria to achieve therapeutic purposes.

Albumin-loaded nanoparticles containing Tanshinone IIA and IR780 exhibit the capability of enhancing drug release under laser irradiation. Effective release of the drug from the formulation is a prerequisite for its action, and stimulus-responsive release nanopreparations possess the function of “on-demand” and “microenvironment-responsive” release. Therefore, we compared the release behaviors of albumin-loaded Tanshinone IIA nanoparticles and albumin-loaded Tanshinone IIA with IR780 nanoparticles, and verified the laser-responsive release characteristics of the latter (Figure 5). As shown in the figure, compared to the composite nanoparticles, albumin-loaded Tanshinone IIA nanoparticles released Tanshinone IIA faster within 24 h (28.1% vs. 20.9% at the 24th hour), and the trend was similar at the 12th hour (23.7% vs. 15.4%). This may be due to the addition of the small molecule IR780 in the composite nanoparticles, which as a cationic hydrophobic molecule, provides additional forces (such as electrostatic adsorption and hydrophobic interactions) during nanoparticle formation, making it more difficult for Tanshinone IIA to be released from the composite nanoparticles. However, after 48 h, there was no significant difference in the release from the two types of nanopreparations, indicating that the state of nanoparticles in the release medium became consistent after 48 h, as did the behavior of drug release. Under laser irradiation, albumin-loaded nanoparticles containing Tanshinone IIA and IR780 showed a significant increase in release, starting from the first hour after irradiation (12.5% at the second h vs. 2.8%; 35.1% at the 12th hour vs. 15.4%; 56.7% at the 48th hour vs. 33.7%). These results demonstrate that albumin-loaded nanoparticles containing Tanshinone IIA and IR780 have excellent laser-responsive release functionality. This experimental section utilizes albumin to deliver two molecules, IR780 and Tanshinone IIA, leveraging both the heat-producing effect of IR780 under laser irradiation and the antimicrobial and anti-inflammatory pharmacological effects of Tanshinone IIA to treat challenging-to-heal wounds. In the early stages of skin damage, due to potential bacterial infections, there is a need to completely eradicate bacteria and other pathogens, thus photothermal action can meet this requirement; simultaneously, the composite nanoparticles release more Tanshinone IIA under laser irradiation, which has excellent antimicrobial and anti-inflammatory pharmacological effects, allowing better penetration into the skin wound, exerting its effects and regulating the immune microenvironment at the wound site to promote rapid healing and recovery (Huang et al., 2022; Xu et al., 2019).

The in vitro antibacterial experiment utilized the CCK-8 assay for measurement. Firstly, we validated the antibacterial properties of Tanshinone IIA. As shown in Figure 6A, both albumin-loaded Tanshinone IIA and free Tanshinone IIA exhibited certain antibacterial effects at high concentrations. At a concentration of 50 μg/mL, the inhibition rate was around 20%, and at 100 μg/mL, the inhibition rate reached 35%–40%. Although there were some antibacterial effects, they were not ideal, hence the addition of photothermal therapy was necessary to further enhance bactericidal activity. From Figure 6B, we observed that the bactericidal capability of the nanoparticles was greatly enhanced with the addition of laser treatment. The laser irradiation group showed significant antibacterial ability even at a Tanshinone IIA concentration of 20 μg/mL (inhibition rate of 40%), and at 100 μg/mL, the inhibition rate reached 93%, indicating that the photothermal effect significantly enhances the antibacterial capability of the nanopreparations. As shown in Figures 3D, 5, the albumin nanoparticles under laser irradiation can more effectively release Tanshinone IIA, which plays an important role in utilizing the antibacterial effects of Tanshinone IIA itself. This is also the main reason why the nanoparticles can achieve a synergistic antibacterial effect under laser irradiation. Upon near-infrared laser irradiation, the IR780 generates localized heat, increasing the temperature of the bacterial environment. The heat generated causes damage to the bacterial cell membrane by increasing membrane fluidity and permeability, ultimately leading to cell lysis. The loss of membrane integrity compromises the bacteria’s ability to maintain homeostasis, which is critical for survival (Xu et al., 2023). This experiment demonstrates that the antibacterial performance of albumin-loaded nanoparticles containing Tanshinone IIA and IR780 primarily relies on the heat generation capability of the near-infrared small molecules, while Tanshinone IIA mainly plays a role in anti-inflammatory responses, scavenging free radicals, and regulating immune therapeutic effects in treatment. The nanoparticles enhance bacterial clearance through the synergistic action of Tanshinone IIA’s antimicrobial properties and the photothermal effect of IR780. Under near-infrared laser irradiation, IR780 generates localized heat that leads to bacterial cell membrane disruption and protein denaturation, while Tanshinone IIA further inhibits bacterial growth (Xu et al., 2023).

We validated the combined effects of nanoparticle-based photothermal therapy and the antibacterial properties of Tanshinone IIA on enhancing wound healing in a mouse wound healing model. The MRSA infected mouse wound model is characterized by its difficulty in healing (Figure 7A Control group), and by the 16th day after establishing the wound model, the relative wound area was 29.9%, confirming the successful establishment of this model. Our treatment group, with albumin-loaded Tanshinone IIA nanoparticles, showed a significantly better therapeutic effect, with a relative wound area of 9.2% on the 16th day, demonstrating the nanoparticles’ excellent capability to promote wound healing and the important role of Tanshinone IIA in wound healing (Figure 7B). In the group treated with albumin-loaded Tanshinone IIA and IR780 nanoparticles, we obtained similar results to the Tanshinone IIA nanoparticle group, with a relative wound area of 4.8% on the 16th day, and statistical results showed no significant difference from the Tanshinone IIA nanoparticle group. In the laser irradiation group, we found that the albumin IR780 nanoparticles without Tanshinone IIA performed no differently from the Control group, proving that photothermal therapy alone under this study’s conditions does not significantly enhance wound healing in mice, with a relative wound area of only 24.7% on the 16th day. However, the final treatment group, albumin-loaded Tanshinone IIA and IR780 nanoparticles combined with laser treatment, leveraging the dual advantages of photothermal sterilization and enhanced release of Tanshinone IIA, exhibited significant antibacterial performance and greatly enhanced the healing of difficult wounds (16th day, relative wound area of only 1.5%). Beyond bacterial clearance, Tanshinone IIA is known for its anti-inflammatory properties. It helps regulate the inflammatory response by reducing the infiltration of inflammatory cells and downregulating pro-inflammatory cytokines, which prevents chronic inflammation and promotes the transition to the proliferation phase of wound healing. This anti-inflammatory effect accelerates tissue repair by reducing tissue damage caused by excessive inflammation (Guo et al., 2020). The results prove that our designed composite nanoparticles have an excellent ability to promote wound healing.