Analgesic-loaded NIR-II photothermal hydrogel for efficient anti-infection and precise pain management in wounds

TL;DR

This study introduces a hydrogel that combines pain relief and infection control for wound healing using light therapy.

Contribution

A novel hydrogel (STB-Lid Gel) that provides sustained analgesia and photothermal anti-infection in wounds.

Findings

STB-Lid Gel provides localized analgesia for over 30 hours through sustained lidocaine release.

NIR-II laser triggers enhanced lidocaine release and bacterial eradication via photothermal therapy.

The hydrogel reduces inflammation and accelerates wound healing by managing pain and infection.

Abstract

Photothermal therapy (PTT) is widely recognized for the treatment of infected wounds. However, the wound hyperpathia caused by infection and heat from PTT is often overlooked, which might affect wound healing significantly. In this study, we systematically investigate the management of pain in bacteria-infected wounds using PTT, and develop a photothermal antibacterial hydrogel capable of sustained and controlled analgesia system (STB-Lid Gel), aiming to simultaneously achieve efficient anti-infection and precise pain management. STB-Lid Gel is constructed from a silk fibroin (SF) hydrogel matrix co-loaded with lidocaine (Lid) as an analgesic and supramolecular stable radicals (TPT-B12) as a novel second near-infrared-window (NIR-II) photothermal agent. During the treatment phase, the sustained release of Lid stored in STB-Lid Gel extends localized analgesic effects beyond 30 h. Under NIR-II laser irradiation, STB-Lid Gel mediates PTT for bacterial eradicating and enhancement of Lid release. These dual effects of anti-infection and pain management, effectively eradicates bacteria, alleviates PTT-induced acute pain, reduces local inflammation, and ultimately accelerates wound healing. This innovative approach highlights the importance of pain relief from PTT-associated acute pain in healing processes, presenting a viable strategy for combining antibacterial and analgesic treatment in infected wounds.

STB-Lid Gel is a silk fibroin-based hydrogel co-loaded with lidocaine and photothermal agent (TPT-B12). STB-Lid Gel achieves efficient photothermal conversion via bacterial in situ reduction of TPT-B12, enabling potent bacteriostasis. STB-Lid Gel elevates pain thresholds through sustained (>30 h) and photothermal-triggered lidocaine release. Collectively, STB-Lid Gel suppresses infection and alleviates treatment-associated pain while accelerating wound healing and attenuating inflammation.Image 1

•Dual-modal lidocaine release for prolonged (>30 h) and NIR-II-triggered on-demand analgesia.•Bacteria-activated supramolecular photothermal agent (TPT-B12) enabling highly efficient, selective NIR-II PTT.•Synergistic antibacterial and anti-hyperalgesic effects promote anti-inflammatory, low-stress wound healing.•Pain alleviation drives wound repair, supporting an integrated antibacterial–analgesic platform.

Dual-modal lidocaine release for prolonged (>30 h) and NIR-II-triggered on-demand analgesia.

Bacteria-activated supramolecular photothermal agent (TPT-B12) enabling highly efficient, selective NIR-II PTT.

Synergistic antibacterial and anti-hyperalgesic effects promote anti-inflammatory, low-stress wound healing.

Pain alleviation drives wound repair, supporting an integrated antibacterial–analgesic platform.

Figures

Characterization of STB-Lid Gel properties and functions. a) Photographs of hydrogels with various compositions. b) FTIR spectra of STB-Lid Gel and its individual components. c) Representative SEM images of the hydrogels. d) Strain-sweep measurements performed at a constant frequency of 1.0 Hz with strain amplitudes ranging from 0.1% to 1000%. e) Frequency-sweep tests conducted at a fixed strain of 1% over the frequency range of 0.1 - 100 Hz. f) Demonstration of hydrogel injectability (inset photograph) and shear-thinning behavior.Fig. 1

Characterizations and photothermal response of the STB-Lid Gel. a) Photographs of STB-Lid Gel after co-culturing with bacterial suspensions at 37 °C for different time periods. b) UV-Vis-NIR absorption spectra of STB-Lid Gel after co-incubation with *S. aureus* at various time points. c) UV-Vis-NIR absorption spectra of SF Gel, STB Gel, and STB-Lid Gel before and after 6 h of co-incubation with *S. aureus*. d) EPR spectra of STB-Lid Gel after 0 h, 2 h, and 6 h of co-incubation with *S. aureus*. e) Temperature evolution curves and f) corresponding infrared thermal images of STB-Lid Gel after di

Immunofluorescence of c-Fos/TRPV1 at 30 h after various treatments in the infection plantar pain model or Blank (without infection and treatment). a) Representative immunofluorescence showing c-Fos (red) and TRPV1 (green) staining. Nuclei were counterstained with DAPI (blue). b) Schematic diagram of tissue sampling for immunofluorescence. c) Statistical analysis of the co-expression proportion of c-Fos^+^ and TRPV1^+^ in TRPV1^+^ neurons among different groups. d) Statistical analysis of MFI of c-Fos in DRG in different groups. Data are shown as mean ± SEM (n = 6) (∗*p<0.05, ∗∗p<0.01, ∗∗∗p<0.0

Fig. 7Histological staining evaluation and RNA-seq analysis of wound tissues. a) H&E staining and Masson's trichrome staining of the skin tissues from the wound edges on day 10; Black arrowheads indicate the epidermis necrosis and dermal separation, red arrowheads indicate the inflammatory cells, and yellow arrowheads indicate the newborn hair follicles. b) Corresponding statistical data of colonies on day 5 after being treated with various groups. c) Statistical analysis of relative inflammatory levels in wound areas on day 10. d) Statistics of relative collagen deposition at the wound site o

Full text

Introduction

1

Wound healing, as a crucial physiological process with a direct impact on clinical outcomes, is influenced by a multitude of factors [1,2]. The most devastating of these is bacterial infection, which can lead to impaired wound healing [3]. Administering antibiotics is an effective strategy for reducing the risk of wound infection, which might induce systemic adverse effects, including microbiota imbalance and antimicrobial resistance [4,5]. By virtue of its highly effective and localized therapeutic action that minimizes systemic toxicity, photothermal therapy (PTT) has gained considerable clinical interest as a treatment strategy for infected wounds [6,7]. The dual stimuli of infection and PTT-induced heat lower the local pain threshold compared to ordinary wounds [8,9], thereby diminishing the patient's healthcare experience and compliance [10]. Most notably, PTT-induced heat hyperpathia during the treatment of infected wounds remains overlooked [11].

The primary approach for pain management in infected wounds, aside from treating the etiology, is the use of systemic or/and localized analgesics [12]. Systemic analgesia, encompassing opioids [13], NSAIDs [14], and paracetamol [15],achieves significant pain relief by inhibiting pain transmission or inflammatory mediators [16]. But, the adverse effects of systemic analgesics span from common gastrointestinal and central nervous system issues to severe [17,18], potentially life-threatening risks such as respiratory depression, organ toxicity, and dependence [19]. In contrast, local anesthetics (lidocaine [20], ropivacaine [21], bupivacaine [22], etc.) work by blocking pain signals at the origin, offering localized pain relief while minimizing systemic side effects [23]. However, the limited duration of local analgesics requires repeated dosing, which might elevate the risk of severe infection, even bacteremia [24]. In light of these circumstances, hydrogel-loaded local anesthetics offer a promising approach by providing sustained drug release and extended analgesic effect in chronic pain management [25]. Notably, lidocaine hydrochloride (Lid) is a widely used local anesthetic in clinical practice, known for its rapid onset of action and potent analgesic efficacy [26]. As a water-soluble salt, Lid dissociates in physiological environments, and the resulting non-ionized lidocaine can permeate neural membranes to exert its anesthetic effect, enabling prompt pain relief [27]. As such, Lid serves as an ideal analgesic component for incorporation into hydrogel-based delivery systems aimed at achieving sustained and controlled drug release.

Among various photothermal agents, organic radicals have emerged as particularly attractive candidates due to their open-shell electronic configuration [28,29], which enables efficient absorption in the second near-infrared window (NIR-II) together with advantages such as low molecular weight, facile synthesis, and outstanding photothermal conversion efficiency [30]. However, their high reactivity and intrinsically short lifetime often result in rapid degradation under physiological conditions, greatly limiting their biomedical applicability and clinical translation [31]. In our previous work [32], we employed a novel assembly motif in supramolecular chemistry that leverages the chaotropic effect, in which closo-dodecaborate anion B_12_H_12_^2−^ acted as superchaotropic anion to drive self-assembly with cationic triazine derivatives, providing a viable strategy for radical stabilization. More interestingly, the chaotropic effect can also act as the driving force to induce the sol-gel transition of hydrogels. We therefore hypothesize that a photothermal-triggered anesthetic release system based on photoresponsive hydrogels could be constructed to manage wound pain and enhance wound healing.

Herein, we developed an injectable hydrogel (STB-Lid Gel) that integrates antibacterial and analgesic functions for treating infected wounds, based on the excellent photothermal conversion efficiency of organic radicals and the analgesic effect of local anesthetics. As shown in Scheme 1, the supramolecular complex self-assembled from 4,4′,4″-(1,3,5-triazine-2,4,6-triyl) tris(1-methylpyridin-1-ium) (TPT^3+^) and B_12_H_12_^2−^ was used as the photothermal agent and co-loaded with Lid into a silk fibroin hydrogel. Under bacterial metabolic stimulation, TPT^3+^ was in situ reduced to radicals with high photothermal conversion capability, enabling local heating (>50 °C) under NIR-II laser irradiation to directly eradicate bacteria at the infection site. Meanwhile, Lid, as an analgesic, provided baseline pain relief through sustained release from the gel network during the initial treatment phase and was rapidly released upon local heating during PTT, precisely alleviating pain hypersensitivity induced by PTT. Importantly, this dual-functional strategy establishes a synergistic therapeutic paradigm in which antibacterial efficacy and precise pain management are simultaneously achieved within a single platform, highlighting the rational design and significant translational potential of this system for infected wound therapy.Scheme 1a) Schematic diagram of the preparation protocol and essential features for STB-Lid Gel. b) Schematic illustration of the strategy for infected wound healing, with dual-release (sustained and PTT-controlled) and dual-functionality (analgesic and antibacterial) capabilities.Scheme 1

Materials and methods

2

Materials and reagents

2.1

Lidocaine hydrochloride was purchased from MP Biomedicals (Aladdin, China). HPLC-grade acetonitrile (FTSCI, China). Yeast extract, casein, and agar were all from BioFrox. Ultrapure water (18.2 MΩ, Eco-S1SUVF; Shanghai High-Tech, China) was used for preparing all aqueous solutions. Calcein-AM/PI Cell Viability (China) was purchased from Beyotime. Antibodies used included TRPV1 (Affinity Biosciences, DF10320) and c-Fos (ServiceBio, GB12069). All bacterial strains were cultured in Luria-Bertani (LB) medium at 37 °C for 12 h before further application. Unless otherwise specified, all materials were obtained from commercial suppliers and used without further purification.

Characterization

2.2

The microstructures of SF Gel and STB-Lid Gel were examined using a scanning electron microscope (SEM, S-4800; Hitachi, Japan). The interactions among functional groups in the samples were analyzed by Fourier transform infrared (FTIR) spectroscopy (UV-1800PC). The rheological properties of the hydrogels were evaluated using a rheometer (Discovery HR-10; TA Instruments, USA). UV-Vis-NIR absorption spectra were obtained using a UV-Vis spectrophotometer (SP-756P; Spectrum Instruments). Photothermal performance under NIR laser irradiation was recorded with a thermal infrared imaging camera (FLIR A35, 60 Hz; FLIR Systems). Fluorescence images of live/dead bacterial staining were captured and processed using a laser scanning confocal microscope (FV1000; Olympus, Japan).

Preparation of STB-Lid Gel

2.3

STB-Lid Gel was prepared from natural silkworm cocoons. Briefly, cocoons were cut into small pieces and boiled in 0.05% (w/v) sodium carbonate solution at 100 °C for 30 min, and this degumming process was repeated three times to remove sericin. The resulting fibroin fibers were thoroughly rinsed with deionized water and dried at 60 °C for 24 h. The dried fibers were then dissolved in 8.0-10.0 M lithium bromide (LiBr) solution at 60 °C for 1-3 h to obtain a silk fibroin (SF) solution, as LiBr disrupts intermolecular hydrogen bonds within the protein. The obtained SF solution was dialyzed against deionized water for 72 h using a dialysis membrane with a molecular weight cut-off (MWCO) of 8–14 kDa to remove residual LiBr, and then centrifuged at 5000 rpm for 10 min to remove insoluble aggregates. The resulting purified SF solution (10 wt%) was stored at 4 °C for further use. 4,4′,4″-(1,3,5-triazine-2,4,6-triyl) tris (1-methylpyridin-1-ium) (TPT^3+^) [33] and closo-dodecaborate anion B_12_H_12_^2−^ were synthesized according to literature methods. TPT^3+^ and B_12_H_12_^2−^ were individually combined with SF solution under gentle stirring. The chaotropic effect of B_12_H_12_^2−^ promoted rapid self-assembly with TPT^3+^, resulting in the formation of an insoluble supramolecular complex, TPT-B_12_. Then, Lid was introduced into the precursor mixture and stirred vigorously to achieve uniform dispersion. The mixture was left undisturbed at room temperature for 2-3 h, resulting in the formation of STB-Lid Gel. In the obtained hydrogel, the final concentrations of TPT^3+^ and Lid were 0.5 mM and 20 mg mL^−1^, respectively.

In vitro lid release test

2.4

To evaluate the sustained-release performance, 2 mL samples of Lid solution, SF-Lid Gel, and STB-Lid Gel, each containing 40 mg of Lid, were loaded into dialysis bags with an MWCO of 14 kDa. The dialysis bags were submerged in 600 mL of PBS buffer (pH 7.4) and maintained at 37 °C under magnetic stirring at 500 rpm. At specific time points, 1 mL of supernatant was collected and replenished with an equal amount of fresh PBS to ensure constant total volume. The collected aliquots were passed through 0.22 μm nylon filters for subsequent analytical measurements. The cumulative in vitro release of Lid was analyzed using high-performance liquid chromatography (HPLC, Shimadzu LC-2050). The mobile phase consisted of acetonitrile-PBS-deionized water (50:20:30, v/v/v), which was sonicated for degassing for 30 min. A C18 column (250 × 4.6 mm, 5 μm) was used with a column temperature of 30 °C, flow rate of 0.6 mL min^−1^, injection volume of 20 μL, and detection wavelength of 210 nm. Each sample was analyzed in triplicate.

In vitro biocompatibility assessment

2.5

Blood samples were obtained from the orbital plexus of rats to perform the hemolysis test. Equal volumes (1:1, v/v) of hydrogel extract and 2% diluted erythrocyte suspension were mixed and incubated at 37 °C for 1 h. The optical density of the mixture at 540 nm was determined using a UV-Vis spectrophotometer, and the hemolysis percentage was subsequently calculated. The MTT assay was employed to assess the concentration-dependent cytotoxicity of B_12_H_12_^2−^, TPT^3+^, TPT-B_12_, and Lid. 3T3 fibroblasts were suspended in complete culture medium and seeded into 96-well plates with 200 μL DMEM per well at a density of 1.5 × 10^4^ cells per well. Following incubation at 37 °C in a 5% CO_2_ atmosphere, 20 μL of MTT dye solution (5 mg mL^−1^) was added to each well, and cells were further incubated for 24 h. After removing the MTT solution, 150 μL of dimethyl sulfoxide (DMSO) was added to dissolve the formazan crystals in each well. Absorbance at 595 nm was recorded using a microplate reader (BIO-RAD 550) to quantify cell viability. The percentage of cell viability was determined from the optical density readings. Following the treatments, cells were subjected to Calcein-AM/PI dual staining as instructed by the manufacturer. Fluorescence images of the stained cells were captured with an inverted fluorescence microscope for further observation.

Photothermal efficiency of STB-Lid Gel

2.6

Equal volumes of the hydrogel samples and bacterial suspensions were mixed and incubated at 37 °C with shaking at 150 rpm for 6 h. Samples were subsequently exposed to a NIR-II laser at intensities of 0.5, 0.8, and 1.0 W cm^−2^ for 10 min, and the temperature evolution was monitored by an infrared thermal imager. As a control, PBS was used instead of the bacterial suspension and mixed with the hydrogel samples; temperature variations under laser irradiation were recorded to evaluate the bacterial reduction-induced response. The photothermal stability of STB-Lid Gel was further assessed by performing five consecutive irradiation cycles on the hydrogel-bacteria mixture after 6 h of incubation. Temperature variations during both the heating and cooling processes were recorded, and the photothermal conversion efficiency (PCE) was determined using the method described in previous studies [34].

In vitro antimicrobial activity assay of STB-Lid Gel

2.7

Escherichia coli (E. coli, ATCC 25922) and Staphylococcus aureus (S. aureus, ATCC 6538) were selected as representative strains of Gram-negative and Gram-positive bacteria, respectively. Bacteria were incubated at 37 °C with shaking for 12 h, then diluted to a concentration of about 1 × 10^6^ CFU mL^−1^ and subsequently mixed with various material samples for further treatment. In the dark group (Dark), samples were incubated at 37 °C for 6 h without light exposure; in the light group (Light), samples were incubated under the same conditions and then irradiated with a NIR-II laser (W cm^−2^) for 10 min. Upon completion of incubation, 100 μL of bacterial suspension from each group was serially diluted, and 100 μL of each dilution was plated evenly on LB agar and incubated at 37 °C for 12 h. Bacterial viability and colony density were quantitatively determined via the LB plate counting assay. To further confirm the antibacterial efficacy of the materials, a live/dead bacterial fluorescence staining experiment was conducted. Bacterial suspensions after various treatments were mixed with PI and Calcein-AM dyes and incubated at 37 °C in the dark for 20 min. Afterward, bacteria were immobilized on glass slides and observed under an inverted fluorescence microscope to evaluate their viability status.

Animals

2.8

All animal experiments were performed in compliance with the ARRIVE guidelines and the Tongji Medical College of Huazhong University of Science and Technology Institutional Animal Care and Use Committee. The study protocol was approved by the Animal Welfare and Ethics Committee of Tongji Medical College, Huazhong University of Science and Technology, and strictly followed the relevant institutional and ethical guidelines for animal experimentation (Approval No. 2022-2685). The sex of the animals used in this study was female, and the influence of sex on the results is discussed in the manuscript. Six-week-old healthy female specific pathogen-free (SPF) Sprague-Dawley (SD) rats were obtained from the Tongji Medical College of Huazhong University of Science and Technology. A 2% equivalent dose of Lid was used in all animal experiments [35,36]. All rats were maintained in a controlled environment (22 ± 2 °C, 40-60% relative humidity) with ad libitum access to food and water, and a 12-h light/dark cycle (12 h light per day).

Von Frey test: Prior to testing, rats were placed individually in transparent boxes with wire mesh bottoms and allowed to acclimate for 30 min Mechanical sensitivity was assessed by stimulating the plantar surface of the left hind paw with a series of calibrated von Frey filaments (0.6, 1.0, 1.4, 2.0, 4.0, 6.0, 8.0, 10.0, 15.0, and 26.0 g). Each filament was applied perpendicularly until it bent into an “S” or “C” shape and was maintained for approximately 5 s to ensure consistent pressure [37]. A positive response was recorded when the rat exhibited a brisk paw withdrawal, licking, or flinching behavior. The 50% paw withdrawal threshold was calculated using Dixon's up-and-down method [38].

Hot plate test: Rats were placed on a thermostatically controlled hot plate maintained at 52 ± 0.5 °C. The latency to the first nociceptive response-such as paw licking, lifting, or jumping-was recorded as the paw withdrawal latency. Each rat was tested five times with 5 min intervals between trials to minimize thermal sensitization, and the mean paw withdrawal latency value was calculated to represent the thermal pain threshold [23].

Infection plantar pain model: Rats in the infection group were injected with 50 μL of S. aureus suspension (1 × 10^6^ CFU mL^−1^) into the plantar surface of the left hind paw, whereas control animals received the same volume of saline. Within 24 h after injection, behavioral observations revealed that infected rats displayed characteristic spontaneous pain behaviors such as frequent licking, biting of the infected site, and paw withdrawal. At the same time, the von Frey and hot plate tests demonstrated that the mechanical pain threshold of infected rats was markedly reduced and their thermal pain latency significantly shortened. This persistent hyperalgesic condition differed significantly from that of the control group, indicating that the plantar infection pain model was successfully established. After 24 h of infection, the rats were randomly divided into six groups (n = 6): Control, Free Lid, STB Gel, STB Gel NIR, STB-Lid Gel, and STB-Lid Gel NIR. Each group received the designated treatment, and pain-related behavioral assessments were carried out at scheduled intervals. After the 6-h evaluation, the STB Gel NIR and STB-Lid Gel NIR groups were subjected to NIR-II laser irradiation (1.0 W cm^−2^ for 5 min) to initiate PTT. After 30 h of treatment, rats were anesthetized by intraperitoneal injection, followed by perfusion with normal saline and 4% paraformaldehyde to fix the tissues. The lumbar spinal cord and L4-L5 dorsal root ganglia (DRG) were dissected, fixed in 4% paraformaldehyde for 24 h, and subsequently dehydrated in 30% (w/v) sucrose. Frozen sections were cut at 20 μm for the spinal cord and 10 μm for the DRG. Immunofluorescence staining was conducted to evaluate the expression of pain pathway-related proteins in the spinal cord and DRG, providing insights into neuronal activation and pain signaling alterations [24].

S. aureus-infected cutaneous wound model: Initially, the dorsal fur of rats was removed using a pet clipper, followed by thorough depilation with a 9% sodium sulfide solution. Subsequently, 200 μL of S. aureus suspension (1 × 10^6^ CFU mL^−1^) was injected subcutaneously into the dorsal skin of each rat. Post-injection, a raised bump was observable at the injection site on the rat's back. Within 24 h post-injection, significant swelling and abscess formation were evident at the dorsal injection area, indicating successful establishment of the dorsal abscess model. At 24 h post-infection, rats were divided into Control, SF Gel, STB Gel, STB Gel NIR, STB-Lid Gel, and STB-Lid Gel NIR groups for respective treatments. Each group contained 6 rats as replicates. The Control, SF Gel, STB Gel NIR, and STB-Lid Gel NIR groups received photothermal therapy with NIR-II laser irradiation (1.0 W cm^−2^) for 5 min at 6 h post-treatment. Mechanical thresholds and thermal latencies were assessed by von Frey test and hot plate assay before and after light irradiation. On day 5, half of the rats were euthanized; dorsal abscesses were collected, suspended, and diluted in saline. 100 μL of the dilution was spread on LB agar plates, incubated at 37 °C for 12 h, and bacterial survival/density was quantified via plate counting. Photographs were taken to document the treatment process of the dorsal abscess wounds over 10 days. On day 10, all remaining rats were euthanized; visceral tissues and dorsal wound skin samples were collected. Tissues were fixed in 4% paraformaldehyde for subsequent analyses. Body weight changes were recorded over 10 days.

*RNA-Seq analysis:*To further investigate the molecular mechanisms underlying accelerated wound healing, tissue samples were collected from the S. aureus-infected cutaneous wound model at 30 h post-treatment. Total RNA extraction and database-building sequencing were performed by Novogene Biotech Co., Ltd. Total RNA was isolated from rat specimens using TRIzol reagent (Tiangen Biotech, China; DP424). RNA purification, reverse transcription, library preparation, and sequencing were conducted on the Illumina NovaSeq X Plus platform (Illumina, USA) according to the manufacturer's protocols. RNA-seq libraries were created using the Fast RNA-seq Library Prep Kit V2 (ABclonal, China). Differential gene expression between groups was analyzed using DESeq2. Transcripts with |log_2_FoldChange| > 1 and p < 0.05 were defined as differentially expressed genes (DEGs). KEGG and GO enrichment analyses of DEGs were performed using the clusterProfiler and org.Rn.eg.db packages.

Statistical analysis

2.9

All data are presented as mean ± standard error of the mean (SEM). Differences were considered statistically significant (∗p<0.05, ∗∗p<0.01, ∗∗∗p<0.001).

Results and discussion

3

Fabrication and characterization of STB-Lid Gel

3.1

Following the reported method [39], a purified high-concentration silk fibroin (SF) solution was obtained. B_12_H_12_^2−^ was directly introduced into the SF solution that had been pre-mixed with TPT^3+^. Owing to the chaotropic effect between B_12_H_12_^2−^ and TPT^3+^, an insoluble supramolecular complex (TPT-B_12_) spontaneously formed in the mixture. Lid was added to the turbid STB suspension, followed by rapid stirring and standing to obtain a homogeneous and stable injectable STB-Lid Gel. As shown in Fig. 1a, we individually mixed the components into the purified SF solution, and then incubated the mixed solutions at room temperature for 3 h. It was observed that SF solutions containing B_12_H_12_^2−^ mixed with either TPT^3+^ or Lid formed hydrogels, while the other groups remained in solution state. Furthermore, except for the SF-BH Gel which remained a homogeneous solution, phase separation was observed in the solutions of other groups. Fig. 1b presents the Fourier transform infrared (FTIR) spectra of the STB-Lid Gel and each of its constituent components. As shown, both the B-H vibration peak of B_12_H_12_^2−^ and the methyl C-H vibration peak of TPT^3+^ are completely retained in the STB-Lid Gel, suggesting structural stability of both species within the composite system and the absence of chemical bond cleavage. For the STB-Lid Gel containing Lid, the amide I absorption peak of SF Gel at 1630 cm^−1^ exhibited a slight red shift to 1650 cm^−1^, influenced by the characteristic peak of Lid at 1830 cm^−1^, accompanied by a marked increase in peak intensity. Additionally, the increased intensity of the broad band in the 3700 - 3100 cm^−1^ range suggests strengthened intermolecular hydrogen bonding interactions in the system. The FTIR spectra demonstrate the presence of intermolecular interactions and the establishment of the hydrogel network structure. To investigate the microstructural features of the hydrogel, scanning electron microscopy (SEM) was employed to observe the hydrogel network morphology of STB-Lid Gel (Fig. 1c). The observations revealed that SF Gel consisted of an irregularly interlaced network of fibers and lamellae forming interconnected pores, whereas STB-Lid Gel exhibited a three-dimensional fibrous network structure with abundant micron-scale pores among the fibers. This open, porous architecture promotes the permeation and diffusion of cells or drugs, thereby offering a favorable structural foundation for biomedical applications.Fig. 1. Characterization of STB-Lid Gel properties and functions. a) Photographs of hydrogels with various compositions. b) FTIR spectra of STB-Lid Gel and its individual components. c) Representative SEM images of the hydrogels. d) Strain-sweep measurements performed at a constant frequency of 1.0 Hz with strain amplitudes ranging from 0.1% to 1000%. e) Frequency-sweep tests conducted at a fixed strain of 1% over the frequency range of 0.1 - 100 Hz. f) Demonstration of hydrogel injectability (inset photograph) and shear-thinning behavior.Fig. 1

The rheological properties of the hydrogels were systematically assessed by means of a rheometer. The strain sweep results (Fig. 1d) revealed that under a fixed strain of 0.1%, the storage modulus (G′) of SF Gel was approximately 1 kPa, whereas that of STB-Lid Gel markedly increased to around 10 kPa, suggesting that its stiffness was enhanced by nearly one order of magnitude. Moreover, the STB-Lid Gel displayed a broader linear viscoelastic region (LVR), indicative of a more stable three-dimensional network capable of tolerating greater deformation without structural disruption. The frequency sweep analysis (Fig. 1e) demonstrated that the storage modulus (G′) and loss modulus (G″) of both hydrogels increased with frequency, with STB-Lid Gel exhibiting a more pronounced rise in G′, corroborating its higher crosslinking density or stronger intermolecular interactions that endow it with superior structural stability. Furthermore, the steady-state viscosity measurement (Fig. 1f) revealed a characteristic shear-thinning behavior for the STB-Lid Gel, in which the apparent viscosity decreased markedly with increasing shear rate, confirming that the hydrogel possesses excellent injectability.

Evaluation of PTT efficacy of STB-Lid Gel

3.2

Building upon our previous findings [40], TPT^3+^ embedded in the STB-Lid Gel is bacterially reduced in situ to TPT∗^2+^, which subsequently binds with B_12_H_12_^2−^ via the chaotropic effect to form the radical-stabilized supramolecular assembly TPT∗-B_12_, leading to robust NIR-II absorption and elevated photothermal conversion efficiency (PCE). To confirm this characteristic, we selected Gram-negative E. coli and Gram-positive S. aureus as representative bacterial models. As illustrated in Fig. 2a, when STB-Lid Gel was co-incubated with an equal amount of bacterial suspension, the solution color progressively darkened over time. UV-Vis-NIR absorption spectra revealed that after co-culturing with S. aureus, the STB-Lid Gel exhibited a new absorption band in the range of 600 - 1200 nm, and the absorbance increased with longer incubation times (Fig. 2b). Electron paramagnetic resonance (EPR) analysis further verified that the sample co-cultured for 6 h displayed a markedly stronger radical signature than that incubated for 2 h (Fig. 2d), suggesting that radicals accumulated over time. To further validate the presence of reduction-generated radicals from a chemical reactivity perspective, a DPPH radical probe assay was conducted. As shown in Fig. S1, the bacteria-reduced TPT∗-B_12_ caused a pronounced decrease in the characteristic DPPH absorption at 550 nm along with visible color fading, whereas the unreduced control TPT-B_12_ showed minimal change. This result indicates that radical-active species were generated after bacterial reduction rather than being intrinsic to the precursor material, consistent with the EPR observations. To elucidate the contribution of each component in STB-Lid Gel, different hydrogels were co-cultured with bacteria under hypoxic conditions for 6 h, followed by comparison of their absorption spectra. The results indicated that only the samples containing the TPT-B_12_ component (namely STB Gel and STB-Lid Gel) and subjected to bacterial reduction displayed prominent absorption peaks in the NIR-II region, while no such phenomenon was observed in the SF Gel or non-reduced control groups (Fig. 2c and S2). These findings suggest that the observed color change and NIR absorption are attributed to the formation of TPT^3+^ radicals, and that Lid incorporation does not compromise the hydrogel's radical generation ability. Collectively, these results establish an experimental foundation for the subsequent investigation of NIR-II induced photothermal conversion.Fig. 2. Characterizations and photothermal response of the STB-Lid Gel. a) Photographs of STB-Lid Gel after co-culturing with bacterial suspensions at 37 °C for different time periods. b) UV-Vis-NIR absorption spectra of STB-Lid Gel after co-incubation with S. aureus at various time points. c) UV-Vis-NIR absorption spectra of SF Gel, STB Gel, and STB-Lid Gel before and after 6 h of co-incubation with S. aureus. d) EPR spectra of STB-Lid Gel after 0 h, 2 h, and 6 h of co-incubation with S. aureus. e) Temperature evolution curves and f) corresponding infrared thermal images of STB-Lid Gel after different co-incubation durations under NIR-II laser irradiation (1.0 W cm^−2^, 10 min). g) Photothermal heating curves of PBS, SF Gel, STB Gel, and STB-Lid Gel after 6 h of co-incubation with S. aureus under NIR-II laser irradiation (1.0 W cm^−2^, 10 min). h) Photothermal stability of STB-Lid Gel evaluated over five consecutive heating and cooling cycles under on/off laser irradiation. i) Cumulative Lid release profiles from Lid sol, SF-Lid Gel, and STB-Lid Gel over 7 days in vitro. j) Thermally responsive Lid release profiles from STB-Lid Gel and corresponding SEM image after incubation at 54 °C for 20 min. k) Laser-triggered Lid release at 2 h and 6 h, where red arrows indicate NIR-II laser irradiation (1.0 W cm^−2^, 10 min). Data are presented as mean ± SEM (n = 3) (∗p<0.05, ∗∗p<0.01, ∗∗∗p<0.001). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)Fig. 2

Next, the photothermal efficiency of STB-Lid Gel was evaluated following incubation with bacteria. Initially, the STB-Lid Gel displayed a distinct power-dependent photothermal response. Upon NIR-II laser irradiation at 1.0 W cm^−2^, the STB-Lid Gel temperature rapidly rose and stabilized at around 54 °C, exhibiting superior photothermal efficiency compared to 0.5 and 0.8 W cm^−2^ (Fig. S3). It is noteworthy that the photothermal efficiency of the STB-Lid Gel was also time-dependent on the duration of bacterial incubation. Under 1.0 W cm^−2^ NIR-II laser irradiation for 10 min, the maximum temperatures of STB-Lid Gel co-cultured with S. aureus for 2, 4, 6, and 12 h reached 41.8, 49.0, 52.9, 54.5, and 54.9 °C, respectively (Fig. 2e and f). Taking into account both photothermal efficiency and biological safety, we identified 6 h as the optimal incubation period for subsequent experiments. Furthermore, the temperature elevation profiles of the hydrogels before and after bacterial incubation were investigated. Hydrogels either unincubated with bacteria or lacking TPT^3+^ exhibited negligible photothermal effects (Fig. 2g and S4). After bacterial incubation, the STB-Lid Gel solution temperature rapidly increased to 54 °C within 5 min and remained stable thereafter, confirming its high efficiency and durable photothermal behavior. Based on cooling kinetics analysis, the PCE of the STB-Lid Gel at NIR-II laser irradiation was determined to be 31.62% (Fig. S5). Five successive laser on/off irradiation cycles (Fig. 2h) produced nearly identical heating-cooling profiles, demonstrating the excellent photothermal stability of the STB-Lid Gel.

In vitro sustained release study of STB-Lid Gel

3.3

To assess the drug-release behavior of STB-Lid Gel as a delivery carrier, we analyzed the in vitro release kinetics of Lid via high-performance liquid chromatography (HPLC). The cumulative in vitro release profile over a 7-day period is presented in Fig. 2i. During the initial 2 h, free Lid solution exhibited a rapid release of 81.54%, whereas SF-Lid Gel and STB-Lid Gel released only 34.33% and 20.31%, respectively, demonstrating the hydrogel's capability to substantially slow down the burst release of Lid. Over the subsequent 4 days, both drug-loaded hydrogels exhibited a slow and continuous increase in cumulative release, further validating their effective sustained-release of Lid. By the seventh day, the cumulative release of Lid from SF-Lid Gel and STB-Lid Gel reached 72.85% and 77.23%, respectively. To confirm that temperature serves as the primary trigger for drug release, the release profiles of the hydrogels were compared under 37 °C and 54 °C conditions within a 24-h period (Fig. 2j). The cumulative release at 54 °C reached 70% after 24 h, markedly exceeding the release observed at physiological temperature (37 °C). SEM observations further revealed that a 10-min exposure to 54 °C caused the hydrogel's three-dimensional network to become loosened and highly porous, suggesting that such thermally induced structural alterations underpin the photothermal-triggered burst release of the drug. Furthermore, in order to demonstrate whether high temperature affects the structure of Lid and thereby influences its pharmacological effect, we will conduct ^1^H NMR on Lid at 60 °C for 10 min, 20 min, and under normal conditions. The results are shown in Fig. S6. No significant changes were observed in the structure. Upon incubation with bacterial suspensions, the STB-Lid Gel underwent in situ reduction, leading to the activation of its photothermal therapy (PTT) capability. To explore the potential of photothermal stimulation in triggering drug release, the STB-Lid Gel was exposed to NIR-II (1064 nm) laser irradiation (1.0 W cm^−2^ for 10 min) at the 2nd and 6th h. The results revealed that each irradiation for 10 min resulted in a temperature increase of 21.1 °C and 29.2 °C respectively. The release amount of Lid immediately increased by 6.46% and 13.9%, indicating that the NIR-II laser irradiation effectively converted into heat and accelerated the release of Lid (Fig. 2k and S7).

In vitro antibacterial effects of STB-Lid Gel

3.4

Before the further biomedical investigation of STB-Lid Gel, hemolysis assays were conducted to evaluate biocompatibility. The hemolysis rate of STB-Lid Gel remained substantially below the 5% safety threshold (Fig. 3a). The cytocompatibility of STB-Lid Gel was further examined in 3T3 cells (mouse embryonic fibroblasts) using MTT (Fig. 3c) and Calcein-AM/PI (Fig. 3d) staining assays. The quantitative data(Fig. 3b and S8) are consistent with the representative images, both hydrogels (SF Gel and STB-Lid Gel) showed no observable cytotoxic effects, while individual components (TPT^3+^, B_12_H_12_^2−^, and Lid) exhibited dose-dependent cytotoxicity. Additionally, Annexin V-FITC/PI staining combined with flow cytometry was employed to evaluate apoptosis of STB-Lid Gel (Fig. S9). The negligible apoptosis in 3T3 cells induced by STB-Lid Gel suggests its cytotoxicity is primarily mediated by its photothermal behavior rather than direct lethal effect.Fig. 3In vitro photothermal antibacterial performance of STB-Lid Gel. a) Hemolysis assay of hydrogels showing hemolytic images and statistical hemolysis ratios. b,d) MTT assay results and fluorescence images (via live/dead staining) of 3T3 cells after 48-h incubation with hydrogels and their components. c) Viability of 3T3 cells after treatment with varying concentrations of B_12_H_12_^2−^, TPT^3+^, TPT-B_12_, Lid, and Lid-B_12_. e) SEM images of E. coli and S. aureus cells with Blank, STB Gel, and STB-Lid Gel. f,h) Photographs of LB plates and g,i) bacterial viability of bacteria following treatment with Blank, STB Gel, and STB-Lid Gel, either with or without NIR-II laser irradiation (1.0 W cm^−2^, 10 min). j) Viability staining (live/dead) of bacterial cells following treatment with STB Gel and STB-Lid Gel under conditions with or without NIR-II laser irradiation (1.0 W cm^−2^, 10 min). Data are shown as mean ± SEM (n = 3) (∗p<0.05, ∗∗p<0.01, ∗∗∗p<0.001).Fig. 3

The antibacterial efficacy of STB-Lid Gel was examined through the LB plate colony counting assay using S. aureus and E. coli. No significant differences in the quantity and morphology of bacterial clones were observed without NIR-II laser irradiation compared to the Blank group. Upon NIR-II laser irradiation (1.0 W cm^−2^ for 10 min), both S. aureus and E. coli treated with STB Gel and STB-Lid Gel showed drastically reduced colony numbers (survival rate below 5%), confirming their potent photothermal antibacterial efficacy (Fig. 3f–i). These findings suggest that STB-Lid Gel does not possess intrinsic “dark toxicity”. Its antibacterial activity is strictly photoactivated, thus minimizing the risk of nonspecific tissue damage associated with traditional antibacterial agents. To further investigate the antibacterial effects of TPT-B_12_, all groups (TPT^3+^, B_12_H_12_^2−^, and TPT-B_12_) showed similar bacterial survival rates of around 90% under non-NIR conditions, while the TPT-B_12_ group was markedly lower than that in the other groups upon NIR-II laser irradiation (Fig. S10). These results indicate that the in-situ reduction of TPT-B_12_ in the bacterial environment results in radical accumulation, the key process responsible for its potent photothermal antibacterial effect. Additionally, free Lid shows pronounced antibacterial efficacy against both S. aureus and E. coli, with inhibition rates exceeding 96.5% (Fig. S11), mainly because of its high concentration (10 mg mL^−1^). With Lid loaded into the hydrogel, the sustained-release property of the matrix attenuated the immediate antibacterial response, suggesting that the antibacterial activity of STB-Lid Gel is predominantly governed by its photothermal effect. To investigate the antibacterial mechanism of STB-Lid Gel, we assessed bacterial membrane integrity using a dual-fluorescence (PI/Calcein-AM) assay and SEM. Fluorescence imaging and quantitative analysis showed that when applied alone, STB Gel and STB-Lid Gel maintained intact cell membranes (green fluorescence), whereas after NIR-II laser irradiation, they exhibited pronounced red fluorescence, indicating widespread bacterial death (Fig. 3j and S12). SEM observations corroborated this, showing that only NIR-II laser irradiated STB-Lid Gel-induced severe cellular deformation and content leakage (Fig. 3e). These findings indicate that the bactericidal effect is mediated by photothermally-induced membrane rupture. Collectively, these results demonstrate that STB-Lid Gel, upon NIR-II laser irradiation, exerts potent antibacterial efficacy by disrupting bacterial membranes.

In vivo analgesic effect of STB-Lid Gel

3.5

The S. aureus-infected rat paw model in rats was constructed to assess the analgesic efficacy of STB-Lid Gel (Fig. 4a). Two separate batches of rats were used for the von Frey filament and hot plate tests, respectively. To determine the intrinsic nociceptive effect of infection, we found that the rats 4 h post-infection exhibited a reduced mechanical threshold by 74.89% and the thermal latency by 42.69%, with the hypersensitive state persisting thereafter, demonstrating that infection itself induces significant hyperalgesia (Fig. 4d and g). At 24 h post-infection, saline (Control), free Lid (Free Lid), STB Gel, or STB-Lid Gel was administered into the infected paw, followed by behavioral evaluations as outlined in Fig. 4a. Notably, PTT during antibacterial treatment typically causes local heating and inflammatory responses, possibly triggering pain sensations. Assessment of PTT-triggered nociception demonstrated that STB Gel under NIR-II laser irradiation significantly lowered the mechanical threshold and thermal latency by 41.4% and 55.8%, respectively, compared to the group without irradiation (Fig. 4b–c). Regarding analgesic effect, rats in the Free Lid group achieved peak analgesic effect at 2 h post-injection, which then diminished significantly after 4 h, consistent with its rapid-onset and short-acting pharmacokinetic profile. In comparison, the STB-Lid Gel group displayed superior sustained-release characteristics, achieving maximal analgesic response at 6 h post-injection and maintaining pain relief for up to 30 h, indicating long-lasting analgesic efficacy. Notably, the STB-Lid Gel NIR group exhibited an “on-demand intensified” analgesic effect after NIR-II laser irradiation (at 8 h), reaching a pain threshold similar to that of the peak analgesic effect of the Free Lid group (Fig. 4e and h). This result indicates that PTT does not reduce the analgesic potency of Lid, as the photothermally triggered release from STB-Lid Gel achieves an analgesic effect comparable to the maximal effect of Free Lid. Furthermore, the analgesic effect of the STB-Lid Gel NIR at 8 h and 30 h was markedly superior to that of STB-Lid Gel (Fig. 4f and i). A key advantage of STB-Lid Gel, as evidenced by these results, is its ability to provide both sustained and on-demand photothermally controlled Lid release, thereby simultaneously overcoming the critical challenge of PTT-induced hyperalgesia.Fig. 4In vivo behavioral assessment of STB-Lid Gel's analgesic effect. a) Schematic diagram of the infection plantar pain model. b) 50% mechanical paw withdrawal threshold of rats in the STB Gel with or without NIR-II laser irradiation (1.0 W cm^−2^, 5 min). c) Thermal paw withdrawal latency of rats in the STB Gel group with or without NIR-II laser irradiation. d) 50% mechanical paw withdrawal threshold 24 h after S. aureus infection. e) 50% mechanical paw withdrawal threshold before and after treatments. f) Comparison of mechanical pain thresholds at 2 h, 8 h, and 30 h in the von Frey test. g) Thermal paw withdrawal latency 24 h after S. aureus infection. h) Thermal paw withdrawal latency before and after treatments. i) Comparison of thermal pain thresholds at 2 h, 8 h, and 30 h in the hot plate test. Data are shown as mean ± SEM (n = 6) (∗p<0.05, ∗∗p<0.01, ∗∗∗p<0.001).Fig. 4

Suppression of pain pathway activation by STB-Lid Gel with NIR-II

3.6

To investigate the effect of STB-Lid Gel on nociceptive neuron activation, L4-L5 dorsal root ganglia (DRG) were harvested 30 h after various treatments in the plantar infection model for immunofluorescence analysis (Fig. 5b). c-Fos, an immediate early gene rapidly expressed after neuronal stimulation, produces the protein Fos, a classical marker reflecting neuronal excitation [41]. TRPV1 serves as a key molecular sensor integrating inflammatory and thermal pain signals [42,43]. The co-expression of c-Fos and TRPV1 serves as a reliable indicator of neuronal subpopulations actively engaged in nociceptive signaling. As illustrated in Fig. 5a and c, the proportion of c-Fos/TRPV1 double-positive neurons in different groups was 94.25% (the Control group), 90.97% (the Free Lid group), 63.83% (the STB-Lid Gel group), 38.90% (the STB-Lid Gel NIR group), and 53.12% (the Blank group). Analysis of neuronal activation subpopulations revealed that bacterial infection potently activated nociceptive neurons. This effect was significantly suppressed by STB-Lid Gel alone (p < 0.05) and further reduced when combined with NIR-II laser irradiation (p < 0.001).Fig. 5. Immunofluorescence of c-Fos/TRPV1 at 30 h after various treatments in the infection plantar pain model or Blank (without infection and treatment). a) Representative immunofluorescence showing c-Fos (red) and TRPV1 (green) staining. Nuclei were counterstained with DAPI (blue). b) Schematic diagram of tissue sampling for immunofluorescence. c) Statistical analysis of the co-expression proportion of c-Fos^+^ and TRPV1^+^ in TRPV1^+^ neurons among different groups. d) Statistical analysis of MFI of c-Fos in DRG in different groups. Data are shown as mean ± SEM (n = 6) (∗p<0.05, ∗∗p<0.01, ∗∗∗p<0.001). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)Fig. 5

Furthermore, the c-Fos mean fluorescence intensity was further analyzed in different groups (Fig. 5d). Compared to the Control and Free Lid groups, the fluorescence intensity of c-Fos was decreased in STB-Lid Gel significantly (p < 0.05) and further reduced when combined with NIR-II laser irradiation (p < 0.01). Collectively, these findings suggested that STB-Lid Gel, particularly with NIR-II laser irradiation, effectively suppressed nociceptive neuron activation, offering a promising strategy for the sustained or/and controlled pain relief over 30 h.

In vivo anti-infective therapy with STB-Lid Gel

3.7

A subcutaneous S. aureus infected cutaneous wound model was established in rats to assess the anti-infective and wound-healing efficacy of STB-Lid Gel, as shown in the flowchart (Fig. 6a). Infrared thermographic analysis (Fig. 6b and d) showed that upon 5 min of NIR-II laser irradiation, the wound temperatures in the STB Gel and STB-Lid Gel groups rapidly rose to 51.6 °C and 52.0 °C, respectively. These findings align well with the in vitro photothermal data, demonstrating that the gels can efficiently initiate photothermal therapy in vivo. Over the 10-day monitoring period, the STB-Lid Gel combined with NIR-II laser irradiation achieved the best healing performance, with the wound healing rate reaching 97% (Fig. 6c and e). On day 5 post-treatment, infected wound tissues were harvested for antibacterial assessment via standard plate colony counting. The results of antibacterial assessment revealed that the residual bacterial counts at the infected sites were minimal in both the STB Gel NIR and STB-Lid Gel NIR groups, with survival rates of 7.84% and 2.10%, respectively (Fig. 7b and Fig. S13), confirming that PTT efficiently eradicated pathogens under in vivo conditions. Regarding pain assessment, von Frey filament analysis demonstrated that STB-Lid Gel treatment markedly elevated the mechanical pain threshold of rats (Fig. 6f), confirming its pronounced analgesic efficacy. Remarkably, the pain threshold of STB-Lid Gel with NIR-II laser irradiation was increased by 28.43%, compared to no irradiation. These results aligned with those observed in the plantar pain model, indicating that the photothermal effect contributed not only to direct antibacterial activity but also to thermally triggered Lid release, ultimately achieving an analgesic effect.Fig. 6In vivo photothermal-mediated antibacterial and analgesic effect of STB-Lid Gel. a) Schematic of the construction and healing process of S. aureus-infected cutaneous wound model. b) Infrared thermographic images and d) respective temperature changes at the wound sites of rats in various groups during NIR-II laser irradiation (1.0 W cm^−2^, 5 min). c) Representative images of the wound-healing process of S. aureus-infected rats with different treatments on days 0, 2, 4, 6, 8, and 10. e) The relative wound sizes of S. aureus-infected rats treated with different treatments. f) Changes of 50% mechanical withdrawal threshold in rat wounds before and after NIR-II laser irradiation (1.0 W cm^−2^, 5 min). Data are shown as mean ± SEM (n = 3) (∗p<0.05, ∗∗p<0.01, ∗∗∗p<0.001).Fig. 6. Fig. 7Histological staining evaluation and RNA-seq analysis of wound tissues. a) H&E staining and Masson's trichrome staining of the skin tissues from the wound edges on day 10; Black arrowheads indicate the epidermis necrosis and dermal separation, red arrowheads indicate the inflammatory cells, and yellow arrowheads indicate the newborn hair follicles. b) Corresponding statistical data of colonies on day 5 after being treated with various groups. c) Statistical analysis of relative inflammatory levels in wound areas on day 10. d) Statistics of relative collagen deposition at the wound site on day 10 (n = 3). e) Volcano plot of differentially expressed genes (DEGs) between the STB-Lid Gel NIR and Control groups (|log_2_FoldChange| > 1.0, adjusted p < 0.05). f) Representative KEGG pathways identified by gene set enrichment analysis (GSEA) based on DEGs. g) Top 20 KEGG pathways and h) top 20 Gene Ontology (GO) terms enriched between the STB-Lid Gel NIR and Control groups, ranked by adjusted p value (n = 4). Data are shown as mean ± SEM (∗p<0.05, ∗∗p<0.01, ∗∗∗p<0.001). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)Fig. 7

Histological examinations provided deeper insight into the marked variations in healing quality across different treatment groups (Fig. 7a). The Control, SF Gel, and STB Gel groups exhibited epidermal necrosis, dermal separation, and extensive infiltration of inflammatory cells (black and red arrows), indicating ongoing inflammatory responses. By contrast, the STB-Lid Gel NIR promoted the formation of an intact epidermis along with abundant newly generated hair follicles (yellow arrows), reflecting markedly improved tissue regeneration. Quantitative evaluation of inflammatory cell infiltration and collagen content (Fig. 7c and d) showed that, compared to the STB Gel, the STB-Lid Gel had substantially fewer inflammatory cells and significantly enhanced collagen deposition. These findings indicate that Lid-mediated analgesic effect mitigated pain-related stress responses, providing a conducive “low-stress” microenvironment for tissue regeneration. In conclusion, by integrating antibacterial and analgesic functions, STB-Lid Gel effectively controlled infection, accelerated wound closure, and facilitated organized collagen deposition and structural regeneration, leading to markedly enhanced wound-healing outcomes.

To further elucidate the potential mechanisms underlying accelerated wound healing, we collected tissue samples at 30 h after treatment in a S. aureus-infected cutaneous wound model and performed RNA sequencing (RNA-seq) analysis. Using the criteria of |log_2_FoldChange| > 1 and p < 0.05, a total of 273 significantly upregulated and 679 downregulated differentially expressed genes (DEGs) were identified in STB-Lid Gel NIR-treated rats compared with the control group (Fig. 7e). This number was markedly higher than that observed between the STB Gel NIR and Control groups (Fig. S14a and S14b).The volcano plot, with the top 10 most significant genes labeled, showed that the STB-Lid Gel NIR regimen downregulated pro-inflammatory genes, including C1s, Cxcl12, and Clec11a, as well as extracellular matrix (ECM)-related genes such as Ctsk, Col5a2, and Bgn, indicating reduced inflammation and ECM damage. Consistently, gene set enrichment analysis (GSEA) demonstrated significant downregulation of pathways related to cytokine–cytokine receptor interaction, PI3K-Akt signaling, and ECM-receptor interaction (Fig. 7f). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis further showed enrichment among the top 20 most significantly downregulated pathways in the STB-Lid Gel NIR group versus controls, including integrin signaling, protein digestion and absorption, Staphylococcus aureus infection, and neuroactive ligand-receptor interaction pathways (Fig. 7g and S14c). Gene Ontology (GO) enrichment analysis similarly indicated attenuated ECM damage in STB-Lid Gel NIR-treated rats compared with controls (Fig. 7h and S14d).Collectively, these transcriptomic results suggest that STB-Lid Gel NIR treatment exerts antibacterial, anti-inflammatory, and pro-healing effects.

Biosafety evaluation of STB-Lid Gel

3.8

To assess the biosafety of STB-Lid Gel, the rats' body weight changes were closely monitored for 10 days during treatment (Fig. S15). No cases of local anesthetic toxicity or mortality were observed. Furthermore, H&E staining was used to evaluate potential damage to major organs (heart, liver, spleen, lung, and kidney) in rats after different treatments, with no significant morphological damage or abnormalities found in all groups (Fig. S16). Serological markers for myocardial [44] (CK-MB, CK), hepatic [45] (AST, ALT), and renal function [46] (UREA, CREA) in all groups remained within normal ranges. These results confirmed that STB-Lid Gel possessed excellent biosafety, promising for combined antibacterial-analgesic treatment in biomedical applications.

Conclusion

4

In summary, we developed an injectable photothermal antibacterial hydrogel (STB-Lid Gel) capable of sustained and heat-triggered Lid release, effectively resolving both bacterial infection and pain management in wounds. In vitro and in vivo antibacterial evaluations demonstrated that STB-Lid Gel possesses excellent NIR-II photothermal conversion efficiency for effective bacterial eradication. In vitro drug release studies and animal pain behavior assessments revealed a dual-mode, sustained-photothermal trigger, drug release of the hydrogel. The hydrogel sustainedly released Lid for over 30 h to relieve persistent infection-induced pain. While local photothermal triggered by NIR-II laser irradiation accelerated drug release, achieving rapid and precise pain management for PTT-induced acute pain relief. Given its effective antibacterial-analgesic effect, the STB-Lid Gel could also reduce the local inflammation and ultimately accelerate infected wound healing. This work underscores the importance of managing pain triggered by PTT and infection in wounds, thereby proposing a potential strategy by combining antibacterial and analgesic treatment to enhance infected wound healing.

CRediT authorship contribution statement

Na Zhou: Conceptualization, Data curation, Methodology, Validation, Writing – original draft. Wenjing Wang: Conceptualization, Data curation, Methodology, Supervision, Writing – original draft, Writing – review & editing. Lian Xu: Conceptualization, Data curation, Investigation, Methodology. Aining Zhang: Data curation, Methodology. Xiaohuan Lu: Data curation, Methodology, Validation. Yu-Pei Chen: Conceptualization, Funding acquisition, Resources. Shiwen Fan: Investigation, Methodology. Tianhao Zhang: Data curation, Validation. Changjiang Yu: Conceptualization, Funding acquisition, Resources, Writing – review & editing. Xiao-Qiang Wang: Data curation, Funding acquisition, Methodology, Project administration, Supervision, Validation, Writing – review & editing. Daan Fu: Conceptualization, Funding acquisition, Investigation, Supervision, Writing – original draft, Writing – review & editing.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.