Introduction

Skin is the largest organ in the human body and plays an essential role in protecting the internal environment. As the body’s foremost barrier, skin shields against damage from temperature fluctuations, pressure changes, chemicals, and pathogens, and helps maintain body fluids, electrolytes, and nutrients1. Skin being the most exposed organ is also susceptible to injuries leading to its damage, commonly occurring in the form of wounds. The management of wounds constitutes a substantial burden on healthcare systems globally2. The repair process can be divided into four progressive stages including hemostasis, inflammation, proliferation, and remodeling. Any defects of these repair processes result in incomplete healing of the wound tissue with impaired structure and function3,4. To accelerate the healing process and reduce any complications5,6,7,8,9, various materials have been developed, including gauze, sponges, hydrogels, and glue10. Among these options, gauze is the most affordable and widely available, but it can stick to the wounds, causing trauma during dressing changes. It also does not maintain a moist environment, which may delay healing. Hydrogels offer moisture and cooling to relieve pain and inflammation, but they have limited absorption and are unsuitable for heavily exuding wounds. Glue dressings are quick to apply and useful in emergencies, however, they are not ideal for large, deep, or highly mobile wounds, as they may not provide sufficient support in areas with constant movement or tension, potentially causing the wound to reopen. Polymer sponge dressings are a promising option because they can quickly absorb tissue exudates and undergo self-gelation in situ to form hydrogels, thus creating a moist environment above the wound11. However, these require a secondary antibacterial dressing for secured application12,13.

Researchers have combined photothermal therapy, which relies on incorporating photothermal agents to transform light energy into thermal energy, establishing a warm healing environment comparable to the natural wound healing milieu14,15,16, which has been shown to facilitate various wound healing processes, including collagen deposition, epithelialization, and angiogenesis17,18,19,20,21. Among the photothermal functional materials, polydopamine nanoparticles (PDA-NPs) are known to work as a natural bio-adhesive and bioactive polymer with intrinsic antioxidant properties and photothermal and antimicrobial activity22,23. They can regulate cell behavior by controlling signal transduction pathways that govern the focal adhesion behavior of cells at the biomaterials interface. These features make PDA-NPs a fascinating biomaterial for wound healing and skin regeneration24. However, the toxicity of the nanoparticles remains a challenge.

Alginate, extracted from brown algae and other marine organisms is a linear polysaccharide with homopolymeric blocks of (1,4)-linked β-D-mannuronate and α-L-guluronate25,26. Its status as a natural polymer for hydrogels is attributed to its wound-favorable characteristics, including non-toxicity, ease of application, hemostatic activity27, biodegradability, mucoadhesion, and non-immunogenicity28,29,30,31. However, its poor mechanical strength, lack of antimicrobial properties, and limited wettability limit its application in wound dressings32.

We developed an innovative self-gelation sponge dressing with both strength and antibacterial properties by combining 3-Aminobenzeneboronic acid-modified alginate (Alg-BA) with PDA-NPs (Fig. 1A). This sponge composite of Alg-BA@PDA shows rapid gelation within 5 s in an aqueous environment, like exudate or blood, by forming reversible borate bonding structures between phenolic hydroxyl and borate bonds (Fig. 1B, C). It also endows the sponge dressing with enhanced mechanical, photothermal, and self-healing properties, which allow for rapid hemostasis, wound repair, and tissue regeneration (Fig. 1D).

Fig. 1: Schematic diagram of Alg-BA@PDA sponge preparation and wound healing process.
Fig. 1: Schematic diagram of Alg-BA@PDA sponge preparation and wound healing process.The alternative text for this image may have been generated using AI.
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A Synthesis of sodium alginate modified with 3-Aminobenzeneboronic acid. B Alg-BA chains crosslink with each other and PDA-NPs via borate ester bonds. C Photos of PDA-NPs, Alg-BA, Alg-BA@PDA, and self-gelation Alg-BA@PDA. D Mild heat-assisted Alg-BA@PDA sponges, significantly kill bacteria, inhibit inflammation, and promote angiogenesis, effectively enhancing the healing of wounds.

Materials and methods

Materials

Sodium Alginate was procured from Aladdin (Shanghai, China). Dopamine hydrochloride, 3-Aminobenzeneboronic acid (BA), 2-(N-Morpholino)ethanesulfonic acid (MES), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), and N-Hydroxysuccinimide (NHS) were purchased from Macklin (Shanghai, China). HUVECs, Raw 264.7 cells was purchased from Meisen CTCC (Zhejiang, China), and healthy Institute of Cancer Research (ICR) mice (8 weeks, 25–30 g) were procured from Charles River (Beijing, China).

Experiments and methods

Preparation of 3-Aminophenyl Boronic Acid Modified Alginate (Alg-BA), Polydopamine Nanoparticles (PDA-NPs), and Alg-BA Loaded PDA-NPs (Alg-BA@PDA)

The Alg-BA was prepared by dissolving the alginate (2 g) in 0.1 M MES buffer (200 mL) solution. Then, the pH of the alginate solution was adjusted to 5.5, followed by addition EDC (460 mg) and NHS (700 mg) to activate the carboxyl groups on the alginate backbone. Subsequently, BA (410, 1640, 3280 mg for different samples) was added and the reaction was allowed to stand for 12 h at room temperature with continuous stirring. The resulting solution was dialyzed using dialysis bag (MWCO: 3500 Da) against ultrapure water for 48 h. The product was named Alg-BA (1), Alg-BA (4), and Alg-BA (8) according to the different BA additions as shown in Table S1 (Supporting Information).

PDA-NPs were synthesized via the oxidation and self-polymerization of dopamine in an alkaline solution14. A mixture of 40 mL ethanol, 90 mL deionized water, and 2 mL ammonia was prepared by thorough mixing. Subsequently, 0.5 g dopamine hydrochloride was dissolved in 10 mL deionized water and gradually added to the prepared solution. The reaction mixture was stirred at room temperature for 24 h to obtain PDA-NPs. The stepwise oxidation of dopamine to dopamine-quinone, followed by intramolecular cyclization, oxidation to leucodopaminechrome, formation of 5,6-dihydroxyindole, and further oxidation to 5,6-indolequinone is illustrated in Fig. 1A and Fig. S1 (Supporting Information). Alg-BA solution and PDA-NPs suspension were mixed at various volume ratios (1 v%, 2.5 v%, 5 v%, 10 v%, and 20 v%) and designated as Alg-BA@PDA (x), where x represents the PDA-NPs volume percentage (1, 2.5, 5, 10, 20).

Characterization of Alg-BA@PDA (Alginate-Boronic Acid, PDA-NPs, and Alg-BA loaded PDA-NPs)

The surface structures of the modified alginate and PDA-NPs were characterized by SEM (Gemini 300, Zeiss, Germany). The degree of substitution (DOS) of the sodium alginate was determined using NMR (Avance neo 600, Bruker, Germany). The size of PDA-NPs was detected using NTA (Nanosight 300, Malvern, UK) and DLS (Zetasizer Ultra, Malvern, UK) techniques. FT-IR (Shimadzu IRTracer-100, Shimadzu Co, Japan.) was used to characterize the changes of chemical bonding in alginate modified process, PDA-NPs formation, and composition of mixed samples. Mechanical and rheological properties of gels were tested by rheometers (HR20 Discovery, Trios, American) using four methods – time sweep, frequency sweep, strain sweep, and cyclic strain sweep, the compressive modulus of the material and its adhesion strength on muscle and skin surfaces were measured using a universal mechanical testing machine (Shimadzu AGS-X with 1000 N load cell, Japan). The swelling ratio of the sponge was measured by immersing the sponges in PBS solution after the sponge formation. In the degradation experiment, 20 mg of PDA-NPs was placed in a centrifuge tube and dispersed in 10 mL of PBS solution. The samples were incubated on a shaker at 37 °C and 120 rpm to assess degradation behavior. The PBS solution was replaced every 3 days. On days 1, 3, 7, 14, 21, and 28, the samples were collected by washing and centrifugation, followed by drying and weighing. The degradation performance was calculated using the following equation:

$$Remaining\,Mass({\rm{ \% }})={M}_{t}/{M}_{0}\times 100{\rm{ \% }}$$

Where Mt is the dried mass at day t, and M0 is the initial mass.

Properties of Alg-BA@PDA sponges

To determine the photothermal properties of the sponges, 200 μL of PBS was added to 20 μg of Alg-BA@PDA for self-gelation. The samples were irradiated with a Near Infrared (NIR) laser (808 nm, 0.8 W/cm2) for 2 min and the real-time temperature was recorded by an infrared thermal imaging system. The antioxidant capacity was evaluated using DPPH and ABTS assays. Intracellular ROS were detected using the DCFH-DA probe.

The extracts of sponge material which were soaked in culture medium, were further used in cellular experiments. The specific details of the preparation process are shown in SI 1.3. Calcein-AM/PI staining and Cell Counting Kit-8 (CCK-8) assay were used to determine the cytotoxicity of sponges. In vitro toxicity of the Alg-BA@PDA sponge was determined using Human Umbilical Vein Endothelial Cells (HUVEC cells). HUVEC cells were seeded into 96-well plates at a density of 5 × 103 cells/well. The cells were cultured for 1, 3, and 5 days using an extract containing the sponge material as a substitute for the original medium. Cell viability was assessed at each time point using the CCK-8 assay, wherein the cell toxicity was determined by measuring the absorbance using a microplate reader (Synergy Neo2, BioTek, USA) at 450 nm. For the Calcein-AM/PI staining experiment, cells were seeded into 24-well plates at a density of 5 × 104 cells/well. The original medium was replaced with the sponge extract at 1, 3, and 5 days. Cells were then stained, and fluorescence images were captured using an inverted fluorescence microscope for observation and documentation. In the scratch assay experiment, cells were cultured in a six-well plate until they covered the entire surface. Create a scratch in the well, and after washing away the detached cells, the cells were cultured in a medium containing the extract. The cell proliferation process was then recorded. The migration rate of the cell was calculated using the equation:

$$Migration\,rate \% =({R}_{0}-{R}_{1})/{R}_{1}\times 100 \%$$

Where R0 was an initial scratch area and R1 remained the unhealed scratch area.

Hemolytic properties test

To test the hemolytic properties of the sponge, rabbit red blood cells (RBCs) were used. Briefly, rabbit blood was washed and centrifuged to extract red blood cells using normal saline three times. Subsequently, the red blood cells were prepared as a 4% red blood cell solution. 200 μL of erythrocyte suspension was mixed and incubated with medium sponge extract as described in 2.2.3 for 30 min and then a sample was taken to test the absorbance at 540 nm by microplate reader.

Real-time Polymerase Chain Reaction

Raw 264.7 cells (1 × 105 cells/well) were seeds in 12-well plates. After 24 h of incubation, the medium was removed. The control group was incubated with normal media while cells in the test group were induced with 100 ng/mL LPS for 6 h. Then, the LPS cell culture medium was removed and the cells were cultured using a medium described in 2.2.3 for 24 h. Subsequently, the Quantitative Real-time Polymerase Chain Reaction (RT-qPCR) assay was used to measure the expression of inflammatory factors, including tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-1β (IL-1β), and inducible nitric oxide synthase (iNOS).

To detect the expression of pro-vascular differentiation factors in HUVECs, cells (5 × 105 cells/well) were incubated in 6-well plates. Then, the cells were incubated with Alg-BA, Alg-BA@PDA (2.5), and PDA-NPs extracts (1 mg/mL) for 48 h. The experiment was divided into NIR and non-NIR groups. The NIR group was irradiated with NIR laser for 3 min every 8 h; the non-NIR group was not treated. Subsequently, the RT-qPCR assay was done to measure the genes regulating angiogenesis, including endothelial nitric oxide synthase (eNOs), vascular endothelial growth factor (VEGF), hypoxia-inducible factor-1 alpha (HIF-1α), and fibroblast growth factor (bFGF). Primers of targeted genes were listed in Table S2.

In vitro antibacterial activity

In vitro antibacterial activity was tested against Gram-negative (E. coli) and Gram-positive (S.aureus) bacteria. First, 5 mg of different sponge samples, like Alg-BA, Alg-BA@PDA (2.5), or PDA-NPs, were mixed with 100 μL of bacterial solution (1 × 103 cfu/mL) in EP tube. As a control, 100 μL of bacterial solution (Control) was also mixed in EP tubes. Non-NIR groups were without any treatment, whereas the NIR groups (Alg-BA, Alg-BA@PDA (2.5), and PDA-NPs) received NIR laser irradiation for 2 min. Finally, 20 μL liquid was taken and plated for colony growth. For the long-term antibacterial performance test, 5 mg of Alg-BA@PDA (2.5) was mixed with 100 μL of bacterial solution (1 × 104 cfu/mL) in a centrifuge tube, followed by treatment with or without NIR laser irradiation. As a control, 100 μL of the bacterial solution was added to a centrifuge tube without any treatment. Subsequently, 20 μL of the bacterial solution was inoculated on an agar plate for incubation over 12 h to observe colony growth. The experiment was conducted on the 3rd, 7th, and 14th days to evaluate the long-lasting antibacterial performance of the material. Bacterial viability was calculated by the following equation:

$$R \% =X/C\times 100 \%$$

Where C was the number of colonies in a Control solution and X was the number of colonies with the treatment of the sponge20.

Effect of the Alg-BA@PDA sponge on wound healing

Healthy Institute of Cancer Research (ICR) mice (8 weeks, 25–30 g) were anesthetized, and four of the full-thickness round wounds (d = 10 mm) were developed on the shaved dorsal side. The mice were divided into four different groups. Group (a) was treated without dressing and irradiation, group (b) was treated without dressing but irradiation, Group (c) was treated with Alg-BA@PDA (2.5) as dressing and without irradiation, and Group (d) was treated with Alg-BA@PDA (2.5) as dressing and irradiation. Photothermal therapy was performed for 4 min per day and the temperature was controlled to be lower than 50 °C. Photographs were taken at 0, 3, 6, 9, 12, and 15 days and tissue samples were taken on 7th day and 14th day for histochemical staining using H&E staining, Masson staining, and immunostaining for CD31, and CD68.

Results and discussion

Preparation and characterization of the PDA-NPs and Alg-BA

Polydopamine nanoparticles (PDA-NPs) and 3-Aminophenyl boronic acid (BA) modified alginate (Alg-BA) were separately synthesized and characterized14,33. As shown in Figs. 2A, B, and S2, the PDA-NPs were intact and uniformly dispersed with a diameter ranging from 150 nm to 250 nm with a concentration of 2 × 1011 particles/mL. The degradation ratio was approximately 40% of the PDA-NPs after about one month (Fig. S3). The degrees of substitution (DOS) of BA on Alg in Alg-BA (1), Alg-BA (4), Alg-BA (8), and unmodified Alg (Table S1) was calculated by the peak area of the benzene ring and shown in Fig. 2C. Four groups of peaks corresponding to four carbon atoms in BA benzene ring appeared in the range of 7.4–7.8 ppm, and the DOS of Alg-BA was 6.47%, 21.66%, and 31.4%, respectively, indicating the successful modification of Alg with BA along with well-controlled DOS of BA on Alg. The morphology and composition of Alg-BA were characterized by SEM-EDS as shown in Fig. 2D and Table S3. The modified polymers exhibited a typical sponge structure, and with increasing DOS and higher nitrogen content, the porous structure appeared denser. The dense porous structures gave higher swelling ratios as observed in Fig. 2E. Alg-BA (8) and Alg-BA (4) sponges exhibited a higher swelling ratio (25.5-fold) compared to Alg-BA (1). However, the higher DOS led to weaker mechanical properties of Alg-BA after gelation. As shown in Fig. 2F, after adding PBS solution to Alg-BA sponges, the viscosity and stress of Alg-BA hydrogel stabilized within 10 s in the time-sweep test, revealing the rapid adsorption and gelation ability of sponges. However, the stress and viscosity of Alg-BA (8) hydrogel was lower than that of Alg-BA (1) and Alg-BA (4) hydrogels, which might be attributed to the restriction of hydrogen bond formation due to higher density and greater spatial site resistance of the BA units. Based on these results, Alg-BA (4) exhibited better overall performances with rapid water absorption capacity while maintaining high mechanical strength.

Fig. 2: Characterization of PDA-NPs and Alg-BA.
Fig. 2: Characterization of PDA-NPs and Alg-BA.The alternative text for this image may have been generated using AI.
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A SEM images of PDA-NPs. B NTA of PDA-NPs. C NMR of Alg, Alg-BA (1), Alg-BA (4), and Alg-BA (8). D SEM-EDS images of Alg-BA (1), Alg-BA (4), and Alg-BA (8). E Swelling ratio of Alg, Alg-BA (1), Alg-BA (4), and Alg-BA. (8). F Time-viscosity-stress curve.

Preparation and characterization of the Alg-BA@PDA Sponges

A variety of composite sponges of Alg-BA@PDA (1), Alg-BA@PDA (2.5), Alg-BA@PDA (5), Alg-BA@PDA (10), and Alg-BA@PDA (20) were fabricated by doping different volume of PDA-NPs into Alg-BA (1 v%, 2.5 v%, 5 v%, 10 v%, and 20 v%, respectively). The structure of composite sponges was characterized by SEM as depicted in Fig. 3A. A uniform dense porous structure interspersed with PDA-NPs was observed in different sponges. The number of PDA-NPs significantly increased with the increase of PDA-NPs doping ratio. The synthesis of PDA-NPs, Alg-BA, and Alg-BA@ PDA-NPs was further characterized by FT-IR as shown in Fig. 3B. In the curve of PDA-NPs (black-line), a broad peak at 3350 cm-1 corresponding to the –OH and N–H indicates the presence of –OH and –NH2. The sharp peak at 1506 cm-1 corresponds to the C = C of the aromatic ring, which is characteristic of the benzene ring in dopamine. The Alg-BA spectrum showed new absorption peaks at 1340 and 1370 cm-1 34, indicating the formation of borate ester bonds within the sponge material. For Alg-BA@PDA, the peak at 1370 cm-1 (associated with B–O bonds) became stronger compared to Alg-BA, as PDA-NPs’s catechol groups also reacted with Alg-BA to form additional borate ester bonds. These observations collectively confirm the interaction between PDA-NPs and Alg-BA. The pH-responsive nature of these dynamic borate ester bonds allows them to reversibly break down in physiological environments, such as the acidic conditions of wounds. In a mouse model of irregular skin wounds, Alg-BA@PDA demonstrated tight conformity to the wound surface via dynamic bond reorganization, highlighting its adaptability to complex wound geometries.

Fig. 3: Characteristics of various Alg-BA@PDA materials with different doped ratios.
Fig. 3: Characteristics of various Alg-BA@PDA materials with different doped ratios.The alternative text for this image may have been generated using AI.
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A SEM of Alg-BA@PDA sponges with pore structures and PDA-NPs. B FT-IR of Alg, Alg-BA, PDA-NPs, and Alg-BA (4)@PDA. C Photothermal effect of the Alg-BA@PDA sponges. D Swelling ratios of Alg-BA@PDA hydrogels (n = 3). E, F Antioxidant properties of Alg-BA@PDA sponges, tested by ABTS and DPPH methods (n = 3). G ROS scavenging efficiency of Alg-BA@PDA sponges (n = 3).

The photothermal capacity and swelling ratio of Alg-BA@PDA during gelation are shown in Fig. 3C, D. A significant positive correlation between the PDA-NPs proportion and temperature was observed, indicating that the PDA-NPs were the primary source of photothermal properties. The temperature corresponding to Alg-BA@PDA (20) with the highest PDA-NPs ratio reached up to 54 °C. The swelling properties improved with the increase of PDA-NPs proportion, which can be attributed to the increased hydrogen bonds with PDA-NPs in the gel network, and led to a swift swelling in the first 2 h and reached a maximum volume (35-fold) in 5 h, which decreased in the next 24 h attributed to the slow hydrolysis of the hydrogen bonds in the PBS solution. The mechanical strength of the Alg-BA@PDA materials was confirmed by rheological measurements (Figs. S4 and S5) and further evaluated using a mechanical testing machine to assess both the bulk mechanical properties and the adhesion strength of the sponge materials (Fig. S6). The G’ of pristine Alg-BA sponge was lower than that of Alg-BA@PDA sponges, indicating that the mechanical strength was improved after PDA-NPs addition. Thus, dynamically reversible borate bonds between the alginate network and PDA-NPs not only ensure the sponge’s biocompatibility but also enhance its mechanical robustness and adaptability to wound irregularity. Moderate PDA-NPs incorporation (5–10%) optimally balances mechanical reinforcement and tissue adhesion, making Alg-BA@PDA sponges promising for biomedical applications such as wound dressings and bioadhesives.

The antioxidant capacity which can reduce oxidative stress and accelerate the wound healing process, was determined by ABTS and DPPH methods, and results for the same are shown in Fig. 3E, F. The PDA-NPs antioxidant capacity stabilized at 98% showing excellent antioxidant properties, and outperformed the vitamin-C positive control group in both testing methods. The antioxidant capacity was positively correlated with the PDA-NPs ratio, as the antioxidant capacity increased from 10% to 80.1% in the ABTS method and increased from 5% to 45.2% in the DPPH method accompanied by the increase of PDA-NPs ratio to 20 v%. Figure 3G displays the scavenging efficiency of intracellular reactive oxygen species (ROS) by sponges. H2O2 and ROSup solutions were employed to stimulate cells and elevate intracellular ROS. These results revealed that the ROS scavenging efficiency depended on the doped ratio of PDA-NPs, and the ROS scavenging ability of the sponge strengthened with the increase of PDA-NPs. The above results collectively indicate that the Alg-BA@PDA sponges were well-formed with enhanced photothermal properties, swelling ratio, antioxidant properties, and ROS scavenging ability.

Effect of Alg-BA@PDA sponges on cell proliferation, migration, and angiogenesis

To confirm the safety and compatibility of Alg-BA@PDA Sponges for wound repair, the biocompatibilities, toxicity, cytocompatibility, and hemolytic properties were further assessed35. The Live/Dead staining assay (Fig. 4A) of HUVECs showed fewer live cells in the PDA-NPs group, owing to nanoparticles’ toxicity. However, a large number of live cells (green fluorescence) with normal morphology were observed in Alg-BA and Alg-BA@PDA groups suggesting the matrix to be safe and not cytotoxic. It suggests that the sponge matrix can restrict the release of PDA-NPs and can effectively mitigate their cytotoxicity. Consistent results were observed in the CCK8 assay test (Fig. 4B), where the Alg-BA@PDA (2.5) group showed higher cell viability (111.8%) than the Alg-BA group (90.3%) and the PDA-NPs group (61.2%) at 24 h, and maintained a consistent trend in the following 5 days, indicating enhanced biocompatibility of Alg-BA@PDA sponges. Cell scratch assays were further performed (Fig. 4C, D) wherein the cell migration rate initially increased and then decreased with the rise of the PDA-NPs ratio in Alg-BA@PDA sponges. Notably, the Alg-BA@PDA (2.5) group exhibited the fastest cell migration rate of around 53.3%, while the PDA-NPs group displayed the slowest migration rate of 18.5%. Considering the interaction of Alg-BA@PDA sponges with blood during applications, the hemolytic properties of Alg-BA@PDA sponges were tested. As shown in Fig. 4E, F, the hemolytic capacity of all tested samples fluctuated within the range of 1–3%, suggesting good hemocompatibility of Alg-BA and Alg-BA@PDA sponges. Through performance testing and biocompatibility assessment of Alg-BA@PDA sponges, a balance was achieved between functionality and biocompatibility by adjusting the doped ratio of PDA-NPs in Alg-BA. Consequently, the Alg-BA@PDA (2.5) sponge with optimal performance was selected as the experimental group for subsequent molecular biology experiments.

Fig. 4: Biocompatibility of Alg-BA@PDA, Alg-BA, and PDA.
Fig. 4: Biocompatibility of Alg-BA@PDA, Alg-BA, and PDA.The alternative text for this image may have been generated using AI.
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A Live/Dead staining of HUVECs after culturing by using media containing sponge extracts. B Cell viability of HUVECs by CCK8 kit after culturing by using media containing Alg-BA@PDA, Alg-BA, and PDA-NPs for 1, 3, and 5 days. C Quantitative results of cell scratch assay which were calculated using the formula in 2.2.3. D Cell scratch assay photos of HUVECs after culturing by using media containing sponge extracts. E Pictures showing the hemolytic activity assay of sponges and PDA-NPs by mixing the red blood cells with the sponges. F Quantitative result of hemolytic activity assay. (All studies used n = 3 per group. Statistical analysis was performed using the one-way analysis of variance (ANOVA). * is compared to Alg-BA, # is compared to PDA-NPs, #, *p < 0.05, ##, **p < 0.01, and ###, ***p < 0.001).

Anti-inflammatory, angiogenic, and anti-bacterial ability of Alg-BA@PDA sponges in vitro

The anti-inflammatory and angiogenic properties of Alg-BA@PDA sponges were further evaluated in the presence and absence of NIR irradiation. We validated inflammatory factors of the RAW 264.7 cells by RT-qPCR after being treated with media containing sponge extracts of Alg-BA, Alg-BA@PDA (2.5), and PDA-NPs. As shown in Fig. 5A, PDA-NPs exhibited anti-inflammatory effects with low expression levels of inflammatory factors including TNF-α, IL-1β, IL-6, and iNOS. The Alg-BA and Alg-BA@PDA also reduced expression levels of TNF-α and IL-1β, but slightly high levels of IL-6 and iNOS, which could be attributed to the irritation effect by the weak acidity of Alg-BA hydrogel on cells. The four pro-angiogenic differentiation factors in HUVECs were tested in the presence and absence of NIR irradiation. Angiogenic potential of Alg-BA@PDA was confirmed by RT-qPCR studies as depicted in Fig. 5B. The gene expression of VEGF, HIF-1α, bFGF, and eNOS was observed to increase in both Alg-BA@PDA and PDA-NPs after NIR irradiation, as compared to the levels in Alg-BA. The upregulation of angiogenic factors induced by PDA-NPs indicates that their photothermal effect facilitates vascular regeneration. The expression level of VEGF, HIF-1α, bFGF, and eNOs in Alg-BA@PDA group was increased compared to the Control group. A slight improvement in relative gene expression levels for Alg-BA@PDA (2.5) group without NIR was observed over the control group (VEGF 115%, HIF-1α 203%, bFGF 221%, and eNOs 123%, indicating relative gene expression levels of Alg-BA@PDA (2.5) group to the relative gene expression value of the control group). A more dramatic increase was observed after treatment with NIR for Alg-BA@PDA (2.5) compared to the control group (VEGF 153%, HIF-1α 213%, bFGF 410%, and eNOs 185%). The PDA-NPs group also showed enhancement in gene expression over the control group without NIR (VEGF 131%, HIF-1α 252%, bFGF 246%, and eNOs 238%), which further increased with NIR (VEGF 155%, HIF-1α 333%, bFGF 506%, and eNOs 442%). However, the toxicity associated with PDA-NPs, as shown previously, precludes its application in wound healing processes independently without it being associated with Alg-BA. These findings underscore the superior angiogenic potential of Alg-BA@PDA (2.5) sponges with the promise of applying them in wound healing.

Fig. 5: Immunoregulatory and antibacterial performances of Alg-BA@PDA sponges.
Fig. 5: Immunoregulatory and antibacterial performances of Alg-BA@PDA sponges.The alternative text for this image may have been generated using AI.
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A RNA expression of pro-inflammatory factors (TNF-α, IL-1β, IL-6, and iNOS) in RAW264.7 macrophages after treatment with different sponges, assessed by RT-qPCR. B Effects of different materials on the expression of key genes (VEGF, HIF, FGF, and eNOs) in HUVECs with or without NIR irradiation. Target gene expression was normalized to GAPDH and presented as the relative expression, calculated as 2^(-ΔΔCt) and expressed as a percentage of the Control group (n = 3). C Photographs of E. coli and S. aureus growth after 12 h of incubation with Alg-BA, Alg-BA@PDA (2.5), and PDA-NPs. D, E Viability of E. Coli and S. aureus after 12 h of culture in Petri dishes with or without Alg-BA, Alg-BA@PDA (2.5), and PDA-NPs, with or without NIR irradiation (808 nm) (n = 3).

To determine the antibacterial activity of Alg-BA@PDA sponges, both Gram-negative (E. coli) and Gram-positive (S. aureus) bacteria were cultured with different sponges in the presence and absence of NIR irradiation for 5 min. As shown in Fig. 5C–E and Fig. S7, Alg-BA did not significantly affect the growth of E. coli and S. aureus, regardless of NIR irradiation. However, the growth of both bacteria types was significantly inhibited especially after NIR irradiation to Alg-BA@PDA and PDA-NPs. In the absence of NIR, the viability of E. coli decreased progressively from 89% to 26% with Alg-BA, Alg-BA@PDA, and PDA-NPs. A similar trend was observed for S. aureus, where bacterial viability decreased from 88% to 32%. After NIR irradiation, the bacterial viabilities dramatically decreased in the groups with Alg-BA@PDA and PDA-NPs, the persistence of antibacterial activity. These results indicated that the Alg-BA@PDA sponge exhibited antimicrobial properties and long-lasting antibacterial activity against both E. coli and S. aureus over a 14-day period through the doping of PDA-NPs with enhanced photothermal effects.

Alg-BA@PDA sponge promotes full-thickness skin wound healing

The longer the wound healing time, the greater the risk of secondary infections36. Therefore, an ideal dressing should promote wound healing and prevent infection. The in vivo wound healing properties of the Alg-BA@PDA sponge were evaluated using a full-thickness skin defect model in Institute of Cancer Research (ICR) mice, following the previously described protocol (Fig. 6A and Section 2.2.7). Figure 6B shows infrared images of ICR mice before and after irradiation with 808 nm NIR light for 180 s. The temperature increased from 33.1 °C to 55.6 °C after NIR treatment, demonstrating the good photothermal properties of the Alg-BA@PDA sponge. As shown in Fig. 6C, the wound area continuously decreased from day 0 to day 15. Wound images are shown in Fig. 6D, and wound closure statistics are presented in Fig. 6E. The wound closure rate after treatment with Alg-BA@PDA was significantly higher than that of the Control group, particularly following NIR application. Photographs of wounds at different time points show that the wound area treated with the Alg-BA@PDA sponge was significantly smaller within the first 3 days compared to the Control group. NIR irradiation of the Alg-BA@PDA sponge exhibited the most effective wound healing, with a significant reduction in wound area. These results suggest that the Alg-BA@PDA sponge can effectively promote wound healing when combined with NIR irradiation.

Fig. 6: Wound closure performance evaluation from day 0 to day 15.
Fig. 6: Wound closure performance evaluation from day 0 to day 15.The alternative text for this image may have been generated using AI.
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A Construction and treatment procedures of a mouse model with full-thickness wound. B Thermal images of wounds before and after 180 s of photothermal irradiation. C Wound healing records. D Representative photographs of full-thickness wounds treated without and with Alg-BA@PDA in the presence and absence of NIR irradiation. Scale bar: 5 mm. E Wound closure rate statistics at different times (n = 3).

Histological analysis was conducted to further assess the regeneration of skin tissue. As shown in Fig. 7A, the wound tissue in the Control group exhibited minimal granulation tissue and abundant blood scabs covering the wound. After treatment with Alg-BA@PDA (2.5) and Alg-BA@PDA (2.5) + NIR, substantial granulation tissues filling the wound were observed. After 14 days of healing, wounds in all groups were nearly healed, but the wound treated with Alg-BA@PDA (2.5) + NIR exhibited the smallest gap of granulation tissue among all groups. Collagen deposition in the wound was also examined by Masson’s trichrome staining. On day 7, increased collagen deposition in the dermis of the wound tissue was observed in the Alg-BA@PDA (2.5) + NIR group compared to the Control, Control+NIR, and Alg-BA@PDA (2.5) groups (Fig. 7B). On day 14, abundant collagen was generated in all groups, with collagen fibers in the wound of the Alg-BA@PDA (2.5) + NIR group being denser and more organized. These results suggest that the combination of Alg-BA@PDA sponge and NIR treatment promotes wound healing and tissue remodeling.

Fig. 7: Photographs of tissue staining with different samples of 7 and 14 days.
Fig. 7: Photographs of tissue staining with different samples of 7 and 14 days.The alternative text for this image may have been generated using AI.
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A H&E staining. B Masson staining. C CD31 (Red). D CD68 (Red) staining.

Immunofluorescent staining was performed to evaluate the wound-healing effect of the Alg-BA@PDA sponge. Angiogenesis in the healed wound was first assessed using CD31 immunofluorescent staining. As shown in Fig. 7C, the Alg-BA@PDA (2.5) + NIR group exhibited higher CD31 expression compared to the other groups. CD68 immunofluorescence staining was also used to assess the level of the inflammatory response in the wound. As shown in Fig. 7D, CD68 expression decreased slightly over time in all groups, consistent with the normal progression of wound healing, where the acute inflammatory response is followed by a reduction in inflammation. Notably, the wound tissue in the Alg-BA@PDA (2.5) + NIR group showed lower CD68 expression than the other groups on both days 7 and 14, indicating that Alg-BA@PDA (2.5) sponge with NIR treatment effectively suppresses inflammatory responses. These results suggest that the Alg-BA@PDA (2.5) sponge with NIR treatment substantially enhances angiogenesis and accelerates the inflammatory phase, thereby promoting wound healing.

Conclusions

In this study, we developed a self-gelation sponge dressing by modifying sodium alginate with 3-Aminobenzeneboronic acid, followed by loading photothermal nanoparticles, for effective wound repair. This sponge dressing absorbs wound exudate and forms an in-situ gel at the wound site. It possesses antioxidant, antibacterial, and photothermal properties, which help neutralize free radicals and reactive oxygen species (ROS), thereby reducing oxidative tissue damage. These properties also enable effective bacterial killing at the wound site, preventing infection, reducing inflammation, and accelerating healing. Dynamic borate ester bonds between Alg-BA and PDA-NPs effectively restrict the release of PDA-NPs, maintaining the material’s functionality while reducing cytotoxicity and ensuring good biocompatibility. Furthermore, the Alg-BA@PDA sponge accelerates in vivo wound healing by restraining the inflammatory response and promoting vascular regeneration. This self-gelling sponge dressing concept provides valuable insights and great potential for developing new anti-infective wound dressings.