An in-situ self-gelation photothermal alginate-based sponge dressing for rapid and effective wound management

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:

Where M_t 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:

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:

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.