Supplies
Dopamine hydrochloride, tris(hydroxymethyl)aminomethane (Tris), methylene blue, 2,2-diphenyl-1-picrylhydrazyl, and ferrous sulfate had been bought from Macklin Biochemical Expertise Co., Ltd. (Shanghai). Hydrogen peroxide, catalase, lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP), stay/lifeless bacterial viability equipment, and three,3′-dipropylthiadicarbocyanine iodide (DiSC3(5)) had been obtained from Sigma-Aldrich. GelMA polymer was bought from Shuhe Biotechnology Co., Ltd. (Wenzhou). Luria–Bertani agar and Luria–Bertani broth had been introduced from Xueyou Biochemical Expertise Co., Ltd. (Guangzhou). ATP assay equipment, stay and lifeless fluorescent equipment, cell counting kit-8, apoptosis detection equipment, Hoechst, ROS assay equipment, and TUNEL apoptosis assay equipment had been obtained from Beyotime Biochemical Expertise Co., Ltd. (Shanghai).
Validation of the ultrafast polymerization response
Dopamine hydrochloride (2 mg), H2O2 (1 M, 2 µL) and catalase (1 mg/mL, 5 µL) had been added to Tris buffer (25 mM, pH 8.5, 200 µL) in a 96-well plate as illustrated. This technique might be scaled up proportionally and ought to be ready contemporary earlier than use [51]. The plate was photographed each 10 min, and the absorbance of the samples was measured (OD652) at 3, 5, 10, 20, 30, 40, 50, and 60 min by a microplate reader (Thermo Fisher). The experiments performed beneath hypoxic situations adopted an analogous protocol.
Bacterial tradition
The MRSA and EC had been each cultured in LB borth medium in a shaking incubator (2000 rpm, 37 °C). The micro organism had been collected on the logarithmic progress section and the focus of micro organism was decided by Nephelometers (BD Biosciences).
Catalysis of H
2
O
2
by MRSA
The oxygen content material was measured utilizing a dissolved oxygen analyzer (JPB-607A) at room temperature. To arrange the samples, MRSA (107 CFU/mL) was launched right into a H2O2 resolution (10 mM), and the combination was stirred constantly. The oxygen focus was recorded each 30 s.
Detection of MRSA by the ultrafast polymerization response
The detection working resolution was ready by introducing dopamine hydrochloride (50 mg) and H2O2 (1 M, 50 µL) into Tris buffer (25 mM, pH 8.5, 5 mL). The working resolution ought to be ready contemporary earlier than use. MRSA (107 CFU/mL), EC (107 CFU/mL), and equal quantity of Tris buffer had been added to the working resolution for 10 min, respectively. The samples had been photographed and measured by a microplate reader.
Snapshot-assisted digital evaluation
A sequence of MRSA focus gradient detections had been performed to evaluate the feasibility of snapshot-assisted digital evaluation. MRSA samples (0.045–450 × 106 CFU/mL) had been added to the working resolution and reacted at room temperature for 10 min. The samples had been then photographed, and their absorbance was measured. All photographs had been imported into the Coloration Acquire APP, the place the “colour gather” mode was utilized to pattern 5 positions in every picture, yielding R, G, and B parameters. The correlation between bacterial focus and these parameters was subsequently plotted.
Stability verification
The ultrafast polymerization response can obtain long-term stability by way of correct aliquoting and storage. Dopamine powder, Tris buffer, and H2O2 resolution had been individually positioned in glass vials and saved at 4 °C, protected against gentle. Samples had been taken at intervals of 0, 3, 7, 15, 30, and 45 days, allowed to equilibrate to room temperature, after which examined in accordance with the described process, utilizing CAT as an ordinary.
Biofilm formation and detection
MRSA bacterial suspension (100 µL) at numerous concentrations had been added to a 96-well plate and incubated at 37 °C for twenty-four h. The supernatant was fastidiously eliminated, revealing a visual biofilm on the backside of the wells. For the detection of biofilm utilizing the ultrafast polymerization response, working resolution was launched into the wells and the plate was incubated at 37 °C for 10 min, adopted by imaging and UV absorption measurements. As a management, biofilm quantification was carried out utilizing the crystal violet methodology. Particularly, methanol (150 µL) was added to the biofilm for fixation for 10 min. After eradicating the surplus methanol, crystal violet resolution (1 wt%) was added and incubated within the wells for 10 min. The surplus crystal violet was gently washed off, and 33% (v/v) acetic acid resolution was added to dissolve the crystal violet-stained biofilm, with absorbance measured at 590 nm. Every pattern was examined in triplicate.
Preparation and characterization of HDG hydrogel
The HDG hydrogel was ready by mixing GelMA resolution (5 wt%), LAP (0.25 wt%) and the H2O2-dopamine containing working resolution, adopted by UV irradiation for crosslinking. For SEM evaluation, MRSA was launched within the HDG hydrogel for 30 min to set off dopamine polymerization. The HDG hydrogel and MRSA-treated HDG hydrogel had been subjected to freeze-drying for SEM statement (Regulus-8100, Hitachi). Rheological checks had been performed on a TA rheometer (TA ARES-G2). Earlier than knowledge assortment, the GelMA hydrogel, HDG hydrogel, and MRSA-treated HDG hydrogel was positioned between two parallel plates (Φ20 mm) respectively at room temperature, and the periphery was sealed with silicone oil to stop water evaporation. The stiffness of the hydrogels was evaluated by an oscillation-frequency take a look at with 1% fixed pressure whereas the frequency was different from 0.1 to 100 rad/s.
Photothermal response of HDG hydrogel
The photothermal response of HDG hydrogel and MRSA-treated HDG hydrogel was assessed utilizing an infrared digital camera (Hikvision). The samples, together with water, HDG hydrogel, and MRSA-treated HDG hydrogel, had been irradiated with an 808 nm laser, with temperature adjustments recorded each 20 s. The heating curves of the HDG hydrogel had been obtained by various the bacterial focus and laser depth (as indicated within the manuscript). The heating and cooling conduct of the HDG hydrogel was additional analyzed by way of 4 cycles of laser ON/OFF.
Antibacterial efficiency of HDG hydrogel
MRSA (102–108 CFU/mL) had been handled with HDG hydrogel for 10 min at room temperature. The mixtures had been then both uncovered to an 808 nm laser (1 W/cm2, 10 min) or left untreated. After remedy, the samples had been plated and incubated at 37 °C for 18 h, adopted by colony counting to evaluate bacterial survival. The same process was used for the dual-color fluorescent assay, besides the handled micro organism had been stained utilizing a stay/lifeless bacterial fluorescent viability equipment in accordance with the producer’s directions.
Evaluation of membrane integrity
The membrane potential adjustments of MRSA handled with HDG hydrogel had been analyzed utilizing a zeta potential analyzer (Malvern) at intervals of 1, 2, 5, and 10 min. The extent of bacterial membrane harm was assessed utilizing the fluorescent probe DiSC3(5). MRSA samples handled with PBS, GelMA hydrogel, HDG hydrogel, and HDG hydrogel + NIR had been centrifuged at 3000 rpm for 3 min and washed with PBS. Subsequently, DiSC3(5) (400 nM) was added to the samples, which had been then incubated at midnight at 37 °C for 15 min in a water tub. Fluorescence measurements had been taken utilizing a microplate reader with excitation and emission wavelengths of 622 nm and 670 nm, respectively.
ATP assay
MRSA samples handled with PBS, GelMA hydrogel, HDG hydrogel, and HDG hydrogel + NIR had been centrifuged at 3000 rpm for 3 min and washed with PBS. The ATP content material of the micro organism was subsequently measured utilizing an ATP assay equipment by figuring out the luminescence.
DPPH scavenging assay
DPPH ethanol resolution (0.5 mg/mL, 200 µL) was added to HDG hydrogel samples at concentrations starting from 0 to 700 µg/mL, bringing the entire quantity to 2 mL. The combination was incubated at midnight at room temperature for 30 min. After incubation, the UV absorption spectrum was recorded utilizing a UV spectrophotometer (300–1000 nm). The absorbance at 519 nm was measured at numerous time factors utilizing a microplate reader.
Hydroxyl radical scavenging assay
To judge the hydroxyl radical scavenging impact of the HDG hydrogel, a combined resolution containing MB (50 µg/mL), FeSO₄ (1 mM), H₂O₂ (2 mM), and ranging concentrations of HDG hydrogel (0–400 µg/mL) was ready to a complete quantity of two mL. The combination was incubated, and the UV absorption spectrum was recorded utilizing a spectrophotometer throughout the vary of 300–1000 nm. The absorbance at 665 nm was measured at numerous time factors utilizing a microplate reader.
Biocompatibility of HDG hydrogel
For the stay and lifeless fluorescent assay, L929 cells had been cultured in a 12-well plate for 12 h, adopted by the addition of PBS or HDG hydrogel for an extra 12 or 24 h. The cells had been then washed with PBS and stained with the stay and lifeless fluorescent equipment in accordance with the producer’s directions.
For the CCK8 assay, L929 cells had been cultured in a 96-well plate with the addition of PBS or HDG hydrogel for numerous incubation occasions. The cells had been subsequently washed with PBS and examined utilizing the CCK8 reagent.
For the apoptosis evaluation, L929 cells had been cultured in a 24-well plate supplemented with PBS or HDG hydrogel for numerous incubation occasions. The cells had been washed with PBS and stained with Annexin V-FITC in accordance with the producer’s directions.
Cell migration assay
The impact of HDG hydrogel on cell migration capability was evaluated by scratch experiment. L929 cells had been co-cultured with hydrogel in 6-well plates at a cell density of 5 × 105 cells/nicely. After 24 h of incubation, a cell monolayer was fashioned, after which three parallel scratches had been made with a 200 μL pipette tip, and the floating cells had been washed thrice with sterile PBS. After 24 h, the cells had been photographed with an inverted microscope (Olympus CKX41, Tokyo, Japan), and the cell mobility was quantitatively calculated utilizing Picture J software program.
In vitro angiogenesis assay
An in vitro angiogenesis assay with HUVECs was carried out. Matrigel Matrix Gel (Corning, 356234, 100 μL) was added to every nicely of a 48-well plate, gently shaken to cowl the underside of the wells, after which the plate was incubated at 37 °C in an incubator for greater than 1 h. The cells had been co-cultured (2 × 104 cells/mL) within the plate at 37 °C for six h beneath 5% CO2. The formation of cell tubes was captured with an inverted microscope. The angiogenic potential was then quantified utilizing Picture J software program.
Intracellular antioxidant assay
To judge the intracellular antioxidant exercise of HDG hydrogel, L929 cells had been seeded in a 24-well plate and incubated for 12 h. After incubation, the cells had been handled with PBS, H2O2 (1 mM), or H2O2 (1 mM) + HDG hydrogel, respectively for six h. To find out intracellular ROS ranges, the cells had been washed with PBS and incubated with DCFH-DA (10 µM) for 30 min at 37 °C at midnight. After incubation, the cells had been washed once more with PBS, stained with Hoechst, and subjected to CLSM imaging. ExHoechst: 405 nm, ExDCF: 488 nm. For the TUNEL assay, the handled cells had been washed with PBS and stained utilizing the TUNEL apoptosis assay equipment in accordance with the producer’s directions. The cells had been additional stained with Hoechst and noticed beneath CLSM. ExHoechst: 405 nm, ExTunel: 561 nm.
Full-thickness contaminated pores and skin wound remedy
All animal procedures had been performed in accordance with the rules of the native ethics committee (Allow Quantity: K2023-01-159). Feminine Balb/c mice (6 weeks outdated) had been shaved and anesthetized utilizing 2% pentobarbital. To validate in vivo MRSA detection, a full-thickness wound was created on the dorsum of every mouse, adopted by an injection of fifty μL of MRSA at completely different concentrations (0, 104, 106, and 5 × 107 CFU/mL) to ascertain an contaminated wound mannequin. The HDG hydrogel was utilized to the contaminated space for 10 min, and images of the hydrogel had been taken. Subsequently, an NIR laser (808 nm, 1 W/cm2, 10 min) was utilized to the contaminated wounds, and temperature adjustments had been recorded utilizing a digital near-infrared photothermal imaging system. To judge the therapeutic efficiency of the HDG hydrogel, mice with contaminated wounds (5 × 107 CFU/mL) had been randomly divided into 4 teams (n = 10 per group) and handled with both PBS, GelMA hydrogel, HDG hydrogel, or HDG hydrogel mixed with NIR. Wound space adjustments had been documented by photographing the injuries on days 1, 7, and 12 post-infections. All surgical procedures had been carried out beneath sterile situations. Wound therapeutic was assessed by measuring wound space adjustments utilizing ImageJ software program, adopted by statistical evaluation to acquire macroscopic wound therapeutic knowledge.
Histological, immunohistochemical, and immunofluorescence evaluation
Full wound specimens had been collected on days 7 and 12 post-surgery for histopathological examination, Masson’s trichrome staining, and immunostaining. These analyses had been performed to judge epidermal regeneration, collagen deposition, irritation, matrix degradation, and macrophage polarization throughout the wound space. After assortment, the specimens had been fastened in 4% paraformaldehyde in a single day, dehydrated utilizing an computerized tissue processor, embedded in paraffin, and sectioned into 4 μm slices. The sections had been stained with H&E and Masson’s trichrome and immunohistochemically labeled for Col-I, IL-1β, MMP-2, CD86, CD206, and F4/80 markers. Observations and documentation had been carried out utilizing an upright microscope (Leica DM2700 M). The center, liver, spleen, lungs, and kidneys had been collected from the mice, and H&E staining was carried out.
Statistical evaluation
All experimental knowledge had been statistically analyzed and expressed as imply ± normal deviation. Statistical variations had been carried out utilizing PRISM software program. Variations had been thought-about statistically vital at p < 0.05. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.
