A multifunctional injectable, self-healing, and adhesive hydrogel-based wound dressing stimulated diabetic wound therapeutic with mixed reactive oxygen species scavenging, hyperglycemia decreasing, and bacteria-killing skills | Journal of Nanobiotechnology

Supplies

4-(Bromomethyl) phenylboronic acid (C₇H₈BBrO, 98%), Poly (vinyl alcohol) 1788 (PVA, 87–90% hydrolyze), 4-(Bromomethyl) phenylboronic acid (C₇H₈BBrO₂, 98), Sodium fusidate (SF, C₃₁H₄₇NaO₆, 98%), metformin hydrochloride (MH, C₄H₁₁N₅·HCl, 97%), Gelatin methacryloyl (GM-30%), phalloidin-TRITC (C₆₀H₇₀N₁₂O₁₃S₂), Span 80 (C₂₄H₄₄O), paraffin oil (AR 98%), Distearoylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG2000, 99%), Lecithin (C₄₂H₈₀NO₈P, 90%), N, N, N’, N’-Tetramethyl-1,3-propanediamine (99%), Ldl cholesterol (C₂₇H₄₆O, 95%), 15-mL centrifuge tubes, iFluor 488 phalloidin, RNAisoPlus equipment (Takara Corp, Shiga, Japan), 200-µL and 1-mL pipette ideas, 4′,6-diamidino-2-phenylindole (DAPI), 24-well and 96-well plates, CCK-8 cell viability reagent, and phosphate-buffered saline (PBS) had been acquired from Macklin (Shanghai, China). Citric acid monohydrate, dihydrate, sodium citrate, and streptozotocin had been bought from Aladdin Firm (Shanghai, China). Micropump injector (LEADFLUID TYD01, Baoding, China), SYBR Inexperienced Actual-Time PCR Grasp Mixes (Thermo Fisher Scientific), SYP-TS hydraulic press (Lith Machine Restricted, Xiamen, China), mannequin NLFY-60 (Tianjin, China), Instron 5943 common materials tester (MA, USA), FT-IR instrument (Thermo Scientific Nicolet iS10 infrared FT-IR spectrometer, MA, USA). Rat L929 cell strains had been procured from Cyagen (Jiangsu, China). ATCC 25923 (MSSA), ATCC 43300 (MRSA), and ATCC 25922 (E.coli) had been obtained from ATCC (Manassas, VA, USA).

Synthesis of PATA-based codelivery hydrogel

Synthesis of nanoliposome

Nanoliposome (LP) was fabricated utilizing the skinny movie dispersion method [30]. The process concerned dissolving 20 mg of soy lecithin, 6 mg of ldl cholesterol, and 6 mg of Distearoylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG 2000) every in 1 mL of chloroform in a 1.5 mL EP tube. The combination was then transferred to a 100 mL round-bottom flask and supplemented with a further 2 mL of chloroform. Utilizing a rotary evaporator (LICHEN, Henan, China) underneath vacuum (110 rpm, 45 °C), the chloroform was evaporated, forming a lipid movie contained in the flask. Subsequently, 5 mL of an aqueous resolution of absolutely dissolved metformin hydrochloride (MH) was added, and the flask was rotated for five min. The ultimate step concerned sonication with a high-intensity probe sonicator (JY92-IIN, Scientz, China) at 25 °C (60 plus/min, 130 W) for five min, leading to single-layer nanoliposomes with decreased particle sizes.

Synthesis of GM (SF/LP) microgels

Gelatin methacryloyl (GM) synthesis adhered to established protocols [31]. The following process concerned crafting GM microgel via a microfluidic emulsion methodology, using a coaxial needle and twin syringe pumps (LEADFLUID TYD 01, Baoding, China). On this course of, sodium fusidate (SF) was homogeneously included into a ten w/v% GM polymer resolution containing MH-enriched LP. The oil part, consisting of paraffin oil, was mixed with the surfactant Span 80 (5 w/v%). Upon integration of the oil part with the aqueous part, the water-to-oil move price ratio was rigorously managed between 0.08 and 0.1 (100 μL/min for the oil, 8–10 μL/min for the aqueous part) [17]. The aqueous part, containing 5 wt% GM, 2.5 wt% MH-loaded LP, and 0.5 wt% photoinitiator dissolved in PBS, alongside the oil part, comprising 5 wt% Span80 in paraffin oil, had been concurrently administered into inlets of the microfluidic gadget utilizing syringes. The formation of emulsion droplets ensued, which had been then subjected to ultraviolet (UV) radiation (64 mW/cm², 360–480 nm) for a minute to provoke photo-crosslinking. The ultimate step entailed centrifugation (6000 rpm, 4 min) to gather the shaped crosslinked microgels containing SF and MH-loaded LP.

Synthesis of ROS-responsive TSPBA linker

TSPBA was synthesized via an acetal response between N, N, N′, N’-Tetramethyl-1,3-propanediamine (TMPDA), and 4-(bromomethyl) phenylboronic acid (4-BPBA), following established strategies [32]. Initially, 1.31 g of 4-BPBA was dissolved in 100 mL of dimethylformamide (DMF). After full dissolution and sonication for 10 min, 260 mg of TMPDA was added to the combination and positioned on a magnetic stirrer (60 °C, 24 h). Including tetrahydrofuran (THF) and centrifuging the answer (10,000 rpm, 5 min) resulted in a precipitate, which was lyophilized to acquire the ultimate TSPBA product.

Preparation of PATA-GM (SF/LP) hydrogel

PATA hydrogels had been made by mixing PVA and TSPBA. Based mostly on earlier research [32], PVA was utilized in various concentrations (3, 6, and 9 w/v%), mixed with TSPBA (3 w/v%), to discover the gelation effectivity at completely different PATA ratios. Upon establishing the optimum PVA focus, the PVA was then built-in with GM containing SF (0.1 w/v%), and MH-loaded LP (1 w/v%). Then 3 w/v% of TSPBA was added to kind a ductile gel matrix [33]. After lyophilization, the structural integrity of the PATA-based hydrogels was scrutinized utilizing an electron scanning microscope (SEM).

Fourier remodel infrared (FT-IR) evaluation of the PATA-based hydrogels

The FT-IR spectroscopy (Horiba, Kyoto, Japan) evaluation was carried out to look at the useful teams throughout the GM, boronic acid bonds, and the PATA-based co-delivery hydrogel. The spectral vary between 4000 and 600 cm⁻¹ was meticulously analyzed in opposition to a backdrop of potassium bromide (KBr) and ambient air.

SEM scan of the PATA-based hydrogels

Morphological and porosity attributes of the hydrogels had been assessed utilizing an SEM (ZEISS Sigma 300 SEM, Jena, Germany). For SEM evaluation, a central part of the freeze-dried hydrogel was ready. ImageJ software program (v1.53) was employed to quantify the typical porosity of the hydrogel.

Rheological take a look at of PATA-based hydrogels

Dynamic monitoring of borate-mediated cross-linking kinetics inside hydrogels was carried out utilizing a Discovery HR-2 Rheometer (TA Devices, DE, UK) outfitted with an 8 mm diameter metal parallel plate. The methodology concerned the fast mixing and deposition of the PVA-containing GM (SF/LP) and TSPBA options onto the plate, adopted by rheological analysis at a constant temperature of 25 °C. Rheological properties had been assessed underneath a continuing pressure of 1% throughout a frequency spectrum of 0.1 to 100 rad/s at 25 °C. The quantitative evaluation of the self-healing capabilities of the samples was carried out by monitoring the adjustments within the storage (G′) and loss (G″) moduli of the PATA-GM (SF/LP) hydrogels all through cycles of harmful shearing (an oscillatory pressure sweep elevating the pressure from 0.1% to 1000% at a continuing frequency of 1 Hz) and subsequent restoration (an oscillatory time sweep at a pressure of 0.5% and a frequency of 1 Hz for 60 s).

Swelling and degradation price of PATA-based hydrogels

Within the experiment, samples had been divided into 4 classes: PATA, PATA-GM (SF), PATA-GM (LP), and PATA-GM (SF/LP). These teams had been equilibrated for two h in PBS at 37 °C, publish which they had been weighed on a microbalance (PUCHUN JA GW 0.001 g SERIES, Shanghai, China), and their lots had been documented as M (s). Following this, they had been pre-cooled in a lyophilizer at − 80 °C for 8 h after which vacuum-dried for 12 h. The mass of the samples after freeze-drying was recorded as M (l). The swelling price of the hydrogel pattern was computed utilizing the formulation:

Submit-equilibration, the hydrogel specimens underwent incubation in PBS with various H₂O₂ concentrations (0 mM, 0.01 mM, 0.1 mM, and 1 mM) at 37 °C. To judge their degradation kinetic, we measured the residual mass of those freeze-dried specimens at designated intervals, recognized as M (b). Subsequently, these measurements had been in contrast with the preliminary dry weight utilizing a delegated formulation for analytical functions.

Characterization of SF and MH launch dynamics

To establish the entire launch of SF and MH from PATA-based hydrogels, baseline calibration was carried out utilizing UV–seen spectroscopy (NanoDrop™ 2000/2000c spectrophotometer, Thermo Fisher Scientific, Waltham, Massachusetts, USA). Calibration concerned measuring the absorbance of various concentrations of SF and MH options (starting from 1 to 200 mg/mL in deionized water). Distinct absorption peaks for SF and MH had been recognized at wavelengths of 290 nm and 233 nm, respectively. Following this, explorations into the discharge dynamics of SF and MH from the hydrogels commenced. The formulation, comprising each SF and MH-loaded LP, was amalgamated with an answer containing PVA (9 w/v%) prepolymer and TSPBA (3 w/v%). The fabricated hydrogels had been allotted into 12-well plates containing PBS, with chosen wells additionally incorporating 100 μM H₂O₂, and had been incubated at 37 °C. Periodically, 1 mL of the answer was extracted from every properly and substituted with an equal quantity of contemporary resolution. Absorbance readings at 290 nm and 233 nm had been subsequently recorded and utilized to find out the discharge portions of SF and MH by comparability with an ordinary curve.

In vitro mobile cultivation

L929 cells maintained as much as the fifth passage, had been employed to find out the biocompatibility of the PATA-based hydrogel [34]. These cells had been propagated in DMEM enriched with 10% fetal bovine serum, in an setting of 5% CO₂ at 37 °C, with the medium being refreshed bi-daily. For biocompatibility evaluation, L929 cells had been incubated alongside the hydrogel specimens, adopted by their evaluation utilizing reside/lifeless cell staining assay and the CCK-8 assay.

Cell viability assay

Cell viability of L929 cells, in co-culture with hydrogel samples, was evaluated utilizing the CCK-8 assay. L929 cells (1 × 10⁵ cells/mL) had been co-cultured with PATA-based hydrogel at 37 °C in a 5% CO₂ setting for intervals of 6 and 12 h. The absorbance of the supernatant was measured at 450 nm utilizing a microplate reader (Tecan Buying and selling AG, Switzerland). Cell viability within the CCK-8 assay was computed utilizing the formulation:

On this formulation, the “clean worth” refers to DMEM enhanced with 10% fetal bovine serum. The “management worth” is related to the spectral knowledge obtained from the cell tradition medium, whereas the “measured worth” corresponds to the readings from the medium co-cultured with PATA-based hydrogels.

To judge cell viability when L929 cells had been co-cultured with varied substrates, a fluorescence-based reside/lifeless viability equipment (Calcein-AM/PI Double Stain Equipment, YEASEN, Shanghai, China) was utilized, adhering to the rules of the producer. Following pre-determined time factors, cells in co-culture with hydrogels had been stained after which incubated for 20 min in darkness. Subsequently, these cells underwent triple washing with PBS. Fluorescence microscopy (ECLIPSE Ti2-E, Nikon, Japan) was used to seize pictures of the stained samples. Viability evaluation, distinguishing reside cells (inexperienced fluorescence) from lifeless cells (crimson fluorescence), was carried out utilizing ImageJ software program (v1.53). The viability of the cell was quantified utilizing a selected formulation:

Cell proliferation assay

In the course of the cell proliferation analysis, cells had been stained in accordance with the required protocols of the producer, utilizing iFluor 488 phalloidin and 4’,6-diamidino-2-phenylindole (DAPI). IFluor 488 phalloidin required a 4-h incubation at 37 °C and DAPI staining required a 30-min incubation. After incubation, the samples had been washed 3 times with PBS. These stained cells had been then noticed utilizing an inverted fluorescence microscope (ECLIPSE Ti2-E, Nikon, Japan). Picture evaluation was carried out utilizing ImageJ software program (v1.53), which was instrumental in analyzing the acquired pictures.

Antibacterial assay

The bacterial strains ATCC 25923 (MSSA), ATCC 43300 (MRSA), and E.coli (ATCC 25922) had been grown in 4 mL of Luria–Bertani (LB) broth for 18 h. Subsequently, an answer with 1 mL of LB and a bacterial focus of 1.0 × 10⁶ CFU/mL was ready and added to a 24-well plate. This association was then co-incubated with varied compounds resembling PATA, PATA-GM (SF), PATA-GM (LP), or PATA-GM (SF/LP) at a continuing temperature of 37 °C for time intervals of 6, 12, and 24 h. After incubation, the micro organism had been harvested and underwent a washing course of utilizing PBS buffer with pH values of 6.3 or 7.4, at a centrifugation velocity of 3000 rpm. The bacterial cultures had been then stained utilizing a reside/lifeless bacterial staining agent for 15 min and visualized underneath an inverted fluorescence microscope (ECLIPSE Ti2-E, Nikon, Japan).

The antibacterial efficacy of the hydrogel samples was evaluated utilizing an inhibition zone assay in opposition to two bacterial strains. After a 24-h incubation at 37 °C, the zones indicative of bacterial development suppression had been captured and quantified digitally, using ImageJ software program (v1.53) for evaluation.

Antibiofilm assay

The efficacy of biofilm eradication was confirmed by way of scanning electron microscopy (SEM) evaluation. For biofilm formation, 1 mL aliquots of MSSA, MRSA, and E. coli suspensions (1 × 10⁸ CFU/mL) had been inoculated into particular person wells of a 24-well plate containing titanium sheets, adopted by a two-day incubation with day by day LB medium substitute and co-culture with PATA-based hydrogels. The titanium sheets with biofilms had been fastened in 2.5% GA at 4 °C for 12 h, dehydrated via an ethanol gradient (50%, 60%, 70%, 80%, 90%, and 100%) for 10 min every at 25 °C [35], freeze-dried, and gold-sputtered. SEM (Sigma 300, ZEISS, Germany) was then employed for visualization.

Analysis of mimetic exercise in superoxide dismutase (SOD) analogs

The superoxide anion scavenging capability of the boronate ester was assessed utilizing the SOD assay kit-WST (Sigma-Aldrich, St. Louis, USA) was utilized. The method concerned immersing the PATA-based hydrogel within the assay resolution, adopted by measuring the optical density of the resultant supernatant at 450 nm. The SOD exercise was calculated utilizing the tactic prescribed by the producer of the equipment. Under is the formulation used for calculating the SOD assay outcomes:

On this formulation, “V complete” denotes the general response quantity, which is 0.2 mL. “V pattern” represents the amount of the pattern launched into the response system, set at 0.02 mL. “V pattern complete” refers back to the complete quantity of the extract added, being 1 mL. The focus of the protein pattern, “Cpr”, is expressed in mg/mL. “W” signifies the mass of the pattern in grams. The numeral 500 corresponds to the entire cell or bacterial rely, equal to five million. Lastly, “F” signifies the dilution issue utilized to the pattern.

Evaluation of catalase (CAT)-like exercise in PATA-based hydrogels

To analyze the CAT-mimetic exercise of PATA-based hydrogels, the Amplex Pink reagent assay equipment (Sigma-Aldrich, St. Louis, USA) was used, drawing on established analysis strategies. The method started with the incubation of PATA-based hydrogel samples in 5 μM hydrogen peroxide. This was adopted by the addition of Amplex Pink and horseradish peroxidase, triggering a response with remaining H₂O₂. Fluorescence emitted by resorufin was quantified at a wavelength of 571 nm:

On this formulation, “V_rt” represents the entire quantity, which is 2 × 10^–4 L. The molar extinction coefficient of H₂O₂, denoted by “ε”, is 43.6 L/mol/cm. The optical path size for a 96-well plate, labeled as “d”, measures 0.6 cm. “V_s” stands for the amount of the pattern added, amounting to 0.01 mL. “V_st” refers back to the quantity of the extract added, which is 1 mL. The length of the response, “T”, is ready at 1 min. The determine 500 signifies the entire cell or bacterial rely, equating to five million. Lastly, the unit conversion issue, represented by “10⁶”, defines the connection the place 1 mol equals 10⁶ μmol.

Mobile ROS scavenging exercise

To judge the intracellular ROS-scavenging effectivity of PATA, PATA-GM (SF), PATA-GM (LP), and PATA-GM (SF/LP) hydrogels, L929 cells had been incubated with these hydrogels in a 12-well format. The tradition medium contained 100 μM H₂O₂ and was sustained for two h. Following this, DCFH-DA staining was utilized to mark intracellular ROS, and the fluorescence depth indicative of ROS ranges was quantitatively assessed utilizing ImageJ software program (v1.53).

In vitro hyperglycemia decreasing assay

The glucose uptake by L929 cells was decided utilizing a modified protocol from a glucose assay equipment (Glucose Assay Equipment with O-toluidine, Beyotime, Shanghai, China). Glucose calibration curves had been established utilizing the glucose requirements from the equipment, starting from 5 to 2000 mg/dL. After establishing the glucose commonplace curve, we analyzed the variations in glucose focus within the supernatant of the medium that had been co-cultured with hydrogels. L929 cells had been cultured in 24-well plates at a density of 4 × 10⁴ cells/properly and incubated for twenty-four h to permit attachment. Cells in 5.5 mM glucose serum-free DMEM had been incubated for 12 h. Submit incubation, L929 cells had been handled with 10% serum DMEM supplemented with PATA, PATA-GM (SF), PATA-GM (LP), and PATA-GM (SF/LP) for twenty-four h. To quantify variations in glucose ranges throughout the tradition medium, a 5 μL quantity of the cell tradition supernatant was allotted right into a 1.5 mL ep tube containing 185 μL of Glucose Assay Reagent. This meeting was then incubated in a water bathtub at 95 °C for 8 min, adopted by fast cooling in an ice bathtub at 4 °C for five min. Subsequently, the response combination from the ep tube was transferred to a 96-well plate for absorbance measurement at 630 nm utilizing a microplate reader (Tecan Buying and selling AG, Switzerland), with the outcomes being meticulously documented. This process was replicated thrice.

Preparation of diabetic SD rats

Animal experiments had been authorised by the Institutional Animal Care and Use Committee at Nanfang Hospital, Southern Medical College (R202009.05, Guangzhou, China). All procedures conformed to the Nationwide Institute of Well being’s Information for the Care and Use of Laboratory Animals (NIH Publications No. 8023, revised 1978). The research concerned 25 male Sprague–Dawley (SD) rats, every weighing roughly 200 g, distributed into 5 teams: Management, PATA, PATA-GM (SF), PATA-GM (LP), and PATA-GM (SF/LP), sourced from the Experimental Animal Heart at Southern Medical College. A sort-1 diabetes mannequin was established in these rats, following established methodologies, by administering intraperitoneal injections of streptozotocin (from Aladdin Firm, Shanghai, China) at doses of fifty–60 mg/kg physique weight. Glucose ranges in rat blood had been monitored on days 0, 1, 3, 7, and 14, with samples collected from the tail vein and analyzed utilizing a Bayer Contour Subsequent EZ meter (Leverkusen, Germany). Rats with blood glucose ranges exceeding 11.1 mmol/L had been recognized as diabetic.

Animal procedures and surgical procedures

On this research, 25 male SD rats, every weighing round 200 g, had been utilized. They had been divided into 5 teams: Management, PATA, PATA-GM (SF), PATA-GM (LP), and PATA-GM (SF/LP), sourced from the Experimental Animal Heart at Southern Medical College. Anesthesia was induced utilizing a 3% pentobarbital sodium resolution, administered intraperitoneally at 100 μL per 100 g of physique weight. Following fur removing and sterilisation, a full-thickness pores and skin wound of 1.0 cm diameter was created on the dorsal floor of every rat. Every wound was then inoculated with 20.0 μL of ATCC 25923 (MSSA) suspension (1 × 10⁸ CFU/mL). A gauze was positioned over the injuries for six–8 h to provoke an infection. Afterwards, the designated hydrogel dressings—PATA, PATA-GM (SF), PATA-GM (LP), and PATA-GM (SF/LP)—had been utilized to the injuries. Wound therapeutic was monitored and photographed on days 0, 1, 3, 7, and 14 post-treatment.

Statistical evaluation

Knowledge from all experiments underwent statistical evaluation utilizing one-way ANOVA and Bonferroni’s publish hoc take a look at, using GraphPad Prism 8.0 software program for the computation. The error bars within the graphical representations point out the imply ± commonplace deviation (SD) of the information collected (*P < 0.05).