Double cross-linked graphene oxide hydrogel for promoting healing of diabetic ulcers

This study explores the synthesis and characterization of a novel double cross-linked hydrogel composed of polyvinyl alcohol (PVA), sodium alginate (SA), graphene oxide (GO), and glutathione (GSH), henceforth referred to as PVA/SA/GO/GSH. This innovative hydrogel system incorporates two distinct types of cross-linking networks and is meticulously engineered to exhibit sensitivity to high glucose and/or reactive oxygen species (ROS) environments. A sequential approach was adopted in the hydrogel formation. The initial phase involved the absorption of GSH onto GO, which was subsequently functionalized with boric acid and polyethylene glycol derivatives via a bio-orthogonal click reaction. This stage constituted the formation of the first chemically cross-linked network. Subsequently, freeze-thaw cycles were utilized to induce a secondary cross-linking process involving PVA and SA, thereby forming the second physically cross-linked network. The resultant PVA/SA/GO/GSH hydrogel retained the advantageous hydrogel properties such as superior water retention capacity and elasticity, and additionally exhibited the ability to responsively release GSH under changes in glucose concentration and/or ROS levels. This feature finds particular relevance in the therapeutic management of diabetic ulcers. Preliminary in vitro evaluation affirmed the hydrogel’s biocompatibility and its potential to promote cell migration, inhibit apoptosis, and exhibit antibacterial properties. Further in vivo studies demonstrated that the PVA/SA/GO/GSH hydrogel could facilitate the healing of diabetic ulcer sites by mitigating oxidative stress and regulating glucose levels. Thus, the developed PVA/SA/GO/GSH hydrogel emerges as a promising candidate for diabetic ulcer treatment, owing to its specific bio-responsive traits and therapeutic efficacy.


Characterization
In order to determine the gel-forming time, the container was placed flat at regular intervals and the sol-gel transition was assessed by observing the deformation of the solution in the container.The time to reach the gel state was recorded as the gel time.
To assess the structure of the lyophilized hydrogels, the cross sections were goldsprayed and examined using scanning electron microscopy (SEM) at 5.00 kV.The water retention capacity of each group was determined by taking the same weight of hydrogel, drying it at 37℃, and weighing it every hour for 8 hours.The water retention ratio was then calculated using a specific formula.asfollows: Water retention rate(%)= hourly hydrogel mass initial hydrogel mass ×100% The swelling property of the hydrogel was evaluated by gravimetric analysis.The weight of the samples was recorded periodically after they were immersed in physiological saline solution.The samples were removed and excess surface water was eliminated with filter paper.The initial weight (W0) and the weight after swelling at various time points (Wt) were recorded to calculate the swelling ratio of the three groups of hydrogels.The swelling ratio was determined using the following formula: For the measurement of electrical conductivity, stick electrodes were attached to both ends of the hydrogel sample, and the resistance was measured using an avometer.This test was performed three times for each sample, and the conductivity was calculated using the following formula: σ = R/(L×S), where σ is the conductivity in Siemens per meter (S/m), R is the resistance in ohms (Ω), L is the distance between the two electrodes in meters (m), and S is the cross-sectional area of the measured hydrogel in square meters (m 2 ).

Drug released
To assess the in vitro release characteristics of glucose in response, 8 g of PVA/SA/GO/GSH hydrogel containing glutathione (24 mg) was taken and divided into four tubes.Then, 40 ml of PBS with varying glucose concentrations of 0.0 mg/ml, 0.4 mg/ml, 1.0 mg/ml, and 3.0 mg/ml (referred to as GBS) was added to each tube, followed by shaking in a 37℃-water bath at specific intervals.At different time points, 3.5 ml of the supernatant was collected and the release profile of glutathione in different GBS was determined by UV-vis analysis at wavelengths of 230 nm, which corresponded to the maximum absorption of glutathione concentration.
For evaluating the cyclic release properties of the PVA/SA/GO/GSH hydrogel in response to changes in glucose concentration, 2 g of the hydrogel (containing 6mg of glutathione) was placed in a tube and 10 ml of 3.0 mg/ml GBS was added.The tube was incubated with shaking in a 37 ℃ water bath for 1h, and the supernatant was collected and analyzed using UV-vis spectrophotometry.The buffer was then poured out, and the hydrogel was washed twice with PBS.The hydrogel was then placed in 0.4 mg/ml GBS, shaken in a water bath at 37℃ for 3 h, and the supernatant was collected.
The process was repeated multiple times to evaluate the cyclic release properties.
For assessing the in vitro release characteristics of ROS in response, H2O2 concentrations of 0.00 mg/ml, 0.01 mg/ml, 0.05 mg/ml, and 0.10 mg/ml (referred to as HBS) were used.The hydrogel samples were incubated with the respective HBS solutions and shaken in a 37℃ water bath.The supernatant was collected at various time intervals and analyzed using UV-vis spectrophotometry to determine the release profile of ROS in different HBS solutions.
To evaluate the cyclic release properties of the hydrogel in response to changes in ROS concentration, a high concentration HBS solution (0.10 mg/ml) and a low concentration HBS solution (0.01 mg/ml) were chosen.The hydrogel was incubated with the high concentration HBS solution, and the supernatant was collected and analyzed using UVvis spectrophotometry.The buffer was then poured out, and the hydrogel was washed twice with PBS.The hydrogel was then placed in the low concentration HBS solution, shaken in a water bath at 37℃ for 3 h, and the supernatant was collected.The cyclic release properties were evaluated by repeating this process multiple times.

In vitro biocompatibility
Hemolysis assay was conducted using freshly collected blood from BALB/c mice.The serum was removed from the blood and it was diluted with saline.The hydrogel sample was then incubated in the erythrocyte suspension.Negative and positive controls were set up by adding the same amount of saline and TritonX-100 (1%), respectively.After incubation and centrifugation, the supernatant was collected and the absorbance was measured at 540 nm to determine the hemolysis rate.The hemolytic quantification was calculated using the following formula: where AS represents the hydrogel group supernatant absorbance, AP represents the positive control absorbance, and AN represents the negative control absorbance.
For in vitro biocompatibility examination, the sterilized hydrogel was immersed in MEM culture solution and incubated at 37°C for 24 and 48 hours to obtain the extract.
The extract was then centrifuged for later use.L929 cells were inoculated into a 96well plate, and after cell adhesion, the medium was replaced with the extract and incubated for 24 hours.The cell viability was measured, and a control group without extract was set.The amount of GSH used in the GSH group was equivalent to the amount used in the hydrogel composition.The metabolic activity of cells treated with hydrogels was tested using the CCK-8 assay.The cell survival rate was calculated using the following formula: Cell survival rate(%)= OD value of experiment group Control dialogue OD value ×100% The cell scratch experiment was conducted by inoculating L929 cells in a 6-well plate at a density of 2 × 10 5 cells per well.After 24 hours of incubation to allow cell attachment, a scratch was made in the cell monolayer using a sterile pipette tip.The cells were washed with PBS to remove the debris and incubated with serum-free hydrogel extract for 24h and 48h.The area of the scratch was measured using microscopy images taken at 0h, 24h, and 48h.The area reduction rate was calculated using the following formula: Area reduction rate(%)= 0h scratch area-48h scratch area 0h scratch area ×100% The Transwell experiment was conducted by first wetting the Transwell chambers with PBS, followed by adding 500 µl of MEM to the lower chamber and 200 µl of L929 cells (4×10 5 cells) to the upper chamber.The cells were incubated for 24h, and then the upper chamber was replaced with MEM containing hydrogel leachate.After 48h of further incubation, the cells were fixed with paraformaldehyde and stained with crystal violet.Random fields of view were selected and images were captured.The number of cells was counted using image j software.

Establishment of the diabetic ulcer damage mouse model
Male BALB/c mice weighing 32±2 g were randomly selected and fasted for 12 hours prior to modeling.On the day of modeling, their body weight was recorded, and blood glucose was measured after blood collection from the tail vein.The mice were then intraperitoneally injected with STZ at a dose of 120 µg/g body weight.Following the injection, the mice were provided with sufficient drinking water and food.Changes in the body weight and blood glucose levels of the mice were recorded for three consecutive days after the injection.Anesthetized mice were immobilized, and the dorsal hair was removed.The surgical site was sterilized using 75% ethanol.A circular, full-thickness skin wound with a diameter of 4 mm was created on the back using a sterile biopsy punch under aseptic conditions.In the case of active bleeding, sterile gauze packing or pressure was applied to stop the bleeding.

In vivo wound oxidative stress levels Assessment
The tissue was rapidly frozen in liquid nitrogen, and a lysis solution ten times the tissue mass was added.After sufficient lysis on ice, the tissue was centrifuged at 4°C for 10 minutes, and the resulting supernatant was collected.
MDA level was assessed by preparing the TBA storage solution and antioxidant into the MDA assay working solution.0.1 ml of the sample was mixed with 0.2 ml of the MDA assay working solution, heated at 100 ℃ for 15 minutes, cooled to room temperature in a water bath, and then centrifuged at room temperature for 10 minutes.
The resulting supernatant was added to a 96-well plate, and the absorbance was measured at 532 nm.The absorbance and MDA content were calculated using the standard curve and tissue weight.
SOD level was measured by mixing the supernatant of the tissue homogenate with the enzyme reaction solution, and incubating the mixture at 30℃ for 30 minutes.The absorbance was measured at 450 nm, and the inhibition rate of SOD activity and SOD enzyme activity units in the samples were calculated using a formula.

Percent inhibition of SOD activity= A blank control 1 -A sample A blank control 1 -A blank control 2 SOD enzyme activity units=
Percent inhibition of SOD activity 1-Percent inhibition of SOD activity × 100% The blank control in SOD activity measurement refers to the absorbance of the enzyme reaction working solution and the reaction starter working solution after incubating the sample for the same time.
3. To determine the level of reactive oxygen species (ROS), the O13 ROS probe was used as a red-light fluorescent probe.The tissue supernatant and O13 probe were mixed well and incubated for 20 minutes away from light.The intensity of tissue ROS was expressed as fluorescence intensity (RFU) per protein concentration (mg protein).For the determination of nitric oxide (NO) level, tissue supernatant was collected and the content of NO was measured by the Griess Reagent method.

Supplementary figures
Assessment of in vitro antimicrobial capacity.Staphylococcus aureus (CMCCB26003)was cultured in a suitable medium.Solid medium was poured into a plate and an Oxford cup was added before it solidified.After spreading the bacterial solution mixed with solid medium evenly on both sides of the Oxford cups, the Oxford cups were removed after solidification.The hydrogel of each group was placed flat in the hole formed by the Oxford cup and incubated.The formation of inhibition circles was observed, and the diameter was recorded and photographed.Assessment of in vitro ROS clearing capacity.Hydrogel groups were incubated with L929 fibroblasts treated with ROS-up agent to stimulate excessive intracellular ROS production.Dichloro-dihydro-fluorescein diacetate (DCFH-DA) was used as a probe to detect ROS levels, and its fluorescence was activated by oxidation of DCFH-DA by intracellular ROS.

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Figure S1: (A) The impact of graphene oxide (GO) on L929 cells viability, (B) The

Figure S4 .
Figure S4.Electrical conductivity of the PVA/SA/GO/GSH hydrogels.The electrical

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Figure S5.(A) The changes in body weight of mice during the modeling period were

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Figure S6.(A) Wound size was measured using a digital caliper, with the longest and

Figure 1 slightly
Figure S7The quantitative analysis of oxidative stress markers in the injured tissues,