Molybdesum selenide-based platelet-rich plasma containing carboxymethyl chitosan/polyvinyl pyrrolidone composite antioxidant hydrogels dressing promotes the wound healing | Journal of Nanobiotechnology

Materials

CMCS was purchased from Shanghai Macklin Biochemical Technology Co., Ltd. (Shanghai, China). 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), and Salicylic acid were purchased from Shanghai Aladdin Reagent Co., Ltd. (Shanghai, China). PVP was purchased from J&K Scientific Biochemical Technology Co., Ltd. (Beijing, China). Hydrogen peroxide was purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Luria-Bertani (LB) was purchased from Yuanye Biotechnology Co., Ltd. (Shanghai, China). Human epidermal growth factor (commercial) was purchased from Guilin Huanowei Gene Pharmaceutical Co. (Guilin, China). 3,3’,5,5’-Tetramethylbenzidine (TMB) was purchased from Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China). Mouse fibroblast cells (L929) were bought from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China). Dulbecco’s Modified Eagle Medium (DMEM) and phosphate buffer saline (PBS) were procured from Gibco Co., Ltd. (Shanghai, China). The Cell counting kit-8 (CCK-8) was purchased from Wellbio Biotechnology Co., Ltd. (Shanghai). Calcein-AM/P.I. Live/Dead kit was purchased from Solarbio Technology (Beijing) Co., Ltd. Kunming (KM) mice (female, 4–6 weeks, 20–25 g) were ordered from Shanghai Slac Laboratory Animal Center (Shanghai, China). All chemicals were used without further purification. All experimental mice were housed under the management of the Experimental Animal Centre of Changhai Hospital, Naval Medical University. All animal experiments were performed in strict compliance with the protocols approved by the Ministry of Health of the People’s Republic of China and the policies of the Ministry of Health.

Preparation of hydrogels

To prepare PRP, the collected blood was separated according to the methods reported in the literature [27, 28], MoSe₂ was prepared according to a previous study [29]. To prepare the hydrogels, 0.1 g of CMCS powder was first dissolved in 2 ml of deionized water in a water bath at 50^◦C and stirred to form a homogeneous CMCS solution, which was then transferred to a magnetic stirrer for homogenous stirring. Then, the activators of NHS and EDC were dissolved in 1 mL of the MoSe₂ aqueous solution (100 mg/mL). After the mixture had been wholly dissolved, it was poured into the CMCS solution with continued stirring. Immediately after that, the PVP solution containing 15% PRP was added to the CMCS solution, and by slow stirring and standing, the CMCS/PVP/MoSe₂/PRP hydrogels were obtained. Based on the above steps, we investigated the synthesis of hydrogels under three different parameters: CMCS/PVP(1): 2 mL of CMCS solution (0.05 g/mL), 1 mL of EDC/NHS solution (0.01 g/mL), and 1 mL of PVP solution (0.2 g/mL); CMCS/PVP(2): 2 mL of CMCS solution (0.05 g/mL), 1 mL of EDC/NHS solution (0.02 g/mL), and 1 mL of PVP solution (0.2 g/mL); CMCS/PVP(3): 2 mL of CMCS solution (0.05 g/mL), 1 mL of EDC/NHS solution (0.02 g/mL), 2 mL of PVP solution (0.2 g/mL). CMCS/PVP/PRP and CMCS/PVP/MoSe₂ hydrogels were prepared based on CMCS/PVP (3) without adding MoSe₂ and PRP, respectively. During the experiments, the digital camera recorded all the images of the hydrogels macroscopic gel formation process.

Characterizations of hydrogels

We used scanning electron microscopy (SEM, Zeiss Sigma 300) to observe the microstructure of hydrogels. The hydrogels were first frozen in a refrigerator before being dried in a lyophilizer and further vacuum dried for 24 h. The solvent in the hydrogels was then wholly evaporated. Next, the dried hydrogels were cut and fixed to the SEM sample stage utilizing conductive adhesive, exposing the cut sections and spraying them with gold. The pore structure of the individual hydrogels was then observed using SEM. Further, the homogeneous distribution of C, O, Se, and Mo in CMCS/PVP/MoSe₂/PRP hydrogels was recorded by elemental mapping images. The structural information of the CMCS/PVP/MoSe₂/PRP hydrogels was scanned and recorded in the range of 4000 –500 cm^− 1 using Fourier transform infrared spectroscopy (FTIR, NICOLET-380, Thermo Fisher Scientific, USA). Finally, the surface chemical state of the doped MoSe₂ in CMCS/PVP/MoSe₂/PRP hydrogels was analyzed by X-ray photoelectron spectroscopy (XPS, ESCAlab 250, Thermo Fisher Scientific, USA).

In vitro antioxidant hydrogels swelling studies

The initial weight of the lyophilized CMCS/PVP hydrogels was first weighed (W₁). The hydrogels were then immersed in PBS buffer and incubated in a 37 °C incubator (n = 3). Finally, we removed the hydrogels from the PBS at the experimental time point, carefully removed the water from the surface of the hydrogels using filter paper, and again recorded the final weight of the hydrogels (W₂). The swelling kinetic curves of hydrogels were obtained from the data, and the swelling ratio of the hydrogels at swelling equilibrium was calculated from Eq. (1).

Degradation of hydrogels

We investigated the in vitro biodegradability of CMCS/PVP/MoSe₂/PRP hydrogels by enzymatic degradation experiments. The initial weight of the lyophilized CMCS/PVP/MoSe₂/PRP hydrogels was recorded as W_a. The hydrogels were then immersed in PBS containing 1000 U/mL lysozyme and water containing 1000 U/mL lysozyme (n = 3), incubated in a shaker at 100 rpm, 37 °C, and removed at days 1, 3, 7, 14, and 21. The hydrogels were lyophilized and weighed as W_b. The degradation rate was calculated using the formula.

Analysis of the mechanical properties of hydrogels

We measured the mechanical properties using a universal material tester (Zwick Roell Z2.5 T.H. with 2.5 kN sensor). The adhesion tensile test of CMCS hydrogels, CMCS/PVP hydrogels, and CMCS/PVP/MoSe₂/PRP hydrogels (width: 2.5 cm, length: 3 cm, thickness: 0.3 cm) was tested first. The two pigskin (6 cm long and 3 cm wide) ends were stuck with the hydrogels, fixed to the test apparatus, and tested by tensile loading at 2 mm/min. The maximum tensile strength of the hydrogels for adhesion can be determined when the ends of the pigskin are separated. Further, the hydrogels were tested in compression tests by making cylindrical CMCS hydrogels, CMCS/PVP hydrogels, and CMCS/PVP/MoSe₂/PRP hydrogels of 1.0 cm in height and 1.6 cm in diameter. To determine their compressive properties, the hydrogels were compressed to 80% of their maximum deformation at a predetermined compression rate of 1 mm/min. All these tests were repeated three times in parallel to obtain the final tensile and compressive stress-strain curves and to calculate the average tensile strength.

Antioxidant properties of hydrogels

·OH scavenging ability

The scavenging ability of CMCS/PVP/MoSe₂/PRP hydrogels for ·OH was determined using the salicylic acid (SA) method. Specifically, CMCS/PVP/MoSe₂/PRP hydrogels doped with different concentrations of MoSe₂ (0, 25, 50, and 100 mg/mL) were mixed with FeSO₄·7H₂O (800 µL, 9 mM), ethanolic salicylic acid (800 µL, 9 mM) and H₂O₂ (800 µL, 8.8 mM). The solutions were incubated at 37 °C for 30 min in a constant temperature incubator (n = 3). The supernatant of these solutions was then collected, and the absorbance curves at 400–800 nm were recorded using a UV-Vis-NIR spectrometer (U-3900 Shimadzu, Japan). The color changes of the solutions at different concentrations were further recorded with a digital camera. The ·OH scavenging ratio was calculated as shown in Eq. (3):

In Eq. (3), ΔA₀ and ΔA_x are the absorbance change values of CMCS/PVP/PRP hydrogels in the control group and CMCS/PVP/MoSe₂/PRP hydrogels, respectively.

O₂
^·
– scavenging ability

We used Electron spin resonance (ESR, ELEXSYS II, Bruker, Germany) spectroscopy to examine the ability of CMCS/PVP/MoSe₂/PRP hydrogels to scavenge O₂·⁻. Specifically, O₂·⁻ was generated by mixing xanthine (5 mM) with 10 µL of xanthine oxidase (0.1 U/mL) in 50 mM of PBS. 5,5-dimethyl-1-pyrroline N-oxide (DMPO) as a superoxide anion trapping agent was added to form the adduct DMPO/·OOH⁻. Finally, adducts were mixed with CMCS/PVP/PRP hydrogels and CMCS/PVP/MoSe₂/PRP hydrogels (MoSe₂: 25, 50, and 100 mg/mL). The solutions were transferred to sample detection tubes to record ESR signals immediately after incubation in an oven at 37 °C for 8 min (n = 3).

DPPH scavenging ability

DPPH (1,1-diphenyl-2-picrylhydrazyl, 1.0 mg) was dissolved in 32 mL of anhydrous ethanol. Then, 2 mL of DPPH solution was mixed with 100 mg of CMCS/PVP/PRP and CMCS/PVP/MoSe₂/PRP hydrogels (MoSe₂: 25, 50, and 100 mg/mL) and incubated in an oven at 37 °C for 15 min (n = 3). Finally, they were placed in a UV-Vis spectrophotometer for spectral scanning in the wavelength range of 400–800 nm, and the color changes of the corresponding solutions were recorded with a digital camera.

Peroxidase (POD) scavenging ability

The POD activity was investigated using the TMB method. In this experiment, we used a reaction solution containing H₂O₂ (1000 µL, 144 mM) and an equal volume of TMB (3.2 mM) mixed with 100 mg of CMCS/PVP/PRP hydrogels and CMCS/PVP/MoSe₂/PRP hydrogels (MoSe₂: 25, 50, and 100 mg/mL). The POD-like activity was investigated by recording the reaction system’s absorbance changes using UV-Vis spectroscopy (n = 3).

3-oxo-2-phenyl-4,4,5,5-tetramethylimidazolidine-1-oxide (PTIO) scavenging ability

Specifically, 3 mg of PTIO powder was added to 30 mL of deionized water. The PTIO working solution was homogenously dissolved by sonication. Then, 2 mL of PTIO working solution was mixed with 100 mg of CMCS/PVP/PRP hydrogels and CMCS/PVP/MoSe₂/PRP hydrogels (MoSe₂: 25, 50 and 100 mg/mL) and incubated in an oven at 37 °C for 15 min, and finally, the change in absorbance was scanned by UV-Vis spectroscopy (n = 3), and the color change of the solution was photographed.

In vitro release of growth factors

We placed CMCS/PVP/MoSe₂/PRP hydrogels in 50 mL of centrifuge tubes with 5 mL of PBS buffer solution (n = 3). At predetermined time points (0.5 h, 2 h, 6 h, 8 h, 12 h, 24 h, 48 h, and 72 h), 250 µL of sample solution was collected from the centrifuge tubes and replaced with the same volume of PBS. The collected sample solution was stored in the refrigerator at -80 °C. Finally, the concentrations of EGF and VEGF in the sample solution were measured using the enzyme-linked immunosorbent assay (ELISA) kit according to the manufacturer’s instructions.

Examination of in vitro cell migration properties

We studied the effect of CMCS/PVP/MoSe₂/PRP hydrogels on the migration of mouse fibroblasts (L929 cells) using a cell scratch assay. The CMCS/PVP/MoSe₂/PRP hydrogels were first sterilized and co-incubated in DMEM medium at 37 °C for 24 h (hydrogels: DMEM medium = 1:10) to obtain the hydrogels extracts and gradient diluted to the desired concentrations (0.25, 0.5, and 1 mg/mL). L929 cells were inoculated into 6-well plates at a cell density of 4 × 10⁴ per well to form a fused monolayer. When the cells were uniformly distributed over the healthy plates at about 80% coverage, the monolayer was scratched with a 20-µL pipette tip to simulate a wound and then incubated with low serum medium for 24 h. After preparation, L929 cells were gently washed twice with PBS to remove cell debris while avoiding blowing up the adherent cells. Finally, 2 mL of hydrogel extracts was added to the above healthy plates, and incubation was continued in an incubator containing 5% CO₂ at 37 °C. Cell migration was observed at 0 h, 12 h, and 24 h, respectively, and photographed with an inverted microscope (DMI4000B. Leica, Germany). Finally, the scratch area at each time point was quantified according to the pictures using ImageJ software. The cell migration ratio was calculated as follows (A₀: the initial scratch area at 0 h, A_t: the remaining scratch area after the specified incubation time):

In vitro compatibility of CMCS/PVP/MoSe₂/PRP hydrogels

We first examined the hemocompatibility of the prepared CMCS/PVP/MoSe₂/PRP hydrogels using whole blood from Kunming (KM) mice provided by the Animal Centre of Changhai Hospital, Naval Medical University. The mice red blood cells (mRBCs) were diluted 100-fold in PBS buffer, and 4 mL of the above PBS-diluted mRBCs suspension was mixed with 5, 25, 50, and 100 mg of CMCS/PVP/MoSe₂/PRP hydrogels in a centrifuge tube. Similarly, mRBCs suspensions treated with PBS and H₂O were defined as positive control and positive control, respectively. The above-mixed solutions were incubated at 37 °C for 2 h. Subsequently, the supernatant was collected by centrifugation (5000 rpm, 5 min), and the absorbance values of the solutions were measured at 541 nm. The corresponding hemolysis ratio was calculated using the following formula, and digital photographs of the corresponding solutions were recorded.

Where A_s represents the absorbance of the hydrogels-treated mRBCs suspension at 541 nm, A_n represents the absorbance of the mRBCs suspension treated with PBS, and A_p represents the H₂O-treated mRBCs suspension.

Further, we determined the cytocompatibility of the hydrogels using the leachate of CMCS/PVP/MoSe₂/PRP hydrogels. L929 cells were inoculated in 96-well plates (4 × 10³ cells/well), and 100 µL of different concentrations of hydrogel extracts was added and co-incubated in a 5% CO₂ incubator. Cell survival was observed on days 1, 3, and 5, respectively, and images were collected using live/dead cell staining and the cell viability of L929 cells was detected using the CCK-8 kit.

In vitro safety and degradation properties of CMCS/PVP/MoSe₂/PRP hydrogels

The animal study protocol was approved by the Institutional Review Board of The First Affiliated Hospital of Naval Medical University of the People’s Liberation Army (SYXK(Shanghai) 2020-0033). CMCS/PVP/MoSe₂/PRP hydrogels (approximately 0.6 cm in diameter and 0.2 cm in thickness, n = 3) were prepared in advance using a mold and aseptically treated. The mice were anesthetized and skin was prepared, and the incision sites were disinfected with iodophor. The hydrogels were implanted into the mice’s dorsal skin and then sutured with simple interrupted sutures before resting for observation. The body weight of the mice was monitored before and after the experiment. At the end of the procedure, the mice were executed at 7, 14, and 28 days after feeding, and the subcutaneous hydrogels were extracted, weighed, and photographed to determine the in vivo biodegradation. During this procedure, mouse blood was extracted for serum biochemical analysis. Meanwhile, skin tissues of mice exposed to the hydrogels and their major organs (heart, liver, spleen, lungs, and kidneys) were dissected, fixed in paraformaldehyde, and analyzed by hematoxylin-eosin (H&E) staining for tissue analysis. H&E staining images were recorded by a German inverted phase contrast microscope (Leica DM IL LED).

Assessment of the wound healing capacity

To evaluate the ability of CMCS/PVP/MoSe₂/PRP hydrogels to scavenge free radicals and promote wound healing, a whole skin wound model was established using Balb/c mice for in vivo studies. The experimental procedure was as follows: after disinfecting the experimental table, the mice were anesthetized, and part of their dorsal hair was removed and disinfected with iodophor. The dorsal skin of the mice was gently pulled upwards, and a full-length wound with a diameter of 0.6 cm was made on the dorsal surface of each Balb/c mouse using a disinfected perforator. CMCS hydrogels, CMCS/PVP hydrogels, commercially available gels (human epidermal growth factor), CMCS/PVP/MoSe₂ hydrogels and CMCS/PVP/MoSe₂/PRP hydrogels (n = 3) were applied to the wounds of the corresponding mice and fixed with sterile gauze. Wound healing in mice was recorded using a digital camera on days 0, 2, 5, 8, 12, and 16, respectively, and the wound area was analyzed using ImageJ software to assess the wound healing-promoting effect of the hydrogels. The change in wound area over time was expressed as the original wound area. The wound healing ratio was calculated as follows.

In the formula, S_s and S_p are the open and original wound areas on the specified date, respectively. On day 16, different groups of mice were executed, and the skin tissue around the wound was collected and fixed in paraformaldehyde and analyzed histologically to assess wound healing [H&E staining, Masson staining, Sirius red staining, Platelet-endothelial cell adhesion molecule (PECAM-1/CD31) staining and α-Smooth muscle actin (α-SMA) staining]. Immunofluorescence staining images were obtained and viewed by fluorescence microscopy, followed by quantitative analysis by Image J software. The serum (day 16) and skin tissues (day 8 and day16) of the experimental mice were also collected to determine TNF-α, EGF, and VEGF levels using ELISA kits and the double antibody sandwich enzyme-linked immunosorbent assay technique. In brief, standards, samples to be tested, and biotinylated detection antibodies are added to the wells of the enzyme plate and incubated at room temperature for 2 h. After removing unbound agents, horseradish peroxidase-labeled streptavidin (Streptavidin-HRP) was added. After washing, the chromogenic substrate TMB was added. The reaction was terminated when the color changed from blue to yellow by adding a termination solution. The absorbance was measured at 450 nm within 30 min to calculate the TNF-α, EGF, and VEGF levels.

Statistical analysis

All results were expressed as mean ± SD. Statistical significance was determined using a one-way analysis of variance (ANOVA), and statistically significant differences were marked with *p < 0.05, **p < 0.01, and ***p < 0.001.