Objective To investigate the effects and mechanism of copper oxide nanozymes on wound healing of full-thickness skin defects in diabetic mice.
Methods (1) Copper oxide nanozymes were synthesized through the reaction of copper chloride and L-ascorbic acid. Transmission electron microscope was used for observing the particle size and morphology of copper oxide nanozymes, and dynamic light scattering particle size analyzers and Zeta potentiometer were used to analyze the hydrated particle size and surface potential of copper oxide nanozymes, respectively. (2) The hydrogen peroxide detection kit, superoxide anion determination kit, and 3, 3′, 5, 5′-tetramethylbenzidine were used to test the hydrogen peroxide, superoxide anion, and hydroxyl radicals scavenging ability of 150 ng/mL copper oxide nanozymes, respectively, and the scavenging proportions of hydrogen peroxide, superoxide anion, and hydroxyl radicals were calculated. The sample numbers were all 3. (3) Mouse fibroblast cell line 3T3 cells were divided into blank control group, simple hydrogen peroxide group, and hydrogen peroxide+ copper oxide group according to the random number table (the same grouping method below), with 3 wells in each group. Cells in hydrogen peroxide+ copper oxide group were pre-treated with copper oxide nanozymes in final mass concentration of 25 ng/mL for 30 minutes, and then hydrogen peroxide in final molarity of 250 μmol/L was added into simple hydrogen peroxide group and hydrogen peroxide+ copper oxide group. Cells in blank control group were routinely cultured. After 24 hours of culture, 2′, 7′-dichlorodihydrofluorescein diacetate fluorescence probe was used to detect the level of reactive oxygen species (indicated by green fluorescence intensity) in cells and cell counting kit-8 assay was performed to detect and calculate the cell survival rate. (4) Ten male BALB/c mice aged 6-8 weeks (the same gender and age below) were divided into phosphate buffer saline (PBS) group and copper oxide group, with 5 mice in each group. The mice in the copper oxide group were injected with 800 ng/kg copper oxide nanozyme at a concentration of 200 ng/mL via the caudal vein, and the mice in PBS group were treated with the same volume of PBS. The mice in the two groups were treated once a day for seven consecutive days. On the eighth day, 5 mice from each group were conducted and blood samples were taken for analysis of blood panel and serum biochemistry, and then the heart, liver, spleen, lung, and kidney were harvested for histopathological observation by hematoxylin-eosin (HE) staining after the mice were sacrificed. (5) Twenty mice were divided into PBS group and copper oxide group, with 10 mice in each group. Diabetes was induced by streptozotocin and high-sugar and high-fat diet and a full-thickness skin defect wound with diameter of 6 mm was reproduced on the back of each diabetic mouse. Immediately after injury, 20 μL PBS and 20 μL copper oxide nanozymes at the concentration of 200 ng/mL were added respectively to the wounds of mice in PBS group and copper oxide group, with the treatment being continued for twelve consecutive days. Three mice were selected from each group, and the wound healing was observed on post injury day (PID) 0 (immediately), 3, 6, 9, and 12 and the un-healed area was calculated. On PID 6, three mice from each group that were not for wound observation were sacrificed, and the content of interleukin 1β (IL-1β), tumor necrosis factor-α (TNF-α), and IL-6 in the wound tissue were determined by enzyme-linked immunosorbent assay. On PID 12, the rest 7 mice in each group were sacrificed for observation of the length of regenerated epidermis in wound tissue by HE staining, and the level of reactive oxygen species (indicated as red fluorescence intensity) in wound tissue by dihydroethidium staining. Data were statistically analyzed with one-way analysis of variance, analysis of variance for repeated measurement, independent sample
t test, and Bonferroni test.
Results (1) The prepared copper oxide nanozymes were uniform in size with an average diameter of 3.5-4.0 nm in dry state, the hydrated particle size of 4.5 nm, and the surface potential of (-9.8±0.3) mV. By comprehensive judgment, copper oxide nanozymes had been successfully prepared. (2) After being treated with copper oxide nanozyme for 2 hours, 10 minutes, and 5 minutes, respectively, the scavenging proportions of hydrogen peroxide, superoxide anion, and hydroxyl radicals were (77±5)%, (45±5)%, and (84±4)%, respectively. (3) After 24 hours of culture, the cells in simple hydrogen peroxide group showed a significantly increased level of reactive oxygen species with abnormal morphology and decrease in cell number, while the cells in hydrogen peroxide+ copper oxide group showed a remarkably decreased level of reactive oxygen species with normal morphology similar to that of blank control group. The cell survival rate in simple hydrogen peroxide group was obviously reduced compared with the rates in blank control group and hydrogen peroxide+ copper oxide group (
P<0.01), while there was no significant difference in cell survival rate between hydrogen peroxide+ copper oxide group and blank control group. (4) After 7 days of injection, there were no obvious differences in liver and kidney function indexes and blood panel indexes between mice in PBS group and copper oxide group. No necrosis, hyperaemia or hemorrhage in heart, liver, spleen, lung, or kidney was observed in mice in copper oxide group, which was similar to that in PBS group. (5) Compared with that of PBS group, wounds of mice in copper oxide group showed an accelerated healing trend with less redness. On PID 6, 9, and 12, the areas of un-healed wound of mice in copper oxide group (28.8±1.9), (17.6±3.8), and (10.4±1.8) mm
2, respectively, significantly lower than (38.0±4.3), (30.2±3.0), and (24.2±3.0) mm
2 in PBS group (
t=3.706, 5.075, 5.558,
P<0.01). On PID 6, the content of IL-1β, TNF-α, and IL-6 in wounds of mice in copper oxide group were significantly lower than that in PBS group (
t=6.115, 11.762, 11.725,
P<0.01). On PID 12, the length of regenerated epidermis in wounds of mice in copper oxide group was obviously longer than that in PBS group, the level of reactive oxygen species in wounds of mice in copper oxide group was obviously lower than that in PBS group.
Conclusions Copper oxide nanozyme not only has good biocompatibility, but also has efficient reactive oxygen species scavenging activity. It can eliminate the over-expressed reactive oxygen species in the full-thickness defect wounds of diabetic mice, reduce oxidative stress and inflammation, thus promoting wound repair.
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