Facile synthesis of nanoparticles-stacked Co3O4 nanoflakes with catalase-like activity for accelerating wound healing

Abstract Delayed wound healing caused by excessive reactive oxygen species (ROS) remains a considerable challenge. In recent years, metal oxide nanozymes have gained significant attention in biomedical research. However, a comprehensive investigation of Co3O4-based nanozymes for enhancing wound healing and tissue regeneration is lacking. This study focuses on developing a facile synthesis method to produce high-stability and cost-effective Co3O4 nanoflakes (NFs) with promising catalase (CAT)-like activity to regulate the oxidative microenvironment and accelerate wound healing. The closely arranged Co3O4 nanoparticles (NPs) within the NFs structure result in a significantly larger surface area, thereby amplifying the enzymatic activity compared to commercially available Co3O4 NPs. Under physiological conditions, it was observed that Co3O4 NFs efficiently break down hydrogen peroxide (H2O2) without generating harmful radicals (·OH). Moreover, they exhibit excellent compatibility with various cells involved in wound healing, promoting fibroblast growth and protecting cells from oxidative stress. In a rat model, Co3O4 NFs facilitate both the hemostatic and proliferative phases of wound healing, consequently accelerating the process. Overall, the promising results of Co3O4 NFs highlight their potential in promoting wound healing and tissue regeneration.


Introduction
Delayed wound healing accompanied by chronic inflammation is a significant challenge in burn injuries, diabetic foot ulcers and surgical wounds.This issue poses a growing burden on the medical system [1,2].Reactive oxygen species (ROS) generated in impaired wounds play a crucial role in cellular homeostasis and physiological regulation.However, excessive ROS accumulation at the wound site leads to oxidative damage, chronic inflammation and hinder tissue repair [3][4][5].Therefore, it is essential to regulate the oxidative microenvironment by scavenging excess ROS to accelerate wound healing [6,7].
Natural antioxidant enzymes such as catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase are effective in scavenging ROS and modulating oxidative stress [8].However, their clinical application faces challenges due to inherent characteristics such as low stability, high cost and sensitivity to environmental conditions [9].On the other hand, nanozymes, which are nanomaterials that mimic enzyme-like functions, offer promising advantages in biomedical applications due to their high stability and costeffectiveness [10].Metal oxide-based nanozymes, including Fe 3 O 4 , CeO 2 , CuO, MnO 2 and Co 3 O 4 , have been developed and extensively studied for their peroxidase-like, oxidase-like, CAT-like and/or SODlike activities in wound healing and tissue repair [11][12][13][14][15][16].
Cobalt (Co) is an essential trace element that forms the active center of vitamin B 12 , crucial for multiple biosynthesis and metabolism processes [17,18].Co-based materials, such as Co alloys, have been widely used as implantable biomaterials due to their excellent biocompatibility [19,20].Recently, Co and its oxide-based nanozymes, particularly Co 3 O 4 nanoparticles (NPs), have demonstrated CAT-like and peroxidase-like activities in various biomedical applications, including glucose [12,21] and hydrogen peroxide (H 2 O 2 ) detection [22], as well as immunohistochemical assays [23].Therefore, it is of great interest to explore the beneficial effects of Co-based nanozymes with enzyme-like properties for facilitating wound healing and promoting tissue regeneration [8, [24][25][26].
In In summary, our study focuses on investigating the advantages of the simply synthesized Co 3 O 4 NFs with CAT-like activity in promoting wound healing and tissue regeneration.The excellent biocompatibility, fibroblast promotion and ability to protect cells from oxidative stress make Co 3 O 4 NFs a promising candidate for enhancing wound healing processes (Figure 1).

Preparation of Co 3 O 4 NFs
To prepare Co 3 O 4 NFs, Co(CH 3 COO) 2 -4H 2 O was placed in a tube furnace and annealed at 600 � C for 120 min in air.Then the system was cooled to room temperature, and Co 3 O 4 NFs were obtained.

Characterization
The scanning electron microscope (SEM) morphology was observed using FEI Inspect F50.The high-resolution transmission electron microscopy (HRTEM) images were taken using FEI Tecnai G2 Spirit TWIN, X-ray powder diffraction (XRD) pattern was recorded with a Rigaku Ultima IV diffractometer using Cu Ka radiation (k ¼ 1.5406 Å).The X-ray photoelectron spectroscopy (XPS) was determined using Thermo Scientific Escalab 250xi.The concentration of Co ions was quantified using Inductively coupled plasma-Mass Spectrometry (ICP-MS, PerkinElmer NexION 2000).The hydrodynamic size of NFs was measured by dynamic light scattering (DLS, Malvern, UK).

Electrochemical measurement
Cyclic voltammetric was performed on a CS150H electrochemical workstation.Then, 5 ll of Co 3 O 4 NFs colloidal solution (3% PTFE in water) was dropped on the surface of the pre-treated carbon cloth (CC) electrode and dried for 3 h under environmental conditions to obtain the Co 3 O 4 NF-modified electrode.The electrocatalytic property was investigated in a three-electrode system comprising the Co 3 O 4 NF-modified electrode as the working electrode, a carbon rod as an auxiliary and a saturated calomel electrode as reference.All experimental solutions were deoxygenated by bubbling highly pure argon for at least 20 min and maintained under argon atmosphere during the measurements.

CAT-like activity of Co 3 O 4 NFs
The CAT-like activity of Co 3 O 4 NFs was assessed using H 2 O 2 assay kit (Solarbio, Shanghai, China).Different concentrations of Co 3 O 4 NFs (0, 0.5, 5, 50, 500, 5000 mg/ml) were incubated with 10 mM H 2 O 2 for 30 min at 37 � C, pH 7.4.The concentration of remaining H 2 O 2 was obtained by calculating the absorbance at 450 nm according to the manufacturer's instructions.Under the same conditions, the generated oxygen in solutions at different reaction times was measured using a dissolved oxygen meter (JPSJ-605F, LEICI Auto Industry Co., Ltd, Shanghai, China).The kinetic assays were performed by testing 50 mg/ml of Co 3 O 4 NFs with different concentrations (0.1, 1, 10, 100 mM) of H 2 O 2 .Michaelis-Menten constants were calculated from the Michaelis-Menten saturation curves of GraphPad Prism 8.0 (GraphPad Software).

pH-switchable peroxidase-like activity of Co 3 O 4 NFs
The peroxidase-like activity of the Co 3 O 4 NFs was measured by TMB colorimetry.Different concentrations of Co 3 O 4 NFs (0, 0.5, 5, 50, 500, 5000 mg/ml) were incubated with 10 mM H 2 O 2 for 30 min at 37 � C, pH 4.0 and pH 7.4, respectively.The supernatant was collected by centrifuging at 6000 rpm for 5 min.The colorless TMB can be converted into blue ox-TMB by �OH.The generated �OH concentration in the supernatant was obtained by calculating the absorbance at 650 nm according to the manufacturer's instructions.

Evaluation of long-term and sterilization stability of Co 3 O 4 NFs
A 30-ml portion of 50 lg/ml Co 3 O 4 NFs aqueous suspension was prepared in PBS at pH 7.4 and the solution was slowly stirred at 37 � C. At time points of 0, 3 and 14 days, respectively, the suspension was centrifuged.Then, 5 ml of supernatant was collected, and 5 ml of fresh PBS was added to maintain a consistent volume.The concentration of Co ions was determined by ICP-MS and filtered with a 0.2-lm filter prior to testing.
The Co 3 O 4 NFs and 2000 U CAT were autoclaved, sterilized in 75% alcohol and allowed to stand for 14 days (37 � C, pH 7.4).The residual enzyme activity of the treated Co 3 O 4 NFs and 2000 U CAT was assayed with a H 2 O 2 assay kit.

Cell viability assessment
L929, ECs and SMCs were first seeded in 96-well plates at 5000 cells/well, respectively.After 24 h, the medium was replaced with fresh medium containing different concentrations of sterilized Co 3 O 4 NFs (0, 0.5, 5, 50, 500, 5000 mg/ml).After 24 and 72 h of incubation, the cell viability was examined by using a CCK-8 kit.
The cell morphology was observed by fluorescence microscopy (CKX53, OLYMPUS, Japan), after three times washing with saline, fixation by 2.5% glutaraldehyde and being stained with DAPI and Rhodamine 123.

Cellular ROS-scavenging assessment of Co 3 O 4 NFs
After 24 h of adhesion of L929 in 96-well plates, fresh medium containing 800 mM H 2 O 2 and 50 mg/ml Co 3 O 4 NFs was replaced, 2000 U CAT was used as positive control.After 6 h of cotreatment, the plates were washed two to three times and cell death was immediately observed under a fluorescent microscope using the Live/Dead Cell Detection Kit (KeyGE, Jiangsu, China).The intracellular ROS content was detected using the fluorescent probe DCFH-DA (Beyotime, Shanghai, China).

Hemolysis assay
The suspension of red blood cells (RBCs) was obtained from fresh (Sprague-Dawley) SD rat blood through centrifugation at 3000 rpm for 15 min.The RBCs were then gently washed and diluted using a saline solution.Subsequently, 20 ll of the RBC solution was mixed with 1 ml of various concentrations of Co 3 O 4 NFs (0.5, 5, 50, 500, 5000 mg/ml) in PBS.Simultaneously, 20 ll of the RBCs solution was mixed with 1 ml of PBS and deionized water to serve as the negative control and the positive control, respectively.After incubating at 37 � C for 3 h, the supernatant from all groups was collected via centrifugation at 3000 rpm for 15 min.The absorbance at 540 nm was measured for each sample using a microplate reader.The hemolysis rate was then calculated using the following equation: where OD test is the OD value of the Co 3 O 4 NFs group, OD neg is the OD value of the negative control group and OD pos is the OD value of the positive control group.

Clotting time assays
Fresh rabbit blood was collected with anticoagulant sodium citrate and centrifuged at 2000 r/min for 10 min to obtain platelet-Regenerative Biomaterials, 2024, Vol.11, rbae006 | 3 poor plasma.The coagulation time of Co 3 O 4 NFs was evaluated using PT and APTT kits.

Wound healing in vivo
All animal experiments were approved by the Animal Research Ethics Committee of Wenzhou Medical University (ethical approval No. xmsq2022-0845).Male SD rats (200 g) were acclimatized to room temperature and normal humidity for 1 week before the experiment.General anesthesia was executed for all rats before the experiment, and dorsal skin was shaved and then sterilized with 70% ethanol.Four circular full-thickness skin wounds of approximately 1 cm in diameter were excised from the back of the rats, then PBS with different concentrations (0, 5, 50 mg/ml) of Co 3 O 4 NFs were applied over the wounds.Elizabethan Collar was applied to each rat, and the wound healing progress was observed on Days 0, 7 and 14.

Histological analysis
Rats were executed on Days 7 and 14, respectively.Tissue samples were collected and fixed with 4% w/v paraformaldehyde.Tissue sections were then prepared by paraffin embedding and stained for collagen fibers (Masson), hematoxylin and eosin (H&E).To evaluate the organ toxicity of Co 3 O 4 NFs, heart, liver, spleen, lung and kidney of rats at Day 14 were also collected for H&E staining.The stained sections were observed and images were captured using light microscopy (CKX53, OLYMPUS, Japan).

Statistical analysis
All results are presented as mean ± standard deviation in this study.Statistical significance was assessed by adopting a oneway analysis of variance via SPSS software.All the tests in this study were executed at least three times with more than four duplicate samples.

Characterization of Co 3 O 4 NFs
Co 3 O 4 NFs were fabricated by a simple one-step method.As the SEM image is shown in Figure 2A-i size distribution of NPs is shown in Figure 2B).The HRTEM images showed that the lattice fringes with spacing of 0.467, 0.286 nm correspond to the (111), (220) planes of Co 3 O 4 (Figure 2A-iii).The hydrodynamic size of Co 3 O 4 NFs in PBS was about �1001 nm (Pdi: 0.33, Supplementary Figure S1).Thus, we speculate that the Co 3 O 4 NFs are stacked by a single layer of Co 3 O 4 NPs.The XPS spectrum of Co 2p (Figure 2D) shows that the difference in binding energy between the peaks of Co 2p3/2 and Co 2p1/2 is 14.99 eV.Among them, the peaks at 780.8 eV and 796 eV correspond to Co 2þ , and the peaks at 779.8 eV and 794.7 eV correspond to Co 3þ , confirming the successful synthesis of Co 3 O 4 NFs [22].XRD result also confirmed the product as a pure cubic spinal structure of Co 3 O 4 NFs (JCPDS no.43-1003, Figure 2E) [27,28], which was consistent with the HRTEM result (Figure 2A-iii).
To assess the electrocatalytic activity of Co 3 O 4 NFs as CAT, its ability to catalyze the reduction of H 2 O 2 was examined.The cyclic voltammogram of the Co 3 O 4 NF-modified CC electrode is shown in Supplementary Figure S2.Notably, a reduction peak at −0.25 V is observed, indicating that Co 3 O 4 NFs possess the capability to catalyze the reduction of H 2 O 2 .

CAT-like activity of Co 3 O 4 NFs
The scavenging activity of Co 3 O 4 NFs for H 2 O 2 was found to be concentration-dependent (Figure 3A).The dissolved O 2 was also measured, which also showed a Co 3 O 4 NFs concentrationdependent O 2 generation (Figure 3B).A large number of bubbles were generated during the reaction, which correspond to the previous results (Figure 3C).TMB colorimetry was used to determine the presence of �OH in the supernatant of the reaction solutions.As shown in Figure 3D, there is no ox-TMB produced in all groups at pH 7.4.That is, the products of H 2 O 2 decomposition by Co 3 O 4 NFs are H 2 O and O 2 without the generation of �OH under physiological conditions.Therefore, Co 3 O 4 NFs possess CAT-like activity under physiological conditions.Moreover, H 2 O 2 decomposition by Co 3 O 4 NFs in an acidic environment (pH 4) was also determined, which showed a significant concentration-dependent �OH generation (Supplementary Figure S3), indicating the pHswitchable peroxidase-like activity of Co 3 O 4 NFs [21].
The CAT-like activity of Co 3 O 4 NFs was further investigated using steady-state kinetics, and Michaelis-Menten curves were used to obtain kinetic data by varying the concentration of one substrate while keeping the concentration of the other substrate constant.By fitting the data to the Michaelis-Menten equation to determine the catalytic parameters (Supplementary Table S1): where V is the initial velocity, [S] is the substrate concentration, K m is the Michaelis-Menten constant and V max is the maximum reaction velocity.It was found that the decomposition of H 2 O 2 induced by Co 3 O 4 NFs followed Michaelis-Menten kinetics (Supplementary Figure S4).Where K m represents the affinity of the enzyme for the substrate and a smaller K m value indicates a stronger affinity between the enzyme and the substrate.This means that Co 3 O 4 NFs have a higher affinity for H 2 O 2 than natural peroxidases and require a lower concentration of H 2 O 2 to obtain the maximum reaction rate.The higher catalytic constant k cat value indicates higher enzyme activity [22,23], thus the catalytic activity of Co 3 O 4 NFs is slightly lower than that of natural CAT (Supplementary Table S1).The possible catalytic mechanism is illustrated in Figure 3E.
Sterilization stability is crucial for ensuring the safety and effectiveness of wound dressings, particularly for clinical use and cost efficiency.Like other natural enzymes, natural CAT has poor stability, which limits its application.By comparing the long-term stability and sterilization stability of CAT with Co 3 O 4 NFs, it was found that Co 3 O 4 NFs can withstand sterilization methods commonly used in medicine, such as autoclaving.In contrast, the activity of CAT was greatly reduced after sterilization (Figure 3E).In addition, after 14-day incubation of Co 3 O 4 NFs and CAT under physiological conditions, the activity of Co 3 O 4 NFs was found to be unaffected (Figure 3F); however, the activity of CAT was reduced to �50% of the original.Meanwhile, there were almost no Co ions released during 14-day incubation (Figure 3G).
Additionally, the comparison of catalytic activities between Co 3 O 4 NFs and commercially available Co 3 O 4 NPs is presented in Supplementary Figure S5.Similarly, the commercial Co 3 O 4 NPs exhibited concentration-dependent generation of O 2 (Supplementary Figure S5A).However, it was observed that the catalytic activities of the Co 3 O 4 NPs significantly decreased at lower concentrations (0.5-50 lg/ml), whereas the Co 3 O 4 NFs remained steady (Supplementary Figure S5B).This difference can be attributed to the enhanced surface area and greater number of catalytic sites present in the Co 3 O 4 NFs.

In vitro cell compatibility and ROS-scavenging activity of Co 3 O 4 NFs
The in vitro cell compatibility of Co 3 O 4 NFs was assessed using L929, ECs and SMCs, all of which play crucial roles in the wound healing process.The cell activity of L929 cells was assessed after co-culturing them with different concentrations (0, 0.5, 5, 50, 500, 5000 mg/ml) of Co 3 O 4 NFs for 1 and 3 days (Figure 4A-C).The statistical analysis revealed that Co 3 O 4 NFs exhibited a promotion in the proliferation of L929 cells when administered at concentrations ranging from 0.5 to 500 mg/ml.However, the group treated with 5000 mg/ml of Co 3 O 4 NFs showed slight cytotoxicity, which was consistent with the observations of cell morphologies using fluorescence microscopy.
The in vitro antioxidant performance of Co 3 O 4 NFs, specifically their cellular ROS-scavenging activity and protective effects under oxidative stress conditions, was further investigated.As depicted in Figure 4D, treatment of L929 cells with H 2 O 2 alone resulted in a significant increase in intracellular ROS levels, leading to reduced cell survival.However, Co 3 O 4 NFs demonstrated a remarkable cytoprotective effect against H 2 O 2induced oxidative stress, evidenced by a reduction in intracellular ROS levels, ultimately promoting enhanced cell viability (Figure 4E).These results strongly indicate that Co 3 O 4 NFs possess the ability to effectively scavenge ROS within a short period of time, consequently shielding cells from oxidative damage (Figure 4F).
Besides, the results indicated that Co 3 O 4 NFs demonstrated good cytocompatibility with ECs and SMCs as well, both of which play a crucial role in vascularization [29,30] (Figure 5).Moreover, the in vitro biosafety of Co 3 O 4 NFs with RBCs was assessed through a hemolytic experiment.The hemolysis rates of Co 3 O 4 NFs at various concentrations were found to be below 3% (Supplementary Figure S6).

Co 3 O 4 NFs accelerated coagulation in vitro
To investigate the impact of Co 3 O 4 NFs on the coagulation process, experiments utilizing PT and APTT clotting time kits were performed.The results demonstrated a notable reduction in the clotting time of fresh plasma when Co 3 O 4 NFs were introduced (Supplementary Figure S7).It was speculated that the accelerated blood coagulation was facilitated by enhanced adhesion and activation of fibrinogen on the surface of Co 3 O 4 NFs.

Co 3 O 4 NFs promoted wound healing in vivo
The effectiveness of Co 3 O 4 NFs in promoting wound healing was assessed in vivo utilizing a full-thickness skin excision SD rat model (Figure 6A).Different concentrations of Co 3 O 4 NFs suspensions were applied to the skin wounds, with PBS serving as the control group.In Figure 6B and C, it can be observed that Co 3 O 4 NFs considerably enhanced the rate of wound healing compared to the PBS treatment.Both 5 and 50 lg/ml of Co 3 O 4 NFs resulted in almost complete closure by Day 14 after treatment (91.7 ± 7.6% and 94.0 ± 5.6%, respectively), whereas the PBS group only achieved 80.7 ± 4.5% closure, indicating a significantly slower healing process.
To evaluate the tissue formation of the healed wounds on Day 14, H&E and Masson staining analyses were performed (Figure 6D).The black double-headed arrow represents the wound edge between the newly formed tissue and the original tissue (Figure 6D).The PBS group has two to three times larger wound edge compared to groups treated with Co 3 O 4 NFs, which is consistent with the previous observation (Figure 6B and C).Specially, all groups exhibited the basic structures of the epidermis and dermis, along with ample capillaries (yellow arrows) and fibroblasts (green arrows) (Figure 6D).Notably, the PBS group displayed less collagen tissue (blue, indicated by Masson staining, Figure 6D) and lacked mature epithelial structures (blue arrows), whereas the Co 3 O 4 NFs treated groups displayed abundant collagen tissue.Additionally, papillary epithelial structures (blue arrows) were observed in the Co 3 O 4 NF-treated groups, indicating the completion of the re-epithelialization stage and the resemblance of the wound tissue to normal tissue.However, hair follicles (black arrows) were found mainly in the group treated with 50 mg/ml Co 3 O 4 NFs.Furthermore, examination of the organs of both normal and wound model mice revealed that Co 3 O 4 NFs were not toxic or accumulated in the organ (Supplementary Figure S8).These findings demonstrate that 50 mg/ml Co 3 O 4 NFs are biocompatible and facilitate the

Conclusion
The Co 3 O 4 NFs with enhanced CAT-like catalytic activity were synthesized through a simple one-step process.Under physiological conditions, these nanozymes effectively decompose H 2 O 2 into water and O 2 without generating harmful �OH.In vitro results indicated that Co 3 O 4 NFs exhibit excellent cytocompatibility, promoting fibroblast growth and protecting cells from oxidative stress.Moreover, they are shown to accelerate wound healing by facilitating both the hemostatic and proliferative phases in a rat model.In addition, we noticed that they also process a pH-switchable peroxidase-like activity, which may benefit future exploration in anti-cancer or anti-microbial applications in acidic environment.In addition, future studies should also aim to investigate the following aspects: this study, we have synthesized Co 3 O 4 NPs-stacked Co 3 O 4 nanoflakes (NFs) with CAT-like activity through a facile onestep synthesis.The closely arranged Co 3 O 4 NPs in the NFs structure provide a large surface area, enhancing the enzymatic activity compared to commercially available Co 3 O 4 NPs.Under physiological conditions, Co 3 O 4 NFs exhibit excellent CAT-like activity and is able to decompose H 2 O 2 into H 2 O and O 2 without generation of harmful �OH.Moreover, Co 3 O 4 NFs demonstrate excellent cytocompatibility with various cells involved in wound healing, significantly promoting fibroblast growth in vitro, and protecting cells from oxidative stress damage in the presence of an oxidative stress environment.Finally, we demonstrate the potential of Co 3 O 4 NFs to accelerate the wound healing process by facilitating both the hemostatic and proliferative phases using a rat model.

Figure 1 .
Figure 1.Schematic illustration of the application of Co 3 O 4 NFs with CAT-like activity and excellent cytocompatibility for promoting wound healing.

Figure 2 .
Figure 2. Characterization of Co 3 O 4 NFs.SEM (A-i) and HRTEM (A-ii to A-iv) images of Co 3 O 4 NFs; (B) size statistics; (C) schematic of the stacked Co 3 O 4 NPs to Co 3 O 4 NFs; XPS analysis (D) and XRD spectrum (E) of Co 3 O 4 NFs.

Figure 3 .
Figure 3. CAT-like ROS-scavenging activities and stability of Co 3 O 4 NFs.(A) Clearance of 10 mM H 2 O 2 by Co 3 O 4 NFs; (B) the generated dissolved oxygen after Co 3 O 4 NFs reacted with 100 mM H 2 O 2 ; (C) the observed O 2 bubble generation; (D) determination of the H 2 O 2 decomposition products �OH at pH 7.4; (E) schematic diagram of the catalytic mechanism; H 2 O 2 scavenging activity of Co 3 O 4 NFs and CAT after autoclaving (F) and 14 days of incubation (G); (H) Co ion release during 14-d incubation.�� p < 0.01, ���� p < 0.0001, n.s: no significance.

Figure 4 .
Figure 4. Co 3 O 4 NFs Promote L929 cell proliferation and reduce cellular oxidative stress.(A) Florescent microscopy of L929 cells co-cultured with different concentrations (0, 0.5, 5, 50, 500 and 5000 mg/ml) of Co 3 O 4 NFs for 1 and 3 days; (B, C) cell viability of the co-cultured L929 cells on Day 1 or Day 3, respectively; (D) expression of intracellular ROS (DCFH-DA) and live-dead state (AO/EB) of L929 cells co-cultured with 50 mg/ml Co 3 O 4 NFs and 800 mM H 2 O 2 for 6 h (live cells were represented by green, dead cells by red, nuclei in blue and intracellular ROS in blue-green); (E) statistical analysis of the number of live cells after incubation; (F) schematic illustration of the ROS-scavenging mechanism of Co 3 O 4 NFs.��� p < 0.001, ���� p < 0.0001.
(i) Enhancing the CAT-like and peroxidase-like activity of Co 3 O 4 NFs through materials design strategies, such as introducing heterostructures.(ii) Systematically evaluating the potential applications of Co 3 O 4 NFs in the biomedical field by comparing