Sprayed PAA-CaO₂ nanoparticles combined with calcium ions and reactive oxygen species for antibacterial and wound healing

Abstract

The most common socioeconomic healthcare issues in clinical are burns, surgical incisions and other skin injuries. Skin lesion healing can be achieved with nanomedicines and other drug application techniques. This study developed a nano-spray based on cross-linked amorphous calcium peroxide (CaO₂) nanoparticles of polyacrylic acid (PAA) for treating skin wounds (PAA-CaO₂ nanoparticles). CaO₂ serves as a ‘drug’ precursor, steadily and continuously releasing calcium ions (Ca²⁺) and hydrogen peroxide (H₂O₂) under mildly acidic conditions, while PAA-CaO₂ nanoparticles exhibited good spray behavior in aqueous form. Tests demonstrated that PAA-CaO₂ nanoparticles exhibited low cytotoxicity and allowed L929 cells proliferation and migration in vitro. The effectiveness of PAA-CaO₂ nanoparticles in promoting wound healing and inhibiting bacterial growth in vivo was assessed in SD rats using full-thickness skin defect and Staphylococcus aureus (S.aureus)-infected wound models based thereon. The results revealed that PAA-CaO₂ nanoparticles demonstrated significant advantages in both aspects. Notably, the infected rats’ skin defects healed in 12 days. The benefits are linked to the functional role of Ca²⁺ coalesces with H₂O₂ as known antibacterial and healing-promoted agents. Therefore, we developed nanoscale PAA-CaO₂ sprays to prevent bacterial development and heal skin lesions.

Graphical abstract

Introduction

Skin is the most significant physical barrier against external pathogens. Nevertheless, skin damage can result from surgical removal of tumors, diabetic ulcers and unintentional wounds. If skin injuries are left untreated, microbial colonization may grow, which could lead to disability or even death [1, 2]. The intrinsic stages of wound repair involve hemostasis, inflammation, proliferation and remodeling [3]. Delay in recovery raises severe concerns about the necessity for additional therapeutic interventions. The patients must take antibiotics to prevent infection until their skin regains its barrier function. Therefore, there is always a risk of infection during the different stages of wound-healing therapy [4]. Furthermore, implementing autologous or allogeneic transplantation is constrained by the limited supply of donors and the high cost, even in clinically prevalent flap transplant therapy. An alternate method for stimulating skin repair at the defect sites is wound dressings. To promote skin healing, fibrous membranes and hydrogel-style wound dressings have been extensively researched [5, 6]. Nevertheless, fiber pads are easily separated from wounds, which could significantly lengthen the time before full ingrowth. Soft hydrogels can fill irregular wounds, yet it impedes gas exchange at the wound site. Therefore, new wound dressing compositions are urgently needed to address the shortcomings.

Recently, Zhang et al. reported the synthesis of SH-CaO₂ nanoparticles demonstrating antitumor therapy. They showed that the SH-CaO₂ nanoparticles decomposed into Ca²⁺ and H₂O₂ under weakly acidic conditions (pH 6.5), appropriate for the tumor microenvironment [7]. H₂O₂, a reactive oxygen species (ROS) family member, would lead to an effective antitumor activity synergistic calcium overload effect. The process of repairing skin injury involves numerous cells and molecular ions. Among them, Ca²⁺ is involved in several signaling cascades as a critical secondary messenger regulating wound healing [8]. The mechanism illustrates that Ca²⁺ can modulate inflammatory cell infiltration, fibroblast proliferation, migration and differentiation, simultaneously enhancing angiogenesis. Moreover, Ca²⁺ acts as a stationary part in maintaining physiological hemostasis. Ca²⁺-incorporated nanofiber films have been employed for wound dressing, demonstrating improved cell proliferation, antibacterial activity and subsequent wound healing [9–11].

When skin damage occurs, H₂O₂ is elevated in the surrounding tissues and accumulates before it gradually declines. The change in H₂O₂ dynamic levels accompanies wound healing, as the concentration of H₂O₂ in the wounds affects trauma recovery to a certain degree. The signified H₂O₂ roles function as a signaling molecule or secondary messenger like Ca²⁺, transmitting stress messages and stimulating effector cells to respond [12, 13]. On the other hand, H₂O₂ acts as an antimicrobial agent and has been widely used for controlling bacterial reproduction [14, 15].

Inspired by these innovative studies, we considered whether nanoparticles with internal CaO₂ cores could enhance cell proliferation and inhibit bacteria growth. However, solitary CaO₂ nanoparticles have the possibility of aggregation [16]. To prevent CaO₂ nanoparticles from congregating and control their particle size or morphology [17], we introduced PAA as a surface modifier to synthesize amorphous CaO₂ nanoparticles (PAA-CaO₂ nanoparticles) that can be applied in curing full-thickness skin defects.

It is reported that the pH of wound sites gradually shifts to an acidic level during the wound-healing process. Local acidification is induced by lactic or acetic acid production by colonized bacteria strains [18]. PAA-CaO₂ nanoparticles will be decomposed into Ca²⁺ and H₂O₂ under a pathological acidic (pH 6.5) wound microenvironment steadily and continuously, which is analogous to a previous report [19]. The released Ca²⁺ and H₂O₂ are potent molecules for impeding bacterial survival. They significantly heightened the levels of calcium, PI3K/AKT and MEK1/2/ERK1/2 signaling pathway-related molecules such as PLC-δ4, PI3K and ERK1/2. These effects not only facilitate antibacterial endeavors but also foster angiogenesis, cell proliferation and migration [20]. Benefiting from the multiple functions, PAA-CaO₂ nanoparticles realize the therapeutic effects on skin wounds.

As shown in Fig. 1, PAA-CaO₂ nanoparticles decomposed into Ca²⁺ and H₂O₂ in the wound acidic surroundings after spraying on the full-thickness skin defect rats. Ca²⁺ coalesces with H₂O₂, effectively maintaining anti-infection wound status and regulating wound healing. PAA-CaO₂ nanoparticles promote fibroblast proliferation and accelerate wound regeneration by contributing to the antimicrobial and appropriate inflammatory response. PAA-CaO₂ nanoparticles were prepared to validate this concept, and their acidic-triggered decomposition was confirmed in vitro. After that, the in vitro antibacterial effects of PAA-CaO₂ nanoparticles were evaluated by employing two typical bacterial strains commonly parasitized in skin wounds. The L929 cells were used for in vitro cell proliferation and migration investigation. Wound-healing efficacy of PAA-CaO₂ nanoparticles was assessed in SD rats using a full-thickness skin defect model (general wounds) in vivo. Further application in the S.aureus-infected wounds (infected wounds) was studied. Overall, this research aimed to provide evidence for the wound-healing catalytic properties of PAA-CaO₂ nanoparticles in different wound-healing sprays.

Figure 1.

Schematic illustration of PAA-CaO₂ nanoparticles for accelerating wound healing.

Experiment section

Synthesis of PAA-CaO₂ nanoparticles

PAA-CaO₂ nanoparticles were fabricated according to the previous report [11]. Briefly, CaCl₂ (100 μl, 1 M) and PAA (100 μl, 0.5 mg/ml) were mixed in the 10 ml absolute ethanol solution under magnetic stirring at room temperature. After that, the above solution was added in NH₃·H₂O (300 μl, 5%) and H₂O₂ (10 μl, 30%) sequentially. After stirring for 10 min, the formed nanoparticles were collected by ultrafiltration, centrifugation and washed with methanol at least three times. The obtained PAA-CaO₂ nanoparticles were then dispersed in ultrapure water to configure as 1 mg/ml nano-spray for study. To obtain unmodified CaO₂ nanoparticles, the PAA solution was replaced by deionized water in the above procedure.

Characterization

The morphology of PAA-CaO₂ nanoparticles was observed using scanning electron microscopy (SEM, SU8010, Hitachi, Ltd, Japan) after freeze-drying, and SEM determined the EDS mapping images with a working voltage of 3 kV. The average size and zeta potential of PAA-CaO₂ nanoparticles were measured with a Zetasizer-ZS90 (Malvern Instruments, Malvern, UK). Thermogravimetric analysis (TGA) was performed using a thermal gravimetric analyzer (TASDT 650, TA, USA) and the measurements were tested using the standard mode. Fourier transform infrared spectroscopy (FTIR) (Thermo Fisher Scientific-CN, USA) spectra were analyzed within the range of 4000–400/cm. Qualitative analysis of the surface elements of PAA-CaO₂ nanoparticles was given by X-ray photoelectron spectroscopy (XPS) PHI5702, Al Kα (1486.7 eV) with monochromatic. The binding energy was calibrated using the C 1 s standard peak of 284.6 S-4eV as a reference [15, 21].

Sprayability analysis of PAA-CaO₂ nanoparticles

Sprayed PAA-CaO₂ nanoparticles solution at 1 mg/ml concentration mixed with a red dye was transferred into a commercial plastic spray bottle, and the sprayability of PAA-CaO₂ nanoparticles was then tested at room temperature [22].

Measurement of the CaO₂ loading capacity in PAA-CaO₂ nanoparticles

The amount of CaO₂ (M_W ≈ 72 g/mol) incorporated in PAA-CaO₂ nanoparticles was determined by using an inductively coupled plasma source mass spectrometer (ICP-MS, Agilent7900, USA) to detect the concentration of Ca²⁺ in the Supernatant after the encapsulation. Firstly, 100 μl of 1M CaCl₂ (C₀) was added to 10 ml absolute ethanol to synthesize PAA-CaO₂ nanoparticles, and the total synthetic system volume was calculated (V). After synthesizing PAA-CaO₂ nanoparticles, the precipitate was obtained by centrifugation and weighed (m_{PAA-CaO2 nanoparticles}) after freeze-drying. Then all the supernatants were collected, and a certain volume of hydrochloric acid (2 M) was added. The concentration of Ca²⁺ in the supernatant (C_s) was measured by ICP-MS. The CaO₂ loading capacity was calculated based on the following formula: CaO₂ loading capacity (%, w/w) = the mass of CaO₂ loaded in PAA-CaO₂ nanoparticles/the mass of PAA-CaO₂ nanoparticles × 100% = [(C₀ − C_s) × V × 72]/m_{PAA-CaO2 nanoparticles} × 100% [23].

Acid-induced Ca²⁺ release profiles

The generation of Ca²⁺ after the decomposition of CaO₂ nanoparticles and PAA-CaO₂ nanoparticles was detected by ICP-MS through the bag filter method. Dialysis bags loaded with 2 ml of 1 mg/ml CaO₂ nanoparticles or PAA-CaO₂ nanoparticles were immersed in 30 ml PBS (calcium-free solution) with different pH values of 6.5 and 7.4 to mimic the environment of chronic wound (pH 6.5) and normal tissues/blood (pH 7.4), respectively [24]. All the samples were oscillated at 100 rpm in a thermostat at 37°C. At varied time points, a 4-ml buffer sample was taken to examine the Ca²⁺ concentration and replaced with an equal volume of fresh medium. The percentage of released Ca²⁺ was then calculated [7].

Blood clotting test

A blood clotting test was used to evaluate the effect of PAA-CaO₂ nanoparticles on blood clotting. Briefly, 0.5 ml of 1 mg/ml PAA-CaO₂ nanoparticles suspension in PBS at pH 7.4 and 6.5 and isometric anticoagulated rabbit whole blood were mixed in a citrate anticoagulated tube, respectively. Each sample was time-recorded and photographed [25].

Hemocompatibility of PAA-CaO₂ nanoparticles

The hemocompatibility of PAA-CaO₂ nanoparticles was investigated by a hemolysis test. Fresh rabbit blood was centrifuged at 1500 rpm to isolate RBCs. Purified RBCs were diluted with PBS to obtain RBC suspension (2%, v/v). One milliliter RBCs suspension mixed with 20 µl PBS as a negative control, 20 µl 0.1% Triton X-100 as a positive control and 20 µl different materials as experimental groups. After being incubated at 37°C for 1 h, the mixture in tubes was centrifuged at 1500 rpm for 15 min. A microplate reader was used to measure the absorbance of the supernatant. OD_t, OD_n and OD_p are the absorbance values of the experimental groups, negative control (PBS) and positive control (1% Triton-X), respectively. HR(%) = [(OD_t − OD_n)/(OD_p − OD_n)] × 100% [15, 26].

In vitro anti-infection properties of PAA-CaO₂ nanoparticles

Gram-positive (S.aureus) and Gram-negative (Escherichia coli (E.coli)) bacteria were used in bacterial experiments. A modified disc diffusion test (K-B) method was used for the antibacterial activity of normal saline (control), CaO₂ and PAA-CaO₂ nanoparticles. The bacterial solution was prepared by inoculating S.aureus and E.coli in Luria–Bertani (LB) liquid medium and oscillating at 37°C (100 rpm) overnight. It was inoculated on agar plates and divided into three groups. Place 6 mm filter paper discs wholly immersed in two spray solutions and normal saline in the center of the plate. After incubation at 37°C for 24 h, the area of the antibacterial ring was measured to evaluate the antibacterial ability [19, 27].

To obtain SEM images of bacteria, bacteria (1 × 10⁹ CFU/ml) were harvested via centrifugation at 12 000 rpm for 3 min, then dispersed to PBS (pH 7.4 and 6.5) and cultured at 37°C for 2 h. After that, bacteria were collected by centrifugation and fixed with 2.5% glutaraldehyde for 4 h at 4°C. The bacteria were rinsed with PBS (pH 7.4) and dehydrated using ethanol. Finally, samples were observed under SEM [15].

In vitro pH-dependent ROS generation

Taking advantage of the fact that PAA-CaO₂ nanoparticles could be decomposed into Ca²⁺ and H₂O₂ in an acidic environment. Since the latter is a type of ROS, which might be attributed to the antibacterial effect of PAA-CaO₂ nanoparticles. Therefore, we examined the generation of ROS in bacteria after treatment with PAA-CaO₂ nanoparticles using DCFH-DA fluorescent probe. Briefly, 1 ml E.coli and S.aureus (10⁸ CFU/ml) were exposed to PBS (pH 7.4 and pH 6.5) after collected by centrifugation, followed by added normal saline (control), CaO₂ and PAA-CaO₂ nanoparticles cultured for 4 h at 37°C with shaking gently. Then, the bacteria were centrifuged at 12 000 rpm for 3 min, and DCFH-DA (10 μM) was sequentially mixed with bacteria solutions and stained in the dark for 30 min. Finally, the bacteria were rinsed with PBS, and samples were photographed under fluorescence microscopy [7].

Cytotoxicity assay

Fibroblast (L929 cell line) was chosen as the model cell in this study because it is closely associated with wound healing [28]. CCK-8 assay was employed to investigate in vitro cytotoxicity of CaO₂ and PAA-CaO₂ nanoparticles. L929 cells were cultured in 96-well plates (5 × 10³ cells/ml) and incubated for 12 h to make the cells adherent. Then, cells of each well were washed with PBS and further incubated in a fresh medium containing 10 μl of different concentration series of spray solutions and normal saline as the control for 24 h. Subsequently, the medium was discarded, the plates were washed with PBS, the culture medium containing 10% CCK-8 was added to each well, and the cells were incubated at 37°C for 2 h. The optical density was determined at a wavelength of 450 nm with a spectrophotometer (Thermo Fisher Scientific-CN, USA) to assess cytotoxicity and cell viability [19].

Cell proliferation assay

Actin staining was carried out to perform the cell proliferation assay. After seeding L929 cells in six-well plates for 24 h, the original culture medium was replaced with a fresh medium containing normal saline (control), CaO₂ and PAA-CaO₂ nanoparticles for another 24 h. Then the medium was discarded, and the plates were washed with PBS. Three hundred microliters of 4% formaldehyde solution were added to each well to fixed cells for 15 min. Next, cells were washed using a shaker (Kylin-Bell Lab Instruments Co., Ltd, 5 min, 50 rpm). Three hundred microliters of 0.2% Triton X-100 solution were added to each well to permeabilization for 15 min. Then, cells were washed with PBS. After that, 600 μl of 3% BSA was added to each well for 1 h. Then 300 μl of diluted FITC-Phalloidin was added and incubated in the dark for 15 min to visualize the cytoskeleton. Finally, 300 μl DAPI staining solution was added to counterstain for 30 s to stain the cell nuclei. The images were observed under a fluorescence microscope (Nikon A1+R-980 confocal microscope).

Cell migration assay

L929 cells were seeded in 6-well plates (5 × 10⁵ cells/ml) and incubated at 37°C for the cell migration assay. After the confluent monolayers were formed in each well, a sterile pipet tip (200 μl) made a straight scratch. Then the cells were incubated in a serum-free medium containing CaO₂, PAA-CaO₂ nanoparticles or the same amount of normal saline as the control for 24 h. The scratches were photographed using a digital camera-equipped inverted microscope for statistical analysis. Image J software analyzed the cell migration rate.

Intracellular ROS and Ca²⁺ detection assays

DCFH-DA and Fluo-4, AM were employed as fluorescent ROS and Ca²⁺ probes to indicate CaO₂ and PAA-CaO₂ nanoparticles induced ROS and Ca²⁺ generation. Briefly, L929 cells were exposed to normal saline (control), CaO₂ and PAA-CaO₂ nanoparticles for 4 h, respectively. Then, the cells were stained with DCFH-DA and Fluo-4, AM.

For DCFH-DA staining: L929 cells were incubated in the 24-well plates for 12 h to adhere. Then 50 μl of the stimulator (CaO₂ or PAA-CaO₂ nanoparticles or normal saline as control) and 150 μl of DAPI solution were added to each well. After 12 h of incubation at 37°C, the original medium was discarded, and the cells were washed using PBS on a shaker. Next, 300 μl of DCFH-DA (10 mM) was added to each well and co-cultured at 37°C for 40 min. Finally, the probe was discarded, and the cells were incubated in DMEM for 20 min at 37°C to fully hydrolyze the probe and washed for further confocal laser scanning microscopy (CLSM) observation and analysis.

For Fluo-4, AM staining: L929 cells were pretreated before staining following the same procedure as above. Then the medium was removed, and the Fluo-4, AM solution was added to each well. Next, the cells were washed to adequately remove residual Fluo-4, AM stain. PBS was added to cover the cells to ensure complete de-esterification of the AM group in the cells. The cells were examined by fluorescence microscopy [7].

General wound healing assay

The experimental scheme and operation have been approved by the Ethics Committee of Chongqing Medical University, and the animals in the experiment are under human care. Adult male SD rats (6–8 weeks) were used as experimental animals. The dorsal surface hairs were shaved after anesthesia with an isoflurane volatile anesthesia machine (RWD Life Science, Shenzhen, China). A circle wound (D = 1 cm) was removed from the back of the skin by a circular perforator. Wounds in all groups were disinfected with iodophor and alcohol. The rats were randomly divided into four groups. All groups were disinfected every 2 days, then the normal saline treated-group as control, the CaO₂ and PAA-CaO₂ nanoparticles group was treated with corresponding sprays, and the SS was treated with sulfadiazine silver cream (SS). The wound areas were photographed every 7 days and calculated by Image J software.

Transcriptomic analysis and western blot assay

Skin wound samples from SD rats on Day 7 were used for RNA sequence (RNA-seq) and western blotting (WB), because the wound healing processes were in the proliferative phase and neovascularization was evident on Day 7 [3].

For transcriptomic analysis: the wounds were treated with normal saline or PAA-CaO₂ nanoparticles for 7 days. Subsequently, differentially expressed genes (DEGs) were determined by DESeq2. DEGs were used for heatmap analysis using bioinformatics (https://www.bioinformatics.com.cn/). Gene set enrichment analysis, including Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG), was conducted using DEGS with clusterProfiler. GO analysis was visualized by the GO plot.

For western blot assay: after grinding the wound tissue at low temperature, it was further treated at 4°C with a glass homogenizer. The supernatant was collected by centrifugation at 700×g (ICEN-24R) for 10 min at a temperature of 4°C and centrifuged again at 14000×g for 30 min to precipitate cell membrane debris. Finally, cytoplasmic proteins were collected. A bicinchoninic acid assay reagent kit was used to determine protein concentration. The expression level of the PLC-δ4, PI3K, AKT, MEK1/2, ERK1/2 and VEGF of wound skin tissue after incubation with different formulas were detected by WB to study antibacterial and wound healing mechanisms. Finally, the signal density of the bands was visualized via the MiniChemi Mini Size Chemiluminescent Imaging System (Beijing Sage Creation Science Co., Ltd) and quantified by ImageJ software.

Staphylococcus aureus-infected wound healing assay

A full-thickness skin defect model of infection was established to evaluate the antibacterial ability of PAA-CaO₂ nanoparticles further. Because CaO₂ nanoparticles spray had no superiority in the healing effect of the general wound since the rats were only randomly divided into three groups in this test: control (normal saline), SS and PAA-CaO₂ to compare the therapeutic efficacy of PAA-CaO₂ nanoparticles and commercial SS cream on S.aureus-infected wounds. SD rats (6–8 weeks) were used to establish an injury model. The back of the mouse was incised, and 100 µl of 1 × 10⁶ CFU/ml S.aureus suspension was injected. During the treatment, bacteria were counted in exudate using the plate dilution method in the first 3 days. Briefly, 12 h after the infection models were established, three rats were randomly selected from each group. After the swab was used to wipe the infected wound exudate, it was immersed in 3 ml of normal saline for 12 h. The solution was coated on agar plates by spread plate method and then incubated at 37°C for 24 h. Dilute the bacteria 10⁴ times before plating for the 12 h-infected. Day 1 and 3 bacteria were diluted 10² times before plating. The bacterial count was then performed by Image J software [29]. Materials were given once a day on the wound in corresponding groups. The wound areas were photographed every 4 days and calculated by Image J software.

Histological analysis

The rats in the general wound group were sacrificed on 3, 7, 14 and 21 days, and the rats in the infected wound group were sacrificed on 3, 7 and 12 days, respectively. The wound skin was taken for tissue staining to observe wound healing. The rats’ organs (heart, liver, spleen, lung, kidney, brain, testis) were obtained for staining to study the pilot toxicity of PAA-CaO₂ nanoparticles. After fixing the tissue in 4% paraformaldehyde for 3 days, the tissue was embedded in paraffin by dehydration. After the embedded tissue was sectioned into 2 μm sections with a micro-chipper and tiled on adhesive glass slides, the slices were incubated overnight at 50°C. Hematoxylin and eosin (H&E) staining of skin tissues was performed to observe the size of the wound bed, and Masson’s trichrome staining was performed to observe collagen deposition. H&E stained the organs sections images were observed and quantified using a confocal fluorescence microscope and Image J software.

Immunofluorescence (IF) staining of CD68, CD31 and proliferating cell nuclear antigen (PCNA) in general wounds was also studied. Tissue sections were treated with 1× EDTA antigen repair solution for antigen extraction, and endogenous HRP enzyme was blocked with 3% H₂O₂. After blocking with 10% normal goat serum, primary antibodies CD68, PCNA, CD31 and secondary antibodies HRP labeled sheep anti-rabbit was added for incubation, respectively. The multiple fluorescence immunohistochemistry kit was used for chromogenic staining. Finally, the images were taken under a fluorescence microscope.

The details regarding reagents used are provided in the Supplementary material.

Results and discussion

Preparation and characterization of PAA-CaO₂ nanoparticles

Based on a previous study, PAA-CaO₂ nanoparticles were created with a loading capacity of 44.25 ± 1% (w/w) of CaO₂ in the nanoparticles. The SEM images showed that many ultra-small black dots represented PAA-CaO₂ nanoparticles. Moreover, these nanoparticles were dispersive and surrounded by irregular nebulosity owing to PAA coating (Fig. 2A). The XPS high-resolution Ca 2p spectrum displayed two prominent peaks and one peak in the O1s spectrum, indicating the valence state of Ca was +2 and O was −1 (Peroxide ions, O–O) in PAA-CaO₂ nanoparticles. Furthermore, the XPS full spectrum verified that O 1 s peaks at 532.5 eV were assigned to O–O, indicating the presence of peroxo groups (Supplementary Fig. S1) [30, 31]. Attributed to the PAA coating and small size, PAA-CaO₂ nanoparticles could be easily dispersed in water and sprayed out from the spray nozzle (Fig. 2B; Supplementary Fig. S2). It would be convenient for the patients to apply PAA-CaO₂ nanoparticles for wound therapy. In Supplementary Fig. S3, the zeta potential of CaO₂ and PAA-CaO₂ nanoparticles was depicted. The initial CaO₂ nanoparticles displayed a zeta potential of approximately +0.24 ± 0.10 mV, while this value decreased to −6.89 ± 1.32 mV for PAA-CaO₂ nanoparticles. This confirmed polyanionic electrolytes-PAA modified CaO₂ nanoparticles by utilizing the attraction between negatively charged ions and positively charged nanocrystals [32]. Therefore, the PAA coating endowed the CaO₂ nanoparticles with an electronegative property, which can effectively facilitate nucleation, prevent aggregation and enhance the biocompatibility of CaO₂ nanoparticles [33]. Moreover, the successful modification was also indicated by the greater mass loss in TGA (Supplementary Fig. S4) [34]. The hydrodynamic diameter of PAA-CaO₂ nanoparticles was ∼106 nm (Fig. 2C), consistent with the one displayed in the SEM images. The 3600–3200/cm broad absorption peak represented the hydrogen-bonded O–H stretching vibration in the FTIR absorption spectrum. It could be attributed to the hydrogen bonding networks in PAA-CaO₂ nanoparticles (Supplementary Fig. S5). Moreover, the existence of peroxo groups was verified by the presence of characteristic peaks at 831, 881 and 1115/cm [35]. Ca and O elements are distributed at the surface of PAA-CaO₂ nanoparticles is also verified by characterization using Energy dispersive spectrometer (EDS) (Fig. 2E).

Figure 2.

(A) The SEM images of PAA-CaO₂ nanoparticles. (B) Re-dispersion of PAA-CaO₂ nanoparticles in ultrapure water after lyophilization and sprayed out from a spray nozzle. (C) The hydrodynamic diameter of PAA-CaO₂ nanoparticles was measured by dynamic light scattering. (D) Time-dependent Ca²⁺ release from CaO₂ and PAA-CaO₂ nanoparticles suspension at different pH values. (E) The EDS spectrum and element mapping of PAA-CaO₂ nanoparticles.

In vitro Ca²⁺ release and blood coagulation

We studied the breakdown of PAA-CaO₂ nanoparticles in acidic conditions by measuring the amount of Ca²⁺ released in vitro. Our findings indicated that PAA-CaO₂ nanoparticles degrade at a pH of 6.5 (Fig. 2D). The results revealed that these nanoparticles are sensitive to changes in pH, as the release of Ca²⁺ increased when the pH decreased from 7.4 to 6.5. The increased release of Ca²⁺ in acidic surroundings was attributed to the degradation of CaO₂ and the reduction of electrostatic interactions between PAA and CaO₂, with less deprotonation of the carboxyl group (–COOH) of PAA as the pH decreased [17]. Interestingly, PAA-CaO₂ nanoparticles exhibited a higher percentage of Ca²⁺ release than CaO₂ nanoparticles under the same conditions. This could be attributed to the PAA modification effectively preventing the aggregation of CaO₂ nanoparticles, making the PAA-CaO₂ nanoparticles more solubility and allowing for a larger surface area in contact with the buffer [32].

Ca²⁺ can cause blood coagulation by catalyzing the conversion of prothrombin to thrombin and is influenced by the concentration of Ca²⁺ [36]. PAA-CaO₂ nanoparticles release Ca²⁺ that may induce blood coagulation, based on the in vitro Ca²⁺ release experiment [25]. The blood clotting effect in the presence of CaO₂, PAA-CaO₂ nanoparticles and normal saline was investigated at the above pH values in vitro (Supplementary Fig. S6). For normal saline, no blood clotting was observed at all pH values. Nevertheless, PAA-CaO₂ and CaO₂ nanoparticles induced blood clotting at pH 6.5 with a blotting time of 1 and 18 min, respectively. The results showed that the PAA-CaO₂ nanoparticles compared to CaO₂ nanoparticles cause faster blood clotting. Similarly, with increased acidity, PAA-CaO₂ nanoparticles evoked more rapid blood clotting at pH 6.5 compared to pH 7.4. Simultaneously, biocompatibility tests confirmed that PAA-CaO₂ nanoparticles were blood compatible at physiological conditions (Supplementary Fig. S7). This unique property of pH-sensitively inducing blood coagulation endows PAA-CaO₂ nanoparticles to be a potential material for adequate hemostasis in wound therapy [8], especially in acidic environments commonly found in pathological wounds.

In vitro antibacterial activity

Bacteria can invade the body from the skin when a wound occurs, likewise in other traumas or surgeries. Invading bacteria and other microorganisms into wounds is the leading cause of severe infections [37]. Antibacterial tests were performed with PAA-CaO₂ nanoparticles to evaluate their antibacterial potential in two representative bacterial strain models (E.coli and S.aureus) coinciding with a previous study [19, 38]. The results are shown in Fig. 3A; PAA-CaO₂ nanoparticles showed the largest area of antibacterial rings for E.coli and S.aureus bacterial strains. We further found that PAA-CaO₂ nanoparticles showed higher antibacterial activity against Gram-positive bacteria (S.aureus) than Gram-negative bacteria (E.coli). In the Gram-negative bacterial group, the inhibition zone of PAA-CaO₂ nanoparticles exhibited a moderate-size ring. In contrast, the inhibition zone of the PAA-CaO₂ nanoparticles treatment group in Gram-positive bacteria was as high as 8.97 ± 0.26 cm², significantly higher than the normal saline group (control). It can be considered that metal peroxide nanoparticles are more virulent against Gram-positive bacteria than Gram-negative bacteria [39]. We speculated that there are different structures between Gram-positive and Gram-negative bacteria. The mechanism of antibacterial activity of metal and metal peroxide nanoparticles involves multiple factors, including membrane damage, metal ions and ROS that affect bacteria metabolism. For example, Gram-positive bacteria have a thicker peptidoglycan layer than Gram-negative bacteria. Nonetheless, Gram-negative bacteria have relatively less permeable outer membranes surrounded by lipids and proteins, which may lead to higher barriers to the penetration of nanoparticles and metal ions into bacteria. In addition, the surface of Gram-positive bacteria is more negatively charged, which helps attract positive ions, such as Ca²⁺ [40–42]. The acid produced in the bacteria caused the release of highly toxic ROS from PAA-CaO₂ nanoparticles, resulting in the massive death of bacteria in contact with the nanoparticles. Additionally, it was confirmed in subsequent WB experiments that PAA-CaO₂ nanoparticles have an antibacterial effect by activating the MEK1/2/ERK1/2 pathway to stimulate neutrophil cells to adhere to endothelium. Meanwhile, S.aureus tends to be a common bacterium on the skin in the existing reports, making it the leading cause of general wound infection [43]. This phenomenon stimulated our interest in further analysis of PAA-CaO₂ nanoparticles in S.aureus-infected wounds.

Figure 3.

(A) The Antibacterial ring of S.aureus and E.coli LB agar plates after treatment with corresponding formulas. (B) SEM images of S.aureus and E.coli after treatment with corresponding formulas at different pH values. Bright-field and DCFH-DA fluorescence images of S.aureus (C) and E.coli (D) bacterial strains after being treated with corresponding formulas at different pH values, respectively.

Simultaneously, the effects of PAA-CaO₂ nanoparticles on bacterial morphology at different pH values were investigated. Not surprisingly, the bacterial membrane surfaces in the control group remained intact in acidic and neutral buffer solutions. However, when the bacteria were treated with PAA-CaO₂ nanoparticles, the bacterial membrane was damaged more severely in an acidic solution than in a neutral solution (Fig. 3B). This is related to a large amount of ROS released by PAA-CaO₂ nanoparticles under acid stimulation, oxidizing the bacterial membrane, resulting in the leakage of bacterial contents [44, 45]. Compared with the control group, PAA-CaO₂ nanoparticles exhibited a more robust and stable antibacterial effect in the wound atmosphere.

We used DCFH-DA as a fluorescent probe to visualize ROS production in bacteria [46]. After incubation of two bacteria with PAA-CaO₂ nanoparticles for 2 h, obvious green fluorescence was observed in acidic buffer, but almost no fluorescence was observed under neutral condition (Fig. 3C and D). This could mimic wound microenvironment [47] and protons produced by the bacteria existing in the wound resulting in certain ROS being produced. The results suggested that releasing toxic ROS is acid-dependent and applying in infected wound is beneficial. The reason is clear that the dissociation rate of PAA-CaO₂ nanoparticles is abruptly accelerated in an acidic environment, depending on the protons concentration around the nanoparticles, compared to the slow hydrolysis process that relies on protons generated by water ionization under neutral conditions. The chemical reaction equation can be written as follows: CaO₂ + 2H⁺ = Ca²⁺ + H₂O₂. Meanwhile, the findings revealed that peroxo groups were present in PAA-CaO₂ nanoparticles, which aligns with the XPS results.

In vitro cell experiments

Given the antibacterial properties and controlled release effects of PAA-CaO₂ nanoparticles, we further explored the impact of PAA-CaO₂ nanoparticles on cell proliferation and migration. Fibroblasts proliferate to form contractile granulation tissues, which are crucial to wound healing [48]. Hence, scratch assays were performed on the mouse epithelioid fibroblasts L929 cells to investigate the effects of PAA-CaO₂ nanoparticles on migration (Fig. 4A). Simultaneously, the migration results were quantified in Fig. 4B. The results revealed that PAA-CaO₂ nanoparticle-treated group had the highest ability to accelerate cell migration.

Figure 4.

In vitro migration, proliferation assays and intracellular Ca²⁺ and ROS detection assays of L929 cells. (A) Migration assay and (B) corresponding statistical analysis by ImageJ (****P < 0.0001). (C) Fluorescence characterization of cell proliferation images. Schematic diagram of intracellular Ca²⁺ (D) detection and ROS (E) detection, respectively.

Furthermore, the CCK-8 assay demonstrated the ability of PAA-CaO₂ nanoparticles to promote cell proliferation and was more robust than CaO₂ nanoparticles at an optimum concentration (Supplementary Fig. S8). Next, fluorescent staining was performed on the L929 cell line and verified that PAA-CaO₂ nanoparticles have a salient promoting effect on cell proliferation (Fig. 4C). This phenomenon is due to the released Ca²⁺ and ROS in PAA-CaO₂ nanoparticles described previously. Ca²⁺ and ROS can modulate fibroblast proliferation/migration, enhancing angiogenesis [8, 11]. To confirm that PAA-CaO₂ nanoparticles did produce Ca²⁺ and ROS that could promote cell proliferation and migration at the cellular level. It was examined by fluorescent probes and monitored by CLSM. The control group exhibited weak green luminescence, while the group cultivated with PAA-CaO₂ nanoparticles exhibited strong intracellular luminescence, indicating the release of the exogenous free Ca²⁺ (Fig. 4D) and ROS (Fig. 4E) from PAA-CaO₂ nanoparticles. The experimental cell results collectively provided a basis for in vivo wound healing. It is reasonable to expect that Ca²⁺ and ROS would play regulatory roles, promoting wound healing.

Accelerated wound healing in full-thickness skin defect rats

We studied the therapeutic effects of PAA-CaO₂ nanoparticles for wound treatment in a full-thickness skin defect rat model. Besides, we used SS as a positive control group for comparison with marketed skin repair drugs [19, 49]. As shown in Fig. 5A and B, the wound region in all groups became smaller while the wounds treated with PAA-CaO₂ nanoparticles healed. On Day 7, the wound area was significantly reduced as PAA-CaO₂ nanoparticles inhibited the inflammatory response in the early stage of healing and regulated the initial effective wound healing. On the 14th day, the defected area of the PAA-CaO₂ nanoparticles group decreased more than the other groups, indicating that PAA-CaO₂ nanoparticles still had superior repair performance in the middle stage of wound repair. On the 21st day, the PAA-CaO₂ nanoparticles group exhibited wound healing. At the same time, visible scabs remained in the other groups. The wound healing rate histogram statistically displayed that the wound repair effects of the PAA-CaO₂ nanoparticles group were the best (Fig. 5C). Meanwhile, we have taken different samples from Day 7 to Day 21 for H&E staining. There were notably larger wound beds in the control, SS and CaO₂ groups than in PAA-CaO₂ nanoparticles group on Day 7 and Day 21 (Fig. 5D). The corresponding quantified statistics of wound beds at 7 (Fig. 5E) and 21 days (Fig. 5F) claim that PAA-CaO₂ nanoparticles possessed satisfactory wound repair performance in vivo.

Figure 5.

(A) Overview of the size change of wound status at 0, 7, 14 and 21 days after differently treating with normal saline (control), SS, CaO₂ and PAA-CaO₂ nanoparticles, respectively. Relative area diagram (B) and statistics (C) of the wound. (D) H&E staining of the wound bed at 7 and 21 days. Corresponding statistics of wound bed length in H&E staining at 7 (E) and 21 (F) days. (G) IF images of the regenerated skin tissues labeled with (i) CD68 on Day 3, and (ii) CD31, (iii) PCNA on Day 7 after treatment with different formulas. (H) The collagen deposition on Day 21 (ns: non-significant, *P < 0.05, **P < 0.01).

Reduced inflammatory reaction

Many biological factors, such as inflammation, angiogenesis and collagen deposition, can influence wound healing [3]. To investigate whether PAA-CaO₂ nanoparticles induced wound healing by impacting inflammation, angiogenesis or collagen deposition. IF and Masson’s trichrome staining were conducted in succession. On Day 3 after injury, the wound area revealed an intense inflammatory response. Excessive inflammation is an additional component that might impede wound healing. Nevertheless, an effective immune response to local wounds during the early phases of recovery promotes the regeneration of the vascular network and the elimination of metabolic byproducts of the lesion [50]. We investigated the state of the inflammatory cell’s density in the early phases using the CD68 IF labeling to identify M1 macrophages in a pro-inflammatory state detrimental to wound healing, characterized by the production of several pro-inflammatory mediators [51]. We demonstrated the PAA-CaO₂ nanoparticles group exhibiting the lowest density of adverse immune cells, indicating that the nanoparticles could alleviate the immunoreaction to a certain extent (Fig. 5G, i). It may be attributed to the PAA-CaO₂ nanoparticles’ initial bacterial elimination ability to mediate it, thereby accelerating wound closure.

Promoted angiogenesis and cell proliferation

Angiogenesis is essential as it requires the nutrients to be transported in the blood to the sites of new tissue creation for wound healing [48]. CD31 IF staining of vasculature in a histologic section was used to characterize the new vessels in wounds (Fig. 5G, ii). The total number of microvessels in the PAA-CaO₂ nanoparticles group indicated a considerable increase compared to the other groups, enhancing angiogenesis. In addition, the formation of new structures is linked to the proliferation of diverse cells within the wound. PCNA is a crucial element in embodying the proliferative capacity of cells in vivo. By labeling cells with an anti-PCNA antibody, we analyzed cell proliferation vigor. The wounds treated with PAA-CaO₂ nanoparticles exhibited the greatest strength of fluorescent highlights (Fig. 5G, iii). These results revealed that PAA-CaO₂ nanoparticles showed anti-inflammatory activity, increased cell proliferation and angiogenesis in vivo [52–55].

Enhanced collagen deposition

Collagen fibers trigger extracellular matrix (ECM) remodeling. Several steps in the healing process involved the deposition of collagen [56]. In this study, we examined that PAA-CaO₂ nanoparticles affect collagen deposition by Masson's trichrome staining. The collagen fibers (stained with aquamarine blue) in the PAA-CaO₂ nanoparticles group were more in number and more likely to form organized networks. The other groups displayed disorganized collagen structure and visible scars (Fig. 5H). We explained that excessive inflammatory reaction would enhance the overexpression of matrix metalloproteinases, destroying the ECM component and inhibiting collagen deposition [21]. While PAA-CaO₂ nanoparticles might reduce inflammation and subsequently improve collagen deposition.

Wound-induced transcriptomic analysis upon PAA-CaO₂ nanoparticles treatment

To better understand the antibacterial and wound-healing mechanisms behind the impact of PAA-CaO₂ nanoparticles, we conducted a broad transcriptome analysis of a wound treated with PAA-CaO₂ nanoparticles as an initial step. Our analysis identified 580 differentially expressed genes in the volcano analysis following treatment with PAA-CaO₂ nanoparticles. Among these genes, 307 were upregulated, while 273 were downregulated (Fig. 6A and B). To gain insights into the impact of PAA-CaO₂ nanoparticles on cell proliferation, we performed a GO-Chord analysis on the enriched items related to this effect, which offered a comprehensive understanding of the functional roles and interactions of the enriched genes in these specific biological processes. The enriched genes identified in the GO-Chord and GO-Bubble plots were assigned to several categories, including ‘cell differentiation’, ‘response to cytokine’, ‘cellular response to endogenous stimulus’ and ‘oxygen carrier activity’ (Fig. 6D and E). These findings provide valuable insights into the molecular changes induced by treatment with PAA-CaO₂ nanoparticles, particularly concerning their effects on wound healing.

Figure 6.

Transcriptomic analysis of rats treated with normal saline (control, ctrl) and PAA-CaO₂ nanoparticles (PAA). (A) Volcano plot of RNA-seq data of wound in the two groups calculated by DESeq2 (n = 3 per group). (B) Heat map of DEGs between the two groups (n = 3 per group). (C) Venn analysis to screen hub genes involved in enrichment analysis and up-regulated DEGs. (D) GO bubble plot and (E) GO chord plot of GO enrichment analysis, displaying the relationship between genes and GO terms. (F) The KEGG enrichment pathway diagram of PAA-CaO₂ nanoparticles enhances calcium, PI3K/AKT and MEK1/2/ERK1/2 signals to regulate wound healing processes. PLC-δ4, phospholipase C Delta 4; DAG, diacylglycerol; IP₃, inositol 1, 4, 5-trisphosphate; PKC, protein kinase C; PI3K, phosphoinositide 3-kinase; AKT, protein kinase B; MEK1/2, mitogen-activated protein kinase kinase (MAPKK); ERK1/2, extracellular signal-regulated kinase; VEGF, vascular endothelial growth factor.

Consistently, we performed KEGG enrichment analysis to explore the initial driver of the wound healing effect of PAA-CaO₂ nanoparticles treatment [57]. It found that these differently expressed genes mainly enriched in the ‘calcium signaling pathway’, ‘PI3K/AKT signaling pathway’ and ‘MEK1/2/ERK1/2 signaling pathway’, including PLC-δ4, PI3K, AKT, MEK1/2 and ERK1/2. Notably, the expression level of wound healing-promoting genes such as PLC-δ4 (an early signal in the calcium signaling pathway) [58], PI3K and ERK1/2 were found to be upregulated (Fig. 6C). These findings shed light on the potential mechanism through which PAA-CaO₂ nanoparticles systemically induce wound repair.

Calcium, PI3K/AKT and MEK1/2/ERK1/2 signaling pathways driven PAA-CaO₂ nanoparticles-mediated wound healing

After analyzing the transcriptome and protein–protein interaction (PPI) network analysis of different formulas treated wounds, we have identified the calcium, PI3K/AKT and MEK1/2/ERK1/2 signaling pathways as crucial events in PAA-CaO₂ nanoparticle-mediated wound healing. The PI3K/AKT pathway is vital in numerous cellular functions, such as signal transmission for cell adhesion, growth, proliferation, migration and angiogenesis [59]. While the activation of intracellular signaling pathways, like mitogen-activated protein kinase (MAPK) signaling, further promotes wound healing. MAPK kinase (MEK1/2), a kinase enzyme, phosphorylates MAPK, leading to the activation of MEK1/2 signaling. The MEK1/2/ERK1/2 signaling pathway is the most common one of the MAPK signaling pathways. Recently reported that catalpol promotes angiogenesis via crosstalk of the MEK1/2/ERK1/2 pathway [60]. Moreover, this signaling pathway also drives cell proliferation/migration of fibroblasts/keratinocytes and differentiation of fibroblasts, which are crucial events in wound healing [61].

Our concept involved the release of Ca²⁺ through the cytoplasmic mechanism, which we call ‘External Calcium Triggering’. This activates the calcium signaling pathway. PPI network analysis revealed that PAA-CaO₂ nanoparticles activate the calcium signaling pathway through PLC-δ4. This pathway relies on PLC-δ4 to hydrolyze phosphatidylinositol 4,5-bisphosphate (PIP₂) to generate two second messenger DAG and IP₃. DAG mediates the activation of PKC, which could mediate multiple downstream cellular events, including transcription, proliferation and differentiation [62]. On the other hand, IP₃ releases Ca²⁺ from intracellular stores, leading to Ca²⁺-dependent MEK1/2 signaling [63] and extracellular P-selectin expression, which endows PAA-CaO₂ nanoparticles with antibacterial ability by enhancing neutrophil cells to adhere to the endothelium [64], subsequently neutrophil cells traveling to the source of infection. At the same time, the H₂O₂ (ROS) released from PAA-CaO₂ nanoparticles can stimulate the MEK1/2 pathway to activate ERK1/2 [65]. In addition, an increase in vascular endothelial cells ROS also caused a release of stored Ca²⁺ from the endoplasmic reticulum into the cytosol. Consequently, Ca²⁺ and ROS can activate numerous molecules involved in wound healing.

WB was used to confirm the pathways of PAA-CaO₂ nanoparticles-activated. In the PAA-CaO₂ nanoparticles treated group, the expression of PI3K, AKT (Fig. 7A, E and F), MERK1/2, ERK1/2 (Fig. 7A, G and H) and PLC-δ4 (Fig. 7B and C) were markedly increased compared to the control group. These results suggested that the PI3K/AKT, MEK1/2/ERK1/2 and calcium signaling pathways were activated. Furthermore, we have proved that the critical molecule on angiogenesis-VEGF expression was significantly increased by PAA-CaO₂ nanoparticles (Fig. 7B and D).

Figure 7.

The protein expression level of the control and PAA-CaO₂ nanoparticles treatment groups on Day 7. The PI3K, AKT, MEK1/2, ERK1/2 (A) and the PLC-δ4, VEGF (B) expression of the wound after incubation with different formulas. Quantification of PLC-δ4 (C), VEGF (D), PI3K (E), AKT (F), MEK1/2 (G) and ERK1/2 (H) expression level by ImageJ (**P < 0.01, ***P < 0.001).

Overall, the present study demonstrated that PAA-CaO₂ nanoparticles can induce angiogenesis, exert cell proliferation, migration subsequently accelerate wound healing in vivo by increasing the expression of PLC-δ4, PI3K, AKT, MEK1/2, ERK1/2 and VEGF. Moreover, activating the MEK1/2/ERK1/2 pathway to stimulate neutrophil cells to adhere to the endothelium enhanced the antibacterial ability of PAA-CaO2 nanoparticles. These biological processes were mediated by calcium, MEK1/2/ERK1/2 and PI3K/AKT signaling pathways together.

Application in S.aureus-infected wound healing

We further evaluated the practical applicability of PAA-CaO₂ nanoparticles in treating common clinical bacterial (S.aureus)-infected wounds due to its favorable properties. Due to the severity and possible delays in the recovery of infected wounds, we increased the frequency of dressing changes in each experimental group to accelerate bacterially infected wounds healing effectively. It is more suitable for spraying reagents’ applicability to infected wounds in clinical practice.

In this experiment, the S.aureus-infected rats in different groups were severally treated with normal saline, SS and PAA-CaO₂ nanoparticles. The wounds were observed and photographed on schedule. As shown, edema and inflammation were observed in the wounds on the back of all rats after the S.aureus infection (Day 0). After 4 days of treatment, compared to the control and SS groups, PAA-CaO₂ nanoparticles-treated wound exuded relatively less, no apparent abscess appeared, and the wound area was much smaller. After 12 days of treatment, we observed that the wounds in the PAA-CaO₂ nanoparticles group were almost healed, while the control and SS groups had distinct wounds (Fig. 8A and B). There was a significant difference between PAA-CaO₂ nanoparticles and the other two treatment groups (Fig. 8D).

Figure 8.

(A) Overview of the size change of S.aureus-infected wound status at 0, 4, 8 and 12 days after treating with normal saline (control), SS and PAA-CaO2 nanoparticles differently. Relative area diagram (B) and statistics (D) of the wound. (C) Infected wounds exudate bacterial colony culture coated plate after being treated with different formulas to post S.aureus-inhibition rate: (i) Day 0 (12 h after infection); (ii) Day 1; (iii) Day 3. (E) Quantitative analysis of the exudate bacterial colony in the wound by ImageJ. (F) H&E staining of the wound bed at 7 and 12 days. Corresponding statistics of wound bed length in H&E staining at 7 (G) and 12 (H) days. (I) The collagen deposition on Day 12 (ns: non-significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

Simultaneously, to quantitatively evaluate the bactericidal effects of PAA-CaO₂ nanoparticles, we collected abscess exudate in the first 3 days to quantify the number of bacteria in the wound area (Fig. 8C). From the grown colony, we found that PAA-CaO₂ nanoparticles exhibited the most effective bacterial elimination ability (Fig. 8E). Infected wounds treated with PAA-CaO₂ nanoparticles had conspicuous power in killing bacteria on the second day, with almost no colony growth. The therapeutic effects of the infected wounds in vivo further confirmed that PAA-CaO₂ nanoparticles have higher feasibility and practical applications in the clinical therapy of infected wounds. It also demonstrated that Ca²⁺ and ROS released by PAA-CaO₂ nanoparticles had higher antibacterial activity than silver ions in SS.

Similarly, we have taken different treatment groups samples for H&E staining. In the control and SS groups, there were large wound beds than the PAA-CaO₂ nanoparticles group (Fig. 8F) with corresponding statistics analysis on Day 7 (Fig. 8G) and 12 (Fig. 8H). Simultaneously, more collagen fibers, organized collagen structure and the smallest scar tissues were discovered in the PAA-CaO₂ nanoparticles group (Fig. 8I). Suggesting PAA-CaO₂ nanoparticles possess satisfactory S.aureus-infected wound repair performance in vivo.

Conclusions

This work developed a novel nanocarrier, PAA-CaO₂ nanoparticles, which are biocompatible, pH-responsive, and have an appropriate size for drug administration. Moreover, they are economically available in large quantities through a one-pot method. PAA-CaO₂ nanoparticles could release Ca²⁺ and H₂O₂ (ROS), and both two can mediate calcium, PI3K/AKT and MEK1/2/ERK1/2 signaling pathways together to antibacterial, reinforce angiogenesis, cell proliferation/migration in the wound microenvironment. Experiments were conducted on general and infected wounds in SD rats to evaluate the healing effects of PAA-CaO₂ nanoparticles in vivo. By the 21st postoperative day, the general wound in injured rats nearly recovered due to the PAA-CaO₂ nanoparticles administration. PAA-CaO₂ nanoparticles owed remarkable antibacterial activity in vivo, mainly when applied in S.aureus-infected rats. The results showed nearly no bacterial growth in the PAA-CaO₂ nanoparticles group 2 days after treatment. Besides, compared with other reported materials involving the exploration of antibacterial and wound healing performance, PAA-CaO₂ nanoparticles are relatively sufficient in these sections (Supplementary Table S1). Therefore, we designed PAA-CaO₂ nanoparticles by fully exploiting Ca²⁺’s distinctive wound-healing mediated mechanism, showing that calcium signaling is as crucial as ROS to wound repair. Because of its pH sensitivity, PAA-CaO₂ nanoparticles progressively break into Ca²⁺ and H₂O₂ in the wound area, antibacterial and accelerating tissue repair. Hence, PAA-CaO₂ nanoparticles will provide promising employment for clinical chronic and infected wound therapy.

Supplementary data

Supplementary data are available at Regenerative Biomaterials online.

Funding

This work was supported by grants from the Natural Science Foundation of Chongqing (cstc2020jcyj-msxmX0330, cstc2021jsyj-yzysbA0057); the National Natural Science Foundation of China (31971242, 12032007); the Science and Technology Innovation Project of Jinfeng Laboratory, Chongqing, China(jfkyjf202203001); the Project of Tutorial System of Medical Undergraduate in Lab Teaching & Management Center in Chongqing Medical University (LTMCMTS202107). We are also thankful for the First Batch of Key Disciplines on Public Health in Chongqing and the Public Experiment Center of State Bioindustrial Base (Chongqing), China.

Conflicts of interest statement. There are no actual or potential conflicts of interest.