Synthesis and characterization of PMMA@PFC-73 membrane
The PMMA@PFC-73 membrane was fabricated by dispersing PMMA and PFC-73 in a mixture of ethanol and chlorobenzene. This solution was then carefully dropped onto the surface of water. Upon contact with the water surface, the solution spread out instantly, forming a continuous film at the gas-liquid interface. The flexible PMMA@PFC-73 membrane was then collected using iron wire rings. X-ray diffraction (XRD) tests were performed to verify the crystal structure of the PMMA@PFC-73 membrane. As shown in Fig. 1a, the XRD patterns of PMMA@PFC-73 membrane closely matched those of solvothermal PFC-73 powders and simulated XRD patterns from single crystal data31,32. Distinct diffraction peaks at 2θ = 3.97, 7.96 and 8.59 can be indexed to (020), (040), and (210) crystal planes, respectively. Fourier transform infrared spectroscopy (FTIR) revealed that PMMA@PFC-73 membrane exhibited characteristic infrared absorption peaks of PFC-73 (1680 cm− 1 for the carboxylic group stretching vibration and 1398 cm− 1 for imine bending vibration), and PMMA (2920 cm− 1 for methylene stretching vibration) as shown in Supplementary Fig. 1. The PMMA@PFC-73 membrane was retrieved from the water surface using a 6 mm diameter wire ring, making it an independent, self-standing film (Fig. 1b). The membrane exhibited the same red color as PFC-73 powder and its absorption capacity in the UV–vis spectrum was consistent with that of PFC-73 (Supplementary Fig. 2). Notably, the PMMA@PFC-73 membrane was extremely flexible and foldable, with a folded thickness of 74 µm (Fig. 1b). Atomic force microscopy (AFM) images revealed a smooth surface with an average roughness (Ra) of 19.8 nm (Fig. 1c, Supplementary Fig. 3). To characterize the microstructure, the membrane was transferred onto a copper mesh for transmission electron microscopy (TEM) analysis. High-resolution TEM (HRTEM) images in Fig. 1d showed a clear lattice spacing of 2.21 nm corresponding to the (020) plane of PFC-73, confirming the crystalline phase of the PMMA@PFC-73 membrane. Additionally, element mapping analysis indicated a uniform distribution of C, N, O, and Ni elements in the PMMA@PFC-73 membrane (Fig. 1e, Supplementary Fig. 4). Scanning electron microscopy (SEM) images also confirmed the uniform distribution of PFC-73 particles within the PMMA@PFC-73 membrane (Fig. 1f).
The freestanding and tissue-conformable PMMA@PFC-73 membrane demonstrates excellent tissue adhesiveness, allowing it to seamlessly conform to tissues and complex organs of various shapes and sizes (e.g. kidney, heart, liver and skin), effectively stopping bleeding and leakage (Fig. 1g-h). It leaves no residue upon removal, which accelerates wound healing and provides high tissue comfort (Supplementary Fig. 5). This indicates that the PMMA@PFC-73 membrane has strong operability and can be used in various irregular wound care applications. Additionally, excellent wound dressings require good breathability to improve wound healing33,34. We estimated the water vapor transmittance rate (WVTR) by measuring the weight loss of water when the PMMA@PFC-73 membrane was attached to the opening of a bottle containing pure water. The results showed that the PMMA@PFC-73 membrane has high air permeability with a WVTR of 369.8 g m− 2 h− 1, comparable to commercial gauze (Fig. 1i). Furthermore, water contact angle measurements indicated that PMMA@PFC-73 membrane has a hydrophobic surface, which facilitates the discharge of wound exudate (Supplementary Fig. 6).
Hemolysis and coagulation effect of PMMA@PFC-73 membrane in vitro
HUVECs were used to evaluate the biocompatibility of PMMA@PFC-73 membranes. Fluorescence imaging and cck-8 assay data showed that the cell survival rate of the PMMA@PFC-73 membrane exceeded 98% after being treated with HUVECs for 48 hours, indicating that the PMMA@PFC-73 nanocomposite membrane is safe for use on skin surfaces (Fig. 2a-b). This high survival rate makes the film particularly effective for covering various trauma sites, making it suitable for wound care applications. Considering that the typical safe hemocompatibility threshold for biomedical materials is less than 5%35,36, further hemocompatibility assessments were performed on the PMMA@PFC-73 membrane using freshly anticoagulated blood. The hemocompatibility of the PMMA@PFC-73 membrane was evaluated at various concentrations of PFC-73, with PBS serving as a negative control and Triton X-100 as a positive control. It was found that PFC-73 induced hemolysis at various concentrations compared to the control group (Supplementary Fig. 7a), with the hemolysis rate increasing with higher concentrations and exceeding the safe range (Supplementary Fig. 7b). However, the hemolysis rate of the PMMA@PFC-73 was lower than that of individual PFC-73 and remained within the safe range (Fig. 2c). Overall, the PMMA@PFC-73 membrane exhibits excellent hemocompatibility and holds significant potential as a composite hemostatic membrane for in vivo applications due to its super-hydrophobic properties. To assess its hemostatic ability, we conducted an in vitro coagulation assay. Initial observations indicated poor coagulation in the PMMA group, with noticeable hemolysis after 10 minutes. In contrast, PMMA@PFC-73 membranes demonstrated superior coagulation ability, achieving a stable state without hemolysis after 120 minutes, comparable to gauze (Fig. 2d).
Furthermore, we evaluated the effect of the PMMA@PFC-73 nanocomposite membranes on in vitro blood coagulation using blood clotting index (BCI) measurements. Lower BCI values indicate faster coagulation rates, serving as an assessment of coagulation performance37,38. The PMMA@PFC-73 nanocomposite membrane exhibited a lower BCI (32.9%) compared to PFC-73 (51.8%) and slightly outperformed the gauze group (36.9%) (Fig. 2e). This result demonstrates that PMMA facilitated blood coagulation by binding with PFC-73, thus exhibiting excellent hemostatic properties. This enhancement can be attributed to the release of nickel ions from the PMMA@PFC-73 nanocomposite membrane, which triggers the coagulation cascade and accelerates clotting39. SEM images provide further insight into the clotting mechanism (Fig. 2f). The PMMA@PFC-73 membrane with hydrogen bond was revealed, which induced erythrocyte fixation and promoted rapid clot formation40 (Fig. 2g).
Effects of cell proliferation and migration by PFC-73 in vitro
Prior to evaluating the effect of the PMMA@PFC-73 nanocomposite membranes on wound closure acceleration in mice, we investigated the effect of PFC-73 on the proliferation and migration of HUVECs during wound healing process. The results demonstrated that different concentrations of PFC-73 exhibited good biocompatibility with HUVECs after co-culture for 24 h, compared to the control group (Supplementary Fig. 8). Furthermore, the cell viability of HUVECs significantly increased by approximately 1.5-fold after 48 h (Fig. 3a-b). Calcein-AM and PI staining were used to detect the cell status of HUVECs co-cultured with PFC-73 for 72 h. There was minimal cell death (PI-positive cells, red fluorescence) following PFC-73 (50 µg/mL) treatment, in contrast to the control group (Supplementary Fig. 9). Additionally, HUVECs maintained good viability and proliferation rates during 5 days of incubation with PFC-73, confirming that PFC-73 significantly enhances HUVECs proliferation (Fig. 3c).
The effects of PFC-73 on the migration of HUVECs were evaluated using scratch assay and transwell migration assay. After 12 h of co-culture, the wound healing rate in the PFC-73 treated group was 23.5% higher than in the control group, with a significant increase observed at 24 h (Fig. 3d-e). The transwell migration assay also corroborated that PFC-73 could significantly promote the migration of HUVECs, which could elevate the number of migrated cells by approximately 10-fold (Fig. 3f-g). These findings elucidate a significant role for PFC-73 in fostering the proliferation and migration of HUVECs, which is pivotal for tissue restoration and regeneration during the wound healing process. Compelling evidence has been presented that PFC-73, we co-cultured HUVECs with PFC-73 for 24 h and detected the expression of cell proliferation marker. The results showed that the PFC-73 treated group significantly promoted the mRNA expression level of Ki67 (Fig. 3h). Immunofluorescence assay further corroborated these findings, revealing a notable increase in the nuclear expression of Ki67 upon treatment with PFC-73 (Fig. 3i). This upregulation suggests that the cells are actively engaged in the growth cycle, thereby accelerating the rate of cell proliferation. In addition, real-time quantitative PCR (RT-qPCR) results also showed that PFC-73 treatment significantly up-regulated the mRNA expression level of VEGF (Supplementary Fig. 10). The collective implications of these results suggest that the release of nickel ions by PFC-73 significantly promoted the proliferation and migration of HUVECs41, potentially expediting the wound healing process.
Effects of improving inflammatory and protecting cells from oxidative stress
During the process of wound healing, a continuous inflammatory response and oxidative stress result in the overproduction of reactive oxygen species (ROS) within tissues, ultimately impairing the healing process42. We developed a cellular model to simulate excess ROS by stimulating HUVECs with lipopolysaccharide (LPS) for 12 h. Simultaneously, we treated the HUVECs with varying concentrations of PFC-73. The results revealed that LPS treatment markedly enhanced the intracellular fluorescence intensity of 2’-7’-dichlorodihydrofluorescein (DCFH-DA), while PFC-73 treatment significantly decreased ROS levels. Moreover, PFC-7 (50 µg/mL) efficiently scavenged the excess ROS produced due to intracellular LPS stimulation (Fig. 4a-b).
We investigated the anti-inflammatory capacity of PFC-73 and its effect on protecting HUVEC from oxidative stress. To explore the anti-inflammatory properties of PFC-73, LPS induced HUVECs were used to form an inflammatory cell model, revealing the underlying mechanism (Fig. 4c). We measured the expression levels of pro-inflammatory factors, specifically interleukin-8 (IL-8), interleukin-6 (IL-6), and interleukin-1b (IL-1β) (Fig. 4d and Supplementary Fig. 11). The mRNA expression levels of these inflammatory factors were markedly elevated in HUVECs treated with LPS for 24 h compared to the control group. Simultaneously, PFC-73 treatment significantly decreased the expression of these inflammatory factors and mitigated the LPS-induced cell inflammatory response. This indicates that PFC-73 acts as an effective scavenger of ROS, reducing excessive ROS production and ameliorating the inflammatory response. Subsequently, we constructed an oxidative stress model in HUVECs by exposing them to H2O2. We then treated the oxidatively stressed cells with varying concentrations of PFC-73 to assess its protective effect against oxidative stress. As shown in Fig. 4e, the cell viability of HUVECs decreased by about 40% after treatment with H2O2 (650 µM). Continuous H2O2 exposure induced cell death, and the protective effect of PFC-73 against excessive oxidative stress induced by H2O2 was demonstrated through a live/dead cell staining assay (Fig. 4f).
Therapeutic effects on wound by PMMA@PFC-73 membrane in vivo
The above results indicate that the PMMA@PFC-73 nanocomposite membrane exhibits self-adaptability and can adsorb erythrocytes and platelets, contributing to effective hemostasis. Additionally, the incorporated PFC-73 not only effectively enhances the proliferation and migration of HUVECs but also possesses excellent anti-inflammatory properties and the ability to protect cells from oxidative stress-induced damage. To assess the impact of the PMMA@PFC-73 nanocomposite membrane on wound healing in an in vitro mice model, we established a mice back wound model involving full-thickness dorsal skin punch wounds with a 10 mm diameter. The mice were randomly assigned to three groups, including control group, PFC-73 (10 µg/mL, 10 µL per wound site), and PMMA@PFC-73 nanocomposite membrane (Fig. 5a). We monitored wound healing every 2 days, and photographs of the wounds revealed that mice in both the PFC-73 and PMMA@PFC-73 nanocomposite membrane-treated groups exhibited accelerated wound healing compared to the control group (Fig. 5b-d). Notably, on day 4, the wound healing rate for the PMMA@PFC-73 group was approximately 1.4-fold higher compared to the control, whereas the PFC-73-treated group exhibited a 1.2-fold increase. By day 9, the wound healing rate of the PMMA@PFC-73 nanocomposite membrane-treated group reached about 97% (Fig. 5c). This indicates that PFC-73 is effective in promoting wound healing in mice, while the PMMA@PFC-73 nanocomposite membrane can completely cover the wound surface, releasing PFC-73 slowly and more effectively than PFC-73 nanoparticles alone, thereby enhancing tissue repair.
On day 9, the mice were sacrificed, and wound tissue samples were harvested for further study of the wound healing process using H&E and Masson staining. H&E staining results revealed that the control group exhibited extensive scar tissue, reduced granulation tissue, and significant inflammatory cell infiltration. The PFC-73 and PMMA@PFC-73 nanocomposite membrane treatment groups showed a significant reduction in inflammatory cell infiltration near the wound tissue, which promoted the re-epithelialization of the wound tissue, and the PMMA@PFC-73 nanocomposite membrane treatment group achieved the best results, with nearly complete epithelial reconstruction (Fig. 5e-f). Additionally, both PFC-73 and PMMA@PFC-73 membrane treatments significantly decreased wound area and width. Masson’s trichrome staining revealed that the wound tissue treated with PFC-73 and PMMA@PFC-73 nanocomposite membrane exhibited significantly higher collagen deposition compared to the control group. Notably, the PMMA@PFC-73 nanocomposite membrane group displayed the highest collagen deposition (Fig. 5f). Subsequently, we evaluated the biosafety of PFC-73 and PMMA@PFC-73 nanocomposite membrane at the end of the animal experiments. The major organs of mice, including heart, liver, spleen, lung and kidney were collected to observe the pathological status by H&E staining. The pathological results showed that no abnormal changes were observed in the histological structure of the major organs, and there was no necrosis in the tissues of these organs (Supplementary Fig. 12a). In addition, the blood of mice was further collected and analyzed for blood parameters. No abnormalities were observed in the blood parameters of mice treated with the PMMA@PFC-73 nanocomposite membrane compared to the control group (Supplementary Fig. 12b). These results indicated that the PMMA@PFC-73 nanocomposite membrane significantly promoted wound healing in mice and had a good biosafety.
Therapeutic mechanism of PMMA@PFC-73 membrane
To investigate the underlying mechanisms of PFC-73 and PMMA@PFC-73 membrane in treating wound healing in mice, we collected wound tissue after the experiment for transcriptome analysis. Hierarchical cluster analysis and principal component analysis (PCA) of gene expression profiles revealed significant differences between the three groups of wound tissue (Supplementary Fig. 13a). Among them, compared with the control group, PFC-73 group had 1995 differential genes, and the PMMA@PFC-73 group had 1827 differential genes. Additionally, the PMMA@PFC-73 membrane group had 460 differential genes compared to the PFC-73 group, with a total of 20 overlapping genes across all three groups (Fig. 6a and Supplementary Fig. 13b). Compared with the control group, the PFC-73 treatment group had 971 down-regulated genes and 1024 up-regulated genes (Fig. 6b), while the PMMA@PFC-73 membrane treatment group had 738 down-regulated genes and 1089 up-regulated genes (Fig. 6c). Based on Gene ontology (Go) analysis, the differential genes in PFC-73 treatment group were mainly enriched in skin development, fatty acid metabolism, epidermal development, cell proliferation, and leukocyte activation and regulation compared with the control group (Supplementary Fig. 14). In the PMMA@PFC-73 membrane treatment group, differential genes were significantly enriched in skin development, epidermal development, fatty acid metabolism, cell proliferation and the regulation of inflammatory response (Supplementary Fig. 15). This suggests that PFC-73 treatment can significantly promote cell proliferation and skin development while regulating the inflammatory response mediated by white blood cells. The PMMA@PFC-73 membrane treatment group showed more effective reduction in the inflammatory response of wound tissue due to the external barrier effect of the membrane. This effect mobilized various inflammatory cells to defend against inflammation by slowing the release of PFC-73, thereby promoting skin development more effectively.
The differential genes were categorized into three groups: cellular components (CC), molecular functions (MF), and biological processes (BP). In the PFC-73 treatment group, differential genes were mainly enriched in leukocyte cell-cell adhesion, epidermis development, and skin development (Fig. 6d). In the PMMA@PFC-73membrane treatment group, differential genes were mainly enriched in epidermis development, skin development, leukocyte migration, cell proliferation and proliferation epidermal cell differentiation (Fig. 6e). CC analysis revealed that the differential genes were primarily enriched in the extracellular matrix region. In MF analysis, these genes were associated with glycosaminoglycan binding, cell adhesion molecules, immune receptors, and cytokines (Supplementary Fig. 16a-b). Next, we examined specific genes involved in the inflammatory response and skin development to further understand the function of the PMMA@PFC-73 membrane. Notably, treatment with the PMMA@PFC-73 membrane significantly up-regulated genes related to skin development and down-regulated pro-inflammatory genes (Fig. 6f-h). Additionally, KEGG analysis indicated changes in other genes and pathways associated with wound healing, such as the cytokine-cytokine receptor binding signaling pathway (Supplementary Fig. 16c), suggesting the involvement of additional mechanisms that promote wound healing.