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Microenvironment-adaptive nanomedicine MXene promotes flap survival by inhibiting ROS cascade and endothelial pyroptosis

Journal of Nanobiotechnology volume 23, Article number: 282 (2025) Cite this article

Abstract

In the field of large-area trauma flap transplantation, preventing avascular necrosis remains a critical challenge. Key mechanisms for improving flap viability include angiogenesis promotion, oxidative stress inhibition, and cell death prevention. Recently, two-dimensional ultrathin Ti3C2TX (MXene) nanosheets have gained attention for their potential contributions to these processes, though MXene’s physiological impact on flap survival had not been previously investigated. This study is the first to confirm MXene's biological effects on the ischaemic microenvironment post-skin flap transplantation. Findings indicated that MXene significantly decreased the necrotic area in ischaemic flaps (37.96% ± 2.00%), with reductions of 30.40% ± 1.86% at 1 mg/mL and 20.19% ± 2.11% at 2 mg/mL in a concentration-dependent manner. Mechanistically, MXene facilitated in situ angiogenesis, mitigated oxidative stress, suppressed pro-inflammatory pyroptosis, and activated the PI3K-Akt pathway, particularly influencing vascular endothelial cells. Comparative transcriptome analysis of skin tissues with and without MXene treatment provided additional evidence, highlighting mechanisms such as pro-inflammatory pyroptosis, ROS metabolic processes, endothelial cell proliferation regulation, and PI3K-Akt signaling pathway activation. Overall, MXene demonstrated biological activity, effectively promoting ischaemic flaps survival and presenting a novel strategy for addressing ischaemic necrosis in skin flaps.

Graphical abstract

Introduction

Advances in surgical techniques have made flap transplantation a standard approach in plastic and reconstructive surgery for preserving skin integrity and function [1, 2]. Despite these developments, distal ischaemic necrosis remains a major postoperative complication, significantly limiting clinical applications [3, 4]. Thus, identifying a novel strategy to markedly enhance the survival rate of distal ischaemic flaps holds substantial theoretical and practical value, particularly in avoiding secondary surgeries [5]. However, no effective clinical treatment currently exists to alleviate distal ischaemic necrosis in flaps. Known mechanisms driving distal ischaemia include severe oxidative stress, vascular insufficiency, cell death, and inflammation, underscoring the need for therapeutic strategies targeting these processes [6,7,8,9]. Skin comprises the epidermis, dermis, and subcutaneous tissue, with various cell types such as endothelial cells, fibroblasts, and macrophages [10, 11]. Further study is required to identify the skin layers and cell types predominantly affected by these ischaemia-induced mechanisms, as well as to understand and mitigate their impact on flap survival. Notably, earlier research has highlighted the critical role of enhancing angiogenesis by protecting dermal endothelial cells in improving flap survival [12, 13], thus underscoring the importance of targeting dermal endothelial cells as potential therapeutic candidates and examining interventions addressing the underlying pathological mechanisms.

Among conductive materials, Ti3C2Tx (MXene), a two-dimensional (2D) nanosheet structure of transition metal elements, alkene oxide, and carbon, has shown effectiveness in enhancing neuronal biological activity and neural network synchronization, accelerating tissue repair [14,15,16]. MXene exhibits excellent biocompatibility, high surface area, and cell proliferation support, making it a promising candidate for drug delivery and other biomedical applications [17]. MXene nanosheets also possess an inherent reactive oxygen species (ROS)-scavenging capability, counteracting hydrogen peroxide, superoxide anions (O2•−), and hydroxyl radicals (•OH) without additional modifications [18]. Studies have demonstrated that MXene can promote endothelial cell migration and angiogenesis through ROS reduction, thereby supporting tissue regeneration [19, 20]. While MXene has shown antioxidant properties and slowed the progression of neurodegenerative diseases and osteoarthritis [15, 21], its effects on ischaemic necrosis in transplanted skin flaps remain largely unexplored. Additionally, vascular endothelial growth factor A (VEGFA), VE-Cadherin, and hypoxia-inducible factor 1α (HIF-1α) are established pro-angiogenic factors [22,23,24]. Investigating MXene’s regulatory effects on these proteins could yield valuable insights into its mechanisms for promoting angiogenesis and improving tissue repair under ischaemic conditions.

Recent progress in understanding programmed cell death offers promising approaches for treating ischaemic flaps [25, 26]. Pyroptosis, a recently discovered pro-inflammatory cell death, influences various tissues, including skin, through inflammasome regulation [13, 27]. The NLRP3 inflammasome, a complex consisting of several components such as NLRP3, Caspase-1, and ASC, triggers the production and activation of GSDMD as well as pro-inflammatory cytokines like IL-18 and IL-1β, which ultimately leads to pyroptosis [28, 29]. Targeting pyroptosis represents a compelling strategy to prevent unintended endothelial cell death in ischaemic flaps [30, 31]. The accumulation of ROS leads to oxidative stress, which can damage proteins and nucleic acids and induce programmed cell death [32, 33]. Key ROS implicated in ischaemic injury include O2•−, hydrogen peroxide, and •OH [34, 35]. Earlier studies have revealed that reducing ROS levels in endothelial cells may effectively inhibit pyroptosis and provide protective effects under ischaemic conditions [36, 37]. Additionally, a significant mechanism of ROS-mediated damage in the ischaemic microenvironment is the increase in MDA levels, which disrupts the balance between oxidative and antioxidant defences [38]. Therefore, mitigating the accumulation of critical ROS effectors can effectively reduce oxidative stress damage and pyroptosis in ischaemic flaps. Given these mechanisms, MXene presents an attractive candidate for preserving the normal microenvironment of ischaemic flaps.

Research has demonstrated that the phosphatidylinositol 3-kinase (PI3K)-protein kinase B (Akt) pathway plays a vital role in promoting angiogenesis, cell proliferation, and survival across various tissues, including skin [39,40,41]. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of RNA-seq data also indicated significant enrichment of this pathway, emphasizing the importance of in vivo experimentation to further explore its activation. Additionally, the PI3K-Akt pathway inhibitor 2–4-morpholinyl-8-phenylchromone (LY294002) was employed for rescue experiment to determine whether MXene promotes angiogenesis and inhibits distal necrosis by activating this signaling pathway in ischaemic flaps [42].

In this study, bioactive MXene were synthesized and characterized. Both in vitro and in vivo, the potential impact of MXene on decreasing ROS levels, inhibiting pro-inflammatory pyroptosis in endothelial cells, enhancing endothelial cell migration and proliferation, and stimulating angiogenesis was systematically examined (Scheme 1). Transcriptome sequencing analysis was further employed to examine gene expression differences in the border area between surviving and necrotic areas of flaps with and without MXene treatment. These findings elucidate MXene’s key characteristics and offer valuable insights to support its potential clinical application in treating distal necrosis in ischaemic flaps.

Scheme 1
scheme 1

Schematic illustration depicting how MXene enhances the survival of ischaemic flaps via the stimulation of angiogenesis, suppression of oxidative stress, and inhibition of pyroptosis, particularly in endothelial cells

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Results and discussion

Characterization and anti-oxidant ability of MXene

The Atomic force microscopy (AFM) and scanning electron microscopy (SEM) were used to characterize the MXene nanosheets’ structure. As shown in the images, MXene displayed a sheet structure with defined lateral dimensions (Fig. 1A and Figure S1A). Energy-dispersive spectroscopy (EDS) analysis confirmed that the characteristic Ti peak overlapped with the O peak, aligning with previous studies [40] (Figure S1B). The primary elements in MXene were C, O, and Ti, with a small amount of F from the surface termination (Fig. 1B). The size distribution of MXene was measured by dynamic light scattering (DLS), which showed a main size range of 0.1–1.5 μm (Fig. 1C). The zeta potential of MXene was also measured, and the results showed an average value of − 5.2 mV (Figure. S1C). Given the role of ROS in various ischaemic conditions, the ROS scavenging capability of MXene was thoroughly examined. The antioxidant properties and ABTS•+ scavenging efficiency were measured using 2,2′-Azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS) (Fig. 1D). Results showed a concentration-dependent increase in the ABTS•+ scavenging ratio with rising MXene concentrations (Fig. 1E). Additionally, 2,2-diphenyl-1-picrylhydrazyl (DPPH) assays, applied for evaluating antioxidant capacity, revealed a significant reduction in the characteristic DPPH peak at 517 nm following MXene treatment, indicating effective DPPH elimination (Fig. 1F). Quantitative analysis confirmed that MXene rapidly reduced DPPH concentrations in solution (Fig. 1G). Further analysis employed UV–Vis spectroscopy to assess MXene’s scavenging ability for hydroxyl radicals (•OH) and superoxide anions (O2•−), two prevalent ROS types. Such a MXene-concentration-dependent ROS scavenging phenomenon could be vividly visualized by the obvious color change of solutions (Fig. 1H, J). Quantitative analysis demonstrated concentration-dependent increases in O2•− and •OH scavenging ratios (Fig. 1I, K). At maximum concentrations, MXene achieved ROS inhibition ratios of approximately 72.78%, 35.36%, 76.61%, and 38.17% for ABTS•+, DPPH, •OH, and O2•−, respectively, highlighting MXene’s antioxidant properties and its potential as an effective ROS scavenger.

Fig. 1
figure 1

Characterization and ROS-scavenging property of MXene. A AFM image of MXene. Scale bar: 1 μm. B The corresponding EDS spectrum of MXene. C Size distribution of MXene. D, F UV–Vis spectra of ABTS and DPPH solutions after treatment with different concentrations of MXene. E, G ABTS (n = 4 per group) and DPPH (n = 3 per group) scavenging activity of MXene at different concentrations. H The UV–Vis spectra of O2•− after treatment with different concentrations of MXene (n = 3 per group). I O2•− scavenging ratio of MXene with different concentrations (n = 3 per group). J Scavenging activities of MXene for •OH studied by the UV–Vis spectra. K The concentration-dependent investigation of •OH in the presence of MXene. SEM error bars are used. Significance (*): p value < 0.05

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MXene promoted the survival and inhibited oxidative stress of OGD-HUVECs

Promoting the survival and proliferation of endothelial cells is critical for the survival of ischaemic flaps, as the distance that new blood vessels can reach determines the survival area of the flap [43]. Human umbilical vein endothelial cells (HUVECs) were used to examine the effects of various treatments on ischaemic flaps vasculature in vitro. The special ROS-scavenging capability of MXene led us to investigate its antioxidant activity in cells. First, MXene toxicity on HUVECs was assessed. The Cell Counting Kit-8 (CCK-8) data indicated no significant variation in cell viability between control and MXene-treated groups at concentrations up to 160 μg/mL (Fig. 2A). HUVECs were subsequently treated with MXene for two days, with daily measurements of cell viability. While treatment with 160 μg/mL MXene significantly reduced cell viability, 80 μg/mL had no impact on cell proliferation (Fig. 2B). Fibroblasts play a beneficial role in the repair of skin tissue damage [44]. Therefore, we also evaluated the toxicity of MXene on L929 fibroblasts. L929 cells were treated with MXene for two days, and cell viability was measured daily. The results showed that a concentration of 80 μg/mL had no effect on cell proliferation, further indicating its good biocompatibility (Figure S2). Based on these results, concentrations of 10, 20, 40, and 80 μg/mL were selected for further study. Glucose and oxygen deprivation (OGD)-treated HUVECs (OGD-HUVECs) served as an in vitro model for endothelial cells in ischaemic flaps. In OGD-HUVECs, cell viability in the OGD-only group was markedly lower than in MXene-treated groups (Fig. 2C). Additionally, as MXene concentration increased from 10 μg/mL to 80 μg/mL, cell viability rose from 55.98% ± 2.69% to 76.48% ± 2.99%. To explore the mechanisms underlying MXene’s protective effect on OGD-HUVECs, oxidative levels were assessed via dichlorodihydrofluorescein diacetate (DCFH-DA) and dihydroethidium (DHE) staining. The ROS scavenging ability of MXene, observed through DCFH-DA staining by confocal microscopy in OGD-HUVECs, displayed a concentration correlation (Fig. 2D, E). DHE staining further confirmed that MXene significantly alleviated oxidative damage (Fig. 2F, G). Collectively, these results demonstrate MXene’s capacity to mitigate cellular oxidative damage, particularly in endothelial cells, underscoring its potential for treating ischaemic skin flaps. Previous studies have demonstrated that nanoscale MXene can be internalized by cells, including via clathrin-mediated endocytosis [45,46,47]. Future studies directly confirming internalization and exploring the mechanisms involved, such as intracellular aggregation sites, will be crucial for elucidating the precise mechanism of action of MXene.

Fig. 2
figure 2

Biocompatibility and antioxidative damage activity of MXene in vitro. A CCK-8 assay for cell viability of HUVECs treated with different concentrations of MXene (n = 5). B Effect of MXene (μg/mL) on the proliferation of HUVECs. (n = 5). C Cell viability of HUVECs treated with different concentrations of MXene under OGD treatment by CCK-8 assay (n = 5). D, E DCFH-DA staining of HUVECs treated with different concentrations of MXene under OGD treatment and their integrated intensity analysis (n = 3). Scale bar: 50 μm. F, G Analysis and quantification of DHE integral absorbance in each group (n = 3) (nuclei: Hoechst 33,342). Scale bar: 20 μm. NC: high-glucose DMEM. SEM error bars are used. Significance (*): p value < 0.05; ns: not significantly different

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MXene acted as a pyroptosis inhibitor and maintained OGD-HUVECs angiogenic functions

The mechanisms underlying MXene’s cytoprotective activity were investigated in ischaemic injury. Increasing evidence links pyroptosis to ROS and highlights its significant role in inflammation and ischaemic diseases [48,49,50]. As a type of programmed cell death, morphological changes during pyroptosis include activation of intracellular inflammasomes, rupture of cell membranes, and release of inflammatory factors [51]. Previous studies have shown that these changes are mainly mediated by NLRP3, activated Caspase-1 (CASP-1), activated Gasdermin D (GSDMD) and other related proteins [52]. Therefore, the expression levels of pyroptosis-related proteins were further quantified to measure the effect of MXene on OGD-HUVECs in the occurrence of pyroptosis. Immunofluorescence staining revealed that MXene treatment significantly reversed the elevated cleaved CASP-1 (C-CASP-1) fluorescence intensity induced by OGD in HUVECs in a dose-dependent manner (Fig. 3A, B). Similarly, MXene demonstrated a protective effect, with GSDMD-N levels markedly lower in MXene-treated groups than in the OGD group (Fig. 3C, D). The expression of CASP-1, GSDMD, and NLRP3 determined cell sensitivity to pyroptosis. Further confirmation via western blotting revealed that the relative levels of NLRP3, CASP-1, and GSDMD-N were elevated in the OGD group but progressively decreased with MXene treatment, reaching the lowest in the MXene 80 μg/mL group (Fig. 3E–H). Additionally, MXene significantly reduced the dead cell rate in a dose-dependent manner, from 35.70% ± 1.45% at 10 μg/mL to 15.61% ± 1.57% at 80 μg/mL, compared to 46.58% ± 2.20% in the OGD-alone group (Fig. 3I, J). These results suggest that MXene protects OGD-HUVECs through the NLRP3/CASP-1/GSDMD pathway. As a critical inflammasome component, NLRP3 activates CASP-1, which initiates pyroptosis via GSDMD-N [53]. CASP-1, GSDMD, and NLRP3 collaboratively orchestrate pyroptosis, forming a pivotal signaling pathway governing cell death and inflammatory responses [54]. Our results demonstrate that MXene effectively shields cells from pyroptosis in OGD-treated cells.

Fig. 3
figure 3

MXene inhibited pyroptosis of OGD-HUVECs. A, B Representative immunofluorescence staining images of C-CASP-1 and its integrated intensity analysis (nuclei: DAPI). Scale bar: 20 μm. C, D Representative immunofluorescence staining images of GSDMD-N and its integrated intensity analysis (nuclei: DAPI). Scale bar: 20 μm. E–H Western blotting analysis of NLRP3, CASP-1 and GSDMD-N in MXene-pretreated HUVECs under OGD conditions. I, J Live/Dead fluorescence staining of HUVECs under OGD treatment with MXene: dead cell rate and representative images for each group. Scale bar: 100 μm. N = 3 per group. NC: high-glucose DMEM. SEM error bars are used. Significance (*): p value < 0.05; ns: not significantly different

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To further investigate whether MXene exerts a protective effect on the angiogenic function of OGD-damaged HUVECs, additional experiments were conducted, including scratch experiments and tube formation assays to evaluate migration and angiogenesis capabilities. OGD-treated and normal oxygen-glucose-treated HUVECs were co-cultured with varying concentrations of MXene for 48 h. A uniform cell-free line was created using a 200 μL pipette tip, and cell healing was recorded at two time points. The scratch assay showed that OGD exposure suppressed the migratory capacity of HUVECs compared to the control group (Fig. 4A). Among the OGD-treated groups, the MXene 80 μg/mL group demonstrated the fastest healing rate, with numerous cells migrating into the scratch area after 12 h, and the wound gap almost caught up with the control group after 24 h of culture, while the OGD-only group showed the slowest healing rate throughout the observation period (Fig. 4C). These results confirmed that MXene significantly enhances HUVEC migration in vitro, particularly under OGD conditions. Tube formation assays further indicated that OGD exposure impaired HUVECs’ ability to form tubular vascular structures. However, treating OGD-damaged HUVECs with various concentrations of MXene significantly restored tube formation (Fig. 4B). The number of junctions in the OGD group was 25 ± 5.13, which increased to 52 ± 5.51 in the 10 μg/mL MXene group, and reached the value of 135.67 ± 5.49 in the 80 μg/mL group (Fig. 4D). Additionally, the total branch length also increased dose-dependently with MXene, from 4353.67 ± 474.43 in the OGD group to 16,879.33 ± 647.33 in the 80 μg/mL MXene group (Fig. 4E). These scratch and tube formation assay results reflect the enhanced angiogenic capacity of the cells, indicating that MXene protects HUVECs function under OGD injury. Furthermore, as shown in Fig. 4G–H, VEGFA protein levels were significantly reduced under OGD treatment, but MXene supplementation resulted in a dose-dependent increase in VEGFA protein expression. Similarly, the level of VE-Cadherin also increased significantly with MXene treatment. These proteins cooperate to regulate the formation, stability, and functionality of blood vessels during the healing process. These results suggest that MXene promotes the angiogenic behavior of HUVECs through the modulation of intracellular VEGFA and VE-Cadherin proteins, showing potential for enhancing distal survival in ischaemic flaps.

Fig. 4
figure 4

MXene maintained the angiogenic function of OGD-HUVECs in vitro. A Cell scratch experiments were performed on HUVECs, and measurements were performed at 0, 12, and 24 h. Scale bars: 100 μm. B HUVECs were subjected to an tube formation assay after treatment for 24 h, and the results yielded 6 h of culture. Scale bars: 100 μm. C Quantification and analysis of migration area of HUVECs on 12 h and 24 h. D, E Quantification of the number of junctions and the total branching length among the 6 groups. F–H Western blotting bands and quantification of VEGFA and VE-Cadherin protein expression in MXene-treated HUVECs under OGD conditions. N = 3 per group. NC: high-glucose DMEM. SEM error bars are used. Significance (*): p value < 0.05

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MXene stimulated angiogenesis and promoted survival of ischaemic flaps

Protecting flaps from ischaemic injury is a critical goal in plastic surgery, yet effective direct therapeutic interventions remain scarce. Following observations that MXene effectively removes ROS and protects cells from OGD injury, its protective effects on ischaemic flaps were further investigated. The biosafety of MXene is a critical factor in determining their clinical application. An in vitro hemolysis assay was performed to assess blood compatibility (Figure S3A). According to previous studies, a hemolysis rate of less than 5% is considered acceptable for biomaterials [55]. The results showed that the hemolysis rate of the red blood cell suspension remained below 5% when incubated with MXene at a concentration of 8 mg/mL, indicating good blood compatibility (Figure S3B). Histochemical analysis using hematoxylin and eosin (H&E) staining of liver and kidney tissues from mice in both the Flap + Saline and 8 mg/mL MXene groups revealed no tissue damage. Specifically, there were no signs of liver damage, such as extensive nuclear shrinkage, inflammatory infiltrates, or fibrosis. Similarly, no irregular tubular atrophy or interstitial inflammation was observed (Figure. S3C). These results suggest that MXene exhibits good biosafety in vivo. To evaluate MXene's therapeutic efficacy in vivo, different concentrations of MXene were injected into skin flaps. The selection of MXene concentrations was based on previous studies, which showed that these concentrations were within the effective range of MXene and ensured therapeutic efficacy and safety in in vivo models [40, 44, 47]. These concentrations were chosen to evaluate dose-dependent effects while minimizing the risk of toxicity. Therefore, we identified treatment groups of 1, 2, 4, and 8 mg/mL to verify the therapeutic potential of MXene. On postoperative day (POD) 7, MXene demonstrated the ability to enhance ischaemic flap survival (Fig. 5A). Statistical analysis revealed that 2 and 4 mg/mL MXene showed significantly stronger therapeutic effects compared to the 1 mg/mL group (Fig. 5B). Subsequently, laser Doppler flowmetry was used to assess the subcutaneous vascular network of the flap, and the results indicated that MXene enhanced blood flow in the ischaemic flap (Fig. 5C). Notably, no significant differences were observed in therapeutic efficacy between the 2 mg/mL and 4 mg/mL concentrations, while the 8 mg/mL MXene group exhibited a reduced ability to promote flap survival and enhance blood flow signals (Fig. 5B, D). The therapeutic effects of MXene at a concentration of 8 mg/mL could be further investigated in future studies to better understand the underlying mechanisms. Based on these results, in vivo experiments were conducted on four groups: a sham group, an ischaemic flap model group, and two experimental groups treated with 1 and 2 mg/mL MXene. Further evaluation using F-CHP staining demonstrated that the 2 mg/mL MXene group significantly reduced collagen damage in the ischaemic flaps, showing better results than the 1 mg/mL group (Fig. 5E, F). Similarly, histochemical Masson's staining revealed that the injection of 2 mg/mL MXene promoted collagen remodeling in the skin, with superior efficacy compared to the 1 mg/mL group (Fig. 5G, H). In general, these findings suggest that MXene administration at appropriate concentrations significantly benefits ischaemic flap survival.

Fig. 5
figure 5

MXene promoted the viability of ischaemic flaps. A Photograph of the flap survival area on POD 7. B Quantification of necrotic area percentage among the five groups on POD 7. C Images of the subcutaneous blood flow network on POD 7. D Quantified blood flow signal intensity in ischaemic flaps on POD 7. E, F F-CHP staining and quantified intensity for damaged collagen detection in the skin on POD 7. Scale bar: 100 μm. G, H Masson staining was used to examine collagen remodeling and its quantification of the collagen deposition ratio in the skin on POD 7. Scale bar: 100 μm. N = 3 per group. SEM error bars are used. Significance (*): p value < 0.05; ns: not significantly different

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Given the favorable survival rate of MXene-treated ischaemic flaps, its ability to promote neovascularization in ischaemic flaps was further examined by co-localizing CD31 (an endothelial cell marker) and α-SMA. Skin tissue from the surviving-necrotic border area of the flaps was harvested for this study. Immunofluorescence staining of angiogenesis markers revealed that, following MXene treatment, the number of CD31/α-SMA-positive blood vessels increased significantly compared to the Flap + Saline group. MXene promoted angiogenesis, with 2 mg/mL MXene showing a more pronounced pro-angiogenic effect than 1 mg/mL (Fig. 6A, B). Histochemical analysis using H&E staining further supported these observations, showing that the average microvessel density in the Flap + Saline group was 41.33 ± 4.98. In contrast, the Flap + MXene groups displayed a increase in microvessel density, with values of 56.19 ± 5.13 and 98.02 ± 5.96, respectively (Fig. 6C, D). To further investigate the underlying molecular mechanisms, western blotting was conducted to examine the levels of VEGFA and VE-Cadherin. The results indicated that MXene reversed the low levels of these pro-angiogenic proteins in ischaemic flaps, consistent with findings from OGD-HUVECs studies in vitro (Fig. 6E–G). Furthermore, enzyme-linked immunosorbent assay (ELISA) results confirmed that MXene promoted the levels of HIF-1α and VEGFA in ischaemic flaps (Figure S4A, B). Previous studies have demonstrated that HIF-1α enhances endothelial cell survival under hypoxic conditions and promotes their proliferation, aiding in the repair of damaged blood vessels. The observed increase in HIF-1α protein levels following MXene treatment further underscores its therapeutic potential. In conclusion, MXene effectively enhances angiogenesis and promotes the survival of ischaemic flaps, demonstrating potential for therapeutic use in ischaemic injury management.

Fig. 6
figure 6

MXene promoted angiogenesis in ischaemic flaps. A Immunofluorescence staining for CD31 (red) and α-SMA (green) in the skin flaps on POD 7. Scale bar: 50 μm. B Quantification of CD31/α-SMA-positive blood vessel density among the 4 groups. C Representative H&E staining images of skin tissues from the 4 groups. Scale bar: 50 μm. D The density of blood vessels (per mm2) was measured. E–G Western blotting bands and quantification of angiogenesis-related proteins in the 4 groups. N = 3 per group. SEM error bars are used. Significance (*): p value < 0.05

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MXene inhibited oxidative stress and pyroptosis in endothelial cells within ischaemic flaps

The discovery of ROS and pyroptosis presents promising targets for preventing undesired endothelial cell death and restoring blood supply in ischaemic flaps. Therefore, the influence of MXene on ROS and pyroptosis was investigated in vivo. MXene was injected into the skin flap during surgery. DHE staining, a tissue ROS probe, demonstrated that MXene effectively reduced ROS accumulation in the Flap + Saline group (2.82 ± 0.18), with ROS levels in the MXene 2 mg/mL group (1.58 ± 0.16) being lower than those in the MXene 1 mg/mL group (2.08 ± 0.16) (Figure S5A, B). In addition, malondialdehyde (MDA), a marker of oxidative stress, was assayed. The MXene group exhibited lower MDA levels than the Flap + Saline group (Figure S5C). To examine whether MXene inhibited pyroptosis, the expression levels of pyroptosis-associated molecules were evaluated. NLRP3, CASP-1, GSDMD-N, and IL-18 are key proteins involved in pyroptosis. Western blotting revealed that the levels of these proteins were significantly higher in the Flap + Saline group than in the Sham group. However, MXene administration reduced pyroptosis-related protein levels, with the most reduction observed at 2 mg/mL MXene concentration (Fig. 7A–E). Furthermore, immunofluorescence staining was used to detect CASP-1 expression in CD31-labeled endothelial cells within the dermis (Fig. 7F). CASP-1 levels were notably higher in the Flap + Saline group (4.74 ± 0.29), but significantly decreased following MXene treatment, with the fluorescent intensity of CASP-1 in the 2 mg/mL MXene group (1.68 ± 0.23) being lower than that in the 1 mg/mL MXene group (2.42 ± 0.25) (Fig. 7G). This trend aligned with the western blotting results from the in vitro assays. Additionally, ELISA detected the expression of GSDMD-N, C-CASP-1, cleaved IL-18, and cleaved IL-1β, with results consistent with the western blot findings. The 2 mg/mL MXene group demonstrated a superior ability to inhibit pyroptosis-related proteins compared to the 1 mg/mL group (Fig. 7H–K). The activated forms of these proteins are essential for pyroptosis, and the cytokines IL-1β and IL-18, released by pyroptotic cells, are key contributors to inflammatory responses. MXene treatment inhibited these cytokines in ischaemic flaps. Overall, these results suggest that MXene effectively inhibits pyroptosis in ischaemic flaps, particularly in dermal vascular endothelial cells, thereby maintaining vascular function and offering valuable insights into MXene's therapeutic potential for preserving vascular health in ischaemic flaps.

Fig. 7
figure 7

MXene inhibited pyroptosis in vascular endothelial cells in ischaemic flaps. A–E Western blotting images and quantitative analysis of pyroptosis-related proteins in the 4 groups. F Immunofluorescence staining for CASP-1 (red) and CD31 (green) on POD 7 detected by confocal microscopy. Scale bar: 20 μm. G Quantitative analysis of CASP-1 level in each CD31-labeled endothelial cell between the four groups. H–K Levels of GSDMD-N, C-CASP-1, cleaved IL-18, and cleaved IL-1β in flaps measured using ELISA kits. N = 3 per group. SEM error bars are used. Significance (*): p value < 0.05

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Differential expression of mRNAs induced by MXene treatment

To investigate the differences between untreated skin flaps and those treated with 2 mg/mL MXene, as well as the potential mechanisms underlying MXene's beneficial effects on ischaemic flap therapy, RNA sequencing (RNA-seq) analysis was performed on skin tissues from both groups on POD 7. The results revealed a strong correlation among the test samples. Compared to the untreated Flap group, MXene treatment led to the differential upregulation of 616 genes and the downregulation of 1,142 genes (Fig. 8A, C). KEGG enrichment analysis and Gene Ontology (GO) analysis were conducted on the differentially expressed genes to elucidate their functions. KEGG analysis identified several signaling pathways associated with the ischaemic flap survival, including the PI3K-Akt signaling pathway, NOD-like receptor signaling pathway, PPAR signaling pathway, and Cytosolic DNA-sensing pathway, among others. Additionally, KEGG analysis revealed relevant indicators affecting flap survival, such as cytokine-cytokine receptor interactions, phagosomes, and ECM-receptor interactions (Fig. 8B). GO enrichment analysis of differentially expressed genes highlighted significant modulation of key biological processes associated with the ischaemic flap survival, including blood vessel maturation (GO:0001955), pyroptosis (GO:0070269), skin development (GO:0043588), wound healing (GO:0042060), negative regulation of hydrogen peroxide-induced cell death (GO:1,903,206), reactive oxygen species metabolic processes (GO:0072593), response to hypoxia (GO:0001666), cell adhesion (GO:0007155), angiogenesis (GO:0001525), response to decreased oxygen levels (GO:0036293), blood vessel diameter maintenance (GO:0097746), regulation of vasculature development (GO:1,901,342), regulation of endothelial cell proliferation (GO:0001936), and regulation of programmed cell death (GO:0043067), among others. Notably, cellular component processes, such as extracellular matrix (GO:0031012), were also significantly modulated (Fig. 8D). Moreover, Gene Set Enrichment Analysis (GSEA) revealed that the neovascularization-related term, positive regulation of sprouting angiogenesis (GO:1,903,672), was significantly upregulated, while the peroxisome term (MMU04146), which is closely associated with ROS, was downregulated in the MXene-treated group (Fig. 8E, F). Taken together, these results indicate that MXene has a unique gene profile that promotes endothelial cell proliferation, inhibits pyroptosis, suppresses ROS accumulation, and reduces ischaemic injury. Collectively, these effects facilitate the improved viability of ischaemic flaps.

Fig. 8
figure 8

Global assessment of the ischaemic microenvironment in flaps with or without MXene treatment by RNA-seq. A A volcano plot was created to show differentially expressed genes. B KEGG analysis was performed on target genes identified from differentially expressed genes between the two groups. C A heatmap showed the up- and down-regulated genes in the MXene group compared to the Flap group (n = 4 per group). D GO functional enrichment analysis of differentially expressed genes between the Flap and MXene groups. E GSEA plots displayed cellular processes associated with positive regulation of sprouting angiogenesis (MXene versus Flap). F GSEA plots displayed cellular processes associated with Peroxisome (MXene versus Flap)

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MXene activated the PI3K-Akt signaling pathway

KEGG analysis suggests that MXene treatment alters the activity of the PI3K/Akt pathway, which is implicated in several biological processes like angiogenesis, cell proliferation, immune responses, and wound healing [56,57,58]. To further explore the mechanisms underlying MXene-induced angiogenesis and flap survival, the activity of this pathway in ischaemic flaps was assessed. Western blotting revealed that the PI3K/Akt signaling pathway was inactivated in the surviving-necrotic border areas of the ischaemic flaps. Additionally, treatment with LY294002, a inhibitor of the PI3K/Akt pathway, further reduced the levels of p-PI3K (phosphorylated Tyr524) and p-Akt (phosphorylated S473). This aligns with previous studies, confirming the effectiveness of LY294002 [59]. However, MXene treatment restored the expression of p-Akt and p-PI3K (Fig. 9A). Band quantification confirmed that MXene did not alter the total levels of PI3K and Akt but effectively reversed the ischaemia-induced decrease in their phosphorylation levels in skin tissues. In contrast, LY294002 administration abolished this effect (Fig. 9B, C). Consistently, the survival rate of ischaemic flaps significantly improved with MXene treatment, an effect that was reversed by LY294002 treatment (Fig. 9D, E). These findings indicate that the PI3K-Akt pathway is crucial for the beneficial effects of MXene on ischaemic flap survival. Data from LDBF measurements also demonstrated that MXene administration significantly enhanced blood flow signal intensity in ischaemic flaps, while co-treatment with LY294002 reversed this enhancement (Fig. 9F, G). These results indicate that MXene activates the PI3K/Akt pathway under ischaemic stimulation, promoting the survival of the distal ischaemic flaps and strengthening the subcutaneous vascular network. This highlights MXene's potential to exert beneficial effects through this signaling pathway in repairing the ischaemic microenvironment of soft tissue transplants. It has been shown that activating the PI3K/Akt pathway in cells can enhance the secretion of VEGFA and promote angiogenesis through HIF-1α-dependent and -independent mechanisms [60]. This is consistent with our in vivo experimental results, which showed elevated protein levels of HIF-1α and VEGFA. Moreover, MXene appears to induce distinct PI3K-Akt pathway activities in different tissues [40, 47]. Further research in this field will offer valuable insights into the complexity of MXene's effects and its potential applications in ischaemic flaps therapy. Although our research explores several critical elements of MXene-based ischaemic flap treatment, gaps remain in our comprehension of the fundamental mechanisms, ideal dosing protocols, and extended consequences. Future studies should focus on determining the ideal dose and application frequency of MXene to enhance its therapeutic benefits while reducing potential adverse effects. Additionally, this study only assessed ischaemic flap activity at postoperative day 7, and long-term follow-up studies are needed to assess the lasting and sustained effects of MXene on ischaemic flap survival. Regarding broader implications, the potential for MXene to be used as a universal treatment in future human trials, as well as its further integration into multifunctional biomaterials to enhance ischaemic flap therapy, needs to be explored in future studies.

Fig. 9
figure 9

MXene enhanced the phosphorylation of PI3K and Akt in skin flaps under ischaemic microenvironment. A Western blotting bands of PI3K, p-PI3K, Akt, and p-Akt proteins. B, C Quantified the ratios of p-PI3K/PI3K and p-Akt/Akt proteins levels among the 5 groups. D, E The necrotic area was imaged on POD 7, and the percentage of the flap's necrotic area was quantified in the graph. F, G The vascular network and corresponding blood flow intensity were quantified using LDBF on POD 7. N = 3 per group. SEM error bars are used. Significance (*): p value < 0.05

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Conclusion

This study successfully prepared multi-enzyme active MXene nanosheets, which are injectable and biocompatible. MXene exhibited exceptional antioxidant activity in both HUVECs and ischaemic flaps. Under in vitro OGD conditions, MXene primarily enhanced the survival of HUVECs by scavenging ROS and inhibiting pyroptosis. Furthermore, MXene promoted HUVECs migration and angiogenic capacity through mechanisms involving the upregulation of VEGFA and VE-Cadherin expression. In vivo, MXene regulated the ischaemic microenvironment by activating the PI3K-Akt signaling pathway, modulating cellular ROS levels, reducing pyroptosis in dermal endothelial cells, and decreasing pro-inflammatory cytokine levels, thereby restoring skin flap vitality. This regulation was accompanied by the secretion of VEGFA and HIF-1α, which promoted neovascularization and facilitated appropriate collagen deposition in skin tissues. In summary, the study offers a crucial theoretical foundation for using MXene to enhance the survival of ischaemic flaps and presents a promising strategy for treating ischaemic flap necrosis and other ischaemia-related tissue injuries. These findings could have a broad impact on the future development of nanomedicine treatments for ischaemic flap necrosis, an area that has previously received limited attention.

Methods

Materials

MXene were purchased from Xfnano Materials Tech Co., Ltd (Nanjing, China). LY294002 (C19H17NO3, purity ≥ 99.86%, 154,447–36-6) was purchased from Med Chem Express (NJ, USA).

Cells culture

The HUVECs and L929 fibroblast cells were obtained from Procell Life Science & Technology Co., Ltd. Cells were grown in an incubator at 37 °C with 5% CO2 and 95% air. The culture medium was Dulbecco's Modified Eagle Medium (DMEM; Procell, PM150210), supplemented with 10% sterile fetal bovine serum (FBS; Gibco, 10099141C) and 1% penicillin–streptomycin solution (Gibco, 1,719,675). To simulate in vivo hypoxia–ischaemia injury, an OGD model was used in vitro. The OGD treatment was conducted as previously described [61]. In brief, HUVECs were incubated in a pre-mixed gas (94% N2, 5% CO2 and 1% O2) incubator (Thermo Fisher Scientific, MA, United States) with glucose-free DMEM (Gibco, 11,966–025) for certain hours. The control group was cultured in normal DMEM with 5% CO2 for the same duration.

Cell viability assay

Cell viability of HUVECs and L929 cells was assessed using the CCK-8 assay (Med Chem Express, HY-K0301) according to the manufacturer’s instructions. In brief, HUVECs and L929 cells were seeded separately at 8 × 103 cells per well in a 96-well plate and cultured for 24 h in DMEM medium supplemented with 10% FBS. Fresh medium with different concentrations of MXene was then used to replace the original medium. HUVECs were treated with either 24 or 48 h of normoxia with DMEM or 24 h of OGD. Similarly, L929 cells were treated with 24 or 48 h of normoxia with DMEM. Each well received fresh DMEM solution containing 10% CCK-8 and was incubated at 37 °C for an additional 2 h. The absorbance (O.D. value) at 450 nm was measured using a microplate reader (Thermo Fisher Scientific, USA). The cells treated with 24 h of normoxia in DMEM were used as the NC group. Calculate relative cell viability according to the formula: Normalized cell viability = O.D. value of treatment group / Average O.D. value of NC group.

Total antioxidant capacity assay

The free radical scavenging capacity of MXene nanosheets was evaluated by incubating 200 μM solutions of DPPH, ABTS, •OH, and O2•− in the dark at 37 °C. UV–visible absorbance was then measured at 517 nm, 734 nm, 510 nm, and 550 nm, respectively, to determine the scavenging efficiency of each free radical. Various concentrations of MXene were tested across all experiments.

DCFH-DA staining

For ROS detection, HUVECs were treated with a 5 μM DCFH-DA solution (Beyotime, S0035S) at 37 °C for 30 min, following the manufacturer’s instructions. Following incubation, the cells were rinsed three times with PBS (Procell, PB180327) and then visualized using a confocal microscope.

Scratch assay

HUVECs were plated in a 6-well plate, and a wound was created in the confluent cell layer using a 200 µL pipette tip. Following the wound, cells were cultured at 37 °C for 24 h. Medium supplemented with different concentrations of MXene (0, 10, 20, 40, or 80 μg/mL) was added to each well, with or without OGD treatment. The migration of HUVECs was monitored and documented at 0, 12, and 24 h using an inverted microscope, and the migration area was quantified using ImageJ software.

Tube formation assay

The in vitro angiogenic activity of HUVECs was assessed using a tube formation assay. Briefly, HUVECs (1.5 × 105 cells/mL) were seeded into Matrigel-coated wells (100 µL Matrigel with growth factors, Corning, 356,234). After treatment, HUVECs were incubated at 37 °C for 6 h. Once tube formation was observed, the cells were stained with calcein AM (Beyotime, C2012) for 30 min. Images of the tube formation were captured using a confocal microscope. The number of junctions and total branching length were quantified using the ImageJ software.

Immunocytochemistry

HUVECs cultured on glass slides were fixed with 4% paraformaldehyde for 30 min and permeabilized with 0.1% Triton X-100 (Aladdin, T109027) for 5 min. The slides were blocked with 10% goat serum (Beyotime, PB180327) for 30 min. Afterward, the slides were incubated overnight at 4 °C with primary antibodies for GSDMD-N (1:200; HuaBio, ER1901-37) and C-CASP1 (1:200; Cell Signaling Technology, 4199), followed by incubation with secondary antibodies at room temperature for 1 h. The secondary antibodies used were goat anti-rabbit IgG H&L (Alexa Fluor®594; Abcam, ab96885) and goat anti-rabbit IgG H&L (Alexa Fluor®488; Abcam, ab150077). Nuclei were stained with DAPI (Abcam, ab285390), and images were captured using a confocal microscope.

Live/dead assay

Live/dead staining was performed to assess the viability of HUVECs. The cells were seeded in a 6-well plate and cultured for 24 h. At the start of the experiment, HUVECs were treated with varying concentrations of MXene with or without OGD treatment. After an additional 24 h, the cells were stained with Calcein-AM and propidium iodide (EFL, Engineering For Life) for 15 min. The stained cells were then observed and imaged using a confocal fluorescence microscope.

Random-pattern skin flap model

All animal experiments in this study were conducted in compliance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals and were approved by the Animal Care and Use Committee of Wenzhou Medical University (wydw2024-0059). Male C57BL/6 J mice (6–8 weeks old, average weight 20–30 g) were obtained from the Experimental Animal Center of Wenzhou Medical University (license no. SCXK [ZJ] 2020–0001). The mice were housed under normal conditions (21–25 °C, humidity: 50–60%, 12-h light/dark cycle) with free access to food and water.

The mouse random-pattern skin flap model was performed as previously described [62]. Briefly, mice were anesthetized with an intraperitoneal injection of 1% (w/v) pentobarbital sodium (50 mg/kg). After hair removal, a 1.5 cm × 4.5 cm flap was created on the back of each mouse. In the control group, both bilateral sacral arteries were preserved, while in the other groups, they were cut. The flap was then sutured using 4–0 silk thread.

The flap was equally divided into 3 areas from its pedicle to the distal part in the following order: area I, area II, and area III. A flap length–width ratio greater than 1.5–2:1 results in the occurrence of necrosis in the skin. On postoperative day 7, the flap necrotic area remains stable; at this time, area I is normally healthy, and area III is normally necrotic without intervention, whereas area II, especially the peri-necrotic area, is usually ischaemic and tends to be necrotic.

Groups and treatments

On postoperative day 7, euthanasia was performed on the flap group and C57BL/6 J mice treated with MXene (n = 4) to collect skin samples for RNA sequencing. The remaining C57BL/6J mice (n = 75) were randomly assigned to 8 groups: Sham group (n = 15), Flap + Saline group (n = 15), Flap + MXene-1 group (n = 15), Flap + MXene-2 group (n = 18), Flap + MXene-4 group (n = 3), Flap + MXene-8 group (n = 3), Flap + LY294002 group (n = 3), and Flap + MXene + LY294002 group (n = 3). In the Saline and MXene treatment groups, different concentrations of 100 µL MXene (1, 2, 4, and 8 mg/mL) were injected subcutaneously with a microneedle during surgery. The LY294002 groups received an intraperitoneal injection of 100 µL LY294002 (25 mg/kg/day) 30 min prior to the flap surgery. The Flap + Saline group was treated with the same volume of saline following the same protocol. Euthanasia was performed on POD 7, and tissues from the peri-necrotic area were collected for further histological analysis.

Flap necrotic area assessment

The macroappearance of the flaps, including color, shape, and hair condition, was monitored daily. On POD 7, high-quality dorsal photographs were taken using a digital camera. Image-ProPlus software (version 6.0; Media Cybernetics, Silver Spring, MD, USA) was used to assess the survival rate of the ischaemic flaps. The percentage of necrotic area was calculated using the formula: necrotic rate = (necrotic area/total area) × 100%.

LDBF imaging

This study employed LDBF imaging technology to observe and analyze the blood supply and vascular network of the flaps. On POD 7, the mice were anesthetized and placed in a safe, quiet environment. A laser Doppler imaging system (Moor Instruments, Axminster, UK) was then used to assess the blood supply and microvascular system of the flaps. The moorLDI Review software (version 6.1; Moor Instruments) was utilized to calculate perfusion units (PU). Each mouse was measured three times, and the average value was used for subsequent statistical analysis.

Western blotting

Tissue samples (5 mm × 5 mm) were collected from the peri-necrotic region of the flaps and homogenized in ice-cold RIPA lysis buffer (Beyotime, P0013B), which was supplemented with a protease inhibitor cocktail (Sigma-Aldrich, P8340) and phosphatase inhibitor cocktail III (Sigma-Aldrich, P0044). The homogenate was then centrifuged at 20,000 g for 30 min at 4 °C to obtain the tissue lysate. The protein concentration was determined using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, 23,227). A total of 30 mg of protein was loaded onto a 12.5% SDS-PAGE gel and transferred to a PVDF membrane. The membrane was blocked with 5% non-fat milk, incubated with primary antibodies at 4 °C for 18 h, and subsequently incubated with HRP-conjugated secondary antibodies at room temperature for 2 h. Protein bands were visualized using the Omni-ECL Pico Light Chemiluminescence Kit (EpiZyme, SQ201) and captured with a ChemiDoc imaging system (Bio-Rad). Band analysis was conducted using Image Lab software (Bio-Rad).

The primary antibodies used included NLRP3 (1:1,000; Cell Signaling Technology, 15,101), IL-18 (1:1,000; Affinity Biosciences, DF6252), β-actin (1:5,000; Servicebio, GB15003-100), CASP1 (1:4,000; HuaBio, ET1608-69), GSDMD-N (1:1,000; Affinity Biosciences, AF4013), PI3K (1:1,000; HuaBio, JE65-38), p-PI3K (1:1,000; HuaBio, HA721672), Akt (1:1,000; HuaBio, ET1609-51), p-Akt (1:1,000; HuaBio, HA722129), VE-Cadherin (1:1,000; Boster, A02632-2), and VEGFA (1:2,000; Abcam, ab214424). The secondary antibodies used were goat anti-rabbit IgG (H + L)-HRP conjugate (Proteintech, SA00001-2) and goat anti-mouse IgG (H + L)-HRP conjugate (Proteintech, SA00001-1).

Immunofluorescence staining

Mouse skin tissues were fixed in 4% paraformaldehyde, followed by dehydration, paraffin embedding, and sectioning to 4 μm thickness. The sections underwent deparaffinization and rehydration, followed by antigen retrieval in sodium citrate buffer. After cooling to room temperature, the sections were blocked with 3% BSA (Beyotime, ST025) for 30 min. Primary antibodies were then applied overnight. On the following day, sections were incubated with secondary antibodies at room temperature for 1 h. Nuclear staining was performed using DAPI.

The primary antibodies used included CASP1 (1:200; HuaBio, ET1608-69), CD31 (1:200; Servicebio, GB12063-100), and α-SMA (1:200; Proteintech, 14,395–1-AP). Secondary antibodies included goat anti-rabbit IgG H&L (Abcam, ab96883) and goat anti-mouse IgG H&L (Abcam, ab96873).

DHE staining

For cell-based experiments, after appropriate treatment, HUVECs were incubated with the DHE probe (Beyotime, S0063) at a concentration of 2.5 μM and 37 °C for 30 min, following the manufacturer's instructions. The cells were then stained with Hoechst (Beyotime, C1028) at 37 °C for 10 min. After washing three times with PBS, the cells were imaged using a confocal microscope.

For tissue analysis, tissue samples from peri-necrotic area were placed in a 30% sucrose solution until they precipitated at the bottom. The dried tissues were then placed on clean filter paper to remove excess water, sealed with OCT, and cooled until completely solidified. The tissues were sectioned into 14 μm thick frozen sections and treated with a spontaneous fluorescence quenching reagent for 5 min following thawing. The DHE solution was diluted with PBS to an appropriate concentration and quickly applied to the tissue samples. These samples were then incubated in a dark water bath at 37 °C for 30 min. After washing the tissue sections three times with PBS at room temperature for 5 min each, DAPI was added, and images were captured using a confocal microscope.

5-FAM-conjugated collagen hybridizing peptide (F-CHP) staining

Collagen damage was assessed using F-CHP staining (3Helix Inc, FLU60). Tissue sections were processed through standard deparaffinization and hydration steps. The CHP solution was diluted with PBS to the appropriate concentration, placed in a sealed microcentrifuge tube, and heated at 80 °C for 5 min. The tube containing the heated CHP solution was immediately immersed in an ice-water bath for 15–90 s to avoid thermal damage to the tissue, and then allowed to cool to room temperature. The tube was centrifuged to restore volume. The solution was rapidly applied to the tissue samples and incubated in the dark at 4 °C for 12 h. After staining, the samples were washed three times with PBS at room temperature and sealed with DAPI. Images were then captured using a confocal microscope.

Hemolysis assay

The blood compatibility of MXene was evaluated through an in vitro hemolysis test. In summary, venous blood was extracted from mice via the retro-orbital sinus and centrifuged to isolate red blood cells (RBCs). These RBCs were washed three times with PBS, then resuspended in PBS to achieve a 5% concentration. Various concentrations of MXene were added to 1 mL of the RBC suspension and incubated at 37 °C with agitation for 2 h in a CO2 incubator. Distilled water was used as a positive control, and saline was used as a negative control. After centrifugation, the absorbance of the supernatant was measured at 545 nm using a spectrophotometer.

Histopathological examination

Hematoxylin–eosin (H&E) staining (Solarbio, G1120) was performed on tissue sections from various experimental groups following the manufacturer's instructions. Bright-field images were captured at 200 × magnification using a light microscope (Olympus, Japan). The mean microvascular density (number of vessels per mm2) in the peri-necrotic area of the flaps was calculated by randomly selecting fields of view. Additionally, an independent pathologist, blinded to the experimental conditions, evaluated hepatocyte morphology, inflammatory activity, liver fibrosis, as well as tubular atrophy and interstitial inflammation. For Masson’s trichrome staining, sections were dewaxed, rehydrated, and stained using the Masson trichrome stain kit (Solarbio, G1340) according to the provided protocol. The slides were observed under a light microscope (Olympus, Japan).

ELISA kit

The protein levels of HIF-1α, VEGFA, GSDMD-N, C-CASP1, cleaved IL-1β, and cleaved IL-18 in skin flaps were measured using ELISA kits (Mlbio, YJ063240, YJ037273, YJ206369, YJ271014, YJ200171, YJ285252). Skin tissues were homogenized in extraction buffer, centrifuged at 1000 g and 4 °C for 20 min, and the supernatants were collected for further analysis. Then, 50 μL of the sample solution and 100 μL of HRP-conjugated corresponding antibodies were added to the sample wells and incubated at 37 °C for 1 h. After washing, 50 μL of substrate A and B were added to each sample well, followed by incubation at 37 °C in the dark for 15 min. The optical density of the samples was measured at 450 nm for protein quantification.

Measurement of malondialdehyde (MDA) level

The total MDA levels were measured following the manufacturer’s protocol after homogenizing mouse flap tissues in 0.9% sterile saline. The MDA Content Assay Kit (BC0025) was obtained from Solarbio Science & Technology (Beijing, PRC).

Transcriptomic analysis

Tissue samples from the surviving-necrotic border area of the skin were collected 7 days post-MXene treatment (MXene group, n = 4), and the corresponding areas from untreated ischaemic flap were used as the control (Flap group, n = 4). RNA purification, reverse transcription, library construction and sequencing were performed at Majorbio Bio-pharm Biotechnology Co., Ltd. (Shanghai, China). Total RNA was extracted from each sample using TRIzol reagent (Invitrogen, USA). mRNA was selected using oligo(dT) beads and fragmented with a buffer. Double-stranded cDNA was then synthesized with random hexamers. The cDNA was processed through end-repair, phosphorylation, and adapter addition. Libraries were size-selected for 300–400 bp fragments using magnetic beads and amplified via PCR for 10–15 cycles. After quantification using Qubit 4.0, the sequencing library was prepared and sequenced on the NovaSeq X Plus platform (PE150) using the NovaSeq Reagent Kit. For data analysis, DESeq2 was utilized to identify differentially expressed genes, with P values < 0.05 and fold changes > 1.5 or < 0.67 considered significant. GO enrichment, KEGG pathway enrichment analysis, and GSEA were conducted for further bioinformatics analysis.

Statistics

Statistical analyses were performed using the SPSS 22 software (USA). Data are presented as mean ± SEM. All data shown here were normalized to correct for potential sources of variability. The sample size (n) for each experimental group/condition is indicated, with n representing independent values (not replicates). For comparisons among 4, 5, or 6 groups, ANOVA was conducted, followed by LSD post hoc tests (assuming equal variances) or Dunnett's T3 (when equal variances were not assumed). A p-value of less than 0.05 was considered statistically significant.

Data availability

No datasets were generated or analysed during the current study.

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Acknowledgements

Graphical abstract was drawn by BioRender.com. We would like to thank EditChecks (https://editchecks.com.cn/) for providing linguistic assistance during the preparation of this manuscript.

Funding

This work was supported by the Natural Science Foundation of Zhejiang Province (LQN25H060010, LY23H060004, LY24H110002), National Natural Science Foundation of China (82372540, 82072192, 81801930), Natural Science Foundation of Ningbo (2023J255, 2022J044), Zhejiang Provincial Medical and Health Science Foundation (2024KY155, 2023RC198), Ningbo Major Science and Technology Research Project (2022Z146).

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Author notes

  1. Ningning Yang, Rongrong Hua, Yingying Lai and Peijun Zhu have contributed equally to this work.

Authors and Affiliations

  1. Affiliated Cixi Hospital, Wenzhou Medical University, Ningbo, 315300, Zhejiang, China

    Jian Ding, Liangliang Yang & Lu Ge

  2. School of Pharmaceutical Sciences, Oujiang Laboratory (Zhejiang Lab for Regenerative Medicine, Vision and Brain Health), Wenzhou Medical University, Wenzhou, 325035, Zhejiang, China

    Ningning Yang, Peijun Zhu, Zhouguang Wang, Hongyu Zhang, Liangliang Yang & Lu Ge

  3. Department of Orthopaedics, Zhejiang Provincial Key Laboratory of Orthopaedics, The Second Affiliated Hospital and Yuying Children’s Hospital, Wenzhou, 325027, Zhejiang, China

    Ningning Yang, Yingying Lai, Jian Ding, Xianhui Ma, Gaoxiang Yu, Weiyang Gao & Kailiang Zhou

  4. The Second Clinical Medical College of Wenzhou Medical University, Wenzhou, 325027, Zhejiang, China

    Yingying Lai, Jian Ding, Xianhui Ma, Gaoxiang Yu, Weiyang Gao & Kailiang Zhou

  5. State Key Laboratory of Macromolecular Drugs and Large-Scale Preparation, School of Pharmaceutical Science, Wenzhou Medical University, Wenzhou, 325000, Zhejiang, China

    Ningning Yang & Zhouguang Wang

  6. Cixi Biomedical Research Institute, Wenzhou Medical University, Ningbo, 315300, Zhejiang, China

    Yiheng Xia, Chao Liang, Hongyu Zhang, Liangliang Yang, Kailiang Zhou & Lu Ge

  7. School of Biomedical Engineering, Wenzhou Medical University, Wenzhou, 325035, Zhejiang, China

    Rongrong Hua

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  1. Ningning Yang
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Contributions

Ningning Yang, Liangliang Yang and Lu Ge conceived the experimental design, conducted both laboratory and theoretical investigations, and drafted the manuscript. Rongrong Hua, Yingying Lai, Peijun Zhu, Jian Ding, Xianhui Ma performed some experiments. Yingying Lai, Gaoxiang Yu, Yiheng Xia, Chao Liang and Weiyang Gao analysed some data. Zhouguang Wang, Hongyu Zhang, and Kailiang Zhou revised the manuscript. Zhouguang Wang, Hongyu Zhang, Kailiang Zhou, Liangliang Yang and Lu Ge assisted in designing research, approved the final version and submitted.

Corresponding authors

Correspondence to Zhouguang Wang, Hongyu Zhang, Liangliang Yang, Kailiang Zhou or Lu Ge.

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Animal studies were conducted in compliance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals and were approved by the Animal Care and Use Committee of Wenzhou Medical University (wydw2024-0059).

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The authors declare no competing interests.

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Yang, N., Hua, R., Lai, Y. et al. Microenvironment-adaptive nanomedicine MXene promotes flap survival by inhibiting ROS cascade and endothelial pyroptosis. J Nanobiotechnol 23, 282 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12951-025-03343-9

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Journal of Nanobiotechnology

ISSN: 1477-3155

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