Strontium-luteolin nanoparticles promote M2 macrophage polarization and accelerate acute wound healing via immune microenvironment regulation

Abstract

Precise spatiotemporal control of inflammation serves as an effective means to regulate the inflammatory microenvironment and promote wound healing. This study constructed strontium‒luteolin (Sr‒Lut) nanoparticles with a spherical morphology, which demonstrated outstanding radical scavenging capacity through synergistic metal‒ligand effects. Specifically, Sr-Lut efficiently scavenges reactive oxygen species, inhibits the TNF-α/NF-κB and JAK-STAT signaling pathways, promotes M2 polarization, and simultaneously reduces the proinflammatory factor IL-1β and increases the anti-inflammatory factor IL-10. Transcriptomic analysis revealed that Sr-Lut reprogrammed macrophages to downregulate Ccl4 and Retnlg while upregulating extracellular matrix remodeling-associated genes. In vivo experiments demonstrated that Sr-Lut accelerated wound closure in a dose-dependent manner, achieving a healing rate of 94.33 % by day 11, which was significantly greater than the 58.54 % reported in the control group, while also enhancing re-epithelialization and collagen deposition. Wound tissue RNA sequencing revealed that Sr-Lut inhibits the IL-17, Toll-like receptor and NF-κB signaling pathways while promoting the expression of genes associated with epidermal structural components. In summary, a dual-function nanocomposite with both anti-inflammatory and tissue-repair capabilities has been developed, offering a promising immunomodulatory strategy for wound treatment.

Graphical abstract

The study introduces strontium-luteolin (Sr-Lut) nanoparticles, which uniquely integrate anti-inflammatory and tissue-repair functions through a synergistic metal-ligand coordination effect.

1. Introduction

The healing of acute wounds is a complex biological process involving the regulation of inflammation, cell proliferation, and matrix remodeling, and the precise temporal control of the inflammatory microenvironment is pivotal in determining the quality of repair [[1], [2], [3], [4]]. Under physiological conditions, the early inflammatory response following trauma eliminates pathogens by recruiting neutrophils and macrophages, thereby creating conditions conducive to tissue regeneration [[5], [6], [7], [8]]. However, in clinical practice, pathological factors such as infection, ischemia, or diabetes frequently cause the inflammatory response to become uncontrolled, evolving into persistent chronic inflammation [[9], [10], [11], [12], [13]]. This pathological state is characterized by excessive infiltration of proinflammatory M1 macrophages and their sustained secretion of cytokines such as TNF-α, IL-1β, and IL-6, resulting in a ‘cytokine storm’ [[14], [15], [16], [17]]. This process not only directly damages newly formed tissue but also activates signaling pathways, including the NF-κB and JAK-STAT pathways, creating a positive feedback loop that severely impedes the recruitment and functional activity of M2-reparative macrophages [[18], [19], [20], [21]]. Although existing therapies, such as silver-ion dressings or growth factors, have made some progress in controlling infection and promoting repair, their single-target intervention strategies struggle to achieve simultaneous inflammation resolution and repair initiation [12,22,23]. Consequently, acute wounds often progress to chronic, difficult-to-heal wounds due to inflammatory dysregulation [[24], [25], [26]]. Therefore, the development of dual-function formulations that combine potent anti-inflammatory properties with enhanced tissue repair capabilities has become a major challenge requiring urgent resolution in the field of wound management.

As central regulators of the transition between inflammation and repair in wounds, macrophages are ideal therapeutic targets because of their phenotypic plasticity [[27], [28], [29]]. M1 macrophages release proinflammatory mediators via the TNF-α/NF-κB pathway, which plays a pivotal role in pathogen clearance [[30], [31], [32], [33]]. However, their sustained polarization may delay wound healing. Conversely, M2 macrophages secrete anti-inflammatory factors and growth factors such as VEGF and TGF-β through the IL-10/STAT6 pathway, thereby promoting angiogenesis and collagen deposition [23,34,35]. However, achieving efficient conversion from M1 to M2 remains a current research bottleneck. Owing to their unique targeted delivery and multitarget regulatory capabilities, nanomedicines have significant potential in reprogramming the immune microenvironment [[36], [37], [38], [39], [40]].

Strontium ions (Sr²⁺), as bioactive metal ions, promote angiogenesis and inhibit osteoclast differentiation by regulating the NFATc1 pathway [[41], [42], [43]]. However, their standalone application is constrained by poor water solubility, low bioavailability, and susceptibility to rapid loss [[44], [45], [46]]. The natural flavonoid luteolin exhibits broad-spectrum antioxidant and anti-inflammatory activity and is capable of blocking the NF-κB pathway by inhibiting IκBα phosphorylation [[47], [48], [49]]. However, its strong hydrophobicity, rapid metabolism, and difficulty in achieving local accumulation at wound sites pose significant limitations [[50], [51], [52]]. On the basis of this, utilizing the coordination chemistry between strontium ions and the phenolic hydroxyl group of luteolin, the construction of nanoscale metal‒organic complexes can achieve synergistic enhancement through the following advantages. (1) The coordination of Sr²⁺ enhances luteolin stability and prolongs its half-life. (2) Luteolin acts as a ligand to promote Sr²⁺ endocytosis and intracellular sustained release. (3) Metal‒ligand synergistic effects concurrently scavenge reactive oxygen species and modulate multiple inflammatory pathways, thereby precisely reprogramming macrophage polarization.

This study successfully synthesized Sr-Lut nanoparticles for acute wound treatment via a coordination-driven self-assembly strategy (Scheme 1). In vitro experiments confirmed that Sr-Lut efficiently scavenges DPPH, ABTS⁺ and ·OH radicals and significantly inhibits LPS-induced ROS accumulation in RAW264.7 macrophages. Transcriptomic analysis revealed that Sr-Lut promotes M1-to-M2 phenotype conversion by synergistically inhibiting the TNF-α/NF-κB, IL-17, and JAK-STAT signaling pathways. In acute wound models, Sr-Lut achieved a wound healing rate of 94.33 % by day 11, representing a 61 % improvement over the control group, while significantly enhancing granulation tissue thickness and collagen cross-linking. Notably, no abnormalities were observed in either histological or hematological parameters within the treatment group, confirming its excellent biological safety profile. The metal–natural product synergistic regulation strategy established in this study offers a promising therapeutic approach for inflammatory dysregulation in wound healing, combining immunomodulatory effects with the promotion of tissue regeneration.

Scheme 1.

(a) Synthesis of Sr-Lut. (b) Application of Sr-Lut in acute wound treatment. (c) Mechanism by which Sr-Lut regulates inflammation. (d) Sr-Lut promotes wound healing.

2. Results and discussion

2.1. Characterization and antioxidant capacity of Sr-Lut

Transmission electron microscopy (TEM) images revealed that the Sr-Lut complex consists of spherical nanoparticles with uniform dispersion and no apparent agglomeration (Fig. 1a–b). X-ray photoelectron spectroscopy (XPS) detected characteristic peaks of Sr, C, and O, confirming the successful incorporation of Sr into the composite (Fig. 1c). The Sr 3d spectrum exhibited a single characteristic peak at 133.8 eV, attributed to the 3d orbital binding energy of Sr²⁺, indicating successful coordination of strontium in its divalent ionic form within the luteolin skeleton (Fig. 1d). The O 1s spectrum exhibited a single characteristic peak at 531.2 eV. This binding energy lies between that of typical metal oxide lattice oxygen (∼530.0 eV) and organic hydroxyl oxygen (∼532.0 eV), indicating that the hydroxyl oxygen in luteolin coordinates with Sr²⁺, leading to homogenization of its chemical environment (Fig. 1e). Fourier transform infrared spectroscopy (FTIR) revealed broadening and redshifting of the phenolic hydroxyl ν(O-H) absorption peak of luteolin (3400–3200 cm⁻¹), confirming the formation of metal‒ligand bonds (Fig. 1f).

Fig. 1.

Characterization and antioxidant capacity of Sr-Lut. (a–b) TEM images of Sr-Lut. (c) X-ray photoelectron spectroscopy (XPS) full spectrum of Sr-Lut. (d) XPS spectra of Sr 3d and (e) O 1s. (f) FTIR spectrum of Sr-Lut. (g) Schematic representation of the antioxidant scavenging mechanism of Sr-Lut. (h) DPPH radical scavenging capacity, (i) ABTS⁺ radical scavenging capacity, and (j) ·OH radical scavenging capacity.

Following the successful construction of Sr-Lut nanoparticles, their antioxidant capacity was evaluated. Fig. 1g schematically illustrates the radical scavenging mechanisms across different detection systems. The DPPH assay revealed that the radical scavenging activity of Sr-Lut was strongly concentration dependent (Fig. 1h). At 517 nm, treatment with 200 μg/mL Sr-Lut for 30 min resulted in a 90 % reduction in the intensity of the characteristic DPPH absorption peak (compared with that of the 0 μg/mL control group). Notably, even at low concentrations (40 μg/mL), Sr-Lut demonstrated sustained scavenging capacity, suggesting favorable antioxidant kinetic properties. The ABTS⁺ assay results further validated the potent antioxidant activity of Sr-Lut (Fig. 1i). At 734 nm, the absorbance of the 10 μg/mL Sr-Lut-treated group nearly disappeared after 30 min. Subsequently, the degradation efficacy of Sr-Lut toward the ·OH radicals generated in the Fenton reaction system was evaluated via the methylene blue (MB) degradation method (Fig. 1j). At 664 nm, the intensity change of the characteristic absorption peak of MB directly reflects the amount of ·OH radicals generated. These results indicate that the ability of Sr-Lut to scavenge ·OH radicals increase in a concentration-dependent manner. The above results collectively demonstrate that the Sr-Lut complex has outstanding radical scavenging capabilities across multiple radical systems through synergistic metal‒ligand effects. It should be noted that this study focuses on evaluating the overall performance advantages of the Sr-Lut complex under a specific synthesis ratio. Although the current data confirm the synergistic effect under this specific ratio, a systematic scan of different Sr: Lut molar ratios was not conducted to determine the optimal ratio.

2.2. Sr-Lut scavenges ROS and promotes M2 macrophage polarization

To evaluate the protective effects of different concentrations of Sr-Lut on LPS-stimulated RAW264.7 cells. Intracellular ROS and RNS were detected via DCFH-DA staining, with DAPI used for nuclear labeling. The results revealed minimal green fluorescence in the control group, which presented significantly lower fluorescence intensity than the other groups did (Fig. 2a). Compared with cells in the LPS group, those treated with low or high concentrations of Sr-Lut presented weaker fluorescence. At a Sr-Lut concentration of 50 μg/mL, fluorescence was almost entirely absent, indicating a positive correlation between the Sr-Lut concentration and the efficiency of ROS and NO scavenging. In the flow cytometry histogram, a marked reduction in the number of cells labeled with DCFH-DA was observed as the Sr-Lut concentration increased (Fig. 2b). These findings indicate that Sr-Lut protects cells from LPS-induced oxidative stress damage. Wound healing is a complex process involving inflammation and repair, with macrophage phenotypic switching serving as a central regulatory mechanism. Macrophages exhibit functional plasticity. M1 macrophages eliminate pathogens and necrotic tissue during the early stages of injury while initiating inflammatory responses, although sustained polarization exacerbates tissue damage. M2 macrophages promote tissue repair by secreting anti-inflammatory and growth factors that stimulate fibroblast proliferation and angiogenesis. On this basis, an LPS-induced macrophage polarization model was established to assess the ability of Sr-Lut to alter macrophage phenotypes. CD86 and CD206, which serve as markers for the M1 and M2 phenotypes of RAW 264.7 cells, respectively, were detected via immunofluorescence staining to assess the impact of Sr-Lut on the LPS-induced expression of CD86 and CD206 in these cells. Following Sr-Lut treatment, CD86 levels were decreased, whereas CD206 expression was markedly increased (Fig. 2c). These results indicate that Sr-Lut treatment promotes the polarization of M1 cells toward M2 cells. By collecting cell supernatants and employing an ELLSA assay kit to detect the proinflammatory cytokine IL-1β and the anti-inflammatory cytokine IL-10, we found that Sr-Lut treatment suppressed the LPS-induced increase in IL-1β (Fig. S1) while simultaneously promoting an increase in the anti-inflammatory factor IL-10 (Fig. S2). In short, Sr-Lut demonstrates exceptional antioxidant capacity by scavenging ROS and exhibits outstanding anti-inflammatory properties by promoting the shift of macrophages from the M1 phenotype to the M2 phenotype while simultaneously reducing the expression of proinflammatory factors such as IL-6 and TNF-α.

Fig. 2.

Sr-Lut alleviates LPS-induced oxidative stress and modulates the expression of macrophage polarization markers. Fluorescence quantitative analysis of (a) RNS levels and (b) ROS levels. (c) Representative immunofluorescence images of CD206 (marker for M2 macrophages, green) with DAPI nuclear staining (blue). (d) Representative immunofluorescence images of CD86 (M1 macrophage marker, red) with DAPI nuclear staining (blue). Experimental groups: 1, control group; 2, LPS-stimulated group; 3, low-dose LPS (Sr-Lut); and 4, high-dose LPS (Sr-Lut). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

To further investigate the mechanism by which Sr-Lut modifies macrophage phenotypes, we performed transcriptomic sequencing analysis on LPS-induced RAW 264.7 cells and conducted differential gene expression analysis between the model group and the material treatment group. A heatmap illustrates the differences in gene expression between these two groups (Fig. 3a). Volcano plots revealed that numerous genes associated with anti-inflammatory pathways were upregulated in the Sr-Lut group (Fig. 3b). KEGG pathway enrichment analysis revealed that, following Sr-Lut treatment, the expression of genes involved in classical inflammatory pathways, such as TNF-α and NF-κB, was significantly reduced, suggesting that pathways influencing cell differentiation were also activated (Fig. 3c). TNF-α, a key proinflammatory factor, drives M1 polarization of macrophages via the NF-κB signaling pathway while simultaneously antagonizing M2 differentiation by inhibiting the activity of STAT6 (a core transcription factor of the M2 phenotype). Consequently, downregulation of the TNF-α signaling pathway helps attenuate the proinflammatory microenvironment and promotes the development of an anti-inflammatory repair phenotype (Fig. 3e). TNF-α is one of the key upstream activators of the NF-κB pathway, whereas NF-κB itself serves as the crucial downstream transcription factor for the TNF-α gene, forming a classic positive feedback loop between them. Upon binding to cells, TNF-α molecules activate the NF-κB pathway, thereby promoting cellular polarization toward the M1 phenotype and the secretion of substantial quantities of inflammatory cytokines. Activation of the NF-κB pathway, in turn, substantially enhances the transcription and expression of the TNF-α gene, leading to increased cellular secretion of TNF-α. This potent positive feedback loop can rapidly and persistently maintain a wound in an inflammatory microenvironment that is unfavorable for M2 macrophage differentiation. GSEA revealed that the NF-κB signaling pathway was also effectively suppressed, demonstrating that Sr-Lut not only blocks the TNF-α signaling pathway but also downregulates the NF-κB pathway. This effectively suppresses the inflammatory microenvironment, thereby promoting M2 macrophage polarization (Fig. 3f). Unlike complex pathways involving multiple-tiered kinases, such as TNF-α and NF-κB, the JAK-STAT signaling pathway represents an exceptionally vital and direct signal transduction pathway that links extracellular signals directly to gene expression within the cell nucleus. Upon stimulation, the pathway rapidly activates receptors within the body, initiating the transcription of target genes to swiftly induce the formation of a proinflammatory microenvironment. The results demonstrated that the JAK-STAT signaling pathway was effectively inhibited (Fig. 3g). In short, Sr-Lut exerts a marked inhibitory effect on both the rapid response phase and the sustained amplification phase of inflammation while effectively modulating the direction of macrophage polarization.

Fig. 3.

Transcriptome sequencing analysis reveals differentially expressed genes and enriched key signaling pathways. (a) Heatmap of differentially expressed genes. (b) Volcano plot displaying the distribution of DEGs between the experimental and control groups. The red dots indicate upregulated genes, the blue dots represent downregulated genes, and the gray dots denote genes whose expression did not significantly differ. (c) Bubble plot depicting the distribution of DEGs across KEGG functional categories. (d) Bar chart of significantly enriched KEGG pathways. (e–f) Gene set enrichment analysis (GSEA) revealing representative signaling pathways with significant negative enrichment. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

2.3. Evaluation of Sr-Lut Therapy for acute wounds

To assess the biocompatibility of Sr-Lut, a CCK-8 assay was employed to determine the viability of L929 cells treated with various concentrations of Sr-Lut. Within the concentration range of 5–60 μg/mL, Sr-Lut had no discernible effect on L929 cell viability, with cell survival rates consistently above 85 % (Fig. S3). An acute wound model was established in mouse skin to systematically evaluate the wound-healing effects of Sr-Lut nanoparticles (Fig. 4a). The images revealed that the wound areas in all the groups tended to decrease over time, yet significant differences were observed between the different treatments (Fig. 4b). On the fourth day post-treatment, the wound healing rate in the Sr-Lut (high-dose) group reached 45.2 %, significantly outperforming those in the control group (17.38 %), the SrCl₂ group (37.42 %), and the luteolin group (34.77 %) (Fig. 4c). This trend persisted through day 7, with the Sr-Lut (high) group exhibiting a healing rate (77.88 %) approximately 35.68 % higher than that of the free luteolin group (50.1 %). By day 11, the Sr-Lut (high) group had achieved near-complete wound closure (94.33 %), whereas the control group had healed only 58.54 %. Notably, the effect of Sr-Lut was markedly dose dependent, with the high-dose group demonstrating superior healing rates at all time points compared with the low-dose group. Nevertheless, the low-dose group still outperformed monotherapy with either SrCl₂ or luteolin alone, suggesting a synergistic effect between strontium and luteolin. Since the experimental design focused on the healing outcome, no relevant tests for inflammatory factors were conducted at extremely early time points such as the 1st and 3rd days. This limitation prevented a precise grasp of the dynamic process of inflammation resolution. Additionally, all groups exhibited a stable increasing trend in body weight throughout the 12-day observation period, with no significant fluctuations or decreases observed, indicating favorable biocompatibility (Fig. 4d). H&E staining on day 11 posttreatment revealed intact, mature stratified squamous epithelial structures in the high-dose Sr-Lut group. The epithelial foot processes extended deeply into the granulation tissue, which appeared dense and highly abundant. Numerous neovascular capillaries (with well-formed lumens) and regularly arranged fibroblasts were visible (Fig. 4e). Masson staining further revealed that the collagen fibers in this group exhibited dense, orderly deposition with pronounced transverse cross-linking, indicating entry into the mature remodeling phase (Fig. 4e). Epidermal thickness measurements revealed that the Sr-Lut high-dose group presented the thickest neocornea, indicating that Sr-Lut accelerated keratinocyte proliferation and differentiation (Fig. S4). Analysis of granulation tissue thickness revealed a thickness of 364.67 μm in the high-dose Sr-Lut group, which significantly exceeded that of the control group. This indicates more mature and stable granulation tissue formation (Fig. S5). Collagen content analysis revealed that the Sr-Lut high-dose group presented the greatest proportion of collagen area, indicating effective promotion of collagen synthesis, deposition, and ordered remodeling (Fig. S6). In brief, the aforementioned findings conclusively validate the synergistic effect of strontium ions and luteolin in the treatment of acute wounds. Their coordinated combination achieves an integrated dual function of promoting repair and counteracting damage, thereby providing an optimized microenvironmental regulation strategy for acute wound healing.

Fig. 4.

Promotion effect of Sr-Lut on acute wound healing and histological evaluation. (a) Schematic timeline of the animal experiments. (b) Photographs and (c) healing rates of wounds across different treatment groups. (d) Body weight changes in the mice over 11 days. (e) H&E and Masson's trichrome staining of skin tissue on day 11 posttreatment. The data are presented as the means ± standard deviations (n = 3).

To investigate the molecular mechanisms by which Sr-Lut promotes wound healing, transcriptomic sequencing analysis was performed on wound tissue on day 11 postsurgery. A heatmap displays the differentially expressed genes (DEGs) (Fig. 5a). The volcano plot indicates that Sr-Lut treatment induced differential expression of 837 genes (372 upregulated, 465 downregulated, with a |log2-fold change| > 1 and P < 0.05). Compared with those in the control group, the expression of key genes, including Retnlg, Ccl4, Col2a1, Chil3, Thbs4, and Icol6a6, was significantly altered in the Sr-Lut treatment group. Among these genes, the inflammatory-related genes Retnlg and Ccl4 were downregulated, whereas the extracellular matrix remodeling-related genes Col2a1 and Thbs4 were upregulated (Fig. 5b). KEGG enrichment analysis revealed that the DEGs were predominantly enriched in inflammation-related regulatory pathways, including the IL-17 signaling pathway, Toll-like receptor signaling pathway, NF-kappa B signaling pathway, and chemokine‒cytokine receptor interactions (Fig. 5c). GO molecular function enrichment analysis revealed the most significant enrichment in cytokine activity, chemokine activity, and receptor binding function, with overall downregulation of related gene expression. These findings indicate that Sr-Lut effectively suppressed excessive inflammatory cascades at the wound site (Fig. 5d). Notably, the functional category “epidermal structural components” was also highly enriched, suggesting that Sr-Lut concurrently promoted keratinocyte differentiation and epidermal barrier repair. GSEA confirmed that Sr-Lut significantly suppressed abnormal activation of the IL-17 signaling pathway (NES = 1.45, P = 0.02) and Jak-Stat signaling pathway (NES = 1.35, P = 0.02) while promoting negative regulation of the cytokine‒cytokine receptor interaction pathway (NES = −1.41, P < 0.01) (Fig. 5e‒f). These findings indicate that Sr-Lut effectively suppresses excessively activated proinflammatory signals within the wound microenvironment at the transcriptomic level by regulating inflammatory responses through multiple targets, thereby promoting macrophage polarization and the resolution of inflammation.

Fig. 5.

Analysis of the Sr-Lut-mediated regulation of acute wound tissue transcriptome profiles. (a) Heatmap of differentially expressed genes. Differences in gene expression between control (Control) and Sr-Lut-treated (Treated) skin tissues are shown. (b) Volcano plot of the differentially expressed genes. The red and blue dots represent significantly upregulated and downregulated genes, respectively, whereas the gray dots denote genes whose expression did not significantly differ. (c) KEGG pathway and (d) GO functional enrichment analysis of significantly altered genes. (e–f) Key pathway enrichment features revealed by GSEA. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

H&E staining revealed intact cardiac, hepatic, splenic, pulmonary and renal structures in all groups on day 11 posttreatment, with no evidence of inflammation, necrosis or pathological damage (Fig. 6a). Compared with the control group, the high-dose Sr-Lut group presented no significant differences in organ histological morphology, indicating that there was no cumulative toxicity. All parameters from the blood counts and biochemical analyses remained within normal physiological ranges, further confirming its favorable biocompatibility (Fig. 6b). These findings confirm that Sr-Lut nanoparticles exhibit no dose-dependent organ toxicity or hematological abnormalities, demonstrating excellent biocompatibility and safe clinical application. This study has preliminarily demonstrated that the Sr-Lut nanoparticles exhibit excellent biocompatibility during the experimental period, providing evidence for their use as a new type of wound immunomodulator. This research marks the first step in moving this nanoparticle from concept to application. The comprehensive preclinical pharmacokinetic and long-term toxicological evaluations of these nanoparticles will be an indispensable next step in advancing their transformation process.

Fig. 6.

In vivo biological safety of Sr-Lut. (a) Representative H&E-stained images of cardiac, hepatic, splenic, pulmonary, and renal tissues from all groups on day 11 posttreatment. (b) Key hematological and biochemical parameters. All the data are presented as the means ± standard deviations, n = 3.

3. Conclusion

The nanozymes constructed based on the coordination interaction between Sr²⁺ and the phenolic hydroxyl groups of luteolin exhibit dual functions of antioxidation and immune regulation through the synergistic effect of their components, suggesting a promising therapeutic strategy for acute wound management. Mechanistically, Sr-Lut reprograms the polarization of wounded macrophages by scavenging reactive oxygen species and inhibiting the abnormal activation of the TNF-α/NF-κB, JAK-STAT, and IL-17 signaling pathways. This facilitates the transition of the inflammatory microenvironment from the proinflammatory M1 phenotype to the reparative M2 phenotype. Transcriptomic analysis confirmed that this complex multitargetedly regulates gene networks associated with inflammation and matrix remodeling, significantly accelerating wound re-epithelialization and collagen deposition in vivo. Importantly, Sr-Lut has excellent biocompatibility and safety, with no systemic toxicity. This study has established a mechanism whereby metal‒natural product nanocomposites synergistically modulate the immune microenvironment to promote tissue repair, offering a promising therapeutic approach for the clinical management of wounds associated with infectious or inflammatory dysregulation. The combination of Sr-Lut with promising hydrogels can further facilitate its progress towards clinical application.

4. Methods

4.1. Chemicals

Polyvinylpyrrolidone (PVP, K30, purity ≥99 %), strontium chloride hexahydrate (SrCl₂·6H₂O, purity ≥99.5 %) and luteolin (purity ≥98 %) were procured from Shanghai Aladdin Biochemical Technology Co., Ltd., Sinopharm Chemical Reagent Co., Ltd. and Shanghai Maclin Biochemical Technology Co., Ltd., respectively.

4.2. Synthesis of Sr-Lut

First, 300 mg of polyvinylpyrrolidone (PVP) was dissolved in 10 mL of methanol. Under continuous stirring, 2 mL of a methanol solution of 100 mg strontium chloride hexahydrate (SrCl₂·6H₂O) was added dropwise. After stirring for 5 min, 2 mL of a methanol solution of 50 mg luteolin was added dropwise. Stirring was continued for 3 h. The resulting solution was transferred to a dialysis bag (MWCO 3500 Da) and dialyzed overnight against deionized water to remove free ligands and impurities. The final strontium‒luteolin nanoparticle mixture was collected for subsequent use.

4.3. Characterization

The microstructures of the samples were observed via transmission electron microscopy (TEM, JEOL JEM-2100F) at an accelerating voltage of 200 kV. The elemental composition and chemical states of the samples were analyzed via X-ray photoelectron spectroscopy (XPS, Thermo Scientific ESCALAB 250Xi). Fourier transform infrared spectroscopy (FTIR) was performed on a Nicolet iS20 infrared spectrometer (Thermo Fisher Scientific, USA) within the scanning range of 400–4000 cm⁻¹.

4.4. Free radical scavenging capacity assay

DPPH radical scavenging assay: Mix 2 mL of Sr-Lut solution at various concentrations (0–200 μg/mL) with 2 mL of 0.1 mM DPPH ethanol solution. The mixture was incubated at 25 °C in the dark for 30 min, after which the absorbance (A) was measured at 517 nm. ABTS⁺ radical scavenging assay: The ABTS⁺ stock solution was prepared by mixing equal volumes of 7 mM ABTS with 2.45 mM potassium persulfate aqueous solution and then incubating at 25 °C in the dark for 12 h. The mixture was diluted with PBS to achieve an absorbance of 0.70 ± 0.02 at 734 nm. Twenty microlitres of Sr-Lut solution (0–10 μg/mL) was mixed with 180 μL of ABTS⁺ working solution. After a 6-min reaction, the absorbance was measured at 734 nm. The hydroxyl radical (·OH) scavenging capacity was determined via the methylene blue (MB) probe method: the reaction system comprised 1 mL of MB (20 μg/mL), 1 mL of FeSO₄ (2 mM), 1 mL of H₂O₂ (2 mM), and 1 mL of Sr-Lut solution. After reacting at 37 °C for 30 min, the absorbance at 664 nm was measured.

4.5. In vitro evaluation of ROS- and RNS-scavenging activity

To evaluate the capacity of Sr-Lut to scavenge intracellular ROS and RNS, confocal scanning microscopy was employed for observation and analysis. RAW264.7 cells (a mouse macrophage line) were seeded in confocal culture dishes and cultured overnight. The cells were subsequently treated with 1 μg/mL endotoxin to induce an intracellular oxidative stress state. The cells were subsequently cultured for an additional 24 h with or without Sr-Lut. Next, the cells were stained for 30 min at 37 °C in the dark with the DCFH-DA probe from the ROS detection kit and the DAF-FM DA probe from the nitric oxide detection kit. The cell nuclei were labeled with DAPI for 5 min. Finally, fluorescence images were acquired via a laser confocal microscope, and the RAW264.7 cells were further analyzed via flow cytometry.

4.6. In vitro anti-inflammatory effects and macrophage polarization

For all in vitro anti-inflammatory evaluations, we employed RAW264.7 cells as a model. These cells were stimulated with LPS to induce phenotypic transformation and promote the secretion of relevant cytokines. For the experiments, various concentrations of Sr-Lut were added to the culture medium, and the cells were cocultured for 12 h. To assess macrophage polarization status, immunofluorescence staining was employed to detect the expression of the M1 macrophage marker (CD86) and M2 marker (CD206) at different Sr-Lut concentrations. Furthermore, to analyze anti-inflammatory activity, we collected the supernatant from polarized cells and quantified the levels of the proinflammatory cytokine IL-1β and the anti-inflammatory cytokine IL-10 by corresponding ELISA kits.

4.7. Cytotoxicity

Following 24 h of culture of L929 cells in a 96-well plate until they were fully adherent, the cells were cocultured with the material for 24 h. Subsequently, 10 μL of CCK-8 solution was added to each well, and the mixture was incubated for 30 min. Finally, the optical density (OD) of each well was measured at 450 nm via an enzyme-linked immunosorbent assay reader to assess cellular viability following Sr-Lut treatment.

4.8. Hemolysis test

Mix 0.5 mL of fresh mouse blood with the anticoagulant, add 10 mL of normal saline and mix well. Centrifuge at 1200 r/min for 15 min, discard the supernatant, wash the red blood cells three times with normal saline and resuspend them in normal saline. Take 800 μL of different concentrations of Sr-Lut nanomaterial solutions (experimental group), 800 μL of normal saline (negative control) and 800 μL of deionized water (positive control), respectively. Add 200 μL of red blood cell suspension to each, incubate at 37 °C for 4 h. Then centrifuge at 1200 r/min for 15 min, take the supernatant and measure the absorbance at 545 nm wavelength to evaluate the degree of hemolysis.

All animal experiments were reviewed and approved by the Animal Care and Use Committee of Anhui Medical University (No. LLSC20220731).

4.9. Acute wound model

Healthy male BALB/c mice (8 weeks old) were housed in an SPF-grade environment maintained at constant temperature and humidity (25 ± 2 °C, 50 % ± 10 % humidity) with a 12-h light‒dark cycle. Following anesthesia via intraperitoneal injection of 3 % pentobarbital sodium (40 mg/kg), a circular full-thickness skin defect measuring 10 mm in diameter was created on the dorsal region of the mice, extending to the fascial layer. The experimental animals were randomly assigned to the following groups: control, SrCl₂, baicalin, low-dose Sr-Lut (25 μg/mL), and high-dose Sr-Lut (50 μg/mL). The mice underwent hair removal and disinfection procedures; the control group was treated with 100 μL of PBS solution, while the other three groups applied 100 μL of solution locally to the wound area every day after the operation. On the 4th, 7th and 11th days after the surgery, the wound healing rate was calculated based on the epithelialized edges of the mice, and complications such as infection were observed. The animals were euthanized by an overdose of anesthetic on day 11. The tissue was excised 5 mm beyond the wound margins, and selected samples were fixed in 4 % paraformaldehyde for H&E and Masson's trichrome staining. Wound closure rate (%) = [ (A₀ - A_t)/A₀ ] × 100 %, A₀: Initial area of the wound; A_t: At a specific time point t.

CRediT authorship contribution statement

Xiaowen Zheng: Conceptualization, Data curation, Formal analysis, Methodology, Writing – original draft. Wenqi Wang: Conceptualization, Data curation, Formal analysis, Methodology. Xiaolong Wei: Conceptualization, Investigation. Jianguo Niu: Conceptualization, Data curation, Formal analysis. Wei Zhang: Data curation, Formal analysis. Zhenxun Lin: Data curation, Formal analysis. Wei Wei: Data curation, Formal analysis. Shipeng Ning: Conceptualization, Funding acquisition, Methodology, Resources, Validation. Min Wang: Conceptualization, Investigation, Supervision. Xianwen Wang: Funding acquisition, Project administration, Resources, Validation, Writing – review & editing. Qingjun Wei: Formal analysis, Funding acquisition, Methodology, Project administration, Resources, Software, Validation, Writing – review & editing.

Declaration of competing interest

All the authors have no conflicts to declare.

Appendix A. Supplementary data

The following is the Supplementary data to this article.

Data availability

Data will be made available on request.