Natural multi-active nanoparticles integrated biological hydrogel microcarriers for wound healing

Abstract

Bioactive substance-integrated hydrogels have demonstrated efficacy in diabetic wound treatment. However, challenges remain in identifying naturally derived, multifunctional active substances capable of addressing the complex pathophysiology of wounds, as well as in tailoring hydrogels to enhance their suitability for wound applications. Here, we present a novel biological hydrogel microcarrier system by integrating Bletilla striata-derived nanoparticles (PdNPs) and polydopamine nanozymes (PDAs) into a hyaluronic acid-methacrylate (HAMA) hydrogel. PdNPs can polarize over-activated macrophages to an anti-inflammatory phenotype and restore fibroblast functionality. Meanwhile, PDAs act as potent reactive oxygen species (ROS) scavengers and protect fibroblasts from oxidative stress-induced apoptosis. When encapsulated into HAMA microcarriers, the PdNP + PDA@HAMA microcarriers significantly accelerate wound healing in a diabetic rat model. These outcomes demonstrate the therapeutic potential of our natural, multifunctional hydrogel microcarriers as a promising wound dressing platform for the treatment of chronic diabetic wounds.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12951-025-03666-7.

Introduction

Chronic non-healing wounds are a significant complication of diabetes, affecting nearly 110 million diabetic patients worldwide. Alarmingly, 84% of these ulcers ultimately lead to non-traumatic lower-limb amputations, posing a severe threat to public health [1, 2]. Wound healing is a complex and dynamic process involving hemostasis, inflammation, proliferation, and remodeling. During hemostasis, platelets release platelet-derived growth factor (PDGF) and transforming growth factor-β (TGF-β) to initiate clot formation and recruit immune cells. The inflammatory phase is mediated by neutrophils and macrophages, which produce pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) to clear debris, followed by a shift to anti-inflammatory cytokines such as interleukin-6 (IL-6) and transforming growth factor-β (TGF-β) to prevent chronic inflammation. In proliferation, fibroblasts are activated to deposit collagen, while vascular endothelial growth factor (VEGF) and epidermal growth factor (EGF) promote angiogenesis and re-epithelialization. Finally, the remodeling phase involves extracellular matrix reorganization through matrix metalloproteinases (MMPs), leading to scar tissue maturation and restoration of tissue integrity [3–5]. The pathogenesis of chronic diabetic wounds is primarily attributed to the overactivation of pro-inflammatory M1 macrophages and the impaired activation and proliferation of fibroblasts, resulting in prolonged inflammation and hindered tissue remodeling [6, 7]. Although synthetic medications such as nonsteroidal anti-inflammatory drugs, antibiotics, and growth factors have demonstrated efficacy in reducing inflammation and promoting tissue regeneration [8–10], their application is often limited by systemic side effects and poor targeting efficiency. Notably, with advances in bioengineering technology, bioactive substance-integrated hydrogel platforms can realize targeted drug delivery to wound sites and sustained therapeutic release, thereby improving treatment outcomes [9, 11–13]. However, these systems usually require specific processing and storage conditions to preserve bioactivity, resulting in increased technical complexity and cost [14, 15]. In addition, conventional hydrogel encapsulation often lacks precise control over drug release and may impede wound breathability, further impairing the healing process. Therefore, developing innovative therapeutic strategies for effective wound regeneration is still demanding.

In this paper, we presented a biological hydrogel microcarrier system integrated with naturally derived multi-active nanoparticles for the treatment of diabetic chronic wounds, as schemed in Fig. 1. Nature-derived formulations are emerging as superior alternatives to conventional medications due to their excellent biocompatibility, multifunctional therapeutic potential, and reduced side effects. These formulations often possess intrinsic anti-inflammatory, antioxidant, and antimicrobial properties, enabling them to target multiple pathological processes simultaneously and better meet clinical demands [16, 17]. Recent research has highlighted the potential of traditional Chinese medicine in modulating various phases of the wound healing process [18]. Among these, Bletilla striata, a well-known traditional traumatology herb, has shown significant efficacy in hemostatic activity, anti-inflammatory properties, and the ability to promote tissue regeneration [19, 20]. Notably, plant-derived nanoparticles are emerging as a new class of therapeutic agents, offering several advantages over conventional drugs, including low toxicity, sustainable plant sources and scalable production [21, 22]. In parallel, polydopamine, a naturally occurring biopolymer with excellent antioxidation properties and high biocompatibility, has been shown to effectively mitigate reactive oxygen species (ROS)-induced inflammation and apoptosis [23, 24]. Moreover, microfluidic technology, with its ability to precisely manipulate fluid dynamics and encapsulate bioactives under mild conditions, offers a promising platform for the fabrication of uniform, functional microparticles for biomedical applications [25, 26]. Therefore, we conceived that integrating plant-derived nanoparticles and polydopamine nanozymes (PDAs) within hydrogel microcarriers via microfluidic synthesis holds great promise for addressing the challenges of non-healing wound regeneration.

Fig. 1.

Co-encapsulation of PdNPs and PDAs into HAMA hydrogel microcarriers for diabetic wound healing. a, Schematic illustration of the extraction of PdNPs from the Chinese medicine Bletilla striata and the synthesis of PDAs, followed by microfluidic encapsulation into HAMA hydrogel microcarriers. b, Overview of the therapeutic mechanisms of PdNP + PDA@HAMA microcarriers in diabetic wound healing, including ROS scavenging, inflammation inhibition and fibroblast function restoration

Herein, we developed microfluidic hyaluronic acid-methacrylate (HAMA) hydrogel microcarriers co-encapsulating Bletilla striata-derived nanoparticles (PdNPs) and PDAs for diabetic wound management. PdNPs were extracted from fresh Bletilla striata juice using a density gradient centrifugation method, while PDAs were synthesized by polymerizing dopamine under alkaline conditions. The PdNPs contained bioactive components capable of polarizing over-activated macrophages to an anti-inflammatory phenotype and restoring fibroblast functionality. Meanwhile, the PDAs exhibited strong anti-oxidative properties, effectively scavenging excessive ROS and inhibiting fibroblast apoptosis. By employing a single-emulsion microfluidic chip, both PdNPs and PDAs were encapsulated into HAMA hydrogel microcarriers (PdNP + PDA@HAMA). Attributing to the biocompatibility and porosity of the HAMA hydrogel, the microcarriers provided additional advantages including wound adhesion, air permeability and sustained nanoparticle release. In a rat diabetic wound model, PdNP + PDA@HAMA microcarriers significantly accelerated wound healing by reducing inflammation, alleviating oxidative stress and promoting wound remodeling. These results highlighted the potential of PdNP + PDA@HAMA as a multifunctional, natural-derived patch for chronic wound management.

Results and discussions

Bletilla striata, a traditional Chinese medicine, has been reported to possess multiple pharmacological properties, including hemostatic, anti-inflammatory, and antioxidant qualities, and the ability to accelerate wound healing [27]. Based on these attributes, we hypothesize that nanoparticles derived from Bletilla striata may retain similar functionalities, thereby facilitating wound repair and regeneration. To investigate this, PdNPs were extracted from fresh Bletilla striata juice using a density gradient centrifugation method (Fig. 2a), and characterized using Dynamic Light Scattering (DLS) and Transmission Electron Microscopy (TEM). DLS analysis indicated that PdNPs had a hydrodynamic diameter of 165 ± 16 nm and a zeta potential of −18 ± 9 mV (Fig. 2c, d). TEM demonstrated that PdNPs exhibited a nearly spherical morphology with a membrane-enclosed vesicle-like structure (Fig. 2b). Given the traditional use of Bletilla striata in treating hemoptysis, hematemesis, edema, and wound related-ailments, we conducted a biochemical composition analysis on PdNPs to identify compounds with potential benefits for diabetic wound healing. Utilizing non-targeted metabolomics, we identified 244 compounds in PdNPs and 256 compounds in Bletilla striata (Fig. 2e, Table S1). The analysis revealed that both PdNPs and Bletilla striata were rich in benzenoids (17.1% and 16.7%) and organic oxygen compounds (12.8% and 10.3%). In contrast to Bletilla striata, the PdNPs showed a higher proportion of organic acids and their derivatives (10.7% vs. 5.4%) and a lower proportion of lipids and lipid-like molecules (4.3% vs. 10.5%) (Fig. 2f, g). These results suggest an enrichment of bioactive compounds during the PdNP extraction process, highlighting the potential of PdNPs as a refined therapeutic agent for wound management.

Fig. 2.

Extraction and characterization of PdNPs. a, Schematic presentation of the PdNPs extraction purification procedure. b, TEM image of PdNPs. Scale bar = 100 nm. c, Particle size distribution of PdNPs measured by DLS. d, Zeta potential distribution of PdNPs measured by DLS. e, Pie chart displaying the biochemical compositions of PdNPs and Bletilla striata. f-g, Top ten metabolites identified in PdNPs and Bletilla striata. Data are presented as mean ± SD in f and g

Chronic inflammation is a hallmark of diabetic wounds, characterized by the persistent polarization of local macrophages into the pro-inflammatory M1 phenotype. This results in the sustained release of pro-inflammatory cytokines, such as TNF-α and IL-6 [28, 29]. Effective tissue repair requires the timely transition of macrophages from the M1 to the anti-inflammatory M2 phenotype at the wound site. To investigate the impact of PdNPs on macrophage polarization, we assessed the expression of phenotypic markers and quantified secreted cytokines. To mimic the resident macrophages in wound tissue, lipopolysaccharide (LPS) was used to drive RAW 264.7 cells to differentiate into M1 macrophages and PdNPs were then incubated for 24 h. Flow cytometry revealed that LPS stimulation increased the population of CD86+ (M1-type) macrophages to 42.3 ± 4.9%, compared to only 1.98 ± 0.41% in the untreated control group, confirming successful M1 polarization. Following PdNPs treatment for an additional 24 h, the M1/M2 macrophage ratio decreased from 5.61 ± 0.30% in a dose-dependent manner, reaching a minimum M1/M2 ratio of 2.57 ± 0.04% at a PdNP concentration of 50 µg/mL (Fig. 3a, b).

Fig. 3.

Effects of PdNPs on macrophage polarization. a, Flow cytometry analysis of RAW 264.7 cells cultured with PdNPs at different concentrations for 24 h, showing expression of M1 phenotype (APC-CD86) and M2 phenotype (PE-CD206) markers. b, Quantification of the M1/M2 ratio in RAW 264.7 cells after culturing with PdNPs at different concentrations (mean ± SD). ****p < 0.0001, one-way ANOVA, Dunnett’s multiple comparisons test, control column: LPS. c, Representative CLSM images of RAW 264.7 cells stained for CD 86 (M1) and CD 206 (M2) after treatment with different concentrations of PdNPs. Scale bar = 100 μm. d, Relative fluorescence intensity of CD86 and CD206 expression (mean ± SD). e-f, Quantification of TNF-α and IL-6 levels in the supernatant of RAW 264.7 cells after PdNP treatment at different concentrations (mean ± SD). ****p < 0.0001, **p < 0.001, n.s. = not significant, one-way ANOVA, Dunnett’s multiple comparisons test, control column: LPS

Furthermore, confocal laser scanning microscopy (CLSM) showed a dose-dependent reduction in M1 macrophages after PdNPs treatment, while the number of M2 macrophages remained relatively unchanged (Fig. 3c, d). Consistently, qPCR analysis of M1 and M2 markers in RAW 264.7 cells showed that PdNPs significantly reduced TNF-α and IL-6 mRNA expression in a dose-dependent manner after LPS stimulation. In contrast, the expression of the M2 marker CD206 remained unchanged (Figure S1). These results confirmed that PdNPs primarily exert an anti-inflammatory effect by downregulating M1 macrophage activation rather than promoting polarization toward the M2 phenotype. Moreover, incubation with PdNPs at 25 µg/mL and 50 µg/mL resulted in a considerable downregulation of the production of inflammatory cytokines TNF-α and IL-6 (Fig. 3e, f). All of these findings suggest the critical role of PdNPs in modulating macrophage-related inflammatory responses.

In addition to macrophages, fibroblasts are essential in supporting wound healing by contributing to wound contraction, extracellular matrix (ECM) synthesis, angiogenesis, and tissue remodeling [30, 31]. Among these functions, fibroblast proliferation and migration are especially critical for effective tissue repair. Thus, we investigated how PdNPs affected 3T3 fibroblast cells. Initially, we evaluated the biocompatibility of PdNPs using the CCK8 and hemolysis assays, confirming that PdNPs did not induce cell damage at concentrations up to 50 µg/mL (Figure S2, Figure S3). This high level of biocompatibility may be attributed to the origin and composition of PdNPs, which are extracted from Bletilla striata and primarily consist of lipids, proteins, and polysaccharides. These components are known for their biodegradability, safety, and low immunogenicity. Subsequently, PdNPs were added to 3T3 cells for 24 and 48 h, and it was shown that PdNPs at 50 µg/mL continuously increased fibroblast proliferation (Figure S4).

Furthermore, the migration ability of fibroblasts following PdNP treatment was evaluated utilizing wound healing and transwell assays. The transwell assay demonstrated that PdNPs enhanced the migration speed of 3T3 cells. As PdNP concentrations rose, more 3T3 cells moved to the bottom side of the transwell membrane, as shown in Fig. 4a-c. Consistently, the wound healing assay further confirmed this effect. As shown in the images in Fig. 4d, f and g, the cell migration rate significantly increased to 38.9 ± 16.3% at 24 h and 76.4 ± 6.6% at 48 h at a PdNP concentration of 50 µg/mL, compared to 6.43 ± 1.21% at 24 h and 43.0 ± 10.9% at 48 h in the control group. To assess the impact of PdNPs on angiogenesis, we performed a tubule formation assay using human umbilical vein endothelial cells (HUVEC). PdNP-treated cells exhibited enhanced blood vessel network formation in a dose-dependent manner compared to the control group (Fig. 4e). Quantification of the tubule network revealed a significant increase in both the number of nodes and total capillary length at all tested PdNP concentrations (Figure h and i). These findings demonstrate that PdNPs not only exhibit high biocompatibility but also effectively promote fibroblast migration and angiogenesis, highlighting their potential for wound healing applications.

Fig. 4.

Effects of PdNPs on fibroblast migration and angiogenesis in vitro. a, Schematic illustration of the transwell assay. b, Crystal violet-stained representative pictures of migrated 3T3 cells. Scale bar = 200 μm. c, Quantification of migrated cells (mean ± SD). ****p < 0.0001, *p < 0.05, Student’s t-test. d, Representative wound healing assay images of 3T3 cells stained with Calcein AM. Scale bar = 400 μm. e, Representative images of tubule formation of HUVEC cells on Matrigel after culturing with PdNPs at different concentrations. HUVEC cells were stained with Calcein AM. Scale bar = 100 μm. f-g, Quantification of cell migration area after different treatments at (f) 24 h and (g) 48 h respectively (mean ± SD). h-i, Quantification of (h) the number of nodes and (i) total capillary length (mean ± SD). ****p < 0.0001, ***p < 0.001, **p < 0.01, one-way ANOVA, Dunnett’s multiple comparisons test, control column: Control

The healing process of diabetic wounds is severely hampered by their high susceptibility to bacterial growth and infection, which can result in chronic inflammation, excessive ROS generation, and severe oxidative stress [32]. To counteract ROS-induced damage, we synthesized a ROS nano-regulator, PDAs, and evaluated its antioxidant properties. PDAs were synthesized via polymerizing dopamine under alkaline conditions. PDAs had a monodispersed spherical morphology, according to transmission electron microscopy (TEM) and scanning electron microscope (SEM) examination (Fig. 5a, b), with an average size of 105 ± 15 nm and a zeta potential of −22 ± 8 mV (Fig. 5c, d). They were demonstrated high biocompatibility up to 200 µg/mL (Figure S2), which can be attributed to their composition—derived from dopamine and inspired by mussel adhesive proteins—ensuring low toxicity, biodegradability, and minimal immune response.

Fig. 5.

ROS scavenging effect of PDAs on fibroblasts in vitro. a, Representative TEM image of PDAs. Scale bar = 500 nm. b, Representative SEM image of PDAs. Scale bar = 500 nm. c, Particle size distribution of PDAs measured by DLS. d, Zeta potential distribution of PDAs measured by DLS. e, Representative CLSM images showing ROS production in H₂O₂-stimulated 3T3 cells after different treatments. Scale bar = 50 μm. f, Flow cytometry analysis of ROS levels in H₂O₂-stimulated 3T3 cells after different treatments. g, Representative flow cytometry images of apoptosis in H₂O₂-stimulated 3T3 cells after different treatments. h, Quantification of relative ROS levels normalized to the control group (mean ± SD). i, Quantification of early and late apoptosis cell populations in H₂O₂-stimulated 3T3 cells after different treatments (mean ± SD)

To assess the ROS-scavenging capability of PDAs, we conducted in vitro studies using 3T3 cells as a model system. The intercellular ROS levels after treatment with different concentrations of PDAs were detected using a dichlorodihydrofluorescein diacetate (DCFH-DA) probe. As shown in Fig. 5e, 3T3 cells pre-treated with H₂O₂ exhibited significant ROS production, indicated by strong green fluorescence signals. However, fluorescence intensity progressively decreased with increasing concentrations of PDAs, suggesting effective ROS scavenging (Fig. 5e). Flow cytometry results further confirmed a reduction in the proportion of ROS-overproducing 3T3 cells after treatment with PDAs (Fig. 5f, h). Additionally, alleviating oxidative stress is beneficial for inhibiting apoptosis and promoting cell proliferation. Notably, PDAs treatment significantly rescued cell viability under high concentrations of H₂O₂, primarily by reducing the late apoptotic rate from 13.7 ± 1.7% to 3.69 ± 1.8% (Fig. 5g, i). These findings suggested that PDAs efficiently alleviate oxidative stress and protect fibroblasts from ROS-induced damage.

After confirming the biological effects of PdNPs and PDAs, we incorporated them into HAMA hydrogel microcarriers using a single-emulsion microfluidic chip. Microscopy images confirmed that the resulting microcarriers had a uniform diameter of 201 ± 20 μm (Figure S5). We further performed SEM and CLSM to characterize the nanoparticles within the HAMA matrix (Figure S6). The results showed that PdNP + PDA@HAMA exhibited micro-structure with nanoparticles embedded. CLSM also revealed that FITC-labeled PDAs and DiD-labeled PdNPs were distributed within the HAMA crosslinked microcarriers. To investigate potential interactions between the nanoparticles and the hydrogel matrix after encapsulation, we lyophilized the PDA + PdNP@HAMA hydrogel microcarriers and performed Fourier transform infrared (FTIR) spectroscopy. A physical mixture of PdNPs, PDAs, and HAMA powder was used as the control. The FTIR spectrum of the PDA + PdNP@HAMA hydrogel microcarriers showed no new absorption peaks compared to the control, indicating that no new chemical interactions occurred between the hydrogel matrix and the loaded nanoparticles (Figure S7). To evaluate the release profile of nanoparticles from the hydrogel, PDA@HAMA and PdNP@HAMA microcarriers were incubated in PBS containing hyaluronidase. The concentration of nanoparticles released into the solution was quantified using nanoparticle tracking analysis (NTA). The results showed that approximately 80% of both PDAs and PdNPs were released from the HAMA microcarriers over a period of 9 days (Figure S8), indicating that the majority of the encapsulated nanoparticles can be effectively released during the treatment window at the wound site.

We further investigated the effect of PdNP + PDA@HAMA microcarriers on macrophages and fibroblasts. We incubated PdNPs, PDAs and PdNP + PDA@HAMA microcarriers with RAW 264.7 cells after LPS stimulation and measured the TNF-α and IL-6 levels in the supernatant. As shown in Figure S9a, b, the PdNP + PDA@HAMA microcarriers resulted in the lowest concentrations of both inflammatory cytokines compared to the single nanoparticle groups, suggesting enhanced anti-inflammatory effects after PdNPs and PDAs encapsulation into HAMA microcarriers. Furthermore, we investigated the ROS-scavenging ability of the different formulations. The results in Figure S9c showed that both PDAs and PdNP + PDA@HAMA microcarriers effectively reduced ROS levels in H₂O₂ stimulated cells, with comparable efficacy between the two groups. In addition, a cell migration assay revealed that both PdNPs and PdNP + PDA@HAMA similarly promoted the migration of 3T3 fibroblasts (Figure S9d, e). These results indicate that PdNPs and PDAs exert synergetic anti-inflammation effects on macrophages when co-delivered, while ROS scavenging and fibroblast migration appear to be primarily attributed to PDAs and PdNPs alone respectively.

To investigate the acute wound healing effects of PdNP + PDA@HAMA in vivo, we established a full-thickness diabetic wound model in mice. Various treatments including PBS, HAMA microcarriers, PdNP@HAMA microcarriers, PDA@HAMA microcarriers and PdNP + PDA@HAMA microcarriers were applied to the wounds and their progression was recorded on Day 0, 3, 6, 9 (Fig. 6a). As shown in Fig. 6b, wounds treated with HAMA, PdNP@HAMA, and PDA@HAMA exhibited improved healing rates compared to the PBS group, with wound closure rates of 65.2 ± 13.1%, 79.9 ± 8.0% and 72.3 ± 16.3% on Day 9, respectively (Fig. 6d). These effects can be attributed to the angiogenetic properties of HAMA and the therapeutic benefits of PDNPs and PDAs. Notably, by day 9, wounds in the PdNP + PDA@HAMA group were nearly completely healed, achieving a healing closure rate of 92.1 ± 5.9%. This remarkable healing outcomes is likely due to the combination effects of PdNPs and PDAs and HAMA.

Fig. 6.

Wound healing effect of PdNP + PDA@HAMA microcarriers. a, Schematic illustration of animal experiment timeline. b, Representation images of diabetic wounds following PBS, HAMA, PdNP@HAMA, PDA@HAMA and PdNP + PDA@HAMA treatments. c, Representative H&E and Masson’s trichrome staining images of the diabetic wound bed on Day 9 after different treatments. Scale bar = 1 mm, 100 μm. d, Quantification of wound areas at Day 3, 6, 9 after different treatments (mean ± SD). e-g, Quantification of granulation gap, granulation thickness and area of collagen area on Day 9 after different treatments (mean ± SD). ****p < 0.0001, one-way ANOVA, Dunnett’s multiple comparisons test, control group: PBS

Further, we histologically examined the newly generated skin tissue using hematoxylin and eosin (H&E) staining. All treatment groups showed signs of granulation tissue development and epidermal regeneration (Fig. 6c). Compared to the PBS control group, the HAMA, PdNP@HAMA, and PDA@HAMA groups exhibited a narrower granulation gap, while the PdNP + PDA@HAMA group demonstrated only a minimal gap between granulation tissue and the surrounding skin (Fig. 6e). However, there were no significant variations in granulation tissue thickness among the groups (Fig. 6f). Collagen deposition was evaluated using Masson’s trichrome staining. The PdNP + PDA@HAMA group exhibited the highest collagen content with more uniformly arranged collagen fibers by Day 9, closely resembling the structure of native skin. In contrast, the other treatment groups showed less collagen deposition and more irregularly oriented collagen fibers (Fig. 6c, g). Furthermore, type III collagen predominates during the early stages of wound healing, while type I collagen becomes increasingly deposited during later phases associated with cell proliferation and tissue remodeling. As shown in Figure S10, the PdNP + PDA@HAMA group exhibited the lowest proportion of type III collagen and the highest deposition of type I collagen, indicating a more advanced stage of wound maturation and tissue remodeling.

Since angiogenesis is a crucial prerequisite in tissue regeneration, CD31 staining was conducted to assess newly formed blood vessels. The PdNP@HAMA and PDA@HAMA groups exhibited a higher blood vessel density compared to the PBS and HAMA groups, while the PdNP + PDA@HAMA group demonstrated the most significant vascularization (Fig. 7a, b), consistent with the findings from H&E staining. These results suggest that PdNP + PDA@HAMA microcarriers not only accelerate wound healing but also promote collagen deposition and angiogenesis, leading to more effective and natural skin regeneration.

Fig. 7.

Angiogenesis and collagen deposition effects of PdNP + PDA@HAMA microcarriers. a, Representative fluorescence images of CD31 and ROS immunofluorescence staining, as well as IL-6 and TNF-α immunohistochemical staining of wound beds on Day 9 after different treatments. Scale bar = 100 μm. b, Quantification of blood vessel density on Day 9 from CD31 staining images (mean ± SD). c, Quantification of mean fluorescence intensity from ROS immunofluorescence staining images on Day 9 (mean ± SD). d-e, Quantification of IL-6 and TNF-α immunohistochemical staining images on Day 9 (mean ± SD). ****p < 0.0001, ***p < 0.001, **p < 0.01, one-way ANOVA, Dunnett’s multiple comparisons test, control group: PBS

Finally, we evaluated the inflammatory levels in diabetic wound tissue, as sustained inflammation is a major factor contributing to delayed wound healing. Since PDAs have demonstrated strong ROS-scavenging capabilities to mitigate oxidative stress and alleviate inflammation, we first assessed ROS levels in the diabetic wound tissues following different treatments. As shown in Fig. 7a and c, only the microcarriers encapsulating PDAs significantly reduced ROS levels, with PdNP + PDA@HAMA microcarriers exhibiting the most pronounced ROS-scavenging effect. Consequently, PdNP + PDA@HAMA microcarriers markedly decreased the levels of IL-6 and TNF-α, two important inflammatory cytokines in the wound tissue, whereas the other treatment groups showed no significant changes (Fig. 7a, d, e). These findings suggest that the accelerated wound healing observed with PdNP + PDA@HAMA microcarriers is primarily attributed to the resolution of inflammation. Additionally, treated mice showed no signs of toxicity from any of the materials used (Figure S11).

Conclusion and discussion

In summary, the integration of multi-active PdNPs and PDAs into HAMA hydrogel microcarriers presents a synergistic, naturally derived therapeutic strategy for diabetic wound healing. By leveraging microfluidic fabrication, this system harnessed the anti-inflammatory and fibroblast-activating properties of PdNPs alongside the ROS-scavenging and anti-apoptotic effects of PDAs. In vivo studies using a diabetic wound model demonstrated that PdNP + PDA@HAMA microcarriers effectively mitigated chronic inflammation, alleviated oxidative stress, and promoted tissue regeneration, leading to accelerated wound closure. Collectively, these findings underscore the potential of PdNP + PDA@HAMA microcarriers as a multifunctional and bioactive wound dressing, offering a promising platform for the clinical management of impaired wound healing.

The novelty of our study lies in the development of nature-derived, multi-active nanoparticles-integrated hydrogel microcarriers for wound healing. We discovered that nanoparticles extracted from Bletilla striata exhibited significant bioactivity, including the ability to polarize M1 macrophages and restore fibroblast function. To address the pathogenesis of chronic diabetic wounds, we further incorporated PDA nanoparticles to scavenge ROS-induced cellular damage. Rather than employing a hydrogel sheet, we utilized microfluidic technology to precisely encapsulate these multifunctional nanoparticles into microcarriers. This approach enables superior wound conformability and coverage, controlled delivery and sustained release of encapsulated nanoparticles.

Owing to their natural origin, nature-derived formulations offer several advantages as therapeutic agents. Firstly, they are generally considered non-toxic and non-immunogenic, as they are derived from edible plant cells. Additionally, these formulations are environmental-friendly due to their renewable and sustainable sources, and they can be easily and cost-effectively cultivated and harvested. Although various nature-derived nanoparticles have been explored as biotherapeutic agents in the treatment of different diseases, some critical issues remain to be addressed. These include: (1) the evaluation of the stability and storage conditions of natural-derived formulations; (2) elucidation of the biogenesis mechanisms of these nanoparticles to further enhance their therapeutic efficacy.

Materials and methods

Extraction of PdNPs

To make Bletilla striata juice, fresh Bletilla striata (bought from a nearby market) was carefully cleaned with PBS and blended. A series of centrifugations were performed on the juice: 1,000 × g for 10 min, 3,000 × g for 20 min, and 10,000 × g for 30 min. The resulting supernatant was collected and ultracentrifuged at 150,000 × g for 90 min. The pellet was resuspended in PBS, and PdNPs were isolated using a sucrose step gradient method [33]. DLS was used to assess the particle size and zeta potential of PdNPs, while TEM was used to characterize their morphology (Malvern, Zetasizer).

In vitro macrophage polarization

To induce M1 macrophage polarization, RAW 264.7 cells were treated for 24 h with LPS (100 ng/mL) (Sigma). After polarization, the cells were incubated for a further 24 h after PdNPs were introduced at doses of 10, 25, and 50 µg/mL. The cells were stained with fluorescence-labeled antibodies against CD86 (M1 marker) and CD206 (M2 marker) (BioLegend) in order to evaluate macrophage polarization. Flow cytometry was used to analyze the fluorescence signals. CLSM (Nikon) was employed to visualize the expression of CD86 and CD206 in RAW 264.7 cells after different treatments. Furthermore, culture supernatants were gathered, and ELISA was used to measure the amounts of two important pro-inflammatory cytokines, IL-6 and TNF-α (CUSABIO). Meanwhile, mRNA levels of TNF-α, IL-6 and CD206 was analyzed by real-time qPCR using Super FastPure Cell RNA Isolation Kit RC102 (Vazyme Biotech Co.,Ltd), ChamQ Universal SYBR qPCR Master Mix (Vazyme Biotech Co.,Ltd) and All-in-one RT SuperMix Perfect for qPCR (Vazyme Biotech Co.,Ltd) using qPCR. TNF-α sense: 5′-CAAGGGACAAGGCTGCCCCG-3′; TNF-α antisense: 5′-GCAGGGGCTCTTGACGGCAG-3′; IL-6 sense: 5′-GCTGGTG ACAACCACGGCCT-3′; IL-6 antisense: 5′-AGCCTCCGACTTGTGAAGTGGT-3′; CD206 sense: 5′-TTCGGTGGACTGTGGACGAGCA-3′; CD206 antisense: 5′-ATAAGCCACCTGCCACTCCGG-3′.

Cell proliferation assay

3T3 cells were cultivated in a 24-well plate and cultured overnight. PdNPs were added at 10, 25, and 50 µg/mL, and incubated for 24 and 48 h. Following treatment, the cells were isolated and allowed to proliferate for two hours in a 10 mM EdU solution (Beyotime). The cells were then permeabilized with Triton X-100 and fixed with 4% paraformaldehyde (PFA). After that, the samples were left in the dark for 30 min while the EdU detection reagents were present. EdU incorporation was quantified by measuring the fluorescence intensity using flow cytometry.

Wound-healing assay

In a 12-well plate, 3T3 cells were cultured until they reached 80% confluence. Following the addition of PdNPs at 10, 25, and 50 µg/mL, the cells were cultured for a full day. A 200 µL pipette tip was used to make a consistent, linear scratch along the well’s diameter after the cells had fully confluenced. To reduce the impact of cell growth, serum-free medium was substituted for the original medium. At 0, 24, and 48 h, the cells were stained with Calcein AM (Beyotime), and the scratched areas were imaged using a fluorescence microscope. The changes in the scratched areas were analyzed using ImageJ software.

Tube formation analysis

HUVEC cells were cultured in a 12-well plate and treated with PdNPs at 10, 25, and 50 µg/mL for 24 h. To assess tube formation, each well of a 24-well plate received 100 µL of Matrigel, which was then left to gel for 30 min at 37 °C. After gelation, the treated HUVEC cells (2 × 10⁵ cells per well) were seeded onto the gel and cultured for 6 h. The cells were then stained with Calcein AM, and the tube formation was imaged using a fluorescence microscope. The tubular structures were quantitatively analyzed using ImageJ software.

Diabetic wound animal model

To establish type I diabetic rats, male SD rats (170–200 g) were administered an intraperitoneal injection of streptozotocin (65 mg/kg) (Aladdin). Blood glucose levels were measured from tail vein samples 72 h post-induction. If blood glucose levels were higher than 16.7 mmol/L for three days in a row, diabetes was diagnosed. Rats meeting this criterion were included in the study as diabetic models. To create diabetic wounds, the dorsal region of the diabetic rats was shaved using a hair clipper, followed by the application of depilatory cream to create a 2 cm × 2 cm hair-free area. In the prepared area, a sterile biopsy punch was then used to make a full-thickness circular skin wound with a diameter of 10 mm. These rats were subsequently split into five groups (12 rats each) and treated with PBS, HAMA microcarriers, PdNP@HAMA microcarriers, PDA@HAMA microcarriers, or PdNP + PDA@HAMA microcarriers (a total treatment volume of 150 µL per rat, PdNP concentration = 1 mg/mL, PDA concentration = 1 mg/mL) [34]^, [35]. Every 3 days, the sites of the wounds were recorded. To assess the inflammatory response, wound tissues from 6 rats per group were taken on Day 4, sectioned, and stained for IL-6 and TNF-α immunohistochemical analysis. The remaining wound tissues were collected on Day 9, sectioned, and stained for H&E, Masson’s trichrome, Collagen Ⅰ, Collagen Ⅲ, CD31, and ROS. In order to assess the biosafety of the various treatments, key organs were also harvested for H&E staining.

Statistical analysis

All biological experiments were conducted in triplicate, and the results are presented as mean ± standard deviation (mean ± SD). Statistical significance was determined using an unpaired Student’s t-test and one-way analysis of variance (ANOVA) with GraphPad Prism software (version 9.0). A p-value of less than 0.05 was considered statistically significant, while p-values of less than 0.01, 0.001, and 0.0001 were considered highly significant.

Author contributions

Y.J.Z. and J.Y.C. conceived the conceptualization and designed the experiment. J.Y.C., G.T.G. and Y.W. carried out the experiments and analyzed the data. J.Y.C., D.Q.H. and Y.J.Z. wrote the paper. All authors reviewed the manuscript.

Data availability

No datasets were generated or analysed during the current study.

Declarations

Competing interests

The authors declare no competing interests.