ROS-responsive hydrogel patch orchestrating macrophage reprogramming and mitochondrial protection for post-MI repair

Abstract

Myocardial infarction (MI) leads to irreversible cardiomyocyte loss accompanied by oxidative stress, inflammation, and fibrotic remodeling, ultimately progressing to heart failure. Using network pharmacology analysis, salvianolic acid B (DB), a major bioactive component of Salvia miltiorrhiza, was identified as a promising therapeutic candidate for MI. Oral administration of DB showed therapeutic efficacy in functional, molecular, and histological assessments, but its clinical translation is limited by poor bioavailability. To overcome this limitation, we developed an injectable reactive oxygen species (ROS)-responsive hydrogel patch for localized MI repair. The hydrogel, composed of whey protein isolate methacrylate (WPI-MA) and o-nitrobenzyl alcohol modified hyaluronic acid (HA-NB), forms a biocompatible and adhesive network through dynamic covalent interactions. ROS-sensitive liposomes encapsulating DB were incorporated into the hydrogel, enabling localized and on-demand drug release in response to the oxidative microenvironment. In vitro studies confirmed that the hydrogel exhibited favorable mechanical strength, selective myocardial adhesiveness, and sustained antioxidant capacity. In a murine MI model, a single administration of the hydrogel patch markedly attenuated fibrosis, promoted angiogenesis, and restored cardiac function. This study establishes a rational design strategy that begins with network pharmacology-based drug discovery, proceeds through validation of therapeutic efficacy, and culminates in the construction of a responsive delivery system, providing a promising approach for localized and sustained post-infarction cardiac repair.

Highlights

• •A ROS-responsive, bioadhesive hydrogel patch was developed for myocardial infarction therapy.
• •The hydrogel integrates WPI-MA and HA-NB to achieve selective myocardial adhesion and in situ gelation.
• •ROS-sensitive liposomes enable localized and on-demand release of Salvianolic acid B.
• •The hydrogel patch exhibits excellent antioxidant activity and promotes angiogenesis and cardiac repair.
• •This multifunctional platform offers a single-dose, minimally invasive strategy for post-MI treatment.

1. Introduction

Myocardial infarction (MI), commonly known as a heart attack, remains a leading cause of death and disability worldwide [1]. It is characterized by the sudden loss of blood supply to the myocardium, which initiates a cascade of biochemical and structural events, including massive oxidative stress, acute and chronic inflammatory responses, and extensive extracellular matrix remodeling [2,3]. These pathological processes contribute to irreversible cardiomyocyte death and adverse ventricular remodeling, ultimately leading to progressive cardiac dysfunction and heart failure. Despite significant advances in reperfusion therapy and pharmacological management [4], current treatments provide limited benefit in regenerating damaged myocardium or reversing fibrotic progression [5,6]. Therefore, there is a critical need for therapeutic strategies that can not only protect the myocardium but also provide sustained and targeted delivery of bioactive agents to the infarct site [7].

Among potential cardioprotective compounds, Salvianolic acid B (DB), a water-soluble phenolic compound extracted from Salvia miltiorrhiza, has gained attention due to its potent antioxidant, anti-inflammatory, and anti-fibrotic properties [8]. DB has been shown to reduce infarct size, inhibit apoptosis, and preserve left ventricular function in preclinical models [9,10]. We also further predicted the therapeutic potential of DB in MI repair through functional, molecular, and histological evaluations. Experimental results demonstrated the cardioprotective effects of DB in the treatment of MI, as evidenced by improved cardiac function, regulation of key repair-related protein expression, and preservation of myocardial tissue structure. However, DB is unstable in aqueous solution, which further complicates its clinical application. Its therapeutic efficacy in clinical practice remains limited owing to its poor oral bioavailability, rapid systemic clearance, and low accumulation in cardiac tissue following systemic administration [11].

To overcome these challenges, stimuli-responsive drug delivery systems have been extensively explored to enable spatiotemporally controlled release in pathological environments [12,13]. In particular, reactive oxygen species (ROS)-responsive nanocarriers have emerged as a promising platform for MI therapy, given the elevated ROS levels that characterize the infarcted myocardium during both acute and subacute phases [14]. By leveraging this oxidative microenvironment, ROS-sensitive carriers can enable site-specific release of therapeutics such as DB, thereby maximizing therapeutic efficacy while minimizing systemic exposure [15,16].

In addition to targeted delivery, local retention of therapeutics at the cardiac surface is crucial for maximizing efficacy. The hydrogel possesses mechanical support, tissue integration, and the ability to serve as a sustained-release reservoir for bioactive substances [17,18]. However, currently used injectable hydrogels or sutured hydrogel patches for the treatment of MI often cause postoperative tissue adhesions, thereby limiting their clinical application. Zheng et al. [19] reported an ROS-responsive liposomal composite hydrogel system for the treatment of myocardial infarction, capable of achieving ROS-triggered release. However, its adhesion mechanism is based on conventional injectable gels and is not specifically designed for selective adhesion to the myocardium or prevention of adhesion to thoracic tissues.

To address this challenge, we focused on engineering a hydrogel with selective adhesiveness—strong attachment to the dynamic myocardial surface while minimizing nonspecific adhesion to surrounding thoracic tissues. This was achieved through the incorporation of hyaluronic acid modified o-nitrobenzyl alcohol (HA-NB) moieties, which generate aldehyde-like groups upon UV irradiation [20]. These groups rapidly react with myocardial amines to form Schiff base linkages, providing robust anchoring during gelation. Once the hydrogel is fully crosslinked, the surface becomes highly hydrated and chemically inert, thereby preventing further adhesion to adjacent tissues. Whey protein isolate (WPI), a biofunctional protein-derived material, exhibits favorable characteristics such as antioxidant activity, immunomodulation, and biocompatibility [21]. Zheng et al. [22] designed an injectable hydrogel microsphere system based on WPI, which protects cardiomyocytes from apoptosis and alleviates oxidative damage in a rat ischemia-reperfusion injury model. Its injectability and mild gelation conditions make it suitable for minimally invasive application. However, its relatively weak adhesiveness limits its performance in dynamic cardiac environments where strong tissue attachment is essential.

To overcome this limitation, we have designed an asymmetric adhesive hydrogel using WPI methylacrylation (WPI-MA) in combination with HA-NB, which enables specific adhesion to cardiac tissue. First, through network pharmacology analysis, we predicted the potential role of DB in the pathophysiology of MI. Subsequently, we encapsulated DB in ROS-responsive liposomes to achieve localized, ROS-triggered drug release following a single administration (see Scheme 1). Finally, we incorporated the liposomal DB@DP into the WPI-MA and HA-NB hydrogel system. Under light exposure, HA-NB undergoes cleavage, releasing aldehyde groups that can form stable covalent bonds with amino groups on both the myocardial tissue surface and WPI-MA [23], thereby promoting gel network polymerization and tissue adhesion. When the light source is removed, the decomposition of the nitrobenzyl alcohol in HA-NB ceases, halting the production of aldehyde groups. This effectively prevents any reaction with amino groups on other tissues, avoiding adhesion to areas outside the application site. This design cleverly integrates physical support, targeted tissue adhesion, and disease-responsive controlled drug release mechanisms, offering a robust strategy for precision therapy. In a murine model of MI, the hydrogel patch demonstrated promising therapeutic efficacy and shows strong potential for clinical translation.

Scheme 1.

Schematic illustration of the preparation of WPIMA/HA-NB/DB@DP hydrogel scaffold and its mechanism for MI treatment.

2. Materials and methods

2.1. Materials and reagents

Whey protein isolate (WPI90), hyaluronic acid, salvianolic acid B, and other chemical reagents were purchased from commercial suppliers (detailed information in Supporting Information). HUVECs and H9C2 cells were obtained from Cellverse Bioscience (Shanghai, China). All reagents were used as received unless otherwise specified.

2.2. Synthesis of functional components

The small-molecule NB was synthesized through a four-step procedure involving etherification, nitration, reduction, and amidation. HA was subsequently conjugated with NB using EDC/HOBt chemistry. Methacrylated WPI (WPI-MA) was prepared by reacting WPI with methacrylic anhydride. Dextran was modified with PBAP to obtain DEX-PBAP, which was further assembled into DB-loaded liposomes (DB@DP). Detailed synthesis procedures are described in Supporting Information.

2.3. Preparation of hydrogels

WPI-MA and HA-NB were dissolved in LAP-containing aqueous solutions, mixed at equal volumes, and crosslinked under 405 nm UV irradiation to form WH hydrogels. For drug-loaded formulations, DB@DP was incorporated into the precursor solution before photocrosslinking to obtain WH/DB@DP.

2.4. Characterization

Chemical structures were confirmed by ¹H NMR spectroscopy. Morphology and size of DB@DP were examined by TEM and DLS. The hydrogels were characterized by rheological tests, compressive modulus, adhesion strength, SEM, swelling, degradation, and DB release profiles. Antioxidant performance was evaluated by hydroxyl radical, ABTS, DPPH, and FRAP assays.

2.5. In vitro studies

Cytocompatibility was assessed using CCK-8 and live/dead staining in HUVECs and H9C2 cells. Pro-angiogenic potential was evaluated by tube formation assay and ELISA. ROS scavenging was analyzed by DCFH-DA staining. Macrophage polarization was determined using flow cytometry and immunofluorescence. Cardiomyocyte functions were examined via immunofluorescence, JC-1 staining, ATP detection, TEM, Western blot, and qPCR. Transcriptome analysis was performed to further reveal underlying mechanisms.

2.6. In vivo MI model

MI was induced in male C57BL/6 mice by left anterior descending ligation, followed by local hydrogel injection. Cardiac function was monitored by echocardiography. Histological and immunohistochemical analyses were performed to evaluate therapeutic outcomes.

2.7. Statistical analysis

In this study, each experiment was performed at least three times, and the results are presented as mean ± SD, with a p-value of less than 0.05 considered statistically significant. The student's t-test was used for comparisons between two groups, while one-way analysis of variance (ANOVA) was used for comparisons across multiple groups. Statistical analyses were performed using GraphPad Prism 9.5e software and Origin software.

3. Results and discussion

3.1. Therapeutic potential of DB in MI repair

To elucidate the therapeutic relevance of DB in MI, we first employed network pharmacology analysis to identify potential molecular targets and signaling pathways associated with DB-mediated cardiac repair. This in silico prediction was then followed by functional, molecular, and histological validation to systematically assess the cardioprotective effects of DB. As illustrated in Fig. 1A, Venn diagram analysis identified 67 overlapping targets between DB and MI, accounting for 4.5 % of DB-related targets and 79.9 % of MI-related targets. This substantial overlap suggests that DB may play a crucial role in the pathophysiological processes underlying MI. To further elucidate the functional relevance of these targets, a protein–protein interaction (PPI) network was constructed (Fig. 1B), revealing extensive molecular interactions that provide insights into the mechanisms by which DB might influence MI. Gene Ontology (GO) enrichment analysis (Fig. 1C) highlighted that the common targets are primarily associated with biological processes such as cell proliferation and migration, cellular components like the extracellular matrix and cell junctions, and molecular functions including protein binding and enzymatic activity-all of which are vital to myocardial tissue repair. Furthermore, KEGG pathway analysis (Fig. 1D) revealed that these targets are involved in several pivotal signaling pathways, including lipid metabolism, atherosclerosis, and the MAPK signaling pathway, suggesting that DB may facilitate MI repair by modulating these pathways. Notably, the KEGG enrichment also identified the PI3K-Akt signaling pathway, cell adhesion molecules, and the MAPK pathway, all of which are implicated in fibrosis. These findings imply that DB may exert anti-fibrotic, anti-inflammatory, and antioxidant effects, with cytokines potentially playing a key role in mediating these actions.

Fig. 1.

Evaluation of the correlation between DB and MI repair. (A) Venn diagram of common targets between the drug DB and the disease MI; (B) PPI (protein–protein interaction) network; (C) GO (Gene Ontology) analysis; (D) KEGG pathway analysis; (E) Echocardiographic images of MI model group and DB oral treatment group; (F) Quantification of EF (ejection fraction); (G) Quantification of FS (fractional shortening), LVIDs, and LVIDd; (H) Western blot bands of MI model group and DB oral treatment group; (I) Quantification of TNF-α, VEGF, and PINK; (J) H&E and Masson staining of MI model group and DB oral treatment group. (n = 5, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001).

Building on these in silico predictions, echocardiographic, molecular, and histological analyses further verified that DB treatment preserves myocardial structure and function following MI. Echocardiographic analysis (Fig. 1E) revealed that DB treatment significantly improved cardiac function compared to the MI model group. Quantitative evaluations showed increased ejection fraction (EF) and fractional shortening (FS), along with reduced left ventricular internal diameters in both systole (LVIDs) and diastole (LVIDd) (Fig. 1F and G), indicating enhanced systolic function and improved ventricular geometry. At the molecular level, Western blot analysis and quantitative results (Fig. 1H and I) demonstrated that DB treatment significantly downregulated TNF-α expression, suggesting an anti-inflammatory effect, while upregulating VEGF and PINK levels, which are associated with angiogenesis and cell survival, respectively-both essential for myocardial tissue repair. Histological assessments, including H&E and Masson's trichrome staining (Fig. 1J), showed that the myocardial architecture in the DB-treated group more closely resembled that of normal tissue, suggesting structural preservation and reparative effects. These histological improvements were consistent with the observed functional and molecular changes. Together, these results demonstrate that DB exerts cardioprotective effects in MI by improving cardiac function, modulating repair-related protein expression, and preserving myocardial architecture. Nevertheless, the therapeutic efficacy achieved through oral administration remains limited due to poor bioavailability and rapid clearance. These limitations provide a clear rationale for engineering a localized and responsive delivery system to optimize DB utilization in cardiac repair.

3.2. Physicochemical characterization of ROS-responsive Liposome/hydrogel patch loaded with DB

To evaluate whether the engineered WH/DB@DP hydrogel patch possesses the essential properties for myocardial repair, we conducted a systematic physicochemical and functional characterization. Fig. 2A–C shows the ¹H NMR spectra of WPI-MA, HA-NB, and DEX-PBAP, confirming successful functionalization of all components. For WPI-MA, characteristic peaks of the methacrylate group were observed at δ 5.4 and 2.8 ppm. HA-NB displayed aromatic proton signals at 7.84 and 7.24 ppm along with the typical peak of N-acetylglucosamine at δ 1.9 ppm, supporting successful NB grafting. Compared with native DEX, DEX-PBAP exhibited additional methyl (δ 2.51 ppm) and aromatic proton peaks (δ 7.38 and 7.67 ppm), confirming efficient PBAP conjugation (The chemical structure of PBAP is shown in Fig. S1). Collectively, these spectral features verify the effective synthesis and modification of the hydrogel building blocks. The presence of characteristic methacrylate peaks in WPI-MA, aromatic signals in HA-NB, and additional methyl and aromatic peaks in DEX-PBAP verified the effective synthesis of each building block, ensuring a reliable foundation for patch fabrication.

Fig. 2.

Characterization of the myocardial patch WH/DB@DP. (A) ¹H NMR spectra of WPI and WPI-MA; (B) ¹H NMR spectra of HA and HA-NB; (C) ¹H NMR spectra of DEX and DEX-PBAP; (D) TEM images of DEX-PBAP and DB@DP; (E) Particle size analysis of DEX-PBAP and DB@DP; (F) Gelation images; (G) SEM images of the hydrogel patch; (H) Adhesion images of the hydrogel patch on different substrates and at different angles; (I) Adhesion stress-strain curves and quantitative analysis of adhesion strength; (J) Rheological properties of the hydrogel over time and frequency; (K) Swelling behavior characterization; (L) Compression performance characterization; (M) Degradation behavior under different ROS conditions; (N) In vitro cumulative release of DB from DB@DP and WH/DB@DP under different ROS conditions; (O) In vitro antioxidant performance of WH/DB@DP hydrogel. (n = 5, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001).

Given the therapeutic promise of DB but it's extremely low oral bioavailability due to instability, rapid clearance, and limited intestinal permeability [24,25], we sought to design a delivery system capable of protecting DB and releasing it selectively at the infarcted myocardium. Since excessive ROS accumulation is a hallmark of myocardial infarction, we incorporated a ROS-responsive liposomal carrier into the hydrogel matrix to enable on-demand, localized drug release. The morphology and size distribution of the constructed nanoparticles were then characterized to verify their suitability for drug delivery. In addition, the encapsulation efficiency of DB in DB@DP liposomes was 72 %, with a loading capacity of 36.05 ± 4.46 % (Fig. S2). TEM images (Fig. 2D) revealed that both DEX-PBAP and DB@DP nanoparticles exhibited uniform morphology and good dispersibility, which are critical for stable circulation. Particle size distribution analysis (Fig. 2E) further confirmed average diameters of 406.9 nm and 427.32 nm, respectively. These nanoscale dimensions are advantageous for prolonging circulation and promoting accumulation at ischemic sites [[26], [27], [28]].

To further construct the myocardial patch, WPI-MA and HA-NB were crosslinked under UV irradiation, resulting in rapid gel formation within 10 s (Fig. 2F & Video 1), confirming the excellent gel-forming capacity of the system. Compared to the CPAMC/PCA Janus hydrogel patch studied by He et al. [29]., the WH/DB@DP hydrogel can be injected into the damaged myocardial area and undergo light-induced in situ polymerization, offering greater flexibility and precision. Unlike traditional hydrogel patches, the WH/DB@DP hydrogel can directly form a firm, three-dimensional structure that conforms to the surface morphology of the heart in vivo. The underlying mechanism is as follows: the aldehyde groups generated by the photolysis of HA-NB can covalently bond with the amino groups on the myocardial tissue surface and those on WPI-MA, promoting the polymerization of the gel network and tissue adhesion. When the light source is removed, the decomposition of nitrobenzyl alcohol ceases, preventing further generation of aldehyde groups and thus avoiding reactions with amino groups on other tissues. This design introduces selective adhesion, ensuring that adhesion occurs only at the targeted site.

Supplementary video related to this article can be found at https://doi.org/10.1016/j.bioactmat.2026.01.015

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SEM images (Fig. 2G&Fig. S3) showed an interconnected porous structure, and the introduction of DB@DP did not significantly affect the hydrogel network structure. This is beneficial for nutrient exchange, cell adhesion, and infiltration—features that are crucial for myocardial tissue regeneration. Adhesion tests demonstrated that the hydrogel patch could firmly attach to diverse substrates, including wet tissue surfaces, and maintained stability even at tilted or inverted positions (Fig. 2H & Video 2). Quantitative analysis of adhesion stress–displacement curves (Fig. 2I) further verified the strong adhesive strength of the hydrogel, with a maximum detachment force reaching 24.80 N and an average adhesion force of 24.49 N. Such robust adhesion ensures secure integration of the patch under continuous cardiac contraction, reducing the risk of displacement or detachment during dynamic myocardial motion.

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Reological analysis (Fig. 2J & Fig. S4) revealed that all hydrogel formulations exhibited G′ values consistently higher than G″, confirming their predominantly elastic nature. Notably, the storage modulus increased with polymer concentration, indicating that higher crosslinking density endowed the hydrogel with greater stiffness and resistance to deformation. Furthermore, the addition of DB@DP did not adversely affect the rheological properties of the hydrogel. Such concentration-dependent reinforcement suggests that the network structure can be tuned to balance mechanical stability with cardiac compliance. Swelling studies (Fig. 2K) demonstrated a rapid water uptake phase followed by equilibrium, but the extent of swelling decreased as polymer concentration increased. This trend is consistent with denser network structures restricting water infiltration, thereby reducing the maximum swelling ratio. Compression testing revealed a clear concentration-dependent trend, with higher concentrations exhibiting greater stiffness and load-bearing capacity, while lower concentrations showed better compliance. Furthermore, the addition of DB@DP did not adversely affect the compressive strength of the hydrogel. All groups-maintained integrity under large deformations, indicating sufficient robustness for cardiac application (Fig. 2L& Fig. S5). These studies indicate that while increasing WPI-MA concentration can modestly improve hydrogel strength, there is no significant difference in maximum swelling rate. Softer hydrogels are more suitable for cell growth, so we ultimately chose 20 % WPI-MA for co-crosslinking with HA-NB for subsequent experiments. In ROS-responsive degradation assays (Fig. 2M), WH/DB@DP exhibited significantly faster mass loss under 10⁻³ M H₂O₂ compared with physiological conditions, confirming its sensitivity to oxidative stress. Correspondingly, drug release studies (Fig. 2N) showed that both DB@DP and WH/DB@DP achieved accelerated and higher cumulative release in the presence of H₂O₂, demonstrating the system's ability to synchronize degradation with pathological ROS levels.

In addition to ROS-triggered release, WH/DB@DP also exhibited intrinsic radical-scavenging activity (Fig. 2O). The hydrogel effectively eliminated 2,2-Diphenyl-1-picrylhydrazyl (DPPH), 2,2′-Azinobis (3-ethylbenzothiazoline-6-sulfonic acid (ABTS), and hydroxyl radicals in a dose-dependent manner, with scavenging efficiencies approaching 80 % at a concentration of 1000 μg/mL. This dual function—both responding to and attenuating oxidative stress—offers synergistic protection: the hydrogel not only delivers DB in a spatiotemporally controlled fashion but also reduces oxidative injury to cardiomyocytes, thereby creating a more favorable microenvironment for myocardial repair and regeneration.

3.3. In vitro evaluation of biocompatibility, antioxidative activity, and pro-angiogenic effects

Cell viability assays confirmed that liposomes displayed dose-dependent cytotoxicity, with viability dropping below 50 % at 800–1000 μg/mL, whereas concentrations ≤400 μg/mL were well tolerated (Fig. S6A). Hydrogel formulations showed good compatibility with both H9C2 and HUVEC cells, maintaining viability above 90 % across 72 h, with WH/DB@DP consistently outperforming WH/DP (Fig. S6B). Calcein-AM/PI staining further corroborated these results, showing abundant live cells and negligible cell death in all hydrogel groups (Fig. S7). These findings establish a safe biological range for subsequent functional studies.

Having established the safe biological range of liposomes and hydrogel formulations, we next investigated their functional performance under oxidative stress conditions [30]. Excessive accumulation of ROS is a hallmark of the infarcted myocardium and a major contributor to cardiomyocyte injury. Intracellular ROS levels in H9C2 cells were therefore evaluated using the DCFH-DA probe (Fig. 3A and B). As expected, the PMA-treated positive control exhibited a marked increase in fluorescence intensity, confirming the reliability of the detection system. Compared with the control group, WH hydrogels induced only a slight elevation in ROS, whereas the WH/DP group significantly suppressed ROS levels, reducing them by approximately 48 % relative to WH hydrogel group. In ROS-rich pathological microenvironments, such as those present in MI, it can be activated to release antioxidant components while simultaneously consuming ROS [31]. Strikingly, WH/DB@DP further decreased ROS to near-control values, demonstrating potent antioxidative efficacy. This effect can be attributed to the ROS-responsive hydrolysis of PBAP, which generates phenolic byproducts and 4-hydroxybenzyl alcohol with intrinsic antioxidative activity, together with the multi-pathway ROS scavenging actions of DB, including hydrogen donation, metal ion chelation, and activation of cellular antioxidant enzymes [32]. These synergistic mechanisms effectively alleviate oxidative stress in cardiomyocytes, thereby creating a microenvironment favorable for myocardial repair. Since oxidative stress not only compromises cardiomyocyte survival but also impairs endothelial function, alleviating ROS is expected to restore endothelial activity and facilitate neovascularization in the ischemic myocardium.

Fig. 3.

In vitro biosafety and performance evaluation of the myocardial patch. (A) ROS staining images of rat cardiomyocytes (H9C2) after hydrogel treatment; (B) Quantification of ROS fluorescence intensity; (C) Tube formation images of cells treated with hydrogel materials; (D) Quantitative analysis of vascular branch points, total length, and branch length; (E) Quantification of growth factors ANG-2, VEGF, and PDGF-BB; (F) Quantification of Ki67 fluorescence intensity; (G) Single immunofluorescence staining of Ki67 in H9C2 cells after hydrogel treatment; (H) Dual immunofluorescence staining of α-actin and CX-43 in H9C2 cells after hydrogel treatment.(n = 5, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001).

To validate this hypothesis, tube formation assays with HUVECs were performed to evaluate the angiogenic potential of different hydrogel systems (Fig. 3C–D). WH hydrogels moderately promoted capillary-like network formation compared with control, while WH/DP significantly increased the number of branches, total tube length, and mean branch length. Notably, WH/DB@DP exhibited the strongest angiogenic response, with quantitative analysis showing a significant increase relative to WH/DP. To further elucidate the mechanism, secretion of angiogenesis-related factors was measured. ELISA results (Fig. 3E) demonstrated that ANG-2, VEGF, and PDGF-BB were mildly upregulated by WH, further elevated by WH/DP, and most significantly increased by WH/DB@DP. These findings indicate that the integration of ROS-responsive DP and DB not only mitigates oxidative stress but also establishes a strongly pro-angiogenic microenvironment, thereby enhancing endothelial cell activity and promoting neovascularization critical for myocardial tissue repair.

Encouraged by the enhanced angiogenic response, we next evaluated whether WH/DB@DP could directly promote cardiomyocyte proliferation and structural remodeling, both of which are essential for myocardial repair. Ki67 immunofluorescence staining (Fig. 3F–G) revealed that WH hydrogels moderately increased proliferative activity in H9C2 cells, while WH/DP further enhanced Ki67 expression. In addition, we conducted primary cardiomyocyte proliferation experiments (Figs. S8 and S9). In the Ki67 and EdU immunofluorescence staining, the WH/DB@DP group showed higher fluorescence intensity, indicating that it better promotes the proliferation of primary cardiomyocytes. The proliferation ability of the WH/DB@DP group was the highest, and quantitative analysis showed a significant increase compared with that of WH/DP, suggesting that the incorporation of DB strongly promoted the proliferation of cardiomyocytes. To assess structural remodeling and intercellular communication, dual immunofluorescence staining of α-actin and CX43 was performed (Fig. 3H). WH hydrogels partially improved cytoskeletal organization and gap junction distribution, WH/DP further upregulated CX43 expression, and WH/DB@DP produced the most pronounced effects, characterized by dense α-actin filaments and continuous CX43 localization at cell-cell interfaces. Quantitative fluorescence analysis (Fig. S10) confirmed that both α-actin and CX43 expression levels were highest in the WH/DB@DP group. These results demonstrate that the composite patch not only promotes cardiomyocyte proliferation but also reinforces structural stability and intercellular connectivity, thereby facilitating the restoration of synchronized myocardial contraction and functional recovery. We used Cav1.2, SERCA2a, p-RyR2, and p-CaMKII markers to create a “comprehensive view" of calcium ion regulation and signaling pathways in cardiac cells. As shown in Fig. S11A–B, the fluorescence intensity of p-CaMKII was significantly reduced in the WH/DB@DP hydrogel group, suggesting that the WH/DB@DP hydrogel may alleviate cellular stress and block key signaling pathways that contribute to the deterioration of heart function, helping to restore calcium regulation to a stable state. Additionally, the reduced protein expression of p-CaMKII further supports this finding (Fig. S11C–D). Furthermore, Fig. S11C and E show that the expression levels of Cav1.2 and SERCA2a were upregulated, indicating enhanced calcium influx ("activation switch") and recovery of cardiac relaxation function. The downregulation of p-RyR2 expression suggests that “abnormal leakage" from the sarcoplasmic reticulum calcium release channel was inhibited, reducing diastolic calcium leakage. This indicates that the WH/DB@DP hydrogel effectively improves the calcium cycling of cardiomyocytes: from calcium influx (Cav1.2) to calcium reuptake (SERCA2a) and the stability of calcium release (p-RyR2), with the entire pathway being improved. By inhibiting the overactive CaMKII signaling pathway, the hydrogel corrects the pathological calcium dysregulation process.

The increase and restoration of intracellular Ca²⁺ concentration reflect the real-time excitation-contraction coupling of cardiomyocytes [33]. In hypoxic conditions, the cellular repair effects of different hydrogels were evaluated by recording the frequency and intensity of instantaneous Ca²⁺ flux signals. We recorded the instantaneous Ca²⁺ flux signals at four points from the Control, H₂O₂, and WH, WH/DP, and WH/DB@DP hydrogel groups after H₂O₂ treatment (Fig. S11F–H). In the H₂O₂ and WH groups, cardiomyocytes with severe hypoxic damage exhibited few scattered changes in Ca²⁺ flux signals and weaker contraction function. The WH/DP group, which contains liposomal DP, showed stronger instantaneous Ca²⁺ flux signals and higher frequencies compared to the first two groups. In the WH/DB@DP hydrogel group, the rhythmic changes in instantaneous Ca²⁺ flux signals were most prominent, with the strongest signals and the highest frequencies. The synchronized change in the overall fluorescence signals demonstrated that the WH/DB@DP hydrogel effectively maintained myocardial electro-mechanical coupling and synchronized the contractile ability of cardiomyocytes. These results suggest that the WH/DB@DP hydrogel can effectively reduce hypoxic damage and maintain the functionalization and synchronized contraction of cardiomyocytes. This suggests that the introduction of DB can activate signaling pathways such as PI3K/Akt and MAPK, regulate endothelial cell proliferation, migration, differentiation, and apoptosis, promote structural remodeling of endothelial cells, enhance cell-cell connections, and thereby facilitate lumen formation and vascular stability [34].

3.4. In vitro immunomodulation, mitochondrial protection and antioxidative mechanisms

Oxidative stress-induced myocardial injury is typically accompanied by excessive M1 macrophage activation, which amplifies the inflammatory cascade and delays repair [35]. Consistent with this, immunofluorescence staining (Fig. 4A–B) and flow cytometry (Fig. 4C–D) showed that H₂O₂ exposure significantly increased the proportion of CD86⁺ M1 macrophages from 8.67 % in Control to 15.74 %, confirming a pro-inflammatory shift. WH hydrogel treatment partially counteracted this effect, modestly elevating CD206⁺ M2 macrophages to 2.15 % and improving the CD206/CD86 ratio to 0.19. Incorporation of DP further enhanced this response, increasing the CD206/CD86 ratio to 0.96. Strikingly, WH/DB@DP treatment produced the strongest effect, with M2 macrophages reaching 14.21 % and the CD206/CD86 ratio rising by 4.2-fold relative to the H₂O₂ group. This stepwise improvement across WH, WH/DP, and WH/DB@DP highlights the synergistic mechanism by which ROS-responsive DP alleviates oxidative triggers of NF-κB signaling, while DB further suppresses pro-inflammatory gene expression and activates the PPARγ pathway to promote reparative polarization [36]. We conducted co-culture experiments to further elucidate the intracellular crosstalk and protective mechanisms between macrophages and cardiomyocytes. As shown in Fig. S12, in the resting state, macrophages exhibited relatively high fluorescence intensity of p-CaMKII, and this intensity increased further upon activation. However, activated macrophages treated with WH/DB@DP showed lower fluorescence intensity, suggesting that when macrophages, whether in a resting or activated state, are co-cultured with cardiomyocytes, they lead to increased calcium leakage and disrupted signaling pathways. In contrast, WH/DB@DP treatment can regulate macrophages and maintain the normal transmission of calcium signals. Furthermore, the expression levels of Bax and caspase-3 in co-cultured macrophages were higher than those in the control group. After treatment with WH/DB@DP, the expression of Bax and caspase-3 in the cells was significantly downregulated. Additionally, after LPS treatment, the expression of Bcl-2 in the cells decreased, while treatment with WH/DB@DP led to a significant increase in Bcl-2 expression. Collectively, these findings demonstrate that WH/DB@DP effectively reprograms macrophages toward an anti-inflammatory phenotype, establishing a regenerative milieu critical for myocardial repair.

Fig. 4.

In vitro antioxidant and immunomodulatory functions of the myocardial patch. (A) Immunofluorescence staining of mouse macrophage polarization after hydrogel treatment; (B) Quantitative analysis of M2/M1 and M1/M2 fluorescence ratios; (C) Flow cytometry plots of macrophage polarization following hydrogel treatment; (D) Quantitative analysis of CD206/CD86 and CD86/CD206 ratios from flow cytometry; (E) Mitochondrial membrane potential staining of H9C2 cardiomyocytes after hydrogel treatment; (F) Quantitative analysis of JC-1 fluorescence intensity (mitochondrial membrane potential); (G) Quantification of ATP production; (H) Dual immunofluorescence staining of PINK and Parkin in H9C2 cardiomyocytes after hydrogel treatment; (I) Quantitative analysis of PINK and Parkin fluorescence intensity; (J) TEM images of mitochondria in H9C2 cells after hydrogel treatment; (K) Quantitative analysis of NRF and 4-HNE fluorescence intensity; (L) Dual immunofluorescence staining of NRF and 4-HNE in H9C2 cardiomyocytes after hydrogel treatment; (M) Western blot bands of key marker proteins in H9C2 cells treated with hydrogel materials (a: Control, b: H₂O₂, c: WH, d: WH/DP, e: WH/DB@DP); (N) Quantification of mitophagy-related protein markers; (O) Quantification of apoptosis-related protein markers; (P) Quantification of oxidative stress-related protein markers; (Q) qPCR analysis of LC3 gene expression in H9C2 cells; (R) qPCR analysis of PINK1 gene expression in H9C2 cells; (S) qPCR analysis of BECN-1 gene expression in H9C2 cells. (n = 5, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001).

Oxidative stress damage and the inflammatory microenvironment are major obstacles to myocardial repair [37]. Studies have shown that ischemia/hypoxia induces mitochondrial damage in cardiomyocytes, which further releases mitochondrial DNA, activating immune cells to participate in the cascade of inflammatory responses in the microenvironment [38]. Given the central role of mitochondria in cardiomyocyte survival, we next evaluated whether the hydrogel system could stabilize mitochondrial function under oxidative stress. JC-1 staining (Fig. 4E and F) showed that H₂O₂ stimulation caused a dramatic loss of mitochondrial membrane potential (ΔΨm), with the red/green fluorescence ratio dropping by 84 % compared to Control. WH hydrogel treatment partially restored ΔΨm, while WH/DP further improved recovery. Notably, WH/DB@DP treatment nearly restored ΔΨm to control levels, demonstrating the strongest protective effect. Consistent with this, ATP quantification (Fig. 4G& Fig. S13) revealed that H₂O₂ exposure reduced ATP production to 2.24 nmol/mg protein, which was gradually rescued by WH (3.88 nmol/mg) and WH/DP (5.73 nmol/mg). Importantly, WH/DB@DP achieved the highest ATP production (15.13 nmol/mg), far exceeding other groups. These results confirm that the composite hydrogel effectively stabilizes mitochondrial bioenergetics under oxidative injury, thereby supporting cardiomyocyte survival and functional recovery.

To further elucidate the mechanism underlying mitochondrial protection, we examined the role of PINK1/Parkin-mediated mitophagy. Immunofluorescence staining (Fig. 4H–I) revealed that H₂O₂ stimulation markedly suppressed PINK1 and Parkin expression, reducing their fluorescence intensities to ∼27 % and ∼23 % of Control, respectively. WH treatment induced a modest recovery (PINK1 ∼57 % and Parkin ∼40 % of Control), while WH/DP produced stronger restoration (PINK1 ∼107 % and Parkin ∼45 % of Control). Strikingly, WH/DB@DP treatment elevated both markers to levels comparable to, or even exceeding, Control (PINK1 ∼155 %, Parkin ∼104 % of Control).

TEM analysis (Fig. 4J) provided ultrastructural confirmation. In the H₂O₂ model group, cardiomyocytes exhibited severe mitochondrial damage, including swelling, disrupted cristae, and vacuolization. WH and WH/DP treatments alleviated some abnormalities but residual defects remained. In contrast, WH/DB@DP markedly preserved mitochondrial ultrastructure, showing intact cristae, dense matrix, and minimal vacuolization. These morphological improvements are consistent with the restored ΔΨm and ATP production, reinforcing that WH/DB@DP promotes clearance of damaged mitochondria and maintains mitochondrial integrity under oxidative stress.

In addition to mitophagy activation, the antioxidant function of WH/DB@DP was systematically evaluated. Dual immunofluorescence staining (Fig. 4K–L) showed that H₂O₂ stimulation in the model group induced a 23.57-fold increase in 4-HNE compared with Control, accompanied by a 45.43-fold decrease in NRF2 nuclear localization. WH treatment partially reversed these changes, reducing 4-HNE by 1.03-fold compared with H₂O₂ group and increasing NRF2 by 1.36-fold vs H₂O₂ group. WH/DP achieved more pronounced effects, lowering 4-HNE by 3.93-fold vs H₂O₂ group and elevating NRF2 by 14.29-fold vs H₂O₂ group (10.51-fold vs WH). Importantly, WH/DB@DP further decreased 4-HNE by 7.33-fold vs H₂O₂ group (1.87-fold vs WH/DP) and markedly increased NRF2 by 34.04-fold vs H₂O₂ group (2.38-fold vs WH/DP). Notably, NRF2 levels in WH/DB@DP approached those of Control (0.75-fold of Control), whereas 4-HNE, although substantially reduced, remained higher than Control (3.22-fold vs Control).

These findings were corroborated at the protein level (Fig. 4M–P). Western blot analysis demonstrated that WH/DB@DP markedly increased LC3 expression by 4.96-fold, PINK1 by 5.63-fold, and Parkin by 4.45-fold compared with the H₂O₂ group, consistent with the enhanced mitophagy observed in imaging studies. Simultaneously, WH/DB@DP significantly upregulated NRF2 (6.03-fold) and its downstream antioxidant proteins HO-1 (4.45-fold), CAT (2.80-fold), NQO-1 (2.76-fold), and SOD2 (3.91-fold), while reducing CYTC release by 0.16-fold, thereby limiting apoptotic signaling. Moderate increases in VDAC expression were also observed (0.31-fold vs H₂O₂ group), indicating partial restoration of mitochondrial material exchange.

Furthermore, qPCR analysis (Fig. 4Q–S) further confirmed at the transcriptional level that WH/DB@DP significantly enhanced the expression of autophagy-related genes: LC3 mRNA (3.58-fold), PINK1 (2.98-fold), and BECN-1 (4.00-fold). Combined with other evidence from the study, we outline a clear synergistic protective mechanism: WH/DB@DP activates the NRF2 signaling pathway on one hand, enhancing the overall antioxidant capacity of the cells and providing a protected “low oxidative stress" environment for the cells (including the autophagic system itself); on the other hand, it directly enhances PINK1/Parkin-mediated mitophagy, actively clearing damaged mitochondria that have become “factories" of ROS. This not only eliminates the source of oxidative damage but also prevents cell apoptosis. Therefore, antioxidant defenses safeguard quality control (autophagy), while effective quality control, in turn, alleviates the burden on the antioxidant system. The two form a positive feedback loop, from the molecular to the functional level, collectively contributing to comprehensive protection against oxidative stress-induced mitochondrial damage.

In summary, the WH/DB@DP hydrogel orchestrates a coordinated protective response by attenuating inflammation, preserving mitochondrial integrity, and activating autophagic and antioxidant pathways, thereby creating a favorable microenvironment for myocardial repair. While these cellular and functional assays establish the hydrogel's therapeutic potential, the precise molecular mechanisms driving these multidimensional effects remain unclear. To gain deeper insights into the signaling networks and transcriptional programs underlying these protective actions, transcriptomic analysis was next performed.

3.5. Transcriptomic profiling reveals molecular pathways regulated by WH/DB@DP hydrogel in myocardial injury

To further elucidate the molecular basis underlying the cardioprotective effects of the WH/DB@DP hydrogel, transcriptomic profiling of H9C2 cardiomyocytes was performed. This unbiased approach allows for the identification of global gene expression changes and signaling pathways that may account for the anti-inflammatory, antioxidant, and mitochondrial-protective activities observed in vitro. In the principal component analysis (Fig. 5A), the H₂O₂ group is clearly separated from the Control and WH/DB@DP groups. The transcriptome of the H₂O₂ treatment group shows significant differences from both the Control and WH/DB@DP groups in the multidimensional space. The Venn diagram in Fig. 5B shows the overlap of differentially expressed genes between the three groups. Additionally, the correlation analysis in Fig. 5C shows a high similarity between the Control group and the WH/DB@DP group, particularly in the overall trend of gene expression patterns. This trend suggests that WH/DB@DP hydrogel treatment can partially restore or maintain the stability of myocardial cells and alleviate the transcriptomic changes induced by H₂O₂ treatment. In the differential expression analysis (Fig. 5D), the gene expression difference between the Control and H₂O₂ groups is significant, with 696 upregulated genes and 1317 downregulated genes. The gene expression difference between the WH/DB@DP group and the H₂O₂ group is also significant, with 1092 upregulated genes and 2042 downregulated genes. The gene expression difference between the WH/DB@DP group and the Control group is smaller, with 142 upregulated genes and 314 downregulated genes. Although there is some gene expression difference between the hydrogel treatment group and the Control group, it suggests that the treatment group may play a role in specific biological processes, such as enhancing antioxidant activity, promoting mitochondrial function, and cell-to-cell communication. Overall, the gene expression pattern of the hydrogel treatment group is similar to that of the Control group, indicating that the hydrogel plays an active role in repairing myocardial function without causing excessive transcriptomic reprogramming. The hierarchical clustering heatmap analysis (Fig. 5E) further confirms the differential distribution of samples, with the gene expression pattern of the H₂O₂ treatment group showing greater differences compared to the Control and WH/DB@DP groups, indicating that oxidative stress induces widespread transcriptomic changes, particularly in genes related to cell damage, inflammation, and antioxidant responses. GO enrichment analysis revealed that the differentially expressed genes were mainly associated with biological processes such as positive regulation of the MAPK cascade, neuron development, connective tissue organization, and establishment of cell-cell junctions (Fig. 5F). These processes are highly relevant to myocardial repair, as they reflect the activation of survival signaling, structural remodeling, and intercellular communication required for functional recovery. Consistently, KEGG analysis identified several classical pathways, including MAPK, calcium (Ca²⁺), cAMP, and Rap1 signaling, together with cytoskeleton regulation and neuroactive ligand-receptor interaction (Fig. 5G). These pathways directly relate to the design rationale of WH/DB@DP: MAPK and Rap1 signaling are linked to anti-inflammatory and pro-survival effects, calcium signaling is essential for mitochondrial homeostasis and contractility, while cAMP signaling supports endothelial proliferation and angiogenesis-matching the in vitro findings of reduced oxidative stress, restored mitochondrial function, and enhanced vascularization. GSEA reinforced this conclusion by showing strong positive enrichment of Ca²⁺ pathways (Fig. 5H) in the WH/DB@DP group, which mechanistically explains the observed improvements in mitochondrial stability, ROS detoxification, and angiogenesis.

Fig. 5.

Transcriptomic analysis of the myocardial patch WH/DB@DP. (A) PCA plot comparing transcriptomes of H9C2 cells treated with control, H₂O₂ group and WH/DB@DP; (B) Venn diagram of transcriptomic profiles; (C) Correlation analysis of samples; (D) Volcano plot showing differentially expressed genes (DEGs); (E) Heatmap comparison of transcriptomic profiles; (F) GO enrichment bubble plot of DEGs; (G) KEGG enrichment bubble plot of DEGs; (H) GSEA plot of DEGs involved in the calcium signaling pathway.

In summary, transcriptomic analysis indicates that the therapeutic benefits of WH/DB@DP are not limited to a single mechanism but arise from coordinated regulation of multiple signaling pathways critical for myocardial repair. This systems-level response reflects the multifunctional design of the hydrogel patch, where ROS-responsive DB release, antioxidative activity, and adhesive support converge to reprogram cellular signaling, thereby providing comprehensive cardioprotection.

3.6. WH/DB@DP hydrogel improves cardiac repair in a mouse MI model

Building on the in vitro findings of immunomodulation, mitochondrial protection, and antioxidant activity, we next investigated whether these benefits translate into functional improvement in vivo. A mouse MI model was employed to systematically evaluate the therapeutic efficacy of the WH/DB@DP hydrogel patch on cardiac performance and post-infarction remodeling. Echocardiographic analysis (Fig. 6A–B & Figure S14) revealed that mice in the MI model group exhibited significant left ventricular dysfunction at both 2 and 4 weeks post-infarction, characterized by markedly reduced EF and FS, as well as significantly increased left ventricular internal diameters during diastole (LVIDd) and systole (LVIDs) In comparison, the WH hydrogel group showed moderate improvements in these parameters, the WH/DP group exhibited further improvements, and the oral DB group demonstrated even greater improvements. It is worth noting that the WH/DB@DP group showed the most significant improvements at both time points—there was an increase in EF and FS levels, and a decrease in LVIDd and LVIDs values. In addition, we compared the effects of oral DB and patch administration on the liver and kidneys of mice (Fig. S15). The HE results indicate that oral administration caused damage to both the liver and kidneys, while the WH/DB@DP heart patch treatment showed no damage. This suggests that the patch treatment has no obvious toxic side effects, significantly enhances cardiac contractile function, and effectively inhibits the progression of left ventricular remodeling.

Fig. 6.

In vivo evaluation of WH/DB@DP in a mouse myocardial infarction (MI) model. (A) Echocardiographic images of different treatment groups (Sham, Model, WH, WH/DP, WH/DB@DP) at various time points during MI treatment; (B) Quantitative analysis of echocardiographic data following MI induction and treatment; (C) Quantification of left ventricular wall thickness and infarct size in each group after MI treatment; (D) Representative H&E and Masson staining images of heart tissue sections post-MI treatment in different groups. (n = 5, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001).

Quantitative analysis of ventricular wall thickness and myocardial fibrosis further confirmed the cardioprotective effect of the WH/DB@DP system (Fig. 6C). Mice in the MI model group exhibited pronounced ventricular wall thinning and extensive fibrotic areas. Both the WH and WH/DP groups showed partial mitigation of these pathological changes, while the WH/DB@DP group demonstrated the most pronounced restoration of wall thickness and significant reduction in fibrosis, with statistically significant differences. Histological analysis through H&E and Masson's trichrome staining (Fig. 6D) revealed consistent findings. The MI model group displayed severe myocardial disorganization and large fibrotic scar formation. Both the WH/DP and WH/DB@DP groups showed improved myocardial architecture and reduced fibrotic areas, with the WH/DB@DP group exhibiting the most substantial repair effect, including nearly intact myocardial structure and markedly reduced collagen deposition.

This enhanced myocardial repair may be largely attributed to DB's antifibrotic activity, which is known to regulate the TGF-β/Smad signaling pathway and thereby suppress fibroblast overproliferation and collagen deposition. At the same time, DB may facilitate microvascular recovery in the infarcted region by promoting angiogenesis, which improves tissue perfusion and provides structural support for remodeling [39]. These mechanisms together explain the marked reduction in fibrosis and improvement of myocardial wall integrity observed in the WH/DB@DP group.

3.7. Restoration of myocardial structure and reduction of fibrosis by WH/DB@DP in vivo

Building upon the observed improvements in cardiac function and attenuation of ventricular remodeling (Fig. 6), further analyses were performed to investigate how WH/DB@DP influences myocardial structure and cellular integrity in the MI model. Dual immunofluorescence staining was used to evaluate the expression of the gap junction protein CX43 and cardiac troponin cTn1 (Fig. 7A, B, and 7D). In the MI model group, expression levels of both CX43 and cTN1 were significantly reduced, indicating severe structural damage. In contrast, the expression of CX43 and cTN1 in the WH/DB@DP group was nearly restored to the levels observed in the sham group, significantly improving myocardial gap junctions and intercellular electrical coupling, both critical for maintaining the structural stability of myocardial tissue. This underscores the key role of DB in promoting myocardial structural repair. Quantitative fluorescence analysis further confirmed that these differences were statistically significant, indicating that WH/DB@DP facilitates the restoration of myocardial structure and intercellular connectivity.

Fig. 7.

Assessment of myocardial infarction and repair in a mouse MI model treated with WH/DB@DP. (A) Dual immunofluorescence staining of gap junction protein CX43 (red) and cardiac troponin cTN1 (green), with nuclear staining by DAPI (blue), in heart tissues from different treatment groups (Sham, Model, WH, WH/DP, WH/DB@DP); scale bar = 50 μm; (B) Quantitative analysis of CX43 fluorescence intensity; (C) Fluorescence staining of the infarction marker wheat germ agglutinin (WGA, green) in cardiac tissues across treatment groups; scale bar = 50 μm; (D) Quantitative analysis of cardiac troponin cTN1 fluorescence intensity; (E) Quantification of WGA fluorescence intensity (infarct area marker); (F) Quantitative analysis of proliferating cell nuclear antigen (PCNA) fluorescence expression; (G) Quantitative analysis of TUNEL fluorescence expression (apoptosis marker); (H) Representative and enlarged images of PCNA (red) fluorescence staining in cardiac tissues from each group after MI modeling and treatment; (I) Representative and enlarged images of TUNEL (red) fluorescence staining in cardiac tissues from each group after MI modeling and treatment. (n = 5, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001).

Additionally, WGA staining was employed to visualize MI and fibrosis (Fig. 7C & E). The MI model group displayed strong WGA fluorescence, indicative of extensive infarction and fibrotic remodeling. In contrast, WH/DB@DP treatment substantially reduced WGA fluorescence intensity, suggesting a smaller infarct area and improved tissue structure. This observation may be attributed to DB's role in promoting repair of the infarcted area and improving myocardial structure through mechanisms such as enhancing angiogenesis, reducing oxidative stress, and inhibiting inflammation.

To investigate cellular proliferation and apoptosis, the expression of PCNA and TUNEL was assessed (Fig. 7F–I). In the MI model group, the number of PCNA-positive cells was significantly reduced, reflecting limited regenerative capacity, while TUNEL-positive cells were markedly increased, indicating extensive apoptosis. Both WH and WH/DP treatments led to partial improvement, but the WH/DB@DP group exhibited the highest PCNA expression and a significant reduction in TUNEL-positive cells. In addition, we performed combined staining of PCNA and cTnI to further confirm myocardial cell proliferation (Fig. S16). These differences were also statistically significant, demonstrating that WH/DB@DP effectively promotes cardiomyocyte proliferation and inhibits apoptosis, thereby contributing to a more favorable microenvironment for cardiac repair.

In summary, the WH/DB@DP composite myocardial patch significantly enhanced myocardial structural integrity, restored gap junction protein expression, reduced infarct size, promoted cell proliferation, and suppressed apoptosis in a mouse MI model. These effects collectively indicate that WH/DB@DP not only preserves myocardial architecture but also creates a favorable cellular environment for regeneration. Given that structural repair is closely linked with vascular support and inflammatory regulation, the subsequent analyses focused on angiogenesis, oxidative stress, and inflammatory responses to further clarify how WH/DB@DP orchestrates a comprehensive myocardial healing process.

3.8. Enhancement of angiogenesis and attenuation of oxidative stress and inflammation by WH/DB@DP

To further clarify how WH/DB@DP supports myocardial repair, we next examined its impact on the post-infarction microenvironment, focusing on angiogenesis, oxidative stress, and inflammation. First, neovascularization was evaluated through immunofluorescence staining of CD31 and α-SMA (Fig. 8A–C). The MI model group exhibited a marked reduction in neovascular density, indicating impaired revascularization. Both the WH and WH/DP groups moderately promoted vascular formation, while the WH/DB@DP group showed the highest expression levels of CD31 and α-SMA, with statistically significant differences. This enhancement is likely attributed to DB activating key signaling pathways, such as PI3K/Akt and MAPK, which promote the proliferation and migration of endothelial cells and surrounding smooth muscle cells, thus accelerating angiogenesis. These findings suggest that the WH/DB@DP patch strongly enhances angiogenesis, thereby improving the ischemic microenvironment.

Fig. 8.

Effects of WH/DB@DP on angiogenesis, antioxidation, and anti-inflammation in a mouse myocardial infarction (MI) model. (A) Immunofluorescence staining of neovascularization at 2 and 4 weeks post-treatment in different groups (Sham, Model, WH, WH/DP, WH/DB@DP), showing platelet-endothelial adhesion molecule CD31 (green), vascular smooth muscle α-SMA (red), and nuclei (DAPI, blue); scale bar = 50 μm; (B) Quantitative analysis of CD31 fluorescence expression at 2 and 4 weeks post-treatment; (C) Quantitative analysis of α-SMA fluorescence expression at 2 and 4 weeks post-treatment; (D) Immunohistochemical staining (overview and magnified views) of oxidative stress marker 4-Hydroxynonenal (4-HNE) in heart tissue at 2 and 4 weeks post-treatment; scale bars: overview = 1 mm, magnified = 50 μm; (E) Quantitative analysis of 4-HNE expression levels at 2 and 4 weeks post-treatment; (F) Immunohistochemical staining of inflammatory cytokines IFN-γ, IL-6, and IL-1β in heart tissue at 2 and 4 weeks post-treatment; scale bar = 50 μm; (G) Quantitative analysis of IFN-γ, IL-6, and IL-1β expression levels at 2 weeks post-treatment; (H) Western blot images of apoptosis- and stress-related proteins (Caspase-3, Cleaved Caspase-3, Bcl-2, BAX, p21, Cyt-c) in heart tissue at 4 weeks post-treatment across treatment groups; (I) Quantification of p21 and Cyt-c expression and relative protein ratios of Bcl-2/BAX and Caspase-3/Cleaved Caspase-3 at 4 weeks post-treatment. (n = 5, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001).

Oxidative stress levels were further evaluated using 4-HNE immunohistochemical staining (Fig. 8D–E). The MI model group displayed elevated 4-HNE expression, indicative of significant oxidative damage. WH/DB@DP treatment notably reduced 4-HNE levels, reflecting effective mitigation of oxidative stress in myocardial tissue. This may be attributed to DB's ability to upregulate the NRF2 pathway, which activates antioxidant enzymes such as SOD and CAT, helping to clear excess ROS and protect cardiomyocytes from oxidative damage, thereby alleviating post-MI injury.

Inflammatory cytokine analysis revealed that IFN-γ, IL-6, and IL-1β were significantly upregulated in the MI model group, indicating a pronounced inflammatory response. Both WH/DP and WH/DB@DP treatments reduced the expression of these cytokines, with WH/DB@DP showing the most significant suppression (Fig. 8F–G, Figure S17), highlighting its potent anti-inflammatory capacity.

To further investigate apoptosis and oxidative damage at the molecular level, Western blot analysis was performed (Fig. 8H–I). Myocardial cell apoptosis is a form of active, programmed cell death that occurs under pathological conditions and plays a crucial role in the progression of MI. Since myocardial cells are terminally differentiated and lack regenerative ability through division [40] reversing myocardial cell damage and preventing apoptosis is critical. Ischemia and hypoxia are primary factors that induce myocardial cell apoptosis [41,42]. Characteristic structural changes of apoptosis, primarily occurring in the nucleus, manifest as nuclear shrinkage. Bcl-2 is a key regulatory gene of apoptosis. Upon apoptotic signal activation, the pro-apoptotic protein Bax translocates from the cytoplasm to the outer mitochondrial membrane, polymerizing to form membrane pores that allow cytochrome C to exit the mitochondria and enter the cytoplasm. Once Caspase-3 is activated in the cytoplasm, cytochrome C facilitates cell death [43].

Western blot analysis revealed that in the MI model group, Cyt-c levels were elevated, the Bcl-2/BAX ratio was reduced, and the Caspase-3/Cleaved Caspase-3 ratio was increased, indicating extensive myocardial cell apoptosis. WH/DB@DP treatment inhibited Cyt-c release, restored the Bcl-2/BAX ratio, and suppressed Caspase-3 activation. These findings suggest that DB alleviated oxidative stress-induced damage to myocardial cells by improving mitochondrial function and regulating the apoptosis signaling pathway [44].

These in vivo findings can be mechanistically explained by the rational design of the hydrogel system. The ROS-responsive PBAP-modified dextran enabled selective activation in the infarcted microenvironment, thereby reducing oxidative stress while triggering DB release. HA-NB contributed to stable myocardial adhesion, ensuring localized retention of the therapeutic payload. DB itself provided dual anti-inflammatory and pro-angiogenic functions, complementing the antioxidative effect of PBAP. Together with the mechanically compliant WPI-based scaffold, these design elements acted in concert to remodel the ischemic microenvironment, facilitating vascular regeneration and suppression of adverse remodeling.

Finally, we evaluated the long-term biosafety and biodegradability of the material. As shown in Fig. S18, after 4 weeks of treatment with the WH/DB@DP hydrogel patch, no obvious damage was observed in the liver, spleen, lungs, or kidneys of the mice, and no significant abnormalities were found in the blood routine results, indicating that the WH/DB@DP hydrogel has good long-term biosafety. Fig. S19 shows that the WH/DB@DP hydrogel was gradually cleared over time, with the area of the hydrogel shrinking, indicating that the material has good biodegradability.

4. Conclusion

This study designed a multifunctional injectable hydrogel patch that selectively covalently binds to myocardial tissue by incorporating HA-NB into the WPI-MA network, thereby avoiding nonspecific adhesion to the thoracic cavity. To improve the bioavailability and stability of rosmarinic acid B, ROS-responsive PBAP-modified liposomes were used, which not only efficiently scavenge excess reactive oxygen species but also release drug molecules on demand within the infarcted microenvironment. Through in vitro cell experiments and a mouse myocardial infarction model, we found that the hydrogel patch could regulate macrophage polarization, stabilize mitochondrial function, activate mitochondrial autophagy, and enhance the antioxidant defense system, ultimately significantly improving heart function while inhibiting fibrosis and cell apoptosis. Therefore, this multifunctional hydrogel patch has significant clinical potential in cardiac repair, as it can be precisely delivered through small incisions or with thoracoscopic assistance. Flexible catheters can accurately control the injection location and size of the patch, enabling more precise and personalized repair treatments. This minimally invasive repair approach not only enhances treatment precision but also overcomes the limitations of traditional treatment methods, reducing the risks of excessive bleeding, infection, and poor bone healing associated with traditional open-heart surgery.

Ethics approval and consent to participate

The animal experiments involved in the study were reviewed and approved by the Animal Experiment Ethics Committee of Guangzhou University of Chinese Medicine. The ethics approval number is 20241009001, with the initial review conducted on October 9, 2024, and the final review completed on March 20, 2025. All experimental procedures strictly adhere to the requirements specified in the Regulations on the Ethical Review of Animal Experiments of Guangzhou University of Chinese Medicine, ensuring compliance with animal welfare and ethical standards.

CRediT authorship contribution statement

Minying Li: Writing – original draft, Conceptualization. Qinghe Wu: Validation, Methodology, Investigation, Formal analysis. Weipeng Sun: Writing – original draft, Data curation. Wenhu Wu: Writing – review & editing. Biyi Zhao: Writing – review & editing, Validation. Yifei Wang: Data curation. Wei Wang: Writing – review & editing. Chun Fan: Writing – review & editing, Supervision, Funding acquisition. Dong Deng: Supervision, Project administration, Funding acquisition, Conceptualization. Fanhang Meng: Writing – review & editing, Supervision, Funding acquisition, Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary data

The following are the Supplementary data to this article: