Abstract Myocardial ischemia-reperfusion (I/R) injury is characterized by oxidative stress, mitochondrial dysfunction, inflammation, and fibrosis, ultimately leading to chronic cardiac dysfunction and heart failure. Current therapeutic strategies that predominantly target single biological pathways exhibit limited long-term efficacy, underscoring the necessity for multi-targeted approaches. In this study, we developed a ROS-responsive hydrogel system, S1&FT/Lipo-QCFT, tailored to deliver drugs for treating various stages of myocardial I/R injury. This system timing of drug release to achieve rapid deployment at early intervention stages and maintain sustained release thereafter. Initially, the hydrogel platform quickly releases the molecular forms of the superoxide inhibitor S1QEL1.1 and tannic acid, specifically targeting the elevated ROS levels at the I/R site to alleviate early oxidative damage and encourage macrophage polarization toward the M2 phenotype. Subsequently, the system gradually releases anti-fibrotic agent FT011, encapsulated in lipid nanocarriers, which actively counters TGF-β1-induced fibrosis and forestalls adverse ventricular remodeling, thereby enhancing long-term cardiac repair. In vivo studies demonstrated that the S1&FT/Lipo-QCFT hydrogel significantly improved cardiac function and reduced adverse ventricular remodeling. This hydrogel system provides a promising multi-targeted therapeutic strategy for comprehensive myocardial I/R injury treatment with strong potential for clinical translation. Keywords: Myocardial ischemia-reperfusion injury, ROS-Responsive hydrogel, Oxidative stress reduction, Multi-targeted therapy, Cardiac repair Graphical abstract [49]Image 1 [50]Open in a new tab Highlights * • ROS-responsive hydrogel enables multi-stage drug delivery for myocardial I/R injury. * • Timed drug release supports early intervention and sustained treatment phases. * • Improves mitochondria, regulates macrophages, and reduces cardiac fibrosis. * • Multi-phase therapy ensures both immediate and long-term cardiac repair. * • Outperforms single-pathway treatments in addressing I/R injury complexity. 1. Introduction Myocardial ischemia-reperfusion (I/R) injury, which occurs during the restoration of blood supply after a period of ischemia, often results in myocardial cell death, decreased cardiac function, fibrosis, and chronic cardiac dysfunction [[51]1,[52]2]. During the I/R process, accumulated succinate is rapidly oxidized, generating a large amount of mitochondrial reactive oxygen species (mROS) that impair mitochondrial function, activates the mitochondrial permeability transition pore (mPTP) [[53]3,[54]4], reduces ATP production, and induces cardiomyocyte apoptosis [[55][5], [56][6], [57][7]]. Additionally, excessive oxidative stress initiates an inflammatory response, exacerbating myocardial infarction and leading to fibrosis and worsening left ventricular remodeling[[58][8], [59][9], [60][10]]. Therefore, developing effective strategies to prevent, mitigate, or reverse the progression from myocardial infarction to heart failure is crucial in clinical practice (see [61]Scheme 1). Scheme 1. [62]Scheme 1 [63]Open in a new tab This schematic illustrates the therapeutic mechanism of the ROS-responsive hydrogel for myocardial ischemia-reperfusion (I/R) injury. By enhancing mitochondrial function, adjusting macrophage polarization, and mitigating cardiac fibrosis, the S1&FT/Lipo-QCFT hydrogel—which includes the superoxide inhibitor S1QEL1.1 and the anti-fibrotic agent FT011—employs a multi-stage cooperative strategy. Initially, the hydrogel clears mitochondrial ROS, reducing early myocardial damage and moderating macrophage polarization to lower inflammatory responses in the early stage. The controlled release of FT011 then inhibits fibroblast-to-myofibroblast transformation, decreasing type I and III collagen secretion, thereby reducing fibrosis and ultimately supporting long-term myocardial repair and functional recovery. DPPC (Dipalmitoylphosphatidylcholine), CHO (Cholesterol), DSPE-mPEG2000 (Distearoylphosphatidylethanolamine conjugated with polyethylene glycol), QCMCS (quaternized carboxymethyl chitosan), and TA (tannin). Current therapies targeting single biological pathways often fail to provide sustained benefits in myocardial I/R injury, highlighting the need for combined and synergistic strategies that address its multifactorial nature [[64][11], [65][12], [66][13]]. Conventional treatments—including antioxidants (e.g., N-acetylcysteine, vitamin C), anti-inflammatory agents (e.g., NSAIDs), and anti-fibrotic drugs (e.g., ACE inhibitors, TGF-β inhibitors)—are limited by their single-target mechanisms, insufficient temporal precision, and potential adverse effects during long-term use, ultimately affecting patient prognosis [[67][14], [68][15], [69][16]]. In the early stages of myocardial I/R injury, the accumulation of mitochondrial superoxide plays a pivotal role in triggering oxidative damage and cell death [[70]17,[71]18]. While existing mitochondrial-targeted antioxidants such as SS31, MitoVitE, and MitoQ rely on high mitochondrial membrane potential and may act as pro-oxidants under pathological conditions, S1QEL1.1 offers a more selective approach [[72][19], [73][20], [74][21]]. It inhibits superoxide generation at the IQ site of mitochondrial complex I, without disrupting electron transport, thereby preserving mitochondrial function and reducing infarct size in preclinical models [[75][22], [76][23], [77][24]]. Moreover, the oxidative microenvironment promotes polarization of macrophages toward the M1 phenotype, prolonging inflammatory responses and impairing repair [[78]25,[79]26]. Tannic acid (TA), a natural polyphenol with potent antioxidative and anti-inflammatory activities, has been shown to modulate macrophage polarization toward the reparative M2 phenotype and promote myocardial healing across various inflammatory disease models [[80][27], [81][28], [82][29]]. In the chronic phase, the post-infarction environment induces fibroblast-to-myofibroblast transition, leading to excessive type I and III collagen deposition and adverse ventricular remodeling[[83][30], [84][31], [85][32]]. FT011, a small-molecule TGF-β receptor I kinase inhibitor and structural analog of pirfenidone, selectively inhibits Smad2/3 phosphorylation and has demonstrated anti-fibrotic efficacy in both preclinical and early-phase clinical studies [[86]33,[87]34]. Considering the distinct temporal roles of these compounds, a spatiotemporally controlled delivery system is essential to maximize their therapeutic potential. S1QEL1.1, currently under preclinical development, functions as a mitochondria-targeted antioxidant that specifically mitigates early-phase superoxide generation [[88]19,[89]21]. FT011, which has completed early-phase clinical evaluation in fibrotic diseases, is released in a sustained manner and inhibits the TGF-β/Smad signaling pathway to prevent fibrosis [[90]35]. Collectively, these agents provide a multi-targeted therapeutic strategy to comprehensively address the pathophysiological progression of myocardial I/R injury. Hydrogels, recognized for their excellent biocompatibility, porous structure, and adjustable mechanical strength, provide an ideal platform for multi-drug delivery in the treatment of myocardial I/R injury[[91][36], [92][37], [93][38]]. These hydrogels create a microenvironment akin to the extracellular matrix (ECM), supporting damaged cardiomyocytes and providing essential mechanical reinforcement to weakened ventricular walls [[94]39]. Additionally, the drugs encapsulated within the hydrogel exhibit varying sizes, leading to differential release rates during delivery[[95][40], [96][41], [97][42]]. For example, Wu et al. engineered a co-assembly system of cisplatin (CDDP) and alginate nanoparticles (AlgNP/IRN) for precise dose control, supporting effective combination therapy [[98]43]. Similarly, Wang et al. incorporated diclofenac sodium (DS) and mangiferin-loaded micelles (MIC@MF) within a hydrogel, achieving both rapid molecular drug release and regulated nanoparticle delivery for chronic diabetic wound treatment [[99]44]. In addition, Sun and colleagues designed a polypeptide hydrogel/nanogel composite to sequentially release regorafenib (REG) and a TGF-β inhibitor, aiming to synergistically suppress colorectal tumor progression and metastasis [[100]45]. Herein, we developed a ROS-responsive hydrogel system to combat the pathological changes of myocardial I/R injury. Utilizing 2-formylphenylboronic acid (2-FPBA) as a crosslinker, alongside quaternized carboxymethyl chitosan (QCMCS) and TA, this system forms a dual dynamic covalent network via Schiff base and boronate ester bonds, creating an injectable ROS-responsive hydrogel. The hydrogel encapsulates the antifibrotic drug FT011 in negatively charged liposomes, integrated with the superoxide inhibitor S1QEL1.1 (S1&FT/Lipo-QCFT). Initially, it rapidly releases S1QEL1.1 and TA in response to ROS, clearing mitochondrial ROS to reduce early myocardial damage and regulate macrophage polarization to decrease inflammatory responses. Subsequently, the gradual release of FT011 inhibits the transformation of fibroblasts to myofibroblasts, diminishing the secretion of type I and type III collagen, significantly alleviating fibrosis and improving cardiac function. This delivery system is engineered to target distinct pathological processes at each treatment stage, ultimately facilitating cardiac repair and functional recovery. 2. Materials and methods 2.1. Materials Carboxymethyl chitosan (CMCS, degree of substitution = 90 %), 2-formylphenylboronic acid (2-FPBA, 98 %), glycidyl trimethylammonium chloride (GTMC, purity ≥95 %), and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) were purchased from Shanghai Aladdin Bio-Chem Technology Co., Ltd. 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-mPEG2000 (DSPE-mPEG2000), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), and 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (PTIO) were obtained from Sigma–Aldrich (USA). Calcein-AM/PI Double Staining Kit was obtained from KEYGEN BIOTECH (Jiangsu, China). The Cell Counting Kit-8 (CCK-8), Reactive Oxygen Species Assay Kit, and Mitochondrial Superoxide Assay Kit with MitoSOX Red were obtained from Beyotime (China). Hoechst 33342, ATP Content Assay Kit, and DPPH Free Radical Scavenging Capacity Assay Kit were purchased from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). Hifair® Ⅱ 1st Strand cDNA Synthesis Kit and Hieff qPCR SYBR® Green Master Mix were purchased from Yeasen (Shanghai, China).Primary antibodies CD86 (ab220188, Abcam, USA), CD206 (ab64693, Abcam, USA), Collagen I Polyclonal Antibody (PA1-26204, Invitrogen, Shanghai, China), Collagen type III (22734-1-AP, Pro-teinTech, China) Alpha-Smooth Muscle Actin Monoclonal Antibody (14-9760-95, Invitrogen, China),and WGA (W7024, Invitrogen, China) were obtained from Abcam(USA), Pro-teintech (China) and Invitrogen(China). All solvents and chemicals were purchased from commercial sources. They were used without further purification. 2.2. Preparation of nanoparticles 2.2.1. Preparation of FT011-Loaded liposomes The preparation of the liposomal formulations has been adapted from the reported procedures, with slight modifications, as follows [[101]46]. Firstly, 15.10 mg DSPC, 1.50 mg CHO and 1.23 mg DSPE-mPEG2000 were dissolved in 10 mL choroform/methanol/H[2]O mixture (15/4/1, v/v/v). A lipid film was formed by rotary evaporation to remove the solvent. Next, 4.1 mg of FT011 dissolved in 6 mL PBS (pH 7.4) was added to the film by sonication. For the preparation of the FT011 loaded nanodrug solution, the mixture was filtered through a 200 nm membrane. To remove any unencapsulated drug and mPEG, the solution was then dialysed against PBS (pH 7.4) using a membrane with a molecular weight cut-off of 140 kDa. Finally, the solution was concentrated through ultrafiltration using a 100 kDa molecular weight cutoff membrane. For future use, the resulting liposomes were stored at 4 °C. 2.2.2. Preparation of DiR-Labeled FT011 liposomes for In vivo imaging For in vivo fluorescence imaging, DiR-labeled FT011 liposomes (S1&DiR-FT/Lipo) were prepared similarly to the above method, except that 0.1 mol% DiR (relative to total lipid) was co-dissolved with DSPC, cholesterol, and DSPE-mPEG2000 during the initial organic phase preparation. The subsequent procedures for film formation, hydration with FT011 solution, sonication, membrane filtration, and dialysis were performed as described. DiR-labeled liposomes were protected from light and stored at 4 °C. 2.3. Preparation of QCMCS The procedure for the preparation of QCMCS was as follows. First of all, 2.18 g of CMCS was dissolved in 100 mL of deionized water. The solution was stirred at 55 °C for 24 h and then 0.45 g GTMC was added. The resultant mixture was then dialyzed for three days by means of a dialysis bag (MwCO: 1000). In the end, the QCMCS product was obtained by lyophilization [[102]47]. 2.4. Synthesis of QCFT hydrogel and S1QEL1.1&FT/Lipo-QCMCS-2-FPBA-TA hydrogel First, 2-FPBA powder was dissolved in a 0.01 M PBS solution (pH 7.4) under ultrasonic agitation at room temperature to prepare a 4 wt% 2-FPBA solution. Next, 2-FPBA/TA solutions with varying concentrations of TA (2 wt% each) were prepared by adding different amounts of TA powder. The QCMCS solution was made by dissolving QCMCS powder in deionized water and stirring at room temperature. Equal volumes of the QCMCS (5 wt%), 2-FPBA (2 wt%), and TA (2 wt%) solutions were then mixed at room temperature. The mixture was rapidly vortexed to form the hydrogel, designated as the QCFT hydrogel. In this naming convention, "QC" refers to QCMCS, "F" to 2-FPBA, and "T" to tannin. FT/Lipo nanoparticles (containing FT011 at a concentration of approximately 10 μM) were first added to a QCMCS solution under constant stirring to obtain the QCMCS@FT/Lipo solution. Separately, S1QEL1.1 (1 μM) was dissolved in a TA solution (2 wt%), which was then mixed with an equal volume of a 2-FPBA solution (2 wt%). The resulting mixtures were thoroughly blended to form the S1QEL1.1&FT/Lipo-QCMCS-2-FPBA-TA hydrogel (abbreviated as S1&FT/Lipo-QCFT hydrogel). This method enabled the formation of two types of dynamic covalent bonds within the hydrogel: (i) a Schiff base bond formed between the amino group of QCMCS and the aldehyde group of 2-FPBA, and (ii) a boronate ester bond established between the boronic acid group of 2-FPBA and the phenolic group of TA. 2.5. Characterization of QCFT hydrogel The morphology of QCFT hydrogel was characterized using scanning electron microscopy (SEM; SU8010, HITACHI, Japan), while the surface elemental composition was analyzed via Energy Dispersive X-ray Spectroscopy (EDS; INCA X-Max50, Oxford, UK). Pore sizes were measured with ImageJ-Pro Plus 6.0 (n = 3). Ultraviolet–visible (UV–Vis) spectra of the hydrogel were recorded using a Shimadzu UV-2450 spectrophotometer (Japan). Fourier transform infrared (FTIR) spectroscopy was conducted on an INVENIO R spectrometer (Bruker, Switzerland) in the wavenumber range of 400–4000 cm^−1. Proton nuclear magnetic resonance (^1H NMR) spectra were obtained by dissolving the hydrogel in deuterium oxide and analyzing them with a Bruker AVANCE III HD spectrometer operating at 400 MHz. X-ray photoelectron spectroscopy (XPS; K-Alpha, Thermo Fisher, USA) was employed to investigate the elemental composition and non-covalent interactions of the QCFT hydrogel. 2.6. In vitro antioxidant activity of QCFT hydrogel The antioxidant capacity of QCFT hydrogel was evaluated in vitro using both 1,1-diphenyl-2-picryl-hydrazyl radical (DPPH•) and 2-phenyl-4,4,5,5-tetramethylimidazolineoxyl-1-oxyl-3-oxide (PTIO•) scavenging assays. For the DPPH• assay, the lyophilized hydrogel was finely powdered and mixed with varying concentrations of a DPPH-ethanol solution. This mixture was incubated in the dark for 30 min. After centrifugation at 3000 rpm for 10 min, the absorbance of the supernatant was measured at 517 nm using a microplate reader. An untreated DPPH• solution was used as the control. In the PTIO• assay, different concentrations of the lyophilized QCFT hydrogel were added to a PTIO• solution and incubated in the dark for 60 min. The absorbance was then measured at 557 nm. The control for this assay was the untreated PTIO• solution. DPPH• or PTIO• scavenging was calculated with the following formula: DPPH• [MATH: (PTIO·)scavenging(%)=[AB−A< /mi>SAB]×100% :MATH] . 2.7. Detection of cytoplasmic ROS (cROS) and mitochondrial ROS (mROS) levels DCFH-DA and MitoSox were used to measure cytoplasmic cROS and mROS, respectively. H9C2 cells were plated at a density of 5 × 10^4 cells per well in 24-well plates. After 4 h of hypoxia, the medium was replaced with fresh medium containing the appropriate hydrogel formulations and incubated for an additional 4 h. The cells were then treated with DCFH-DA or MitoSox solutions at 37 °C for 20 min. Following this incubation, the cells were washed three times with PBS and stained with Hoechst-33342. Fluorescence images were acquired using an inverted fluorescence microscope (TCS SP8, Leica, Germany). 2.8. Assessment of mitochondrial function 2.8.1. Analysis of mitochondrial permeability transition pore (mPTP) opening Calcein AM staining in conjunction with CoCl[2] quenching assays was used to assess mitochondrial mPTP opening, as previously described. H9C2 cells were seeded in 24-well plates at a density of 5 × 10^4 cells per well and allowed to incubate for 24 h. After 4 h hypoxic exposure, the medium was replaced with fresh medium containing hydrogel extracts from different groups and the cells were cultured for another 4 h mPTP opening was visualised and analyzed using an inverted fluorescence microscope (TCS SP8, Leica, Germany). The cells were then treated with 5 mM CoCl[2] and stained with 1 μM calcein AM for 15 min at 37 °C. 2.8.2. Assessment of mitochondrial membrane potential JC-1 staining was used to assess the mitochondrial membrane potential with the Mitochondrial Membrane Potential Assay Kit. In brief, H9C2 cells were seeded in a 24-well plate with a density of 5 × 10^4 cells per well and cultured for 24 h. After hypoxia for 4 h, the medium was replaced with fresh medium containing hydrogel extracts from different groups and the cells were cultured for another 4 h. The cells were stained with JC-1 according to the manufacturer's instructions. The cells were washed twice with JC-1 washing liquid before being captured by inverted fluorescence microscope (TCS SP8, Leica, Germany). 2.8.3. Mitochondrial DNA (mtDNA) copy number assay To evaluate the effect of drug-loaded hydrogels on mitochondrial function under hypoxic and reoxygenation conditions, the mtDNA copy number was measured using qRT-PCR. After 4 h of hypoxia, the cells were incubated for an additional 12 h in fresh medium containing hydrogels from each experimental group. The mitochondrial gene MT-CO2 (mitochondria-encoded cytochrome C oxidase II) cycle threshold (Ct) values were quantified by qRT-PCR and compared to those of the nuclear gene GAPDH to estimate the mtDNA copy number. 2.8.4. ATP generation assay ATP production was measured using the ATP Visible Spectrophotometry method according to the manufacturer's instructions. After 4 h of hypoxia, the cells were incubated for an additional 12 h in fresh medium containing hydrogels from each experimental group. then lysed with RIPA buffer, and centrifuged. The supernatant was collected and used for ATP quantification. 2.9. Annexin V-FITC/PI assay The Annexin V-FITC apoptosis kit was used to detect the apoptotic protection of H9C2 gel samples against hypoxia-induced apoptosis. H9C2 cells were seeded in a 24-well plate at a density of 5 × 10^4 cells/well. After 12 h, H9C2 cells were treated with different hydrogel sample extracts for 24 h. Then, H9C2 cells were harvested and stained with the Annexin V-FITC apoptosis detection kit according to the manufacturer's recommendations. Then, the apoptosis rate of H9C2 cells was determined by flow cytometry (Agilent). 2.10. Determination of succinate dehydrogenase (SDH) and lactate dehydrogenase (LDH) activity H9C2 cells were seeded in 24-well plates at a density of 5 × 10^4 cells per well and allowed to incubate for 24 h. After 4 h hypoxic exposure, the medium was replaced with fresh medium containing hydrogel extracts from different groups and the cells were cultured for another 4 h to detect the SDH and LDH activity. The culture medium was collected and the LDH activity was detected by the LDH assay kit (S03034) and SDH assay kit (BC0955). 2.11. Macrophage polarization assessment To evaluate the effect of various hydrogel formulations on macrophage polarization in vitro, flow cytometry, immunofluorescence staining, and ELISA were employed. 2.11.1. Flow cytometry Flow cytometry was used to analyze macrophage polarization markers. RAW 264.7 cells were seeded into 12-well plates at a density of 1 × 10^5 cells per well and activated with LPS (100 ng/mL) for 6 h. Control cells were non-inflammatory macrophages. After LPS activation, cells were exposed to PBS, QCFT hydrogel, S1-QCFT hydrogel, FT011-Lipo-QCFT hydrogel, or S1&FT-Lipo-QCFT hydrogel. Cells were first incubated in serum-free medium for 4 h, then in medium with 10 % FBS for an additional 20 h. Subsequently, macrophages were collected and incubated with anti-mouse CD16/32 on ice for 10 min, followed by staining with a mixture of FITC-conjugated anti-mouse CD80 and APC-conjugated anti-mouse CD86 antibodies for 30 min on ice. After fixation and permeabilization, cells were stained with PE-Cy7-conjugated anti-mouse CD206 for 30 min at room temperature. Cells were then resuspended in 300 μL PBS and analyzed using flow cytometry on a NovoCyte Advanteon (Agilent, China). 2.11.2. ELISA test ELISA was performed to quantify levels of pro-inflammatory and anti-inflammatory cytokines. RAW 264.7 cells were plated into 12-well plates at a density of 1 × 10^5 cells per well and treated as described. After treatment, the culture supernatants were collected, and the concentrations of pro-inflammatory cytokines (IL-6 and TNF-α) and the anti-inflammatory cytokine (IL-10) were measured using ELISA kits (Proteintech, China). 2.12. Quantitative real-time polymerase chain reaction Real-time quantitative PCR (RT-qPCR) was used to assess the relative mRNA expression levels of M1 macrophage markers (IL-6 and TNF-α) and the M2 macrophage marker (IL-10). Total RNA was extracted from macrophages using TRIzol (Takara, Japan) following the manufacturer's instructions. cDNA was prepared using the iScript gDNA Clear cDNA Synthesis Kit (Yeasen) according to the manufacturer's instructions. DNA (qPCR) and cDNA (qRT-PCR) were analyzed with Hieff qPCR SYBR® Green Master Mix (Yeasen). Gene expression related to M1 and M2 macrophage phenotypes was analyzed with SYBR GREEN reagent (Thermo, USA) using the Gentier 96E Fully Automated Medical PCR Analysis System (Tianlong, China). Gene expression data were normalized to GAPDH as an internal control. The sequences of the primers are listed in [103]Table S1. 2.13. Assessment of fibroblast fibrosis 2.13.1. Immunofluorescence assay Rat cardiac fibroblasts (RCF) were plated in 48-well plates at a density of 4.8 × 10^3 cells per well and cultured in 500 μL of Dulbecco's Modified Eagle's Medium (DMEM/F12) for 12 h. The medium was then replaced with 500 μL of serum-free DMEM/F12 for 1 h. After this, cells were treated with one of the following conditions: (i) PBS (control), (ii) 10 ng/mL TGF-β1 (TGF-β1), (iii) 10 ng/mL TGF-β1 with FT011/Lipo-Hydrogel (FT-Lipo-QCFT hydrogel), or (iv) 10 ng/mL TGF-β1 with S1&FT011/Lipo-Hydrogel (S1&FT011/Lipo hydrogel). Following treatment, cells were fixed using 4 % formaldehyde, permeabilized with 0.1 % Triton X-100, and blocked with 5 % BSA. They were then incubated overnight with primary antibodies against Alpha-Smooth Muscle Actin Monoclonal Antibody (1:1000, 14-9760-95, Invitrogen, China), Collagen I Polyclonal Antibody (1:1000, PA1-26204, Invitrogen, China), Collagen type III (1:500,22734-1-AP, Pro-teinTech, China). After washing with PBS, cells were incubated with secondary antibodies for 1 h, followed by additional washing with PBS. Nuclei were stained with DAPI. Observations were made using a microscope (TCS SP8, Leica, Germany), and quantitative analysis was performed using ImageJ-Pro Plus 6.0. 2.13.2. Quantitative real-time polymerase chain reaction The expression levels of α-SMA, collagen type I, and collagen type III mRNA were quantified using real-time quantitative PCR. Total RNA was extracted from macrophages using TRIzol (Takara, Japan) following the manufacturer's instructions. cDNA was prepared using the iScript gDNA Clear cDNA Synthesis Kit (Yeasen) according to the manufacturer's instructions. DNA (qPCR) and cDNA (qRT-PCR) were analyzed with Hieff qPCR SYBR® Green Master Mix (Yeasen). Gene expression related to M1 and M2 macrophage phenotypes was analyzed with SYBR GREEN reagent (Thermo, USA) using the Gentier 96E Fully Automated Medical PCR Analysis System (Tianlong, China). Gene expression data were normalized to GAPDH as an internal control. The sequences of the primers are listed in [104]Table S2. 2.14. Establishment and treatment of rat myocardial ischemia/reperfusion (I/R) injury model using hydrogel Male Sprague-Dawley rats, each weighing approximately 200 g, were sourced from Guangdong Weitonglihua Laboratory Animal Technology Co., Ltd. All animals were housed under standard conditions (12-h light/dark cycle, temperature 25 ± 2 °C with free access to food and water. All procedures followed the ethical guidelines approved by the Animal Research Committee of Jinan University (Animal Ethics ID: No. IACUC-20240108-05). All animal experiments were conducted in compliance with the ARRIVE guidelines. Animals were randomly assigned to treatment groups using a computer-generated randomization schedule to ensure unbiased group allocation. Only male Sprague-Dawley rats were used in this study to minimize variability caused by hormonal fluctuations, ensuring consistency in the experimental model. Considering the complexity of myocardial ischemia-reperfusion experiments, ethical requirements, and possible animal attrition (e.g., model failure, animal death, etc.), we considered three animals per group to be feasible and appropriate. With the above approach, we sought to ensure the scientific validity of the experimental design and the reliability of the results while following the 3R principles (Replacement, Reduction, and Optimization). Researchers performing the outcome assessments and data analysis were blinded to the treatment groups to minimize experimental bias. To establish myocardial I/R injury model, the rats were anesthetized with isoflurane, and thoracotomies were performed at the fourth intercostal space to expose the heart and 6–0 prolene suture was passed underneath the LAD at 2–3 mm distal to its origin between the left auricle and conus arteriosus [[105]48]. After 1.5 h of ischemia, the suture was removed to allow for reperfusion. ST-segment elevation was observed after ligation of the LAD (left anterior descending artery) during the ischemic phase, which progressively decreased during the reperfusion phase. These electrocardiographic alterations validate the successful establishment of the myocardial ischemia-reperfusion injury model in rats. The rats were randomly assigned to different treatment groups for the myocardial I/R injury model, including I/R combined with PBS, I/R treated with QCFT hydrogel, I/R treated with S1QEL1.1-loaded QCFT hydrogel, I/R treated with FT011 liposome-loaded QCFT hydrogel, or I/R treated with a combined S1QEL1.1 and FT011 liposome-loaded QCFT hydrogel. A separate sham-operated group underwent open-chest surgery without ischemia/reperfusion. According to previous studies [[106]49], hydrogel treatments were applied to the ischemic region and five adjacent sites, with 20 μL administered to each location. 2.15. RNA sequencing and transcriptomic analysis of animal myocardial tissue Myocardial tissues were collected from experimental animals following treatment protocols. After euthanasia, the left ventricle was excised under sterile conditions, frozen in liquid nitrogen, and stored at −80 °C. Total RNA was extracted using the RNeasy Mini Kit (Qiagen, Germany), and samples with RNA Integrity Numbers (RIN) > 7.0, assessed by an Agilent 2100 Bioanalyzer, were selected for sequencing. Library preparation was performed using the NEBNext® Ultra™ II RNA Library Prep Kit for Illumina (New England Biolabs, USA). mRNA was enriched, fragmented, and reverse-transcribed to cDNA, which was amplified and purified into sequencing libraries. RNA sequencing was conducted on an Illumina NovaSeq 6000 platform, generating 150-bp paired-end reads. Raw data were quality-checked with FastQC, cleaned with Trimmomatic, and aligned to the reference genome using Hisat2. Differentially expressed genes (DEGs) were identified via DESeq2 (version 1.40.0), applying |log2(FC)| ≥ 1 and adjusted P < 0.05 as selection criteria. Genes with consistently low expression were excluded, and multiple testing corrections were applied using the Benjamini-Hochberg method. Functional annotation and enrichment analyses were performed with ClusterProfiler (version 4.8.1) in R software (version 4.3.0), focusing on Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. GO and KEGG pathway analyses identified terms and pathways with P < 0.05 as significant, ensuring reliable and biologically meaningful findings. 2.16. Histochemistry and immunofluorescence analysis Cardiac tissues were collected at designated time points, embedded in Optimal Cutting Temperature (OCT) compound, and sectioned into 6 μm thick slices. These sections were subjected to various staining procedures, including Hematoxylin and Eosin (H&E), Masson's trichrome, and TUNEL assay (C1098, Beyotime). Additionally, immunofluorescence staining was performed using antibodies for CD86 (1:200, ab220188, Abcam, USA), CD206 (1:200, ab64693, Abcam, USA), Collagen I Polyclonal Antibody (1:1000, PA1-26204, Invitrogen, Shanghai, China), Collagen type III (1:500, 22734-1-AP, Pro-teinTech, China),and WGA (1:200, W7024, Invitrogen, China), Cardiac Troponin T Polyclonal antibody(1:500,5513-1-AP Pro-teinTech, China) as well as TGF-β1 (1:100,ab179695, Abcam, USA) following established protocols. Furthermore, cardiac frozen sections were stained with 2′,7′-Dichlorodihydrofluorescein diacetate (DCFH-DA) solution (10 μM, Sigma-Aldrich, D7008) to assess the levels of ROS in the cardiac tissue. Fluorescence and immunohistochemical images were acquired using an inverted fluorescence microscope (EVOS M5000, Invitrogen, USA) and analyzed with ImageJ-Pro Plus 6.0, with three sections per animal being examined. All staining procedures were carried out by a pathologist who was blinded to the treatment group assignments. 2.17. Statistical analysis Data processing was performed using SPSS 22.0 statistical software, and the measurement data were expressed as mean ± standard deviation (X ± S) using one-way ANOVA (one-way ANOVA), and the difference was considered statistically significant with P < 0.05. Data that conformed to normal distribution were expressed as mean ± SD, and data that did not conform to normal distribution were expressed as median ± interquartile spacing. All samples were from at least three independent experiments. When two groups were compared, statistical significance was calculated by using the post-hoc tests test for p-values. Statistical analysis was performed with GraphPad Prism 10 software. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 and ∗∗∗∗P < 0.0001 were considered statistically significant. 3. Results and discussion 3.1. Preparation of FT/Lipo and QCMCS/2-FBPA/TA hydrogel (QCFT hydrogel) To enhance the delivery efficiency of FT011, which has a short plasma half-life and limited aqueous solubility, the drug was incorporated into the lipid bilayer of liposomes to form FT/Lipo nanoparticles. Transmission electron microscopy (TEM) images showed that these nanoparticles were uniformly spherical and exhibited relatively consistent size distribution ([107]Fig. 1A). Dynamic light scattering (DLS) measurements indicated a polydispersity index (PDI) of less than 0.3, reflecting a narrow size distribution of the FT/Lipo nanoparticles ([108]Fig. 1B). The size of these nanoparticles remained stable during a 7-day period in saline ([109]Fig. 1C). The average hydrodynamic diameter increased from about 126 nm for empty liposomes to approximately 146 nm for FT011-loaded liposomes ([110]Fig. 1D). The zeta potential of the empty liposomes was −5.65 ± 0.53 mV and changed to −6.42 ± 0.45 mV after loading with FT011 ([111]Fig. S1C). These results confirm that FT/Lipo nanoparticles were successfully prepared and demonstrate their excellent stability. Fig. 1. [112]Fig. 1 [113]Open in a new tab Preparation and characterization of liposome nanoparticles and hydrogels. A) TEM image of FT011/Lipo (scale bar: 500 nm). B) Average size and size distribution of FT011/Lipo as determined by DLS. C) Stability of FT011/Lipo incubated in PBS (pH 7.4) for 7 days, monitored by DLS (n = 3). D) Size distribution of blank liposomes compared to FT011/Lipo. E) Photographs of QCFT hydrogel prepared by mixing TA solution, 2-FPBA solution, and QCMCS solution. F) SEM images of QCFT hydrogel (scale bar: 200 μm). G) FT-IR spectra of QCFT hydrogel. H) UV–vis spectra of QCFT hydrogel. I) C 1s XPS spectra of freeze-dried QCFT hydrogel. J) O 1s XPS spectra of freeze-dried QCFT hydrogel. K) Mapping images of C, O, N, and B elements in the freeze-dried QCFT hydrogel (scale bar: 200 μm). The preparation of the S1QEL1.1&FT/Lipo-QCMCS-2-FPBA-TA hydrogel was carried out as follows: FT/Lipo nanoparticles were first added to a QCMCS solution under stirring to obtain the QCMCS@FT/Lipo solution. S1QEL1.1 was then dissolved in a TA solution, which was combined with an equal volume of 2-FPBA solution. The resulting mixtures were subsequently blended, forming the S1QEL1.1&FT/Lipo-QCMCS-2-FPBA-TA hydrogel (abbreviated as S1&FT/Lipo-QCFT hydrogel). Representative images illustrating the pre- and post-gelation stages are shown in [114]Fig. 1E. SEM analysis revealed that the hydrogel exhibited interconnected cavities and a uniform porous architecture ([115]Fig. 1F). The chemical structures of S1QEL1.1 and FT011 are provided in [116]Supplementary Fig. S1A–B. To investigate the construction mechanism of QCFT hydrogel, a series of analyses were performed, including proton nuclear magnetic resonance (^1H NMR), Fourier-transform infrared (FT-IR), Ultraviolet–visible spectroscopy (UV–Vis), and X-ray photoelectron spectroscopy (XPS). [117]Fig. S2 in the Supporting Information shows a new imine proton signal at 8.47 ppm in the ^1H NMR spectrum, indicating the reaction between QCMCS and 2-FPBA. FT-IR analysis ([118]Fig. 1G) identified a stretching vibration at 1684 cm^−1 corresponding to the imine bond in QCMCS/2-FPBA and QCFT hydrogel. The phenolic hydroxyl (-OH) peak of TA shifted from 3352 cm^−1 to 3372 cm^−1, and the B─O bond peak shifted from 1350 cm^−1 to 1338 cm^−1, indicating the formation of boronate ester bonds. The UV–Vis spectra ([119]Fig. 1H) revealed characteristic peaks for TA, 2-FPBA/TA, and QCFT hydrogel at 277, 296, and 305 nm, respectively, which demonstrates redshift due to the interaction between TA and 2-FPBA, affecting TA's resonance structure via boronate ester bond formation. XPS spectra ([120]Fig. 1I and J and [121]Supplementary Fig. S3) showed a peak at 401.7 eV for the Schiff base bond (C═N) in the N 1s spectrum and another peak for the boronate ester bond (B─O) in the O 1s spectrum, confirming the presence of both bonds in the QCFT hydrogel. Additionally, EDS mapping confirmed the uniform distribution of elements C, N, O, and B (see [122]Fig. 1K and [123]Supplementary Fig. S4), supporting the homogeneous structure of the QCFT hydrogel. 3.2. Mechanical, injectable, and antioxidant properties of the QCFT hydrogel The hydrogel matrix plays a critical role not only in drug delivery but also in providing mechanical support and therapeutic bioactivity during myocardial repair. The physiological stiffness of healthy myocardium ranges from 10 to 15 kPa and can increase to approximately 50 ± 15 kPa following fibrosis [[124]41,[125]50]. The synthesized QCFT hydrogel exhibited a Young's modulus of ∼29.85 kPa ([126]Fig. S5A), placing it within the range suitable for cardiac tissue applications. Rheological analysis demonstrated that the QCFT hydrogel possesses favorable mechanical resilience and shear-responsive behavior. Frequency and amplitude scans showed that the storage modulus (G′) consistently exceeded the loss modulus (G″), indicating elastic solid-like behavior ([127]Fig. 2A–S5B –D). The hydrogel maintained structural stability under low strain but transitioned to a fluid-like state when G′ fell below G″ at high strain levels ([128]Fig. 2B and C), reflecting the reversible disruption of Schiff base and boronate ester bonds. Notably, upon strain reduction, both G′ and G″ rapidly recovered, demonstrating self-healing ability ([129]Fig. 2C). This dynamic reversibility, confirmed by cyclic and complex viscosity measurements ([130]Fig. 2D), supports both injectability and network restoration, key for vivo application. Fig. 2. [131]Fig. 2 [132]Open in a new tab Properties of ROS-Responsive Hydrogels. A) Frequency sweep of S1&FT/Lipo-QCFT and QCFT hydrogels. B) Amplitude sweep of S1&FT/Lipo-QCFT hydrogel and QCFT hydrogel. C) Cyclic strain sweep of S1&FT/Lipo-QCFT and QCFT hydrogels between 1 % and 300 %. The test alternated between 1 % (2 min) and 300 % strain (1 min) at 6.28 rad/s to assess self-healing performance. D) Complex viscosity measurements of S1&FT/Lipo-QCFT and QCFT hydrogels. E) Swelling behavior of hydrogels in PBS (n = 3). F) Degradation curves of the hydrogels in PBS with different concentrations H[2]O[2] stimulation (n = 3). Release kinetics of S1QEL1.1(G), TA (H) and FT011 (I) from S1&FT/Lipo-QCFT hydrogel under different conditions(n = 3). J-K) UV–Vis spectra of DPPH J) and K) PTIO exposure to different concentrations of the QCFT hydrogels. M) The color change of each reaction solution of DPPH• and PTIO• exposed to different concentrations of QCFT Hydrogel. N) Injectability of S1&FT/Lipo-QCFT hydrogel. O) Self-healing properties of S1&FT/Lipo-QCFT hydrogel. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001, ns: not significant. (For interpretation of the references to color in this figure legend, the reader is referred to