Adaptive bioactivable nanosystems for synergistic myocardial infarction therapy using traditional pharmaceutics

Abstract

Heart failure resulting from myocardial infarction (MI) is a leading global health concern. Current revascularization therapies cannot fully restore the infarcted myocardium or prevent maladaptive ventricular remodeling. Traditional Chinese medicine with its multitarget regulation and favorable biosafety shows a promising therapeutic potential. Tanshinone IIA (TIIA) and formononetin (FM), two bioactive compounds derived from Salvia miltiorrhiza and Astragalus membranaceus, respectively, exhibit antioxidant, anti-inflammatory, and proangiogenic effects. Herein, a neutrophil-targeted nanomedicine (TF-5NP) was developed to deliver TIIA and FM to the infarcted myocardium for mitigating oxidative damage and promoting angiogenesis. TF-5NP was synthesized by coassembling bis-5-hydroxytryptamine-modified 1,2-distearoyl-sn-glycero-3-phosphoethanolamine–polyethylene glycol–carboxylic acid with cholesterol and lipid 1,2-distearoyl-sn-glycero-3-phosphoglycerol, which binds to troponin in the infarcted myocardium. This nanomedicine reduces inflammation and cardiomyocyte damage and improves cardiac function in porcine MI models, with therapeutic effects lasting for ∼28 d. These findings suggest that TF-5NP use is a promising approach for treating post-MI maladaptive remodeling and heart failure.

Graphical abstract

Schematic develop an inflammation-activated size variable nanoparticle TF-5 NP to achieve effective long-term retention of TIIA and FM. (A) A neutrophil-targeting polymer is synthesized by modifying the DSPE-PEG-COOH with bis-5-hydroxytryptamine (bis-5HT), which is then used to construct lipid nanoparticles (TF-5 NP) loaded with TIIA and FM through a co-assembly strategy with cholesterol and the lipid DSPG. TF-5 NP were guided by the bis-5HT target head to the inflammatory microenvironment (IME) after myocardial infarction. The local inflammatory microenvironment formed in the infarction area after myocardial infarction and a large amount of neutrophils accumulated. A neutrophils-specific enzyme, MPO can facilitated the convertion of phenolic residues into the form of radicals, which could undergo dimerization or bind to tyrosine residues-based proteins. The radical bis-5HTs can aggregate to increase the volume of drugs and bis-5HT can bind with local troponin and forming stronger retention of liposomes in the myocardium. (B) In the infarcted area, TIIA and FM can be slowly released TIIA played an important role in inhibiting ROS production, improving mitochondrial function, reducing oxidative stress, and alleviating inflammation. FM stimulates the release of pro-angiogenic growth factors VEGF, PDGF, FGFR1 and CD31 to activate angiogenesis. Collectively, these findings demonstratedthat TF-5 NP have a long-term therapeutic effect on improving heart function and treating myocardial infarction by regulating inflammatory response and promoting angiogenesis.

Highlights

• •Neutrophil/troponin-targeted nanosystem enables sustained co-delivery of TIIA/FM for heart repair.

1. Introduction

Heart failure resulting from myocardial infarction (MI), a leading cause of death worldwide, is a major challenge to global healthcare. MI caused by coronary artery occlusion induces the apoptosis of numerous cardiomyocytes (CMs), which contributes to maladaptive ventricular remodeling and ultimately heart failure [1,2]. Currently, revascularization therapies (the gold standard) such as percutaneous coronary intervention [3,4] can effectively reopen the occluded coronary artery but delay maladaptive ventricular remodeling and do not restore the infarcted myocardium [5,6]. Various inflammatory cells such as neutrophils infiltrate the infarcted heart tissue, releasing proinflammatory factors and cytokines, which exacerbate CM damage [7,8]. Moreover, the reduced oxygen and nutrient supply in the infarcted area disrupts CM metabolism, triggering apoptosis. The disrupted CM metabolism elevates the levels of reactive oxygen species (ROS), which further damages CMs and amplifies inflammatory responses, worsening CM damage [9,10]. The limited regenerative capacity of CMs significantly limits their self-repair function, leading to maladaptive ventricular remodeling. Therefore, improving CM survival in the infarcted area is crucial to inhibiting maladaptive ventricular remodeling [[11], [12], [13]].

Traditional Chinese medicine (TCM) exhibits unique advantages in protecting CM and preventing post-infarction pathophysiological remodeling owing to its multitarget regulation and favorable biosafety [14,15]. It is a promising approach to combine the use of TCM to optimize therapeutic outcomes. Tanshinone IIA (TIIA), a bioactive compound extracted from Salvia miltiorrhiza, can scavenge ROS and reduce oxidative stress, thereby alleviating inflammation [16,17]. Formononetin (FM), predominantly derived from the root of Astragalus membranaceus, exhibits proangiogenic properties [18,19]. These properties make the combination of TIIA and FM a promising drug candidate for MI treatment. However, effective drug delivery to the beating heart is challenging [20,21]. The unique structure of the heart, low solubility of small-molecule drugs, and nontargeted distribution of these drugs limit the delivery of high doses required for effective post-MI treatment [22]. Nanomaterial-based drugs have the potential to address these issues. However, simultaneously achieving active targeting and prolonged retention in the infarcted myocardium remains a huge challenge for developing nanomaterial-based delivery strategies for MI therapy [8,23,24].

Herein, we developed a neutrophil-targeted nanomedicine that prolongs retention and sustains the release of TIIA and FM in the infarcted myocardium. It effectively mitigates CM oxidative damage and promotes angiogenesis, ultimately alleviating maladaptive ventricular remodeling [25,26] (Scheme 1). A neutrophil-targeting polymer (TF-5NP) was synthesized by modifying 1,2-distearoyl-sn-glycero-3-phosphoethanolamine–polyethylene glycol–carboxylic acid (DSPE–PEG–COOH) with bis-5-hydroxytryptamine (bis-5HT), which was used to synthesize lipid nanoparticles (5NP) to load TIIA and FM through a coassembly strategy with cholesterol (Chol) and lipid 1,2-distearoyl-sn-glycero-3-phosphoglycerol (DSPG). TF-5NP either self-aggregates or binds to troponin in the infarcted myocardium via radical–radical quenching, catalyzed by the neutrophil-specific enzyme myeloperoxidase (MPO) [27,28] (Scheme 1a). The released TIIA protects against mitochondria oxidative damage in CMs by scavenging ROS and alleviating the inflammatory response. The released FM promotes angiogenesis in infarcted heart tissues by upregulating the levels of vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), and fibroblast growth factor (FGF) (Scheme 1b). In a zebrafish myocardial injury model, TF-5NP considerably promotes angiogenesis and cardiac repair. It significantly eases CM damage, improves cardiac systolic function, reduces inflammatory responses, and promotes angiogenesis. These effects last ∼28 d in a porcine myocardial infarction model.

Scheme 1.

Development of an inflammation-activated size variable NP TF-5NP to achieve effective long-term retention of TIIA and FM.

2. Materials and methods

2.1. Materials and reagents

All chemicals were purchased from Sigma-Aldrich unless otherwise specified. TIIA and FM were purchased from Aladdin Chemistry Co. Ltd. (Shanghai, China). Primary antibodies against Caspase-3, Bax, Bcl-2, VEGF, PDGF, FGF, CD31, α-smooth muscle actin (SMA), Caspase-1, IL-1β, TOM20, TOM40, and Flotillin-1 were purchased from Cell Signaling Technology (Danvers, MA, USA). Secondary antibody fluorescein-conjugated goat antirabbit and fluorescein-conjugated goat antimouse were purchased from Sigma-Aldrich (St. Louis, MO). Atropine was purchased from Shanghai Tonge Pharmaceutical Co., Ltd., sutaine was procured from France Vic Co., Ltd., and propofol was obtained from Guangdong Gabor Pharmaceutical Co., Ltd.

2.2. Preparation of DSPE–PEG–bis-5HT

• Step 1(9H-Fluoren-9-yl)methyl(1,5-bis{[2-(5-hydroxy-1H-indol-3-yl)ethyl]amino}-1,5-dioxopentan-2-yl)carbamate(bis-5HT-Glu-Fmoc). A solution of Fmoc-Glu-OH (831 mg, 2.25 mmol), EDCI (892 mg, 4.8 mmol), and 1-hydroxybenzotriazole (HOBt, 644 mg, 4.8 mmol) was prepared in dimethylformamide (DMF,10 mL) (Solution A). A solution of 5-HT hydrochloride (5HT·HCl, 957 mg, 4.5 mmol) and N,N-diisopropylethylamine (DIPEA,1.05 g, 9.1 mmol) in DMF (5 mL) was added dropwise to Solution A. After stirring at room temperature for 2 h, the reaction mixture was quenched with saturated ammonium chloride (NH₄Cl) solution (25 mL) and water (10 mL) and extracted with ethyl acetate (EA, 3 × 10 mL). The organic phase was washed with brine, dried over anhydrous sodium sulfate (Na₂SO₄), and concentrated in a vacuum. The residue was purified via silica gel column chromatography using methanol (MeOH)/dichloromethane (DCM) as the eluent (v/v = 1/10–1/3) to afford the desired product bis-5HT-Glu-Fmoc (802 mg, 52 %) as a gray solid.
• Step 22-Amino-N¹,N⁵-bis(2-(5-hydroxy-1H-indol-3-yl)ethyl)pentanediamide DSPE–PEG–bis-5HT. Piperidine (993 mg, 11.7 mmol) was slowly added to a solution of bis-5HT–Glu–Fmoc (800 mg, 1.17 mmol) in MeOH (25 mL) at room temperature. The mixture was stirred for 1 h, and the solvent was removed under reduced pressure.The residue was purified via silica gel column chromatography using MeOH/DCM as the eluent (v/v = 1/20–1/10) to afford the desired product bis-5HT–Glu–NH₂(DSPE–PEG–bis-5HT) (443 mg, 82 %) as a brown solid.

2.3. Preparation of TF-5NP

NPs with variable sizes for drug-carrying inflammatory activation were prepared via thin-film dispersion (TF-5NP). The lipids 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), Chol, and DSPE–PEG–bis-5HT were dissolved in a solution of methanol and chloroform (5:1) at a molar ratio of 58:38:5. FM (2 mg) and TIIA (1 mg) dissolved in dimethyl sulfoxide were added to the lipid substance solution, and the solution was rotavaporized. A uniform film was formed at the bottom of the flask, and ultrasonic hydration was performed with deionized water. The solution was dialyzed at 4 °C (MWCO = 3500 Da) for 4 h.

2.4. Characterization

The morphology and size of LPs were investigated using an EOL JEM-1011 transmission electron microscope (TEM). The size distribution and zeta potential of LPs were determined via dynamic light scatting analysis (Malvern Nano ZS90, UK). The absorbance and fluorescence spectra of the liposomes were obtained using an ultraviolet–visible spectrometer (UV–Vis, UV-2450PC, Shimadzu, Japan) and a fluorescence spectrometer (LS50B, PerkinElmer, USA), respectively. To determine the release profiles of TP and FM, TF-5NP solutions were subjected to the following conditions: (1) 50 μg mL⁻¹ MPO and 100 μM H₂O₂; (2) 50 μg mL⁻¹ MPO; (3) 100 μM H₂O₂; (4) 50 μg mL⁻¹ MPO, 100 μM H₂O₂, and 50 μM 4-ABAH; (5) activated neutrophils; and (6) PBS solution. To investigate neutrophil-mediated activation of TF-5NP, neutrophils were isolated from mouse bone marrow using standard density gradient centrifugation. The cells were resuspended in RPMI-1640 medium at a density of 1 × 10⁶ cells/mL. Neutrophil activation was induced by treatment with 100 ng/mL phorbol 12-myristate 13-acetate (PMA) for 30 min at 37 °C. The solutions were transferred into dialysis bags (MWCO = 3500 Da) and dialyzed against 100 mL of the corresponding release medium at 37 °C under constant shaking. At predetermined time intervals, 2 mL of the release medium was collected for subsequent analysis, and an equal volume of fresh medium was replenished to maintain a constant total volume. The amounts of TP and FM released were quantified using high-performance liquid chromatography.

2.5. Cellular uptake

Human umbilical vein endothelial cells (HUVEC) (5 × 10⁵) were seeded in 6-well plates with 1640 medium and cultured for 12 h. After treatment with PBS, cy5.5, cy5.5-NP, and cy5.5-5NP for 6, 12, 24, and 48 h respectively, the cells were fixed with 4 % (w/v) paraformaldehyde for 30 min. The cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, blue) for 5 min. The actin cytoskeleton of cells was stained with fluorescein isothiocyanate (FITC) phalloidin (green) for 30 min. The cells were examined via confocal laser scanning microscopy (CLSM).

2.6. Cell viability studies

HUVECs (5 × 10³) were seeded in 96-well plates in a 1640 medium and cultured for 12 h. The medium was replaced with fresh culture medium with different drugs: TIIA, FM, TIIA + FM, TF-NP, and TF-5NP at concentrations of 0.00625–50 μM. After 48 h of incubation, 5 mg mL⁻¹ cell counting kit-8 (CCK-8) solution in PBS was added, and the plates were incubated for another 2 h at 37 °C. Finally, the absorbance of the above solution was measured on a microplate reader (Infinite M1000 Pro, Tecan, Switzerland) at 450 nm.

2.7. Apoptosis analysis

HUVECs (5 × 10⁵) were seeded in 6-well plates in a 1640 medium and cultured for 48 h. After treatment with a fresh medium containing TIIA (with a concentration of 5 μM), FM (with a concentration of 10 μM), TIIA + FM (TIIA concentration: 5 μM, FM concentration: 10 μM), TF-NP (TIIA concentration: 5 μM, FM concentration: 10 μM) and TF-5NP (TIIA concentration: 5 μM, FM concentration: 10 μM), the cells were harvested and stained with Annexin-V FITC and propidium iodide (PI) following the manufacturer's protocol for flow cytometry (FCM). The data were analyzed using FlowJo software.

2.8. Cell scratch

All instruments used in the experiment were sterilized. On a super-clean table, the bottom of the culture plate was marked evenly with a marker, with an interval of 1 cm, and at least three lines were drawn across the hole for each hole. The HUVECs at the logarithmic growth stage were digested with trypsin, prepared into a cell suspension, seeded into a culture plate, and cultured overnight until the cells were overgrown. On Day 2, a 200-μL pipette tip was used, perpendicular to the transverse line at the bottom of the culture plate to scratch. The cells were washed twice with PBS to remove the scratched cells. Drugs were added to each group at the same concentrations used in Section 2.7 (Apoptosis analysis), and culture continued. Finally, the culture plates were removed at 0 h, serving as control, and later observed and photographed under an inverted microscope at 12 and 24 h.

2.9. Cell canalization

HUVEC cells were selected and cultured until the cells reached the logarithmic growth phase. The cells were digested with pancreatic enzymes and counted, and the cell suspension was adjusted to 1 × 10⁵ mL⁻¹. A layer of substrate with a thickness of 100 μm was applied to the bottom of the Petri dish and allowed to set for 30 min. The cell suspension was added to the cured matrix, usually 200 μL of cell suspension per well. Drugs were added to each group at the same concentrations used in Section 2.7 (Apoptosis analysis), the culture was continued for 24 h. The pipeline or network structure formed by the cells in the matrix was observed and photographed using a microscope, and the relationship between cell migration, pipeline formation, and treated substance was recorded. Image analysis software was used to quantitatively analyze the length, the number of tubes, and the structure of the tubes to assess the tube formation ability of the cell.

2.10. Animal studies

The animal protocols underwent rigorous review and comprehensive assessment and received explicit approval from the Institutional Animal Care and Use Committee (IACUC) at Guangzhou University of Chinese Medicine, with the assigned IACUC ethical review number of IACUC-2024-7903-01. These guidelines are rooted in internationally recognized ethical principles and regulatory standards, guaranteeing the welfare, care, and appropriate handling of animals involved in scientific investigations.

2.11. Maintenance of zebrafish

This study utilized the AB line, transgenic zebrafish expressing Tg (cmlc2:GFP) for labeling CMs and Tg (fli1:EGFP) for the vascular endothelium. Zebrafish were maintained at the China Zebrafish Resource Center following standard laboratory protocols and housed in circulating freshwater tanks at 28 °C, pH 6.0–6.5, conductivity of 450–550 μS cm⁻¹, and hardness of 50–100 mg L⁻¹ calcium carbonate (CaCO₃). The fish were also subjected to a 14-h light:10-h dark cycle.

2.12. Morphological and functional quantification of the zebrafish heart

Wild-type AB-strain zebrafish embryos at 48 hpf were chosen at random and placed in 6-well plates, with each well containing 30 zebrafish. After treatment with a fresh medium containing PBS, TIIA + FM (TIIA concentration: 5 μM, FM concentration: 10 μM), TF-NP (TIIA concentration: 5 μM, FM concentration: 10 μM) and TF-5NP (TIIA concentration: 5 μM, FM concentration: 10 μM), all experimental groups were given 100 μM of verapamil hydrochloride to induce model cardiac dysfunction. Following this, 10 zebrafish from each group were randomly selected and photographed using dissecting and fluorescence microscopes. The resulting data were analyzed using the NIS-Elements D 3.20 advanced image processing software to assess the cardiac area and venous siltation area of the examined zebrafish. Additionally, 10 zebrafish from each group were randomly chosen for heart rate and blood flow measurements, and video data were recorded. The collected information was processed using ViewPoint Application Manager software to evaluate the heart rate, blood flow rate, and cardiac output. The data obtained were processed using NIS-Elements D 3.20 advanced image processing software to analyze the area of cardiomyocytes and the distance of the sinus venosus–bulbus arteriosus (SV–BA) in the heart.

2.13. Neovascularization in zebrafish

Zebrafish carrying the Tg (fli1:EGFP) transgene were treated with PBS, TIIA + FM (TIIA concentration: 5 μM, FM concentration: 10 μM), TF-NP (TIIA concentration: 5 μM, FM concentration: 10 μM), or TF-5NP (TIIA concentration: 5 μM, FM concentration: 10 μM) at 28 °C from 3 hpf to 48 hpf. The viability and changes in the morphology of blood vessels were observed at 48 hpf using a fluorescence microscope (Axio Imager Z1, Zeiss, Oberkochen, Germany). The analysis focused on the number and mean length of intersegmental vessels and angiogenesis. This experiment was performed thrice independently with 10 embryos in each group.

2.14. MI model in pigs and treatment administration

Male Tibetan mini pigs, weighing 40–50 kg were chosen as the primary animal model for this study owing to their physiological similarities to human cardiac anatomy and function. The induction of anesthesia (4 mg kg⁻¹) for these pigs involving a combination of tiletamine hydrochloride, zolazepam hydrochloride, propofol, and xylazine was administered to ensure a controlled and safe sedation process. Before experimental MI induction, the cardiovascular health of animals was thoroughly assessed through transthoracic echocardiographic measurements using Simpson's method. MI was induced by ligating the first diagonal branch (D1) of the anterior descending branch. Rigorous randomization procedures were applied to allocate the pigs into five distinct treatment groups. Sham: Thoracotomy + D1 threading without ligation + saline (i.v.). Model: D1 ligation + saline (i.v.). TIIA + FM: D1 ligation + TIIA and FM solution (0.45 mg kg-1, n = 3, i.v.). TF-NP: D1 ligation + TIIA/FM-loaded non-bioresponsive NPs (0.45 mg kg-1, n = 3, i.v.). TF-5 NP: D1 ligation + TIIA/FM-loaded bioresponsive NPs (0.45 mg kg-1, n = 3, i.v.). Each group was administered 3 mL of either the drug or saline per injection and these injections were administered at post-MI induction immediately. To maintain a state of light anesthesia conducive to the procedure, a continuous administration of sevoflurane (30–40 μg kg⁻¹ min⁻¹) was used. Simultaneously, a comprehensive monitoring system was in place to observe and record electrocardiography readings, heart rate fluctuations, and arterial pressure changes to guarantee the stable condition of the pigs throughout the MI induction. After completion of the experimental procedures, a vigilant monitoring regime was implemented to observe the animals until they fully recovered from anesthesia, ensuring their well-being and stability. Earlier empirical studies have demonstrated the reliability and consistency of the coronary artery ligation technique in inducing uniform and reproducible MI [29].

2.15. In vivo pharmacokinetic study of TF-5NP

Using cy5.5-labeled 5HT-LPs and LPs, we examined the pharmacokinetic profile of cy5.5-5NP after intravenous injection in pigs. cy5.5-5 NP and cy5.5-NP were administered to pigs at a dose of 1 mg kg⁻¹. Pigs in the control group were treated with the same dose of cy5.5. At predefined time points, an equal volume of whole blood samples was collected in 96-well black plates. An in vivo imaging system (IVIS) spectrum system (PerkinElmer, USA) was used to determine the mean fluorescence intensity of blood samples. The targeting and tissue distribution of TF-5NP after intravenous injection in pigs, cy5.5, cy5.5-NP, and cy5.5-5 NP were administered by intravenous injection at 1 mg kg⁻¹. Fourteen days and 28 d after administration, the pigs were euthanized, and the heart and main organs were isolated. The biodistribution of cy5.5 in cardiac tissue was observed via fluorescence imaging on an IVIS Lumina III Imaging System (Caliper, USA) with an excitation filter of 670 nm and emission filter of 700–800 nm.

2.16. Cardiac magnetic resonance

Animals were allowed to fast for 8 h in advance and intramuscularly injected atropine (0.04 mg kg⁻¹) 15 min before the induction of anesthesia, followed by sutaine (5  mg kg⁻¹) for the induction of anesthesia. Endotracheal intubation and intravenous access were performed after reaching the ideal depth of anesthesia, and propofol (6–10 mg kg⁻¹ h⁻¹) was used to maintain anesthesia during the operation. After anesthesia, the animals were placed in the supine position on a magnetic resonance imaging (MRI) operating bed and affixed with a chest electrode sheet, which was then connected to an MRI electrocardiogram (ECG) signal sensor. The positioning phase and T1, enhanced, and delayed sequences were scanned. After scanning, the animals were revived in a constant-temperature recovery room and monitored until they could stand on their own, which were returned to the animal room for routine feeding.

2.17. Histological hematoxylin and eosin (HE) and Masson staining

Heart tissues were fixed and embedded in paraffin after dehydration, followed by paraffinization and dewaxing. For HE staining, sections were initially stained with hematoxylin solution for 3–5 min, followed by treatment with hematoxylin differentiation solution, hematoxylin Scott Tap Bluing, 85 % ethanol for 5 min, and 95 % ethanol for 5 min. Finally, the sections were stained with eosin dye for 5 min and, after dehydration, sealed with neutral gum. For Masson staining, slices were soaked in Masson dye solution overnight. Masson B and Masson C were mixed at a ratio of 1:1 to prepare the Masson solution. Tissues were stained with Masson solution for 1 min and differentiated with 1 % HCl-alcohol. Slices were immersed in Masson D for 6 min, Masson E for 1 min, and Masson F for 2–30 s. Next, the slices were rinsed with 1 % glacial acetic acid, followed by dehydration with two cups of anhydrous ethanol and treatment with 100 % ethanol for 5 min and xylene for 5 min. Finally, the sections were sealed with neutral gum and subjected to microscopic examination, image acquisition, and analysis.

2.18. Histological wheat germ agglutinin (WGA) staining

The histological sections of the model and TF-5NP groups were prepared and fixed with formaldehyde in the border and infarction areas, respectively. The samples were treated with Triton X-100 and incubated with Alexa Fluor 488 labeled WGA at 10 μg mL⁻¹ for 30 min. The sample was gently washed twice with PBS to remove unbound WGA. The nucleus was stained using DAPI or other nuclear staining agents, and the stained sample was sealed and subjected to microscope observation.

2.19. Mitochondrial electron microscope

The fresh tissue was dissected into small tissue blocks of 1 mm³ and fixed with osmic acid at room temperature for 2 h. The tissues were dehydrated for 20 min each time and 100 % acetone twice for 15 min each time. After dehydration, the tissues were infiltrated with acetone before embedded in resin and polymerized in an oven at 60 °C for 48 h. The resin block was sectioned into an ultrathin slice (60–80 nm) and placed on 150 mesh copper grids. The copper mesh grid was stained in the dark for 8 min and stained with 2.6 % lead citrate solution in a carbon dioxide–free environment for another 8 min. The grids were placed in a copper mesh box and dried overnight at room temperature. Images were captured and analyzed via TEM.

2.20. TUNEL analysis

The sections were deparaffinized and rehydrated. Following liquid removal, the objective tissue was marked with a liquid blocker pen. The tissues were covered with proteinase K working solution and incubated at 37 °C for 25 min. After the removal of excess liquid, the permeabilization solution was added and incubated at room temperature for 20 min. The sections were allowed to slightly dry before adding a buffer to tissues within the marked circle at room temperature for 10 min. An appropriate amounts of terminal deoxynucleotidyl transferase enzyme, deoxyuridine triphosphate pyrophosphatase (dUTP), and buffer from the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) kit were used according to the number of slices and tissue size mixed at a 1:5:50 ratio, and incubated at 37 °C for 2 h. The sections were incubated with DAPI at room temperature for 10 min in the dark and washed thrice with PBS. The liquid was removed, and sections were mounted using an antifade mounting medium. All sections were examined under a fluorescence microscope, and images were obtained for analysis.

2.21. Immunofluorescence analysis

Slides were deparaffinized, rehydrated, then immersed in ethylenediamine tetraacetic acid (EDTA) antigen retrieval buffer (pH 8.0), and maintained initially at sub-boiling temperature for 8 min, followed by exposing them to room temperature standing for 8 min, and further incubation at sub-boiling temperature for another 7 min. After the removal of the liquid, the objective tissue was marked with a liquid blocker pen. The marked tissue sections were incubated with 3 % bovine serum albumin (BSA) to block nonspecific binding for 30 min. The tissue area was immersed in 10 % donkey serum or 3 % BSA. Slides were incubated with the primary antibody overnight at 4 °C, followed by a secondary antibody at room temperature for 50 min in the dark. The slides were treated with DAPI at room temperature for 10 min in the dark. After incubation with a spontaneous fluorescence quenching reagent for 5 min, the slides were washed in running tap water for 10 min. The liquid was subsequently removed, and the slides were mounted with antifade mounting medium for analysis via fluorescence microscopy.

2.22. Immunoblot analysis

Homogenates of LV myocardium samples were analyzed using immunoblot analysis. Frozen samples were homogenized in ice-cold lysis buffer and proteins per lane were separated on 8–15 % SDS-polyacrylamide gels and transferred to PVDF membranes. Protein lysates were probed with primary antibodies diluted in 5 % BSA overnight at 4 °C. Next, blots were rinsed, incubated with horseradish peroxidase-conjugated secondary antibodies in 5 % BSA for 1 h, and developed using a chemiluminescent substrate. Densitometric analysis was conducted using Image J software (version 1.44, Bethesda, Maryland, USA).

2.23. Statistical analysis

Experimental data (expressed as mean ± SD) were assessed via one-factor analysis of variance (SPSS software, version 28.0, SPSS Inc.). Differences among various experimental and control groups were analyzed with ordinary one-way ANOVA. Differences were considered statistically significant at ∗ p < 0.05, ∗∗p < 0.01 and ∗∗∗p < 0.001.

3. Results and discussion

3.1. Establishing the inflammation-activated size variable and cTnT-anchored NPs

A neutrophil-targeting polymer was synthesized by modifying DSPE–PEG–COOH with bis-5HT, as shown in Fig. S1. The nuclear magnetic resonance spectra of DSPE–PEG–bis-5HT are presented in Fig. S2–S5. 5NPs were synthesized to load TIIA and FM through a coassembly strategy with DSPE–PEG–bis-5HT, Chol, and TF-5NP. The encapsulation efficiencies of TIIA and FM by TF-5NP are 87.8 % and 90.6 %, respectively. The drug-loading contents of TIIA and FM are1.4 % and 3.1 %, respectively, proving that TF-5NP exhibits good drug-carrying properties.

MPO can convert the phenolic residues of DSPE–PEG–bis-5HT into radicals. The radical-based substrates can dimerize, allowing bis-5HT radicals to form bonds (Fig. 1A). We examined the morphology of TF-5NP before and after MPO enzyme activation via TEM, which exhibits a spherical structure before MPO activation, while after MPO enzyme activation, the liposomes exhibit a linked structure (Fig. 1B). Liposomes without bis-5HT modification were prepared as the control group (TF-NP). The zeta potentials of TF-NP and TF-5NP are −21.14 and −15.02 mV (Fig. 1C), respectively. To further confirm the MPO enzyme activation behavior of TF-5NP, we treated the drug with the MPO enzyme and H₂O₂. The hydrodynamic diameter of TF-5NP is 135.65 nm, as obtained via dynamic light scattering, while the addition of MPO and incubating with H₂O₂ increase the diameter of TF-5NP to 527.46 nm. In contrast, no significant increase was observed when TF-5NP was treated with MPO alone, H₂O₂ alone, or with MPO/H₂O₂ in the presence of an MPO inhibitor (Fig. S6). To address whether activated neutrophils promote TF-5NP cross-linking and drug release in an MPO-dependent manner, we incubated TF-5NP with activated neutrophils in vitro. Dynamic light scattering (DLS) analysis revealed a significant increase in the hydrodynamic diameter of TF-5NP to 420.62 nm upon neutrophil stimulation (Fig. 1D). These findings confirm that liposome crosslinking and enhanced drug retention occur following activation of neutrophil-derived MPO, thereby improving drug persistence within the cardiac inflammatory microenvironment (IME).

Fig. 1.

Inflammation-activated size variable and cTnT anchored NPs. (A) Schematic diagram of MPO converting the phenolic residues of DSPE–PEG–bis-5HT to radicals, enabling the bis-5HT radicals to form bonds. (B) TEM images of TF-5NP in PBS or PBS catalyzed by MPO in the presence of H₂O₂, scale bar: 100 nm. (C) Zeta potentials of TF-NP and TF-5NP. (D) Size distributions of TF-NP and TF-5 NP under different conditions: PBS alone, MPO/H₂O₂ activation, and neutrophil treatment. (E) Schematic diagram of MPO converting the phenolic residues of DSPE–PEG–bis-5HT and bis-5HT radicals, anchoring to the cysteine residue of cardiac cTnT. (F) Confocal fluorescence images of MI tissue slices after intravenous administration of cy5.5, cy5.5-NP, and cy5.5-5-NP for 14 d. Blue channel: nucleus; green channel: cTnT; red channel: cy5.5-labeled MPO-responsive NPs (cy5.5-5 NP) or cy5.5-labeled nonMPO-responsive NPs (cy5.5-NP) or cy5.5. Scale bar: 50 μm. (G) Release profiles of TIIA from TF-5 NP under different conditions: untreated, +MPO, +H₂O₂, +MPO/H₂O₂, +MPO/H₂O₂ + 4-ABAH (MPO inhibitor), and +neutrophils. All data are presented as mean ± SD (n = 3). Statistical significance was determined via one-way ANOVA. ∗∗∗P < 0.001.

We further investigated whether bis-5HT-modified NPs could anchor on cTnT. Cy5.5 was encapsulated as a tracer in liposome particles with (cy5.5-5NP) or without (cy5.5-NP) bis-5HT modification. After intravenous administration of the labeled NPs in MI pigs for 14 d, the CLSM images of the sectioned slices from the MI tissue were examined. A comparison of the cy5.5- and cy5.5-NP-treated groups indicates that the cy5.5-5NP group exhibits a stronger red fluorescence signal that completely overlaps with the green fluorescence signal of the cTnT antibody, which confirm that bis-5HT-modified NPs can be anchored to the cysteine residues of cardiac cTnT (Fig. 1F).

To assess the environment-dependent drug release, TF-5NP was exposed to PBS at varying pH levels (7.4, 6.5, and 4.0) over 8 d. The resulting TIIA release percentages are 50.81 %, 78.8 %, and 93.82 % at pH 7.4, 6.5, and 4.0, respectively (Fig. S7A). After MI, the local environment becomes acidic owing to necrosis and the inflammation of CMs, facilitating the composition change of liposomes and triggering drug release. Our findings suggest that a low-pH environment significantly accelerates the rate and extent of drug release. To assess the long-term release kinetics of TIIA from TF-5NP, we investigated the influence of MPO and H₂O₂ on drug release profiles. With the absence of both MPO and H₂O₂, TF-5NP exhibited a biphasic release pattern with 79.28 % cumulative TIIA release observed within 10 days, followed by a gradual plateauing of release rate. Notably, neither MPO nor H₂O₂ alone significantly altered this release profile. However, when both MPO and H₂O₂ were present, a distinct sustained release pattern emerged, with 74.36 % TIIA released at day 10 and a continuous, gradual increase in drug release observed up to 30 days. This MPO/H₂O₂-dependent sustained release was abolished by the MPO-specific inhibitor 4-ABAH, confirming the catalytic requirement for MPO activity. Concomitantly, neutrophil activation induced a sustained drug release profile from TF-5NP, consistent with MPO/H₂O₂-mediated cross-linking (Fig. 1G). These results collectively demonstrate that activated neutrophils trigger TF-5NP aggregation and drug release primarily through MPO-dependent mechanisms, a conclusion strongly supported by the identical responses observed with the purified MPO/H₂O₂ system and its specific inhibition by 4-ABAH. This finding aligns well with the study's rationale of neutrophil-targeted delivery. Similar results are observed for FM release (Fig. S7B and S7C). In summary, the combined effects of inflammation and the acidic microenvironment in myocardial infarction promote both particle size increase and cardiac troponin T (cTnT) anchoring of TF-5NP, enabling sustained high-concentration drug release for up to 30 days post-MI. This sustained release profile suggits promising therapeutic potential.

3.2. Delivery of NPs facilitates the endocytosis of drugs, while sustained drug release from NPs increases cardiac cell survival and invigoration under hypoxia mimicking

The intracellular uptake behavior of bis-5HT-modified NPs was evaluated in the HUVEC cell line. Cy5.5 was used as a tracer to replace TIIA and FM, it was encapsulated in the core of NPs with 5NP (cy5.5-5NP) or NP (cy5.5-NP). For examined the consistency in key biophysical properties and drug release behavior between Cy5.5-loaded NPs (Cy5.5-5 NP) and TIIA/FM-loaded NPs (TF-5 NP). Dynamic light scattering (DLS) analysis confirmed comparable hydrodynamic diameters and size distributions for both formulations. Measurements revealed no significant difference in surface charge (zeta potential) between the NPs and closely matched release kinetics for the two NP types (Fig. S8A–D). Cy5.5 fluorescence was used to track the internalization of NPs via CLSM in HUVEC cells. cy5.5-5NP exhibits stronger red fluorescence in the first 2 h, indicating better uptake, while cy5.5-NP and cy5.5 groups exhibit a slow uptake. This effect lasts for 48 h (Fig. S9). The results confirm that TF-5NP has a good CM targeting ability and biocompatibility.

To further evaluate the effectiveness of TF-5NP, we assessed its impact on cell proliferation in HUVEC cells via the CCK-8 assay (Fig. 2A). The TF-5NP group more effectively reverses the inhibition of cell proliferation under hypoxic conditions compared with the free drug FM or TIIA groups. To determine apoptosis, HUVECs were incubated with the same experimental and control agents, which showed opposite trends compared with the CCK-8 results (Fig. 2B). TF-5NP significantly reduces cell death (9.85 % cell apoptosis) compared with TF-NP (14.21 % cell apoptosis) and TIIA + FM (27.04 % cell apoptosis) in 2 d (Fig. 2C), confirming that TF-5NP can effectively alleviate CM death in a hypoxic environment.

Fig. 2.

Evaluation of cell activity and apoptotic in vitro. (A) Relative cell viability of HUVECs in the anoxic environment after 48 h of incubation with TIIA, FM, TIIA + FM, TF-NP, and TF-5NP. Annexin-V/PI apoptosis assay of HUVECs treated with each group for 48 h measured via (B) FCM and (C) statistics analyses. (D) Representative fluorescence micrographs showcasing the expression of Ki67 (green) in NRCMs, with sarcomeric α-actinin (α-SA) labeling (red). Scale bar: 100 μm. (E) Quantification of Ki67+ cells using ImageJ. (F) Representative fluorescence micrographs from the LIVE/DEAD assay assessing the viability of NRCMs. Scale bar: 100 μm. (G) NRCM viability measured using ImageJ. (H) Representative fluorescence micrographs of cell apoptosis detected using the TUNEL assay. Scale bar: 100 μm. (I) Percentage of TUNEL + NRCM determined using ImageJ. Western blotting (J) and quantification (K) of cleaved Caspase-3, Bax, and Bcl-2. All data are presented as mean ± SD (n = 3 independent experiments). Statistical significance was determined by one-way ANOVA. ∗∗P < 0.01, ∗∗∗P < 0.001.

To further validate the efficacy of TF-5NP in HUVECs, double immunofluorescence staining was performed to investigate the in vitro protective effects of TF-5NP against hypoxic injury in neonatal rat CMs (NRCMs). Proliferating NRCMs were identified through Ki67+ staining, and cell viability was examined via Calcein-AM expression. Incubation with TF-5NP promotes NRCM proliferation, increases cell viability and reduces cell death, which increases the cell numbers (Fig. 2D–G and S10A–S10B). Furthermore, coculture with TF-5NP inhibits the anoxia-induced apoptosis of NRCMs, as proved via the TUNEL assay (Fig. 2H and I and S10C). Further studies reveal that TF-5NP regulates apoptotic signaling cytokines such as cleaved Caspase-3, Bax, and Bcl-2 to alleviate the apoptosis of CMs (Fig. 2J and K). The above results confirm that TF-5NP can effectively improve the viability of CMs under hypoxic conditions and reduce the apoptosis of CMs. The reduction of apoptosis can potentially mitigate the loss of cardiac tissue after MI, which is beneficial for the long-term protection of cardiac function.

3.3. NPs promote angiogenesis and mitochondrial function in vitro

To investigate the mechanism of TF-5NP in MI treatment, we first examined the priming effects of TF-5NP on HUVECs in vitro through cell migration and tube formation assays (Fig. 3A). In endothelial cell migration assays, the addition of TF-5NP significantly promotes the migration of HUVECs, indicating improved cell mobility (Fig. 3B and C). In addition, the results of the Matrigel tube formation assays demonstrate that the vessel-forming capability is significantly enhanced in TF-5NP-treated HUVECs, evidenced by enhanced tube branching (Fig. 3D and E). Immunoblotting analyses performed to evaluate the effects of TF-5NP on angiogenesis reveal that TF-5NP significantly affects angiogenesis. Further findings show a significant upregulation of angiogenesis-related proteins including VEGF, PDGF, and FGF in TF-5NP-treated HUVECs (Fig. 3F–I). The migration of endothelial cells, the main constituent cells of the inner wall of blood vessels, promotes angiogenesis and provides blood supply to the infarction area, thus promoting the repair and regeneration of myocardial tissue.

Fig. 3.

Effects of TF-5NP on angiogenesis and mitochondrial function in vitro. (A) Experimental diagram of TF-5NP intervention in HUVEC migration and tubule formation. (B) Images of HUVEC migration. scale bar: 200 μm. (C) Quantitative analysis of migration rates for 12 and 24 h. (D) Tube formation assay of HUVECs. Scale bar: 200 μm. (E) Quantitative analysis of the number of junctions. (F) Western blotting and quantification analysis of (G) VEGF, (H) PDGF, and (I) FGF expression levels. (J) Quantitative analysis of the ATP level. (K) Evaluation of mitochondrial membrane potential using MitoTracker staining. Scale bar: 100 μm. (L) Evaluation of intracellular ROS levels using MitoSOX staining. Scale bar: 100 μm. (M) Mitochondrial membrane potential levels were evaluated by Mito-JC-1 staining. Scale bar: 100 μm. All data are presented as mean ± SD (n = 3 independent experiments). Statistical significance was determined via one-way ANOVA. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

Mitochondria are the main energy source in CMs, and the loss of mitochondrial integrity and function is an important pathological factor that affects the structure and function of the heart [30]. Therefore, we investigated the effects of TF-5NP on mitochondria. Mitochondria mainly produce adenosine triphosphate (ATP) through oxidative phosphorylation to provide the necessary energy for cells. The results show that TF-5NP-treated cells exhibit a significantly higher ATP level than the other cell groups (Fig. 3J), indicating an enhanced function of mitochondria. We assessed intracellular and mitochondrial activity, mitochondrial membrane potential, and mitochondrial ROS production using Mito Tracker, MitoSox, and Mito-JC-1 red staining, respectively. TF-5NP-treated cells show significantly elevated levels of MitoTracker red fluorescence, indicating improvement in mitochondrial activity (Fig. 3K–S11A, and S11D). Furthermore, the reduced levels of MitoSOX red fluorescence suggest low mitochondrial ROS production and enhanced mitochondrial function (Fig. 3L–S11B, and S11E). The elevated levels of Mito-JC-1 red fluorescence show an increase in mitochondrial membrane potential, indicating an improvement in mitochondrial energy metabolism (Fig. 3M–S11C, and S11F). These outcomes confirm the critical role of TF-5NP in regulating mitochondrial activity, membrane potential, and ROS production, safeguarding endothelial cells.

3.4. NPs improved cardiac morphology and function in zebrafish

The verapamil-induced zebrafish cardiac dysfunction model has proved to be an effective tool for assessing the efficacy of therapeutic interventions in cardiac remodeling [31,32]. In this study, we used this model to assess the effects of TF-5NP on cardiac morphology and function. The cardiac function and level of angiogenesis of the zebrafish treated with different groups of drugs were observed through images and videos (Fig. 4A). The analysis of phenotypic changes reveals that TF-5NP significantly alleviates the venous congestion and reduces heart enlargement induced by verapamil (Fig. 4B). Quantitative analysis further confirms that TF-5NP reduces the enlarged heart area (Fig. 4C) and venous congestion in zebrafish (Fig. 4D). Heart enlargement can lead to myocardial hypertrophy or heart chamber dilation, impairing the systolic and diastolic functions of the heart, reducing the ability of the heart to pump blood and causing irreversible damage. Thus, TF-5NP can prevent heart enlargement and associated dysfunction.

Fig. 4.

TF-5 NP protects against cardiac dysfunction and effect on angiogenesis in zebrafish. (A) Schematic diagram of Verapamil-induced cardiac dysfunction zebrafish administration and detection. (B) Photomicrographs of zebrafish with red and yellow-dotted lines marking the pericardial sac and areas of venous congestion. Scale bar: 200 μm. (C) Statistical analysis of pericardial sac area in zebrafish. (D) Statistical analysis of venous congestion area in zebrafish. (E) Representative images of zebrafish hearts showing verapamil-induced heart dilation, which was mitigated by treatment with TF-5 NP. Scale bar: 200 μm. (F) Quantification of cardiomyocyte area in zebrafish across each experimental. (G) Measurement of the distance between sinus venosus and bulbus arteriosus (SV-BA distance), in zebrafish across each group. (H) Analysis of blood flow velocity in zebrafish. (I) Heart rates recording and statistical analysis in each group. (J) Cardiac output of zebrafish in each experimental group was determined and subjected to statistical analysis. (K) The name of each blood vessel of zebrafish is marked with schematic diagram and fluorescence images utilized to observe the structural characteristics of ISVs in 72-hpf Tg (fli1: EGFP) zebrafish. Scale bar: 200 μm in low-magnification images, Scale bar: 50 μm in high-magnification images. (L) Statistics of the number of Intersegmental vessels. (M) Fluorescence imaging was employed to assess ISV length in 72-hpf Tg (fli1: EGFP) zebrafish. (N) Quantification of ISV numbers. (O) Quantification of the area of SIV branch points in 72-hpf zebrafish embryos. (P) Quantification of PCV diameters in 72-hpf zebrafish. All data are presented as mean ± SD (n = 10). Statistical significance was determined via one-way ANOVA. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

To assess the effects on the CM morphology, transgenic zebrafish Tg (cmlc2:eGFP) expressing the green fluorescent protein in their hearts were used. Zebrafish in the model group exhibited more dilated CMs and longer SV–BA distance than those of the control group. In contrast, TF-5NP treatment improves the CM morphology and restores the SV–BA distance (Fig. 4E–G). The heart area is a key indicator of heart function, and variations in the SV–BA distance reflect changes in the heart chamber and ventricle position. A small CM area and the restoration of the SV–BA distance suggest a good protective effect of the drug on cardiac function and morphology. Moreover, cardiac function parameters such as the blood flow velocity (Fig. 4H), heart rate (Fig. 4I), and cardiac output (Fig. 4J) are significantly reduced in verapamil-treated zebrafish and partially restored after drug treatment. Notably, TF-5NP shows the best therapeutic effect among all groups. Notable enhancements in cardiac morphology and function are observed with the administration of TF-5NP to zebrafish. Subsequently, we elucidated the mechanisms underlying the therapeutic effects of TF-5NP.

3.5. Improved angiogenesis level can improve the heart function of zebrafish

To assess the angiogenic effects of TF-5NP on zebrafish blood vessel development, we used Tg (fli1:EGFP) transgenic zebrafish from shield stage 3 hpf to 48 hpf. The development of the intersegmental vessel (ISV), dorsal aorta (DA), and posterior cardinal vein (PCV) was examined at 48 hpf via in vivo fluorescence imaging (Fig. 4K). Quantitative analysis reveals that the ISVs in the TF-5NP group (24.0 ± 0.5) are significantly larger than those in the model (18 ± 1) and TF (19 ± 1) groups (Fig. 4L). Moreover, the mean ISV length (246 ± 5 pixels) in the TF-5NP treated group significantly increases more than that of the model group (227 ± 2 pixels; Fig. 4M). TF-5NP treatment significantly enhances subintestinal vessel (SIV) formation, and SIV branches (8.1 ± 0.5) and areas (35,000 ± 2000 pixels) are significantly increased compared with those of the model group (5.6 ± 0.7 and 22,000 ± 1000 pixels) (Fig. 4N and O).

Consistent with the changes in the mean ISV length and area, the diameters of the TF-5NP-treated DA and PCV are wider by 31 ± 1 and 61 ± 1 pixels than those of the model group of 25 ± 1 (Fig. S12A and S12B) 54 ± 2 pixels (Fig. 4P), respectively. As VEGF plays a crucial role in angiogenesis during zebrafish development, an investigation was conducted to determine if VEGFR1 and vegfaa expressions during angiogenesis were impacted by TF-5NP treatment. The results show a significant upregulation of VEGFR1 and vegfaa mRNA expression, indicating that TF-5NP promotes angiogenesis (Fig. S12C and S12D). Thus, the angiogenic potential of TF-5NP during zebrafish development showcases its crucial role in promoting cardiac repair.

3.6. NPs prolong drug metabolism in miniature pigs for up to 28 d after MI

Miniature pigs have several advantages as animal models of cardiovascular diseases. Compared with humans, miniature pigs have similar heart size, specific gravity, coronary artery flow, hemodynamics, and myocardial contraction [33,34]. We established an MI model of pigs (Fig. S13A), The permanent coronary artery ligation model was employed to specifically replicate the persistent inflammatory microenvironment and chronic ventricular remodeling observed in MI patients who fail to achieve timely reperfusion, thereby enabling the evaluation of TF-5NP's long-term retention and sustained therapeutic efficacy over 28 days. This model choice aligns with our therapeutic focus on mitigating maladaptive remodeling in non-reperfused MI, a high-risk subgroup, where the sustained inflammatory niche provided optimal conditions for neutrophil-targeted TF-5NP accumulation and prolonged drug release. (a) The pigs were anesthetized and mechanically ventilated, (b) the heart was exposed (c) with pericardium cut, and (d) the left anterior descending coronary artery was ligated to create a left ventricular (LV) infarction, except for the sham group, which underwent left anterior descending threading, as previously described. (e and g) After ligation, 600 μL of cy5.5, cy5.5-NP, or cy5.5-5-NP was injected into the border and center regions of the infarct position, (f) followed by the surgical closure of the incision in the chest of pigs. All pigs were anesthetized and evaluated via echocardiography 2 d after ligation. Only pigs with an infarction of >25 % of the LV free wall were included. After the MI model was prepared, blood samples were collected after 6 and 12 h, 1, 3, 5, 7, 14, and 28 d for blood fluorescence detection. Tissue fluorescence staining was conducted to observe the in vivo pharmacokinetics of cy5.5-5-NP after 14 and 28 d (Fig. 5A). The fluorescence analysis of blood samples reveals that cy5.5-5-NP significantly enhances the retention of cy5.5 in the heart until 28 d than cy5.5 and cy5.5-NP (Fig. 5B and C).

Fig. 5.

Effects of TF-5 NP on pharmacokinetics in pigs. (A) Pigs were intravenouslyinjected with cy5.5 and cy5.5-NP, cy5.5-5 NP with the same dosage of cy5.5 (2 mg kg-1). (B) The blood samples were collected at predetermined intervals and the fluorescence intensity was subsequently measured by in vivo imaging system. (C) Quantitative analysis of fluorescence intensity. Histological sections of the infarct region were immunolabeled with the (D) Endothelial marker CD31 (green), (F) Cardiomyocytes marker α-SMA (green), (H) Neutrophil marker CD11b (green), (J) MPO (green), cy5.5 (white) and DAPI (blue). Scale bar: 100 μm. Quantitative analysis of (E) CD31, (G) α-SMA, (I) CD11b and (K) MPO fluorescence intensity was conducted using Image J. All data are presented as mean ± SD (n = 3). Statistical significance was determined via one-way ANOVA. ∗∗∗P < 0.001.

To examine the penetration ability of the drug, drug retention was assessed in the heart section through CD31 and α-SMA staining and colocalization of the drug. The cy5.5-5-NP drug exhibits long retention in the heart tissue; specifically, the cy5.5-5-NP group shows more retention in myocardial tissue and vascular smooth muscle tissue than cy5.5 or cy5.5-NP on Days 14 and 28 after injection (Fig. 5D–G). To examine the targeting specificity of the drug, drug retention was assessed in the heart section through CD11b and MPO staining and colocalization of the drug. Cy5.5-5 NP effectively targeted the neutrophil-infiltrated infarcted region and exhibited significant colocalization with MPO (Fig. 5H–K). These findings not only validate the targeting specificity of TF-5NP but also corroborate the proposed mechanism of TF-5NP responding to MPO enzyme activity for localized drug release within the infarcted myocardium. The above results demonstrate the slow-release effect of cy5.5-5-NP and the excellent permeability of MI tissue in the MI pig model, which achieved retention up to 28 d. The liposome delivery system with the 5HT targeting head enables the drug to be target-released in vivo at a controlled rate, avoiding excessive fluctuations in the blood concentration caused by frequent administration, thereby improving the therapeutic effect and patient experience.

3.7. NPs improved cardiac function in pigs after MI

To demonstrate the effect of TF-5NP on improving cardiac function, pigs in the MI model were immediately treated with TF-5NP via intravenous administration. A cardiac magnetic resonance examination was performed to determine the MI size and cardiac function of the pigs, followed by pathological staining on Day 28 (Fig. 6A). A comparative analysis of global cardiac remodeling and shape provides important insights into the post-MI treatment effects. Cardiac tissues were sampled to observe the cardiac conditions (Fig. 6B and S13B). The TF-5NP group shows a higher capacity to mitigate global cardiac remodeling than the model group (Fig. S13D). To investigate the histological changes in the TF-5NP-treated heart, we performed H&E staining on samples collected at the end of the experiment. Necrotic muscle fibers in the infarct area are replaced by loose fibrous connective tissue, loosening the structure of the infarcted area. The capillaries are dilated, and the surviving CMs are scattered along the edge of the infarction area, suggesting hypertrophy and fibroblast proliferation in the post-MI heart (Fig. 6C). Notably, these changes are partially reversed in the TF-5NP group, proved by the significant reduction in the infarction area in the TF-5NP-treated group (11.49 %) on Day 28 post-MI compared with that in the model group (31.42 %), TIIA + FM group (23.01 %) and TF-NP group (21.35 %) (Fig. 6E). Masson staining reveals the substantial deposition of myocardial collagen fibers in the infarct area (mainly including Types I and II collagen fibers), which is significantly reduced in the TF-5NP treated group (Fig. 6D). The percentages of collagen fiber areas are 1.06 %, 34.33 %, 29.79 %, 24.49 % and 12.5 % in the sham, model, TIIA + FM, TF-NP and TF-5NP groups, respectively (Fig. 6F), which demonstrate that the infarct size is significantly smaller in the TF-5NP-treated group than those in the untreated MI groups. Furthermore, the WGA staining of CMs reveals a notable increase in the CM cross-sectional area in post-MI hearts, while treatment with TF-5NP exhibits an effective reduction of hypertrophy (Fig. 6G).

Fig. 6.

TF-5 NP attenuated adverse LV remodeling in pigs. (A) MR imaging and heart sectioning were performed on pigs on day 28. (B) Representative images of hearts harvested 28 days post-MI, with and without TF-5 NP treatment. Representative histological analysis of the infarcted myocardium was performed among different treatment groups using (C) H&E staining and (D) Masson's trichrome staining. (scale bars: 3 mm in overall tissue image, 200 μm in low-magnification images, and 50 μm in high-magnification images). Quantitative analysis of the (E) Percentage of Infarction area heart area, and (F) Percentage of collagen fiber area. (G) Representative images showcasing WGA immunofluorescence staining. Scale bar: 50 μm. (H) Representative MR images in short (upper pannel) and long (bottom) axis views at the area of the infarct scar at 28 days post-MI. The infarct area appears in white (circled), while the non-infarcted myocardium appears black. (I) Representative MR images in Systole and diastole. Quantitative analysis of (J) Left Ventricular End Systolic Volume, (K) left ventricular end-diastolic volume, (L) Body surface area, (M) indexed left ventricular end-systolic volumes, (N) indexed left ventricular end-diastolic volumes, (O) the left ventricular ejection fraction. All data are presented as mean ± SD (n = 3). All data are presented as mean ± SD (n = 3). Statistical significance was calculated via one-way ANOVA. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

The size and volume of the heart play an important role in heart function, influencing its ability to contract and pump blood efficiently after MI. The direct visualization and organized observation of heart tissue have provided convincing data on the improvement of ventricular remodeling post-MI by TF-5NP. Baseline MRI data show no differences in cardiac function between the studied groups. TF-5NP-treated pigs exhibit small MI size and good cardiac function 28 d post-MI (Fig. 6H and I). In contrast, untreated animals showed a decrease in BSA and signs of dilation, as indicated by the increase in end-diastolic and systolic volumes of the left ventricle (LVESV, LVEDV, iLVESV, and iLVEDV) (Fig. 6J–N). Moreover, TF-5NP treated pigs significantly improve the LVEF (25.38 % in the Model group, 48.07 % in the Sham group, and 39.13 % in the TF-5NP-treated group) (Fig. 6O). Thus, TF-5NP synergistic therapy enhances cardiac function based on the assessment of LVEF, LVEDV, and LVESV. While our study demonstrated attenuated ventricular remodeling via improved LVEF, reduced fibrosis, and enhanced angiogenesis, we lacked tissue-level biomechanical characterization. Future studies will include fresh-tissue biomechanical assays and in vivo pressure-volume loop analyses to comprehensively evaluate therapeutic efficacy [[35], [36], [37], [38]]. In terms of safety, after 28 d of treatment, the pigs were weighed. No significant change in body weights of each group was observed (Fig. S13C). We also stained the liver, spleen, lungs, kidneys, intestines, and muscles of pigs with hematoxylin and eosin (H&E) The experimental treatment modalities reveal that the treated pigs exhibited no signs of inflammatory response to the administered TF-5NP (Fig. S17), highlighting the biosafety of TF-5NP within the complicated biological environment of the heart, which is essential for future clinical applications. The comprehensive analysis shows that TF-5NP treatment after MI reduces scar expansion, interstitial fibrosis, cardiac remodeling, improves cardiac function, and exhibits favorable safety profiles.

3.8. Mechanism underlying the NP-related improvement of ventricular remolding

To elucidate the mechanism underlying the improvement of ventricular remodeling by TF-5NP, we conducted RNA-seq analysis on cardiac ischemic tissues with and without TF-5NP treatment. Eight-hundred and eighty-eight out of the 16856 differentially expressed genes (DEGs) were identified in the model group (505 downregulated and 383 upregulated) compared to the sham group, while TF-5NP treatment reveals 742 downregulated and 2346 upregulated genes according to RNA-seq analysis (Fig. 7A and C). Using P < 0.05 and fold change (FC) ≥ 5 or FC ≤ 0.2 as the threshold of significant difference, 29 transcripts in pig hearts were identified. One-hundred and seventy-five downregulated and 190 upregulated transcripts are presented in the TF-5NP-treated group compared to those in the model group (Fig. 7B). The gene ontology biological process and Kyoto encyclopedia of genes and genomes pathway enrichment analyses show that TF-5NP treatment primarily impacts the pathways related to VEGF signaling, extracellular matrix (ECM)–receptor interaction, mammalian target of rapamycin signaling, nitrogen metabolism, and ferroptosis (Fig. S14C). A comparison of the main components of the sham, model, and TF-5NP groups reveals notable differences between the components of the TF-5NP group and the other two groups (Fig. 7D). The DEG is primarily involved in angiogenesis, immune system process, apoptotic chromosome condensation, collagen-containing ECM, mitochondrion, and other classical biological processes that are strongly associated with macrophage functional metabolism and neovascularization (Fig. 7E and S14B). The gene set enrichment analysis indicates that TF-5NP-bound genes are largely associated with the regulation of angiogenesis and wound healing (Fig. S14A). Thus, TF-5NP might promote angiogenesis and regulate the inflammation.

Fig. 7.

Mechanism underlying the effects of TF-5 NP on regulate the inflammation and promoting angiogenesis. (A) The volcano plot illustrates the discovery of differentially expressed transcripts (DETs) using a significance threshold of P < 0.05 and a fold change (FC) of ≥5 or ≤0.2. (B) Venn diagrams from DETs analysisidentified 365 transcripts of interest. (C) A heatmap depicting selected proteins that represent significantly altered signaling pathways. (D) Principal component analysis between sham, model and TF-5 NP group. (E) Gene Ontology (GO) enrichment results. (F) Tanshinone IIA inhibits ventricular remolding after MI. (G) Representative images showcasing TUNEL staining. Scale bar: 0.5 μm. (H) Western blot of TOM20 Flotillin-1 and TOM40 expression levels. (I) Histological sections of the infarct region and border region were immunolabeled with the pyroptosis specific protein, Caspase-1, GSDMD, IL-1β, IL-4 (orange), DAPI (blue). Scale bar: 20 μm. (J) Schematic diagram of Formononetin regulating angiogenesis through apoptosis. (K) Representative electron microscopic picture of mitochondria. scale bars: 50 μm in low-magnification images, and 20 μm in high-magnification images. (L) Western blot of VEGF, PDGF, FGFR1 and CD31 expression levels. (M) Histological sections of the infarct region and border region were immunolabeled with the angiogenesis specific protein, CD31 and α-SMA (green), DAPI (blue). Scale bar: 50 μm. All data are presented as mean ± SD (n = 3).

To validate the sequencing results, the mitochondrial structure was examined via TEM. MI can cause the accumulation of damaged mitochondria and the appearance of mitochondrial fold fracture. However, TF-5NP treatment can effectively reduce mitochondrial damage, and the mitochondrial folds are considerably completed and arranged regularly (Fig. 7G). The effect of mitochondrial structural changes on function was investigated by measuring the mitochondrial oxidative stress indexes. The Western blot analysis of TOM20, TOM40, and Flotillin-1 levels shows that TF-5NP can significantly improve mitochondrial function (Fig. 7H). After MI, excessive ROS production impairs mitochondrial function and inhibits the expression of TOM20, TOM40, and Flotillin-1, while TF-5NP treatment can reverse the effect. It can increase the expression of mitochondrial proteins such as TOM20, indicating improved mitochondrial function.

Functional damage caused by mitochondrial oxidative stress can induce the pyroptosis of cells, releasing inflammatory mediators to trigger the inflammatory response of the body. During MI, moderate inflammation clears the necrotic myocardial tissue. However, excessive inflammatory response in the early stage of MI enlarges the MI area and forms large scar tissues (Fig. 7F) [39,40]. The rigid scar tissue damages cardiac function. After MI, the accumulation of pyroptosis cells induces a strong inflammatory response in the body. To investigate the effect of drugs on cytopyroptosis protein and inflammatory protein, immunofluorescence staining was performed. Compared with the model group, the TF-5NP treated group significantly reduces the expression of pyroptosis protein Caspase-1 and Gasdermin D, proinflammatory factor IL-1β, and increases the expression of inhibitory inflammatory factor IL-4 in the border and infarction areas (Fig. 7I). As VX765 is a potent Caspase-1 inhibitor known to reduce IL-1β production, it was used as a positive control. Notably, immunofluorescence staining revealed that TF-5NP treatment surpassed VX765 and other groups in suppressing IL-1β expression (Fig. S15A and S15B). M1/M2 macrophage polarization critically relates to pyroptosis. We assessed macrophage polarization status via immunostaining of specific M1 (pro-inflammatory) and M2 (anti-inflammatory/pro-resolving) surface markers CD68 and CD163. Results showed TF-5NP effectively inhibited M1 markers while promoting M2 marker accumulation around and within the infarct core, compared to other groups (Fig. S16A–16D). These data demonstrate TF-5 NP's potential to modulate pyroptosis. After MI, the local blood flow of the heart is interrupted, and the oxygen supply of myocardial tissue is severely compromised. Therefore, mitochondria accumulate in the infarcted area to provide energy. However, they also produce excessive ROS, which lead to oxidative stress, cell damage, cell pyrosis, and excessive inflammation. The results show that TF-5NP can effectively inhibit excessive cellular inflammatory response and pyroptosis. Pyroptosis functional assays were assessed only at day 28 due to serial sampling challenges in large animal models. While literature strongly indicates peak activity occurs at 24h with decline by day 7 [[41], [42], [43]]. Future studies should include acute-phase timepoints to fully resolve treatment kinetics.

During the repair of MI, angiogenesis is essential for restoring the blood supply and heart function. Apoptosis can stimulate angiogenesis, as tissue growth and repair require the formation of new blood vessels, which inhibit the further occurrence and development of apoptosis (Fig. 7J) [44,45]. To explore the effect of TF-5NP on angiogenesis, we measured the apoptosis level using TUNEL staining on cardiac tissue sections. CMs display a characteristic apoptotic morphology in the model group, while the TF-5NP group shows a significant decrease in apoptotic cells at the infarction border and infarction area, suggesting the potential antiapoptotic properties of TF-5NP (Fig. 7K). The Western blot analysis of the LV tissue obtained from pigs with MI suggested that angiogenesis-related proteins (such as VEGF, PDGF, FGFR1, and CD31) significantly increased (Fig. 7L). CD31 and α-SMA are well-established biomarkers for identifying and characterizing vessels. With immunofluorescence staining, we observe higher levels of CD31 and α-SMA in the infarction border and area of the TF-5- NP-treated group than the model group (Fig. 7M). Thus, TF-5NP can effectively inhibit apoptosis and activate angiogenesis. The activation of angiogenesis releases proangiogenic growth factors (VEGF, PDGF, FGFR1, and CD31). Collectively, these findings demonstrate that TF-5NP treatment can improve heart function and treat MI by regulating inflammatory response and activating angiogenesis.

4. Conclusion

Post-MI, neutrophils are released and accumulated in the IME for secreting MPO. We developed a neutrophil-targeted nanomedicine (TF-5NP) that delivered TIIA and FM to the infarcted myocardium. TF-5NP was synthesized through the coassembly of bis-5-HT-modified DSPE–PEG–COOH with Chol and DSPG, allowing it to bind to troponin in the infarcted tissue and ensuring a strong retention of liposomes in the myocardium. This enhanced the accumulation of TF-5NP in IME and sustained the release of TIIA and FM. Notably, TF-5NP could produce a long-acting therapeutic effect for up to 28 d, solving the key problem of retaining drugs in the heart under its vigorous contractions, thereby improving the local drug concentration and therapeutic effect. TF-5NP slowly released TIIA and FM, which mitigated oxidative damage and promoted angiogenesis to improve cardiac function.

CRediT authorship contribution statement

Shuai Mao: Writing – original draft, Resources, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization. Yubin Liang: Visualization, Validation, Methodology, Investigation, Conceptualization. Zikang Chen: Methodology, Investigation. Lei Wang: Writing – review & editing, Conceptualization. Quanfu Chen: Methodology. Zhuting Fang: Writing – review & editing, Supervision, Project administration. Qifan Zheng: Writing – review & editing, Project administration. Wen Ma: Writing – review & editing, Supervision. Hanping Zhang: Writing – review & editing, Investigation, Conceptualization. Zhiqiang Yu: Writing – review & editing, Investigation, Conceptualization. Ling Yu: Writing – review & editing, Supervision, Resources, Investigation, Funding acquisition, Conceptualization.

Ethics approval and consent to participate

All animal experiments and care were carried out in accordance with protocols approved by the Regional Ethics Committee for Animal Experiments at Guangzhou University of Chinese Medicine (Approval No. IACUC-2024-7903-01).

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 is the Supplementary data to this article.