Targeting efferocytosis with carbonized polymer dots from a natural anti-inflammatory drug to alleviate excessive inflammatory responses in the acute phase of myocardial infarction

Abstract

Excessive inflammation serves as a central pathological driver of adverse outcomes following myocardial infarction (MI). However, current anti-inflammatory interventions have demonstrated limited efficacy and safety in clinical trials. A key instigator of secondary inflammatory cascades post-MI is impaired efferocytosis—the insufficient recognition and clearance of apoptotic cells by macrophages. Concurrently, NLRP3/Caspase-1/GSDMD-mediated pyroptosis exacerbates both acute and persistent inflammatory responses. Modulating efferocytosis to suppress pyroptosis thus represents a promising therapeutic strategy for managing acute-phase inflammation. In this study, we developed pharmacologically active carbonized polymer dots derived from the natural anti-inflammatory drug curcumin (Cur-CPDs) with high water dispersibility and biocompatibility to modulate post-MI inflammation. Compared with free curcumin, Cur-CPDs significantly enhanced efferocytosis and facilitated inflammation resolution by activating the NOTCH-1/CD47 signaling pathway. Both in vitro and in vivo experiments revealed that Cur-CPDs restored the clearance of apoptotic cardiomyocytes, suppressed pyroptotic activation, and improved cardiac function during the early phase of MI. Collectively, these findings establish Cur-CPDs as a novel class of bioactive nanotherapeutics and underscore efferocytosis modulation as a viable target for mitigating residual inflammation and limiting cardiac injury following MI.

Graphical abstract

Highlights

• •Cur-CPDs are synthesized through a facile alkali-assisted carbonization approach.
• •Cur-CPDs target efferocytosis and regulate pyroptosis in acute myocardial infarction.
• •Cur-CPDs break the vicious cycle between inflammation and myocardial injury.
• •Cur-CPDs provide an immunomodulatory cardioprotective strategy for MI.

1. Introduction

Despite substantial advances in reperfusion strategies [[1], [2], [3]] and secondary prevention [4,5], acute myocardial infarction (AMI) remains a leading cause of mortality and recurrent cardiovascular events worldwide [[6], [7], [8]]. Real-world evidence indicates persistently high 30- and 90-day readmission rates of 11.6% and 18.8%, respectively [6], underscoring the continued vulnerability of patients during the early post-infarction phase. Cardiomyocyte necrosis following AMI elicits a robust sterile inflammatory response. When excessive or sustained, this response exacerbates myocardial injury [9], accelerates adverse ventricular remodeling [[10], [11], [12]], and ultimately contributes to the development of heart failure [13,14]. Clinically, residual inflammatory risk serves as a potent predictor of poor prognosis, as reflected by the strong association between elevated hsCRP [15], increased levels of inflammatory biomarkers such as S100A8/A9 [16], and higher incidence of major adverse cardiovascular events. Post-AMI inflammation is orchestrated as a tightly regulated biphasic process [17,18], comprising an initial pro-inflammatory phase [19] followed by an active resolution phase [[20], [21], [22], [23]]. A pivotal mechanism underlying inflammation resolution is efferocytosis, the phagocytic clearance of apoptotic cells by macrophages, which prevents secondary necrosis and curtails ongoing inflammation [[24], [25], [26], [27], [28], [29]]. In the infarcted myocardium, however, efferocytosis is frequently impaired, leading to the accumulation of dying cardiomyocytes and persistent inflammatory signaling [25,[30], [31], [32]]. This defective clearance perpetuates inflammatory forms of programmed cell death, most notably pyroptosis [18,33,34].

Pyroptosis, driven by NLRP3 inflammasome activation and gasdermin D-mediated membrane pore formation, promotes the release of pro-inflammatory cytokines such as IL-1β and IL-18, thereby amplifying the inflammatory cascade and exacerbating infarct expansion, adverse remodeling, and cardiac dysfunction [[34], [35], [36]]. Despite numerous efforts to suppress post-MI inflammation or inhibit pyroptosis directly [17,37], therapeutic strategies aimed at restoring efferocytosis as an endogenous resolution pathway remain largely underexplored. Curcumin, a natural polyphenol derived from Curcuma longa, possesses well-documented anti-inflammatory and antioxidant properties [38,39], yet its clinical translation remains severely constrained by poor aqueous solubility and systemic bioavailability [[40], [41], [42]]. Of note, curcumin-based nanoplatforms have recently been shown to potentiate efferocytosis in diverse inflammatory disease models [43]. Carbonized polymer dots (CPDs) have emerged as particularly attractive nanocarriers in biomedicine, owing to their broad precursor availability, high biosafety profile, and chemically programmable bioactivities [44]. CPDs synthesized from functional precursors can inherit precursor-derived structural motifs and functional groups, a property that enables direct translation of molecular features into nanoscale functionalities [[45], [46], [47], [48], [49]]. When fabricated in the ultrasmall size regime, rationally engineered CPDs exhibit markedly improved in vivo behavior, characterized by prolonged circulation persistence and high tissue-targeting specificity [[50], [51], [52], [53], [54], [55], [56]]. Based on these advantages, we hypothesized that converting curcumin into CPDs, while preserving its pharmacophoric moieties and achieving optimized biodistribution, would effectively mitigate the excessive inflammatory response during acute myocardial infarction, thereby affording robust cardioprotective efficacy.

Here, we report the synthesis of Cur-CPDs from the natural anti-inflammatory drug curcumin via an alkali-assisted carbonization strategy and demonstrate their therapeutic efficacy in the early phase of AMI. Compared with native curcumin, Cur-CPDs exhibit markedly improved solubility and bioavailability. Mechanistically, Cur-CPDs restore efferocytosis by modulating the NOTCH-1/CD47 axis, thereby suppressing inflammatory pyroptosis and excessive post-infarction inflammation (Scheme 1). By engaging the endogenous “clearance and resolution” program rather than merely blocking inflammatory mediators, this study establishes an efferocytosis-promoting nanotherapeutic platform that reduces inflammatory burden, limits adverse remodeling, and improves cardiac function after AMI. More broadly, the precise regulation of “eat-me” and “don't-eat-me” signals by nanomedicine offers a new paradigm for treating diseases characterized by defective inflammatory clearance, including myocardial infarction and heart failure.

Scheme 1.

Schematic illustration of the alkali-assisted synthesis of curcumin-derived carbonized polymer dots (Cur-CPDs) and their mechanism of action in myocardial infarction. (A). Preparation of Cur-CPDs via alkali-assisted carbonization. (B). Cur-CPDs restore defective efferocytosis by regulating the NOTCH-1/CD47 axis and then inhibit inflammatory pyroptosis, thereby reprogramming the inflammatory microenvironment in the early phase after myocardial infarction.

2. Results

2.1. Synthesis and characterization of Cur-CPDs

Curcumin was converted into carbonized polymer dots (Cur-CPDs) via an alkali-assisted carbonization strategy. Transmission electron microscopy (TEM) revealed well-dispersed spherical nanoparticles with an average diameter of 17.08 nm (Fig. 1A and B). The ultraviolet-visible near-infrared (UV-Vis-NIR) absorption spectrum of Cur-CPDs in water exhibited a characteristic peak at approximately 280 nm, which can be attributed to the aromatic domains formed during carbonization (Fig. 1A). Free curcumin is practically insoluble in water, while Cur-CPDs exhibit excellent water dispersibility. Conducting a comparative UV-Vis-NIR measurement across different solvents would be inappropriate. The UV-Vis-NIR spectrum is primarily presented to demonstrate the optical features of the final Cur-CPDs, rather than to serve as the sole evidence of structural transformation, and the successful formation of Cur-CPDs has been well confirmed by the multiple independent characterization techniques described below. Fourier transform infrared (FT-IR) spectroscopy indicated that most of the characteristic functional groups of curcumin were preserved in Cur-CPDs, suggesting the partial inheritance of the molecular structure of the precursor (Fig. 1C). Notably, a newly emerged absorption band at around 1700 cm⁻¹ was observed, corresponding to carboxyl groups generated through carbonization-induced oxidation. The spectrum of free curcumin exhibits characteristic bands at ca. 3500 cm⁻¹ (O–H stretching of phenolic hydroxyl), 1627 cm⁻¹ (C=O stretching), 1602 and 1509 cm⁻¹ (aromatic C=C stretching vibrations of the benzene rings), 1429 cm⁻¹ (C–H bending), 1281 cm⁻¹ (C–O stretching), and <1200 cm⁻¹ (C–O–C stretching and C–H bending). Compared with free curcumin, the Cur-CPDs spectrum exhibits markedly reduced C–O–C and C–H bands, indicating substantial structural reorganization during carbonization, with partial loss of C–O–C and C–H moieties upon formation of the carbonized core. This observation confirms the occurrence of carbonization and the formation of a carbon core structure. X-ray photoelectron spectroscopy (XPS) further confirmed the surface chemical composition of Cur-CPDs. High-resolution C 1s and O 1s spectra revealed abundant C=O, C–O, and O–C=O species, consistent with the FT-IR results (Fig. 1D–F, Figs. S1–S6). In addition, the alkali-assisted carbonization process resulted in the formation of nitrogen-doped graphitic carbon cores featuring N–C₃ configurations with a high carbon content (Fig. 1G and H). The characteristic D and G bands observed in Raman spectra, the standard and most sensitive tool for graphitic structure analysis, already offered reliable evidence for a partially graphitized core (Fig. 1I). In contrast, X-ray diffraction analysis (XRD) and high-resolution TEM yielded essentially featureless patterns, owing to the small size, poor crystallinity, and outer polymeric moieties of Cur-CPDs (data not shown). To improve their biological applicability, Cur-CPDs were further PEGylated via hydrophobic interactions between the phospholipid moieties of mPEG-DSPE and the graphitic carbon core. PEGylation led to a slight increase in the ζ potential compared with bare Cur-CPDs (Figs. S7–S9) and markedly enhanced their colloidal stability. The PEGylated Cur-CPDs exhibited excellent dispersion stability in water, phosphate-buffered saline (PBS), and Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (Fig. S10). Moreover, the colloidal stability of PEGylated Cur-CDs in saline and PBS was evaluated via ζ potential measurements over a 7-day period (Fig. S11), which revealed excellent stability in both media, as anticipated. These results demonstrate the successful construction of a well-defined curcumin-derived nanomedicine featuring a nitrogen-doped graphitic carbon core and curcumin-inherited surface functionalities, providing a structural basis for subsequent biological investigations.

Fig. 1.

Characterization of Cur-CPDs. (A) UV-Vis-NIR spectrum and representative TEM image of Cur-CPDs. (B) Size distribution profile of Cur-CPDs. (C) FT-IR spectra of Curcumin and Cur-CPDs. (D) XPS survey spectra of Curcumin and Cur-CPDs. High-resolution XPS spectra in the C 1s (E), O 1s (F), and N 1s regions (G). (H) Atomic percentages of C, O, and N in Curcumin and Cur-CPDs based on XPS deconvolution. (I) Raman spectrum of Cur-CPDs.

2.2. Biodistribution and biocompatibility of Cur-CPDs

To investigate the in vivo biodistribution of Cur-CPDs, the carbonized polymer dots were covalently labeled with Cy5 to generate a near-infrared fluorescent probe (Cy5-Cur-CPDs). 6∼8-week-old C57BL/6 mice were intravenously injected with Cy5-Cur-CPDs via the tail vein, and near-infrared fluorescence imaging was performed at multiple time points (0, 1, 3, 12, 24, 48, and 72 h post-injection) to dynamically track their whole-body and organ-level distribution (Fig. 2A and B). Strong fluorescence signals were detected in the liver, spleen, lung, kidney, and heart shortly after administration. Ex vivo imaging of major organs revealed predominant accumulation in the liver and kidneys, indicating that Cur-CPDs are mainly cleared through hepatic and renal pathways. Notably, fluorescence signals in the heart became evident at approximately 1 h post-injection and gradually declined, becoming barely detectable by 48 h. By 72 h, fluorescence signals in all organs were nearly undetectable, suggesting rapid accumulation in cardiac tissues followed by efficient systemic clearance. After intraperitoneal administration, cardiac fluorescence was detectable as early as 1 h and persisted for up to 24 h, a temporal profile comparable to intravenous delivery yet indicative of robust and prolonged myocardial retention, a pharmacokinetic advantage unattainable with native curcumin given its rapid metabolic clearance (Fig. S12A and B). In addition, the pharmacokinetics of Cy5-Cur-CPDs revealed the fluorescence intensity peaked at approximately 0.5 h post-injection, with a subsequent gradual decline; notably, sustained fluorescence signal remained detectable at 8∼12 h and persisted above baseline until 72 h (Fig. S13).

Fig. 2.

Biodistribution and biocompatibility of Cur-CPDs. (A) Representative in vivo and ex vivo fluorescence images of major organs at indicated time points after intravenous administration of Cy5-Cur-CPDs. (B) Quantification of mean fluorescence intensity (MFI) in vivo. (C) Colocalization of Cy5-Cur-CPDs with lysosomes in H9C2 cells (scale bar, 100 μm). (D) H&E staining of heart, liver, spleen, lung, and kidney tissues from indicated groups (scale bar = 200 or 500 μm). (E-F) Serum levels of alanine aminotransferase (ALT) and creatinine (CRE) across groups. (G-J) CCK-8 assay evaluating cytotoxicity of curcumin or Cur-CPDs under normoxia (G-H) and hypoxia (I-J) at indicated concentrations. Data are presented as mean ± SD (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by one-way ANOVA.

To further assess the intracellular fate of Cur-CPDs, the subcellular localization of Cy5-Cur-CPDs was examined by fluorescence staining. Partial colocalization of Cy5-Cur-CPDs with lysosomes was observed at 1 h, whereas markedly reduced colocalization was detected at 4 h, indicating effective lysosomal escape of Cur-CPDs after cellular internalization (Fig. 2C). This property is favorable for maintaining the bioactivity of Cur-CPDs in cells.

Furthermore, a comprehensive evaluation of the biosafety of Cur-CPDs administration was conducted. To assess the in vivo toxicity of Cur-CPDs, healthy C57BL/6 mice were administered a single intraperitoneal dose of 5, 10, or 20 mg kg⁻¹. Histological analysis by hematoxylin and eosin (H&E) staining revealed no treatment-related toxicological changes in major organs, including the heart, liver, spleen, lung, and kidneys, following Cur-CPDs administration at various concentrations (Fig. 2D). Consistently, serum biochemical analysis showed no significant alterations in alanine aminotransferase (ALT) or creatinine (CRE) levels, indicating preserved liver and kidney function after different treatments (Fig. 2E and F). In vitro cytotoxicity was assessed using H9C2 cells under normoxic and hypoxic conditions. Cells were exposed to varying concentrations of Cur-CPDs or Curcumin (2, 5, 10, and 100 μg mL⁻¹), and cell viability was evaluated by CCK-8 assay. Cur-CPDs exhibited negligible cytotoxicity across all tested concentrations under normoxia, whereas curcumin induced a dose-dependent reduction in cell viability (Fig. 2G and H). Under hypoxic conditions, Cur-CPDs significantly improved the viability of injured H9C2 cells, while increasing concentrations of curcumin further exacerbated hypoxia-induced cell death (Fig. 2I and J). The above results demonstrate that Cur-CPDs exhibit favorable biodistribution characteristics, efficient clearance, effective lysosomal escape, and excellent biocompatibility. The safety and pharmacokinetic profiles support their suitability as a nanotherapeutic platform for myocardial infarction treatment.

2.3. Cur-CPDs alleviate cardiac injury and improve myocardial functional recovery after MI

The therapeutic efficacy of Cur-CPDs was evaluated in a murine model of myocardial infarction induced by permanent ligation of the left anterior descending (LAD) coronary artery. Following successful model establishment, mice received a 5-day therapeutic regimen with intraperitoneal injections of vehicle, curcumin, or Cur-CPDs every other day. Transthoracic echocardiography and invasive hemodynamic assessments were performed on day 5 post-MI to evaluate myocardial structure and function (Fig. 3A). Histopathological analysis by H&E staining revealed severe myocardial injury in the MI group, characterized by extensive inflammatory cell infiltration and disrupted myocardial architecture within the infarct region. Curcumin treatment exerted minimal protective effects, as inflammatory infiltration and structural disorganization remained prominent. In contrast, Cur-CPDs treatment markedly reduced inflammatory cell recruitment and preserved myocardial structural integrity (Fig. 3B). Consistently, serum biomarkers of cardiac injury, measured by ELISA, were significantly elevated following MI but were substantially attenuated by Cur-CPDs administration, whereas curcumin showed limited efficacy (Fig. 3C–E). These findings indicate that Cur-CPDs effectively mitigate acute ischemia-induced myocardial damage.

Fig. 3.

Cur-CPDs alleviate cardiac injury and improve functional recovery after MI. (A) Experimental flowchart: MI induction by LAD ligation and intraperitoneal injection every other day; samples collected on day 5. (B) H&E staining of heart tissue (Scale bar = 625 or 500 μm). Serum markers of cardiac injury: cTnI (C), CK-MB (D), and NT-proBNP (E). (F) Masson's trichrome staining of heart tissue (Scale bar = 625 or 500 μm). (G) Picrosirius Red staining of heart tissue sections showing collagen fibrils type I (yellow-orange) and type III (green) deposition (scale bar = 50 μm). (H) Representative echocardiography and quantification of LVEF, LVFS, and LVIDs. (I) Hemodynamic parameters: LVESP, +dP/dt max, and −dP/dt max. Data are mean ± SD (n = 3); *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; one-way ANOVA.

To further assess post-infarction pathological remodeling, Masson's trichrome staining was performed. MI hearts exhibited extensive collagen deposition, pronounced left ventricular wall thinning, and ischemic pallor in the infarct zone. Remarkably, Cur-CPDs treatment significantly reduced collagen accumulation and preserved ventricular wall thickness more effectively than free curcumin, indicating effective suppression of adverse ventricular remodeling (Fig. 3F). Quantitative analysis of Masson's trichrome-stained sections revealed a marked increase in collagen volume fraction (CVF) in the MI group relative to Sham controls. Notably, Cur-CPDs treatment conferred a significantly greater reduction in CVF than native curcumin (Fig. S14B). Picrosirius Red staining under polarized light further distinguished collagen subtype distribution among groups: the MI group exhibited predominant deposition of thick, densely packed type I collagen fibers (yellow-orange birefringence), whereas the Cur-CPDs group displayed a shift toward thin, loosely arranged type III collagen fibers (green birefringence), along with a tendency to a reduced collagen I/III ratio (Fig. 3G, S14C-E). This histological observation was corroborated by Western blot analysis of cardiac tissue, which demonstrated elevated type I collagen and reduced type III collagen in MI hearts, with Cur-CPDs treatment effectively reversing this imbalance by promoting type III collagen synthesis (Fig. S14F and S14G, S15). Collectively, these findings indicate that Cur-CPDs facilitate a favorable collagen remodeling process during post-MI repair, favoring the formation of cardioprotective, compliant tissue over rigid, scar collagen type I deposition, thereby preserving ventricular compliance and attenuating adverse cardiac remodeling. Functional recovery was further evaluated by echocardiography. Quantitative analysis demonstrated that Cur-CPDs treatment significantly improved left ventricular ejection fraction (LVEF) and fractional shortening (LVFS), while reducing left ventricular internal dimension at systole (LVIDs), compared with both untreated MI and curcumin-treated groups (Fig. 3H). These improvements indicate a robust restoration of systolic function in the early post-MI phase. To corroborate these findings, invasive hemodynamic measurements were performed. MI resulted in marked reductions in left ventricular end-systolic pressure (LVESP), maximal rates of pressure rise (+dP/dt max), and relaxation (−dP/dt max). Cur-CPDs treatment significantly reversed these impairments, yielding the most pronounced recovery of left ventricular contractile and diastolic performance among all groups (Fig. 3I). These results demonstrate that Cur-CPDs confer potent cardioprotection in the early phase of myocardial infarction by attenuating myocardial injury, suppressing adverse ventricular remodeling, and substantially improving cardiac functional recovery, outperforming native curcumin treatment.

2.4. Cur-CPDs suppress inflammatory pyroptosis and restore efferocytosis capacity

To delineate whether ROS suppression contributes to the cardioprotective efficacy of Cur-CPDs, we first evaluated myocardial ROS accumulation post-MI, given the pivotal role of ROS burst in initiating the inflammatory cascade. Free curcumin markedly attenuated oxidative stress and curtailed excess ROS production in the ischemic myocardium. Strikingly, Cur-CPDs exerted only modest ROS-inhibitory effects, with antioxidant capacity substantially inferior to that of the free counterpart (Fig. S16A). These findings effectively exclude ROS scavenging as the principal mechanism underlying Cur-CPDs-mediated cardioprotection. Consequently, we hypothesized that Cur-CPDs may confer myocardial protection through alternative pathways and subsequently interrogated inflammatory pyroptosis, a critical executor of MI pathology, as the putative target. We next examined the effects of Cur-CPDs on canonical pyroptosis signaling in infarcted hearts. Western blot analysis revealed that myocardial infarction markedly increased the expression of NLRP3 inflammasome components and downstream pyroptosis-related proteins, including cleaved Caspase-1, GSDMD-N, IL-1β, and IL-18. In contrast, Cur-CPDs treatment significantly suppressed the activation of this pyroptotic cascade, as evidenced by reduced expression of these key mediators (Fig. 4A–F, Fig. S17 and Fig. S18). Consistently, serum lactate dehydrogenase (LDH) levels, a surrogate marker of membrane rupture associated with pyroptotic cell death, were markedly elevated following MI but were significantly reduced by Cur-CPDs administration (Fig. 4G). In parallel, serum levels of pro-inflammatory cytokines, including IL-6, TNF-α, IL-1β, and IL-18, were markedly elevated in the MI group relative to Sham, whereas Cur-CPDs treatment significantly attenuated their expression compared with natural curcumin (Fig. S16B). Notably, the anti-inflammatory cytokine IL-10 was concurrently upregulated in the Cur-CPDs group, suggesting a shift toward an inflammation-resolving phenotype. These data collectively substantiate the potent anti-inflammatory efficacy of Cur-CPDs and corroborate pyroptosis suppression as the underlying mechanism of Cur-CPDs-mediated cardioprotection. As unresolved inflammation is closely linked to defective clearance of dying cells, we next investigated whether Cur-CPDs modulate efferocytosis, a central mechanism of inflammation resolution [26]. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining of ventricular sections revealed a marked accumulation of TUNEL⁺/DAPI⁺ cells after MI. While this finding is consistent with impaired efferocytic clearance, we acknowledge that TUNEL staining alone cannot distinguish between increased cardiomyocyte apoptosis and reduced apoptotic cell clearance. To dissect these possibilities, we simultaneously carried out F4/80 staining. Notably, the absolute number of macrophages (F4/80⁺/DAPI⁺ cells) serves as a proxy for phagocytic capacity, whereas the proportion of macrophages that have engulfed apoptotic cells (F4/80⁺/TUNEL⁺/DAPI⁺ triple-positive cells) directly indicates the efficiency of apoptotic cell recognition and clearance. Strikingly, Cur-CPDs treatment substantially reduced the abundance of TUNEL-positive apoptotic cells while concurrently enhancing their recognition by macrophages, without significantly altering overall phagocytic capacity (Fig. 4H–K). These findings collectively demonstrate that Cur-CPDs promote efferocytosis by augmenting apoptotic cell recognition rather than merely suppressing cardiomyocyte death, thereby facilitating post-infarction inflammation resolution. To further delineate the molecular basis of efferocytosis regulation, we assessed the expression of key efferocytosis-related molecules. Immunofluorescence staining demonstrated that Cur-CPDs selectively decreased the expression of the anti-phagocytic “don't-eat-me” signal CD47 in infarcted myocardium, whereas immunofluorescence staining of cardiac tissue sections revealed that the pro-efferocytic receptors MerTK and MFGE8 remained unaltered across MI and Cur-CPDs treatment groups, highlighting the selective modulation of CD47 by Cur-CPDs (Fig. 4L and Fig. S19). Western blot analysis confirmed these observations, showing a significant reduction in CD47 protein levels following Cur-CPDs treatment, with no detectable alterations in MerTK or MFGE8 expression (Fig. 4M, Figure S20, S21). Collectively, these results indicate that Cur-CPDs alleviate excessive post-infarction inflammation through a dual mechanism: suppression of inflammatory pyroptosis and restoration of defective efferocytosis. Notably, this effect is achieved predominantly by downregulating the anti-phagocytic checkpoint CD47, thereby favoring apoptotic cell clearance and promoting inflammation resolution in the infarcted heart.

Fig. 4.

Cur-CPDs suppress inflammatory pyroptosis and restore efferocytosis capacity. (A-F) Western blot and quantification of NLRP3, GSDMD-N, Caspase-1, IL-1β, and IL-18 in cardiac tissue. (G) Serum LDH (fold change) levels. (H-K) TUNEL/F4/80 staining of heart tissue (blue: nuclei; red: apoptotic cells; green: macrophages; Scale bar = 100 μm) and quantification. (L) Immunofluorescence of CD47 (red: CD47⁺ cells; blue: nuclei; Scale bar = 50 μm) and quantification. (M) Western blot and analysis of MerTK, MFGE8, and CD47 expression. Data are mean ± SD (n = 3); statistical significance by one-way ANOVA; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

2.5. Cur-CPDs reshape the transcriptomic and proteomic landscape in the infarcted heart and selectively modulate the NOTCH-1/CD47 axis

To gain insight into the biological functions and signaling pathways associated with Cur-CPDs-mediated cardioprotection, integrative multi-omics profiling was performed (Fig. 5A–D and Fig. S22A–D). Principal component analysis (PCA) of RNA sequencing revealed clear separation among the four experimental groups, with high intragroup consistency, indicating robust transcriptomic alterations induced by Cur-CPDs treatment (Fig. S22A). Differential gene expression analysis between the MI and MI + Cur-CPDs groups identified extensive transcriptional reprogramming. A total of 357 genes were significantly upregulated, and 643 genes were downregulated following Cur-CPDs treatment, as visualized by volcano plot analyses (Fig. S22B). These data indicate that Cur-CPDs exert broad regulatory effects on gene expression in the infarcted myocardium. Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) enrichment analyses of transcriptomic data revealed significant enrichment of pathways related to inflammation regulation, immune signaling, tissue repair, and cell death (Fig. 5A and B). Notably, the NOTCH, MAPK, and Wnt signaling pathways emerged as prominently enriched, all of which have been implicated in immune homeostasis and inflammatory responses after myocardial infarction. Strikingly, further annotation identified significant enrichment of terms associated with phagocytosis, apoptotic cell clearance, and CD47-related membrane recognition, directly implicating efferocytic machinery in the transcriptomic reprogramming induced by Cur-CPDs.

Fig. 5.

Cur-CPDs reshape the transcriptomic and proteomic landscape in the infarcted heart and selectively modulate the NOTCH-1/CD47 axis. Top 20 KEGG (A) and GO enrichment (B) analyses of filtered differentially expressed genes (DEGs). (C-D) Top 20 KEGG and GO enrichment analyses of filtered differentially expressed proteins (DEPs). (E) Western blot and quantification of WNT-1, P38 MAPK, p-P38 MAPK, and NOTCH-1 in heart tissue (β-tubulin as control) (n = 3). Data are mean ± SD; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; one-way ANOVA or Student's t-test.

To validate these transcriptomic findings at the functional level, quantitative proteomic analysis was performed on infarcted myocardial tissues. PCA demonstrated clear separation between experimental groups, indicating distinct proteomic profiles following Cur-CPDs treatment (Fig. S22C). Comparative analysis revealed substantial alterations in protein expression, with 100 proteins significantly upregulated and 66 proteins downregulated in response to Cur-CPDs treatment (Fig. S22D). Remarkably, KEGG and GO pathway analyses of the proteomic dataset converged on the same functional categories identified in the transcriptome, namely, immune regulation, inflammation, and cell death, with NOTCH, Wnt, and MAPK pathways again prominently enriched (Fig. 5C and D). This multi-omics convergence substantiates that Cur-CPDs coordinately reprogram inflammatory and immune-related gene networks at both transcriptional and translational levels.

To validate the omics findings and identify the dominant effector pathway, key components of the enriched signaling cascades were further examined by Western blot analysis (Fig. 5E, Figure S23, and Figure S24). Strikingly, Cur-CPDs treatment led to a significant upregulation of NOTCH-1 protein expression in infarcted hearts, whereas no appreciable changes were observed in representative components of the Wnt or MAPK pathways. These results demonstrate that Cur-CPDs selectively activate NOTCH-1 signaling rather than broadly stimulating multiple inflammatory pathways. Integration of the multi-omics datasets with protein validation data further identified the NOTCH-1/CD47 axis as the principal pathway underlying Cur-CPDs-mediated cardioprotection. Combined with the observed downregulation of the anti-phagocytic checkpoint CD47 and suppression of inflammatory pyroptosis, these multi-layered findings establish a NOTCH-1/CD47 regulatory axis as the selective molecular mechanism by which Cur-CPDs restore immune homeostasis and enhance efferocytic clearance during the acute inflammatory phase of myocardial infarction.

2.6. Disruption of the NOTCH-1/CD47 axis abolishes the therapeutic efficacy of Cur-CPDs in post-MI cardiac repair

To establish the functional necessity of the NOTCH-1/CD47 axis in Cur-CPDs-mediated cardioprotection, complementary loss-of-function and gain-of-function rescue experiments were performed (Fig. 6A). Pharmacological blockade of NOTCH-1 signaling by DAPT, as well as genetic hyperactivation of CD47 via adeno-associated virus-mediated cardiac-specific overexpression, markedly reversed the histological and functional benefits of Cur-CPDs, reinstating myocardial necrosis, inflammatory infiltration (H&E), collagen deposition (Masson's trichrome and picrosirius red staining), left ventricular systolic dysfunction, and myocardial injury markers to levels comparable to untreated MI mice (Fig. 6B–I, Figure S25, Figure S26).

Fig. 6.

Disruption of the NOTCH-1/CD47 axis abolishes the therapeutic efficacy of Cur-CPDs in post-MI cardiac repair. (A) Experimental schematic: Male C57BL/6 mice (6∼8 weeks old, 20 g) underwent LAD ligation-induced MI or sham operation on Day 1. Mice with AAV9-mediated cardiac CD47 overexpression received retro-orbital injection 3 weeks before MI. Beginning on Day 2, mice were randomized to receive intraperitoneal injections of DMSO + Cur-CPDs + DAPT (10 mg/kg, every other day) or DMSO + Cur-CPDs according to the groups. Echocardiography, hemodynamic monitoring, and tissue harvesting were performed on Day 5. (B) H&E staining of heart tissue (Scale bar = 625 or 500 μm). (C-E) Serum markers of cardiac injury. (F) Masson's trichrome staining of heart tissue (Scale bar = 625 or 500 μm). (G) Picrosirius Red staining of heart tissue sections (scale bar = 50 μm). (H) Representative echocardiography and quantification. (I) Representative hemodynamic parameters. Data are mean ± SD (n = 3); *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; one-way ANOVA.

To map the relationship between the NOTCH-1/CD47 axis and inflammation resolution, western blot analysis was applied to solve the hierarchical relationship within this axis. In MI mice receiving Cur-CPDs, NOTCH-1 was significantly upregulated, accompanied by downregulation of CD47 and inflammatory pyroptosis markers (NLRP3, GSDMD-N, Caspase-1, IL-18, and IL-1β). However, DAPT co-administration completely reversed these molecular alterations: NOTCH-1 upregulation was abolished, with concomitant reinstatement of CD47 expression and pyroptotic factors to MI levels (Fig. 7A–B, Fig. S27). Similarly, in AAV9-OE-CD47 mice, Cur-CPDs failed to suppress CD47 and pyroptosis markers, yet NOTCH-1 upregulation remained intact (Fig. 7C–D, Fig. S28). These reciprocal patterns demonstrate that NOTCH-1 operates upstream of CD47, with Cur-CPDs initiating anti-inflammatory cardioprotection through NOTCH-1-dependent CD47 downregulation. Concomitantly, serum levels of pro-inflammatory cytokines (IL-6, TNF-α, IL-1β, and IL-18) and pyroptosis marker LDH (Fig. 7I) were increased, and the anti-inflammatory cytokine IL-10 was suppressed in both rescue groups, mirroring the inflammatory profile of untreated MI mice (Fig. S29).

Fig. 7.

Disruption of the NOTCH-1/CD47 axis aggravates inflammatory pyroptosis and impairs efferocytosis capacity. (A-D) Western blot and quantification of NOTCH-1, CD47, NLRP3, GSDMD-N, Caspase-1, IL-1β, and IL-18 in cardiac tissue. (E-H) DAPI/TUNEL/F4/80 staining of heart tissue (blue: nuclei; red: apoptotic cells; green: macrophage; scale bar = 100 μm) and quantification. (I) Serum LDH levels. Data are mean ± SD (n = 3); statistical significance by one-way ANOVA or Student's t-test; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

In cardiac tissue sections, immunofluorescence staining of cardiac sections revealed that macrophage abundance remained comparable across all groups, indicating that basal phagocytic capacity was unaltered by either DAPT or CD47 overexpression. However, the proportion of efferocytic macrophages was significantly increased by Cur-CPDs, and this enhancement was abolished by both DAPT and AAV9-OE-CD47 interventions, reducing apoptotic cell clearance to baseline MI levels (Fig. 7E–H). Notably, the dissociation between unchanged macrophage numbers and impaired efferocytic efficiency directly implicates defective apoptotic cell recognition—rather than diminished phagocytic capacity—as the functional consequence of NOTCH-1/CD47 axis disruption. Collectively, these rescue results indicate that Cur-CPDs restore efferocytosis and thus attenuate inflammation in vivo through modulating the NOTCH-1/CD47 axis.

2.7. Cur-CPDs mitigate hypoxia-induced inflammation in H9C2 cells via the NOTCH-1/CD47 axis

Building upon the robust anti-inflammatory regulation properties of Cur-CPDs in vivo, we investigated their potential in a cell model of hypoxia that mimics the inflammatory microenvironment following MI. H9C2 cells cultured under normoxic conditions are called Group Con, while cells that have been hypoxic for 12 h are classified as Group H to better simulate the pathological progression of MI. Depending on the different treatment methods, they are further subdivided into Group Con + Cur-CPDs, Group H + Cur, and Group H + Cur-CPDs. Consistent with the in vivo ROS staining data, in vitro experiments further corroborated that Cur-CPDs confer potential cytoprotection through mechanisms independent of ROS suppression (Fig. S30). The Cur-CPDs treatment significantly reduced the TUNEL⁺ cell rate, suggesting its role in enhancing the capacity of efferocytosis (Fig. 8A and B). The anti-phagocytosis protein CD47 and inflammatory pyroptosis-related factors (NLRP3, Caspase-1, GSDMD-N, IL-18, and IL-1β) were increased in the H group, suggesting that a successful in vitro hypoxia model is established. As anticipated, the expression level of CD47 and pyroptosis-related indicators was significantly reduced after Cur-CPDs treatment. Meanwhile, the expression of NOTCH-1 is higher in hypoxic H9C2 cells treated with Cur-CPDs compared with the H or H + Cur group (Fig. 8C–J, Figure S31 and Figure S32). These results consistently confirm Cur-CPDs’ therapeutic potential to mitigate inflammatory pyroptosis, activate NOTCH-1, and inhibit CD47 in hypoxia-induced injury.

Fig. 8.

Cur-CPDs mitigate hypoxia-induced inflammation in H9C2 cells. (A-B) Representative TUNEL staining (red: apoptotic cells; blue: nuclei; Scale bar = 500 μm) and quantification. (C-J) Western blot and analysis of NOTCH-1, CD47, NLRP3, GSDMD-N, Caspase-1, IL-1β, and IL-18. Data are mean ± SD (n = 3); *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; one-way ANOVA.

To further evaluate the role of NOTCH-1 in anti-CD47 and anti-inflammation, we transfected H9C2 cells with NOTCH-1 siRNA or NOTCH-1 overexpressing lentivirus or CD47 overexpressing lentivirus. To clarify the effects of transfection, a Western blot assay was performed, and it turned out that the protein expression levels of NOTCH-1 and CD47 changed as expected (Figs. S33 and S35). After anaerobic treatment, we found that the apoptotic cell rate represented by positive TUNEL staining was effectively reduced in the NOTCH-1 overexpression cell group; conversely, the rate was obviously increased in H9C2 cells transfected with si-NOTCH-1 (Fig. 8A and B). Additionally, cells transferred with si-NOTCH-1 or OE-CD47 abolished the effects of anti-inflammation and efferocytosis restoration of Cur-CPDs treatment (Fig. 9A–Q, Figs. S33–S35), with the expression of markers of efferocytosis and inflammatory pyroptosis (CD47, NLRP3, GSDMD-N, Caspase-1, IL-1β, IL-18, and LDH) increased compared to the H + Cur-CPDs group. To assess whether Cur-CPDs enhance apoptotic cell recognition, macrophages were co-cultured with hypoxia-induced apoptotic cardiomyocytes. Fluorescence microscopy showed that Cur-CPDs increased the proportion of macrophages colocalized with apoptotic cells, indicating enhanced recognition (Fig. 9R–S). These in vitro findings are consistent with the in vivo rescue experiments, wherein NOTCH-1/CD47 axis disruption impaired efferocytic recognition, collectively supporting a unified mechanism by which Cur-CPDs promote apoptotic cell clearance through CD47-mediated recognition enhancement.

Fig. 9.

Cur-CPDs mitigate inflammation in H9C2 cells via the NOTCH-1/CD47 axis. Western blot and quantification in cells transfected with si-NOTCH-1 (A-H)/OE-CD47 (I-P). (Q) LDH release in vitro. (R-S) Immunofluorescence staining and quantification of RAW264.7 macrophages phagocytize hypoxia H9C2 cells (blue: H9C2 cells, red: RAW264.7 macrophages, scale bar = 500 μm). Data are mean ± SD (n = 3); *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; one-way ANOVA or Student's t-test.

Collectively, these results confirm that the protective and anti-inflammatory effects of Cur-CPDs are modulated through the NOTCH -1/CD47 signal pathway. By restoring efferocytosis, Cur-CPDs suppress inflammatory pyroptosis and promote inflammation resolution, thereby attenuating post-infarction cardiac injury.

2.8. Activated NOTCH-1 promotes lysosomal degradation of CD47

To elucidate the molecular mechanism by which NOTCH-1 suppresses CD47 expression, we next investigated CD47 protein stability in hypoxic H9C2 cells. Following inhibition of protein synthesis with cycloheximide, CD47 protein levels declined more rapidly in hypoxic cells than in NOTCH-1-overexpressing cells, suggesting enhanced CD47 degradation under these conditions (Fig. 10A, Figure S36 and Figure S37). As intracellular protein degradation primarily occurs via the ubiquitin-proteasome or autophagy-lysosome pathways, we next assessed the contribution of these systems. Treatment with the proteasome inhibitor MG132 did not significantly alter CD47 levels in either hypoxic or NOTCH-1-overexpressing cells (Fig. 10B, Figure S36 and Figure S37). In contrast, inhibition of the autophagy-lysosome pathway with 3-methyladenine (3-MA) markedly restored CD47 expression in both conditions (Fig. 10C, Figure S36 and Figure S37), indicating that NOTCH-1 facilitates CD47 degradation predominantly through a lysosomal pathway, consistent with the omics findings. To substantiate the proposed mechanism whereby NOTCH-1 activation promotes CD47 lysosomal degradation, we investigated CD47 trafficking and subcellular localization. Immunofluorescence staining was performed using antibodies against CD47 and LAMP-1, a well-established lysosomal marker. Quantitative colocalization analysis revealed reduced CD47/LAMP-1 overlap in the H group compared with normoxic controls, whereas Cur-CPDs treatment significantly increased their colocalization. This enhanced lysosomal targeting was abolished by siRNA-mediated NOTCH-1 knockdown or CD47 overexpression, indicating that Cur-CPDs promote CD47 lysosomal degradation in a NOTCH-1-dependent manner (Fig. 10D). These findings are consistent with CD47 protein levels across groups, supporting a model in which Cur-CPDs facilitate NOTCH-1-dependent CD47 internalization and subsequent lysosomal degradation. To confirm the physiological relevance of this mechanism, identical immunofluorescence analysis was performed on cardiac tissue sections from the murine MI model. The in vivo results mirrored the in vitro findings: CD47/LAMP-1 colocalization was diminished in MI hearts, restored by Cur-CPDs, and reversed by DAPT or AAV9-OE-CD47 (Fig. S38), establishing that NOTCH-1-driven CD47 lysosomal trafficking operates in the intact infarcted myocardium. To additionally explore the potential interaction between NOTCH-1 and CD47, molecular docking analysis was performed. Structural modeling revealed a stable binding interface between NOTCH-1 and CD47, with alanine at position 102 of NOTCH-1 interacting with glutamine at position 165 of CD47 (Fig. 10E). Consistently, co-immunoprecipitation assays in hypoxic H9C2 cells overexpressing NOTCH-1 confirmed an association between NOTCH-1 and CD47 (Fig. 10F). These findings demonstrate that Cur-CPDs activate NOTCH-1, which directly interacts with CD47 and accelerates its lysosomal degradation. This NOTCH-1-dependent removal of the anti-phagocytic checkpoint CD47 provides a mechanistic basis for enhanced efferocytosis and resolution of hypoxia-induced inflammation.

Fig. 10.

Activated NOTCH-1 promotes lysosomal degradation of CD47. (A) Western blot and quantification of CD47 stability under hypoxia ± CHX (100 μg/mL) in H9C2 cells or NOTCH-1 overexpression H9C2 cells (n = 3). Effects of MG132 (15 μM) (n = 4) (B) or 3-MA (5 mM) (n = 4) (C) on CD47 protein levels. (D) Immunofluorescence staining and quantification of CD47 and LAMP-1 in H9C2 cells (red: CD47; green: LAMP-1; scale bar = 500 μm) (n = 3). (E) Molecular docking of NOTCH-1 (green) with CD47 (brown), showing key interacting residues. (F) Co-immunoprecipitation of NOTCH-1 and CD47 (Input: total lysate; IP: anti-NOTCH-1 or IgG). Data are mean ± SD; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; one-way ANOVA.

3. Discussion

Excessive cardiomyocyte death and the ensuing uncontrolled inflammatory cascade following acute myocardial infarction are major determinants of adverse ventricular remodeling and poor clinical outcomes [17,18,34,57]. Although anti-inflammatory strategies have been intensively explored, their clinical translation remains limited due to systemic adverse effects and inconsistent efficacy, underscoring the need for alternative approaches that target endogenous inflammation-resolution programs rather than broadly suppressing immune responses [17].

Curcumin, a naturally derived polyphenol with well-documented anti-inflammatory activity, has been extensively investigated in cardiovascular diseases [38,58]. However, its clinical utility is severely constrained by poor aqueous solubility, low bioavailability, rapid metabolism, and pronounced dose dependency [59]. Recent advances in bioactive molecule-derived CPDs, an emerging class of zero-dimensional carbon-based nanomaterials, offer a promising strategy to overcome these limitations by improving solubility, stability, and pharmacokinetic behavior while preserving bioactivity [60]. In this study, the natural anti-inflammatory drug curcumin was transformed into pharmacologically active CPDs, enabling efficient anti-inflammatory intervention during the early inflammatory phase of MI. Importantly, Cur-CPDs restore efferocytosis by modulating the NOTCH-1/CD47 axis, thereby activating an endogenous inflammation-resolution pathway rather than relying on exogenous immunosuppression. This strategy may ultimately complement current reperfusion and pharmacological therapies.

We synthesized Cur-CPDs via an alkali-assisted carbonization strategy. Compared with native curcumin, Cur-CPDs exhibited superior aqueous dispersibility, nanoscale uniformity, and structural stability. Consistent with previous reports, the biological activity of CPDs is closely associated with particle size and homogeneous nanostructure [61], which facilitates cellular uptake and intracellular trafficking. Indeed, Cur-CPDs readily penetrated cell membranes, escaped lysosomal sequestration, and demonstrated enhanced intracellular stability. Biodistribution analysis further revealed improved accumulation and prolonged retention of Cur-CPDs in major organs, including the heart (myocardial retention exceeding 24 h), compared with curcumin and other nanodrugs reported in earlier studies [62,63]. Pharmacokinetic analysis revealed that plasma concentrations of Cur-CPDs peaked at 0.5 h post-intraperitoneal injection, declining to approximately 50% of peak levels by 8 h. This prolonged cardiac retention and slow terminal elimination contrast sharply with free curcumin, which exhibits rapid metabolic clearance (plasma half-life of minutes to hours) and negligible cardiac residence [[64], [65], [66], [67]]. Given that systemically administered drugs rarely accumulate efficiently in cardiac tissue, whereas local delivery strategies require invasive procedures, the favorable pharmacokinetics of Cur-CPDs represent a distinct translational advantage. Comprehensive in vivo and in vitro safety evaluations further confirmed their excellent biocompatibility.

It has been established in our previous work that CPDs derived from pharmaceutical agents can exhibit enhanced bioactivities compared to their parent compounds [68]. Based on these observations, we hypothesized that Cur-CPDs possess intrinsic immunomodulatory potential in MI. This hypothesis was validated through integrated in vivo and in vitro experiments. NLRP3 inflammasome-dependent pyroptosis has been recognized as a critical driver of post-MI inflammation and cardiac dysfunction [34,35]. Upon activation, the NLRP3 inflammasome triggers caspase-1-mediated cleavage of gasdermin D, leading to membrane pore formation and the release of pro-inflammatory cytokines IL-1β and IL-18 [69]. Consistent with previous reports, activation of the NLRP3/Caspase-1/GSDMD pathway was observed in MI hearts in this study [33,37]. Remarkably, Cur-CPDs effectively suppressed pyroptotic signaling, which was accompanied by reduced myocardial injury and improved cardiac function.

Efficient efferocytosis, the prompt clearance of apoptotic cells, is essential for limiting secondary necrosis, resolving inflammation, and initiating tissue repair [32,70]. Impaired efferocytosis has been widely documented in cardiovascular diseases, leading to the accumulation of dying cardiomyocytes, sustained inflammation, and adverse remodeling [[70], [71], [72]]. Our findings corroborate these observations, demonstrating defective efferocytosis during the acute phase of MI. Apoptotic cells regulate their clearance by balancing “eat-me” signals, such as phosphatidylserine recognized by MFGE8 and MerTK, and “don't-eat-me” signals, most notably CD47 [57,71,73,74]. Upregulation of CD47 on injured cardiomyocytes after MI has been shown to inhibit phagocytic clearance and exacerbate inflammation [[70], [71], [72]]. Therapeutic blockade of CD47 or its ligands restores efferocytosis, reduces infarct size, and improves cardiac function [70,[75], [76], [77]].

Cur-CPDs selectively downregulated CD47 expression without significantly altering MFGE8 or MerTK levels, distinguishing their mechanism from conventional approaches aimed at enhancing phagocytic receptors. By reducing the “don't-eat-me” signal on apoptotic cardiomyocytes, Cur-CPDs enhanced their recognition and subsequent engulfment by macrophages, thereby augmenting local efferocytic capacity and driving inflammation resolution. Compared with free curcumin, Cur-CPDs more effectively restored local efferocytosis, thereby interrupting the vicious cycle between persistent inflammation and myocardial injury in the acute post-MI phase. These findings position Cur-CPDs as a novel endogenous anti-inflammatory strategy centered on efferocytosis restoration rather than broad immune suppression. Whether this approach confers long-term benefits in chronic heart failure with heightened inflammatory burden warrants further investigation.

To further elucidate the molecular basis underlying the anti-inflammatory effects of Cur-CPDs, integrated transcriptomic and proteomic profiling was performed. These analyses identified multiple enriched pathways, including NOTCH, Wnt, and MAPK signaling cascades, all of which have been implicated in post-MI inflammation and tissue repair [12]. NOTCH-1 was prioritized for mechanistic validation based on the following considerations. First, NOTCH-1 exhibited the most robust enrichment across both omics layers, with consistent upregulation at the transcript and protein levels in Cur-CPDs-treated hearts. This temporal induction aligns with the established post-MI expression pattern of NOTCH-1, which peaks around day 4 after infarction [12], precisely coinciding with our acute-phase sampling window. Western blot validation showed that representative Wnt and MAPK components remained unaltered at the protein level in Cur-CPDs-treated hearts, whereas NOTCH-1 exhibited consistent upregulation. The evolutionarily conserved NOTCH signaling pathway plays a pivotal role in diverse cellular processes, including proliferation, differentiation, and tissue repair [78]. Accumulating evidence indicates that activation of NOTCH-1 confers cardioprotection by limiting myocardial injury and improving cardiac function [79,80], as well as by promoting angiogenesis [81,82] and attenuating fibrotic remodeling [79,83]. Beyond its established role in cardiac repair, NOTCH-1 has also been implicated in the regulation of efferocytosis. Hee Ja Kim and colleagues demonstrated that NOTCH-1 activation maintains optimal efferocytic capacity while suppressing pathological cell migration and invasion [82], suggesting that the cardioprotective effects of NOTCH-1 may be partially mediated through enhanced apoptotic cell clearance and modulating immunoregulatory function. This functional linkage provided the rationale for investigating whether Cur-CPDs-induced NOTCH-1 upregulation drives the observed enhancement of macrophage-mediated clearance in our model. In line with these scientific hypotheses, complementary loss-of-function experiments demonstrated that both pharmacological inhibition of NOTCH-1 by DAPT in vivo and siRNA-mediated NOTCH-1 knockdown in vitro abolished the protective effects of Cur-CPDs, upregulating CD47 expression, impairing efferocytic clearance, and aggravating pyroptosis. Interestingly, CD47 overexpression reversed Cur-CPDs-induced benefits despite ongoing NOTCH-1 activation by Cur-CPDs, establishing that CD47 downregulation is the obligatory downstream step for NOTCH-1-mediated efferocytosis restoration and inflammation resolution. However, this prioritization does not exclude potential contributions from Wnt or MAPK pathways. The Mertk/MAPK axis participates in inflammatory responses and vascular remodeling in injury settings [[84], [85], [86]], suggesting that MAPK signaling may contribute to post-MI tissue repair beyond the acute efferocytosis phase. Furthermore, NOTCH-Wnt crosstalk has been implicated in efferocytosis-dependent tissue remodeling and fibroblast reprogramming [87]. In atherosclerosis models, Wnt/β-catenin activation promotes macrophage lipid accumulation and impairs apoptotic cell clearance [88]. By analogy, Cur-CPDs-mediated NOTCH-1 activation may similarly modulate Wnt signaling dynamics to influence cardiac fibroblast behavior and scar maturation. Whether MAPK and Wnt pathways act in concert with or parallel to the NOTCH-1/CD47 axis during later-stage remodeling, and whether such crosstalk influences long-term cardiac remodeling beyond the acute window examined here, warrants future investigation.

Mechanistically, activated NOTCH-1 promoted efferocytosis by regulating CD47 protein stability via the autophagy-lysosome pathway. Quantitative colocalization analysis revealed that Cur-CPDs treatment significantly increased CD47/LAMP-1 overlap in hypoxic cardiomyocytes and infarcted myocardium alike, indicating enhanced trafficking of CD47 to lysosomes. This effect was abolished by NOTCH-1 inhibition or CD47 overexpression, confirming that NOTCH-1 activation drives CD47 internalization and subsequent lysosomal degradation. Consequently, membrane-resident CD47 was rapidly depleted, attenuating the “don't-eat-me” signal presented to macrophages, facilitating efficient recognition and engulfment of apoptotic cells, and limiting secondary inflammatory amplification. By coupling restoration of efferocytosis with suppression of pyroptosis, Cur-CPDs reprogram the post-inflammatory milieu toward resolution rather than persistence. Collectively, this study presents a curcumin-derived nanomedicine with favorable cardiac retention that selectively activates the NOTCH-1/CD47 axis to coordinate efferocytosis enhancement and pyroptosis inhibition, offering a mechanistically distinct anti-inflammatory strategy for early-stage myocardial infarction.

Several therapeutic strategies have been explored for ligation-induced MI, including broad anti-inflammatory agents, CD47 pathway blockade, macrophage reprogramming, efferocytosis-enhancing drugs, and nanomedicine-based delivery systems. However, these approaches are limited by systemic immunosuppression [89], disruption of hematologic homeostasis [72], non-specific manipulation of macrophage biology [90], significant off-target side effects, or poor cardiac targeting efficiency with rapid clearance [91]. Cur-CPDs offer a mechanistically distinct profile: by activating NOTCH-1 signaling, they suppress CD47 expression through lysosomal degradation, thereby enhancing recognition and clearance of apoptotic cardiomyocytes without directly depleting systemic CD47 or broadly manipulating macrophage function. This upstream restoration of efferocytosis prevents debris-driven NLRP3 inflammasome activation and subsequent pyroptosis, positioning Cur-CPDs as a multi-mechanistic, cardiac-targeted, endogenous-modulating platform with favorable biocompatibility and translational potential.

It is also worth noting that, during the pyrolysis synthesis, a substantial fraction of the original curcumin functional groups is preserved on the surface of the Cur-CPDs. These retained pharmacologically active moieties serve as the direct molecular basis for the bioactivity, engaging the NOTCH-1/CD47 pathway and mediating the therapeutic effects observed in the present study. The carbonized cores, combined with PEGylation, endow Cur-CPDs with superior pharmacokinetic properties that are unattainable by free curcumin. Specifically, the nanocarrier architecture enables prolonged systemic circulation and sustained cardiac retention. These properties ensure that the retained pharmacologically active moieties reach the target tissue at therapeutically relevant concentrations over a clinically meaningful time window.

Despite these promising findings, several limitations warrant consideration. While intraperitoneal injection was used in this study to establish proof-of-concept efficacy and pharmacokinetics, intravenous or intracoronary delivery would be more appropriate for clinical translation in acute MI. Intravenous administration offers rapid systemic availability during the critical reperfusion window, whereas intracoronary infusion during percutaneous coronary intervention provides targeted myocardial accumulation with reduced off-target exposure. Post-reperfusion local injection or sustained-release formulations may also be explored to prolong therapeutic effects. These alternative delivery strategies, together with long-term safety assessment in large animal models, will be systematically evaluated in future studies to facilitate clinical development of Cur-CPDs. Additionally, this study primarily interrogated the acute inflammatory phase within the first 5 days post-MI, a window during which efferocytosis and pyroptosis are most dynamically regulated. The long-term consequences of Cur-CPDs treatment on ventricular remodeling, fibrosis progression, and the eventual development of heart failure remain to be determined. Future studies employing chronic post-MI surveillance models will be necessary to evaluate whether the acute benefits of enhanced efferocytosis translate into sustained functional recovery and attenuated adverse remodeling. Moreover, further optimization of the nanostructure may extend myocardial residence time and facilitate scalable production. Future studies incorporating gene-edited MI models and advanced synthesis strategies will be essential to comprehensively evaluate the therapeutic potential of Cur-CPDs in post-infarction cardiac repair.

4. Conclusion

In summary, this study demonstrates that carbonized polymer dots from the natural anti-inflammatory drug curcumin (Cur-CPDs) are more effective than native curcumin in promoting inflammation resolution during the early phase of myocardial infarction by modulating the NOTCH-1/CD47-mediated efferocytosis pathway. By enhancing efferocytosis and suppressing inflammatory pyroptosis, Cur-CPDs reprogram the post-infarction inflammatory microenvironment toward resolution of inflammation. Collectively, these findings establish Cur-CPDs as a mechanistically defined and rapidly acting anti-inflammatory nanotherapeutic with significant translational potential for acute myocardial infarction.

CRediT authorship contribution statement

Yue Yang: Conceptualization, Investigation, Methodology, Validation, Writing – original draft. Weiwei Chen: Conceptualization, Investigation, Methodology, Validation. Huiling Luo: Investigation, Methodology, Validation. Zhixi Yu: Investigation, Methodology, Validation. Weisi Yin: Investigation, Methodology, Validation. Xingtong Wang: Methodology. Huan Wang: Conceptualization, Funding acquisition, Supervision, Writing – review & editing. Beibei Du: Funding acquisition, Supervision, Writing – review & editing. Yuquan He: Supervision. Ping Yang: Funding acquisition, Supervision, Writing – review & editing.

Funding sources

This study was supported by the National Natural Science Foundation of China [82470400, 22475210].

Declaration of competing interests

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:

Data availability

Data will be made available on request.