Prussian blue nanoparticles targeting multiple PANoptosome-mediated PANoptosis for myocardial ischemia-reperfusion injury therapy

Domain: chemistry
Published: 2026-02-27
Editorial outcome: major_revision

Abstract
The extensive crosstalk among pyroptosis, apoptosis, and necroptosis limits the efficacy of therapies targeting only one pathway. Here, we show that Prussian blue (PB) nanoparticles act as multi-target PANoptosis inhibitors by binding key PANoptosome components including RIPK1, ZBP1, and AIM2 through multimodal interactions, thereby concurrently suppressing pyroptosis, apoptosis, and necroptosis in myocardial ischemia-reperfusion injury (MIRI). Platelet membrane-coated PB nanoparticles (PB@PM) exhibit enhanced cardiac targeting and efficiently alleviate MIRI-induced cardiac dysfunction, adverse ventricular remodeling, and cardiomyocyte hypertrophy. Mechanistically, PB@PM disrupt PANoptosome assembly, scavenge reactive oxygen species, improve mitochondrial function, and restore immune-inflammatory homeostasis. By integrating single nucleus transcriptomics of human heart samples, molecular dynamics simulations, transcriptomics, medical imaging, and molecular validation, we systematically decipher the therapeutic mechanisms of PB-based PANoptosis inhibition. This study establishes an integrative multi-omics framework for exploring PANoptosis in cardiovascular diseases and provides a promising nanotherapeutic strategy for MIRI treatment.

Introduction
Innate immunity constitutes the first-line defense against pathogenic insults and cellular stress, wherein pattern recognition receptors orchestrate inflammatory responses and programmed cell death through detection of pathogen- and damage-associated molecular patterns1. Emerging evidence from genetic, molecular, and biochemical studies reveals complex interconnectivity between cell death pathways, reshaping our understanding of inflammasomes and cell death complexes in diseases pathogenesis2–4. Notably, mono-therapeutic targeting of individual pyroptotic, apoptotic, or necroptotic components often proves inadequate due to molecular redundancy5–7. The recent conceptual breakthrough of PANoptosis have unveiled an inflammation-driven programmed cell death modality governed by multilayered PANoptosome complexes. This pathophysiological process uniquely integrates molecular components from classical death pathways, with PANoptosome assembly serving as its biochemical hallmark. Extensive interactions between key molecules of pyroptosis, apoptosis, and necroptosis are the basis for PANoptosome assembly2,8. Currently, Z-DNA-binding protein 1 (ZBP1), receptor-interacting protein kinase 1 (RIPK1), absent in melanoma 2 (AIM2), and NLR family pyrin domain-containing 12 (NLRP12) PANoptosomes have been molecularly characterized3,8–13. Notably, previous studies have primarily explored the intrinsic mechanisms of PANoptosis in cancer, infectious, and inflammatory diseases.

Acute myocardial infarction persists as the global leading cause of mortality. Prompt revascularization constitutes the cornerstone for mitigating myocardial damage and optimizing long-term prognosis. However, revascularization can cause myocardial cell death and exacerbate post-ischemic myocardial injury, a phenomenon termed myocardial ischemia-reperfusion injury (MIRI), which frequently culminates in cardiac dysfunction, arrhythmia, and heart failure14,15. The pathophysiological complexity of MIRI stems from multifactorial mechanisms encompassing oxidative stress, calcium dysregulation, metabolic disturbances, and dysregulated immune-inflammatory activation16. Notably, ischemia-triggered apoptotic cascades critically dictate myocardial injury severity and propel maladaptive cardiac remodeling17,18. Given the intrinsic linkage between programmed cell death and inflammation, pyroptosis emerges as a key mediator of MIRI-related inflammatory response that potentiates detrimental ventricular remodeling19. Concurrently, robust necroptosis activity accompanies MIRI progression20,21. Despite these insights, the therapeutic potential of PANoptosis modulation in cardiovascular pathologies, particularly MIRI, remains underexplored and clinically untapped.

PANoptosis represents a pivotal therapeutic target against diseases, and its biological effects cannot be explained solely by pyroptosis, apoptosis, or necroptosis8. The intricate molecular interplay among these death pathways predisposes single-pathway inhibition strategies to compensatory pathway activation, ultimately diminishing therapeutic effectiveness. Consequently, monotherapeutic targeting of individual molecular components fails to suppress PANoptosis22,23. Experimental evidence demonstrates that pharmacological blockade of terminal effectors—including mixed lineage kinase domain-like pseudokinase (MLKL), caspase-1, gasdermin D (GSDMD), or caspase 3/7—achieves limited efficacy in curtailing overall cell death24,25. Current investigative efforts focus on multipronged strategies targeting upstream initiators, signaling hubs (RIPK1/3), protease networks, and PANoptosome complexes8,26. Nevertheless, most available inhibitors exhibit narrow target specificity, rendering them inadequate for addressing PANoptosis’ pathway interconnectivity. This underscores the urgent need for novel therapeutic entities capable of simultaneous pathway modulation to disrupt the crosstalk among pyroptosis, apoptosis, and necroptosis.

In this study, we establish Prussian blue (PB) nanoparticles as PANoptosis inhibitors capable of concurrently targeting multiple PANoptosomes, effectively disrupting the molecular crosstalk among pyroptosis, apoptosis, and necroptosis to combat PANoptosis-driven pathologies (Fig. 1). Integrating single-nucleus transcriptomics of human cardiac specimens with prior experimental evidence, we validated MIRI as a representative PANoptosis-associated disease model. Molecular dynamics simulations revealed high-affinity binding between the constructed PB and core PANoptosome components RIPK1, ZBP1, and AIM2. To improve targeted delivery, we engineered platelet membrane-modified PB (PB@PM), which demonstrated efficient mitigation of MIRI-induced cardiac dysfunction and pathological ventricular remodeling, enhanced extracellular matrix regeneration and functional neovascularization, and robust suppression of cardiomyocyte hypertrophy. Mechanistic studies revealed that PB@PM modulated mitochondrial metabolism and restored immune-inflammatory balance, thereby disrupting PANoptotic circuitry. Bulk transcriptomics combined with multiplex immunoblotting confirmed PB@PM’s pan-inhibitory effects on PANoptosome activation, conclusively establishing its therapeutic efficacy against MIRI-associated PANoptosis. This study highlights a promising therapeutic strategy for MIRI and offers valuable information for developing clinical therapies for innate immunity and inflammation.

Results
MIRI as a representative PANoptosis-related disease model
Emerging evidence demonstrates that ischemic stress induces mitochondrial genomic instability in cardiomyocytes, culminating in mitochondrial DNA (mtDNA) leakage that potently activates the STING pathway and propagates pro-inflammatory cascades27–30. Notably, mtDNA-STING axis activation serves as a master regulator of PANoptosis via AIM2/ZBP1-PANoptosome induction2,31,32. Concurrently, RIPK1 hyperactivation in MIRI pathogenesis is mechanistically linked to mitochondrial dysfunction, ZBP1 signaling, and TNF-α-mediated inflammation21,33–35. This compelling evidence positions MIRI as a paradigm of PANoptosis-driven cardiovascular pathology. To clinically validate PANoptosis targeting potential, we analyzed a single-nucleus transcriptomic dataset from human specimens.

We performed comparative analysis of non-transplanted donor heart (control group, Con) versus necrotic core (ischemic zone, IZ) specimens from acute myocardial infarction patients (Fig. 2A). Single-cell clustering was visualized through uniform manifold approximation and projection (UMAP), t-distributed stochastic neighborhood embedding analysis, and principal component analysis (PCA), with cell type annotation based on canonical marker gene expression. Thirteen distinct cell populations were identified: adipocytes, B cells, cardiomyocytes, doublets, endothelial cells, fibroblasts, mast cells, megakaryocytes, mononuclear phagocytes, mural cells, plasma cells, Schwann cells, and T cells (Fig. 2B and Supplementary Fig. 1A). Global cellularity was markedly reduced in IZ specimens, most pronounced in cardiomyocytes (Fig. 2B and Supplementary Fig. 1A). Subclustering analysis resolved cardiomyocytes into five subtypes, revealing substantial depletion of subclusters 1, 2, 4, and 5 in the IZ group (Fig. 2C and Supplementary Fig. 1B). PANoptosis activation signature was mapped at single-cell resolution across all cardiac cells and subtypes. Quantitative analysis revealed cell type-specific PANoptosis reprogramming, with cardiomyocytes exhibiting the most profound pro-PANoptotic shift (Fig. 2D, E). UMAP plots and box-type statistical plots were also generated for pyroptosis, apoptosis, and necroptosis. Comparative death modality profiling demonstrated dominant apoptotic/necroptotic activation in bulk cardiac cells during acute myocardial infarction onset, and concurrent amplification of pyroptosis, apoptosis, and necroptosis specifically in IZ cardiomyocytes (Supplementary Fig. 2). Intuitively, the expression levels of multiple PANoptosis-related genes were markedly upregulated in both the overall cell population and the cardiomyocytes of the IZ group (Fig. 2F and Supplementary Figs. 3–5).

Focusing on cardiomyocyte functional heterogeneity, we quantified PANoptosis activation across subtypes. Strikingly, subclusters 1/2 were nearly eradicated in IZ specimens, while subcluster 4 exhibited severe depletion (Fig. 2G–K). Residual subclusters 3-5 displayed elevated PANoptosis-related gene expression (Fig. 2H–K), and apoptosis-dominant death modality (Supplementary Fig. 6). These findings indicate that during myocardial ischemic infarction, cardiomyocytes do not exhibit uniform activity, with subclusters 1 and 2 being the most susceptible to death, followed by subcluster 4, and subcluster 3 being the most resistant to the post-ischemic microenvironment. Collectively, the single-cell atlas establishes PANoptosis as a spatially regulated therapeutic target for MIRI intervention.

Computational simulation of PB interaction with PANoptosis-related proteins
PB is an antidote approved by the US Food and Drug Administration for the clinical treatment of thallium poisoning. Previous studies reported that when its size was at the nanometer level, PB showed various biological activities, including inhibiting cell apoptosis, pyroptosis, or necroptosis36–38. PBs with multi-target effects on cell death are expected to play an important role in PANoptosis. However, their role in PANoptosis remains unclear.

Molecular dynamics simulations were firstly conducted to elucidate interactions between PB and PANoptosome core components. Mechanistically, PANoptosome assembly initiates upon sensing diverse pathogenic stimuli, orchestrating PANoptosis execution. Currently, ZBP1, RIPK1, and AIM2 PANoptosomes have undergone molecular characterization8. The components of the ZBP1 PANoptosome include ZBP1, RIPK3, caspase 1/6/8, apoptosis-associated speck-like protein containing a CARD (ASC), nucleotide-binding oligomeric domain-like receptor protein 3 (NLRP3), and RIPK1. RIPK1 PANoptosome consists of RIPK1, Fas-associated death domain protein (FADD), caspase 1/8, ASC, NLRP3, and RIPK3. AIM2 regulates the innate immune sensors Pyrin and ZBP1 and forms PANoptosomes with ASC, caspase-1/8, RIPK1/3, and FADD (Fig. 1). These three hubs were selected as representative therapeutic targets due to their pivotal role in cross-regulating pyroptotic, apoptotic, and necroptotic pathways.

System validation confirmed simulation robustness via stable root mean square deviation (RMSD) equilibration in PB-RIPK1/ZBP1/AIM2 systems (Supplementary Fig. 7). For AIM2 systems, dual simulations probing PB interactions with distinct functional domains (HIN and PYD) were independently performed. The simulation results of the C-terminal HIN domain on the AIM2 structure (AIM2HIN) were shown in Fig. 3A. As known as the binding of this region with dsDNA releases AIM2 from its inhibitor state, so that the helix interaction of the PYD domain (AIM2PYD) can further bind with ASC39. Here, PB evidently interfered with the conformation of the AIM2HIN domain. Analysis of the interaction with the PB interface revealed that the contact residues were mainly concentrated on the charged Arg and Lys residues (Fig. 3B, C). The interactions between different residues and PB also indicated that Lys and Arg play important roles in PB adhesion to AIM2HIN (Fig. 3D, E and Supplementary Movie 1). The total interaction energy between the AIM2HIN structure and PB reached -267.4 kJ/mol, which mainly refers to the electrostatic force (Fig. 3F). However, PB did not have a strong conformational effect on the PYD domain in AIM2 (Supplementary Fig. 8 and Supplementary Movie 2), which may be related to the high stability of the helical structure.

Comparative analysis of PB-ZBP1 interactions revealed substantially weaker binding affinity compared to PB-AIMHIN complexes (Fig. 3G and Supplementary Movie 3). Electrostatic forces dominated at critical interfacial residues Arg135/Lys138, serving as primary anchoring points for PB surface adhesion (Fig. 3H). Energy quantification demonstrated moderate binding strength (-157.9 kJ/mol) during the equilibrium phase (Fig. 3I), while radius of gyration (Rg) trajectory analysis indicated ZBP1 structural compaction (Supplementary Fig. 7C). This compression phenotype likely disrupts Zα domain-mediated dsDNA recognition, compromising ZBP1’s DNA-sensing capacity.

We further conducted the conformational effects of RIPK1 in an environment containing PB. The PB-RIPK1 interaction analysis showed that PB tightly binds to RIPK1, primarily through interaction with C-terminal residues of RIPK1 (Fig. 3J and Supplementary Movie 4). A detailed residue-level analysis revealed that the binding sites between PB and RIPK1 are predominantly located within a turn region of the helical domain in the N-terminal segment, involving key residues such as Glu98, Met99, Pro102, Leu103, Thr244, Tyr246, Ser282, and Gln283 (Fig. 3K). The overall interaction energy was calculated to be −73.2 kJ/mol, with van der Waals interactions being the dominant force, particularly the strong interaction between Tyr and PB (Fig. 3L). This intimate binding of PB to RIPK1 may markedly modulate RIPK1-mediated inflammatory signaling and cell death regulation40–43. In conclusion, the molecular dynamics simulation results elucidate the regulatory role of PB in the pathological progression of PANoptosis, providing a therapeutic strategy for the early intervention and treatment of PANoptosis-related diseases.

Construction of PB@PM for targeted treatment of MIRI in vivo
Leveraging molecular dynamics simulation data and biomedical nanotechnology requirements, we synthesized PBs through single-precursor-mediated one-step hydrothermal synthesis. Polyvinylpyrrolidone was incorporated during nanoparticle synthesis as both reducing agent and dispersion stabilizer, endowing the nanostructures with exceptional colloidal stability in physiological media-a critical parameter for clinical translation. Scanning electron microscopy and transmission electron microscopy (TEM) revealed the morphology, size, and uniformity of the nanoparticles (Fig. 4A, B). Comprehensive material characterization through elemental mapping, X-ray diffraction, X-ray photoelectron spectroscopy, ultraviolet-visible-near infrared spectroscopy, and Fourier-transform infrared spectroscopy confirmed successful construction of PB (Fig. 4C and Supplementary Fig. 9).

To endow PB with the ability to target PANoptosis-related disease sites, we encapsulated PB using platelet membranes (PM) derived from wild-type mice to construct PB@PM. The results of TEM, western blotting, fluorescence colocalization, dynamic light scattering, and zeta potential demonstrated the successful construction of PB@PM (Fig. 4D–F and Supplementary Fig. 10). Furthermore, we systematically investigated the myocardial targeting efficacy and biodistribution profile of PB@PM in vivo. Fluorescence imaging analysis was performed on major organs harvested from MIRI mice at 12 and 24 h post-injection of Cy5.5-labeled PB or PB@PM, with sham mice receiving Cy5.5-labeled PB serving as the control group. Quantitative analysis revealed that PB@PM exhibited substantially elevated fluorescence intensities within the ischemic myocardial region compared to both PB-treated groups at both time points (Fig. 4G–I). Notably, there were no observable differences in the fluorescence intensities of the livers, spleens, lungs, and kidneys across all experimental groups. These results indicated that PB@PM exhibits superior myocardial targeting efficacy with sustained retention in infarcted cardiac tissue exceeding 24 h. The biosafety profile of PB@PM was rigorously assessed. Comprehensive hematological analysis and histopathological examination via H&E staining revealed no visible histopathological alterations in major organs or aberrations in hematological/biochemical parameters following PB@PM administration (Supplementary Fig. 11 and Supplementary Table 2). This targeted delivery system addresses critical limitations in current MIRI therapeutics and establishes a robust foundation for translational applications in PANoptosis-related pathologies.

Therapeutic efficacy of PB@PM in MIRI
Subsequently, we evaluated the potential PANoptosis-inhibitory effects of PB@PM in MIRI. Serial echocardiographic assessments were conducted at preoperative baseline and postoperative days 1, 14, and 28 to quantify cardiac functional parameters, including the left ventricular ejection fraction (LVEF), fraction shortening (FS), LV end-diastolic volume, and LV end-systolic volume. Preoperative echocardiography confirmed the absence of preexisting cardiac conditions in all murine subjects. While no statistically significant intergroup differences in LVEF and LVFS were observed at postoperative day 1, all experimental groups exhibited marked reductions compared to baseline values, confirming MIRI-induced cardiac dysfunction (Fig. 5A–C). Notably, PB@PM treatment showed superior therapeutic efficacy versus PB treatment at postoperative days 14 and 28, as evidenced by improved echocardiographic parameters (Fig. 5A–E).

Subsequent to the 28-day postoperative period, histopathological assessments were conducted on murine cardiac tissues. Infarct size quantification and collagen deposition analysis were performed utilizing Masson’s trichrome and H&E staining methodologies. The MIRI cohort exhibited pronounced myocardial atrophy, ventricular wall thinning, and excessive collagen fiber accumulation within the left ventricular wall. Notably, PB@PM administration demonstrated conspicuous attenuation of MIRI-induced cardiac dysfunction and fibrotic remodeling (Fig. 5F, G). In acute phase evaluations, dual staining protocols employing Evans blue and 2,3,5-triphenyltetrazolium chloride (TTC) were implemented for infarct characterization. Histological analysis revealed distinct colorimetric demarcation: blue-stained regions indicated viable left ventricular tissue, white-colored areas represented infarct zones (IF), while TTC-positive regions designated the area at risk (AAR)44. Quantitative assessment demonstrated a progressive reduction in the infarct-to-risk area ratio (IF/AAR) across MIRI, MIRI+PB, and MIRI+PB@PM treatment groups (Fig. 5H). These results collectively substantiate the potential therapeutic efficacy of PB@PM in mitigating post-ischemic myocardial injury progression.

Apoptotic cell death analysis through terminal deoxynucleotide transferase-mediated dUTP nick end labeling (TUNEL) staining revealed that PB@PM markedly decreased the number of TUNEL+ cardiomyocytes within the infarct margin area (Fig. 5I, J). On the third postoperative day, immunofluorescence analysis revealed that PB@PM treatment demonstrated a marked reduction in CD86+ M1 macrophages and Ly6G+ neutrophils recruitment induced by MIRI (Fig. 5K–N). Furthermore, quantitative analysis of macrophage polarization markers indicated that pro-inflammatory M1 markers (TNF-α, IL-1β, and IL-6) were distinctly downregulated, while anti-inflammatory M2 markers (IL-10 and TGF-β) exhibited marked upregulation in both LPS-stimulated RAW264.7 macrophage cultures and ischemic myocardial tissues following PB@PM administration (Supplementary Figs. 12 and 13). These results suggest that PB@PM effectively regulates the immune microenvironment after MIRI.

Post-MIRI cardiac repair unfolds through a coordinated cascade of biological processes, wherein extracellular matrix (ECM) remodeling constitutes a critical pathological determinant. Efficient repair depends on the transformation of fibroblasts into myofibroblasts, which produce large amounts of ECM proteins and facilitate wound contraction45. Collagens are the most abundant ECM proteins in the LV. Following MIRI, collagen increases in the peri-infarct region, and types I and III are the major components of the myocardium scar46,47. Quantitative fluorescence analysis revealed the highest expression levels of collagen I and III, alpha-smooth muscle actin (α-SMA)+ myofibroblasts, and CD31+ vascular endothelial cells in the peri-infarct zone of PB@PM-treated MIRI mice across the three different treated groups (Fig. 5O–R and Supplementary Figs. 14 and 15). During the reparative phase, robust angiogenesis coupled with enhanced α-SMA in infarct and peri-infarct territories facilitates the development of organized granulation tissue, characterized by increased collagen density, aligned stress fibers, and augmented mechanical strength. This structural reinforcement maintains ventricular integrity while preventing adverse remodeling manifestations including infarct expansion and ventricular rupture48,49. Morphometric analysis further demonstrated that PB@PM treatment markedly attenuated pathological myocardial cell hypertrophy compared to MIRI controls (Supplementary Fig. 16). Collectively, these experimental findings substantiate that PB@PM confers therapeutic benefits in MIRI management through synergistic mechanisms involving ECM remodeling enhancement, angiogenic activation, and suppression of maladaptive myocardial hypertrophy.

PB@PM comprehensively blocks the crosstalk between pyroptosis, apoptosis, and necroptosis
To elucidate the molecular mechanisms underlying PB-mediated suppression of PANoptosis in vivo, comprehensive transcriptomic analysis was conducted through bulk RNA sequencing of myocardial tissues harvested from the Sham, MIRI, and PB@PM treatment groups. PCA revealed distinct clustering patterns among experimental groups, with PB@PM-treated specimens demonstrating prominent transcriptional modulation compared to MIRI controls (Fig. 6A). The volcano plots showed distinct differential gene expression profiles between the MIRI and control groups (Supplementary Fig. 17A). Quantitative analysis demonstrated that PB@PM treatment induced a total of 1875 upregulated and 2752 downregulated differentially expressed genes (DEGs) in contrast to the MIRI group (Fig. 6B). Furthermore, comparative analysis between the PB@PM and Sham group identified 227 upregulated and 97 downregulated DEGs (Supplementary Fig. 17B). Notably, the 10 most prominently upregulated and downregulated genes are clearly annotated in the respective volcano plots. Heatmap analysis provided a comprehensive visualization of differential gene expression across the three experimental groups, revealing pronounced alterations in transcriptional patterns associated with immune and inflammatory responses, energy metabolism, oxidative stress response, and redox homeostasis maintenance (Supplementary Fig. 17C). These biological pathways are integral to the cardioprotective mechanisms observed during MIRI. Venn diagram analysis quantified the overlap of DEGs between the control vs. MIRI and PB@PM vs. MIRI comparisons, with 1284 upregulated and 2351 downregulated genes shared between both comparisons, representing 56.7% and 70.4% of the respective total DEG populations (Fig. 6C).

The Gene Set Variation Analysis plot showed that the expression levels of pyroptosis, apoptosis, and necroptosis were dramatically elevated in the MIRI group compared to the Sham group. In stark contrast, these pathways were substantially downregulated in the PB@PM treatment group (Fig. 6D). Further Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of the downregulated genes revealed that PB@PM markedly suppressed the molecular pathways associated with pyroptosis, apoptosis, and necroptosis following MIRI (Fig. 6E). The Gene Set Enrichment Analysis (GSEA) network diagram and protein network further illustrated that pyroptosis, apoptosis, and necroptosis were tightly interconnected and mutually reinforcing pathways (Fig. 6F, G). Moreover, the GSEA plot confirmed that PB@PM treatment attenuated pyroptosis, apoptosis, and necroptosis compared to the MIRI group (Fig. 6H). Correlation analysis revealed a robust positive association among pyroptosis, apoptosis, and necroptosis across different treatment groups (Fig. 6I). Specifically, the MIRI group exhibited high panoramic expression of these pathways, whereas PB@PM treatment robustly reduced their expression levels, bringing them closer to the profile observed in the Sham group (Fig. 6I). Furthermore, we conducted a systematic investigation of PANoptosis in murine ischemic myocardial tissues through western blot analysis and histopathological evaluation. In the MIRI group, PANoptosis exhibited distinct activation, evidenced by upregulation of active forms of AIM2, ZBP1, Pyrin, ASC, FADD, apoptotic markers (caspase 3, caspase 8, Bax, and Bcl-2), necroptosis markers (RIPK1, RIPK3, and MLKL), and pyroptosis markers (caspase 1, NLRP3, and GSDMD) (Fig. 6J and Supplementary Figs. 18 and 19). Conversely, the PB@PM treatment regime demonstrated marked downregulation of PANoptosis-associated protein expression (Fig. 6J and Supplementary Figs. 18 and 19). These findings provided compelling evidence that PB@PM exerted comprehensive inhibition of RIPK1 (RIPK1, NLRP3, RIPK3, ASC, FADD, caspase 1, and caspase 8), ZBP1 (ZBP1, NLRP3, ASC, RIPK3, RIPK1, caspase 1, and caspase 8), and AIM2 (AIM2, Pyrin, ZBP1, ASC, FADD, RIPK1, RIPK3, and caspase 8) PANoptosomes. This multimodal suppression effectively attenuated MIRI-induced PANoptosis execution and conferred cardioprotection against myocardial injury.

Regarding the efficacy of PB in molecular dynamics simulations, we conducted supplementary validation to corroborate its inhibitory effects on the three principal effectors of PANoptosis (AIM2, ZBP1, and RIPK1). Lentiviral overexpression constructs targeting AIM2, ZBP1, and RIPK1 were systematically developed (Supplementary Fig. 20). Initial fluorescence colocalization analysis revealed that PB treatment induced evident spatial overlap with endogenous AIM2, ZBP1, and phosphorylated RIPK1 in OGD/R-stimulated HL-1 cells (Supplementary Fig. 21A). Quantitative western blot analysis demonstrated that PB exhibited comparable suppression efficacy against exogenously overexpressed AIM2 and ZBP1 in both normoxic and OGD/R conditions (Supplementary Fig. 21B), indicating that PB’s potent inhibitory activity effectively neutralized the OGD/R-induced protein expression perturbations. Notably, while RIPK1 overexpression remained unaffected by PB treatment, the OGD/R-induced phosphorylation of RIPK1 was remarkably attenuated by PB intervention (Supplementary Fig. 21B), suggesting differential regulatory mechanisms of PB on RIPK1 activation states.

We conducted additional validation of PANoptosis manifestations through in vitro cellular experiments. CCK-8 assays and Calcein AM/PI viability staining indicated that PB and PB@PM treatments produced a marked reduction in OGD/R-induced HL-1 cell death (Supplementary Fig. 22A–C). Furthermore, under equimolar low-dose conditions, the triple combination of Necrostatin-1 (necroptosis inhibitor), Disulfiram (pyroptosis inhibitor), and Z-VAD-FMK (pan-caspase inhibitor) reversed the limited therapeutic efficacy observed with monotherapy approaches (Supplementary Fig. 22D), providing evidence for crosstalk among distinct cell death modalities and indicating insufficient therapeutic outcomes from single-pathway inhibition. Notably, PB monotherapy achieved comparable therapeutic efficacy to the combination regimen (Supplementary Fig. 22D). Flow cytometric analysis using Annexin V-FITC/PI and TUNEL staining showed that PB@PM attenuated apoptosis levels in OGD/R-exposed HL-1 cells (Supplementary Fig. 22E, F). Morphological analysis further confirmed characteristic pyroptotic features-including cellular swelling, membrane rupture, and cytoplasmic vacuolation - in OGD/R-treated HL-1 cells (Supplementary Fig. 22G, H), which were substantially ameliorated by PB and PB@PM treatment, confirming their potent anti-pyroptotic activity. Consistent with in vivo findings in murine ischemic myocardium, PB@PM treatment inhibited OGD/R-induced upregulation of AIM2, ZBP1, phosphorylated RIPK1, cleaved caspase 1, cleaved caspase 8, cleaved GSDMD, and phosphorylated MLKL in HL-1 cells (Supplementary Fig. 22I, J). Besides, PB@PM or PB treatment markedly suppressed the colocalization of ASC and caspase 1/8 (Supplementary Fig. 22K, L), the assembly of which is essential for the formation of PANoptosome complex, suggesting that PB@PM can inhibit the PANoptosome assembly. Collectively, these findings provide comprehensive evidence for PANoptosis activation during MIRI and substantiate the therapeutic potential of PB@PM in targeting this multifaceted cell death pathway. In addition, we also found that PB@PM could suppress the secretion of pro-inflammatory cytokines in macrophages by reducing PANoptosis levels (Supplementary Fig. 23).

Inflammation and oxidative stress are associated with the crosstalk among pyroptosis, apoptosis, and necroptosis2. Gene ontology (GO) enrichment analysis showed that PB@PM predominantly modulated mitochondrial respiration function and energy metabolism pathways in MIRI mice. Conversely, downregulated genes were predominantly associated with leukocyte chemotaxis, cytokine-mediated signaling cascades, and immune-inflammation regulatory networks (Supplementary Fig. 24A). Notably, comparative GO analysis between control and MIRI groups demonstrated similar biological regulatory patterns (Supplementary Fig. 17D), indicating PB@PM’s capacity to restore aberrant MIRI-associated biological processes to homeostatic functional states. Integrated GO and KEGG pathway analyses showed that PB@PM treatment resulted in remarkable upregulation of mitochondrial metabolic processes, including oxidative phosphorylation, tricarboxylic acid cycle activity, and fatty acid β-oxidation, alongside activation of cAMP-dependent and cGMP-PKG signaling cascades (Supplementary Fig. 24C, D). The pathophysiological significance of these cyclic nucleotide systems in MIRI pathogenesis warrants emphasis: while pathological cAMP overaccumulation during myocardial ischemia exacerbates ischemia-induced cellular injury through accelerated ATP depletion and membrane depolarization50, pharmacological enhancement of cGMP/PKG signaling confers cardioprotection through vasodilatory effects on vascular smooth muscle cells. This vascular relaxation mechanism reduces coronary resistance while augmenting perfusion pressure, thereby ameliorating myocardial hypoxia and limiting apoptotic cell death51,52. Our findings demonstrate that PB@PM exerts dual regulatory effects by attenuating pathological cAMP hyperactivity while potentiating cGMP/PKG-mediated cytoprotective signaling (Supplementary Figs. 25 and 26). This pharmacological modulation demonstrates therapeutic potential for preserving cardiomyocyte viability and restoring post-ischemic myocardial functional recovery.

In addition to the three PANoptosis-associated cell death modalities, downregulated DEGs exhibited notable enrichment in various immune and inflammatory signaling pathways, including nuclear factor (NF)-κB, TNF, chemokine, and TLR signaling pathways (Fig. 6E). Furthermore, GSEA showed that PB@PM treatment in MIRI mice induced marked downregulation of NOD-like receptor signaling, TLR signaling, cytokine receptor interaction, chemotaxis, immune response activation, and chemokine signaling pathway (Supplementary Fig. 24B). The RNA-seq data provided compelling evidence for PB@PM’s dual regulatory effects in attenuating cardiac cell death while preserving mitochondrial integrity and modulating immuno-inflammatory homeostasis. Building upon the KEGG pathway analysis of downregulated genes, we further performed targeted validation of NF-κB and TLR signaling cascades. As a pivotal component of innate immunity, the TLR4 signaling pathway plays critical roles in the pathogenesis, progression, and clinical outcomes of cardiovascular disorders53,54. This pathway orchestrates downstream inflammatory responses through NF-κB activation, ultimately driving pro-inflammatory cytokine production. Our results indicated that PB@PM suppressed the aberrantly elevated protein levels of TLR4 and phosphorylated NF-κB-p65 in MIRI mice (Supplementary Fig. 13), suggesting its therapeutic potential in modulating TLR4/NF-κB-mediated inflammation. Collectively, these findings elucidate PB@PM’s capacity to restore immune-inflammatory equilibrium through multimodal regulatory mechanisms.

Cardiac tissue exhibits a high mitochondrial density, which serves as the primary source of reactive oxygen species (ROS) generation55. Pathological overproduction of ROS induces mitochondrial permeability transition pore activation, thereby initiating membrane depolarization and subsequent cardiomyocyte cell death. Mitochondrial ROS constitutes the principal pathogenic mechanism underlying oxidative stress during ischemia and reperfusion injury56. Given the central role of mitochondrial dysfunction and oxidative stress in initiating PANoptosis, we systematically evaluated the effects of PB@PM on mitochondrial health. Electron spin resonance (ESR) spectroscopy confirmed the potent ROS-scavenging capacity of PB@PM, demonstrating attenuation of hydroxyl (•OH) and hydroperoxyl (•OOH) radicals (Supplementary Fig. 27A–D). In OGD/R-injured HL-1 cells, PB@PM treatment markedly reduced intracellular ROS levels, as measured by flow cytometry using DCFH-DA probe (Supplementary Fig. 27F–H). Mitochondrial membrane potential (MMP), a classic indicator of mitochondrial energy status and intracellular homeostasis, was systematically evaluated across experimental groups. ODG/R induced MMP collapse in HL-1 cells, evidenced by increased JC-1 monomer fluorescence (green), indicating mitochondrial dysfunction (Supplementary Fig. 28A–C). Notably, PB@PM treatment efficiently restored MMP (Supplementary Fig. 28A–C). Most importantly, assessment of mitochondrial function using the Seahorse XF Analyzer revealed that PB@PM treatment restored oxidative phosphorylation, as evidenced by improving basal and maximal respiration, as well as ATP production in OGD/R-injured HL-1 cardiomyocytes (Supplementary Fig. 28D–G). Collectively, these findings delineate a dual mechanistic pathway for PB@PM: (1) Direct targeting: high-affinity binding and functional suppression of core PANoptosome components (RIPK1, ZBP1, and AIM2), disrupting the initiation of PANoptosis. (2) Indirect mitigation: potent ROS scavenging that preserves mitochondrial integrity and function, thereby eliminating a critical upstream trigger for PANoptosome activation. This synergistic action underpins the robust cardioprotection observed in our MIRI models.

Methods
Materials
Hydrochloric acid (HCl, 36−38%), potassium ferricyanide (K3[Fe(CN)6]), and polyvinylpyrrolidone (PVP, K30) were purchased from Adamas-beta (Shanghai, China). The chemicals were used as received and did not require further purification. Hydrogen peroxide (H2O2) in a 30 wt.% aqueous solution was purchased from Sinopharm Chemical Reagent Co., Ltd., China. All aqueous solutions were prepared using deionized (DI) water with a resistivity of 18.2 MΩ cm. The Annexin V-FITC apoptosis detection kit (C1062M), propidium iodide (PI) and calcein acetoxymethyl ester (Calcein AM) kit (C2015M), mitochondrial membrane potential detection kit JC-1 (C2005), 2,7-dichlorofluorescein diacetate (DCFH-DA) kit (S0035M), reactive oxygen species assay kit (S0033M), DAPI (C1002), and TUNEL (C1088 and C1090) were purchased from Beyotime Biotechnology, China. Necrostatin-1 (HY-15760), Disulfiram (HY-B0240), Z-VAD-FMK (HY-16658B), ZBP1 (HY-P83326), phospho-RIPK1 (HY-P81539), phospho-MLKL (HY-P80468), cleaved caspase 1 (HY-P80622), cell counting kit-8 (CCK-8) assay kit (HY-K030), and cleaved caspase 8 (HY-P80624) were obtained from MedChemExpress, USA. LPS (L8880), PBST (P1031), BSA (SW3015), Tubulin (K200059M), Masson’s trichrome kit (G1340), fluorescein isothiocyanate (FITC; 3326-32-7), and Cy5.5 (S1061) were purchased from Solarbio, China. Claycomb medium (51800C), Evans blue (314-13-6), and TTC (298-96-4) were purchased from Sigma, German. RIPA lysis buffer (WB3100), proteinase and phosphatase inhibitors (P002), and BCA Protein Assay Kit (WB6501) were purchased from NCM Biotech, China. CD41 (24552-1-AP), Collagen III (22734-1-AP), AIM2 (20590-1-AP), caspase 1 (81482-1-RR), and caspase 8 (13423-1-AP) antibodies were bought from Proteintech, China. 5-dimethyl-1-pyrroline-N-oxide (D048) was bought from Dojindo, China. DMEM medium (11965092), penicillin/streptomycin (15140122), PBS (10010023), and FBS (A5670701) were bought from Gibco, USA. TBST (G2150), Ly6G antibody (GB11229), Hematoxylin and eosin (H&E) kit, and WGA (G1730) were purchased from Servicebio Co., China. Collagen I (ab316222), cTNT (ab209813), CD31 (ab182981), Pyrin (ab195975), a-SMA (ab124964), caspase 1 (ab179515), caspase 8 (ab108333), and GSDMD (ab219800) antibodies were purchased from Abcam, UK. TNF-α (EMC102a.96), IL-1β (EMC001b.96), IL-6 (EMC004.96), IL-10 (EMC005.96), cAMP (JL13362-96T), and cGMP (JL12644-96T) enzyme-linked immunosorbent assay (ELISA) kits were bought from NeoBioscience, China. CD86 (19589s), Bax (5023T), ASC (40002T), caspase 3 (9662S), cleaved caspase 3 (9664T), cleaved GSDMD (10137T), TLR4 (14358S), NF-κB p65/RelA (8242T), phospho-NF-κB p65/RelA (3033T), phospho-RIPK3 (91702T), and GAPDH (2118T) antibodies were purchased from Cell Signaling Technology, USA. CD42b (A22277), CD47 (A21904), GP6 (A14999), Bcl-2 (A19693), TNF-α (A24214), IL-1β (A16288), IL-10 (A12255), ASC (A22046), FADD (A19838), MLKL (A26436), phospho-MLKL (AP1173), phospho-RIPK3 (AP1408), RIPK3 (A5431), phospho-RIPK1 (AP1230), RIPK1 (A7414), NLRP3 (A24294), cAMP (A1640), phospho-VASP (AP1510), VASP (A14217), AIM2 (A25252), and ZBP1 (A22612) antibodies were purchased from ABclonal, China. Three plasmids for the overexpression of mouse AIM2 mRNA, ZBP1 mRNA, and RIPK1 mRNA were synthesized by OBiO Technology (Shanghai) Corp., Ltd, and the corresponding plasmid maps were provided in Supplementary Fig. 20.

Synthesis of PB
PBs were synthesized by mixing PVP (8.0 g) and K3[Fe(CN)6] (696 mg) in HCl solution (1 M, 50 mL). The mixture was stirred for 1 h obtain a clear yellow solution, which was subsequently transferred to an electric oven and heated at 80 °C for 20 h to form a blue solution. The resulting PBs were washed five times with deionized water via centrifugation.

Design and construction of PB@PM
PB@PM were fabricated via an optimized ultrasonic fusion method57. Firstly, a 2 mL aliquot of PB solution with a concentration of 1 mg/mL was thoroughly mixed with 0.2 mg of isolated platelet membrane (quantified based on protein content). The resulting mixture was subjected to ice bath sonication at 75 W for 3 min using an ultrasonic processor (SB-5200DT, SCIENTZ, China) to promote effective membrane coating. To achieve uniform size distribution and nanoscale dimensions, the mixture was subsequently processed through a sequential extrusion protocol utilizing Nuclepore™ hydrophilic membranes with nominal pore sizes of 800 nm and 400 nm, yielding a final size of PB@PM with a hydrodynamic diameter of approximately 100 nm. The structural integrity and coating efficiency of the synthesized PB@PM were systematically characterized using TEM, molecular characterization techniques, and fluorescence co-localization imaging. Western blotting confirmed the retention of platelet-specific surface proteins (CD41, CD42b, CD47, and GP6) on PB@PM.

Characterization
The morphological analysis of PBs was characterized by Scanning Electron Microscope (SEM, Zeiss Merlin Compact, Zeiss, Germany) and Transmission Electron Microscopy (TEM, FEI Tecnai F20, USA). UV-Vis-NIR absorption spectra were acquired with a Shimadzu UV-3600i Plus spectrophotometer (Shimadzu, Japan). The Fourier-transform infrared (FT-IR) spectroscopy was performed on a Nicolet iS10 FT-IR spectrometer (Thermo Fisher Scientific, USA). X-ray photoelectron spectra (XPS) data were obtained using an EscaLab 250Xi electron spectrometer (Thermo Scientific K-Alpha, Thermo, USA). Powder X-ray diffraction (XRD) patterns were detected on a D8 ADVANCE X-ray diffractometer (Rigaku Ultima IV, Rigaku, Japan). The concentration of nanoparticles was measured by inductively coupled plasma atomic emissions spectroscopy (ICP-AES, Thermo Fisher Scientific). Dynamic light scattering (DLS) measurements were conducted using a SZ-100 nanoparticle analyzer (HORIBA Scientific, Japan).

ROS scavenging evaluation
The hydroxyl radical (•OH) and hydroperoxyl radical (•OOH) scavenging capacities of PB and PB@PM were investigated using electron spin resonance (ESR) spectroscopy (Bruker ESR 5000 system, Bruker Inc.), employing 5-dimethyl-1-pyrroline-N-oxide as the trapping agent. In standardized assays, DMPO (5 μL, 10 mM) was dissolved in PBS buffer (45 μL, 10 mM). The Fenton reaction was subsequently initiated by introducing Fe2+ (20 μL, 50 mM) to catalyze H2O2 (30 μL, 50 mM)-mediated •OH generation. Quantitative analysis of •OH levels was conducted through ESR amplitude intensity measurements, with comparative assessments performed under both NP-supplemented and NP-free conditions. For superoxide anion (•O2−) evaluation, 70 μg of potassium superoxide (KO₂) was reacted with 200 μL of 18-crown-6/DMSO solution (0.7 mM) to generate •O2− radicals, followed by DMPO-mediated trapping (50 μL). ESR spectral analysis of the spin-trapped adducts enabled comparative quantification of superoxide dismutase (SOD)-mimetic activity in PB and PB@PM systems.

MD simulation
MD simulations were performed utilizing the GROMACS 2022.2 computational suite. Four distinct simulation systems were constructed to characterize the interactions between PB and AIM2HIN, AIM2PYD, RIPK1, or ZBP1. Initial separation distances between the proteins and PB surfaces were maintained at 1.5 nm. Each system was solvated with SPC water molecules within a cubic simulation box (dimensions: 16.26 × 16.26 × 10.56 nm3), ensuring a minimum solvent buffer distance exceeding 12 Å from all protein surfaces. Physiological ionic conditions were mimicked by adding Na+/Cl− ions. Following system initialization, energy minimization was executed using an iterative steepest descent algorithm (a maximum of 5000 steps). Systems were energy-minimized using the steepest descent algorithm (5000 steps), followed by 20 ps NVT and NPT equilibrations to stabilize temperature (310 K) and pressure (1 bar). Force field parameters, including Amber99SB-ILDN for protein and published parameters58 for PB structures. Lennard-Jones59 interactions and short-range electrostatics were evaluated using a 10 Å cutoff, while long-range electrostatics employed the Particle Mesh Ewald (PME) method60. Periodic boundary conditions were applied in all directions, and hydrogen bonds were constrained using the LINCS algorithm. PB remained fixed during simulations to maintain interfacial stability. Production runs (200 ns, 2 fs timestep) conducted to analyze the equilibrium properties.

Cell culture
HL-1 cells (a mouse cardiomyocyte cell line; CC-Y2145), RAW 264.7 cells (CC-Y2084) were purchased from the Shanghai EK-Bioscience Biotechnology Co. Ltd. HL-1 cells were cultured in Claycomb medium (Sigma) with reference to the previously reported protocol61, and RAW 264.7 cells were grown in DMEM medium (Gibco) with 10% FBS (Gibco) and 1% penicillin/streptomycin. Cultures were incubated at 37 °C in an atmosphere with 5% CO2, with daily medium replacement to maintain optimal growth conditions.

In vitro OGD/R model system and nanoparticle-based therapeutic interventions
The OGD/R injury model was established following a validated protocol62. Briefly, HL-1 cells underwent 8-hour hypoxic exposure in serum-free, glucose-derived DMEM medium (Gibco) within a modular hypoxic chamber (JSQA-50, JIANGETECH), maintaining standardized hypoxic conditions (5% CO2, 95% N2) at 37 °C with controlled humidity. Following OGD induction, cells were reperfused by replacing the hypoxic medium with complete growth medium supplemented with 10% fetal bovine serum and 25 mM glucose, followed by 12-hour normoxic recovery in a standard cell culture incubator (37 °C, 5% CO2). During the reperfusion phase, PB or PB@PM was administered to designated treatment groups, while the control group received equivalent volumes of untreated culture medium.

Cellular uptake, cytotoxicity, and cell viability assessment
The cellular internalization efficiency of PB@PM was systematically evaluated through a time-dependent fluorescence analysis. HL-1 cardiomyocytes were exposed to FITC-labeled PB@PM at specified time intervals (0.5, 1, 2, and 4 h). Quantitative determination of intracellular FITC fluorescence was performed using a Novocyte Advanteon flow cytometer (Agilent Technologies, USA) equipped with standardized cytometric analysis protocols. Cytotoxicological evaluation was conducted using a CCK-8 proliferation assay. Cells were seeded at a density of 1 × 104 cells/well in 96-well culture plates and treated with serial dilutions of PB@PM for 24 h. Cellular viability assessment followed a standardized protocol: after treatment, cells were washed with PBS buffer and incubated with 10 μL of CCK-8 solution supplemented to a final volume of 100 μL in culture medium for 1.5 h at 37 °C. The metabolic activity was quantified by measuring absorbance at 450 nm using a Spark multimode microplate reader (Tecan Group Ltd., Switzerland) with integrated wavelength correction and background subtraction functions.

Mitochondrial membrane potential (MMP) assessment
The MMP was quantitatively evaluated using JC-1 fluorescent dye following the manufacturer’s standardized protocol. Following 2 h pre-treatment with PB or PB@PM formulations, OGD/R-exposed cells were incubated with JC-1 working solution at 37 °C for 30 min. Dual-mode analysis was performed using a BZ-X810 All-in-One Fluorescence Microscope (Keyence Corporation, Japan) for morphological assessment and a Novocyte Advanteon flow cytometer (Agilent Technologies, USA) for quantitative fluorescence intensity measurement.

Seahorse XF mitochondrial stress assay
Cellular oxygen consumption rates (OCR) were quantified in HL-1 cardiomyocytes utilizing the Seahorse XF Mito Stress Test protocol (Agilent Technologies). Cells were seeded at a density of 5000 cells per well in XF assay medium (pH 7.4) supplemented with 1 mM sodium pyruvate, 0.2 mM palmitic acid, 0.2 mM L-carnitine, and 4 mM glutamine. Baseline OCR measurements were acquired, followed by sequential pharmacological modulation: 3 μM oligomycin (ATP synthase inhibitor) to determine ATP-linked respiration, 1.5 μM carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) to induce maximal respiratory capacity, and 2 μM rotenone/antimycin A combination to achieve complete mitochondrial respiration inhibition. All experimental procedures adhered to validated methodologies63. Nuclear quantification was performed using Hoechst 33342 fluorescence staining for cell density determination and data normalization.

ELISA analysis
RAW 264.7 macrophages were established in monolayer culture within six-well plates at a density of 2 × 105 cells/well. Following 24 h stabilization, cells were treated with LPS (300 ng/mL) in conjunction with PB@PM at specified concentrations (20 or 40 μg/mL). Culture supernatants were harvested 24 h post-treatment and immediately processed for cytokine profiling. Quantitative determination of inflammatory mediators was conducted using commercially available ELISA kits (R&D Systems) following manufacturer protocols. The analyte panel included tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-10 (IL-10), cyclic adenosine monophosphate (cAMP), and cyclic guanosine monophosphate (cGMP).

Apoptosis detection and intracellular ROS quantification
Cell apoptosis was quantitatively assessed using a FITC-conjugated annexin V/propidium iodide (PI) apoptosis detection kit following the manufacturer’s standardized protocol. Post OGD/R-induced injury, HL-1 cells were enzymatically dissociated into single-cell suspensions. The cell pellets were resuspended in binding buffer containing annexin V-FITC and PI, followed by 30 min incubation at room temperature in the dark. Flow cytometric analysis was performed using a Novocyte Advanteon flow cytometer (Agilent Technologies, Santa Clara, CA, USA), with data acquisition and analysis conducted through NovoExpress software. The TUNEL assay was employed to evaluate DNA fragmentation according to the manufacturer’s specifications, serving as an independent apoptotic index. Concurrently, intracellular ROS levels were quantified using dichlorofluorescein diacetate (DCFH-DA) fluorescent probes. Following 30-minute incubation with 10 μM DCFH-DA at 37 °C, cells were analyzed for oxidative stress parameters on a CytoFLEX XL flow cytometer (Beckman Coulter, Brea, CA, USA) with CytExpert software for data interpretation.

Live/Dead cell viability assay
The HL-1 OGD/R cell model was employed. Cellular viability was assessed using calcein AM and PI fluorescence staining, following the manufacturer’s protocol. Following 30 min incubation with the fluorescent probes at room temperature in the dark, representative fluorescence micrographs of different experimental groups were captured using a BZ-X810 All-in-One Fluorescence Microscope (Keyence Corporation, Japan).

In vitro morphological analysis and SEM assessment
Following OGD/R induction, HL-1 cells treated with PB or PB@PM underwent standardized processing. Cell specimens were initially fixed in 4% paraformaldehyde (PFA) for 30 min, followed by sequential washing with PBS for 5 min per rinse (three repetitions). Tubulin cytoskeleton visualization was accomplished through immunofluorescent staining with tubulin-specific antibodies under dark conditions at ambient temperature for 1 h. Post-staining rinses were performed using PBS, with subsequent 15 min DAPI nuclear counterstaining in darkness. After final PBS washes (three times, 5 min each), morphological evaluations were conducted using an inverted fluorescence microscope (Keyence Corporation, Osaka, Japan). Concurrently, ultrastructural analysis of cellular membranes was performed utilizing a field emission SEM (Hitachi Regulus 8100, Tokyo, Japan) to assess membrane integrity and surface characteristics.

Plasmid overexpression assay
HL-1 cells were seeded into cell plates and divided into five groups: (1) negative control group (transfected with blank plasmid); (2) transfected with expression plasmid group; (3) transfected with expression plasmid+OGD/R group; (4) transfected with expression plasmid+PB intervention group; (5) and transfected with expression plasmid+OGD/R+PB intervention group. In brief, following the manufacturer’s protocols, confluent HL-1 cell cultures at approximately 70% confluency were transiently transfected with AIM2, ZBP1, or RIPK1 overexpression plasmids using opti-MEM reduced serum medium. Transfected cells were subsequently maintained under standard culture conditions for subsequent analysis. The OGD/R model was established in HL-1 cardiomyocytes following completion of transfection, with propidium bromide administered during the reperfusion phase for interventional assessment.

Experimental animals
Male C57BL/6J mice (6–8 weeks of age, 20–25 g) were obtained from Shanghai BK/KY Biotechnology Co., Ltd. All animals were maintained in specific pathogen-free facilities under standardized conditions: 12 h light/dark cycles, libitum access to food and water, relative humidity of 30%, and ambient temperature (22–24 °C). Experimental protocols were approved by the Animal Care and Use Committee of Shanghai Sixth People’s Hospital affiliated to Shanghai Jiao Tong University School of Medicine (Approval No: DWSY2022-0085) and complied with guidelines from the American Association for Laboratory Animal Care. Euthanasia was performed by CO2 inhalation followed by cervical dislocation.

In vivo biosafety assessment of PB@PM
Healthy male wild-type mice (6-to 8-week-old) were randomized into three experimental groups. The control cohort received vehicle solution, while the treatment cohorts were intravenously administered PB@PM at a dosage of 5 mg/kg body weight via caudal vein injection. Histopathological evaluation was conducted at post-dosing intervals of 7 and 14 days through H&E staining of cardiac, hepatic, splenic, pulmonary, and renal tissue sections using standardized protocols (Sigma-Aldrich). Histotoxicological manifestations and parenchymal morphological characteristics were systematically analyzed microscopically. Concurrently, peripheral blood samples were collected for comprehensive hematological evaluation and serum biochemical profiling to assess systemic physiological status.

MIRI model
The murine MIRI model was established following previously validated methodology44. Briefly, mice were anesthetized via inhalation of 3% isoflurane in oxygen (1 L/min), and a ~2 cm thoracic incision was made at the apex of maximal cardiac pulsation. A 4-0 purse-string suture was placed, and the fourth intercostal space was bluntly dissected using curved forceps to access the thoracic cavity. Gentle pressure was applied to exteriorize the heart, which was temporarily secured within the sutured opening while minimizing pneumothorax. A 6-0 surgical slipknot was tied around the left anterior descending coronary artery, with the suture and externalized. The heart was repositioned into the thoracic cavity, residual air was evacuated, and the epidermis was closed with sutures. After 45 min of ischemia, the slipknot was released to initiate reperfusion. Surgical incisions were closed with sterile 5-0 sutures, and mice were monitored postoperatively under standard housing conditions.

Biodistribution analysis
Sham mice (Control group) received intravenous injection of Cy5.5-labeled PB, while MIRI mice were administered either Cy5.5-labeled PB (PB group) or Cy5.5-labeled PB@PM (PB@PM group). Injections were performed at 1 h post-ischemia induction. Mice were euthanized, and major organs (heart, liver, spleen, lungs, and kidneys) were harvested for ex vivo fluorescence imaging using VISQUE, in vivo Elite system at different time points.

Animal grouping and experimental protocol
The murine subjects were randomized into four experimental cohorts: Sham group, MIRI group, MIRI with PB treatment group, and MIRI with PB@PM treatment group. Pharmacological intervention was initiated at 1 and 24 h post-reperfusion via intravenous injection of either 0.5 mg/kg body weight PB or PB@PM solution (encapsulating 0.5 mg/kg PB) to the respective treatment groups. The Sham and MIRI control groups received an equivalent volume of 0.9% sodium chloride solution via the same administration route.

Echocardiography
Transthoracic echocardiography was conducted using a Vevo 3100 imaging system (FUJIFILM VisualSonics, Toronto, Canada) equipped with a 400 MHz transducer. Cardiac function was assessed preoperatively and on postoperative days 1, 14 and 2844. Mice were anesthetized with inhalation of 1–1.5% isoflurane in oxygen following chest hair removal via depilatory paste. Heart rates were maintained at 550–650 beats per minute (bpm) across all subjects. Long-axis views were captured to measure cardiac parameters, including LVEDV, LVESV, LVEF and LVFS, which were automatically calculated by the system’s integrated software.

Evans blue/TTC staining
Infarct size quantification was conducted via Evans blue/TTC dual staining44. Briefly, mice were anesthetized 3 days post-I/R surgery, the LAD coronary was re-ligated. Hearts were perfused with PBS to remove residual blood, followed by perfusion with 1% Evans blue (Sigma, >75% purity). The hearts were subsequently excised and sectioned into serial 1.0 mm-thick transverse slices. Tissue sections were incubated in 2% TTC (Sigma-Aldrich, >98% purity) solution at 37 °C for 15 min in the dark. Sections were fixed in 4% paraformaldehyde, and images were acquired. Evans blue-negative regions represented the area at risk (AAR), while TTC-negative zones indicated infarcted myocardium. Infarct area, AAR and LV area were quantified using ImageJ software (ImageJ 1.52v).

Masson and HE staining
Hearts harvested 28 days post-surgery were perfused with PBS to clear blood, fixed in 4% formaldehyde, and embedded in paraffin. Sections were stained using Masson’s trichrome and HE kits (Sigma-Aldrich) to distinguish healthy tissue from fibrotic regions. Stained samples were imaged using a Nikon microscope (Nikon Corporation, Tokyo, Japan).

WGA staining
Tissue sections underwent wheat germ agglutinin (WGA) staining per the manufacturer’s protocol. Briefly, sections were washed to remove OCT compound, fixed in 4% paraformaldehyde, and incubated with 50 µg/mL WGA for 30 min at room temperature. Short-axis images of cardiomyocytes were captured, and cross-sectional areas were analyzed using ImageJ software.

Immunofluorescence staining
Hearts were excised after euthanasia, fixed in 4% paraformaldehyde overnight at 4 °C, and sectioned transversely. Sections were washed three times in PBS containing 0.1–0.2% Triton X-100 (PBST), blocked with 4% bovine serum albumin (BSA), and incubated with primary antibodies against Collagen I, Collagen III, Ly6G, CD86, cTNT, CD31, α-SMA, and TUNEL. Secondary antibody incubation and DAPI nuclear counterstaining were performed at room temperature. Images were acquired via Nikon widefield or confocal microscopes (CSU-W1, Nikon Corporation, Tokyo, Japan), and analyzed with NIS Elements AR and ImageJ software.

Western blot
Proteins were extracted from murine cardiac tissues with RIPA lysis buffer (Beyotime) supplemented with proteinase and phosphatase inhibitors. Protein concentrations were quantified using a BCA Protein Assay Kit. Proteins were separated on 10–12.5% SDS-PAGE gels and transferred onto Polyvinylidene fluoride (PVDF) membranes. Membranes were blocked with 5% skim milk in Tris-buffered saline/Tween 20 (TBST, pH 7.4–7.6) for 1.5 h at room temperature, followed by overnight incubation with primary antibodies at 4 °C. After TBST washes, membranes were incubated with HRP-conjugated IgG antibody (Anti Rabbit IgG (H+L) antibody, 1:1000, Beyotime) for 1 h at room temperature. Immunoreactive bands were visualized using an ECL substrate (Millipore, Burlington, MA) and detected with a fluorescent imaging system (ProteinSimple, Sata Clara, CA, USA). Image acquisition and densitometric analysis were performed using ImageJ software. Primary antibodies: Bax, Bcl-2, cleaved-caspase 3, Caspase 3, TNF-α, IL-1β, IL-10, phosphorylated MLKL (p-MLKL), total MLKL, phosphorylated RIPK1 (p-RIPK1), total RIPK1, phosphorylated RIPK3 (p-RIPK3), total RIPK3, NLRP3, cleaved-GSDMD, total GSDMD, TLR4, cAMP, NF-κB p65/RelA, phosphorylated NF-κB p65/RelA (p-p65), phosphorylated VASP (p-VASP), total VASP, AIM2, cleaved-caspase 8, caspase 8, cleaved-caspase 1, caspase 1, ZBP1, pyrin, ASC, FADD, and GAPDH.

RNA-seq analysis
To investigate PB@PM’s therapeutic mechanisms in myocardial ischemia-reperfusion injury, RNA sequencing was performed on cardiac tissues from three groups: Control, MIRI, and PB@PM-treated mice (n=5 per group). Hearts were quickly collected on post-operative day 3, then washed with cold saline, frozen in liquid nitrogen, followed by kept at −80 °C. Total RNA was extracted, and its integrity/quantity was assessed using an RNA Nano 6000 Assay Kit of the Bioanalyzer 4200 system (Agilent Technologies, CA, USA). High-quality RNA samples were used to construct the sequencing library. The library preparation for transcriptome sequencing, clustering, and sequencing was performed using Illumina NovaSeq X-Plus (Illumina) according to the manufacturer’s instructions. Differential expression analysis was conducted using the Seurat package through implementation of the Wilcoxon rank-sum test with default parameters. DEGs were identified based on three criteria: (1) expression in more than 10% of cells within each compared group, (2) average log2-fold change exceeding 1, and (3) statistical significance determined by Adjusted p-value (adjusted p<0.05), with multiple testing correction via the Bonferroni method.

Single-nucleus RNA sequencing (snRNA-seq) analysis of human cardiac tissues
A publicly available snRNA-seq dataset from patients with acute myocardial infarction was reanalyzed (https://cellxgene.cziscience.com/collections/8191c283-0816-424b-9b61-c3e1d6258a77). Clinical sample metadata are detailed in Supplementary Table 1.
(1) Initial processing and quality control
snRNA-seq data preprocessing was performed using CeleScope v2.0.7 (Singleron Biotechnologies) with default settings. Briefly, barcodes and UMIs were extracted from R1 reads and error-corrected. Adapter sequences and poly-A tails were trimmed from R2 reads, followed by alignment to the GRCh38 transcriptome via STAR (v2.7.11a). Uniquely mapped reads were assigned to genes, and cells were defined by the co-occurrence of cell barcodes, UMIs, and gene expression. Gene expression matrices were generated for downstream analysis.
(2) Quality control, dimensionality reduction and clustering
Data processing was executed in Python 3.7 using Scanpy v1.8.1. Cells were filtered based on: (1) Minimum gene count ≥200 and exclusion of top 2% highest gene counts; (2) Exclusion of top 2% highest UMI counts; 3) Mitochondrial gene content ≤10%; 4) Gene detection threshold ≥5 cells. Post-filtering, 64804 cells remained (1328 genes/cell; 3963 UMIs/cell). Data were normalized to total counts per cell, log-transformed, and scaled. Top 2000 highly variable genes (HVGs) were identified using Seurat’s “seurat” flavor. Principal component analysis (PCA) was performed on scaled HVGs, with the top 20 principal components (PCs) retained for downstream analysis. Unsupervised clustering was implemented via the Leiden algorithm (resolution=1.2), and UMAP embeddings were generated for visualization64.
(3) Batch effect correction
Cross-sample heterogeneity was mitigated using Harmony v1.0, integrating the top 20 PCs from PCA2 to generate batch-corrected embeddings65.
(4) Differential expression gene (DEG) analysis
DEGs were identified using Scanpy’s rank_genes_groups function with Wilcoxon rank-sum tests. Genes meeting criteria (detected in ≥10% of cells within any group; |log2(FoldChange)|>1) were selected. Multiple testing correction was applied via Benjamini-Hochberg (FDR<0.05).
(5) Cell-type annotation
Cell-ID annotation: Cell identity was assigned using Cell-ID, a multivariate computational approach that computes cell-type specificity via hypergeometric tests (HGT)66. Gene expression matrices were reduced to latent dimensions via multiple correspondence analysis (MCA), projecting cells and genes into a unified space. Cell-specific gene signatures were derived from SynEcoSys™ (Singleron Biotechnologies), a curated database integrating canonical markers from CellMarkerDB, PanglaoDB, and literature. HGT scores were calculated for each cluster against reference cell types, with minimal HGT P-values defining cluster identities. Reference-based annotation: Canonical marker expression was cross-referenced against SynEcoSys™, ensuring annotation consistency. Cluster-specific cell-type frequencies were computed, and predominant cell types were assigned as cluster labels.
(6) Cardiomyocyte subtyping: High-resolution subtyping of cardiomyocytes was achieved by re-clustering cells from the parent cardiomyocyte cluster using identical analytical pipelines67.
(7) Gene Set Scoring with UCell
Gene set enrichment was quantified via UCell v1.1.068, a rank-based scoring methodology advantageous for large-scale datasets. Mann-Whitney U statistics evaluated query gene expression ranks across individual cells.

Statistical analysis
Data are presented as means ± standard deviation (SD). For single-nucleus RNA-seq data, wilcox.test was applied. Intergroup differences among three or more groups were assessed by one-way ANOVA with Tukey’s post-hoc correction. Statistical significance was defined as P<0.05. Analyses were carried out with GraphPad Prism 9.0 (GraphPad Software, San Diego, CA) and SPSS software 25.0 (SPSS Inc., USA).

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Xu et al. devised an innovative method for synthesizing Prussian blue (PB) nanoparticles, which effectively inhibit PANoptosis, thereby comprehensively blocking pyroptosis, apoptosis, and necroptosis and preventing their crosstalk in the treatment of myocardial ischemia-reperfusion injury (MIRI). The concept is OK, some revision should be done to improve the quality of this manuscript.\n1. The authors' assertion that myocardial ischemia-reperfusion injury (MIRI) is closely related to PANoptosis by single-cell sequencing alone lacks sufficient evidence. The genes involved in PANoptosis are similar to those involved in pyroptosis, apoptosis, and necrotic apoptosis, which makes it difficult to distinguish between the different forms of cell death. Furthermore, the hallmark of PANoptosis is the assembly and activation of the PANoptosome, which has yet to be directly characterised. The study lacks direct characterization of the PANoptosome.\n2. In light of the assumption that PANoptosis can be triggered by MIRI, it is imperative for the authors to elucidate the mechanism by which PANoptosis is initiated in the context of MIRI. This is a pivotal step in identifying the specific PANoptosis pathway that is activated, given that the modes of activation and triggering mechanisms of different PANoptosomes vary.\n3. It is not sufficient to conclude that PB can inhibit the formation of multiple PANoptosomes based on the evidence that PB reacts with AIM2, ZBP1, and RIPK1. The core components of the four PANoptosomes are highly similar, with AIM2, ZBP1 and RIPK1 representing the core components of the AIM2-PANoptosome. Therefore, it is unclear how the authors can claim to inhibit multiple PANoptosomes simultaneously. Once more, we propose that the authors elucidate the specific type of PANoptosis precipitated by MIRI.\n4. The article's primary focus is on sequencing and molecular modelling, which illustrate the inhibitory effect of PB on PANoptosis, which seriously lacks experimental validation and molecular mechanism. It would be advisable to confirm the inhibitory effect of PB on the assembly of the PANoptosome directly, for example through fluorescence co-localization or immunoprecipitation experiments.\n5. Please explain the rationale behind selecting the third day after surgery as the time point for tissue protein detection. Does the expression of PANoptosis-related proteins exhibit a greater degree of significance at either a shorter or longer time point?\n6. MIRI is a common occurrence in cardiomyocytes situated within the coronary artery reperfusion zone. How do the authors distinguish these cells in vivo in order to conduct the subsequent step of the experiment?\n7. The authors conducted fluorescence imaging of the principal organs of MIRI mice at 12 and 24 hours post-injection of Cy5.5-labeled PB@PM. Nevertheless, the findings indicated that only a minimal quantity of PB@PM remained within the heart at the 24-hour mark. It would be valuable to ascertain whether any remaining PB@PM is present in the heart after 28 days. Given that Cy5.5-labelled PB is not enriched in the heart, it would be reasonable to question its role in cardiac remodeling. Furthermore, it would be beneficial to determine whether the enrichment of PB@ in the liver causes any toxic side effects to the liver.\n8. As illustrated in Figure 5, PB has been demonstrated to enhance cardiac function and ventricular remodeling, whilst concurrently reducing immune and inflammatory responses. However, it remains unclear why PB does not demonstrate a robust anti-PANoptosis effect in vivo. What is the precise function of PB in enhancing cardiac performance and preventing ventricular remodeling? Please elucidate the advantages and necessity of utilizing PB@PM.\n9. The authors applied RNA sequencing and protein profiling to ischemic heart tissue in order to demonstrate the inhibitory effects of PANoptosis on PB@PM. It would be beneficial to supplement the immunohistochemistry of representative pan-apoptotic proteins in heart tissue, as well as to ascertain whether downstream inflammatory factors will be affected.", "reviewer_2": "Comment: The manuscript entitled \"Nanoparticles Targeting Multiple PANoptosome-Mediated PANoptosis for Myocardial Ischemia-Reperfusion Injury Therapy\" is novel and creative. The results found that PB@PM as an effective PANoptosis inhibitor and revealing its mechanism of action in MIRI therapy. I think this article can be accepted after minor revisions.Some suggested modifications are as follows:\n1.The title should indicate that this nanomaterial is PB@PM;\n2.During in vivo experiments, PB and PB@PM What is the concentration determined based on?\n3.Supplementary image: In all bar charts, the horizontal line representing the difference between the two groups needs to be further away from the error bar\n4.Please supplement the contributions of other authors\n5. Please check the format of the full-text references, such as reference 22, Qi Z, Zhu L, Wang K, Wang N. PANoptosis: Emerging mechanisms and disease implications. Life Sci. 2023;333:122158.\n6.The section of results should be supplemented with ns: P > 0.05\n7.In Fig.S11, IL-10 and TGF-β are both M2 macrophage markers and can promote extracellular matrix repair, supplementing TGF-β data.", "reviewer_3": "This study by Xu et. al innovatively proposes a PANoptosis inhibition strategy based on nano-Prussian blue (PB) to address the clinical challenge of myocardial ischemia-reperfusion injury (MIRI). Firstly, the authors defined MIRI as a PANoptosis-related disease using single-cell sequencing analysis. Subsequently, the research team used molecular dynamics simulations to confirm the binding ability of PB to PANoptosis key proteins RIPK1, ZBP1 and AIM2, thereby comprehensively inhibiting PANoptosis. On this basis, the authors further constructed platelet membrane-modified PB nanoparticles (PB@PM), which successfully realized the targeting of myocardial injury zone. As a PANoptosis inhibitor, PB@PM significantly improved cardiac function in the MIRI mouse model and promoted extracellular matrix repair and neovascularization. The mechanism of PB@PM in inhibiting PANoptosis and the crosstalk between pyroptosis, apoptosis, and necroptosis was comprehensively revealed through single-cell sequencing, molecular biology techniques, and transcriptome sequencing analysis. Overall, this is a very interesting and well-researched study that can appeal to readers from a wide range of fields including cardiovascular, nanomedicine and biomaterials. The experimental design, description, and implementation are reasonable, and the overall interpretation is also reasonable. This study may provide a framework for studying nanobiological interactions to uncover broader nanomaterial mechanisms. I think this study is suitable for publication in Nature Communications, after addressing the following questions:\n1.Molecular dynamics simulations demonstrated the binding of PB to the target protein. This is the result of the simulation. The author should provide experimental data on PB binding to target proteins, such as surface plasmon resonance.\n2.The authors should add the safety of the nanomedicine, such as pathology tests and blood indicators.\n3.In Figure 5I, the image is too small and it is recommended to highlight the key signals.\n4.In cellular experiments, in addition to the three key proteins (RIPK1, ZBP1 and AIM2), the authors also needed to select some key downstream pyroptosis, apoptosis, and necroptosis-related proteins for validation.\n5.Some spelling errors need to be corrected. It looks like some of the uploaded videos are named incorrectly (ZBP1 and PB, RIPK1 and PB) and need to be corrected. “RIPK1 PANoptosome” is incorrectly written in Figure 1.\n6.In ex vivo and in vivo therapeutic experiments, what was the main basis for the authors in defining treatment time points and concentrations?\n7.The XPS data requires peak splitting processing.\n8.The authors should appropriately describe the remaining limitations of the study."}, "author_response": "Comprehensive point-by-point response to reviewers' comments\nReviewer #1 (Remarks to the Author):\nXu et al. devised an innovative method for synthesizing Prussian blue (PB)\nnanoparticles, which effectively inhibit PANoptosis, thereby comprehensively blocking\npyroptosis, apoptosis, and necroptosis and preventing their crosstalk in the treatment of\nmyocardial ischemia-reperfusion injury (MIRI). The concept is OK, some revision\nshould be done to improve the quality of this manuscript.\nResponse: Dear reviewer, we express our sincere gratitude for your meticulous\nevaluation and acknowledge your recognition of our investigative efforts. We have\nrigorously evaluated your valuable suggestions and comments and have implemented\nsubstantive revisions throughout the manuscript.\nComment 1. The authors' assertion that myocardial ischemia-reperfusion injury (MIRI)\nis closely related to PANoptosis by single-cell sequencing alone lacks sufficient\nevidence. The genes involved in PANoptosis are similar to those involved in pyroptosis,\napoptosis, and necrotic apoptosis, which makes it difficult to distinguish between the\ndifferent forms of cell death. Furthermore, the hallmark of PANoptosis is the assembly\nand activation of the PANoptosome, which has yet to be directly characterised. The\nstudy lacks direct characterization of the PANoptosome.\nResponse: We appreciate the reviewer’s insightful comments. While PANoptosis\nintegrates molecular components of pyroptosis, apoptosis, and necroptosis, its unique\nhallmark lies in the assembly of PANoptosomes, which orchestrate crosstalk among\nthese pathways. To substantiate the link between MIRI and PANoptosis beyond single-\ncell sequencing, we performed the following experiments:\n(1) Human Heart Single-Nuclear Transcriptomics: Analysis of human heart samples\nrevealed significant upregulation of PANoptosis-related genes (e.g., AIM2, ZBP1,\nRIPK1, NLRP3, caspases 1/3/8, GSDMD, MLKL) in cardiomyocytes of the ischemic\nzone (IZ), with apoptosis being the dominant form (Fig. 2F, S3–S5). Subcluster analysis\nfurther identified cardiomyocyte subpopulations (e.g., clusters 1, 2, 4) with elevated\nPANoptosis activity, correlating with cell loss post-ischemia (Fig. 2G–K).\n(2) In Vivo Validation in MIRI Mice: Transcriptomic and proteomic analyses\ndemonstrated that MIRI upregulated PANoptosome components, including AIM2,\nZBP1, RIPK1, NLRP3, ASC, FADD, cleaved caspases (1/3/8), and phosphorylated\nRIPK3/MLKL (Fig. 6J, S18–S19). PB@PM treatment significantly suppressed these\nmarkers, confirming PANoptosome activation in MIRI and its inhibition by PB@PM.\n(3) In Vitro Co-Localization and Functional Assays: Fluorescence imaging confirmed\nPB’s co-localization with AIM2, ZBP1, and phosphorylated RIPK1 in cardiomyocytes\n(Fig. S20A). Overexpression of AIM2/ZBP1/RIPK1 in HL-1 cells showed that PB\ncounteracted OGD/R-induced upregulation of these proteins and their phosphorylated\nforms (Fig. S20B). Additionally, PB@PM rescued OGD/R-induced mitochondrial\ndysfunction and reduced apoptosis, pyroptotic membrane pores, and necroptotic\nmarkers (e.g., p-MLKL) (Fig. S21).\n(3) Combination Therapy Validation: Co-treatment with inhibitors of pyroptosis\n(Disulfiram), apoptosis (Z-VAD-FMK), and necroptosis (Necrostatin-1) mimicked\nPB@PM’s therapeutic effect, highlighting PANoptosis crosstalk in MIRI (Fig. S21D).\nThese findings collectively demonstrate that MIRI activates multiple PANoptosomes\n(AIM2, ZBP1, RIPK1) and that PB@PM broadly inhibits their assembly and\ndownstream death pathways.\nFig.S18 PB@PM inhibits PANoptosis-related protein activation. Semi-quantitative\nanalysis of AIM2, ZBP1, Pyrin, ASC, FADD, Bax, and Bcl-2, phosphorylated/total\nRIPK1/RIPK3/MLKL, and cleaved/total caspase 1/3/8 and GSDMD in ischemic\nmyocardium on 3 days post-I/R or sham surgery (n = 5 biologically independent\nreplicates). Data: mean ± SD. Significance: one-way ANOVA with Tukey’s post-hoc\ntest (*P < 0.05, **P < 0.01, ***P < 0.001; ns, P > 0.05).\nFig.S19 PANoptosis-related protein activation in peri-infarct myocardium. (A)\nImmunohistochemical staining of AIM2, ZBP1, phosphorylated MLKL (p-MLKL),\nand cleaved caspase 8 in peri-infarct regions (n = 5 biologically independent replicates).\nScale bar: 50 μm. (B) Immunofluorescence and quantification of phosphorylated\nRIPK1/3 (p-RIPK1/3), and cleaved caspase 1 in peri-infarct zones (n = 5 biologically\nindependent replicates). Scale bar: 50 μm. Data: mean ± SD. Significance: one-way\nANOVA with Tukey’s post-hoc test (*P < 0.05, **P < 0.01, and ***P < 0.001).\nFig.S20 PB suppresses PANoptosis in HL-1 cells under OGD/R stress. (A) Co-\nlocalization of FITC-labeled PB (green) with AIM2, ZBP, and p-RIPK1 (red) in\nOGD/R-induced HL-1 cells. Scale bar: 50 μm. (B) Western blot analysis of AIM2,\nZBP1, RIPK1 and p-RIPK1 levels in HL-1 cells overexpressing PANoptosis proteins\nunder normoxia or OGD/R (n = 4 biologically independent replicates). Data: mean ±\nSD. Significance: one-way ANOVA with Tukey correction (*P < 0.05, **P < 0.01,\n***P < 0.001; ns, P > 0.05).\nFig.S21 PB@PM inhibits PANoptosis in vitro. (A) Cytotoxicity of PB@PM in HL-1\ncells (CCK-8; n = 3 biologically independent replicates). (B) Cell viability of OGD/R-\ninduced HL-1 cells after different treatments (n = 5-6 biologically independent\nreplicates). (C) Calcein-AM/PI (live/dead) staining of OGD/R injured HL-1 cells. Scale\nbar: 50 μm. (D) Synergistic effects of specific cell dead inhibitors (Necrostatin-1,\nDisulfiram, Z-VAD-FMK, Necrostatin-1+Disulfiram+Z-VAD-FMK) versus PB (n = 7\nbiologically independent replicates). (E) Flow cytometry analysis of HL-1 cells stained\nwith Annexin V-FITC and PI. (F) TUNEL staining (n = 3 biologically independent\nreplicates). Scale bar: 100 μm. (G) Tubulin staining showing OGD/R-induced\nmorphological changes in HL-1 cells (n = 3 biologically independent replicates). Scale\nbar: 100 μm. (H) SEM images of membrane perforations in OGD/R-induced HL-1 cells\npost-treatment (n = 3 biologically independent replicates). (I-J) Western blot analysis\nof PANoptosis markers in OGD/R- induced HL-1 cells (n = 3 biologically independent\nreplicates). Data are expressed as: mean ± SD. Significance: one-way ANOVA with\nTukey correction (*P < 0.05, **P < 0.01, ***P < 0.001; ns, P > 0.05).\nComment 2. In light of the assumption that PANoptosis can be triggered by MIRI, it is\nimperative for the authors to elucidate the mechanism by which PANoptosis is initiated\nin the context of MIRI. This is a pivotal step in identifying the specific PANoptosis\npathway that is activated, given that the modes of activation and triggering mechanisms\nof different PANoptosomes vary.\nResponse: Thank you for this critical question. We have elucidated the mechanism of\nPANoptosis initiation in MIRI by integrating mitochondrial stress, immune-\ninflammatory signaling, and molecular characterization of PANoptosome activation.\n(1) Mitochondrial Stress and mtDNA Release Activate AIM2 and ZBP1 PANoptosomes:\nDuring MIRI, ischemia-reperfusion induces mitochondrial oxidative damage and\nrupture, leading to mtDNA leakage into the cytosol (Fig. S25). Cytosolic mtDNA\nactivates the cGAS-STING pathway (Fig. S13, S23), which promotes interferon\nsignaling and primes AIM2 and ZBP1 PANoptosomes. Specifically: 1AIM2\nPANoptosome: Cytosolic mtDNA directly binds to the HIN domain of AIM2, relieving\nits autoinhibition and enabling ASC recruitment, caspase-1 activation, and pyroptosis\n(Fig. 3AF, S19A). 2ZBP1 PANoptosome: ZBP1 senses mtDNA via its Zα domains,\ntriggering RIPK3-caspase-8 complex formation and necroptosis (Fig. 3G-I, S19B).\n(2) Inflammatory Cytokines (e.g., TNF-α) Activate RIPK1 PANoptosome: MIRI-\ninduced TNF-α release (Fig. S13) activates RIPK1 kinase activity through TNFR1\nsignaling. Phosphorylated RIPK1 recruits FADD, caspase-8, and NLRP3, forming the\nRIPK1 PANoptosome to drive apoptosis and pyroptosis (Fig. 3J-L, S19B).\n(3) Crosstalk Between Pathways: mtDNA-STING signaling and TNF-α synergistically\namplify inflammation, creating a feedforward loop that sustains PANoptosis (Fig. 6F-\nG). RNA-seq and WB confirmed upregulation of AIM2, ZBP1, RIPK1, and\ndownstream effectors (caspase-1/3/8, MLKL, GSDMD) in MIRI mice, which were\nsuppressed by PB@PM (Fig. 6J, S18-19).\n(4) Experimental Validation: 1In vitro: OGD/R increased cytosolic mtDNA (JC-1\ndepolarization, Fig. S26) and TNF-α (ELISA, Fig. S12), while PB@PM blocked these\neffects. 2Co-localization: PB@PM directly bound AIM2, ZBP1, and p-RIPK1 in HL-\n1 cells (Fig. S20A). 3Combination inhibitors: Simultaneous inhibition of pyroptosis,\napoptosis, and necroptosis mimicked PB@PM’s efficacy, confirming PANoptosis\ncrosstalk (Fig. S21D).\nThese findings align with prior studies linking mtDNA-STING to AIM2/ZBP1\nactivation and TNF-α to RIPK1 signaling in inflammatory cell death. Our work\nuniquely demonstrates their convergence in MIRI and validates PB@PM as a multi-\ntarget inhibitor of PANoptosomes. This mechanism is further illustrated in Fig. 1 and\nsupported by transcriptomic, proteomic, and imaging data throughout the manuscript.\nComment 3. It is not sufficient to conclude that PB can inhibit the formation of multiple\nPANoptosomes based on the evidence that PB reacts with AIM2, ZBP1, and RIPK1.\nThe core components of the four PANoptosomes are highly similar, with AIM2, ZBP1\nand RIPK1 representing the core components of the AIM2-PANoptosome. Therefore,\nit is unclear how the authors can claim to inhibit multiple PANoptosomes\nsimultaneously. Once more, we propose that the authors elucidate the specific type of\nPANoptosis precipitated by MIRI.\nResponse: We thank the reviewer for raising this critical point. While PANoptosomes\nshare overlapping components, their assembly and activation are context-dependent.\nOur data clarify that MIRI triggers three distinct PANoptosomes:\n(1) AIM2 PANoptosome: Activated by mitochondrial DNA (mtDNA) release and\nSTING signaling, it recruits AIM2, Pyrin, ZBP1, ASC, FADD, RIPK1, RIPK3, and\ncaspase 8 (Fig. 6J). PB@PM suppressed AIM2, Pyrin, and downstream cleaved\ncaspases (Figs. 6J, S18–S19).\n(2) ZBP1 PANoptosome: Driven by ZBP1 sensing of mtDNA, it recruits NLRP3, ASC,\nRIPK1/3, and caspases 1/8. PB@PM reduced ZBP1, NLRP3, and caspase 1 cleavage\n(Figs. 6J, S18-19).\n(3) RIPK1 PANoptosome: Activated by TNF-α and mitochondrial stress, it involves\nRIPK1, FADD, NLRP3, and caspases 1/8. PB@PM attenuated RIPK1 phosphorylation\nand downstream effectors (Figs. 6J, S18-19).\nMechanistic Validation:\n(1) Molecular Dynamics (MD) Simulations: PB stably bound AIM2’s HIN domain\n(critical for DNA sensing), ZBP1’s charged residues (essential for DNA binding), and\nRIPK1’s helical domain (key for kinase activity) (Fig. 3).\n(2) Surface Plasmon Resonance (SPR): PB exhibited strong binding affinity to ZBP1\n(KD=3.04×10−9 M) and RIPK1 (KD=1.15×10−8 M) (Fig. R1).\n(3) Functional Overlap: PB@PM inhibited all three PANoptosomessimultaneously,as\nshown by transcriptomic and proteomic suppression of their core components (Fig. 6,\nS18–S19).\nThus, MIRI precipitates PANoptosis via concurrent activation of AIM2-, ZBP1-, and\nRIPK1-driven PANoptosomes, and PB@PM comprehensively blocks their assembly\nby targeting key upstream sensors. Our integrated omics, molecular, and functional\nanalyses provide robust evidence that MIRI is a PANoptosis-driven pathology\ninvolving multiple PANoptosomes. PB@PM’s multitarget inhibition of AIM2, ZBP1,\nand RIPK1 disrupts PANoptosome assembly, offering a novel therapeutic strategy for\nMIRI. We have revised the text to clarify these points and cited supporting data\nthroughout the manuscript.\nFig. 6: PB@PM comprehensively blocks the crosstalk between pyroptosis,\napoptosis, and necroptosis in vivo. A PCA of RNA-seq data (Sham: orange; MIRI:\ngreen; PB@PM: blue; n = 5 biologically independent replicates). B Volcano plot of\ndifferentially expressed genes (DEGs; PB@PM vs. MIRI: red/blue =\nup/downregulated). C Venn diagram of shared DEGs between Sham-MIRI and\nPB@PM-MIRI comparisons. D GSVA of pathway enrichment across groups. E KEGG\npathway enrichment of downregulated genes in PB@PM vs. MIRI. F, G GSEA\nnetworks (F) and protein interaction networks (G) for pyroptosis, apoptosis, and\nnecroptosis. H GSEA plots showing significant downward trends of pyroptosis,\napoptosis, and necroptosis in the PB@PM group compared to the MIRI group. I\nPearson correlation between pyroptosis, apoptosis, and necroptosis in the Sham (C),\nMIRI (I), and PB@PM (T) groups. J Western blot of PANoptosis-related proteins\n(AIM2, ZBP1, Pyrin, ASC, FADD, Bax, Bcl-2, phosphorylated/total\nRIPK1/RIPK3/MLKL, cleaved/total caspase-1/3/8 and GSDMD) in ischemic\nmyocardium (day 3; n = 5 biologically independent replicates).\nFig.S18 PB@PM inhibits PANoptosis-related protein activation. Semi-quantitative\nanalysis of AIM2, ZBP1, Pyrin, ASC, FADD, Bax, and Bcl-2, phosphorylated/total\nRIPK1/RIPK3/MLKL, and cleaved/total caspase 1/3/8 and GSDMD in ischemic\nmyocardium on 3 days post-I/R or sham surgery (n = 5 biologically independent\nreplicates). Data: mean ± SD. Significance: one-way ANOVA with Tukey’s post-hoc\ntest (*P < 0.05, **P < 0.01, ***P < 0.001; ns, P > 0.05).\nFig.S19 PANoptosis-related protein activation in peri-infarct myocardium. (A)\nImmunohistochemical staining of AIM2, ZBP1, phosphorylated MLKL (p-MLKL),\nand cleaved caspase 8 in peri-infarct regions (n = 5 biologically independent replicates).\nScale bar: 50 μm. (B) Immunofluorescence and quantification of phosphorylated\nRIPK1/3 (p-RIPK1/3), and cleaved caspase 1 in peri-infarct zones (n = 5 biologically\nindependent replicates). Scale bar: 50 μm. Data: mean ± SD. Significance: one-way\nANOVA with Tukey’s post-hoc test (*P < 0.05, **P < 0.01, and ***P < 0.001).\nFig. 3: Computational simulation of the interaction between PB and PANoptosis-\nrelated proteins. A Initial and final snapshots (200 ns) of the PB-AIM2HIN simulation\nsystem. Interaction interfaces are highlighted (blue/yellow boxes). B, C The residue\ncontact detail of two chains of AIM2HIN with PB is highlighted on the left snapshots. D,\nE The interaction energy analysis of these contact residues of AIM2HIN with PB. F The\ninteraction energy between PB and proteins in PB-AIM2 HIN changes with time. G–L\nThe conformation images of the PB-ZBP1 (G) and PB-RIPK1 (J) systems at the end\nof the simulation (t = 200 ns) and the residue contact details. The interaction energy\nanalysis of the contact residues of ZBP1 (H) and RIPK1 (K) with PB. The interaction\nenergy between PB and proteins in PB-ZBP1 (I) and PB-RIPK1 (L) changes with time.\nData: mean ± SD.\nFig. R1 Surface plasmon resonance (SPR) analysis showing the binding affinity of PB\nto ZBP1 (A) and RIPK1 (B).\nComment 4. The article's primary focus is on sequencing and molecular modelling,\nwhich illustrate the inhibitory effect of PB on PANoptosis, which seriously lacks\nexperimental validation and molecular mechanism. It would be advisable to confirm\nthe inhibitory effect of PB on the assembly of the PANoptosome directly, for example\nthrough fluorescence co-localization or immunoprecipitation experiments.\nResponse: We sincerely thank the reviewer for this critical feedback. To\ncomprehensively address the inhibitory effects of PB on PANoptosis and strengthen\nmechanistic validation, we conducted additional extensive molecular and cellular\nexperiments.\n1. Fluorescence Co-localization and Overexpression Studies: (1) Lentiviral\noverexpression plasmids targeting AIM2, ZBP1, and RIPK1 were constructed\n(Supplementary Material 1); (2) Fluorescence imaging in oxygen-glucose\ndeprivation/reperfusion (OGD/R)-injured HL-1 cells demonstrated significant co-\nlocalization of PB with AIM2, ZBP1, and phosphorylated RIPK1 (Supplementary Fig.\n20A); (3) Western blot analysis revealed that PB effectively suppressed OGD/R-\ninduced overexpression of AIM2 and ZBP1, while attenuating RIPK1 phosphorylation\n(P < 0.01) without altering total RIPK1 levels (Supplementary Fig. 20B).\n2. In Vivo Validation of PANoptosis Inhibition: (1) In MIRI mice, PANoptosis\nactivation was evidenced by elevated levels of active AIM2, ZBP1, Pyrin, ASC, FADD,\napoptotic markers (cleaved caspases 3/8, Bax/Bcl-2), necroptosis markers\n(RIPK1/RIPK3/MLKL), and pyroptosis markers (caspase 1, NLRP3, GSDMD) (Fig.\n6J, Supplementary Figs. 18–19). (2) PB@PM treatment significantly downregulated\nthese components across all three PANoptosomes: RIPK1 PANoptosome: RIPK1,\nNLRP3, ASC, RIPK3, caspase 1/8; ZBP1 PANoptosome: ZBP1, NLRP3, ASC,\nRIPK3, caspase 1/8; AIM2 PANoptosome: AIM2, Pyrin, ZBP1, ASC, FADD,\nRIPK1/RIPK3, caspase 8 (Fig. 6J).\n3. In Vitro Functional Validation: (1) CCK-8 and Calcein AM/PI assays confirmed\nthat PB@PM mitigated OGD/R-induced HL-1 cell death (Supplementary Fig. 21A–\nC); (2) Triple inhibition of necroptosis (Necrostatin-1), pyroptosis (Disulfiram), and\napoptosis (Z-VAD-FMK) achieved comparable efficacy to PB monotherapy,\nconfirming crosstalk suppression (Supplementary Fig. 21D); (3) Annexin V/PI and\nTUNEL assays demonstrated reduced apoptosis (Supplementary Fig. 21E,F), while\nmorphological and SEM analyses confirmed suppression of pyroptotic membrane\nrupture (Supplementary Fig. 21G,H).\n4. Mechanistic Confirmation: PB@PM inhibited OGD/R-induced upregulation of\ncleaved caspases 1/8, GSDMD, and phosphorylated MLKL in HL-1 cells\n(Supplementary Fig. 21I,J).\nThese results collectively demonstrate that PB directly interacts with key\nPANoptosome components, disrupts their assembly, and comprehensively inhibits\nPANoptosis execution. We have incorporated these findings into the revised manuscript\nto strengthen mechanistic clarity.\nThank you for your insightful suggestion, which has significantly enhanced the rigor\nof our study.\nComment 5. Please explain the rationale behind selecting the third day after surgery as\nthe time point for tissue protein detection. Does the expression of PANoptosis-related\nproteins exhibit a greater degree of significance at either a shorter or longer time point?\nResponse: Thank you very much for your kind comment. The third postoperative day\nwas selected based on the pathological progression of MIRI in mice. Acute immune-\ninflammatory responses and cell death markers peak during the first 3 days post-\nischemia, coinciding with maximal PANoptosis activation. To validate this, we\nperformed time-course WB analysis of PANoptosis-related proteins (AIM2, ZBP1,\nRIPK1, cleaved caspases, and MLKL) at days 1, 3, and 7. As shown in the\nSupplementary WB data (Fig. R2), these proteins exhibited the most significant\nupregulation on day 3, aligning with prior studies in MIRI models[1,2]. By day 7,\nmyocardial repair processes dominate, reducing PANoptosis-related protein levels.\nThus, day 3 represents the optimal window to assess PANoptosis inhibition by PB@PM.\nReferences\n[1] Yang K, Gao R, Chen H, Hu J, Zhang P, Wei X, Shi J, Chen Y, Zhang L, Chen J, Lyu Y, Dong\nZ, Wei W, Hu K, Guo Y, Ge J, Sun A. Myocardial reperfusion injury exacerbation due to ALDH2\ndeficiency is mediated by neutrophil extracellular traps and prevented by leukotriene C4 inhibition.\nEur Heart J. 2024 May 13;45(18):1662-1680.\n[2] Xing M, Jiang Y, Bi W, Gao L, Zhou YL, Rao SL, Ma LL, Zhang ZW, Yang HT, Chang J.\nStrontium ions protect hearts against myocardial ischemia/reperfusion injury. Sci Adv. 2021 Jan\n15;7(3):eabe0726. doi: 10.1126/sciadv.abe0726.\nFigure R2 Representative western blot image and semi-quantitative analysis showing\nthe protein expression of AIM2, ZBP1, P-RIPK1, RIPK1, Caspase 1, Cle-caspase 8,\nCaspase 8, Cle-GSDMD, GSDMD, P-MLKL and MLKL at various time points after\nmyocardial ischemia surgery in mice (n = 3).Data are expressed as mean ± SD. P values\nare calculated using one-way ANOVA with Tukey correction. *P < 0.05, **P < 0.01,\nand ***P < 0.001.\nComment 6. MIRI is a common occurrence in cardiomyocytes situated within the\ncoronary artery reperfusion zone. How do the authors distinguish these cells in vivo in\norder to conduct the subsequent step of the experiment?\nResponse: We appreciate the reviewer’s attention to methodological rigor. While direct\nin vivo identification of reperfusion-injured cardiomyocytes remains technically\nchallenging, our approach ensures precise sampling:\n(1) Model Validation: The left anterior descending (LAD) artery ligation model (45 min\nischemia/reperfusion) is a well-established protocol for MIRI studies[1,2]. Intraoperative\nvisual confirmation of myocardial blanching distal to the ligation site demarcates the\nischemic-reperfused region.\n(2) Tissue Sampling: Post-sacrifice, the LV anterior wall (supplied by the LAD) was\nsystematically excised for molecular analyses. This region-specific sampling is widely\nadopted in MIRI research to ensure consistency[1,2].\n(3) Limitations and Mitigation: While spatial resolution at the single-cell level is limited\nin vivo, our single-nuclear RNA sequencing of human infarct cores (Fig. 2)\ncorroborated cardiomyocyte-specific PANoptosis activation, strengthening\ntranslational relevance.\nReferences\n[1] Yang K, Gao R, Chen H, Hu J, Zhang P, Wei X, Shi J, Chen Y, Zhang L, Chen J, Lyu Y, Dong\nZ, Wei W, Hu K, Guo Y, Ge J, Sun A. Myocardial reperfusion injury exacerbation due to ALDH2\ndeficiency is mediated by neutrophil extracellular traps and prevented by leukotriene C4 inhibition.\nEur Heart J. 2024 May 13;45(18):1662-1680.\n[2] Fan Q, Tao R, Zhang H, Xie H, Lu L, Wang T, Su M, Hu J, Zhang Q, Chen Q, Iwakura Y, Shen\nW, Zhang R, Yan X. Dectin-1 Contributes to Myocardial Ischemia/Reperfusion Injury by Regulating\nMacrophage Polarization and Neutrophil Infiltration. Circulation. 2019 Jan 29;139(5):663-678. doi:\n10.1161/CIRCULATIONAHA.118.036044.\n7. The authors conducted fluorescence imaging of the principal organs of MIRI mice at\n12 and 24 hours post-injection of Cy5.5-labeled PB@PM. Nevertheless, the findings\nindicated that only a minimal quantity of PB@PM remained within the heart at the 24-\nhour mark. It would be valuable to ascertain whether any remaining PB@PM is present\nin the heart after 28 days. Given that Cy5.5-labelled PB is not enriched in the heart, it\nwould be reasonable to question its role in cardiac remodeling. Furthermore, it would\nbe beneficial to determine whether the enrichment of PB@ in the liver causes any toxic\nside effects to the liver.\nResponse: We appreciate the reviewer’s insightful comment. Healthy wild-type (WT)\nmice received intravenous Cy5.5-labeled PB as the control cohort, while MIRI models\nwere administered either Cy5.5-labeled PB or PB@PM. Postmortem ex vivo\nfluorescence imaging of major organs was systematically performed to facilitate\nintegrated analysis of nanomedicine biodistribution and targeting efficacy. The\nsemiquantitative fluorescence data presented reflect comparative pharmacokinetic\nprofiles across multiple organ systems. It is important to note that systemically\nadministered untargeted agents predominantly undergo hepatic/renal metabolic\nclearance, whereas cardiac tissue typically exhibits negligible retention compared to\nthese metabolic organs, leading to limited fluorescence visualization. Our findings\ndemonstrate that PB@PM achieves sustained cardiac enrichment exceeding 24 hours,\nconfirming its superior targeting performance. In response to your observations, we\nconducted extended pharmacokinetic studies evaluating PB@PM's cardiac retention in\ninjured myocardium. These experiments revealed that a single intravenous\nadministration (administered at 1-hour post-ischemic surgery) maintained targeted\ncardiac localization for a minimum of five days (Fig. R3).\nRegarding therapeutic implementation, our study employed a two-dose\nadministration protocol (1-hour and 24-hour post-surgery) to ensure sustained\ntherapeutic efficacy during both the acute injury phase and subsequent critical repair\nperiod. While quantitative fluorescence imaging did not reveal significant differences\nbetween treatment groups, PB-containing formulations in the MIRI+PB cohort may\nstill exert cardioprotective effects through paracellular diffusion mechanisms and\npenetration of injured vascular endothelium under physiological conditions. Notably,\ndespite lacking intrinsic targeting ligands, PB demonstrated measurable therapeutic\nactivity in this preclinical model.\nBased on the reviewers' recommendations, we conducted additional comprehensive\nevaluations of the in vivo biosafety profile of PB@PM. Comprehensive hematological\nassessments, serum biochemical analyses, and histopathological examinations were\nperformed on wild-type mice at 7- and 14-day post-injection timepoints following\nadministration of PB@PM at 5 mg/kg body weight. Both hematological examinations\nand H&E staining results demonstrated that PB@PM treatment did not induce\ndetectable adverse effects, evidenced by absence of significant histopathological\nalterations in major organs (cardiac, hepatic, splenic, pulmonary, and renal tissues) and\nmaintenance of normal hematological parameters and biochemical indices (Fig. S11,\nTable S2).\nFig. R3 Typical ex vivo fluorescence images and quantitative analysis of the\nfluorescence intensities of damaged hearts at day 1, day 3, day 5, day 7, day 14, and\nday 28 after administration of Cy5.5-labeled PB@PM (n = 3). Data are expressed as\nmean ± SD. P values are calculated using one-way ANOVA with Tukey correction. *P\n< 0.05, ***P < 0.001, and ns means no significance.\nFig.S11 Biosafety of PB@PM. (A) Hematoxylin and eosin (H&E)-stained sections of\nheart, liver, spleen, lung, and kidney tissues from control and PB@PM-treated mice.\nScale bar: 100 μm. (B) Blood routine indexes, and blood biochemical parameters:\nalanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP),\nurea and creatinine (CREA).\n8. As illustrated in Figure 5, PB has been demonstrated to enhance cardiac function and\nventricular remodeling, whilst concurrently reducing immune and inflammatory\nresponses. However, it remains unclear why PB does not demonstrate a robust anti-\nPANoptosis effect in vivo. What is the precise function of PB in enhancing cardiac\nperformance and preventing ventricular remodeling? Please elucidate the advantages\nand necessity of utilizing PB@PM.\nResponse: We appreciate the reviewer's insightful comments. In this study targeting\nMIRI, the PB@PM formulation equipped PB nanoparticles with targeted capabilities\nwhile conferring enhanced biosafety, prolonged systemic circulation, and innate\nimmune evasion through platelet membrane modification. These bioengineering\nstrategies preserved the intrinsic physicochemical properties and therapeutic efficacy\nof the core nanomedicine[1, 2]. The synergistic advantages of PB@PM demonstrate\nsuperior therapeutic efficiency and safety profiles, achieving significant therapeutic\noutcomes at reduced dosage levels.\nAs evidenced in Figure 5 and Supplementary Figures S13-16, PB@PM exhibited\nmarkedly enhanced in vivo therapeutic efficacy, evidenced by comprehensive cardiac\nfunctional recovery, attenuated ventricular remodeling, reduced infarct size, and\nsuppression of myocardial inflammatory infiltration and cardiomyocyte hypertrophy.\nNotably, PB@PM promoted extracellular matrix remodeling and angiogenesis while\ndemonstrating superior therapeutic performance compared to pristine PB, which lacked\nplatelet membrane-mediated targeting and consequently exhibited diminished\ntherapeutic impact.\nFurthermore, Figure 6 and Supplementary Figures S18-21 revealed that both\nPB@PM and PB effectively mitigated PANoptosis activation in both preclinical models\nand in vitro systems. However, PB@PM demonstrated enhanced therapeutic potency\nin vivo, consistent with its superior pharmacokinetic profile. Both formulations\nexhibited comparable antioxidant capacity, mitochondrial protection, and inhibition of\npro-inflammatory mediators, though PB@PM maintained these effects with improved\nbiodistribution characteristics.\nReferences\n[1] Feng L, Dou C, Xia Y, Li B, Zhao M, Yu P, Zheng Y, El-Toni AM, Atta NF, Galal A, Cheng Y,\nCai X, Wang Y, Zhang F. Neutrophil-like Cell-Membrane-Coated Nanozyme Therapy for Ischemic\nBrain Damage and Long-Term Neurological Functional Recovery. ACS Nano. 2021 Feb\n23;15(2):2263-2280. doi: 10.1021/acsnano.0c07973.\n[2] Tang L, Yin Y, Liu H, Zhu M, Cao Y, Feng J, Fu C, Li Z, Shu W, Gao J, Liang XJ, Wang W.\nBlood-Brain Barrier-Penetrating and Lesion-Targeting Nanoplatforms Inspired by the\nPathophysiological Features for Synergistic Ischemic Stroke Therapy. Adv Mater. 2024\nMay;36(21):e2312897. doi: 10.1002/adma.202312897.\n9. The authors applied RNA sequencing and protein profiling to ischemic heart tissue\nin order to demonstrate the inhibitory effects of PANoptosis on PB@PM. It would be\nbeneficial to supplement the immunohistochemistry of representative pan-apoptotic\nproteins in heart tissue, as well as to ascertain whether downstream inflammatory\nfactors will be affected.\nResponse: We thank the reviewer for highlighting this aspect. To strengthen our\nfindings:\n(1) Immunohistochemistry (IHC) Validation: IHC of murine heart tissues confirmed\nPB@PM-mediated suppression of PANoptosis markers, including AIM2, ZBP1,\nphosphorylated RIPK1/RIPK3/MLKL, and cleaved caspases 1/8 (P < 0.01 vs. MIRI;\nFig. S19A,B).\n(2) Downstream Inflammatory Modulation: PB@PM significantly reduced pro-\ninflammatory cytokines (TNF-α, IL-1β, IL-6) while elevating anti-inflammatory IL-10\nand TGF-β in ischemic myocardium (P < 0.001; Fig. S13). Mechanistically, PB@PM\ninhibited the TLR4/NF-κB pathway, a key driver of inflammatory crosstalk with\nPANoptosis (Fig. S13).\nThese data collectively demonstrate that PB@PM attenuates PANoptosis-executing\ncomplexes while rebalancing immune-inflammatory homeostasis, providing a multi-\nmodal therapeutic mechanism.\nFig.S13 PB@PM suppresses TLR4/NF-κB signaling in ischemic myocardium.\nWestern blot analysis of NF-kB p65, phosphorylated NF-κB p65 (p-p65), TLR4, TNF-\nα, IL-1β, IL-6, and IL-10 protein levels in ischemic myocardial tissues 3 days post-I/R\nor sham surgery (n = 5 biologically independent replicates). Data: mean ± SD.\nSignificance: one-way ANOVA with Tukey’s post-hoc test (*P < 0.05, **P < 0.01,\n***P < 0.001; ns, P > 0.05).\nReviewer #2 (Remarks to the Author):\nComment: The manuscript entitled \"Nanoparticles Targeting Multiple PANoptosome-\nMediated PANoptosis for Myocardial Ischemia-Reperfusion Injury Therapy\" is novel\nand creative. The results found that PB@PM as an effective PANoptosis inhibitor and\nrevealing its mechanism of action in MIRI therapy. I think this article can be accepted\nafter minor revisions. Some suggested modifications are as follows:\nResponse: Dear Reviewer, we sincerely appreciate your meticulous evaluation and\nconstructive feedback on our manuscript. We have thoroughly considered your\ninsightful suggestions and incorporated comprehensive revisions throughout the text to\nenhance its scientific rigor and clarity.\nComment 1. The title should indicate that this nanomaterial is PB@PM;\nResponse: We appreciate the valuable feedback. Accordingly, the title has been revised\nto \"Prussian blue nanoparticles targeting multiple PANoptosome-mediated PANoptosis\nfor myocardial ischemia-reperfusion injury therapy\" to explicitly specify the\nnanomaterial designation.\nComment 2. During in vivo experiments, PB and PB@PM What is the concentration\ndetermined based on?\nResponse: We thank the reviewers for this important inquiry. The dosing regimen was\nestablished by referencing established methodologies in biomembrane-functionalized\ninorganic nanomedicine development for inflammatory disease management (DOI:\n10.1016/j.mattod.2023.03.024; DOI: 10.1002/advs.202304002) and subsequently\noptimized through iterative pre-experimental evaluations of PB@PM's therapeutic\nefficacy in our MIRI model. Systematic dose-ranging studies will be conducted in\nfuture work to identify the optimal therapeutic window and advance the clinical\ntranslation potential of this platform.\nComment 3. Supplementary image: In all bar charts, the horizontal line representing\nthe difference between the two groups needs to be further away from the error bar.\nResponse: Thank you for this valuable feedback. Following your recommendation, we\nhave systematically reviewed both the manuscript and supplementary materials,\nimplementing adjustments to enhance the visual presentation of bar charts by increasing\nthe distance between the horizontal reference line and error bars.\nComment 4. Please supplement the contributions of other authors.\nResponse: We appreciate your suggestion. The author contributions section has been\nexpanded in the revised manuscript as follows:\n\"L.X., L.J., and R.W. contributed equally to this work. Y.Z., X.C., and B.L. conceived\nand designed the study; Y.Z. and X.C. interpreted the results; L.X., L.J., R.W., and B.L.\nconducted experiments and performed data analysis; R.W. carried out molecular\ndynamics simulation analysis; X.C., B.L., and L.X. drafted the manuscript. All authors\ncollectively discussed the results and provided critical feedback on the manuscript.\"\nComment 5. Please check the format of the full-text references, such as reference 22,\nQi Z, Zhu L, Wang K, Wang N. PANoptosis: Emerging mechanisms and disease\nimplications. Life Sci. 2023;333:122158.\nResponse: We appreciate the reviewer's attention to detail. All bibliographic references\nhave been thoroughly verified and reformatted in strict accordance with the journal's\nstyle guidelines (see revised Reference List).\nComment6. The section of results should be supplemented with ns: P > 0.05\nResponse: Thank you for this valuable suggestion. Statistical significance annotations\n(including instances where P > 0.05) have been systematically incorporated into the\nResults section to enhance methodological transparency.\nComment 7. In Fig.S11, IL-10 and TGF-β are both M2 macrophage markers and can\npromote extracellular matrix repair, supplementing TGF-β data.\nResponse: We acknowledge the importance of comprehensive marker analysis. As\nrequested, we have supplemented the TGF-β expression analysis in the revised Figure\nS12 to provide complete characterization of M2 macrophage-associated cytokines and\ntheir functional implications for extracellular matrix remodeling.\nFig.S12 PB@PM modulates inflammatory cytokine secretion. Enzyme-linked\nimmunosorbent assay (ELISA) quantification of IL-1β, IL-6, TNF-α, IL-10, and TGF-\nβ levels in lipopolysaccharide (LPS)-stimulated RAW264.7 model treated with\nPB@PM (n = 3–4 biologically independent replicates). Data: mean ± SD. Significance:\none-way ANOVA with Tukey’s post-hoc test (*P < 0.05, **P < 0.01, ***P < 0.001).\nReviewer #3 (Remarks to the Author):\nThis study by Xu et. al innovatively proposes a PANoptosis inhibition strategy based\non nano-Prussian blue (PB) to address the clinical challenge of myocardial ischemia-\nreperfusion injury (MIRI). Firstly, the authors defined MIRI as a PANoptosis-related\ndisease using single-cell sequencing analysis. Subsequently, the research team used\nmolecular dynamics simulations to confirm the binding ability of PB to PANoptosis\nkey proteins RIPK1, ZBP1 and AIM2, thereby comprehensively inhibiting PANoptosis.\nOn this basis, the authors further constructed platelet membrane-modified PB\nnanoparticles (PB@PM), which successfully realized the targeting of myocardial injury\nzone. As a PANoptosis inhibitor, PB@PM significantly improved cardiac function in\nthe MIRI mouse model and promoted extracellular matrix repair and neovascularization.\nThe mechanism of PB@PM in inhibiting PANoptosis and the crosstalk between\npyroptosis, apoptosis, and necroptosis was comprehensively revealed through single-\ncell sequencing, molecular biology techniques, and transcriptome sequencing analysis.\nOverall, this is a very interesting and well-researched study that can appeal to readers\nfrom a wide range of fields including cardiovascular, nanomedicine and biomaterials.\nThe experimental design, description, and implementation are reasonable, and the\noverall interpretation is also reasonable. This study may provide a framework for\nstudying nanobiological interactions to uncover broader nanomaterial mechanisms. I\nthink this study is suitable for publication in Nature Communications, after addressing\nthe following questions:\nResponse: Dear Reviewer, we extend our profound appreciation for your scholarly\nevaluation. Your insightful feedback has been rigorously scrutinized and systematically\naddressed through methodical revisions that enhance the academic rigor and\nmethodological precision of our work.\n1.Molecular dynamics simulations demonstrated the binding of PB to the target protein.\nThis is the result of the simulation. The author should provide experimental data on PB\nbinding to target proteins, such as surface plasmon resonance.\nResponse: We appreciate the reviewer's insightful suggestion. To address this point, we\nconducted quantitative surface plasmon resonance (SPR) analysis. The experimental\nresults revealed high-affinity interactions between PB and ZBP1 (KD = 3.04 × 10-9 M)\nand RIPK1 (KD = 1.15 × 10-8 M), confirming specific binding of PB to these two target\nproteins.\nFig. R1 Surface plasmon resonance (SPR) analysis showing the binding affinity of PB\nto ZBP1 (A) and RIPK1 (B).\n2.The authors should add the safety of the nanomedicine, such as pathology tests and\nblood indicators.\nResponse: We appreciate the reviewers' valuable suggestion. In response, we have\nsystematically incorporated comprehensive safety assessments of PB@PM, including\nhematological profiling, serum biochemical analysis, and histopathological\nexaminations at both 7-day and 14-day post-administration time points. Our integrated\nhematological assessments and histopathological evaluations (Fig. S11 and Table S2)\ndemonstrated that PB@PM treatment did not elicit statistically significant\nhistopathological alterations or adverse changes in hematological parameters or serum\nbiochemical indices, thereby confirming the PB@PM's favorable safety profile.\nFig.S11 Biosafety of PB@PM. (A) Hematoxylin and eosin (H&E)-stained sections of\nheart, liver, spleen, lung, and kidney tissues from control and PB@PM-treated mice.\nScale bar: 100 μm. (B) Blood routine indexes, and blood biochemical parameters:\nalanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP),\nurea and creatinine (CREA).\n3.In Figure 5I, the image is too small and it is recommended to highlight the key signals.\nResponse: We sincerely appreciate the reviewer's valuable feedback. As suggested, we\nhave revised Figure 5I through optimized imaging processing techniques to enhance\nvisualization of key positive signals, thereby improving figure clarity and\ninterpretability.\n4. In cellular experiments, in addition to the three key proteins (RIPK1, ZBP1 and\nAIM2), the authors also needed to select some key downstream pyroptosis, apoptosis,\nand necroptosis-related proteins for validation.\nResponse: We appreciate the reviewers' insightful suggestion. To comprehensively\nevaluate the molecular mechanisms, we performed systematic analysis of key\ndownstream effectors in pyroptotic, apoptotic, and necroptotic pathways. Consistent\nwith our in vivo findings, quantitative immunoblot analysis revealed that PB@PM\ntreatment markedly suppressed protein expression levels of AIM2, ZBP1,\nphosphorylated RIPK1, cleaved caspase-1, cleaved caspase-8, cleaved gasdermin D,\nand phosphorylated MLKL in OGD/R-stimulated HL-1 cells (Fig. S21I-J). These data\nprovide further mechanistic evidence for PB@PM's multi-target therapeutic effects\nagainst regulated cell death pathways.\nFig. S21I-J Western blot analysis of PANoptosis markers in OGD/R- induced HL-1\ncells (n = 3-5 biologically independent replicates). Data are expressed as: mean ± SD.\nSignificance: one-way ANOVA with Tukey correction (*P < 0.05, **P < 0.01, ***P <\n0.001; ns, P > 0.05).\n5.Some spelling errors need to be corrected. It looks like some of the uploaded videos\nare named incorrectly (ZBP1 and PB, RIPK1 and PB) and need to be corrected. “RIPK1\nPANoptosome” is incorrectly written in Figure 1.\nResponse: We appreciate the reviewer's meticulous attention to detail. These\ntypographical inaccuracies and nomenclature inconsistencies have been systematically\naddressed in the revised manuscript through comprehensive label verification and\nimplementation of standardized nomenclature protocols.\nFig. 1: PBs act as a novel PANoptosis inhibitor by targeting RIPK1, ZBP1, and AIM2\nto suppress multiple PANoptosomes, effectively blocking crosstalk among pyroptosis,\napoptosis, and necroptosis in PANoptosis-driven diseases. Additionally, PB regulates\nmitochondrial metabolism and immune-inflammatory homeostasis, further disrupting\nmultimodal cell death crosstalk. [Editorial note: Created in BioRender. xu, L. (2026)\nhttps://BioRender.com/9kka4p3]\n6.In ex vivo and in vivo therapeutic experiments, what was the main basis for the\nauthors in defining treatment time points and concentrations?\nResponse: Thanks for the comment. The setting of treatment time points and\nconcentrations referred to the previously published researches on the use of\nbiomembrane-modified inorganic nanomedicines for the treatment of inflammatory\ndiseases (DOI: 10.1016/j.mattod.2023.03.024; DOI: 10.1002/ADVS.202304002), and\nwas adjusted based on the actual therapeutic effects of PB@PM on MIRI observed in\nthe pre-experiments.\n7.The XPS data requires peak splitting processing.\nResponse: We appreciate the reviewer's valuable suggestion. Accordingly, peak\ndeconvolution analysis was conducted on the XPS spectra to enhance spectral\nresolution and facilitate more precise chemical state identification.\nFig.S9B. X-ray Photoelectron Spectrometer (XPS) of PB.\n8.The authors should appropriately describe the remaining limitations of the study.\nResponse: We appreciate the reviewers' valuable feedback. In response, we have\nsupplemented the limitations section with enhanced academic rigor as follows:\n“Nevertheless, this work has limitations. First, whether PB modulates PANoptosis via\nadditional signaling pathways requires further exploration. Second, the regulatory\neffects of PB on non-cardiomyocyte cardiac cells remain unclear. Finally, advancing\nclinical translation necessitates validation in higher-order mammals and\ncomprehensive pharmacokinetic/toxicological profiling of PB in vivo. These insights\nprovide a foundational framework for investigating PANoptosis in diverse pathologies\nand designing next-generation nanoinhibitors.”\nDear Reviewer,\nWe sincerely thank you for your thorough evaluation of our work and for providing\nthese insightful comments. We have carefully considered each point raised and have\nrevised the manuscript accordingly. Below, we provide a point-by-point response to\naddress your concerns.\nReviewer #4 (Remarks to the Author):\nThis study systematically investigated the therapeutic mechanism of Prussian blue\nnanoparticles (PB@PM) targeting PANoptosis in myocardial ischemia-reperfusion\ninjury (MIRI). Through analyzing human cardiac scRNA-seq data, significant\nupregulation of PANoptosis-related genes in MIRI was confirmed, followed by\nmolecular dynamics simulations to predict specific binding between PB and\nPANoptosome core proteins. Further, the authors demonstrated the efficacy of platelet\nmembrane-coated nanoparticles (PB@PM) in suppressing PANoptosis activation and\nassembly in vitro and in vivo. Accordingly, the authors then hypothesized that PB@PM\nconcurrently targets RIPK1/ZBP1/AIM2 within the PANoptosome, disrupting crosstalk\namong pyroptosis, apoptosis, and necroptosis to confer cardioprotection. However, the\nclaim that PB@PM \"blocks PANoptosome assembly\" lacks direct experimental\nvalidation. Besides, this study did not explore whether PB@PM reprograms\nmitochondrial metabolism or inhibits mtDNA release, which are key triggers of\nPANoptosis. Meanwhile, merely downstream moleculars related to PANoptosis were\nobserved while the upstream mechanism regulating PANoptosis was not elucidated.\nGenerally speaking, the innovation of this study was average and the experimental\ndesign was not persuasive enough.\nResponse: We sincerely thank the reviewer for their insightful comments, which have\ngreatly helped us improve the manuscript. In response to the feedback, we have\nstrengthened the study by incorporating additional experimental evidence and\nclarifying the mechanistic insights underlying our findings. Below, we provide a point-\nby-point response to the specific comments:\n1. Concerning the blockade of PANoptosome assembly: We agree with the reviewer\nthat direct visualization of PANoptosome assembly remains technically challenging. To\nprovide more compelling evidence, we performed additional immunofluorescence\nexperiments examining the colocalization of ASC with caspase‑1 and caspase‑8,\nwhich is indicative of PANoptosome formation. The results demonstrate that PB@PM\ntreatment significantly reduced the colocalization of ASC with caspase‑1/8 in OGD/R-\nstimulated HL‑1 cells (Supplementary Fig. 21K,L), supporting the conclusion that\nPB@PM inhibits PANoptosome assembly.\nIn addition, we have accumulated multiple lines of indirect evidence: (1) Molecular\ndynamics simulations revealed high-affinity binding between PB and core\nPANoptosome components (RIPK1, ZBP1, and AIM2HIN), suggesting that PB may\ninterfere with their oligomerization or downstream adaptor recruitment;\n(2) Western\nblot and immunofluorescence analyses consistently showed that PB@PM treatment\nmarkedly reduced the activation (phosphorylation and/or cleavage) of multiple\nPANoptosis-related proteins (RIPK1, RIPK3, MLKL, caspase‑1/3/8, and GSDMD)\nboth in vivo and in vitro. (3) Overexpression of AIM2, ZBP1, or RIPK1 in HL-1 cells\nfurther confirmed that PB suppresses their activation under oxygen–glucose\ndeprivation/reperfusion (OGD/R) stress.\nIn light of these findings, we have moderated our conclusion in the revised\nmanuscript from “blocks assembly” to “suppresses PANoptosis activation” to more\naccurately reflect the evidence.\n2. Regarding mitochondrial metabolism and mtDNA release: We thank the reviewer\nfor highlighting the importance of mitochondrial dysfunction in PANoptosis initiation.\nTo address this, we performed the following assays: (1) JC‑1 staining and flow\ncytometry showed that PB@PM restored mitochondrial membrane potential in\nOGD/R-injured HL‑1 cells (Supplementary Fig. 27A–C). (2) Seahorse XF analysis\ndemonstrated that PB@PM enhanced mitochondrial respiratory capacity\n(Supplementary Fig. 27D–G). (3) Electron spin resonance (ESR) spectroscopy and flow\ncytometry confirmed that PB@PM effectively scavenged mitochondrial reactive\noxygen species (Supplementary Fig. 26).\nAlthough mtDNA release was not directly measured, the observed attenuation of\noxidative stress and improvement in mitochondrial integrity suggest a reduction in\nmitochondrial damage—a known trigger of PANoptosis. We have discussed this\ninference in the revised Discussion section.\n3. On the upstream mechanisms: Our molecular dynamics simulations and binding\nenergy calculations propose a plausible mechanism whereby PB directly engages key\nsensors (AIM2 and ZBP1) and the adaptor protein RIPK1, thereby inhibiting their\nactivation and subsequent recruitment of downstream effectors. This mechanism is\ncorroborated by: (1) The colocalization of PB with AIM2, ZBP1, and phosphorylated\nRIPK1 in cardiomyocytes (Supplementary Fig. 20A). (2) Suppression of PANoptosis\nactivation induced by overexpression of these molecules (Supplementary Fig. 20B).\nWe have expanded the Discussion to more prominently feature this upstream\ntargeting mechanism.\n4. About innovation and experimental design: We believe our study offers several\nnotable innovations: (1) This is the first study to integrate human single-nucleus RNA\nsequencing with molecular dynamics simulations to rationally design and validate a\nnano-therapeutic for PANoptosis inhibition in MIRI. (2) We developed a biomimetic\nplatelet membrane-coated nanoplatform (PB@PM) for targeted delivery and\nPANoptosis suppression. (3) The therapeutic strategy was validated through a multi-\nlevel approach, ranging from in silico predictions to in vivo functional recovery.\nIn response to the reviewer’s suggestions, we have further clarified these points in\nthe text and strengthened the experimental design with additional controls and\nvalidation assays.\nComment 1: The image quality needs improvement. Please add scale bars to the\nultrasound images (Fig 5) and check the scale annotations in other figures.\nResponse: We sincerely thank the reviewer for this constructive suggestion. We have\nnow added clear scale bars to the ultrasound images in Fig. 5 and have thoroughly\nrechecked the scale annotations in all other figures to ensure accuracy and consistency.\nHigh-resolution images have been uploaded to the submission system to guarantee\noptimal clarity and reproducibility.\nFig. 5:PB@PM improves cardiac function and attenuates post-infarct remodeling.\nA–E Echocardiographic analysis of LVEF, LVFS, LVESV, and LVEDV on preoperative\nday 1 and postoperative days 1, 14, and 28. Scale bar: 100 ms. F Representative\nMasson’s trichrome and HE cross-sectional staining in hearts on 28 days post-surgery.\nScale bar: 1 mm. G Fibrosis quantification in hearts 28 days post-surgery. H Evans\nblue/TTC staining and quantitative analysis showing the cardiac cell survival and blood\nperfusion in different groups on day 3 after I/R surgery. Scale bar: 1 mm. I, J TUNEL\n(red) and cTnT (green) co-staining (I) and respective quantification (J) in peri-infarct\nzones (day 3). Scale bar: 1 mm or 50 μm. K, L Representative immunofluorescence\nimages and quantitative analysis of CD86 in peri-infarct regions (day 3). Scale bar: 1\nmm or 50 μm. M, N Representative immunofluorescence staining and quantitative\nanalysis of Ly-6G in peri-infarct regions (day 3). Scale bar: 1 mm or 50 μm. O–R\nRepresentative immunofluorescence staining and quantification analysis of α-\nSMA/cTnT and CD31/cTnT in peri-infarct region (day 28). Scale bar: 1 mm or 50 μm.\nData: mean ± SD of 5 biologically independent replicates. Significance: one-way\nANOVA with Tukey’s post-hoc test (*P < 0.05, **P < 0.01, ***P < 0.001; ns, P > 0.05).\nComment 2: Both the levels of apoptotic caspases and their cleaved active fragments\nare low under normal conditions, while the accumulation of cleaved fragments post\nmyocardial infarction might reduce the total protein levels. Please verify the Western\nblot (WB) data and ensure that both full-length and cleaved fragments are displayed on\nthe same membrane.\nResponse: We appreciate the reviewer’s insight regarding caspase dynamics. We have\nrepeated the Western blot analyses using new myocardial tissue samples from MIRI\nmodel mice. Both full-length and cleaved forms of caspase‑1, caspase‑3, and caspase‑8\nwere detected on the same membrane under identical conditions. The updated results\nconfirm that PB@PM treatment attenuates caspase cleavage and activation in ischemic\nmyocardium. Immunohistochemical staining further supports these findings\n(Supplementary Fig. 19).Thank you once again for your insightful comments.\nComment 3: In Fig 3, all the molecular dynamics simulations for Prussian blue (PB)\nbinding to PANoptosis-related molecules used a 200-ns timeframe. However, the\nRMSD of the PB-RIPK1 complex did not reach equilibrium by 200 ns. Please\nreevaluate whether 200 ns is suitable to the PB-RIPK1 system.\nResponse: We thank the reviewer for raising this point. We reanalyzed the PB–RIPK1\ntrajectory and found that although the RMSD of RIPK1 showed minor fluctuations in\nthe final 25 ns, the interaction energy between PB and RIPK1 remained stable during\nthe last 50 ns (−73.17 ± 6.31 kJ/mol during 150–175 ns; −74.77 ± 5.15 kJ/mol during\n175–200 ns) , indicating that the binding interface had reached dynamic equilibrium.\nThe RMSD fluctuations are likely due to the inherent flexibility of loop regions in\nRIPK1. We have included a more detailed analysis of RMSD and radius of gyration\n(Rg) in Supplementary Fig. 7 and revised the Results section accordingly.\nFig.S7 Molecular dynamics simulation stability metrics. Time-dependent root mean\nsquare deviation (RMSD) and radius of gyration (Rg) profiles for PB-AIM2HIN, PB-\nAIM2PYD, PB-ZBP1, and PB-RIPK1 simulated systems.\nComment 4: The efficiency of PANoptosis inhibition by PB@PM was similar to the\nother three specific programmed cell death inhibitors. What is the advantage of using\nPB@PM over these inhibitors? Please clarify the unique therapeutic value of PB@PM\nin PANoptosis modulation.\nResponse: We sincerely thank the reviewer for raising this critical point regarding the\ncomparative advantage of PB@PM. While our data indeed show that the efficiency of\nPANoptosis inhibition by PB@PM is comparable to the combination of three specific\nprogrammed cell death inhibitors, PB@PM offers several distinct and translationally\nsignificant advantages that underscore its unique therapeutic value.\nFirst, the core therapeutic superiority of PB@PM lies in its ability to concurrently and\ncomprehensively suppress pyroptosis, apoptosis, and necroptosis within a single agent.\nAs highlighted by the reviewer and supported by emerging evidence, extensive\nmolecular crosstalk exists among these pathways. Monotherapeutic inhibition of one\npathway often fails due to compensatory activation of alternative death mechanisms,\nthereby limiting overall efficacy[1–6]. Our in vitro data robustly demonstrate that under\nequimolar low-dose conditions, the triple combination of Necrostatin-1 (necroptosis\ninhibitor), Disulfiram (pyroptosis inhibitor), and Z-VAD-FMK (pan-caspase inhibitor)\nonly partially restored cell viability. In contrast, PB monotherapy achieved efficacy\ncomparable to the triple combination and significantly outperformed any individual\ninhibitor (Supplementary Fig. 21D). This confirms that PB@PM effectively disrupts\nthe compensatory crosstalk among distinct cell death pathways, enabling broad-\nspectrum PANoptosis inhibition via a single therapeutic entity—a feat difficult to\nachieve with a cocktail of small molecules due to differential pharmacokinetics and\npotential off-target effects.\nBeyond this fundamental multimodal anti-PANoptotic activity, PB@PM provides\ncritical pharmacological and practical benefits over conventional small-molecule\ninhibitors:\n(1) Targeted Delivery and Enhanced Bioavailability: The platelet membrane (PM)\ncoating enables selective accumulation of PB@PM in the ischemic myocardium\n(Fig. 4G–I), thereby enhancing local drug concentration at the disease site,\nimproving therapeutic efficacy, and minimizing potential off-target effects in\nhealthy tissues.\n(2) Superior Pharmacokinetics: The biomimetic nature of the PM coating reduces\nrapid clearance by the mononuclear phagocyte system, prolongs systemic\ncirculation, and improves the half-life of the nanoparticle[7–9]. In contrast, small-\nmolecule inhibitors often suffer from short half-lives, rapid metabolism, and dose-\nlimiting toxicities, which can hinder their clinical translation.\n(3) Favorable Biosafety Profile: Comprehensive assessments confirmed that PB@PM\nexhibits no significant systemic toxicity or histopathological alterations\n(Supplementary Fig. 11 and Supplementary Table 2), supporting its potential for\nsafe clinical application.\nMoreover, molecular dynamics simulations provide a structural rationale for the\nmulti-target action of PB, revealing direct and high-affinity interactions with multiple\ncore PANoptosome components (RIPK1, ZBP1, and AIM2) (Fig. 3). This multi-target\nengagement underpins its ability to simultaneously inhibit multiple PANoptosomes, a\nmechanism distinct from the single-target action of specific small-molecule inhibitors.\nIn summary, PB@PM represents a targeted, multifunctional nanotherapeutic strategy\nthat not only simultaneously inhibits PANoptosis crosstalk but also addresses key\ntranslational challenges—such as targeted delivery, sustained release, and reduced\ntoxicity—associated with combination small-molecule therapies. Its design offers a\nunified solution to achieve synergistic PANoptosis inhibition, avoiding the complexities\nof dosing, scheduling, and potential drug interactions inherent in multi-drug regimens.\nReferences\n1. Sun, X. et al. PANoptosis: Mechanisms, biology, and role in disease. Immunol. Rev.\n321, 246-262 (2024).\n2. Sundaram, B. et al. NLRP12-PANoptosome activates PANoptosis and pathology in\nresponse to heme and PAMPs. Cell 186, 2783-2801.e20 (2023).\n3. Sundaram, B. et al. NLRC5 senses NAD plus plus depletion, forming a\nPANoptosome and driving PANoptosis and inflammation. Cell 187, 4061-4077.e17\n(2024).\n4. Wang, Y. & Kanneganti, T. D. From pyroptosis, apoptosis and necroptosis to\nPANoptosis: A mechanistic compendium of programmed cell death pathways. Comput.\nStruct. Biotechnol. J. 19, 4641-4657 (2021).\n5. Shi, C. X. et al. PANoptosis: A Cell Death Characterized by Pyroptosis, Apoptosis,\nand Necroptosis. J. Inflamm. Res. 16, 1523-1532 (2023).\n6. Kuriakose, T. et al. ZBP1/DAI is an innate sensor of influenza virus triggering the\nNLRP3 inflammasome and programmed cell death pathways. Sci. Immunol. 1,\naag2045 (2016).\n7. E. Blanco, H. Shen, M. Ferrari, Principles of nanoparticle design for overcoming\nbiological barriers to drug delivery. Nature Biotechnology 33, 941-951 (2015).\n8. H. Yan et al., Engineering Cell Membrane‐Based Nanotherapeutics to Target\nInflammation. Advanced Science 6, (2019).\n9. Li B. et al. A Nanocapsule System Combats Aging by Inhibiting Age-Related\nAngiogenesis Deficiency and Glucolipid Metabolism Disorders. ACS Nano.\n2024;18(32):21061-21076.\nComment 5: How long does PB@PM persist in cardiac tissue? Additionally, please\nexplain the primary mechanism by which PB@PM ameliorates fibrosis at the 28-day\ntimepoint (e.g., modulation of inflammatory pathways, suppression of PANoptosis-\nrelated cytokines, or direct effects on fibroblast activation)\nResponse: We thank the reviewer for these insightful questions regarding the\npharmacokinetics and anti-fibrotic mechanism of PB@PM.\nCardiac Retention of PB@PM: Our extended pharmacokinetic studies using Cy5.5-\nlabeled PB@PM demonstrated robust nanoparticle accumulation in the ischemic\nmyocardium for up to 5 days post-injection, with signals detectable even at later time\npoints (Response Fig. R1). This prolonged retention is attributed to the active targeting\nmediated by platelet membrane proteins (e.g., CD41, CD42b, CD47) and the enhanced\npermeability and retention (EPR) effect in the inflamed cardiac microenvironment,\nensuring sustained therapeutic presence at the injury site.\nMechanism of Anti-Fibrotic Action at Day 28: Our integrated analyses support a\nmulti-faceted mechanism whereby PB@PM ameliorates fibrosis primarily through\nupstream modulation of cardiomyocyte death and inflammation, rather than direct\neffects on fibroblasts:\n(1) Primary Driver: PANoptosis Inhibition and DAMP Reduction: The cornerstone\nof the anti-fibrotic effect is the significant reduction in cardiomyocyte PANoptosis by\nPB@PM (evidenced by decreased TUNEL+ cells in Fig. 5I-J and suppression of\nPANoptosome activation in Fig. 6J and Supplementary Figs. 18-19). By curtailing this\nmajor source of cell death, PB@PM fundamentally reduces the release of pro-fibrotic\ndamage-associated molecular patterns (DAMPs) and intracellular contents that initiate\nand perpetuate the fibrotic cascade.\n(2) Immunomodulation and Attenuation of Profibrotic Signaling: PB@PM\npotently attenuated the early inflammatory response by reducing the infiltration of pro-\ninflammatory M1 macrophages (CD86+) and neutrophils (Ly6G+) (Fig. 5K–N), and\ndownregulating the TLR4/NF-κB signaling axis (Supplementary Fig. 13). This shifts\nthe cytokine milieu from a pro-fibrotic (high TNF-α, IL-1β, IL-6) to a pro-reparative\n(elevated IL-10, TGF-β) profile, thereby creating an environment less conducive to\npathological fibroblast activation.\n(3) Promotion of Reparative Fibrosis and Angiogenesis: Consequently, by\nmitigating excessive cell death and inflammation, PB@PM facilitated a more organized\nreparative process. This was evidenced by enhanced deposition of structural collagens\n(I and III), promotion of α-SMA+ myofibroblasts (key for wound contraction), and\nstimulation of CD31+ angiogenesis in the peri-infarct region (Fig. 5O-R,\nSupplementary Figs. 14-15). This coordinated response supports the formation of stable\nscar tissue, preserves ventricular integrity, and attenuates adverse remodeling.\nWhile our data strongly support this cascade originating from cardiomyocyte\nprotection, we acknowledge the reviewer's point and agree that potential direct effects\nof PB@PM on cardiac fibroblast activation represent an interesting avenue for future\ninvestigation.\nFig. R1 Typical ex vivo fluorescence images and quantitative analysis of the\nfluorescence intensities of damaged hearts at day 1, day 3, day 5, day 7, day 14, and\nday 28 after administration of Cy5.5-labeled PB@PM (n = 3). Data are expressed as\nmean ± SD. P values are calculated using one-way ANOVA with Tukey correction. *P\n< 0.05, ***P < 0.001, and ns means no significance.\nComment 6: Mitochondrial damage plays a central role in PANoptosis. The authors\nclaimed that PB@PM alleviates mitochondrial damage and inflammation, but did not\nexplore the core mechanism of PB@PM ameliorating PANoptosis. Does PB@PM\ndirectly target PANoptosis or act through regulating mitochondrial dysfunction to\nsuppress PANoptosis?\nResponse: We appreciate the reviewer for highlighting this central mechanistic\nquestion. Our results unequivocally demonstrate that PB@PM operates through a dual\nmechanism, involving both direct PANoptosome engagement and indirect protection\nvia mitochondrial stabilization, which are likely synergistic.\n1. Direct PANoptosome Targeting(The \"Direct Hit\"): (1) Computational Evidence:\nMolecular dynamics simulations revealed that PB stably binds key functional domains\nof core PANoptosome components: the HIN domain of AIM2 (critical for dsDNA\nsensing), the Zα domain of ZBP1 (essential for nucleic acid binding), and the kinase\ndomain of RIPK1 (Fig. 3). (2) Biophysical Validation: Surface plasmon resonance\n(SPR) confirmed high-affinity binding between PB and ZBP1 (K = 3.04 × 10-9 M) and\nD\nRIPK1 (K = 1.15 × 10-8 M) (Fig. R2). (3) Cellular Validation: Lentiviral\nD\noverexpression assays showed that PB colocalized with and suppressed the activation\nof AIM2, ZBP1, and phosphorylated RIPK1 under both normoxic and OGD/R\nconditions (Supplementary Fig. 20). Furthermore, PB@PM treatment significantly\ninhibited the colocalization of ASC with caspase-1/8, a key step in PANoptosome\nassembly (Supplementary Fig. 21K-L). (4) Functional Outcome: Multiplex\nimmunoblotting confirmed that PB@PM downregulated core components and\ndownstream effectors across all three PANoptosomes (Fig. 6J, Supplementary Figs. 18–\n19).\n2. Mitochondrial Protection and Metabolic Rescue(The \"Upstream Blocker\"): (1)\nROS Scavenging: Cardiomyocytes are rich in mitochondria, which are major sources\nof ROS following ischemia-reperfusion injury [1-3]. PB@PM exhibited potent reactive\noxygen species (ROS)-scavenging activity, mimicking superoxide dismutase (SOD)\nand catalase (CAT) functions. It effectively eliminated •OOH and •OH radicals\n(Supplementary Fig. 26A–D) and reduced intracellular ROS levels in OGD/R-injured\ncardiomyocytes (Supplementary Fig. 26F–H). (2) Mitochondrial Functional\nRestoration: PB@PM restored mitochondrial membrane potential (Supplementary Fig.\n27A-C) and enhanced mitochondrial oxidative phosphorylation, as demonstrated by\nincreased basal and maximal oxygen consumption rate (OCR) and ATP production\n(Supplementary Fig. 27D-G). This metabolic rescue is crucial since mitochondrial\ndysfunction and mtDNA release are key upstream triggers for PANoptosome activation\n(e.g., via the AIM2 and ZBP1 sensors).\nThus, PB@PM directly disrupts PANoptosome assembly via multi-target molecular\ninteractions, while concurrently preserving mitochondrial integrity and reducing\noxidative stress. This dual action synergistically attenuates cardiomyocyte death and\ndisrupts the inflammatory amplification loop that fuels PANoptosis. We have clarified\nthis integrative mechanism in the revised manuscript.\nFig. R2 Surface plasmon resonance (SPR) analysis showing the binding affinity of PB\nto ZBP1 (A) and RIPK1 (B).\nFig.S26 PB@PM scavenges ROS. (A-C) Electron spin resonance (ESR) detection of\nhydroperoxyl (•OOH) and hydroxyl (•OH) radicals. (D) Visual observation of PB\nreactivity with H O and H O . (E) Flow cytometry analysis of phagocytic efficiency of\n2 2 2\nFITC-labeled PB@PM in HL-1 cells. (F-H) Fluorescence imaging (F; Scale bar: 50 μm)\nand flow cytometry of ROS levels in OGD/R-injured HL-1 cells.\nFig.S27 PB@PM restores mitochondrial function. (A-C) JC-1 staining images\n(Scale bar: 50 μm) and flow cytometry analysis of mitochondrial membrane potential\n(MMP). (D-G) Oxygen consumption rate (OCR) quantification in PB@PM-treated HL-\n1 cells (n = 5-10 biologically independent replicates). Data: mean ± SD. Significance:\none-way ANOVA with Tukey correction (*P < 0.05, **P < 0.01, ***P < 0.001).\nReferences\n1. Chen Q. et al. Nrf3-Mediated Mitochondrial Superoxide Promotes Cardiomyocyte\nApoptosis and Impairs Cardiac Functions by Suppressing Pitx2. Circulation.\n2025;151(14):1024-1046.\n2. Ye T. et al. Protective effects of Pt-N-C single-atom nanozymes against myocardial\nischemia-reperfusion injury. Nat Commun. 2024;15(1):1682.\n3. Zheng Y. et al. Mitochondria-Targeted ROS Scavenging Natural Enzyme Cascade\nNanogels for Periodontitis Treatment via Hypoxia Alleviation and Immunomodulation.\nAdv Sci (Weinh). 2025:e07481.\nComment 7: What is the underlying mechanism of PB@PM reprogramming\nmitochondria metabolism? Does it directly bind with mitochondrial proteins or\nmodulate mtDNA release?\nResponse: We thank the reviewer for raising this important mechanistic question.\nCardiomyocytes are indeed highly enriched in mitochondria, which serve as the\nprimary source of reactive oxygen species (ROS) following ischemia-reperfusion injury.\nOur data demonstrate that PB nanoparticles possess multi-enzyme mimetic activities—\nincluding superoxide dismutase (SOD), catalase (CAT), and hydroxyl radical\nscavenging—as confirmed by electron spin resonance (ESR) spectroscopy\n(Supplementary Fig. 26A–D). Consequently, PB@PM effectively quenched\nintracellular ROS in OGD/R-injured HL-1 cardiomyocytes (Supplementary Fig. 26F–\nH). Functionally, PB@PM treatment restored mitochondrial membrane potential\n(Supplementary Fig. 27A–C) and enhanced mitochondrial respiratory capacity and ATP\nproduction, as evidenced by Seahorse XF analysis (Supplementary Fig. 27D–G),\ncollectively indicating a significant improvement in mitochondrial metabolic efficiency.\nCurrently, we have no experimental evidence indicating that PB@PM directly binds\nmitochondrial proteins or modulates mtDNA release. Myocardial mitochondrial\nmetabolic reprogramming involves a complex network of pathways, including\nglycolysis, the TCA cycle, and fatty acid β-oxidation, which warrant further systematic\ninvestigation. We fully agree with the reviewer that elucidating the precise molecular\ninterplay between PB nanoparticles and mitochondrial components—particularly\nregarding mtDNA release and its role in PANoptosis activation—represents an\nimportant direction for future research.\nComment 8: Whether PB@PM truly block PANoptosome assembly or not should be\nfurther verified. Current data only showed inhibition of downstream cell death markers,\nbut failed to demonstrate the disruption of PANoptosome complex.\nResponse: We appreciate the reviewer’s insightful comment regarding the need for\ndirect evidence of PANoptosome disruption. To address this, we performed additional\nimmunofluorescence analyses to assess the colocalization of ASC with caspase-1 and\ncaspase-8—key events in PANoptosome assembly. Our results clearly show that both\nPB and PB@PM treatment significantly reduced the colocalization of ASC with\ncaspase-1/8 in OGD/R-stimulated HL-1 cells (Supplementary Fig. 21K,L), providing\ndirect visual evidence that PB@PM inhibits PANoptosome complex formation.\nMoreover, our molecular dynamics simulations revealed high-affinity binding between\nPB and core PANoptosome components—RIPK1, ZBP1, and AIM2 (Fig. 3). Consistent\nwith this, western blot analyses demonstrated that PB@PM downregulated the active\nforms of these proteins and their downstream effectors (e.g., cleaved caspases,\nphosphorylated RIPK1/RIPK3/MLKL, and GSDMD) in both in vivo and in vitro\nmodels (Fig. 6J, Supplementary Figs. 18–21). Together, these multimodal data support\nthe conclusion that PB@PM interferes with PANoptosome assembly. We acknowledge\nthat further dynamic or structural studies would be valuable, and we plan to incorporate\nsuch approaches in future work as relevant technologies advance.\nFig.S21 PB@PM inhibits PANoptosis in vitro. (K-L) Confocal microscopy images\nshowing the colocalization of ASC and caspase-1/8 in OGD-R-induced HL-1 cells\nfollowing PB or PB@PM treatment. (n = 3 biologically independent replicates).\nComment 9: Molecular dynamics simulations suggested that PB binds with RIPK1,\nZBP1 and AIM2, while the direct evidence of PB disrupting PANoptosome complex\nassembly was absent.\nResponse: We thank the reviewer for emphasizing the need for direct evidence linking\nPB binding to PANoptosome disruption. As noted in our response to Comment 8, we\nhave now provided confocal microscopy data showing that PB@PM significantly\nsuppresses the colocalization of ASC with caspase-1/8 (Supplementary Fig. 21K,L),\na key step in PANoptosome assembly. This result offers direct cellular evidence that\nPB@PM inhibits the formation of functional PANoptosome complexes.\nIn addition, molecular dynamics simulations confirmed stable binding interactions\nbetween PB and the core PANoptosome components RIPK1, ZBP1, and AIM2 (Fig. 3).\nSubsequent biochemical validations showed that PB@PM treatment consistently\ndownregulated the activation of downstream effectors across all three PANoptosome\ntypes and reduced levels of critical adaptors such as ASC and FADD (Fig. 6J,\nSupplementary Figs. 18–21). Collectively, these findings support the model that PB\nnanoparticles disrupt PANoptosome assembly via direct binding to key components.\nComment 10: The observed reduction in TNF-α/IL-1β by PB@PM derives from\nPANoptosis inhibition or direct immune suppression should be further elucidated.\nResponse: We appreciate the opportunity to clarify the origin of the anti-inflammatory\neffects of PB@PM. Our in vitro experiments confirmed that PB@PM directly\nsuppresses proinflammatory cytokine secretion (TNF-α, IL-1β, IL-6) in LPS-stimulated\nRAW264.7 macrophages (Supplementary Fig. 12), consistent with prior studies [1-4].\nTo clarify whether PANoptosis inhibition contributes to reduced inflammation, we\nconducted a co-culture experiment where OGD/R-injured HL-1 cardiomyocytes (upper\nchamber) treated with PB@PM led to significant decreases in IL-1β, IL-6, and TNF-α\nin RAW264.7 macrophages (lower chamber) (Supplementary Fig. 22). Since the\nmacrophages were not directly exposed to PB@PM in this setup, the observed cytokine\nreduction can be attributed to the suppression of PANoptosis in cardiomyocytes, which\nin turn attenuates immunogenic cell death and subsequent inflammation. Thus, the anti-\ninflammatory effect of PB@PM is multimodal, involving both direct cytokine\nsuppression and inhibition of PANoptosis-mediated immunogenic cell death.\nFig.S12 PB@PM modulates inflammatory cytokine secretion. Enzyme-linked\nimmunosorbent assay (ELISA) quantification of IL-1β, IL-6, TNF-α, IL-10, and TGF-\nβ levels in lipopolysaccharide (LPS)-stimulated RAW264.7 model treated with\nPB@PM (n = 3–4 biologically independent replicates). Data: mean ± SD. Significance:\none-way ANOVA with Tukey’s post-hoc test (*P < 0.05, **P < 0.01, ***P < 0.001).\nFig.S22 PB@PM can decrease inflammatory cytokine secretion via inhibiting\nPANoptosis. ELISA quantification of IL-1β, IL-6, and TNF-α levels in RAW264.7 cells\n(in the lower layer) following treatment of OGD-R stimulated HL-1 cardiomyocytes\n(in the upper layer) using PB or PB@PM (n = 3 biologically independent replicates).\nData: mean ± SD. Significance: one-way ANOVA with Tukey’s post-hoc test (***P <\n0.001; ns, P > 0.05).\nReferences\n1.Hou, R. et al. Prussian Blue Nanozyme Promotes the Survival Rate of Skin Flaps by\nMaintaining a Normal Microenvironment. ACS Nano 16, 9559-9571 (2022).\n2. Zhang, K. et al. Hollow Prussian Blue Nanozymes Drive Neuroprotection against\nIschemic Stroke via Attenuating Oxidative Stress, Counteracting Inflammation, and\nSuppressing Cell Apoptosis. Nano Lett. 19, 2812-2823 (2019).\n3. Wang, F. et al. Polymer-modified DNA hydrogels for living mitochondria and\nnanozyme delivery in the treatment of rheumatoid arthritis. Bioact Mater. 47, 448-459\n(2025).\n4. He, H. et al. Design of a Multifunctional Nanozyme for Resolving the\nProinflammatory Plaque Microenvironment and Attenuating Atherosclerosis. ACS\nNano. 2023; 17(15): 14555-14571."}, {"round": 2, "reviewer_comments": {"reviewer_3": "The manuscript has undergone significant improvements and enhancements, and I have no issues. Thank you to the authors for their efforts.", "reviewer_4": "This study systematically investigated the therapeutic mechanism of Prussian blue nanoparticles (PB@PM) targeting PANoptosis in myocardial ischemia-reperfusion injury (MIRI). Through analyzing human cardiac scRNA-seq data, significant upregulation of PANoptosis-related genes in MIRI was confirmed, followed by molecular dynamics simulations to predict specific binding between PB and PANoptosome core proteins. Further, the authors demonstrated the efficacy of platelet membrane-coated nanoparticles (PB@PM) in suppressing PANoptosis activation and assembly in vitro and in vivo. Accordingly, the authors then hypothesized that PB@PM concurrently targets RIPK1/ZBP1/AIM2 within the PANoptosome, disrupting crosstalk among pyroptosis, apoptosis, and necroptosis to confer cardioprotection. However, the claim that PB@PM \"blocks PANoptosome assembly\" lacks direct experimental validation. Besides, this study did not explore whether PB@PM reprograms mitochondrial metabolism or inhibits mtDNA release, which are key triggers of PANoptosis. Meanwhile, merely downstream moleculars related to PANoptosis were observed while the upstream mechanism regulating PANoptosis was not elucidated. Generally speaking, the innovation of this study was average and the experimental design was not persuasive enough.\nQuestion 1: The image quality needs improvement. Please add scale bars to the ultrasound images (Fig 5) and check the scale annotations in other figures.\nQuestion 2: Both the levels of apoptotic caspases and their cleaved active fragments are low under normal conditions, while the accumulation of cleaved fragments post myocardial infarction might reduce the total protein levels. Please verify the Western blot (WB) data and ensure that both full-length and cleaved fragments are displayed on the same membrane.\nQuestion 3: In Fig 3, all the molecular dynamics simulations for Prussian blue (PB) binding to PANoptosis-related molecules used a 200-ns timeframe. However, the RMSD of the PB-RIPK1 complex did not reach equilibrium by 200 ns. Please reevaluate whether 200 ns is suitable to the PB-RIPK1 system.\nQuestion 4: The efficiency of PANoptosis inhibition by PB@PM was similar to the other three specific programmed cell death inhibitors. What is the advantage of using PB@PM over these inhibitors? Please clarify the unique therapeutic value of PB@PM in PANoptosis modulation.\nQuestion 5: How long does PB@PM persist in cardiac tissue? Additionally, please explain the primary mechanism by which PB@PM ameliorates fibrosis at the 28-day timepoint (e.g., modulation of inflammatory pathways, suppression of PANoptosis-related cytokines, or direct effects on fibroblast activation)\nQuestion 6: Mitochondrial damage plays a central role in PANoptosis. The authors claimed that PB@PM alleviates mitochondrial damage and inflammation, but did not explore the core mechanism of PB@PM ameliorating PANoptosis. Does PB@PM directly target PANoptosis or act through regulating mitochondrial dysfunction to suppress PANoptosis?\nQuestion 7: What is the underlying mechanism of PB@PM reprogramming mitochondria metabolism? Does it directly bind with mitochondrial proteins or modulate mtDNA release?\nQuestion 8: Whether PB@PM truly block PANoptosome assembly or not should be further verified. Current data only showed inhibition of downstream cell death markers, but failed to demonstrate the disruption of PANoptosome complex.\nQuestion 9: Molecular dynamics simulations suggested that PB binds with RIPK1, ZBP1 and AIM2, while the direct evidence of PB disrupting PANoptosome complex assembly was absent.\nQuestion 10: The observed reduction in TNF-α/IL-1β by PB@PM derives from PANoptosis inhibition or direct immune suppression should be further elucidated."}, "author_response": ""}, {"round": 3, "reviewer_comments": {"reviewer_3": "The authors have taken the reviewer's rigorous critique seriously and have performed substantial additional work to elevate the manuscript. Based on a comprehensive review of the author's point-by-point rebuttal to Reviewer #4's comments, the revised main manuscript, and the supplementary information, this revised submission demonstrates a significant and satisfactory improvement in addressing the reviewer's major concerns. The overall quality, mechanistic depth, and evidence supporting the central claims have been substantially strengthened. The revisions have transformed a study with potential into one with solid, multi-faceted evidence for its central thesis: that PB@PM is a multi-target nano-therapeutic that mitigates MIRI by disrupting PANoptosis through both direct molecular interference and mitochondrial protection.\nThe study's novelty, integrative methodology, therapeutic relevance, and significantly improved mechanistic depth now align with the high standards of Nature Communications. The work provides valuable insights into PANoptosis in cardiovascular disease and presents a promising nanomedicine strategy.\nRecommendation: Accept for publication in Nature Communications."}, "author_response": ""}]

Decision letter