Targeting the ENO1 metabolic checkpoint via microenvironment-responsive nanotherapeutics promotes postinfarction cardiac repair

Figures

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  Fig. 1.. Schematic of acid-responsive PECS targeting macrophages in MI for siENO1-mediated anti-inflammatory cardiac repair. Created in BioRender. Deng, X. (2026) https://BioRender.com/2xc9lii.

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  Fig. 2.. ENO1 is highly expressed in post-MI macrophages and modulates inflammatory polarization. (A) Venn diagram showing the intersection of glycolysis-related enzymes with DEGs in post-MI macrophages (day 3 and 7 versus sham), revealing 14 intersecting genes. (B) Protein-protein interaction (PPI) network of intersecting genes constructed via STRING. (C) Top 10 hub genes ranked by degree centrality using CytoHubba: Tpi1, Gapdh, Pgam1, Eno2, Aldoa, Ldha, Pgk1, Eno1, Pkm, and Aldoc. (D) UMAP visualization of ENO1 expression in human MI single-cell transcriptomic data (GSE270788). Left: global ENO1 expression across cell clusters. Right: ENO1 expression specifically in macrophage clusters from donor versus MI hearts. (E) Quantification of ENO1 expression in macrophages from donor versus MI human heart tissues (P = 0.00485). (F) Representative immunofluorescence staining of ENO1 in heart sections at sham, 3, 7, and 28 days after MI. Blue, 4′,6-diamidino-2-phenylindole (DAPI; nuclei); green, MAC2 (macrophages); red, ENO1. Scale bars, 20 μm. (G) Quantification of ENO1+ macrophages density over time (****P < 0.0001 versus sham; ^^^^P < 0.0001 versus 7 days; n = 5). (H) Eno1 mRNA expression in…

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  Fig. 3.. Physicochemical characterization and acid-responsive behavior of siENO1@PECS. (A) Schematic of NP behavior under different pH conditions (the schematic was created by the authors and is original to this work). (B) 1H NMR spectra showing the time-dependent cleavage of CDM linkers under acidic conditions. ppm, parts per million. (C) GPC confirming the degradation of mPEG-CDM-PCL into block components. (D) Agarose gel electrophoresis analysis of siRNA complexation efficiency at varying siRNA: C12-PEI mass ratios. (E) Hydrodynamic diameter and (F) zeta potential of siENO1@PECS at the indicated pH values. (G) Representative TEM images showing spherical morphology before and after PEG detachment. Scale bar, 100 nm. (H) Nanoflow cytometry analysis and (I) quantification of PS exposure on PECS under different pH conditions (****P < 0.0001 versus pH = 7.4; n = 5). Data are presented as mean ± SEM. Statistical significance was determined by an unpaired Student’s t test.

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  Fig. 4.. Acid-responsive PECS enable targeted delivery and lysosomal escape in macrophages. (A) Cell viability of BMDMs and H9c2 cardiomyocytes after 24-hour incubation with the indicated NPs (CCK-8 assay). (B) Representative confocal microscopy images showing cellular uptake in BMDMs and H9c2 cells cultured at pH 6.8 or 7.4 for 4 hours. Blue, DAPI; red, Cy5-si. Scale bars, 20 μm. (C) Quantification of cellular uptake (****P < 0.0001 versus Cy5-si; ^^^P < 0.001 and ^^^^P < 0.0001 versus Cy5-si@PECS in BMDMs at pH 6.8; n = 5). (D) Flow cytometry histograms and (E) quantification of the mean fluorescence intensity (MFI) of Cy5-si in BMDMs and H9c2 cells at pH 6.8 and 7.4 (4 hours) (****P < 0.0001 versus Cy5-si; ^^^^P < 0.0001 versus Cy5-si@PECS in BMDMs at pH 6.8; n = 4). (F) Representative confocal images tracking lysosomal escape of naked Cy5-si and Cy5-si@PECS in BMDMs over 2, 4, and 6 hours. Green, LysoTracker (lysosome); red, Cy5-si; blue, DAPI. Scale bars, 10 μm. (G) Pearson’s correlation coefficient (r) quantifying colocalization between Cy5-si and LysoTracker to assess lysosomal escape efficiency (****P < 0.0001 versus naked Cy5-si; ^^P < 0.01 and ^^^^P < 0.0001 versus Cy5-si@PECS…

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  Fig. 5.. siENO1@PECS mediates efficient gene silencing and inhibits inflammatory macrophage polarization in vitro. (A) Eno1 mRNA expression in BMDMs treated with PBS, naked siENO1, or the indicated NPs (****P < 0.0001 versus PBS; ^P < 0.05, ^^P < 0.01, and ^^^^P < 0.0001 versus siENO1@PECS; n = 4). (B) Representative Western blot images of ENO1 protein levels in BMDMs after the NP incubation treatments. (C) Quantification of ENO1 protein normalized to β-actin (**P < 0.01 and ****P < 0.0001 versus PBS; ^P < 0.05, ^^^P < 0.001, and ^^^^P < 0.0001 versus siENO1@PECS; n = 3). (D to F) mRNA expression of inflammatory markers (Il-1β, Tnf-α, and Nos2) in BMDMs (***P < 0.001, and ****P < 0.0001 versus control; ^P < 0.05, and ^^^P < 0.001 versus LPS/IFN-γ; n = 3). (G) Representative Western blot images of ENO1 and NOS2 in BMDMs under the conditions described in (D). (H and I) Quantification of ENO1 and NOS2 protein levels normalized to β-actin (*P < 0.05 and ***P < 0.001 versus control; ^^P < 0.01 versus LPS/IFN-γ; n = 3). (J) Representative immunofluorescence staining of BMDMs. Blue, DAPI; green, MAC2 (macrophages); red, NOS2 (inflammatory macrophages). Scale bars, 20 μm. (K) Quantification of…

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  Fig. 6.. Integrated metabolic and transcriptomic profiling reveals the ENO1-KLF4 axis in macrophage polarization. (A) Real-time ECAR profiles of BMDMs treated with LPS/IFN-γ + siNC@PECS (black) or siENO1@PECS (red). Arrows indicate sequential injections of glucose, oligomycin, and 2-DG. (B to D) Quantification of glycolytic parameters derived from ECAR: (B) glycolysis, (C) glycolytic capacity, and (D) glycolytic reserve. (E) Real-time OCR profiles. Arrows indicate sequential injections of oligomycin, FCCP, and rotenone/antimycin A (ROT/AA). (F to H) Quantification of mitochondrial parameters derived from OCR analysis: (F) basal respiration, (G) maximal respiration, and (H) spare respiratory capacity (*P < 0.05, **P < 0.01, and ***P < 0.001 versus LPS/IFN-γ + siNC@PECS; n = 4). (I) Heatmap showing the top 10 up- and down-regulated DEGs in LPS/IFN-γ + siENO1@PECS-treated BMDMs versus controls (LPS/IFN-γ + siNC@PECS). (J) Venn diagram intersecting identified DEGs with a dataset of transcription factors known to regulate macrophage polarization transcription factors. (K) Top nine candidate transcription factors identified in (J). (L) Klf4 expression in BMDMs under indicated conditions (^^^P <…

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  Fig. 7.. siENO1@PECS regulates macrophage glycolysis and KLF4 expression by modulating lactate production. (A) Intracellular and extracellular lactate concentrations in control, LPS/IFN-γ–treated, and LPS/IFN-γ + siENO1@PECS–treated macrophages (****P < 0.0001 versus control; ^^^^P < 0.0001 versus LPS/IFN-γ; n = 5). (B) Representative Western blot images showing the protein expression of KLF4 and ENO1 in macrophages under different treatments including control, LPS/IFN-γ, LPS/IFN-γ + siENO1@PECS, LPS/IFN-γ + siENO1@PECS + lactate, and LPS/IFN-γ + lactate alone (n = 5). (C) Quantification of ENO1 and KLF4 protein levels, normalized to β-actin (*P < 0.05, ***P < 0.001, and ****P < 0.0001 versus control; ^P < 0.05, ^^P < 0.005, ^^^P < 0.001, and ^^^^P < 0.0001 versus LPS/IFN-γ group; n = 5). (D) Quantification of intracellular and extracellular lactate concentrations in the full treatment groups (****P < 0.0001 versus control; ^P < 0.05 and ^^^^P < 0.0001 versus LPS/IFN-γ group; n = 5). Data are presented as mean ± SEM. Statistical significance was determined by unpaired Student’s t test or one-way ANOVA with Sidak’s post hoc test.

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  Fig. 8.. Acid-responsive CDM linker and PS modification facilitate specific accumulation of siENO1@PECS in the infarcted heart. (A) In vivo experimental timeline including MI/sham surgery, NP injection, IVIS imaging, echocardiography, and histological analysis, created in BioRender. Deng, X. (2026) https://BioRender.com/pi46g9m. (B) Representative in vivo IVIS images of MI mice following intravenous injection of naked Cy5-siRNA or various Cy5-siRNA–loaded NPs at indicated time points (4, 8, 12, 24, and 48 hours). (C) Quantification of fluorescence intensity in the cardiac region over time (n = 3). (D) Ex vivo IVIS images of excised hearts from mice treated with naked Cy5-siRNA (8 hours) and Cy5-siRNA@PECS (4, 8, 12, 24, and 48 hours). (E) Quantification of ex vivo cardiac fluorescence intensity (*P < 0.05 and **P < 0.01 versus naked siRNA; ^P < 0.05 and ^^P < 0.01 versus 8-hour siRNA@PECS; n = 3). (F) Representative immunofluorescence images showing the distribution of naked Cy5-siRNA and Cy5-siRNA@PECS in the infarcted myocardium. Macrophages were labeled with MAC2 (green), cell nuclei with DAPI (blue), and Cy5-siRNA with red fluorescence. Scale bars, 20 μm. (G) Quantification of the…

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  Fig. 9.. siENO1@PECS attenuates inflammatory macrophage polarization in vivo and protects against cardiomyocyte apoptosis. (A) Representative immunofluorescence staining of heart sections 7 days post-MI. Green, MAC2 (macrophages); red, ENO1 (ENO1+ cells). Scale bar, 20 μm. (B) Quantification of the proportion of ENO1+ macrophages within the total MAC2+ population in the infarcted region (****P < 0.0001 versus sham group; ^^^P < 0.001 and ^^^^P < 0.0001 versus siENO1@PECS group; n = 5). (C) Representative immunofluorescence staining of heart sections 7 days post-MI. Green, MAC2 (macrophages); red, CD86 (inflammatory macrophages). Scale bar, 20 μm. (D) Quantification of the portion of CD86+ macrophages within the total MAC2+ population in the infarcted region (****P < 0.0001 versus sham; ^^^P < 0.001 and ^^^^P < 0.0001 versus siENO1@PECS; n = 5). For both (A) and (C), treatment groups included sham and MI mice treated with various NPs. (E) Representative TUNEL staining of apoptotic cells in the infarcted region. Scale bar, 20 μm. (F) Quantification of TUNEL+ cells in the infarcted region (**P < 0.01 and ****P < 0.0001 versus sham; ^^P < 0.01, ^^^P < 0.001, and ^^^^P < 0.0001 versus…

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  Fig. 10.. siENO1@PECS ameliorates cardiac dysfunction and reduces fibrosis in MI mice. (A) Representative H&E and (B) Masson’s trichrome staining of heart sections 28 days post-MI. Scale bars, 1 mm. (C) Quantification of infarct size (H&E) (****P < 0.0001 versus sham; ^^^P < 0.001 and ^^^^P < 0.0001 versus siENO1@PECS; n = 7). (D) Quantification of fibrosis area (Masson’s trichrome) (***P < 0.001 and ****P < 0.0001 versus sham; ^^^^P < 0.0001 versus siENO1@PECS; n = 5). (E) Representative echocardiography images at 7 and 28 days post-MI. (F to I) Quantification of echocardiographic parameters: (F) LVEF, (G) LVFS, (H) LVIDs, and (I) LVIDd (*P < 0.05, **P < 0.01,***P < 0.001, and ****P < 0.0001 versus sham; ^P < 0.05, ^^P < 0.01, ^^^P < 0.001, and ^^^^P < 0.0001 versus siENO1@PECS; n ≥ 9). Data are presented as mean ± SEM. Statistical significance was determined by one-way ANOVA with Sidak’s post hoc test.