A closed-loop myocardial infarction theranostic platform activated by macrophage-derived nitric oxide and acidic microenvironment

Figures

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  Scheme 1. Schematic illustration of the PM720@NRP theranostic platform and its mechanism of action. (Top) Schematic depicting the synthesis of NRP and PM720@NRP via platelet membrane coating. (Middle) PM720@NRP homes to the infarcted myocardium by hitchhiking on circulating monocytes, where it promotes repolarization of pro-inflammatory macrophages toward an anti-inflammatory phenotype and synergistically enhances angiogenesis through platelet-derived growth factors. (Bottom) By exploiting the defining biochemical features of pro-inflammatory macrophages—elevated NO production and localized acidic microenvironment—PM720@NRP establishes a closed-loop intelligent theranostic paradigm for cardiac diagnosis and treatment. This paradigm enables NO-activated NIR-II imaging, machine learning-assisted quantitative diagnosis, and pH-gated on-demand drug release, thereby realizing a guided, on-demand therapeutic strategy.

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  Fig. 1. Fabrication, characterization, and functional validation of PM720@NRP. (A) Cryo-TEM images of NRP, PM@NRP and PM720@NRP. (B, C) Hydrodynamic diameters and zeta potentials of NRP, PM@NRP and PM720@NRP (n = 3). (D) Hydrodynamic diameter stability of NRP, PM@NRP and PM720@NRP monitored over 7 days (n = 3). (E) pH-dependent release profile of FTY720 from PM720@NRP (n = 3). (F) Western blot analysis of five key membrane proteins in PLTs, PMVs, NRP, and PM720@NRP (n = 6). (G) Wheat germ agglutinin (WGA) staining of PMVs, NRP, and PM720@NRP, confirming the right-side-out orientation of glycosylated membrane proteins (n = 6). (H) UV-vis absorbance spectra of PM720@NRP before and after addition of an NO donor (20 μM). Inset: Photographs of the solution before and after NO donor treatment. (I) Fluorescence emission spectra of PM720@NRP incubated with increasing concentrations of the NO donor (0∼40 μM). (J) Fluorescence emission spectra (n = 6) obtained after mixing the NO donor with PM720@NRP at various NRP concentrations (0∼150 μM). (K) Relative fluorescence intensity of PM720@NRP in the presence of various biologically relevant analytes (100 μM) (n = 3). Data are presented as mean ± SD.

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  Fig. 2. Evaluation of PM720@NRP adhesion to monocytes and its effect on monocyte homing. (A) Confocal microscopy images showing adhesion of PM720@NRP to THP-1 cells under inflammatory (LPS + IFN-γ) and non-inflammatory conditions. Lower right panels present fluorescence colocalization analysis. (B) Adhesion of non-coated NRP to THP-1 cells under inflammatory and non-inflammatory conditions. Lower right panels display fluorescence co-localization analysis. (C) Pearson's correlation coefficient analysis of red-green fluorescence colocalization from (A) and (B) (n = 6). (D) Transwell migration assay assessing the homing capability of THP-1 cells toward an MCP-1 gradient across an endothelial barrier after adhesion to PM720@NRP. The bar graph quantifies migrated cells in the lower chamber (n = 6). Data are presented as mean ± SD. Statistical comparisons were performed using one-way ANOVA followed by Tukey's multiple-comparisons test.

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  Fig. 3. Modulation of macrophage polarization and in vitro NIR-II imaging efficacy. (A) Representative images showing iNOS and CD206 expression, NO production, and NIR-II fluorescence intensity in macrophages under different polarization states. (B) Correlation analyses between macrophage iNOS levels, CD206 levels, NO production, and NIR-II fluorescence intensity, as well as the correlation between iNOS levels and NO production under different polarization states (n = 65). (C) NIR-II fluorescence images and quantitative analysis of relative fluorescence intensity in macrophages treated with PBS, PMVs, PM@NRP, or PM720@NRP, with or without pro-inflammatory stimulation (n = 9). (D) Representative NIR-II fluorescence images and quantitative analysis of pro-inflammatory macrophages treated with PM720@NRP in the presence or absence of HUVECs (n = 3). (E) Immunofluorescence staining of macrophages treated with PBS, PMVs, PM@NRP, free FTY720, or PM720@NRP. Bar graphs show quantitative analysis of iNOS and CD206 proportions (n = 6). (F) Western blot analysis and relative densitometric quantification of p-STAT3, STAT3, iNOS, and CD206 expression in macrophages treated with PBS, PMVs, PM@NRP, free…

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  Fig. 4. Protective effects of PM720@NRP on endothelial cells in an in vitro OGD model. (A) Heatmap of DEGs in HUVECs identified by RNA sequencing under OGD conditions (n = 3) versus OGD + PM720@NRP treatment (n = 6). (B, C) GSEA enrichment plots for selected pathways based on the identified DEGs. (D) GO and KEGG pathway enrichment analyses of the DEGs. (E) Cell viability of HUVECs measured at 0, 6, and 12 h after OGD treatment (n = 6). (F) Western blot analysis and relative quantification of p-ERK1/2, total ERK1/2, p-AKT, and total AKT in HUVECs after OGD treatment (n = 6). (G) Representative images and quantitative analysis of tube formation assays in OGD-treated HUVECs following different treatment (n = 6). (H) Representative images and quantitative analysis of cell migration assays in OGD-treated HUVECs following different treatment (n = 6). Data are presented as mean ± SD. Statistical comparisons were performed using one-way ANOVA followed by Tukey's multiple-comparisons test.

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  Fig. 5. In vivo targeting efficiency and real-time NIR-II imaging. (A) Timeline of tail vein injection of PM720@NRP in MI mice. (B) Ex vivo DiR fluorescence images and quantitative analysis of hearts harvested 6 h after PM720@NRP injection (n = 5). (C) Schematic illustrating samples injection at 3 days post-MI. (D) Ex vivo DiR fluorescence images and quantitative analysis of hearts collected 6 h after injection (n = 5). (E, F) In vivo and ex vivo NIR-II fluorescence imaging of MI (day 3) and sham mice 48 h after PM720@NRP injection, with corresponding quantitative analysis (n = 8). (G, H) Longitudinal in vivo and ex vivo NIR-II fluorescence imaging of MI mice (day 3) before injection and at 6, 12, 24, and 48 h post-injection, with corresponding quantitative analysis (n = 8). Data are presented as mean ± SD. Statistical comparisons were performed using one-way ANOVA followed by Tukey's multiple-comparisons test.

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  Fig. 6. Immunomodulatory effects on macrophages in the infarcted myocardium. (A-D) Immunofluorescence staining of CD206 and iNOS in the infarct zone of MI mice after treatment with different samples. Bar graphs show the relative quantification of the CD206/F4/80 ratio and the iNOS/F4/80 ratio(n = 8). (E) Western blot analysis and relative densitometric quantification of p-STAT3, total STAT3, iNOS, and CD206 in infarct zone tissue from mice treated with PBS, PMVs, PM@NRP, NRP-720, or PM720@NRP (n = 8). (F) Correlation analysis between cardiac NIR-II fluorescence intensity and iNOS expression, CD206 expression, and the iNOS/CD206 ratio in the PM720@NRP and PM@NRP groups. (G, H) ELISA quantification of TNF-α, IL-1β, IL-10, and TGF-β levels in cardiac infarct zone homogenates (n = 8). Data are presented as mean ± SD. Statistical comparisons were performed using one-way ANOVA followed by Tukey's multiple-comparisons test.

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  Fig. 7. Machine learning-based prediction of NO and iNOS levels using in vitro and in vivo NIR-II signals. (A) Scatter plots comparing the performance of six machine learning models for predicting NO levels in the validation dataset. (B) Lollipop plots summarizing model performance on the validation dataset, evaluated by the coefficient of determination (R2) and root mean square error (RMSE). (C) Cumulative distribution curves of absolute prediction errors for each model, showing the proportion of samples predicted within increasing error thresholds. (D) Density plots of residuals (predicted minus observed NO) for each model, depicting the distribution of prediction errors. (E) Lasso coefficient trajectories as a function of log(λ), with the optimal regularization parameter selected by cross-validation. (F) SHAP summary plots demonstrating the relative contributions of CD206, mean fluorescence intensity (MFI), NIR-II signal intensity, and iNOS level to NO prediction. (G) Scatter plot of NIR-II signal intensity versus measured NO levels in the in vitro validation dataset, fitted with a univariate linear regression line. (H) Scatter plot of in vivo NO levels inferred from NIR-II signal…

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  Fig. 8. Therapeutic efficacy of PM720@NRP in mitigating cardiac injury and promoting angiogenesis. (A-C) Dual immunofluorescence staining for CD31 and WGA in the infarct border zone at 14 days post-myocardial infarction. Bar graphs show capillary density per cardiomyocyte and cardiomyocyte cross-sectional area quantified from the staining (n = 8). (D-H) Representative echocardiographic images at baseline (pre-MI) and at 3 and 14 days post-MI, along with quantification of EF, FS, LVDd, and LVEDV (n = 8). EF, ejection fraction; FS, fractional shortening; LVDd, left ventricular end-diastolic dimension; LVEDV, left ventricular end-diastolic volume. (I) Masson's trichrome staining and fibrosis quantification of cardiac cross-sections from each group at 14 days post-MI. Data are presented as mean ± SD. Statistical comparisons were performed using one-way ANOVA followed by Tukey's multiple-comparisons test.

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  Fig. 9. Pharmacokinetics and biosafety profile of PM720@NRP. (A) Representative fluorescence images of venous blood collected at 2, 4, 8, 16, 24, 36, and 48 h after intravenous injection of DiR-labeled PMVs and PM720@NRP, used to assess metabolic clearance. (B) Quantitative analysis of fluorescence intensity at the indicated time points from (A) (n = 6). (C–E) Analysis of blood biochemical markers for liver function (γ-GT, AST, ALT), renal function (BUN, CREA, UA), and coagulation function (PT, APTT) 48 h after injection (n = 6). (F) H&E staining of major organs (lung, liver, spleen, and kidney) collected 11 days after treatment. Left panels show low-magnification overviews; right panels show high-magnified views. Data are presented as mean ± SD. γ-GT, gamma-glutamyl transferase; AST, aspartate aminotransferase; ALT, alanine aminotransferase; BUN, blood urea nitrogen; CREA, creatinine; UA, uric acid; PT, prothrombin time; APTT, activated partial thromboplastin time.