Platelet and Erythrocyte Membranes Coassembled Biomimetic Nanoparticles for Heart Failure Treatment

Abstract

Cardiac fibrosis is a prevalent pathological process observed in the progression of numerous cardiovascular diseases and is associated with an increased risk of sudden cardiac death. Although the BRD4 inhibitor JQ1 has powerful antifibrosis properties, its clinical application is extremely limited due to its side effects. There remains an unmet need for effective, safe, and low-cost treatments. Here, we present a multifunctional biomimetic nanoparticle drug delivery system (PM&EM nanoparticles) assembled by platelet membranes and erythrocyte membranes for targeted JQ1 delivery in treating cardiac fibrosis. The platelet membrane endows PM&EM nanoparticles with the ability to target cardiac myofibroblasts and collagen, while the participation of the erythrocyte membrane enhances the long-term circulation ability of the formulated nanoparticles. In addition, PM&EM nanoparticles can deliver sufficient JQ1 with controllable release, achieving excellent antifibrosis effects. Based on these advantages, it is demonstrated in both pressures overloaded induced mouse cardiac fibrosis model and MI-induced mouse cardiac fibrosis that injection of the fusion membrane biomimetic nanodrug carrier system effectively reduced fibroblast activation, collagen secretion, and improved cardiac fibrosis. Moreover, it significantly mitigated the toxic and side effects of long-term JQ1 treatment on the liver, kidney, and intestinal tract. Mechanically, bioinformatics prediction and experimental validation revealed that PM&EM/JQ1 NPs reduced liver and kidney damage via alleviated oxidative stress and mitigated cardiac fibrosis via the activation of oxidative phosphorylation activation. These results highlight the potential value of integrating native platelet and erythrocyte membranes as a multifunctional biomimetic drug delivery system for treating cardiac fibrosis and preventing drug side effects.

Keywords: cardiac fibrosis; erythrocyte membrane; heart failure; hybrid membrane; nanotherapy; platelet membrane.

Figure 1

Schematic design of PM&EM/JQ1 NP-mediated delivery of JQ1 to myofibroblast for the treatment of heart failure. (a) Schematic illustration of the preparation of PM&EM/JQ1 NPs. (b) Schematic diagram of intravenous injection of PM&EM/JQ1 NPs to the heart failure mice.

Figure 2

Preparation and characterization of PM&EM/JQ1 NPs. (a) The lipid membrane is labeled with FRET dye for DiO and Dil and then fused with the platelet membrane. The fluorescence intensity was recorded. (b) The particle size was measured by dynamic light scattering (DLS) (mean ± SD, n = 3). (c) Zeta potential distribution (mean ± SD, n = 3 independent experiments) of platelet membrane vesicles, erythrocyte membrane vesicles, JQ1 NPs, and PM&EM/JQ1 NPs. (d) Representative TEM image of PM&EM/JQ1 NPs. Scale bar = 100 nm. (e) CLSM image of PM&EM/JQ1 NPs. Two membranes and a physical mixture of PLGA coated with DiI fluorescein were used as controls. Scale bar = 20 μm. (f) Principal component analysis (PCA) and the 2D score plots display repertoires of PM vesicles, EM vesicles, and PM&EM vesicles. Each point represents a sample, and ellipses represent 95% confidence regions (mean ± SD, n = 3 independent experiments). (g) Classification of PM&EM/JQ1 NPs proteins by the biological process. (h) Classification of PM&EM/JQ1 NP proteins by cellular component. (i) Heatmap of protein levels from PM&EM NPs, PM NPs, and EM NPs. (mean ± SD, n = 3). (j) Protein composition of PM&EM/JQ1 NPs was shown by SDS-PAGE analysis. (k) Analysis of PM&EM/JQ1 NPs through Western Blot to achieve targeting and immune evasion of key protein receptors.

Figure 3

Targeted delivery and immune evasion performance of PM&EM/JQ1 NPs. (a) After 4h incubation with in vitro collagen, the ability of collagen to adhere to DiI NPs, PM&EM/JQ1 NPs, gray is collagen, red is encapsulated DiI fluorescent molecules, scale bar = 10 μm. (b) Fibroblasts and their collagen production stimulated by angiotensin II, green for type I collagen, blue for nuclei; scale bar = 20 μm. (c) After 4 h incubation of activated fibroblasts (stimulated with angiotensin II), cell adhesion, uptake of DiI nanoparticles, PM&EM/JQ1 NPs scale bar = 25 μm. (d) FC analysis of activated fibroblasts after 4 h incubation of DiI NPs, PM&EM/JQ1 NPs, and DiI-positive fibroblasts as a percentage of total fibroblasts (mean ± SD, n = 4 independent experiments). (e) RAW 264.7 cells were coincubated with DiI NPs and PM&EM/JQ1 NPs nanoparticles for 4 h and then subjected to laser confocal analysis, blue for nuclei, green for lysosomes, red for encapsulated DiI fluorescent molecules, scale bar = 25 μm. (f) DiI-positive macrophages as a percentage of total macrophages after coincubation with DiI-labeled nanoparticles for 4 h (mean ± SD, n = 3 independent experiments). ***p < 0.001, one-way ANOVA, Tukey’s multiple comparison test.

Figure 4

PM&EM-NP-mediated delivery to the heart failure mice and biodistribution in other organs. (a) CLSM images of different DiI-labeled nanoparticles at fibrotic sites of the heart in the heart failure mice at 24 h postintravenous injection, scale bar = 5 μm, 20 μm. (b) CLSM images of different DiI-labeled nanoparticles in kidney and liver of the heart failure mice at 24 h postintravenous injection, scale bar = 15 μm. (c) Representative ex vivo fluorescence images of DiD fluorescence dye accumulated in different organs at 24 h postintravenous injection of DiD-labeled nanoparticles. (d) Heatmap of DiD fluorescence dye in different organs (n = 3). (e) Quantitative data of DiD fluorescence dye in the heart (mean ± SD, n = 3) **p < 0.01, one-way ANOVA, Tukey’s multiple comparison test.

Figure 5

Evaluation of the therapeutic effect of novel drug-loaded systems in TAC-induced cardiac insufficiency in mice. (a) Experimental protocol and dosing protocol of pre-established TAC-induced mouse stress overload model. (b) Representative plot of mouse cardiac ultrasound 45 days after construction of the TAC model. (c) Left ventricular ejection fraction quantified by echocardiography. (d) Left ventricular minor axis contraction (mean ± SD, n = 6). (e) Quantification of fibrotic area in the sham group, TAC model group, and TAC with different treatment group (mean ± SD, n = 6). (g) Representative cross sections from remote LV stained with Sirius red; scale bar = 100 μm. (f) Statistical plot of changes in cardiomyocytes area stained with wheat germ agglutinin (WGA) (mean ± SD, n = 6) and (h) representative LV cross-section stained with WGA, scale bar = 20 μm. *p < 0.05, **p < 0.01, and ***p < 0.001, one-way ANOVA, Tukey’s multiple comparison test.

Figure 6

Evaluation of the therapeutic effect of novel drug-loaded systems in MI-induced cardiac insufficiency in mice. (a) Experimental protocol and dosing protocol of MI-induced mouse cardiac insufficiency model. (b) Representative plot of mouse cardiac ultrasound 30 days after the construction of the MI model. (c) Left ventricular ejection fraction quantified by echocardiography. (d) Left ventricular minor axis contraction (mean ± SD, n = 6). (e) Quantification of fibrotic area in the sham group, MI model group, and MI with different treatment group. (g) Representative cross sections from remote LV stained with Masson; scale bar = 100 μm. (f) Statistical plot of changes in cardiomyocytes area stained with WGA (mean ± SD, n = 6) and (h) representative LV cross-section stained with WGA, scale bar = 20 μm. *p < 0.05, **p < 0.01, and ***p < 0.001, one-way ANOVA, Tukey’s multiple comparison test.

Figure 7

Potential mechanisms and therapeutic targets for PM treatment of myocardial infarction-induced cardiac fibrosis. (a) Volcano plots show the differentially expressed genes (DEGs) between the PM&EM/JQ1 NPs and free JQ1 in bulk RNA-seq data sets. (b) Top five upregulated (red) and downregulated (green) KEGG pathways by normalized enrichment score (NES) identified by GSEA in PM&EM/JQ1 NPs. (c) GSEA-based KEGG analysis of Oxidative phosphorylation. (d) GSEA-based KEGG analysis of the ECM-receptor interaction. (e) Heatmap showing the genes of Oxidative phosphorylation. Heatmap scale is a Z score. (f) Heatmap showing the genes of the Oxidative ECM-receptor interaction. Heatmap scale is a Z score. (g) Heatmap of BRD4- bound enhancers in free JQ1 and PM&EM/JQ1 NPs therapy heart covering 3kb upstream and downstream of the enhancer summit, where enhancers are grouped by increased or decreased BRD4 binding in PM&EM/JQ1 NPs therapy heart compared to free JQ1 therapy. (h) ATAC-seq from the fibrosis sites of MI mosue with free JQ1 and PM&EM/JQ1 NPs therapy at the Plcg1 locus. (i) Down-regulated pathway enrichment analysis of PM&EM/JQ1 NPs therapy relative to Free JQ1 in ATACseq.

Figure 8

Biosafety analysis of JQ1, JQ1 NPs and PM&EM/JQ1 NPs. (a) Representative cross section from liver and kidney stained with H&E and intestines with Alcian blue, scale bar = 100 μm. (b) Venn diagrams of JQ1, nephrotoxicity, gastrointestinal toxicity, and hepatoxicity related gene from GENCARDS. (c) GO pathway enrichment analysis (biological process, BP) of the common gene from different groups. (d) Immunofluorescence staining of mitochondria (Mito, red) and reactive oxygen species (ROS, green) in the kidney and liver. Nuclei were counterstained with DAPI (blue); scale bar = 20 μm.