Cardiac-targeted delivery of miRNA via antioxidant nanozymes ameliorates cardiac dysfunction and fibrosis after myocardial infarction

Scheme 1. Schematic representation of the therapeutic mechanism by which the nanozyme CCTP-miR@CTP mitigates oxidative damage and fibrosis in the heart following myocardial infarction. (A) Schematic diagram of CCTP-miR@CTP synthesis and administration in myocardial infarction mice. CCTP-miR@CTP accumulates in the infarct region of the heart via the bloodstream. (B) In cardiomyocytes, CCTP-miR@CTP exerts CAT- and SOD-like activities to scavenge harmful free radicals, reduce ROS levels, improve mitochondrial membrane potential, and mitigate apoptosis and excessive autophagy. (C) CCTP-miR@CTP exerts anti-inflammatory effects by reducing the proportion of pro-inflammatory M1 macrophages and increasing the proportion of anti-inflammatory M2 macrophages. (D) CCTP-miR@CTP inhibits excessive activation of fibroblasts, thereby alleviating fibrosis. (E) CCTP-miR@CTP promotes angiogenesis

Fig. 1. Synthesis, characterization, and enzyme-mimicking activities of the CCTP. (A) Hydrodynamic size distributions and (B) zeta potentials of CCT, CCTP, and CCTP@CTP (n = 3). (C) FTIR spectra of the synthesized materials. (D) Representative TEM image of CCTP. (E) Elemental mapping images of CCTP showing the uniform distribution of C, O, and Co. (F) XPS survey of CCTP. (G) High-resolution Co 2p XPS spectrum of CCTP. (H) Relative SOD-like activity of CCTP at different concentrations. (I) ESR spectra of •O₂⁻ signal after different treatments. (J) CAT-mimicking activity evaluated by O₂ generation. (K) ESR spectra of •OH signal after different treatments. (L) Mechanism of ROS elimination

Fig. 2. Cardiac targeting capabilities of the CCTP@CTP. (A) The particle sizes of CCTP and CCTP-miR26a. (B) The zeta potentials of CCTP and CCTP-miR26a (n = 3). (C) SDS-PAGE analysis of CCTP and CCTP-miR26a. (D) Degradation of miRNA, either in free form or in CCTP-miR26a, when exposed to purified RNase A for increasing amounts of time. (E-F) Fluorescence microscopy images of H9C2 cells after co-incubation with Cy3-labeled CCTP-miR26a for 0, 2, 4, 6 and 8 h (n = 4). Scale bar = 50 μm. (G) qRT-PCR analysis of the expression of miR-26a in H9C2 cells after different treatments (n = 6). (H) Confocal microscopy images of H9C2 after varied treatments stained with lysosomes. Lysosomes were stained with Lysotracker Green (green), CCTP-miR26a with Cy3 (red) and cell nucleus with DAPI (blue), Scale bar = 10 μm. (I) Scheme for the experimental arrangement of in vivo targeting. (J-K) In MI or Sham mice injected with Cy5.5, CCTP+Cy5.5 or CCTP@CTP+Cy5.5 via the tail vein, in vivo imaging (J) of the fluorescence intensity of Cy5.5 and in vitro imaging (K) of the major organs heart, liver, spleen, lung and kidney were performed at the specified time points. (L) Representative confocal microscopy images…

Fig. 3. The antioxidant activity and mitochondrial protective effect of CCTP-miR26a under H2O2 stimulation in vitro. (A-D) The cell viabilities of H9C2 (A), NMCMs (B), PMCFs (C) and HUVECs (D) after incubation with CCTP for 48 h (n = 4). (E-F) Live/Dead staining images of cardiomyocytes H9C2 treated with CCTP or CCTP-miR26a, followed by H2O2 treatment (n = 4), scale bar = 200 μm. (G) The analysis of cell viability in the indicated groups (n = 6). (H-I) Fluorescence images and quantification analysis of intracellular ROS level after different treatments measured by DCFH-DA indicator (n = 4), scale bar = 100 μm. (J) The MDA content in H9C2 cells (n = 3). (K-L) Nrf2 and SOD2 mRNA expression in H9C2 cell was detected by qRT-PCR (n = 4). (M-N) Membrane potential (MMP) were observed by fluorescence microscopy (n = 5), scale bar = 100 μm. (O) Analysis of ATP content in mitochondria of H9C2 cells (n = 4)

Fig. 4. CCTP-miR26a restores autophagy and anti-apoptosis under oxidative stress. (A) H9C2 transfected with the GFP-mRFP-LC3 plasmid were treated with CCTP, CCTP-miR26a and H2O2. The images were obtained using a confocal microscope. The yellow and red puncta represent autophagosomes and autolysosomes, respectively. (B-D) Autophagy-related proteins expression in the indicated groups. β-actin served as a loading control (n = 3). (E-F) Representative flow cytometric images of apoptosis (E) and quantified data showing percentage of apoptotic cells (F) in H9C2 cells (n = 3). (G-J) Representative Western blots and quantified data showing apoptosis-related proteins expression in the indicated groups (n = 3)

Fig. 5. The anti-inflammatory and angiogenesis effects of CCTP-miR26a under LPS and H2O2 stimulation. (A) Schematic diagram of the in vitro anti-inflammatory activity of CCTP. (B) Optical microscope images of RAW264.7 cells treated with CCTP or CCTP-miR26a, followed by LPS treatment, scale bar = 50 μm. (C-F) The quantification of RAW264.7 differentiated into M1 (CD86) and M2 (CD206) macrophages after different treatment stimuli was detected by flow cytometry (n = 3). (G-I) The mRNA expression levels of pro-inflammatory cytokines, IL-1β, IL-6 and TNF-α in RAW264.7 cells were measured by qRT-PCR assay (n = 5). (J-L) The expression levels of IL-1β (n = 4), IL-6 (n = 6) and TNF-α (n = 6) were evaluated by ELISA. (M-N) After treating HUVEC cells with CCTP or CCTP-miR26a for 24 h, representative images were collected and quantitatively analyzed to evaluate the migration rate (n = 3). Scale bar = 200 μm. (O) Cell viability of HUVEC cells under different conditions (n = 6). (P-R) Images were captured to document the tubes formed in vitro by HUVEC cells, and the total tube length (Q) and the number of junctions (R) were quantitatively analyzed (n = 6)

Fig. 6. CCTP-miR@CTP improves cardiac function and reduces infarct size in acute MI mice. (A) Scheme for the experimental arrangement of the animal experiments. (B) Representative M-mode echocardiograms. (C) H&E staining of heart sections. Scale bar = 200 μm. (D-E) TTC-stained heart sections and quantified data showing myocardial infarct size 48 h post-MI in mice injected with CCTP@CTP or CCTP-miR@CTP (n = 5). Scale bar = 2 mm. (F-G) Quantitative analysis of EF% and FS% at 48 h post-MI (n = 5). (H-I) Serum levels of CK-MB and LDH (n = 5). (J-K) Relative miR-26a, caspase-3, Bcl-2, and Bax expression in the infarct zone (n = 5). (L-M) Western blot analysis of apoptosis-related proteins (n = 3). (N-O) Western blot analysis of autophagy-related proteins (n = 3). (P) Immunofluorescence staining for CD86 (red), CD206 (green) and DAPI (blue) in the peri-infarct area. Scale bar = 50 μm. (Q-S) Quantification of IL-1β, IL-6 and TNF-α levels in the mice serum after MI (n = 5)

Fig. 7. CCTP-miR@CTP improves cardiac structural remodeling, delays fibrosis process, and promotes vascular regeneration after 28 days of MI. (A) Scheme for the experimental arrangement of the animal experiments. (B) Representative M-mode images 2 weeks or 4 weeks after treatment. (C-D) Quantitative statistics of left ventricular EF% and FS% in echocardiography results (n = 5). (E) Hematoxylin-eosin staining images of mouse hearts. Scale bar = 200 μm. (F) Representative Masson’s trichrome images. Scale bar = 200 μm. (G) Representative Sirius red staining images after treatment. Scale bar = 200 μm. (H) Cell apoptosis was detected by Tunel staining. Scale bar = 100 μm. (I) Immunofluorescence images of cTnT and CD31 staining in the infarct marginal area 28 days after different treatments. cTnT is labeled with GFP (green), CD31 is labeled with RFP (red), and the cell nucleus is stained with DAPI (blue). Scale bar = 100 μm. (J-N) Relative miR-26a, Acta2, FN1, col1a1 and col3a1 expression in the infarct zone was detected by qRT-PCR assay (n = 5). (O-P) Representative Western blot results and quantitative statistical results of cardiac tissue (n = 3). (Q) Immunohistochemical images of collagen…