Intercellular NETwork-facilitated sarcoplasmic reticulum targeting for myocardial ischemia-reperfusion injury treatment

Figures

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  Fig. 1.. Intercellular NETwork-facilitated SR-targeted drug delivery system for myocardial and skeleton muscle injury treatment. (A) Lut-loaded p-toluenesulfonamide-modified nanoparticles (L-P-NPs) were prepared through a nanoprecipitation method and SR-targeted neutrophil carriers (L-P-NPs@NEs) were prepared by co-incubating L-P-NPs with NEs for 1 hour. L-P-NPs@NEs were administered intravenously for the treatment of MIRI of male C57BL/6 mice and skeleton muscle inflammation injury of female BALB/c mice. (B) Intercellular NETwork-facilitated L-P-NPs transport: L-P-NPs@NEs were recruited to the inflammatory site in response to the chemotactic gradients and transported L-P-NPs to damaged myocardial cells through the generation of NETs. (C) L-P-NPs targeted the SR of injured muscle cells and released Lut, triggering a series of beneficial events, including restored activity of SERCA, reciprocal inhibition of Ca2+ overload and oxidative stress, and inhibition of muscle cell apoptosis. Created with BioRender.

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  Fig. 2.. L-P-NPs target the SR. (A and B) Size distribution and particle morphology of L-P-NPs (A) and L-M-NPs (B). Scale bars, 100 nm. (C to F) Representative confocal laser scanning microscopy (CLSM) images and colocalization ratios of nanoparticles in SR of H9C2 cells (C), C2C12 cells (D), primary myocardial cells (E), and primary skeletal myoblasts (F). Cells were detected after incubation with Cou6-M-NPs or Cou6-P-NPs at 37°C for 4 hours. HRC was marked with red fluorescence to delineate the SR. Scale bars, 20 μm. Blue: nucleus; green: Cou6-loaded nanoparticles; red: SR. n = 3 biologically independent samples per group. (G) Real-time fluorescence imaging of MIRI model of C57BL/6 mice treated with DID-M-NPs or DID-P-NPs at 1, 4, and 8 hours after intravenous injection and ex vivo fluorescence image of heart (He), liver (Li), spleen (Sp), lung (Lu), and kidney (Ki) at 8-hour post-injection. MIRI model of male C57BL/6 mice was established by ligating the left descending branch of the coronary artery for 45 min, followed by reperfusion and intravenous injection of formulation. The position of the heart was marked with a red circle. (H) Corresponding semiquantitative analysis of major…

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  Fig. 3.. Intercellular NETwork mediation enables L-P-NPs@NEs to target the SR. (A) Illustration of L-P-NPs@NEs. (B) Representative flow cytometry plots of the purity of isolated NEs (CD11b+/Ly6G+). (C) Representative flow cytometry of NEs before (left)/after (right) treated with DID-P-NPs for 1 hour. SSC-A, side scatter-area. (D) Representative CLSM image of DID-P-NPs@NEs. Blue: nucleus; green: cell membrane; red: DID-loaded NPs. Scale bar, 10 μm. (E) Representative transmission electron microscope (TEM) image of NEs (left) and L-P-NPs@NEs (right). Scale bar, 2 μm. (F) Schematic illustration of the chemotaxis function of L-P-NPs@NEs using a transwell system. Cell numbers of NEs, Lut@NEs, P-NPs@NEs, and L-P-NPs@NEs in the lower chamber after incubation at 37°C for 1 hour. (G) Determination of the quantity of Lut released from and retained in L-P-NPs@NEs in the absence of fMLP and PMA and in the presence of fMLP (10 nM) or PMA (100 nM) over time. (H) Representative fluorescent spectra of normal DII-P-NPs, DIO-P-NPs, DII/DIO-P-NPs, and released DII/DIO-P-NPs, which were incubated with NEs for 1 hour and then treated with PMA (100 nM) for 6 hours. Excitation wavelength is 460 nm, and emission…

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  Fig. 4.. L-P-NPs@NEs target the IR heart and SR in vivo. (A) Schematic illustration showing the establishment of the MIRI model of male C57BL/6 mice by ligating the left descending branch of the coronary artery for 45 min, followed by reperfusion and intravenous injection of DID-loaded formulation. (B) Real-time fluorescence imaging of C57BL/6 mice treated with free DID, DID-P-NPs, or DID-P-NPs@NEs at 1, 4, 8, 12, and 24 hours after intravenous injection and fluorescence imaging of ex vivo hearts at 24-hour post-injection. Fluorescence intensities were normalized to the same scale. The position of the heart was marked with a red circle. n = 3 animals per group. (C) Corresponding semiquantitative analysis of major organs. Heart-related data are magnified. n = 3 animals per group. (D) Representative CLSM images of heart slices from sacrificed MIRI mice 24-hour post injection of DID-P-NPs@NEs. The white dotted line separates injured area from normal area. Blue: nucleus; green: cTnT; red: HIF-1α; violet: DID-labeled nanoparticles. Scale bar, 20 μm. (E) Representative CLSM images of SR and Cou6-M-NPs@NEs or Cou6-P-NPs@NEs in the heart tissue at 24-hour post-injection. HRC was marked with red…

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  Fig. 5.. L-P-NPs@NEs alleviated oxidative stress injury in vitro by restoring the activity of SERCA. H2O2-injured H9C2 cells were treated with free Lut, L-M-NPs, L-P-NPs, or L-P-NPs@NEs at a Lut equivalence of 5 μg/ml for 12 hours. L-P-NPs@NEs were pre-incubated with PMA (100 nM) for 6 hours before being treated with cells. (A) SERCA activity was detected using a Ca2+-ATPase Assay kit by extracting SR. (B and C) Quantitative results of Ca2+ level (B) and intracellular ROS level (C) in H2O2-injured H9C2 cells were investigated by flow cytometry. (D) Representative CLSM images of intracellular ROS level in H2O2-injured H9C2 cells. Scale bars, 20 μm. Blue: nucleus; green: DCFH-DA marked ROS. (E and F) MPTPs openness (E) and quantitative results of Ca2+ level (F) in mitochondria of H2O2-injured H9C2 cells were investigated by flow cytometry. (G) Intracellular ATP level in H2O2-injured H9C2 cells was measured by quantifying the luminescence emitted by firefly luciferase. (H) Cell viability in H2O2-induced acutely injured H9C2 cells was measured by flow cytometry. (I) Representative CLSM images of H2O2-injured H9C2 cells. Red: apoptotic cells stained with PI; green: live cells stained with…

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  Fig. 6.. Therapeutic effects of L-P-NPs@NEs in the MIRI model (short-term efficacy). (A) Schematic illustration showing the short-term treatment schedule. To be specific, the MIRI models of male C57BL/6 mice were established by ligating the left descending branch of the coronary artery for 45 min, followed by reperfusion and intravenous injection of free Lut, L-P-NPs, L-M-NPs@NEs, or L-P-NPs@NEs at a Lut equivalence of 750 μg/kg. Relevant parameters were measured after 1 day. (B) SERCA activity was detected using a Ca2+-ATPase Assay kit by extracting SR from injured myocardial cells. (C) Representative TEM images of myocardial cells in Sham group (left), Untreated group (medium) and L-P-NPs@NEs group (right). Scale bars, 2 μm (low magnification) and 500 nm (high magnification). The yellow arrows refer to SR. (D) Representative TUNEL staining for cardiomyocyte apoptosis and serum levels of CK-MB, BNP, and cTnT. Scale bar, 100 μm. Blue: nucleus; green: TUNEL-positive cells. (E) Representative images of two-lead ECGs and time of QRS and QT interval. n = 6 animals per group (B, D, and E). The experiments in (B) to (E) were repeated twice independently with similar results. Data are presented…

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  Fig. 7.. Therapeutic effects of L-P-NPs@NEs in the MIRI model (long-term efficacy). (A) Schematic illustration showing the long-term treatment schedule. To be specific, the MIRI models of male C57BL/6 mice were established by ligating the left descending branch of the coronary artery for 45 min, followed by reperfusion and intravenous injection of free Lut, L-P-NPs, L-M-NPs@NEs, or L-P-NPs@NEs at a Lut equivalence of 750 μg/kg at 0, 2, and 4 days. Relevant parameters were measured at 2 and 4 weeks. (B) Representative echocardiographic images and changes in the EF, FS, LVEDV, and LVESV for different groups at 2 and 4 weeks post-injection. (C) Representative Masson’s trichrome staining of myocardial sections after 4-week treatment and quantitative assay of fibrosis area in total left ventricle area and infarct wall thickness. Myocardial sections were sliced from the ligation site to apex at 600-μm intervals. Scale bars, 1 mm (low magnification). Scale bars, 100 μm (high magnification). n = 6 animals per group (B and C). The experiments in (B) and (C) were repeated twice independently with similar results. Data are presented as means ± SD. Statistical analysis was performed using one-way…

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  Fig. 8.. L-P-NPs@NEs target the inflammation-injured skeletal muscle and SR. (A) Schematic illustration showing the establishment of the skeletal muscle inflammation model by injecting LPS (1 mg/kg) into the right biceps femoris muscle of femal BALB/c mice and intravenous injection of formulation. (B) Real-time fluorescence imaging of BALB/c mice treated with free DID, DID-P-NPs, or DID-P-NPs@NEs at 1, 4, 8, 12, and 24 hours after intravenous injection. Fluorescence intensities were normalized to the same scale. The position of the right legs was marked with a red circle. n = 3 animals per group. (C) Fluorescence imaging of ex vivo left and right legs and corresponding semiquantitative analysis of left and right legs at 24-hour post-injection. Fluorescence intensities were normalized to the same scale. n = 3 animals per group. (D) Representative CLSM images of SR and Cou6-M-NPs@NEs or Cou6-P-NPs@NEs in inflamed skeleton muscle tissue at 24-hour post-injection. HRC was marked with red fluorescence to delineate the SR. Blue: nucleus; green: Cou6-loaded NPs; red: SR. Scale bars, 20 μm. (E) Fluorescence intensity of isolated SR from inflammation injured skeleton muscle cells at 24-hour…

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  Fig. 9.. Therapeutic effects of L-P-NPs@NEs in skeletal muscle inflammation model. (A) Schematic illustration showing the establishment of the skeletal muscle inflammation model by injecting LPS (1 mg/kg) into the right biceps femoris muscle of femal BALB/c mice and intravenous injection of free Lut, L-P-NPs, L-M-NPs@NEs or L-P-NPs@NEs at a Lut equivalence of 750 μg/kg. Relevant parameters were measured after 1 day. (B) SERCA activity was detected by extracting SR from injured muscle cells. (C) Serum levels of CK and LD on day 1. (D) Tissue homogenate levels of TNFα, IL-1β, and IL-6 on day 1. (E) Hematoxylin and eosin (H&E)–stained slices of right biceps femoris muscle with different treatment. Scale bars, 100 μm (low magnification) and 50 μm (high magnification). (F) Representative single contraction curve and quantitative results of Pt, IT, and HRT of right biceps femoris muscle by a tension sensor. Left biceps femoris muscle (L) and right biceps femoris muscle (R) of the same mouse were independently measured (B and D). n = 6 animals per group (B to D). n = 3 animals per group (F). The experiments in (B) to (F) were repeated twice independently with similar results. Data are presented…