Nanoparticle-Mediated Simultaneous Targeting of Mitochondrial Injury and Inflammation Attenuates Myocardial Ischemia-Reperfusion Injury

Abstract

Background The opening of mitochondrial permeability transition pore and inflammation cooperatively progress myocardial ischemia-reperfusion (IR) injury, which hampers therapeutic effects of primary reperfusion therapy for acute myocardial infarction. We examined the therapeutic effects of nanoparticle-mediated medicine that simultaneously targets mitochondrial permeability transition pore and inflammation during IR injury. Methods and Results We used mice lacking cyclophilin D (CypD, a key molecule for mitochondrial permeability transition pore opening) and C-C chemokine receptor 2 and found that CypD contributes to the progression of myocardial IR injury at early time point (30-45 minutes) after reperfusion, whereas C-C chemokine receptor 2 contributes to IR injury at later time point (45-60 minutes) after reperfusion. Double deficiency of CypD and C-C chemokine receptor 2 enhanced cardioprotection compared with single deficiency regardless of the durations of ischemia. Deletion of C-C chemokine receptor 2, but not deletion of CypD, decreased the recruitment of Ly-6C^high monocytes after myocardial IR injury. In CypD-knockout mice, administration of interleukin-1β blocking antibody reduced the recruitment of these monocytes. Combined administration of polymeric nanoparticles composed of poly-lactic/glycolic acid and encapsulating nanoparticles containing cyclosporine A or pitavastatin, which inhibit mitochondrial permeability transition pore opening and monocyte-mediated inflammation, respectively, augmented the cardioprotection as compared with single administration of nanoparticles containing cyclosporine A or pitavastatin after myocardial IR injury. Conclusions Nanoparticle-mediated simultaneous targeting of mitochondrial injury and inflammation could be a novel therapeutic strategy for the treatment of myocardial IR injury.

Keywords: cardioprotection; drug delivery system; ischemia‐reperfusion injury; nanotechnology.

Figure 1. Double deficiency of cyclophilin D and CCR2 showed additive cardioprotection over single deficiency against ischemia‐reperfusion injury.

A‐D, Ratio of infarct size/area at risk in mice subjected to 30, 45, 60, or 90 minutes of myocardial ischemia followed by 24 hours of reperfusion. The horizontal lines represent mean±SD (n=8 per group). *P<0.01, **P < 0.05, 1‐way ANOVA. E, Relationship between the duration of ischemia and infarct size. Redrawn from the data in Figure 1A through 1D. Error bars indicate SD. #1, P<0.0001 vs 30 minutes, #2; P<0.0001 vs 30 minutes, P<0.05 vs 45 minutes; #3, P<0.0001 vs 30 minutes, P<0.001 vs 45 minutes; #4 P<0.01 vs 30 minutes; #5, P<0.0001 vs 30 minutes, P<0.0001 vs 45 minutes; #6, P<0.0001 vs 30 minutes, P<0.0001 vs 45 minutes, P<0.001 vs 60 minutes; #7, P<0.01 vs 30 minutes; #8, P<0.0001 vs 30 minutes; #9, P<0.0001 vs 30 minutes, P<0.001 vs 45 minutes, P<0.01 vs 60 minutes; #10, P<0.0001 vs 30 minutes, P<0.0001 vs 45 minutes; #11, P<0.0001 vs 30 minutes, P<0.0001 vs 45 minutes, P<0.001 vs 60 minutes, 2‐way ANOVA. CCR2‐KO indicates C‐C chemokine receptor 2‐knockout mice; CypD‐KO, cyclophilin D‐knockout mice; Double‐KO, cyclophilin‐D/C‐C chemokine receptor 2‐double knockout mice; mPTP, mitochondrial permeability transition pore; and WT indicates wild‐type mice.

Figure 2. Inflammation was attenuated in CCR2‐KO or double‐KO, while CypD‐KO showed residual inflammation.

A, Dual channel fluorescence molecular tomography demonstrating cell death (AnnexinVivo750) and protease activity (ProSense680) after ischemia‐reperfusion injury. Bar graphs show quantitative data represented as mean±SD (n=8 per group). *P<0.01, 1‐way ANOVA. B, Ischemia‐reperfusion‒injured myocardium was subjected to flow cytometric analysis. The numbers of monocytes were normalized by IS. Ly6C^high activated monocytes were decreased in CCR2‐KO or double‐KO mice, whereas CypD‐KO showed larger numbers of total monocytes. The data are mean±SD (n=4 per group). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, 1‐way ANOVA. *P<0.05 vs wild‐type, #P<0.05 vs CypD‐knockout, 1‐way ANOVA. C, IL‐1β protein levels in the ischemia‐reperfusion‒injured myocardium measured by ELISA. The data are mean±SD (n=4–7 per group). CCR2‐KO indicates C‐C chemokine receptor 2‐knockout mice; CypD‐KO, cyclophilin D‐knockout mice; Double‐KO, cyclophilin‐D/C‐C chemokine receptor 2‐double knockout mice; IL‐1β, interleukin‐1β; IR, ischemia‐reperfusion; IS, infarct size; and WT indicates wild‐type mice.

Figure 3. Deletion of cyclophilin D augmented releases of IL‐1β and IL‐18 in bone marrow‐derived macrophages.

A through C, Bone marrow‐derived macrophages were pre‐treated with the antioxidant Mito‐TEMPO (500 mM) for 15 minutes before stimulation with ATP (2 mM). Culture supernatants were collected 1 hour later and IL‐1β, IL‐18 and TNF‐α protein levels were measured by ELISA. The data are mean±SD (n=4 per group). CypD‐KO indicates cyclophilin D‐knockout mice; IL‐1β, interleukin‐1β; IL‐18, interleukin‐18; TNF‐α, tumor necrosis factor‐α; and WT; wild‐type mice. *P<0.001, #P<0.001, 2‐way ANOVA.

Figure 4. The effects of IL‐1β neutralizing antibody on myocardial inflammation and infarct size.

*P<0.05, unpaired t tests. A, IR‐injured hearts harvested from CypD‐KO mice were subjected to flow cytometric analysis. The number of monocytes were normalized by IS. The data are mean±SD (n=5 per group). *P<0.05, unpaired t tests. B, The therapeutic effects of IL‐1β neutralizing antibody on the infarct size in WT, CypD‐KO or CCR2‐KO mice subjected to 60 minutes of myocardial ischemia followed by 24 hours of reperfusion. The horizontal lines represent mean±SD (n=4–8 per group). CCR2‐KO indicates C‐C chemokine receptor 2‐knockout mice; Ctrl, control; CypD‐KO, cyclophilin D‐knockout mice; IgG, immunoglobulin G; IL‐1β, interleukin‐1β; IR, ischemia‐reperfusion; IS, infarct size; and WT indicates wild‐type mice.

Figure 5. Nanoparticle‐mediated simultaneous targeting of CypD and C‐C chemokine receptor 2 confer superior cardioprotection against IR injury.

*P<0.05, **P<0.01, 1‐way ANOVA. A, Gross appearance of left ventricle myocardial sections after Evans blue and triphenyltetrazolium chloride staining. B, The therapeutic effects of CsA‐NP, Pitava‐NP or CsA/Pitava‐NP on the infarct size in wild‐type mice with 30 or 60 minutes of ischemia followed by 24 hours of reperfusion. The data represent the mean±SD (n=8–9 per group). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, 1‐way ANOVA. C, The infarct‐sparing effects of CsA‐NP or Pitava‐NP in CypD‐knockout or CCR2‐knockout with 45 minutes of ischemia followed by 24 hours of reperfusion. The horizontal lines represent mean±SD (n=8 per group). CCR2‐KO indicates C‐C chemokine receptor 2‐knockout mice; CypD‐KO, cyclophilin D‐knockout mice; CsA‐NP, nanoparticles containing Cyclosporine A; FITC‐NP, nanoparticles containing fluorescein‐isothiocyanate; IR, ischemia‐reperfusion; Pitava‐NP, nanoparticles containing pitavastatin.

Figure 6. The effects of CsA‐NP or Pitava‐NP on inflammation.

*P<0.05, 1‐way ANOVA. A, Ischemia‐reperfusion‒injured myocardium was subjected to flow cytometry. The number of monocytes were normalized by IS. Ly6C^high activated monocytes were decreased in CsA/Pitava‐NP treated mice. The data are mean±SD (n=4 per group). *P<0.05, **P<0.01, 1‐way ANOVA. B, IL‐1β protein levels measured 24 hours after reperfusion and normalized by infarct size. The horizontal lines represent mean±SD (n=5 per group). *P<0.05, **P<0.01, 1‐way ANOVA. C, Dual channel FMT imaging revealed that CsA‐NP reduced cell death (AnnexinVivo750), whereas protease activities (ProSense680) were comparable with saline group. Treatment with Pitava‐NP or CsA/Pitava‐NP reduced both Annexin750 and ProSense680 signals. The data are mean±SD (n=7–8 per group). CsA‐NP, nanoparticles containing Cyclosporine A; FMT, fluorescence molecular tomography; IL‐1β, interleukin‐1β; IR, ischemia‐reperfusion; IS, infarct size; Pitava‐NP, nanoparticles containing pitavastatin.