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Letter Cite This: Nano Lett. 2018, 18, 5885−5891

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Nanoparticle Delivery of miRNA-21 Mimic to Cardiac Macrophages Improves Myocardial Remodeling after Myocardial Infarction Tzlil Bejerano,† Sharon Etzion,‡ Sigal Elyagon,‡,§ Yoram Etzion,‡,§ and Smadar Cohen*,†,‡,∥ †

Avram and Stella Goldstein-Goren Department of Biotechnology Engineering, ‡Regenerative Medicine and Stem Cell (RMSC) Research Center, §Department of Physiology and Cell Biology, and ∥The Ilse Katz Institute for Nanoscale Science and Technology, Ben-Gurion University of the Negev, Beer-Sheva 8410501, Israel

Nano Lett. 2018.18:5885-5891. Downloaded from pubs.acs.org by UNIV OF SUNDERLAND on 10/19/18. For personal use only.

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ABSTRACT: MicroRNA-based therapy that targets cardiac macrophages holds great potential for treatment of myocardial infarction (MI). Here, we explored whether boosting the miRNA-21 transcript level in macrophage-enriched areas of the infarcted heart could switch their phenotype from proinflammatory to reparative, thus promoting resolution of inflammation and improving cardiac healing. We employed laser capture microdissection (LCM) to spatially monitor the response to this treatment in the macrophage-enriched zones. MiRNA-21 mimic was delivered to cardiac macrophages post MI by nanoparticles (NPs), spontaneously assembled due to the complexation of hyaluronan-sulfate with the nucleic acid mediated by calcium ion bridges, yielding slightly anionic NPs with a mean diameter of 130 nm. Following intravenous administration, the miRNA-21 NPs were targeted to cardiac macrophages at the infarct zone, elicited their phenotype switch from pro-inflammatory to reparative, promoted angiogenesis, and reduced hypertrophy, fibrosis and cell apoptosis in the remote myocardium. Our work thus presents a new therapeutic strategy to manipulate macrophage phenotype using nanoparticle delivery of miRNA-21 with a potential for use to attenuate post-MI remodeling and heart failure. KEYWORDS: miRNA-21, laser capture microdissection, macrophages, myocardial infarction, nanoparticles yocardial infarction (MI), commonly known as a “heart attack” causes the formation of noncontractile scar tissue and left ventricular (LV) remodeling that further deteriorate cardiac function. MI is associated with excessive risk of heart failure and death.1 In response to cardiomyocyte cell death and matrix degradation after MI, the innate immune system is robustly activated; myeloid cells, including neutrophils, monocytes and macrophages are accumulated within the injured myocardium.2 These cells produce inflammatory and oxidative responses and contribute to scar expansion, LV remodeling, and LV systolic dysfunction. Central to the cellular events after MI is the increase in macrophage number in the heart through the combined effects of massive recruitment of bone marrow-derived cells and local self-renewal of macrophages that predominately function to remove by phagocytosis the apoptotic/necrotic cardiomyocytes and produce important mediators, such as tumor necrosis factor α (TNF-α), that establish crosstalk with other cardiac cell types.3 During inflammation resolution within a few days, reparative macrophages replace and dominate the tissue, upregulating signals that direct endothelial cells, fibroblasts, and local progenitor cells to rebuild damaged tissue.4 The extent and coordination of these two macrophage responses are critical for the appropriate myocardial healing after MI; in cases where inflammation is persistent and/or resolution is

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delayed, the implications on infarct size and LV remodeling are devastating. Realizing this, we5 and others6,7 have suggested that manipulation of innate immune responses may be sufficient to reduce cardiac tissue damage and improve outcomes. In the present study, we investigated whether targeting postMI inflammation status could be achieved by nanoparticle delivered miRNA-21 mimic to cardiac macrophages post MI. Our strategy was prompted by recent findings showing miRNA-21 upregulation following the phagocytosis of apoptotic cells by peritoneal macrophages, consequently leading to inflammation resolution (reviewed by Sheedy et al.8). Thus, miRNA-21 mimic was delivered in nanoparticles (NPs), spontaneously assembled due to the complexation of hyaluronan-sulfate (HAS) with the nucleic acid mediated by calcium ion bridges.9,10 We locally monitored the therapeutic effect of the nanoparticle delivered miRNA-21 mimic to the macrophage-enriched area at the infarct border vs its effect on the remote area, that is, the LV posterior wall, by employing laser capture microdissection (LCM) system. The use of the LCM system enabled researching the cardiac macrophages in Received: June 25, 2018 Revised: August 10, 2018 Published: August 24, 2018 5885

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Figure 1. Isolation of macrophage-enriched area at the infarct border area using LCM. MI was induced in mice, and heart samples were collected 3 and 7 days post MI. (A) Frozen sections of the cardiac tissue were stained with cresyl violet to histologically define the infarct (i) and with CD11b (red) and nuclei (blue) (ii) to define a macrophage enriched area (the infarct border area). The infarct border area and a remote area were cut and captured using LCM (iii), and RNA was extracted from these areas. (B) Relative levels of inflammatory gene expression in the MI border area and a remote area. Both areas were normalized to LV samples from healthy hearts. qPCR readout of inflammatory genes TNF-α (i) and iNOS (ii) and anti-inflammatory genes Arg1 (iii) and HO-1 (iv). (C) Relative miRNA-21 expression levels at the MI border area (i) and a remote area (ii). Both areas were normalized to healthy hearts. Data represent mean ± SEM (n = 3) *p < 0.05, **p < 0.01.

however, is unavailable. Furthermore, various findings have reported conflicting effects of miRNA-21 on macrophage polarization, as reviewed by Essandoh et al.12 Some studies reported that miRNA-21 is up-regulated in macrophages after the engulfment of apoptotic cells3 augmenting inflammation resolution,13 while others reported upregulation of pri-miR-21 to control the early stages of inflammation.14 To address this knowledge gap, we used LCM to collect the macrophage-enriched tissue samples from the infarct border as

their natural microenvironment with no need for cell processing or in vitro culturing, which have been previously shown to greatly affect their activation.11 At first, we aimed to quantify miRNA21 expression in the heart and specifically in cardiac macrophages following coronary ligation in mice. Previous reports on miRNA-21 expression in macrophages were mainly obtained in peritoneal and bone marrow derived cells; information on miRNA-21 expression in myocardial macrophages of infarcted hearts, 5886

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Figure 2. In vivo up-take and accumulation of CY5-tagged NPs in cardiac macrophages of mice post-MI. NPs were injected i.v. to the tail vein of mice 3 days post-MI or to healthy control mice. Five hours after injection, the animals were sacrificed and the organs were examined ex vivo by IVIS (A) to evaluate accumulation of NPs in the heart (i). Signal to noise ratio (ii) of the fluorescent signal to autofluorescence was significantly higher at the infarcted hearts, (iii) biodistribution of NPs (IVIS images of the kidneys, spleen, and liver are shown in Supplementary Figure S2). Cryosections of the hearts were stained for CD11b (green) and nuclei (blue) and imaged using confocal microscopy (B). Full cross-section of the heart shows accumulation of the NPs (red) mainly in the infarct area (i) and close-up images show the NPs in cardiac macrophages (ii,iii). Scale bars in (B): i, 500 μm; ii, 100 μm; iii, 50 μm. (n = 4, per group. p = 0.0094, two-tailed Student’s t test).

7 post-MI where it reached a statistical difference compared to healthy tissue. Importantly, we found that the level of miRNA21 detected in the macrophage-enriched area from the infarct border (Figure 1Ci) is one-order of magnitude greater compared to the remote area (Figure 1Cii). The changes in miRNA-21 expression levels with time after MI together with the perception that prolonged inflammatory macrophage activity after MI impairs infarct healing led us to explore the effect of boosting miRNA-21 expression on macrophage phenotype switch from pro-inflammatory to anti-inflammatory state, for promoting resolution of inflammation and improving cardiac healing. We further explored whether we could deliver miRNA-21 mimic to cardiac macrophages post-MI, using NPs coassembled from miRNA-21, Ca2+ and hyaluronan-sulfate (HAS). The HAS-Ca2+-miRNA NPs had a slightly anionic surface charge (ζ potential = −10 mV). Their mean average size was 130 nm according to dynamic light scattering (DLS) analysis (Supplementary Figure S1) and they reveal NP population with a narrow size distribution, as judged by nearly overlapping peaks of the unweighted vs weighted results (Supplementary Figure S1). The NPs efficiently encapsulate nucleic acid and could be loaded up to 1.36 mg miRNA/mg HAS. Suspensions of the NPs have been stable for several days and were cytocompatible in physiologically relevant environments.9 The NP ability to target cardiac macrophages post-MI was first evaluated by injecting CY5-labeled siRNA/NPs intravenously (i.v.) into the tail vein of mice 3 days post-MI (n = 4). The selection of this time point is based on our results, shown in Figure 1B as well as previous studies,4,5 showing the greatest number of cardiac macrophages in infarcted hearts at this time point. Five hours after the injection, the mice were sacrificed,

well as from samples from the LV posterior wall (the remote area). Initially, the macrophage-enriched area was identified on cryo-sections from the infarcted hearts (Figure 1Ai) by laser scanning confocal microscope (LSCM) after immunostaining the cryo-sections (Figure 1Aii), for CD11b, a surface marker of macrophages(red) and DAPI (blue) for nuclei. Adjacent sections collected by LCM (Figure 1Aiii) were further quantitatively analyzed by qRT-PCR for macrophage-related genes in the infarct border compared to the remote myocardium and healthy LV myocardial tissue, 3 and 7 days post-MI (Figure 1B). On day 3, the transcription levels of TNF-α, iNOS, Arg1, and HO-1 were all significantly up-regulated in the infarct border area compared to their levels in the remote or healthy myocardium, indicating macrophage accumulation at the infarct zone. On day 7, the transcription levels were reduced but they were still greater in the infarct border area compared to the remote myocardium. The kinetics and spatial location of macrophage accumulation in infarcted heart, as revealed here by the LCM strategy, is in agreement with previous reports using robust techniques involving tissue mincing, cell isolation, and analysis,3,4 thus offering credibility to this methodology for monitoring macrophages, which is vital in order to track the success of macrophage-targeting therapies. Following identification of the cardiac macrophage-enriched area, miRNA-21 expression in this area was quantified by qRTPCR compared to the remote myocardium area and to healthy myocardial tissue at day 1, 3, and 7 after MI. This information is critical for planning the timing of treatment with miRNA-21 NPs after MI. As seen in Figure 1Ci, a marked time-dependent increase in miRNA-21 level is seen in the MI border zone; it started on day one after MI and intensified with time up to day 5887

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Figure 3. Modulation of cardiac macrophages post-MI by delivery of miRNA-21 NPs. MI was induced in mice, and 24, 48, and 72 h after the MI the mice were i.v. injected with either miRNA-21 NPs, control NPs, or saline. Twenty four hours after the last injection, the animals were sacrificed and RNA was extracted from the infarct border area (A) or a remote area (B), using the LCM. Gene expression levels of TGFβ (i), Socs1 (ii), and TNFα (iii). Data represent mean ± SEM (n = 3−6 in each group). * p < 0.05.

48, and 72 h after the MI) into the tail vein. Twenty-four hours after the last injection (collectively 96 h post MI) the mice were sacrificed. Using the LCM, RNA was collected and extracted from the infarct border area and the remote area for qRT-PCR analysis. For proper comparison, two control groups were included in this experiment: saline-treated infarcted animals in which the triple injection of miRNA-21 NPs was replaced by volumetric saline injections (saline group) and control NP-treated infarcted animals in which the triple injection of miRNA-21 NPs was replaced by NPs containing nontargeting siRNA (control-NPs group). At the infarct border area, miRNA-21 NP treatment caused a significant increase in the transcriptional level of the anti-inflammatory gene TGF-β compared to its level in the saline treated group with a clear tendency of increase compared to the control-NPs group as well (Figure. 3Ai). A significant increase in the level of Socs1, another anti-inflammatory gene, was also noted in this area compared to the saline treated group (Figure 3Aii). Concomitantly, miRNA-21 NP treatment reduced the transcriptional level of TNFα (Figure 3Aiii), indicating a clear and specific anti-inflammatory effect of the miRNA-21-mimic in this context, while mice treated with the control-NPs showed high levels of the pro-inflammatory cytokine TNFα in the infarct border area. A rather similar picture was also noted in the remote myocardium area. The miRNA-21 NPs treatment caused an increase in the level of TGF-β compared to its level in saline-treated or control NP-treated infarcted animals (Figure 3Bi) and a tendency of increase in Socs1, was also noted in this area compared to both sham groups (Figure 3Bii). However, in the remote myocardial area the basal level of TNF-alpha was low and rather similar in all of the three treatment groups (Figure 3Biii). Collectively, our results support the notion that boosting miRNA-21 expression level in post MI cardiac macrophages elicits their phenotype switch from pro-inflammatory to anti-inflammatory state. Anti-inflammatory or reparative macrophages can upregulate signals that direct endothelial cells, fibroblasts, and

their major organs (heart, liver, spleen, kidneys) were retrieved, and imaged (ex vivo) with an IVIS imaging system. To avoid background noise, each organ’s signal was normalized to the autofluorescence of the same organ from an animal injected with saline. We found a significant accumulation of the NPs in infarcted hearts, compared with healthy hearts (n = 4) (Figure 2Ai,ii). Additionally, we detected large signals in organs such as kidney, spleen, and liver (Figure 2Aiii, imaging results for these organs are presented in Supplementary Figure S2) most likely due to the role of these organs as monocyte reservoir and a destination of monocytes/macrophages leaving the infarct post MI.15 To verify the accumulation of CY5-labeled siRNA/NPs in cardiac macrophages, cross sections from the hearts of mice which were injected i.v. with these NPs were immunostained for CD11b (macrophage marker, green stain). LCSM images revealed colocalization of the CY5-labeled siRNA/NPs (red) and CD11b positive cells, indicating targeting and uptake of the NPs by cardiac macrophages at the infarct area (Figure 2B). These results are not surprising as medium-sized NPs (∼10−300 nm in diameter) have been shown to be efficiently taken up and accumulated in diseased tissues into which macrophages have been recruited and reside (for example, ischemic myocardium).16 The mechanism by which the HASCa2+-miRNA NPs are uptaken by macrophages is most likely phagocytosis, and it is not mediated via activated CD44, as the sulfation of HA changes the polymer and it is no longer recognized by the HA receptors on cells.9 Next, we examined the capability of the nanoparticles to deliver miRNA-21-mimic to post-MI cardiac macrophages after their administration by the i.v. route and the consequences of boosting miRNA 21 level in cardiac macrophages on their phenotype. We decided to intervene during the first 4 days post-MI, as in this time period there is the greatest accumulation of macrophages, concomitant with a major change in miRNA-21 expression level (Figure 1Ci). MiRNA-21 NPs were injected on a daily basis for 3 days (24, 5888

DOI: 10.1021/acs.nanolett.8b02578 Nano Lett. 2018, 18, 5885−5891

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Figure 4. Histological assessment of angiogenesis and macrophage accumulation 7 days post-MI in mice treated with miRNA-21 NPs. MI was induced in mice, and 24, 48, and 72 h after the MI the mice were i.v. injected with either miRNA-21 NPs, control NPs, or saline. A week later, the harvested hearts were sectioned and stained with antibodies against CD31 for endothelial cells (A) and CD11b for macrophages (B). Data represent mean ± SEM (n = 3 per group). Representative images of the other groups are presented in the Supplementary Figure S3. * p < 0.05.

Figure 5. Effect of miRNA-21 NPs on cardiac function and remodeling after MI. MI was induced in mice, and 24, 48, and 72 h after the MI the mice were i.v. injected with either miRNA-21 NPs, control NPs, or saline. Twenty four hours and a month after the MI, echocardiography was performed. Echocardiography results of (A) ejection fraction on day 30, (B) LV posterior wall thickness in diastole at day 30, and (C) change in LV mass. The hearts were then harvested, sectioned, and stained with Sirius Red and quantified for collagen (D) or with an antibody against activated caspase-3 for apoptosis. Apoptotic cells were counted at the MI border area (E) and at the LV posterior wall (F). Data represent mean ± SEM * p < 0.05. Representative images of the other groups are presented in the Supplementary Figure S4.

miRNA-21 promotes reparative macrophages, we examined the effect of miRNA-21 NP treatment on the extent of angiogenesis in the infarcted heart area, a week after the MI, by

local progenitor cells to rebuild damaged tissue. Manifestation of this process is enhanced angiogenesis, as reflected by the increase in blood vessel density at the infarct site.4 As boosting 5889

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accelerating the switch of inflammatory macrophages to the reparative state. This conclusion is substantiated by the finding of marked upregulation of TGFβ in the remote area (the LV posterior wall), upon treatment with the triple injections of miRNA-21 NPs (Figure 3Bi). Increasing TGF-β is a double edged sword. On the one hand, TGF-β is known to be upregulated in anti-inflammatory macrophages and it exerts potent matrix-preserving actions by suppressing the activity of matrix metalloproteinases and by inducing synthesis of protease inhibitors.18,19 However, it is also known that by acting on cardiac fibroblasts, TGF-β induces the differentiation of fibroblasts to myofibroblasts20 promoting fibrosis.21 As the nanoparticle-based delivery of miRNA-21 was targeted to cardiac macrophages at the infarct, it is unlikely that the exogenous delivered miRNA-21 acts directly on cardiac fibroblasts in the remote myocardial area. The good or bad outcome of TGF-β up-regulation depends on the timing and dosage.22 In our case, it had a positive effect on the remodeling process in the remote area. miRNA-21 expression has been found to be upregulated in several pathologies, including carcinogenesis (reviewed by Feng and Tsao23) and boosting its expression may potentially raise major safely concerns. In our experiments, we did not observe any macroscopic indication for tumor formation in the animals treated with miRNA-21 NPs up to 1 month following the exposure to the treatment. Nevertheless, our study was not designed to thoroughly target safety issues and future studies should address this concern more directly with long-term safety studies. Importantly, a study was performed by Harlan Biotech Israel Ltd. (Rehovot, IL), to assess the potential toxic effects of HASCa2+-siRNA (NC5) NPs following a single slow intravenous injection of 3.3 mg/kg NPs to either male and female ICR(CD-1) (unpublished data). No mortality or clinical signs occurred throughout the entire study period. In terms of body weight, all animals made positive body weight gain at the end of the 14 day observation period and body weight curves were within the normal range. In terms of gross macroscopic evaluation at necropsy, no abnormalities were noted. In view of the reported findings and under the conditions of this study, it was concluded that acute administration of HAS-Ca2+-siRNA (NC6) NPs at a dose of 3.3 mg/kg NPs via the intravenous (IV) route is not associated with toxic risk. In summary, our results demonstrate that nanoparticle-based targeted delivery of miRNA-21 to cardiac macrophages after MI can induce their modulation toward an anti- inflammatory, reparative state. The consequences of this modulation are the increased angiogenesis, lower number of apoptotic cells, and attenuation in left ventricle remodeling after MI. Our current modulation did not lead to improvement of systolic function and will probably require further refinement of this new methodology in order to reach such outcome as well. From a technical point of view, this study also demonstrates the utility of LCM as a tool to gain knowledge about cell-type specificity of regulated miRNAs, such as suggested for miR-21, under a given pathophysiological condition. This eventually would help to optimize strategies of targeted delivery of miRNAs mimics or their inhibitors (antagomirs) to the celltype or tissue of interest for systemic application in terms of a therapeutic approach.

immuno-histochemistry. Post-MI mice were randomly assigned to three consecutive daily i.v. injections of miRNA-21 NPs, control NPs, or saline, as described in the previous section. Quantification of blood vessels, stained for CD31 (endothelial cells marker), on full cross sections of the heart, showed a significant increase in the number and density of blood vessels under 10 μm in diameter, in mice treated with miRNA-21 NPs, compared with the saline-treated group, and a trend of increase in density compared to the control NPstreated group (Figure 4A) (Supplementary Figure 3S shows the immunohistochemistry results for control groups). Assessment of macrophage accumulation on full cross sections of the heart, by quantification of CD11b positive cells, showed no significant difference between the different groups (Figure 4B). Because both pro-inflammatory and reparative macrophages are positive for CD11b, the interpretation is that our treatment did not change the number of macrophages but it did affect their phenotype as revealed in Figure 3. The finding that i.v. administration of miRNA-21 NPs can target macrophages in infarcted hearts, leading to upregulation of anti-inflammatory genes and angiogenesis, encouraged us to test the efficacy of this strategy to improve infarct repair after MI. Infarcted mice were randomly subjected to three daily i.v. injections of either miRNA-21 NPs (n = 6), control NPs (n = 7) or saline (n = 7). Echocardiography studies were performed 1 day after MI, prior to the treatment and 1 month later, to examine the effect of treatment on infarct repair. Overall the echocardiography analysis indicated no differences between the groups in regard to functional parameters as ejection fraction (the percentage of blood volume ejected from the heart with each contraction) (Figure 5A, Supplementary Table S4). However, a marked effect of the miRNA-21 NP treatment was detected on the diastolic thickness of the LV posterior wall (Figure 5B) and on the LV mass (Figure 5C). Both parameters were reduced in the miRNA-21 NPs-treated mice compared with the saline-treated group and demonstrated similar tendency compared to control NPs group, although the latter did not reach statistical significance. Elevated global LV mass and increased thickening of the noninfarcted myocardium are typical indicators of adverse LV remodeling associated with pathological cardiac hypertrophy, fibrosis, and apoptosis.1,17 Thus, the echocardiographic findings are consistent with reduced LV remodeling in the miRNA-21 NPs-treated mice. To further explore this possibility, cross sections of the hearts were stained with Sirius Red and quantified for the presence of collagen in the LV posterior wall as an indicator for fibrosis (Supplementary Figure S4). In addition, hearts were stained for activated caspase-3, a marker for cellular apoptosis (Supplementary Figure S4). Indeed, miRNA-21 NPs treated mice demonstrated reduced collagen in the posterior wall myocardium compared to the saline-treated mice (Figure 5D). Control NP treatment had a similar inhibitory effect on collagen deposition in this regard (Figure 5D). In terms of cell apoptosis, activated caspase-3, which was elevated in the infarcted zone in all of the groups as expected (Figure 5E), demonstrated a clear trend of decrease in the posterior remote zone of miRNA-21 NP treated mice relative to both saline and control NP groups (Figure 5F). Collectively, these results support that boosting miRNA-21 expression in cardiac macrophages at the infarct site during the first days after MI (1−3 days) has a notable effect on disease progression mainly in the remote myocardium, possibly by 5890

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(6) Courties, G.; Heidt, T.; Sebas, M.; Iwamoto, Y.; Jeon, D.; Truelove, J.; Tricot, B.; Wojtkiewicz, G.; Dutta, P.; Sager, H. B.; Borodovsky, A.; Novobrantseva, T.; Klebanov, B.; Fitzgerald, K.; Anderson, D. G.; Libby, P.; Swirski, F. K.; Weissleder, R.; Nahrendorf, M. J. Am. Coll. Cardiol. 2014, 63 (15), 1556−66. (7) Ben-Mordechai, T.; Kain, D.; Holbova, R.; Landa, N.; Levin, L.P.; Elron-Gross, I.; Glucksam-Galnoy, Y.; Feinberg, M. S.; Margalit, R.; Leor, J. J. Controlled Release 2017, 257, 21−31. (8) Sheedy, F. J. Front. Immunol. 2015, 6, 19. (9) Forti, E.; Kryukov, O.; Elovic, E.; Goldshtein, M.; Korin, E.; Margolis, G.; Felder, S.; Ruvinov, E.; Cohen, S. J. Controlled Release 2016, 232, 215−227. (10) Korin, E.; Bejerano, T.; Cohen, S. J. Controlled Release 2017, 266, 310−320. (11) Nahrendorf, M.; Swirski, F. K. Circ. Res. 2016, 119 (3), 414− 417. (12) Essandoh, K.; Li, Y.; Huo, J.; Fan, G.-C. Shock 2016, 46 (2), 122−131. (13) Sheedy, F. J.; Palsson-McDermott, E.; Hennessy, E. J.; Martin, C.; O’Leary, J. J.; Ruan, Q.; Johnson, D. S.; Chen, Y.; O’Neill, L. A. Nat. Immunol. 2010, 11 (2), 141−7. (14) Wang, Z.; Brandt, S.; Medeiros, A.; Wang, S.; Wu, H.; Dent, A.; Serezani, C. H. PLoS One 2015, 10 (2), 0115855. (15) Leuschner, F.; Rauch, P. J.; Ueno, T.; Gorbatov, R.; Marinelli, B.; Lee, W. W.; Dutta, P.; Wei, Y.; Robbins, C.; Iwamoto, Y.; Sena, B.; Chudnovskiy, A.; Panizzi, P.; Keliher, E.; Higgins, J. M.; Libby, P.; Moskowitz, M. A.; Pittet, M. J.; Swirski, F. K.; Weissleder, R.; Nahrendorf, M. J. Exp. Med. 2012, 209 (1), 123. (16) Weissleder, R.; Nahrendorf, M.; Pittet, M. J. Nat. Mater. 2014, 13 (2), 125−138. (17) Sam, F.; Sawyer, D. B.; Chang, D. L. F.; Eberli, F. R.; Ngoy, S.; Jain, M.; Amin, J.; Apstein, C. S.; Colucci, W. S. American Journal of Physiology - Heart and Circulatory Physiology 2000, 279 (1), H422. (18) Schiller, M.; Javelaud, D.; Mauviel, A. J. Dermatol. Sci. 2004, 35 (2), 83−92. (19) Mauviel, A. Methods Mol Med. 2005, 177, 69−80. (20) Desmoulière, A.; Geinoz, A.; Gabbiani, F.; Gabbiani, G. J. Cell Biol. 1993, 122 (1), 103. (21) LEASK, A.; ABRAHAM, D. J. FASEB J. 2004, 18 (7), 816− 827. (22) Bujak, M.; Frangogiannis, N. G. Cardiovasc. Res. 2007, 74 (2), 184−195. (23) Feng, Y. H.; Tsao, C.-J. Biomed. Rep. 2016, 5 (4), 395−402.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.nanolett.8b02578. NPs DLS measurement, representative histological images for assessment of angiogenesis and macrophage accumulation 7 days post-MI in mice treated with miRNA-21 NPs, representative histological images for assessment of apoptotic cells and fibrosis 30 days post MI in mice treated with miR mimic-21 NPs, TaqMan assay data for quantitative RT-PCR, siRNA/miRNAmimic sequences, echocardiography summary, and methods (PDF)



AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Tzlil Bejerano: 0000-0001-7029-9704 Smadar Cohen: 0000-0002-3765-8354 Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Funding

The research was supported by a grant from the Israel Science Foundation (1637/12) and the Baruch Stem Cell Fund. Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS Tzlil Bejerano gratefully acknowledges the generous fellowship from the late Mr. Daniel Falkner and his daughter Ms. Ann Berger. This work was done in partial fulfillment of the requirements for a PhD degree (T.B.) at the Avram and Stella Goldstein-Goren Department of Biotechnology Engineering, Ben-Gurion University of the Negev, Israel. Prof. Cohen holds the Claire and Harold Oshry Professor Chair in Biotechnology.



ABBREVIATIONS HAS, hyaluronan sulfate; iNOS, inducible nitric oxide synthase; Arg1, arginase 1; HO-1, heme oxygenase 1; qRTPCR, quantitative reverse transcription polymerase chain reaction; LCM, laser capture microdissection; LSCM, laser scanning confocal microscope; LV, left ventricle; miRNA, microRNA; MI, myocardial infarction; NPs, nanoparticles; TNF-α, tumor necrosis factor-α; TGF-β, transforming growth factor β.



REFERENCES

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DOI: 10.1021/acs.nanolett.8b02578 Nano Lett. 2018, 18, 5885−5891