Nanoparticle-Mediated Delivery of Irbesartan Induces Cardioprotection from Myocardial Ischemia-Reperfusion Injury by Antagonizing Monocyte-Mediated Inflammation

Myocardial ischemia-reperfusion (IR) injury limits the therapeutic effect of early reperfusion therapy for acute myocardial infarction (AMI), in which the recruitment of inflammatory monocytes plays a causative role. Here we develop bioabsorbable poly-lactic/glycolic acid (PLGA) nanoparticles incorporating irbesartan, an angiotensin II type 1 receptor blocker with a peroxisome proliferator-activated receptor (PPAR)γ agonistic effect (irbesartan-NP). In a mouse model of IR injury, intravenous PLGA nanoparticles distribute to the IR myocardium and monocytes in the blood and in the IR heart. Single intravenous treatment at the time of reperfusion with irbesartan-NP (3.0 mg kg−1 irbesartan), but not with control nanoparticles or irbesartan solution (3.0 mg kg−1), inhibits the recruitment of inflammatory monocytes to the IR heart, and reduces the infarct size via PPARγ-dependent anti-inflammatory mechanisms, and ameliorates left ventricular remodeling 21 days after IR. Irbesartan-NP is a novel approach to treat myocardial IR injury in patients with AMI.


Flow cytometry
Peripheral blood was drawn via a cardiac puncture, and red blood cells were lysed with VersaLyse Lysing solution for 10 minutes at room temperature. Hearts were removed and digested with a cocktail of 450 U mL -1 collagenase type I, 125 U mL -1 collagenase type XI, 60 U mL -1 DNase I and 60 U mL -1 hyaluronidase (all enzymes were obtained from Sigma-Aldrich) in PBS containing 20 mM Hepes at 37 °C for 1 h. The cell suspension was The leukocytes were also incubated with appropriate isotype controls (BD Pharmingen, San Diego, CA, USA).

Distribution of FITC-nanoparticles
The distribution of FITC in the heart and peripheral blood leukocytes was examined. After myocardial IR, animals were sacrificed, and peripheral blood was drawn by means of cardiac puncture with EDTA (ethylenediaminetetraacetic acid) as an anticoagulant, and red cells were lysed with VersaLyse Lysing Solution (Becton Dickinson Biosciences, San Jose, CA, USA). The distribution of FITC-NP in the peripheral blood leukocytes was analyzed with a FACSCalibur cytometer (Becton Dickinson Biosciences). The heart was harvested and fixed in 10% phosphate-buffered formalin (pH 7.4), and the distribution of FITC-NP was analyzed in 5-µm OCT-embedded sections. Sections of the heart were evaluated by fluorescence microscopy.

Histological and immunohistochemical analyses
Twelve hours after reperfusion, hearts were harvested and fixed overnight in 10% buffered-formalin. After fixation, the tissue was embedded in paraffin. Serial cross-sections (5-µm thick) were used for analysis. The sections were subjected to immunohistology using anti-MCP-1 antibodies (1:200, Santa Cruz Biotechnology). The degree of MCP-1 expression in the AAR 12 hours after reperfusion was evaluated. Digital images of 5 fields in the AAR per heart were stored, and the MCP-1 staining area was assessed using a 20 × objective.

Western blot analysis
Homogenates of IR myocardium were analyzed with immunoblotting. At predetermined time points, ischemic myocardium was isolated and analyzed as previously reported 10 . Briefly, frozen samples were homogenized in lysis buffer, and proteins were separated on SDS-polyacrylamide gels and then blotted to PVDF membranes. The following antibodies were used as primary antibodies: cytochrome c (cytosol fraction; 1:1000, Santa Cruz Biotechnology), VDAC (1:1000, Cell Signaling), and GAPDH (1:2500, Santa Cruz Biotechnology). liquid nitrogen and were sliced into sequential 1-mm-thick cross-sections. The sections were incubated with 2% 2,3,5-triphenyltetrazolium chloride (TTC, Sigma Aldrich) for 10 minutes at 37 °C and were photographed with a stereomicroscope (Nikon, HC-2500) 13,14 .

In vitro drug release kinetics
Irbesartan-NP was dissolved in PBS (10 mM, pH 7.4) (0.1 mg mL -1 ) and loaded to 10 k MWCO Slide-a-lyzer dialysis cassettes (0.1-0.5 mL, Thermo Scientific) and dialyzed against 1000 mL PBS (10 mM, pH 7.4) at 37 °C. At each time point, 50 µL were removed from inside of the cassette for the measurement of remaining irbesartan 15 .

Chemotaxis assay
THP-1, a human monocyte cell line, was obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ; Brauschweig, Germany). The cells were cultured in RPMI 1640 with 10% FBS at 37 °C in a 5% CO 2 environment until they were subconfluent. The growth medium was replaced with starvation medium with irbesartan-NP containing 0.1303 to 13.03 µg mL -1 of PLGA and 0.01 to 1.0 µM of irbesartan, FITC-NP containing 0.1303 to 13.03 µg mL -1 of PLGA, or vehicle alone for 24 hours. The chemotactic activity of THP-1 cells in response to 10 ng mL -1 MCP-1 was measured in a 96-well microchemotaxis Boyden chamber (ChemoTx; Neuroprobe), as described previously 16 .
Monocytes that had transmigrated through the micropore were stained with trypan blue. The number of monocytes that migrated in response to MCP-1 was counted.

Measurements of irbesartan concentrations in the plasma and tissues
Irbesartan concentrations in the plasma and tissues were measured at predetermined time points by liquid chromatography coupled to tandem mass spectrometry (LC/MS/MS). Briefly, the high-performance liquid chromatography (HPLC) analysis was performed using the   Data are expressed as the mean ± SD (n=3 each)