The mice were divided into 5 groups (n=8C9/group); each group received an intravenous injection of the following drugs at the time of reperfusion: (1) vehicle (5.0?mL/kg saline), (2) FITC\NP (1.4?mg PLGA in 5.0?mL/kg saline), (3) CsA\NP (PLGA that contained 1.0?mg/kg cyclosporine in 5.0?mL/kg saline), (4) Pitava\NP (PLGA that contained 1.0?mg/kg pitavastatin in 5.0?mL/kg saline), or cocktail containing CsA/Pitava\NP (PLGA that contained 1.0?mg/kg CsA and PLGA that contained 1.0?mg/kg pitavastatin in 5.0?mL/kg saline). 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. murine myocardial IR injury model with various durations of ischemia. Another possible barrier is insufficient local drug concentrations at the target cells or organelle when it is administered at the time of reperfusion. From a clinical perspective, it is essential to deliver drugs to target tissues, for example, by using a drug delivery system (DDS). We have recently developed a nanoparticle\mediated DDS that uses bioabsorbable poly\lactic/glycolic acid (PLGA) nanoparticles.18, 19, 20 Nano\sized materials accumulate to the injured tissues, including the IR\injured myocardium, where vascular permeability is enhanced.21, 22 We reported that intravenous administration of PLGA PDE9-IN-1 nanoparticles containing cyclosporine (CsA\NP), a potent inhibitor of CypD, at the time of reperfusion increased the concentrations of cyclosporine in mitochondrial fraction of cardiomyocytes and protected the heart from PDE9-IN-1 myocardial IR injury.23 PLGA nanoparticles were also taken up by circulating monocytes after intravenous administration. We have reported that PLGA nanoparticles containing pitavastatin (Pitava\NP) inhibited recruitment of Ly6Chigh activated monocytes by inhibiting monocyte chemoattractant protein\1/C\C motif chemokine 2?mediated pathway and conferred cardioprotection against IR injury.24, 25 Therefore nanoparticle\mediated DDS can be a promising therapeutic modality to simultaneously target mitochondria and monocytes in the IR\injured heart. Thus, the goals of this study were (1) to determine the contributions of mitochondrial injury (CypD) and inflammation (CCR2) in the myocardial IR injury; (2) to examine whether nanoparticle\mediated simultaneous targeting of both mitochondrial injury and inflammation confer additive cardioprotection against myocardial IR injury. METHODS The data that support the findings of this study are available from the corresponding author upon reasonable request. Reagent Evans Blue and 2,3,5\triphenyltetrazolium chloride (TTC), collagenase type I, collagenase type XI, DNase I, hyaluronidase, Mito\TEMPO, Adenosine 5’\triphosphate (ATP) (Sigma Aldrich, St. Louis, MO), AnnexinVIvo750, ProSense680 (PerkinElmer, MA), VersaLyse Lysing solution (Beckman Coulter, Brea, CA), Fc receptor with anti\CD16/32 monoclonal antibodies, Rat anti\mouse CD90\PE (53\2.1), Rat anti\mouse B220\PE (RA3\6B2), Rat anti\mouse CD49b\PE (DX5), Mouse anti\mouse NK1.1\PE (PK136), Rat anti\mouse Ly6G\PE (1A8), Rat anti\CD11b\APC (M1/70), Rat anti\Ly6C\FITC (AL\21) (BD Biosciences, CA), non\immune immunoglobulin G (mabg1\ctrlm), neutralizing monoclonal mouse antibody against mouse IL\1 (mabg1\mil1b), lipopolysaccharide from serotype 0111: B4 (InvivoGen, San Diego, CA), RPMI 1640, UltraGlutamine (Lonza, Basel, Switzerland), Fetal bovine serum, Macrophage colony\stimulating factor (R&D Systems, MN) were purchased commercially. Nanoparticle Preparation PLGA, an average molecular weight of 20?000 and a copolymer ratio 75:25 lactide to glycolide (Wako Pure Chemical Industries Ltd., Osaka, Japan), was used as a matrix for the nanoparticles; PDE9-IN-1 polyvinyl alcohol (PVA\403; Kuraray, Osaka, Japan) was used as a dispersing agent. PLGA nanoparticles incorporating a fluorescent marker fluorescein\isothiocyanate (FITC; Dojin Chemical, Tokyo, Japan) (FITC\NP), cyclosporine (Sigma Aldrich) (CsA\NP) or pitavastatin (Kowa Pharmaceutical Co Ltd, Tokyo, Japan) (Pitava\NP) were prepared by an emulsion solvent diffusion method as previously described.18, 19, 20 The FITC\NP contained 4.06% (wt/vol) FITC, the CsA\NP contained 2.67% (wt/vol) cyclosporine and the Pitava\NP contained 12.0% (wt/vol) pitavastatin. The IL17RA diameters of FITC\NP, CsA\NP, and Pitava\NP were 231?nm, 175?nm, and 159?nm, respectively. The surface charges (zeta potential) were also analyzed using a Zetasizer Nano (Sysmex, Hyogo, Japan) and were ?20.3?mV (FITC\NP), ?20.2?mV (CsA\NP), and ?4.0?mV (Pitava\NP), respectively. Mouse Myocardial IR Model The study protocol was reviewed and approved by the committee on the Ethics of Animal Experiments, Kyushu University Faculty of Medicine and was conducted in accordance with the American Physiological Society guidelines and National Institutes of Health guidelines. Male wild\type (WT) mice (C57Bl/6J background) were PDE9-IN-1 purchased from CLEA Japan, Inc. (Tokyo, Japan), and CypD\knockout (CypD\KO) mice were purchased from Jackson Laboratories (Stock#: 009071, Bar Harbor, ME). CCR2\knockout (CCR2\KO) mice (C57Bl/6J and 129/svJae hybrids)26 backcrossed with C57Bl/6J mice at least 10 times were used for experiments. The animals were maintained on a 12\hour light\dark cycle with free.