reomicrographs of whole hearts 3 hours after intravenous injection of FITC-NP. Scale bar: 5 mm. Fluorescence microscopic images of the cross sections of the IR hearts treated with FITC-NP. Cardiomyocytes are identified by anti-troponin T antibody and nuclei by DAPI. Merged image shows colocalization of troponin T and FITC-NP. Scale 
bar: 100 nm., Representative light and fluorescence stereomicrographs of cross-sections of the IR hearts 3-hour after intravenous injection of vehicle, FITC alone, or FITC-NP. In the light images, hearts were double-stained with Evans blue and TTC to determine the area at risk. Scale bar: 5 mm., Quantification of FITC fluorescence MS 275 chemical information intensity in AAR and non-ischemic area 3-hour after intravenous injection of vehicle, FITC alone, or FITC-NP. N = 4 each. Data are compared using one-way ANOVA followed by Bonferroni’s multiple comparison tests., Fluorescence stereomicrographs of the IR hearts from rats co-treated with Evans blue dye and FITC-NP. Evans blue and FITC fluorescence signals PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19741364 were co-localized in IR myocardium. Scale bar: 5 mm. Close correlation between the intensity of FITC and Evans blue. Thirty ROIs were placed on the fluorescence images of heart sections per animal at random. Values of mean fluorescence intensity of both FITC and Evans blue were determined in the same ROI. Pearson’s correlation was used to investigate relationships between the fluorescence intensity of FITC and Evans blue., Flow cytometric histograms of CD11b-positive leukocytes in the IR hearts and the blood 24-hour after intravenous injection of FITC-NP. Cells were labeled with anti-CD11b antibodies. White indicates control fluorescence in cells derived from uninjected animals. Green indicates fluorescence in cells derived from FITC-NP injected mice. Right graphs show mean fluorescence intensity in CD11b-positive leukocytes in ischemic myocardium and blood. Data are meanSEM. Data are compared using unpaired t tests. doi:10.1371/journal.pone.0132451.g002 we examined the localization of FITC and Evans blue and found that the distribution of Evans blue was closely correlated with the distribution of FITC, suggesting a role of enhanced vascular permeability in the mechanism of PLGA nanoparticle delivery to the IR cardiomyocytes. Flow cytometry of the IR heart and blood 24-hour after IR and FITC-NP treatment revealed that CD11b-positive leukocytes in the IR heart and blood had significant FITC signals, suggesting that PLGA nanoparticle were delivered to CD11b-positive leukocytes in the circulation and in the IR myocardium. 8 / 23 Nanomedicine for Myocardial Reperfusion Injury Data are expressed as the meanSEM. P<0.05 versus pitavastatin group. #P<0.05. versus non-ischemic myocardium by the unpaired t-test. Values below measurable limits are replaced with the one-half value of the limits. The statistical analysis of differences between two groups, in which one of values was replaced by the complementary value, was assessed with the unpaired t-test after transforming values into a natural logarithm. doi:10.1371/journal.pone.0132451.t001 Plasma and tissue concentrations of pitavastatin were measured in IR animals. In PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19741295 the Pitavastatin-NP group, the myocardial concentrations of pitavastatin were 2- to 3-fold higher in IR myocardium than in non-ischemic myocardium at 30 min, 3 hours, and 24 hours of reperfusion. There were no differences in myocardial concentrations of pitavastatin in IR myocardium between pitavastatin and pitavastatin-NP grou