Detection of Vascular Reactive Oxygen Species in Experimental Atherosclerosis by High-Resolution Near-Infrared Fluorescence Imaging Using VCAM-1-Targeted Liposomes Entrapping a Fluorogenic Redox-Sensitive Probe

Figure 5

Analysis of VLp-RB700 targeted delivery of RB700 into the aortic atherosclerotic lesions in ApoE-/- mice. Fluorescently labeled VLp-RB700 were administrated to mice via retroorbital injection, and after 2 hours, the mice were sacrificed and the aortas analyzed using the IVIS™ imaging system. (a) Representative images of the epifluorescence signal induced by NBD-DSPE are shown, indicating the concentration of RB700 within the atherosclerotic lesions. Spectral unmixing analysis was done to delineate the specific NBD-DSPE signal (green) from the tissue autofluorescence (red). Arrows indicate the specific signal of NBD-DSPE. (b) Colocalization (indicated by arrows) of the labeled RB700-Lp with the atherosclerotic lesions assessed by mirroring of the NBD-DSPE signal with the oil red O (ORO) staining of the atherosclerotic aorta. (c, d) Representative spectral analysis of the VCAM-1-targeted Lp incorporating PBS solution or RB700. Note the emission spectra generated by Lp loaded with PBS (maximum emission peak in the green spectral range) and Lp loaded with RB700 (one maximum emission peak in the green spectral range related to NBD-DSPE and one maximum emission peak in the near-infrared spectral range related to RB700 are observed). (e) Comparative spectral analysis between aortas derived from atherosclerotic ApoE-/- mice injected with Lp (PBS) or Lp (RB700) shows that the localization of the oxidized RB700 near-infrared fluorescence signal along the aorta. (f) Representative images depicting the coincidence (arrows) between VLp-RB700 signal with the ORO staining of the aortic atherosclerotic lesions.

(a)

(b)

(c)

(d)

(e)

(f)