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. 2009;48(2):299-303.
doi: 10.1002/anie.200804851.

Hydrocyanines: a class of fluorescent sensors that can image reactive oxygen species in cell culture, tissue, and in vivo

Kousik Kundu  1 , Sarah F Knight, Nick Willett, Sungmun Lee, W Robert Taylor, Niren Murthy
Affiliations

Hydrocyanines: a class of fluorescent sensors that can image reactive oxygen species in cell culture, tissue, and in vivo

Kousik Kundu et al. Angew Chem Int Ed Engl. 2009.
No abstract available

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Figures

Figure 1
Figure 1
a) Synthesis of hydrocyanines by a one-step reduction with NaBH4. Reaction with superoxide or the hydroxyl radical oxidizes the hydrocyanines to produce the fluorescent cyanine dyes. b) Hydro-IR-676 has negligible fluorescence emission; however, oxidation with superoxide causes a 100-fold increase in fluorescence (λex = 675 nm, λem = 693 nm).
Figure 2
Figure 2
Hydro-Cy3 has high specificity for superoxide and radical oxidants over other reactive oxygen and nitrogen species. (See the Supporting Information for details). RFU = relative fluorescence unit, TBHP = tert-butyl hydroperoxide, TBO· = tert-butyl peroxyl radical, NOC-5 = 3-(aminopropyl)-1-hydroxy-3-isopropyl-2-oxo-1-triazene, SIN-1 = 3-(4-morpholinyl)sydnone imine hydrochloride, GSH = γ-l-glutamyl-l-cysteinyl-glycine.
Figure 3
Figure 3
Sensitivity of hydro-Cy3, hydro-Cy7, and DHE towards the hydroxyl radical. Both hydro-Cy3 and hydro-Cy7 have nanomolar sensitivity towards the hydroxyl radical and are significantly more sensitive than DHE.
Figure 4
Figure 4
Stability profile of hydro-Cy3, hydro-Cy7, and DHE towards autoxidation. The stability was measured in PBS buffer (pH 7.4) at 37°C (see the Supporting Information for details).
Figure 5
Figure 5
Detection of ROS in live cells and tissue explants by hydrocyanines: a–c) confocal fluorescent images of live RASM cells: a) RASM cells incubated with 100 µm hydro-Cy3; b) RASM cells treated with Ang II (100 nm) for 4 h and incubated with 100 µm hydro-Cy3; c) RASM cells incubated with Ang II (100 nm) for 4 h followed by 5 mm TEMPOL, prior to the addition of hydro-Cy3 (100 µm); d–f) confocal fluorescent images of rat aortic tissue. Fluorescent image of the aorta section of d) the untreated mouse incubated with hydro-Cy3 for 15 min, e) the mouse treated with LPS incubated with hydro-Cy3 for 15 min, and f) the mouse treated with LPS followed by incubation with 5 mm TEMPOL and hydro-Cy3 for 15 min. Slides (d–f) were stained with 4′,6-diamidino −2-phenylindole (DAPI) to identify cell nuclei (blue dots).
Figure 6
Figure 6
In vivo imaging of ROS production from the peritoneal cavity of mice with hydro-Cy7 during an LPS-mediated inflammatory response: a) I) LPS was injected into the peritoneal cavity of the mice, followed by an i.p. injection of hydro-Cy7; II) saline was injected in the i.p. cavity of mice, followed by i.p. injection of hydro-Cy7; III) negative control, neither LPS nor hydro-Cy7 was injected. Mice from each group were imaged at the same time after hydro-Cy7 injection. b) Quantification of fluorescence intensities from LPS-treated mice, saline-treated mice, and a control (I, II, and III from (a), respectively). Oxidized hydro-Cy7 fluorescence intensity is increased in LPS-treated mice relative to saline-treated mice (n = 3, *p < 0.001).
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