We have recently reported that a red-emitting iridium complex (btp)2 Ir (acac) (BTP) serves as a hypoxia-sensing
probe for tumor imaging in living mice. BTP exhibits oxygen-sensitive phosphorescence that can be utilized to
monitor oxygen levels in living cells and to visualize hypoxic tissues. To improve the tissue penetrance of BTP, we
designed and synthesized near-IR emitting iridium complexes by two different approaches: extension of the π-
conjugated system of benzothienyl-pyridinato ligand in BTP and introduction of substituents into suitable
positions of ligands. The former approach was successful, and near-IR emitting iridium complexes were obtained
without reduction in the emission quantum yield. Cellular uptake of BTP was greatly improved by introducing a
hydrophilic group into the acetylacetonato ligand. Using these improved probes, in-vivo lifetime measurements
were made to substantiate the hypoxia of tumor tissues in SCC-7 tumor-bearing mice. The second-harmonic (532 nm) of Nd3+:YAG laser was used to excite iridium complexes in tissues, and the phosphorescence lifetime was measured using the time-correlated single photon counting technique. The phosphorescence emitted from the tumor region actually gave longer lifetimes compared to those emitted from the normal tissues, demonstrating the hypoxic nature of tumor tissues.
Iridium complex, a promising organic light-emitting diode material for next generation television and
computer displays, emits phosphorescence. Phosphorescence is quenched by oxygen. We used this
oxygen-quenching feature for imaging tumor hypoxia. Red light-emitting iridium complex
Ir(btp)2(acac) (BTP) presented hypoxia-dependent light emission in culture cell lines, whose intensity
was in parallel with hypoxia-inducible factor (HIF)-1 expression. BTP was further applied to imaging
five nude mouse-transplanted tumors. All tumors presented a bright BTP-emitting image as early as 5
min after the injection. The BTP-dependent tumor image peaked at 1 to 2 h after the injection, and was
then removed from tumors within 24 h. The minimal BTP image recognition size was at least 2 mm in
diameter. By morphological examination and phosphorescence lifetime measurement, BTP is
presumed to localize to the tumor cells, not to stay in the tumor microvessels by binding to albumin.
The primary problem on suse of luminescent probe for tumor imaging is its weak penetrance to deep
tissues from the skin surface. Since BTP is easily modifiable, we made BTP analogues with a longer
excitation/emission wavelength to improve the tissue penetrance. One of them, BTPHSA, displayed
560/720 wavelength, and depicted its clear imaging from tumors transplanted over 6-7 mm deep from
the skin surface. We suggest that BTP analogues have a vast potential for imaging hypoxic lesions
such as tumor tissues.
Iridium complexes exhibit highly-efficient phosphorescence that is quenched notably by ambient molecular oxygen. We
utilized the oxygen-sensitive properties of the phosphorescence to visualize tumors in vivo because the levels of oxygen
in tumors may be significantly below those of normal tissue. We used (btp)2Ir(acac) (BTP) as an oxygen probe because
the phosphorescence of BTP appears in the red to near-infrared region and has a relatively long lifetime (5.8μs) and high
quantum yield (0.32) in solution. The oxygen-quenching rate constants were determined to be 5.7 x 104 and 1.2 x 104
mmHg-1s-1 in n-hexane and in lipid bilayer of DMPC in Tris-HCl buffer solution, respectively, at 35°C. We took the
phosphorescence image of HeLa cells that had been incubated under 5% and 20% O2 conditions. In 20% O2 culture
condition, HeLa cells did not exhibit notable phosphorescence, while in 5% O2 culture condition, they emitted bright red
phosphorescence due to BTP. Then we tested this probe to image tumors transplanted in nude mice. After 5 minutes of
the BTP injection, tumor moieties began to emit red phosphorescence and after one hour each tumor was visualized very
clearly by the BTP phosphorescence that could be seen only in a low oxygen state.
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