Optical fluorescence imaging is increasingly used to monitor biological functions of specific targets in small animals (1-3). However, the intrinsic fluorescence of biomolecules poses a problem when fluorophores that absorb visible light (350–700 nm) are used. Near-infrared (NIR) fluorescence (650–900 nm) detection avoids the natural background fluorescence interference of biomolecules, providing a high contrast between target and background tissues. NIR fluorophores have wider dynamic range and minimal background fluorescence as a result of reduced scattering compared with visible fluorescence detection. They also have high sensitivity, resulting from low background fluorescence, and high extinction coefficients, which provide high quantum yields. The NIR region is also compatible with solid-state optical components, such as diode lasers and silicon detectors. NIR fluorescence imaging is a noninvasive complement to radionuclide imaging in small animals or with probes in close proximity to the target in humans (4). Among the various optical imaging agents, only indocyanine green (ICG), with NIR fluorescence absorption at 780 nm and emission at 820 nm, is approved by the United States Food and Drug Administration for clinical applications in angiography, blood flow evaluation, and liver function assessment (5-8). It is also under evaluation in several clinical trials for other applications, such as optical imaging and mapping of both the lymphatic vessels and lymph nodes in cancer patients for surgical dissection of tumor cells and endoscopic imaging of the pancreas and colon.
Integrins are a family of heterodimeric glycoproteins on cell surfaces that mediate diverse biological events involving cell–cell and cell–matrix interactions (9). Integrins consist of an α and a β subunit and are important for cell adhesion and signal transduction. The αvβ3 integrin is the most prominent receptor affecting tumor growth, tumor invasiveness, metastasis, tumor-induced angiogenesis, inflammation, osteoporosis, and rheumatoid arthritis (10-17). Expression of the αvβ3 integrin is strong on tumor cells and activated endothelial cells, whereas expression is weak on resting endothelial cells and most normal tissues. The peptide sequence Arg-Gly-Asp (RGD) has been identified as a recognition motif used by extracellular matrix proteins (vitronectin, fibrinogen, laminin, and collagen) to bind to a variety of integrins, including αvβ3. The αvβ3 antagonists are being studied as anti-tumor and anti-angiogenic agents (12, 16, 18). Various radiolabeled (such as 18F, 64Cu, 68Ga, and 99mTc) and NIR fluorescence-labeled (such as Cy5.5, Cy7, and Cypate) RGD peptides have been introduced for imaging of tumors and tumor angiogenesis (19). ICG derivative 02 (ICG-Der-02), a hydrophilic dye, contains one carboxyl functional group for covalent conjugation to the amino group of biomolecules. Cao et al. (20) conjugated ICG-Der-02 via the α-amino group of Glu residue of Glu-c(RGDyK)2 (dimer) peptide to form ICG-Der-02-c(RGDyK)2. ICG-Der-02 was also conjugated to the Ɛ-amino group of the lysine residue of RGD (linear) and c(RGDyK) (monomer) to form ICG-Der-02-RGD and ICG-Der-02-c(RGDyK), respectively. The three ICD-Der-02-labeled conjugates were evaluated for in vivo NIR optical imaging in tumor-bearing mice.
ICG-Der-02-N-hydroxysuccinimide ester (0.01 mmol) was reacted with RGD (linear), c(RGDyK), or Glu-c(RGDyK)2 (0.01 mmol) in sodium borate buffer (pH 8.3) for 18 h at room temperature (20). The ICG-Der-02 conjugates were purified with high-performance liquid chromatography and verified with mass spectroscopy. There is one dye molecule per conjugate. The yields and purities of ICG-Der-02-RGD, ICG-Der-02-c(RGDyK), and ICG-Der-02-c(RGDyK)2 were not reported. ICG-Der-02-RGD, ICG-Der-02-c(RGDyK), and ICG-Der-02-c(RGDyK)2 displayed spectral properties similar to those of ICG-Der-02 (maximum absorption at 783 nm and maximum emission at 830–834 nm), with quantum yields of 13.6%, 14.3%, and 13.9%, respectively. The quantum yield of ICG-Der-02 was 13.0%.
In Vitro Studies: Testing in Cells and Tissues
In vitro photostability studies of ICG-Der-02, ICG-Der-02-RGD, ICG-Der-02-c(RGDyK), and ICG-Der-02-c(RGDyK)2 showed that the three ICG-Der-02 conjugates (22.7%–26.1% reduction) exhibited higher photostability than ICG-Der-02 (45.4%–46.8% reduction) in borate buffer and human plasma after 60 min of irradiation at 765.9 nm (20). Rhodamine (RhB)-labeled RGD probes showed high fluorescence signals in αvβ3-expressing MDA-MB-231 human breast cancer and U87MG human glioblastoma with intensity order dimer > monomer > linear. On the other hand, αvβ3-negative MCF-7 human breast tumor cells exhibited much weaker fluorescence than MDA-MB-231 and U87MG cells with the three RGD-based probes. Laser confocal microscopy showed binding of RGD-based probes in the cell surface and cytoplasm of the cells. Excess non-conjugated RGD peptides blocked the fluorescence signals. RhB was used because the investigators do not have confocal fluorescence microcopy with NIR capability.
Cao et al. (20) performed in vivo whole-body NIR fluorescence imaging studies in normal nude mice (n = 5) at 0–24 h after intravenous injection of 2 nmol ICG-Der-02, ICG-Der-02-RGD, ICG-Der-02-c(RGDyK), or ICG-Der-02-c(RGDyK)2. Images showed that NIR fluorescence spread over the body at 1 h and then concentrated in the kidneys and urinary bladder. Most NIR fluorescence was cleared from the body by 24 h. The kidney/muscle ratios peaked at 4 h, with 9–10 for the RGD-based probes and 8 for the dye alone. The liver/muscle ratios peaked at 1 h, with ~3 for the dye and RGD-based probes. Ex vivo imaging of the main organs (the liver, kidneys, lung, intestine, heart, and spleen) of mice was performed at 6 h. NIR fluorescence was detected mainly in the kidneys, whereas little NIR fluorescence was observed in the other organs. Blood NIR fluorescence levels of the dye and RGD-based probes peaked at 1 h and then gradually declined to background by 6 h. No obvious differences were observed in the biodistribution of the dye and RGD-based probes, which were mainly excreted by the kidneys.
Cao et al. (20) performed in vivo whole-body NIR fluorescence imaging studies in nude mice (n = 5/group) bearing MDA-MB-231, U87MG, or MCF-7 xenografts at 0, 1, 4, 6, 12, 24, and 48 h after injection of 2 nmol ICG-Der-02, ICG-Der-02-RGD, ICG-Der-02-c(RGDyK), or ICG-Der-02-c(RGDyK)2. For the MDA-MB-231 tumors, all three probes and ICG-Der-02 identified the tumors, with maximum fluorescence intensity at 4 h, and then cleared through the renal pathway. The tumor/muscle ratios at 4 h were 3.0, 3.6, 5.5, and 6.8 for ICG-Der-02, ICG-Der-02-RGD, ICG-Der-02-c(RGDyK), and ICG-Der-02-c(RGDyK)2, respectively. At 48 h, the tumor/muscle ratios were 1.2, 2.3, 3.0, and 3.5, respectively. Blocking experiments were performed with co-injection of excess corresponding non-conjugated RGD peptides with 63%, 70%, and 75% reduction of fluorescence intensity in the tumor for ICG-Der-02-RGD, ICG-Der-02-c(RGDyK), and ICG-Der-02-c(RGDyK)2, respectively. Ex vivo imaging at 4 h showed the tumor/background ratios were 10, 17, 28, and 34 for ICG-Der-02, ICG-Der-02-RGD, ICG-Der-02-c(RGDyK), and ICG-Der-02-c(RGDyK)2, respectively. The organ with the highest tissue/background ratio was the kidney (~22), followed by the liver (~7), lung (~7), intestine (~6), spleen (~3), and heart (~3). No obvious differences were observed in the normal organs amongst the dye and probes. For the U87MG tumor, whole-body NIR fluorescence imaging scans showed slightly weaker NIR fluorescence signals (dimer > monomer > linear RGD peptides) than the MDA-MD-231 tumor, whereas minimal NIR fluorescence signals were observed in the αvβ3-negative MCF-7 tumor.
Other Non-Primate Mammals
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Created: September 24, 2012; Last Update: December 28, 2012.
National Center for Biotechnology Information (US), Bethesda (MD)
Leung K. Indocyanine green derivative 02-Glu-c(RGDyK)2. 2012 Sep 24 [Updated 2012 Dec 28]. In: Molecular Imaging and Contrast Agent Database (MICAD) [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2004-2013.