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Molecular Imaging and Contrast Agent Database (MICAD) [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2004-2013.

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Molecular Imaging and Contrast Agent Database (MICAD) [Internet].

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, PhD
National Center for Biotechnology Information, NLM, NIH
Corresponding author.

Created: ; Last Update: May 30, 2006.

Chemical name:NIR2-Folateimage 11110527 in the ncbi pubchem database
Abbreviated name:
Synonym:NIR2-Folic acid
Agent category:Folic acid
Target:Folate receptor
Target category:Receptor
Method of detection:Optical, near-infrared (NIR) fluorescence imaging
Source of signal:NIR2
  • Checkbox In vitro
  • Checkbox Rodents
Click on the above structure for additional information in PubChem.



Optical fluorescence imaging is increasingly used to monitor biological functions of specific targets (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 (700-1,000 nm) detection avoids the background fluorescence interference of natural biomolecules, providing a high contrast between target and background tissues. NIR fluorophores have a wider dynamic range and minimal background as a result of reduced scattering compared with visible fluorescence detection. They also have high sensitivity, resulting from low infrared background, 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 becoming a non-invasive alternative to radionuclide imaging in small animals.

Folic acid is a water-soluble B vitamin (4) that is essential for methylation and DNA synthesis. The primary pathway for entry of folate into cells is through the facilitated transporter, which has a low affinity for folate (Michaelis constant (Km) = 1-5 μM). Some cells in the choroid plexus, kidney, lung, thyroid, spleen, placenta, and thymus also possess a higher affinity (dissociation constant (Kd) = 0.5 nM) receptor that allows folate uptake via receptor-mediated endocytosis. Some human epithelial tumor cells were found to overexpress folate-binding protein (5). More than 90% of human ovarian and endometrial cancers express the high-affinity receptor, which is absent in normal tissues. Breast, colorectal, renal, and lung carcinomas also overexpress the folate receptor but at lower frequencies (20-50%). Activated macrophages, but not resting macrophages, have been also found to have folate receptor (6). Several folate-based conjugates (111In-DTPA-folate and 99mTc-EC-folate) have been studied in tumor imaging.

Lin et al. (7) reported the synthesis of four new, water-soluble NIR cyanine fluorophores that have superior chemical stability and optical properties. One of the NIR dyes (NIR2) was conjugated to amino-derivatized folic acid to form NIR2-folate, which has an excitation maximum at 665 nm and an emission maximum at 686 nm. NIR2-folate is being developed as an optical imaging agent for detection of folate receptors in vivo.



Tung et al. (8) first reacted folic acid with a hydrophilic spacer, 2,2-(ethylenedioxy)-bis(ethylamine) (EDBEA) in anhydrous dimethyl sulfoxide with diisopropylcarbodiimide/N-hydroxysuccinimide (NHS) as the coupling agent. This converted the carboxyl group of folic acid into a functional amino group. The folate-EDBEA conjugate was purified by high-performance liquid chromatography (HPLC). The NHS ester of NIR2 was then coupled to the folate-EDBEA in 0.1 M NaHCO3/dimethylformamide. The final product, NIR2-folate, was purified by HPLC and was confirmed by mass spectroscopic analysis ([M]+: calculated, 1,401; found, 1,402). The absorption and fluorescence spectra of NIR2-folate showed the characteristics of both folic acid and NIR2.

In Vitro Studies: Testing in Cells and Tissues


The human nasopharyngeal carcinoma cell line KB (12 pmol/106 cells) and ovarian cancer cell line OVCAR3 (1.4 pmol/106 cells) have putative folate receptors, whereas cells from the human lung carcinoma A549 and fibrosarcoma HT1080 have no detectable folate receptors as determined by [3H]folate binding and internalization in cultures (8, 9). Cellular uptake of 50 nM folate by KB and OVCAR3 cells was saturable within 10-20 min of incubation and was completely blocked by excess folate or NIR2-folate. A549 and HT1080 cells (folate-receptor negative) did not show any specific uptake of folate. Fluorescence microscopy experiments to determine cellular distribution of NIR2-folate revealed bright signals only in the KB and OVCAR3 cells but not in the A549 and HT1080 cells.

Animal Studies



Tung et al. (8) performed a preliminary tumor imaging study of NIR2-folate in nude mice (n = 3) bearing an OVCAR3 xenograft model. Whole-body small-animal planar fluorescence reflectance imaging performed after injection of NIR2-folate (2 nmol/mouse) revealed intense tumor signals as early as 1 h after injection that reached a plateau at 24 h. No tumor signals were obtained with NIR2 alone at 1 or 24 h. Moon et al. (9) performed more extensive studies in KB and HT1080 tumors transplanted in nude mice (n = 36). The folate-receptor-positive KB tumors (870 ± 93 absorbance units (AU)) showed a 2.4-fold (P < 0.01) higher signal than the receptor-negative HT1080 tumors (366 ± 41 AU) at 24 h after NIR2-folate injection. Tumor/background ratios remained elevated for at least 24-48 h with a peak at 4 h. Tumor signals returned to background (115 ± 17 AU) at 120 h. Injection of NIR2 alone did not produce tumor signals above background. Injection of a mixture of NIR2-folate (2 nmol/mouse) and folate (600 nmol/mouse) reduced the tumor signals to background at 24 h.

Chen et al. (10) tested NIR2-folate for in vivo optical imaging of arthritis, using a lipopolysaccharide (LPS) intra-articular injection model. The intensities of the fluorescence signals for NIR2-folate (n = 12) and free NIR2 (n = 5) were compared between LPS-treated and control joints. The intensity of the fluorescence signal for NIR2-folate in inflammatory joints was significantly higher than in the normal control joints (2.3-fold; P < 0.001) at 24 h after intravenous (i.v.) NIR2-folate injection (2 nmol/mouse). The group receiving an i.v. injection of NIR2-free dye showed a persistently lower enhancement ratio (1.6-fold) than the group receiving an NIR2-folate injection (2 nmol/mouse). Folate pretreatment (1,200 nmol/mouse 5 min before injection of NIR2-folate) significantly lowered (P < 0.05) NIR2-folate accumulation in the inflamed joint, suggesting that the binding is specific to folate receptors. Fluorescence microscopy, histology, and immunohistochemistry validated the optical imaging results and indicated that activated macrophages were the primary source of NIR2-folate signals. Chen et al. (11) also reported detection of NIR2-folate signals from macrophages in dysplastic intestinal adenomas of APCΔ468-knockout mice.

Other Non-Primate Mammals


No publication is currently available.

Non-Human Primates


No publication is currently available.

Human Studies


No publication is currently available.

NIH Support

N01 C017014, P01 A154904, P50 CA86355, R24 CA92782, R33 CA88365


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