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Gadolinium-tetraazacyclododecane tetraacetic acid coupled with folate via bis(aminoethyl)ethylene glycol linker

, PhD
National Center for Biotechnology Information, NLM, NIH

Created: ; Last Update: April 19, 2011.

Chemical name:Gadolinium-tetraazacyclododecane tetraacetic acid coupled with folate via bis(aminoethyl)ethylene glycol linkerImage GdDOTAFolate.jpg
Abbreviated name:Gd.DOTA.Folate
Agent Category:Compounds
Target:Folate receptor
Target Category:Receptor
Method of detection:Magnetic resonance imaging (MRI)
Source of signal / contrast:Gadolinium (Gd3+)
  • Checkbox In vitro
  • Checkbox Rodents
Structure of Gd.DOTA.Folate (1).



Gadolinium-tetraazacyclododecane tetraacetic acid (Gd.DOTA) coupled with folate via the bis(aminoethyl)ethylene glycol linker, abbreviated as Gd.DOTA. Folate, was synthesized by Kalber et al. for folate receptor (FR)-targeted magnetic resonance imaging (MRI) of FR-positive tumors (1).

Folate (folic acid) is an essential vitamin for cell synthesis of nucleotide bases. Cellular uptake of the folate is mediated either by membrane transport proteins or by FR. The human FR has three isoforms: FR-α-isoform, FR-β-isoform, and FR-γ-isoform. The FR-α isoform is expressed on the apical (luminal) surface of epithelial cells in limited tissues, and because of no vascular supply, it is inaccessible to exogenous folate conjugates with the exception of the FR-α isoforms on the proximal tubules of kidneys (1-3). The FR-β isoform is observed mainly on activated macrophages, whereas the FR-γ isoform is rarely detected in human tissues (1, 4). Several features of FR make it a valuable target for developing FR-targeted imaging and therapeutic agents (2, 3, 5). First, FR has a high affinity for the exogenous folate conjugates (Kd = ~100 pM), and the conjugates do not bind to most normal cells. Second, the FR-α isoform is overexpressed in ~40% of human cancer tissues, where it is completely accessible to folate conjugates. Third, folate conjugates are taken up by cancer cells via FR-mediated endocytosis, and FR recycles actively to the cell surface with a frequency of 5.7–20 h, depending on cell types. Fourth, conjugation of imaging labels via the γ-carboxyl group of folate has no apparent effects on the ligand-binding affinity to FR. Furthermore, folate is a small molecule (MW = ~441) and exhibits rapid and complete penetration of solid tumors and rapid clearance from FR-negative tissues (t1/2 = <10 min). However, the MRI contrast can be limited by the relatively low density of FR on the tumor cell surface (1–3 million FR/cell) and by the rapid saturation of binding (2, 3). Nevertheless, most studies demonstrate that cellular uptake of the Gd- or iron oxide-folate conjugates in FR-positive tumors is much higher than in FR-negative tumors. Normal organs except for the kidneys have minimal uptake (1, 2, 6).

Kalber et al. synthesized the novel compound Gd.DOTA.Folate by coupling folate to Gd.DOTA via bis(aminoethyl)ethylene glycol, a very short linker (1). MRI with Gd.DOTA.Folate in tumor-bearing mice showed an increase in R1 with clear enhancement on the MRI images in a human ovarian carcinoma (IGROV-1) xenograft overexpressing the FR-α isoform, but no change was observed in a FR-α–negative human ovarian carcinoma (OVCAR-3) xenograft. However, contrast enhancement with Gd.DOTA.Folate was not observed in vitro in either IGROV-1 cells or OVCAR-3 cells (i.e., the in vitro MRI data did not support the in vivo findings (1)). A similar discrepancy has been reported in other studies when comparing in vitro and in vivo results with FR-targeted agents (7, 8). Studies with 153Gd-folate dendrimer and P866 low molecular weight dimeric Gd.DOTA indicate that the discrepancy may be caused by the potential sensitivity limitation in vitro, perhaps due to the marked cellular variability in FR expression (5, 8). Miotti et al. have shown that the degree of internalized folate is not completely proportional to the degree of expression for the FR-α isoform because cell lines with low expression of the FR-α isoform are still capable of low levels of uptake (7). This appears to be supported by the in vitro studies by Kalber et al. in that, although the FR-α–positive IGROV-1 cells had a much higher degree of Gd.DOTA.Folate internalization than did the FR-α–negative OVCAR-3 cells, there was still a degree of uptake within the OVCAR-3 cells as measured with inductively coupled mass spectrometry (ICP-MS) (1).



Kalber et al. described the details of Gd.DOTA.Folate synthesis (1). Solid-phase synthesis was utilized for the synthesis of the folate precursor (yield = 83%). The precursor was then reacted with activated DOTA-N-hydroxysuccinimide ester to produce the DOTA.Folate ligand (yield = 57%). Addition of Gd chloride to the ligand resulted in the target molecule, Gd.DOTA.Folate. The purity of Gd.DOTA.Folate was confirmed with high-performance liquid chromatography. The isotopic Gd peaks were clearly visible in the mass spectrometry. A xylenol orange assay did not indicate the presence of free Gd ions to any significant degree.

The MRI efficacy of Gd.DOTA.Folate in aqueous solution was measured, and a 70% reduction in the bulk water T1 value was observed compared to water alone and DOTA.Folate control solutions. The r1 relaxivity for Gd.DOTA.Folate was calculated to be 1.28 mM-1s-1, which was slightly lower than that of the non-conjugated Gd.DOTA (3.66 mM-1s-1) (1).

In Vitro Studies: Testing in Cells and Tissues


In vitro MRI (4.7 T) was performed with agarose phantoms that contained cells obtained at 2, 8, and 14 h after incubation with 0.5 mM Gd.DOTA.Folate, DOTA.Folate, Gd.DOTA, or buffer alone (control) (1). Images did not show any signal enhancement or changes in R1 for any compound compared to control cells for either FR-α–positive IGROV-1 cells or FR-α–negative OVCAR-3 cells. However, ICP-MS studies of the cell pellets after 1 h incubation showed that IGROV-1 cells had a concentration of Gd that was approximately double that observed in the OVCAR-3 cells (P < 0.01), suggesting FR-α–mediated uptake in the IGROV-1 cells (1).

Competitive binding experiments were carried out with an excess of 1 mM folate that was added 15 min before the addition of Gd.DOTA.Folate (1). IGROV-1 cells showed a significant reduction in the Gd uptake (P < 0.01). OVCAR-3 cells also showed a reduction, although not to the same extent. Free folate did not completely inhibit the uptake of the folate conjugate in either cell line, suggesting that another mechanism of uptake rather than FR-mediated endocytosis also took place in both cell lines.

None of the agents (Gd.DOTA.Folate, DOTA.Folate, Gd.DOTA) had a significant effect on cell viability (1).

Animal Studies



In vivo MRI was performed in mice bearing either IGROV-1 or OVCAR-3 tumor xenografts at 2 h and 14 h after administration of Gd.DOTA.Folate, Gd.DOTA, or DOTA.Folate (control) (n = 3 mice/group) (1). MRI images with Gd.DOTA.Folate showed clear tumor enhancement in the IGROV-1 tumor xenografts compared to the baseline image, whereas OVCAR-3 tumor xenografts did not show any enhancement on the images. This observation was reflected in the R1 values. An increase in the R1 value was observed at 2 h in the IGROV-1 tumors, and this value increased further at 14 h (P < 0.05). No change in the R1 values was noted with any of the other groups. The gradual increase in R1 values over the 14 h in the IGROV-1 mice treated with Gd.DOTA.Folate suggests that the compound is not washed out of the tumor over this time period; consequently, the compound is either bound to the FR or internalized into the tumor cells through the receptor-mediated transportation. Liver and kidneys showed a slight increase in R1 values; however, the results were not significant, and MRI images were not consistently enhanced in all animals (data not shown).

The presence of Gd within the IGROV-1 tumors in mice treated with Gd.DOTA.Folate was confirmed at 14 h with laser ablation-ICP-MS (1). 157Gd was mainly situated in the rim of the tumor. No 157Gd was detected in the necrotic areas of the tumor core or in tumors from mice injected with the Gd-free control agent, DOTA.Folate. Blocking studies were not reported.

Other Non-Primate Mammals


No references are currently available.

Non-Human Primates


No references are currently available.

Human Studies


No references are currently available.


Kalber, T.L., N. Kamaly, P.W. So, J.A. Pugh, J. Bunch, C.W. McLeod, M.R. Jorgensen, A.D. Miller, and J.D. Bell, A Low Molecular Weight Folate Receptor Targeted Contrast Agent for Magnetic Resonance Tumor Imaging. Mol Imaging Biol, 2010. [PubMed: 20809208]
Sega E.I., Low P.S. Tumor detection using folate receptor-targeted imaging agents. Cancer Metastasis Rev. 2008;27(4):655–64. [PubMed: 18523731]
Low P.S., Kularatne S.A. Folate-targeted therapeutic and imaging agents for cancer. Curr Opin Chem Biol. 2009;13(3):256–62. [PubMed: 19419901]
Low P.S., Henne W.A., Doorneweerd D.D. Discovery and development of folic-acid-based receptor targeting for imaging and therapy of cancer and inflammatory diseases. Acc Chem Res. 2008;41(1):120–9. [PubMed: 17655275]
Zhao X., Li H., Lee R.J. Targeted drug delivery via folate receptors. Expert Opin Drug Deliv. 2008;5(3):309–19. [PubMed: 18318652]
Wang Z.J., Boddington S., Wendland M., Meier R., Corot C., Daldrup-Link H. MR imaging of ovarian tumors using folate-receptor-targeted contrast agents. Pediatr Radiol. 2008;38(5):529–37. [PMC free article: PMC2745549] [PubMed: 18357444]
Miotti S., Bagnoli M., Ottone F., Tomassetti A., Colnaghi M.I., Canevari S. Simultaneous activity of two different mechanisms of folate transport in ovarian carcinoma cell lines. J Cell Biochem. 1997;65(4):479–91. [PubMed: 9178098]
Konda S.D., Aref M., Wang S., Brechbiel M., Wiener E.C. Specific targeting of folate-dendrimer MRI contrast agents to the high affinity folate receptor expressed in ovarian tumor xenografts. MAGMA. 2001;12(2-3):104–13. [PubMed: 11390265]
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