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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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Lys-Thr-Leu-Leu-Pro-Thr-Pro-cross-linked iron oxide-Cy5.5

PTP-CLIO-Cy5.5
, PhD
National Center for Biotechnology Information, NLM, NIH, Bethesda, MD

Created: ; Last Update: February 22, 2011.

Chemical name:Lys-Thr-Leu-Leu-Pro-Thr-Pro-cross-linked iron oxide-Cy5.5
Abbreviated name:PTP-CLIO-Cy5.5
Synonym:
Agent category:Peptide
Target:Plectin-1
Target category:Receptor
Method of detection:Magnetic resonance imaging (MRI), optical (near-infrared (NIR) fluorescence)
Source of signal\contrast:Iron oxide, Cy5.5
Activation:No
Studies:
  • Checkbox In vitro
  • Checkbox Rodents
Click on protein, nucleotide (RefSeq), and gene for more information about Plectin-1.

Background

[PubMed]

Optical fluorescence imaging is increasingly being used to obtain images of biological functions of specific targets in vitro and in small animals (1, 2). Near-infrared (NIR) fluorescence (700–900 nm) detection avoids the background fluorescence interference of natural biomolecules, providing a high contrast between target and background tissues. NIR fluorescence imaging is becoming a non-invasive alternative to radionuclide imaging in vitro and in small animals.

Magnetic resonance imaging (MRI) maps information about tissues spatially and functionally. Protons (hydrogen nuclei) are widely used to create images because of their abundance in water molecules, which comprise >80% of most soft tissues. The contrast of proton MRI images depends mainly on the density of the nucleus (proton spins), the relaxation times of the nuclear magnetization (T1, longitudinal; T2, transverse), the magnetic environment of the tissues, and the blood flow to the tissues. However, insufficient contrast between normal and diseased tissues requires the use of contrast agents. Most contrast agents affect the T1 and T2 relaxation of the surrounding nuclei, mainly the protons of water. T2* is the spin–spin relaxation time composed of variations from molecular interactions and intrinsic magnetic heterogeneities of tissues in the magnetic field (3). Cross-linked iron oxide (CLIO) and other iron oxide formulations affect T2 primarily and lead to a decreased signal.

A multimodal nanoparticle probe that consists of a contrast agent and a NIR fluorochrome may provide consistent information. CLIO nanoparticles can be internalized by cells of the reticuloendothelial system and have long circulating times in vivo. The blood half-life of CLIO is ~10 h in mice (4). The accumulation of nanoparticles in cells causes a reduction in signal intensity with T2-weighted (T2*W) spin-echo pulse sequences. NIR fluorochromes (e.g., Cy5.5) provide an improved optical (NIR) signal from tissue. CLIO-Cy5.5 has been developed as a multimodal probe for imaging (5).

Patients with pancreatic ductal adenocarcinoma (PDAC) have a median survival of 6 months and a 5-year survival rate of only 3% (6). PDAC is an aggressive cancer with early metastasis in >80% of patients. A method for early detection would be particularly useful for monitoring people at high risk of developing pancreatic cancer. Using phage display screenings, the linear peptide Lys-Thr-Leu-Leu-Pro-Thr-Pro (Plectin-1 targeting peptide, PTP) (7) has been found to specifically bind to Plectin-1, a cytoskeletal protein (8, 9) that is present both inside and on the membrane of human and mouse PDAC cells but only on the inside of normal pancreatic cells. Kelly et al. (7) conjugated PTP to CLIO-Cy5.5 to image Plectin-1 expression in PDAC. PTP-CLIO-Cy5.5 is a multimodal agent that consists of CLIO (MRI) and Cy5.5 (NIR imaging).

Synthesis

[PubMed]

The synthesis of PTP-CLIO-Cy5.5 was described by Kelly et al. (7). The amino-CLIO was labeled with Cy5.5-monofunctional N-hydroxysuccinimide ester to yield CLIO-Cy5.5, which had a diameter of 39 nm as determined with light scattering and R1 and R2 values of 21 and 63 mM-1s-1, respectively. CLIO-Cy5.5 was purified with column chromatography and reacted with succinimidyl iodoacetic acid for 15 min. PTP-Cys was conjugated with the activated CLIO-Cy5.5 at room temperature for 1 h to yield the multimodal PTP-CLIO-Cy5.5, which had 3.5 PTP molecules and 2.3 Cy5.5 molecules per nanoparticle as determined with spectrophotometry.

In Vitro Studies: Testing in Cells and Tissues

[PubMed]

Kelly et al. (7) performed cell-binding assays with fluorescein isothiocyanate (FITC)-PTP-phage using human and mouse PDAC cell lines. FITC-PTP-phage and FITC-control phage bound in a similar manner to individual cells with populations of normal human ductal cells. Binding of FITC-PTP-phage to PDAC cells had an average ratio of PTP phage/control phage of 141. Frozen pancreas section studies showed that FITC-PTP-phage bound to PDAC tumor lesions but not to normal regions. Anti–Plectin-1 antibody reduced FITC-PTP-phage binding to PDAC cells by 97%.

Animal Studies

Rodents

[PubMed]

Kelly et al. (7) performed biodistribution studies in 9-wk-old Kras/p53L/+ mice, which harbor small, focal PDAC as well as regions of normal pancreas, ductal metaplasia, and fibrosis. Intravital confocal microscopy revealed discrete areas of fluorescence in the abdominal region of these mice 24 h after injection of 15 mg Fe/kg PTP-CLIO-Cy5.5. The in vivo fluorescence correlated with surface reflectance imaging of the excised pancreas where discrete foci of signal were found. In contrast, control peptide-CLIO-Cy5.5 failed to highlight any regions of the pancreas. The PDAC tumors accumulated 3.13% of the injected dose at 24 h. Accumulation in the liver was similar to that of the tumors. The kidney, spleen, and lung showed moderate accumulation, and the muscle, intestine, normal pancreas, heart, and skin showed little to minimal accumulation. Control peptide-CLIO-Cy5.5 accumulated 1.9% of the injected dose in the tumors at 24 h with a tissue distribution pattern similar to PTP-CLIO-Cy5.5. Tumor uptake relative to normal pancreas was 10-fold and 4-fold higher for PTP-CLIO-Cy5.5 and control peptide-CLIO-Cy5.5, respectively. MRI showed a reduction in MR signal in focal regions of the pancreas. Histological analysis confirmed that the loss of signal associated with PTP-CLIO-Cy5.5 uptake occurred primarily in PDAC regions but not in normal regions or regions of ductal metaplasia. Fluorescence microscopy of the sections demonstrated PTP nanoparticle accumulation in areas of PDAC but not in areas of normal pancreas. No blocking experiment was performed.

Other Non-Primate Mammals

[PubMed]

No publication is currently available.

Non-Human Primates

[PubMed]

No publication is currently available.

Human Studies

[PubMed]

No publication is currently available.

NIH Support

RO1 CA104647, PO1 CA117969, P50 CA86355

References

1.
Achilefu S. Lighting up tumors with receptor-specific optical molecular probes. Technol Cancer Res Treat. 2004;3(4):393–409. [PubMed: 15270591]
2.
Ntziachristos V., Bremer C., Weissleder R. Fluorescence imaging with near-infrared light: new technological advances that enable in vivo molecular imaging. Eur Radiol. 2003;13(1):195–208. [PubMed: 12541130]
3.
Wang Y.X., Hussain S.M., Krestin G.P. Superparamagnetic iron oxide contrast agents: physicochemical characteristics and applications in MR imaging. Eur Radiol. 2001;11(11):2319–31. [PubMed: 11702180]
4.
Wunderbaldinger P., Josephson L., Bremer C., Moore A., Weissleder R. Detection of lymph node metastases by contrast-enhanced MRI in an experimental model. Magn Reson Med. 2002;47(2):292–7. [PubMed: 11810672]
5.
Kircher M.F., Weissleder R., Josephson L. A dual fluorochrome probe for imaging proteases. Bioconjug Chem. 2004;15(2):242–8. [PubMed: 15025519]
6.
Li D., Xie K., Wolff R., Abbruzzese J.L. Pancreatic cancer. Lancet. 2004;363(9414):1049–57. [PMC free article: PMC3062508] [PubMed: 15051286]
7.
Kelly K.A., Bardeesy N., Anbazhagan R., Gurumurthy S., Berger J., Alencar H., Depinho R.A., Mahmood U., Weissleder R. Targeted nanoparticles for imaging incipient pancreatic ductal adenocarcinoma. PLoS Med. 2008;5(4):e85. [PMC free article: PMC2292750] [PubMed: 18416599]
8.
Andra K., Kornacker I., Jorgl A., Zorer M., Spazierer D., Fuchs P., Fischer I., Wiche G. Plectin-isoform-specific rescue of hemidesmosomal defects in plectin (-/-) keratinocytes. J Invest Dermatol. 2003;120(2):189–97. [PubMed: 12542521]
9.
Sonnenberg A., Liem R.K. Plakins in development and disease. Exp Cell Res. 2007;313(10):2189–203. [PubMed: 17499243]
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