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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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Annexin A5-Gd-micelles-Cy5.5

AnxA5-micelles
, PhD
National Center for Biotechnology Information, NLM, NIH

Created: ; Last Update: April 27, 2011.

Chemical name:Annexin A5-Gd-micelles-Cy5.5
Abbreviated name:AnxA5-micelles
Synonym:
Agent category:Protein
Target:Phosphatidylserine
Target category:Phospholipid
Method of detection:Multimodality imaging: 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 annexin V.

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 nuclear (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 (3). On the other hand, paramagnetic T1 agents, such as gadolinium (Gd3+) and manganese (Mn2+), accelerate T1 relaxation and lead to brighter contrast images.

A multimodal nanoparticle probe that consists of a contrast agent and a NIR fluorochrome may provide consistent imaging information. CLIO nanoparticles can be internalized by cells of the reticuloendothelial system and have long circulating times within an animal body. 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.

Apoptosis (programmed cell death) plays an important role in the pathophysiology of many diseases, such as cancer, neurodegenerative disorders, vascular disorders, atherosclerosis, and chronic hepatitis, as well as in the biology of normal cells like epithelial cells and immune cells (5). During apoptosis, there is rapid redistribution of phosphatidylserine (PS) (5) from the inner membrane leaflet to the outer membrane leaflet, exposing the anionic head group of PS. PS is also accessible for annexin V (or annexin A5) binding in necrosis because of disruption of the plasma membrane. Annexin V, a 36-kDa protein, binds to PS with high affinity (Kd = 7 nM). Annexin V has been radiolabeled with 123I, 124I, and 99mTc for single-photon emission computed tomography (SPECT) imaging (6-8). Annexin V was successfully labeled with 18F (18F-labeled annexin V (4-[18F]FBA)) (9, 10) and is currently being developed as a positron emission tomography agent for imaging apoptosis as well as necrosis (11). Cy5.5-Annexin V has been evaluated as an optical probe in small animals. Annexin V-CLIO-Cy5.5 (AnxCLIO-Cy5.5) is a multimodal agent that consists of CLIO nanoparticles labeled with annexin V and Cy5.5 (12). van Tilborg et al. (13) incorporated annexin A5 into di-stearoyl-polyethylene glycol-phosphatidylethanolamine 2000 (DSPE-PEG2000) micelles labeled with Gd-DTPA and Cy5.5 (Annexin A5-Gd-micelles-Cy5.5 (AnxA5-micelles)) for multimodality imaging of atherosclerotic plaques.

Synthesis

[PubMed]

DSPE-PEG2000, Gd-DTPA-bisstearylamide, and maleimide-PEG2000-DSPE in a molar ratio of 40:50:10 were mixed in chloroform (13). Cy5.5-PEG2000-DSPE (1 mol %) was added to the mixture, and the solution was dried by rotary evaporation for 3 min at 40°C. The lipid film was subsequently hydrated with 4 ml HEPES-buffered saline to form micelles. Annexin A5-cys was conjugated to the maleimide-PEG2000-DSPE-micelles by sulfhydryl-maleimide bonding. AnxA5-Micelles were purified with dialysis. Control micelles without annexin A5 were prepared similarly. The hydrodynamic diameters of AnxA5-micelles and control micelles were determined to be 22.3 ± 0.2 nm and 19.5 ± 0.7 nm, respectively. There were two annexin A5 molecules, one Cy5.5 molecule, and 40 Gd molecules per AnxA5-micelle. AnxA5-micelles and control micelles exhibited r1 relaxivity values of 12.4 mM-1s-1 and 13.4 mM-1s-1 in HEPES-buffered saline at 1.5 T and 37°C, whereas Gd-DTPA exhibited an r1 relaxivity value of 3.3 mM-1s-1 under the same conditions.

In Vitro Studies: Testing in Cells and Tissues

[PubMed]

van Tilborg et al. (13) performed cell-binding assays with AnxA5-micelles and control micelles in apoptotic human Jurkat T cells with the use of confocal laser scanning microscopy. The apoptotic cells incubated with AnxA5-micelles showed a clear cell-associated fluorescence signal, whereas negligible fluorescence signal was obtained with control micelles.

Animal Studies

Rodents

[PubMed]

van Tilborg et al. (13) used a 9.4-T MRI scanner to perform in vivo T1-weighted MRI in apolipoprotein E–deficient (ApoE-/-) mice, which exhibit severe atherosclerosis in the abdominal area. AnxA5-Micelles (n = 3, 2.5 µmol lipid, 50 µmol Gd/kg, 53 nmol annexin A5) and control micelles (n = 3, 2.5 µmol lipid, 50 µmol Gd/kg) were injected in ApoE-/- mice. Pre-injection scans showed extensive atherosclerotic lesions in the aorta in ApoE-/- mice but not in wild-type mice (n = 2). Contrast enhancement was observed within the width of the aortic wall in ApoE-/- mice at 24 h after injection of AnxA5-micelles and persisted for up to 7 days. No contrast change was observed in the aorta of wild-type mice at 24 h after injection of control micelles. Furthermore, atherosclerotic lesions in ApoE-/- mice injected with control micelles showed less signal enhancement at 24 h after injection. Ex vivo biodistribution of AnxA5-micelles and control micelles in ApoE-/- mice was assessed in the liver, spleen, kidney, and lung by measurement of Gd content at 24 h after injection. The kidney and lung showed <5 µg/g Gd concentration, and results were similar with both micelles. On the other hand, Gd content was 50 µg/g in the liver and 42 µg/g in the spleen with AnxA5-micelles, whereas Gd content was 25 µg/g in the liver and 12 µg/g in the spleen with control micelles. Mean MRI signal in the aortic wall was calculated to be increased by 10.7 ± 1.7% and 6.7 ± 3.4% with AnxA5-micelles and control-micelles, respectively. The total NIR photon measurement in the entire aorta was enhanced by 257.1 ± 9.1% and 113.1 ± 4.4% with AnxA5-micelles and control micelles, respectively. No blocking studies were 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.

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.
Thompson C.B. Apoptosis in the pathogenesis and treatment of disease. Science. 1995;267(5203):1456–62. [PubMed: 7878464]
6.
Blankenberg F.G. Recent advances in the imaging of programmed cell death. Curr Pharm Des. 2004;10(13):1457–67. [PubMed: 15134569]
7.
Lahorte C., Slegers G., Philippe J., Van de Wiele C., Dierckx R.A. Synthesis and in vitro evaluation of 123I-labelled human recombinant annexin V. Biomol Eng. 2001;17(2):51–3. [PubMed: 11163751]
8.
Keen H.G., Dekker B.A., Disley L., Hastings D., Lyons S., Reader A.J., Ottewell P., Watson A., Zweit J. Imaging apoptosis in vivo using 124I-annexin V and PET. Nucl Med Biol. 2005;32(4):395–402. [PubMed: 15878509]
9.
Toretsky J., Levenson A., Weinberg I.N., Tait J.F., Uren A., Mease R.C. Preparation of F-18 labeled annexin V: a potential PET radiopharmaceutical for imaging cell death. Nucl Med Biol. 2004;31(6):747–52. [PubMed: 15246365]
10.
Zijlstra S., Gunawan J., Burchert W. Synthesis and evaluation of a 18F-labelled recombinant annexin-V derivative, for identification and quantification of apoptotic cells with PET. Appl Radiat Isot. 2003;58(2):201–7. [PubMed: 12573319]
11.
Medarova Z., Pham W., Farrar C., Petkova V., Moore A. In vivo imaging of siRNA delivery and silencing in tumors. Nat Med. 2007;13(3):372–7. [PubMed: 17322898]
12.
Schellenberger E.A., Sosnovik D., Weissleder R., Josephson L. Magneto/optical annexin V, a multimodal protein. Bioconjug Chem. 2004;15(5):1062–7. [PubMed: 15366960]
13.
van Tilborg G.A., Mulder W.J., Deckers N., Storm G., Reutelingsperger C.P., Strijkers G.J., Nicolay K. Annexin A5-functionalized bimodal lipid-based contrast agents for the detection of apoptosis. Bioconjug Chem. 2006;17(3):741–9. [PubMed: 16704213]
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