Annexin A5-Gd-micelles-Cy5.5

Optical fluorescence imaging is increasingly being used to obtain images of biological functions of specific targets 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 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 (Gd) and manganese (Mn), 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 ( = 7 nM). Annexin V has been radiolabeled with I, I, and Tc for single-photon emission computed tomography (SPECT) imaging (6-8). Annexin V was successfully labeled with F (F-labeled annexin V (4-[F]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.