Cy5.5-Annexin V

Excerpt

Apoptosis, or programmed cell death, is a fundamental component of tissue development and differentiation and plays an important role in a great variety of diseases, including cancer. Excessive apoptosis is observed in diseases such as AIDS, Alzheimer’s, and progressive heart failure, whereas insufficient apoptosis often occurs in tumor growth. It also plays a central role in the pathology of type 1 and type 2 diabetes (1). Imaging of apoptosis may be a valuable tool for quantifying the development (or regression) of such diseases and for monitoring the effects of chemotherapies (2), anti-hormonal therapeutics, and anti-angiogenic therapies.

The highly regulated mechanism of cell apoptosis involves an externalization process of amino-phospholipids, primarily phosphatidylserine (PS), that normally face the cytoplasm. Through this process, PS residues are exposed at the outer plasma membrane and face the extracellular fluid. At this stage, Annexin V (36 kDa; 10−¹⁰ < K_d < 10−⁹m (3)) may be used to recognize the surface- exposed PS by strongly and specifically binding to the phospholipid, reflecting a biological role as an anti-coagulant (4-6).

Imaging of apoptosis through PS exposure using Annexin V removes the need for cellular internalization. This in vivo imaging method, which has increased in popularity in recent years, has led to the development of both fluorescent and magnetic annexins for various applications (7-9). Tagging Annexin V with a fluorophore produces a probe that acts similarly to native Annexin V and preserves the mass, high affinity, and specificity of the protein.

Nevertheless, Annexin V can be retained in normal tissues and interfere with the imaging of apoptotic uptake. Because the changes in signal are usually small in the case of apoptosis, the sensitivity of the imaging technique is therefore an important parameter to consider (10). Several studies have also shown the ability of Annexin V to bind to PS exposed on the cell membrane in other pathological conditions, such as widespread cutaneous necrosis resulting from vascular damage (e.g., antiphospholipid antibody (aPL) syndrome associated with dermal microvascular thrombosis) (11, 12).

Cy5.5 is a fluorophore with low hydrophobicity and high photostability that can be used as a probe for near-infrared fluorescence (NIRF) imaging of tumor apoptosis using Annexin V (13). Its absorbance and emission maxima are 683 nm and 707 nm respectively (measured in 0.1 m triethylammonium acetate (TEAA) buffer, pH 7).