Cy5.5-Amino-terminal fragment of urokinase-type plasminogen activator conjugated to magnetic iron oxide nanoparticles

Magnetic resonance imaging (MRI) maps information about tissues spatially and functionally. Protons (hydrogen nuclei) are widely used in imaging because of their abundance in water molecules. Water comprises ~80% of most soft tissue. The contrast of proton MRI depends primarily on the density of the nucleus (proton spins), the relaxation times of the nuclear magnetization (, longitudinal, and , transverse), the magnetic environment of the tissues, and the blood flow to the tissues. However, insufficient contrast between normal and diseased tissues requires the development of contrast agents. Most contrast agents affect the T1 and relaxation times of the surrounding nuclei, mainly the protons of water. * is the spin–spin relaxation time composed of variations from molecular interactions and intrinsic magnetic heterogeneities of tissues in the magnetic field [1]. Cross-linked iron oxide (CLIO) nanoparticles and other iron oxide formulations affect primarily and lead to decreased signals. On the other hand, the paramagnetic agents, such as gadolinium (Gd), and manganese (Mn), accelerate relaxation and lead to brighter contrast images. Optical fluorescence imaging is increasingly used to obtain biological functions of specific targets [2, 3] in small animals. However, the intrinsic fluorescence of biomolecules poses a problem when visible light (350-700 nm) absorbing fluorophores are used. Near-infrared (NIR) fluorescence (700-1000 nm) detection avoids the background fluorescence interference of natural biomolecules, providing a high contrast between target and background tissues. NIR fluorophores have wider dynamic range and minimal background as a result of reduced scattering compared with visible fluorescence detection. They also have high sensitivity, resulting from low infrared background, and high extinction coefficients, which provide high quantum yields. The NIR region is also compatible with solid-state optical components, such as diode lasers and silicon detectors. NIR fluorescence imaging is becoming a non-invasive alternative to radionuclide imaging in small animals. Extracellular matrix (ECM) adhesion molecules consist of a complex network of fibronectins, collagens, chondroitins, laminins, glycoproteins, heparin sulfate, tenascins, and proteoglycans that surround connective tissue cells, and they are mainly secreted by fibroblasts, chondroblasts, and osteoblasts [4]. Cell substrate adhesion molecules are considered essential regulators of cell migration, differentiation, and tissue integrity and remodeling. These molecules play an important role in inflammation and atherogenesis, but they also participate in the process of invasion and metastasis of malignant cells in the host tissue [5]. Invasive tumor cells adhere to the ECM, which provides a matrix environment for permeation of tumor cells through the basal lamina and underlying interstitial stroma of the connective tissue. Overexpression of matrix metalloproteinases (MMPs) and other proteases by tumor cells allows intravasation of tumor cells into the circulatory system after degradation of the basement membrane and ECM [6]. Several families of proteases are involved in atherogenesis, myocardial infarction, angiogenesis, and tumor invasion and metastasis [7-10]. Urokinase-type plasminogen activator (uPA) is a serine protease [11, 12]. The uPA and uPA receptor (uPAR) system is responsible for tissue degradation after plasminogen activation to plasmin, which leads to a cascade of proteolysis or thrombolysis depending on the physiological conditions. uPA also directly activates MMPs, vascular endothelial growth factor, and human growth factor [13]. Malignant tumors and tumor-associated stromal cells (macrophages, endothelial cells and fibroblasts) often express high levels of uPA and uPAR [14]; therefore, the uPA/uPAR system is linked to vascular diseases and cancer. The amino-terminal fragment (ATF, 1-135 amino acids) of uPA binds with high affinity to uPAR [15]. Yang et al. [16] have developed a multimodality imaging probe by conjugation of Cy5.5-ATF to iron oxide nanoparticles to form Cy5.5-ATF-IO nanoparticles for NIR imaging and MRI of uPAR expression in tumor. Cy5.5 is a NIR fluorescent dye with absorbance maximum at 675 nm and emission maximum at 694 nm with a high extinction coefficient of 250,000 Mcm.