Background

[PubMed]

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 (1). Cross-linked iron oxide (CLIO) and other iron oxide formulations affect T2 primarily and lead to a decreased signal. On the other hand, the paramagnetic T1 agents, such as gadolinium (Gd³⁺), and manganese (Mn²⁺), accelerate T1 relaxation and lead to brighter contrast images.

Endothelial cells are important cells in inflammatory responses (2, 3). Bacterial lipopolysaccharide, virus, inflammation, and tissue injury increase tumor necrosis factor α (TNFα), interleukin-1 (IL-1), and other cytokine and chemokine secretion. Emigration of leukocytes from blood is dependent on their ability to adhere to endothelial cell surfaces. Inflammatory mediators and cytokines induce chemokine secretion from endothelial cells and other vascular cells and increase their expression of cell surface adhesion molecules, such as intracellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), integrins, and selectins. Chemokines are chemotactic toward leukocytes and toward sites of inflammation and tissue injury. The movements of leukocytes through endothelial junctions into the extravascular space are highly orchestrated through various interactions with different adhesion molecules on endothelial cells (4).

VCAM-1 is found in very low amounts on the cell surface of resting endothelial cells and other vascular cells, such as smooth muscle cells and fibroblasts (5-9). VCAM-1 binds to the very late antigen-4 (VLA-4) integrin on the cell surface of leukocytes. IL-1 and TNFα increase expression of VCAM-1 and other cell adhesion molecules on the vascular endothelial cells, which leads to leukocyte adhesion to the activated endothelium. Furthermore, VCAM-1 expression is also induced by oxidized low-density lipoproteins under atherogenic conditions (10). Overexpression of VCAM-1 by atherosclerotic lesions plays an important role in their progression to vulnerable plaques, which may erode and rupture. CLIO nanoparticles targeted with anti-VCAM-1 antibody are being developed as a noninvasive agent for VCAM-1 expression in vascular endothelial cells during different stages of inflammation in atherosclerosis (11). A cyclic peptide, Cys-Asn-Asn-Ser-Lys-Ser-His-Thr-Cys (R832), was identified with phage screening against VCAM-1 (12). Gd-Tetraazacyclododecane-N,N’,N’’,N’’’-tetraacetic acid (Gd-DOTA) was conjugated to the N-terminus of R832 to form Gd-DOTA-R832 with a squarate group as a linker. Gd-DOTA-R832 is being developed as a non-invasive MRI agent for VCAM-1 expression in vascular endothelial cells in atherosclerosis.

Synthesis

[PubMed]

The synthesis of Gd-DOTA-R832 was described by Burtea et al. (12). R832 with the Lys residue protected with trifluoroacetic acid (250 mg) was added to Gd-DOTA-diethylsquarate (210 mg) at pH 8.5 and incubated for 4 days. The peptide was deprotected by hydrolysis (pH 9.5) at room temperature for 2 h. Gd-DOTA-R832 was isolated with high-performance liquid chromatography and confirmed with mass spectroscopy. A scrambled peptide (R832.Sc) was also conjugated with Gd-DOTA as a control. Gd-DOTA-R832, Gd-DOTA-R832.Sc, and Gd-DOTA exhibited longitudinal (r₁) relaxivity values of 8, 8, and 3 mM^-1s^-1 at 60 MHz, respectively.

In Vitro Studies: Testing in Cells and Tissues

[PubMed]

Burtea et al. (12) measured the binding affinity of R832 to human VCAM-1 with a dissociation constant (K_d) of 103 nM. In vitro cell adhesion assays to human VCAM-1 with R832 using Jurkat cells exhibited a 50% inhibition concentration (IC₅₀) value of 630 nM. Anti-VCAM-1 antibody showed an IC₅₀ value of 1,350 nM.

Animal Studies

Human Studies

[PubMed]

No publication is currently available.

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