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 movement 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 levels 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 heptapeptide, cyclo(Cys-Asn-Asn-Ser-Lys-Ser-His-Thr-Cys) (R832), was identified with phage screening against VCAM-1 (12). Burtea et al. conjugated R832 to polyethylene glycol (PEG)–coated ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles. USPIO-R832 (also known as P03011) was evaluated as a noninvasive MRI agent for VCAM-1 expression in vascular endothelial cells in atherosclerosis, vascular inflammation, and cerebral ischemia in mice (13-15).
The synthesis of USPIO-R832 was described by Burtea et al. (13). A solution of 8-amino-3,6-dioxaoctanoyl-R832 with the Lys residue protected by reaction with trifluoroacetic acid to form amides (10 mg) and N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDCI) was added to USPIO nanoparticles and incubated for 20 h. The nanoparticles were isolated with ultrafiltration. A solution of the nanoparticles, EDCI, and aminoPEG750 was incubated for 17 h. The peptide was deprotected by hydrolysis (pH 10) for 6 h at room temperature. The product, USPIO-R832, was isolated with ultrafiltration. A nonspecific peptide (NSP) was also similarly conjugated to USPIO for use as control. The hydrodynamic diameters of USPIO-R832, USPIO-NSP, and USPIO-PEG (also known as P03007) were 26–27 nm. USPIO-R832, USPIO-NSP, and USPIO-PEG exhibited longitudinal (r1) relaxivity values of 1.9–2.2 mM−1s−1 at 7 T, and transverse (r2) relaxivity values of 85.9, 75.6, and 74.6 mM−1s−1 at 7 T, respectively. The number of peptides per USPIO-R832 nanoparticle was not reported. Michalska et al. (15) reported that there was one R832 peptide per P03011 nanoparticle.
In Vitro Studies: Testing in Cells and Tissues
Burtea et al. (12) measured the binding affinity of R832 to human VCAM-1 with a dissociation constant (Kd) value of 103 nM. Saturation binding assays to human VCAM-1 were performed with various concentrations of USPIO-R832, which exhibited a Kd value of 37.2 nM (13). In vitro cellular accumulation of USPIO-R832 (37.2 nM, 4 mM Fe) and USPIO-PEG (4 mM Fe) assays were performed in human umbilical vein endothelial cells (HUVEC) before and after stimulation with TNFα. The cellular Fe level of USPIO-R832 was 170% higher in the stimulated cells than that in the non-stimulated cells and four- to six-fold higher than that of USPIO-PEG.
Burtea et al. (13) performed in vivo 4.7-T MRI in apolipoprotein E–deficient (apoE−/−) mice (n = 15) before and after injection of 0.1 mmol Fe/kg USPIO-R832, USPIO-NSP, or USPIO-PEG. There was −52% contrast in MRI signal intensity (ΔSNR) in the aortic wall at 32 min after injection of USPIO-R832. The aortic ΔSNR remained constant for up to 92 min of imaging and decreased to −18% at 24 h. The liver signals were −34%, −31%, and −63% at 33 min, 92 min, and 24 h, respectively. UPSIO-NSP exhibited aortic ΔSNR of −20% during the 92-min scans and decreased to −16% at 24 h. The liver signals were −11%, −20%, and −33% at 33 min, 92 min, and 24 h, respectively. USPIO-PEG produced aortic ΔSNR of −37% and −6% at 221 min and 24 h, respectively. The liver signals were 4%, 12%, and −30% at 33 min, 92 min, and 24 h, respectively. Immunohistological staining of the aorta sections of apoE−/− mice confirmed the presence of atherosclerotic lesions with extensive expression of VCAM-1. R832 colocalized with VCAM-1 and expression (r2 = 0.939). The blood elimination half-lives for USPIO-R832 and USPIO-PEG were 285 min and 443 min, respectively. Michalska et al. (15) reported a marked MRI signal loss (17.6 T) in the aortic root in apoE−/− mice injected with P03011. No blocking studies were performed.
Frechou et al. (14) performed in vivo 7-T MRI in mice with cerebral ischemia induced by occlusion of the left common carotid artery, followed by perfusion. P03011 (USPIO-R832) (n = 4) or P03007 (USPIO-PEG) (n = 4) (0.1 mmol Fe/kg) was injected intravenously at 5 h after the induction of cerebral ischemia. A lesion was located in both the cortex and striatum, with maximal VCAM-1 expression in the lesion at 24 h post-ischemia. In vivo MRI (T2*-weighted) was performed in the brain at 24 h after nanoparticle injection. The volume of hypointense pixels in the control normal mice was 272.6 ± 99.3 µl. Injection of P03007 had no effect on this volume, whereas injection of P03011 increased this volume by 1.6-fold (P < 0.05). Ex vivo T2*-weighted imaging exhibited a similar profile, with P03011 increasing the volume by four-fold (P < 0.05).
Other Non-Primate Mammals
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Created: March 3, 2013; Last Update: May 9, 2013.
National Center for Biotechnology Information (US), Bethesda (MD)
Leung K. Ultrasmall superparamagnetic iron oxide-cyclo(Cys-Asn-Asn-Ser-Lys-Ser-His-Thr-Cys) 2013 Mar 3 [Updated 2013 May 9]. In: Molecular Imaging and Contrast Agent Database (MICAD) [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2004-2013.