Magnetic microbubbles conjugated with anti-vascular cell adhesion molecule-1 monoclonal antibody 429

MBvM

Leung K.

Publication Details

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Table

In vitro Rodents

Background

[PubMed]

Ultrasound is a widely used imaging modality (1), and its role in noninvasive molecular imaging with ligand-carrying microbubbles (MBs) is expanding (2). MBs are comprised of spherical cavities encapsulated in a shell and filled with a gas. The shells are made of phospholipids, surfactants, denatured human serum albumin, or synthetic polymers. Ligands and antibodies can be incorporated into the MB shell surface. MBs are usually 2–8 μm in size. MBs of this size provide a strongly reflective interface and resonate to ultrasound waves. They are used as ultrasound contrast agents in imaging of inflammation, angiogenesis, intravascular thrombus, and tumors (2-4). They also can potentially be used for drug and gene delivery (5).

Endothelial cells are important cells in inflammatory responses (6, 7). Bacterial lipopolysaccharide (LPS), virus, inflammation, and tissue injury increase tumor necrosis factor α (TNFα), interleukin-1 (IL-1), and other cytokine and chemokine secretion. Leukocyte emigration from blood is dependent on their ability to roll along endothelial cell surfaces and subsequently 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 (8).

VCAM-1 is found in very low amounts or nod-detectable on the cell surface of resting endothelial cells and other vascular cells, such as smooth muscle cells and fibroblasts (9-13). VCAM-1 binds to 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 was also induced by oxidized low-density lipoproteins under atherogenic conditions (14). Overexpression of VCAM-1 by atherosclerotic lesions plays an important role in their progression toward vulnerable plaques, which may erode and rupture. MBs targeted with monoclonal antibody against VCAM-1 are being developed as a noninvasive agent for VCAM-1 expression in vascular endothelial cells during different stages of inflammation in atherosclerosis (15). However, MBs tend to remain close to the axial center of blood vessels and have a small rate of adhesion to the target endothelium (16). Wu et al. (17) prepared magnetic MBs coupled with rat anti-mouse VCAM-1 monoclonal antibody (mAb) 429 (MBvM) to evaluate the efficacy of MBvM to enhance the ultrasound contrast of atherosclerosis in the aorta using a magnetic field guidance system. Their studies showed that the use of MBvM resulted in greater attachment to atherosclerotic aortas in apolipoprotein E (APOE)–deficient mice on a hypercholesterolemic diet (HCD) than did the use of nonmagnetic targeted MBs.

Synthesis

[PubMed]

Wu et al. (17) prepared biotinylated MBs by sonication of an aqueous dispersion of decafluorobutane gas, dipalmitoyl phosphatidylcholine, polyoxyethylene-40-stearate, and distearoylphosphatidylethanolamine-polyethylene glycol(PEG2000)-biotin. MBs were combined with magnetic streptavidin, washed, and conjugated with 0.33 nmol/108 MBs biotinylated rat mAb 429 against mouse VCAM-1 (MBvM) or control inactive mAb (MBiM). Nonmagnetic MBs coupled with mAb 429 (MBv) were prepared similarly using nonmagnetic streptavidin. The three types of MBs were ~2.2 μm in diameter. The streptavidin:mAb:MB ratio was estimated to be ~100,000:300,000:1.

In Vitro Studies: Testing in Cells and Tissues

[PubMed]

Wu et al. (17) performed perfusion of MBvM, MBv, or MBiM (5 × 106/ml) through the flow chamber coated with VCAM-1-Fc chimera at wall shear rates of 1.0–24.0 dynes/cm2 (n = 5/group). In the absence of magnetic field guidance, both MBvM and MBv showed ~50 MBs per field at 1 dyne/cm2, whereas MBiM showed minimal attachment. The MB attachment decreased with increasing shear rates, with minimal attachment at 8 dynes/cm2. In the presence of magnetic field guidance, attachment of MBvM was significantly higher than that of MBv at all shear rates (P < 0.05). There were 260, 120, and 10 MBs at 4 dynes/cm2 for MBvM, MBv, and MBiM, respectively. At 20 dynes/cm2, there were 80, 0, and 0 MBs for MBvM, MBv, and MBiM, respectively. After termination of magnetic field guidance, MBvM remained firmly attached even at higher shear rates. All experiments were performed in a double-blinded manner.

Animal Studies

Rodents

[PubMed]

Wu et al. (17) determined MBvM, MBv, or MBiM attachment to excised aorta at 10 min after intravenous injection in wild-type (C57) or APOE-deficient mice on either regular diet (RD) or hypercholesterolemic diet (HCD) (n = 10–14/group). The first 5 min after MB injection was under magnetic field guidance, and then 5 min passed without this guidance. Aortas were removed at 10 min after injection for contrast-enhanced ultrasound (CEU) molecular imaging. Median CEU video intensity signals for MBvM were 28, 15, 9, and 3 for APOE-HCD, APOE-RD, C57-HCD, and C57-RD, respectively. Median CEU video intensity signals for MBv were 10, 6, 3, and 2 for APOE-HCD, APOE-RD, C57-HCD, and C57-RD, respectively. Median CEU video intensity signals for MBiM were 3, 3, 2, and 1 for APOE-HCD, APOE-RD, C57-HCD, and C57-RD, respectively. MBvM provided greater video intensity signals than those of MBv (P < 0.001), which provided greater video intensity signals than MBiM in the four groups of mice (P < 0.001). The aortas from APOE-HCD mice provided the greatest video intensity signals for MBiM compared to the aortas from the other three groups of mice (P < 0.001). Immunohistochemical analysis of the aorta sections showed expression of VCAM-1 in the rank order of APOE-HCD >> APOE-RD > C57-HCD > C57-RD. All experiments were performed in a double-blinded manner. No blocking studies were performed with non-biotinylated mAb 429.

Other Non-Primate Mammals

[PubMed]

No publication is currently available.

Non-Human Primates

[PubMed]

No publication is currently available.

Human Studies

[PubMed]

No publication is currently available.

NIH support

R21/33 CA102880

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