Background

[PubMed]

Optical fluorescence imaging is increasingly used to monitor biological functions of specific targets in small animals (1-3). However, the intrinsic fluorescence of biomolecules poses a problem when fluorophores that absorb visible light (350–650 nm) are used. Near-infrared (NIR) fluorescence (650–1,000 nm) detection avoids the background fluorescence interference of natural biomolecules, providing a high contrast between target and background tissues. NIR fluorophores have a 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 noninvasive alternative to radionuclide imaging in small animals (4, 5).

Photoacoustic imaging (PAI) is an emerging hybrid biomedical imaging modality based on photoacoustic effects (6-9). In PAI, non-ionizing optical pulses are delivered into biological tissues. Some of the delivered energy is absorbed and converted into heat, leading to transient thermoelastic expansion and thus ultrasonic emission. The generated ultrasonic waves are then detected by ultrasonic transducers to form images. It is known that optical absorption is closely associated with physiological properties, such as hemoglobin concentration and oxygen saturation. As a result, the magnitude of the ultrasonic emission (i.e., photoacoustic signal), which is proportional to the local energy deposition, reveals physiologically specific optical absorption contrast and tissue structures. However, exogenous NIR contrast agents are necessary to overcome the intrinsic low tissue and hemoglobin absorption and the scattering of signal in tissue. On the other hand, these small molecules exhibit fast clearance, small optical absorption cross section, and non-targeted specificity. Therefore, there is a need for contrast agents with long blood circulation and targeted specificity.

Gold (Au) nanoparticles have been studied as molecular imaging agents because of their bright NIR fluorescence emission of 700–900 nm and low toxicity (10). They can be tuned to emit in a range of wavelengths by changing their sizes, shapes, and composition, thus providing broad excitation profiles and high absorption coefficients. They can be coated and capped with hydrophilic materials for additional conjugation with biomolecules, such as peptides, antibodies, nucleic acids, and small organic compounds for in vitro and in vivo studies. Use of Au nanoparticles has been approved by the United States Food and Drug Administration for the treatment of patients with rheumatoid arthritis. Au nanoparticles have been studied as contrast agents in X-ray/computed tomography, NIR optical coherence tomography, PAI, and photoacoustic tomography (PAT) (3). NIR Au nanocages (AuNCs) are biocompatible, have low toxicity, and are tunable to strong NIR absorption (11). They have an outer edge of ~50 nm and an inner edge of ~42 nm, with a wall thickness of ~4 nm. Yang et al. (12) performed PAT of the cerebral cortex of rats with polyethylene glycol–coated AuNCs (PEG-AuNCs) as an optical contrast agent. The investigators observed an enhanced optical contrast in the vasculature in the cerebral cortex. Song et al. (13) demonstrated the use of Au nanocages as a PAI probe for detection of sentinel lymph nodes in rats.

Endothelial cells are important cells in inflammatory responses (14, 15). 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 (16).

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 (16-20). 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 (21). Overexpression of VCAM-1 by atherosclerotic lesions plays an important role in their progression to vulnerable plaques, which may erode and rupture. Rouleau et al. (22) developed gold nanoshells coated with polyethylene glycol (PEG) and anti-VCAM-1 antibody (AuNS-PEG-VCAM-1Ab) for use with in vivo PAT imaging of atherosclerotic plaques in mice.

Synthesis

[PubMed]

Rouleau et al. (22) reported the synthesis of AuNS by incubation of HAuCl₄ (9.5 µM) and cobalt nanoparticles in degassed water with stirring for 10 min at 25°C. The mixture was exposed to ambient conditions to allow oxidation of cobalt nanoparticles as the AuNS formed. AuNS nanoparticles were isolated with ultracentrifugation. To prepare AuNS-PEG-VCAM-1Ab, a solution of bi-functional OPSS-PEG-NHS (2 kDa) and rat anti-mouse VCAM-1 antibody (eBioscience, clone 429) was incubated overnight at 4°C. The reaction was stopped by addition of water to an antibody concentration of 0.13 nM that was mixed with AuNS. The mixture was incubated overnight at 4°C. The final product, AuNS-PEG-VCAM-1Ab nanoparticles, was isolated with centrifugation and ultrafiltration. The AuNS-PEG-VCAM-1Ab solution had a maximum absorbance at 710 nm. The external diameter was 38.7 ± 1.9 nm, with a shell thickness of 4.5 ± 0.2 nm as determined with transmission electron microscopy. There were ~1,500 antibody molecules per AuNS-PEG-VCAM-1Ab. The number of Ab molecules per nanoparticles decreased by ~25% after storage for 3 months at 4°C.

In Vitro Studies: Testing in Cells and Tissues

[PubMed]

In vitro cellular accumulation assays of AuNS-PEG-VCAM-1Ab and AuNS-PEG were performed in 2H11 mouse endothelial cells before and after stimulation with TNFα (22)_. Fluorescence confocal imaging showed that AuNS-PEG-VCAM-1Ab bound to the activated 2H11 cells with marginal binding to nonactivated 2H11 cells. AuNS-PEG exhibited little binding to both activated and nonactivated 2H11 cells.

Animal Studies

Human Studies

[PubMed]

No publication is currently available.

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