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 (350–700 nm) are used. Near-infrared (NIR) fluorescence (700–1,000 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 fluorescence 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 (NIRF) imaging is becoming a noninvasive alternative to radionuclide imaging in small animals (4, 5).

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 (6). Cell substrate adhesion molecules are considered essential regulators of cell migration, differentiation, and tissue integrity and remodeling. These molecules play a role in inflammation and atherogenesis, but they also participate in the process of invasion and metastasis of malignant cells in the host tissue (7). ECM 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 degrading the basement membrane and ECM (8).

Several families of MMPs are involved in atherogenesis, myocardial infarction, angiogenesis, and tumor invasion and metastases (9-12). MMP expression in normal cells, such as trophoblasts, osteoclasts, neutrophils, and macrophages, is highly regulated. Elevated levels of MMPs have been found in tumors associated with a poor prognosis for cancer patients (13). A peptide, Gly-Pro-Leu-Gly-Val-Arg-Gly-Cys-NH₂, was found to be a MMP substrate and is cleaved between Leu and Gly residues. Lee et al. (14) used this sequence with a Cy5.5 NIR dye molecule to attach to AuNPs to form fluorescence-quenched Cy5.5-Gly-Pro-Leu-Gly-Val-Arg-Gly-Cys-AuNPs (Cy5.5-MMP-AuNPs). The Cy5.5 molecules are in close proximity, resulting in fluorescence quenching because of efficient fluorescence resonance energy transfer to Au. The NIR fluorescence signal will increase when the Leu-Gly bond is cleaved by MMPs, releasing fragments containing Cy5.5. 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 M−¹cm−¹. Cy5.5-MMP-AuNPs are being developed for NIR fluorescence imaging of MMP expression in tumors, atherosclerosis, myocardial infarction, and other diseases.

Ferritin is composed of 24 subunits of heavy and light chains that self-assemble to form a cage-like nanoparticle (nanocage) at physiological pH (7.4) with internal and external diameters of 8 nm and 12 nm (15, 16), respectively. The outer surface of ferritin can be chemically or genetically modified with ligands, and the cavity of ferritin can capture metal ions with high affinity (17). Lin et al. (18) chemically coupled Cy5.5-Gly-Pro-Leu-Gly-Val-Arg-Gly-Cys and black hole quencher-3 (BHQ-3) onto heavy chain ferritin to form C-Fn and B-Fn, respectively. Hybridization of C-Fn and B-Fn (1:1) resulted in the formation of C/B-Fn nanocages. The Cy5.5 molecules are in close proximity to BHQ-3, resulting in fluorescence quenching. NIR fluorescence signal will increase when the Leu-Gly bond is cleaved by MMPs, releasing fragments containing Cy5.5. C/B-Fn nanocages have been developed for NIRF imaging of tumor vasculature to study in vivo biodistribution of the tracer in tumor-bearing mice.

Synthesis

[PubMed]

Recombinant Fn subunits were purified from transfected Escherichia coli cell lysates (18). Fn nanocages were conjugated with a bifunctional compound, N-succinimidyl-4-maleimidobutyrate, and then with Cy5.5-Gly-Pro-Leu-Gly-Val-Arg-Gly-Cys to form C-Fn nanocages. BHQ-3 was conjugated to a different set of Fn nanocages to form B-Fn nanocages. There was one dye/quencher per Fn subunit. To prepare C/B-Fn nanocages, C-Fn and B-Fn nanocages (1:1 ratio) were broken down to subunits by adjusting pH to 2. The pH was then adjusted back to pH 7.4 with 1 M NaOH, and the mixture was incubated for 30 min to form C/B-Fn nanocages. C/B-Fn nanocages were isolated with column chromatography. The diameter of C/B-Fn nanocages was determined to be ~10 nm with dynamic light scattering analysis at pH 7.4

In Vitro Studies: Testing in Cells and Tissues

[PubMed]

C-Fn (430 nM) and B-Fn nanocages (430 nM) were decomposed in acidic buffer (pH 2) into C-Fn and B-Fn subunits (18). There was a maximal NIRF intensity (excitation at 675 nm, emission at 690 nm). The pH was then adjusted back to pH 7.4 with 1 M NaOH. There was a marked decrease in NIRF signal as the subunits reassembled into C/B-Fn nanocages. Addition of MMP13 (100 nM) restored the NIRF as MMP13 cleaved the Leu-Gly bond, releasing fragments containing Cy5.5. Incubation of C/B-Fn nanocages (C/B = 1:1) with 0.1–5 nM MMP13 for 30 min at 37°C showed that the activation of NIRF intensity depended on MMP concentration.

Animal Studies

Human Studies

[PubMed]

No publication is currently available.

NIH Support

Intramural Research Program

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