Background

[PubMed]

Optical fluorescence imaging is increasingly used to monitor biological functions of specific targets in small animals (1, 2). 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 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 (NIRF) imaging is becoming a non-invasive alternative to radionuclide imaging in small animals.

Hainfeld et al. (3) performed experiments in mice using gold nanoparticles (AuNPs; 1.9 nm in diameter, ~50 kDa) as a computed tomography contrast agent to enhance imaging contrast in the vasculature, kidneys, and tumor tissue in mice. No physiological complications were observed in the mice for up to a year. James et al. (4) showed that gold nanoshells (110 nm in diameter) coated with polyethylene glycol (PEG) had no toxic effects in mice for up to 28 days. PEG is found to minimize nonspecific adsorption of proteins onto nanoparticles and to reduce their uptake by the liver (5). Surface-enhanced Raman scattering (SERS) involves surface plasmons of silver and gold as excited by a NIR laser (785 nm), and the resulting electric fields cause other nearby molecules (reporter molecules) to become Raman active (6, 7). The result is amplification of the Raman signals (by up to 10¹⁴), allowing detection and identification of single molecules. Qian et al. (8) found that SERS PEG-AuNPs (60–80 nm) are >200 times brighter (NIR signal) than quantum dots on a particle-to-particle basis. A single-chain anti-EGFR B10 antibody fragment (~25 kDa) consisting of the variable V_H and V_L domains for antigen binding (scFvB10) was conjugated to SERS nanoparticles (MGITC-AuNPs-scFvB10) for tumor imaging in a small animal model. The reporter molecules are malachite green isothiocyanate (MGITC). MGITC-AuNPs-scFvB10 has electronic transitions at 633 nm or 785 nm.

Synthesis

[PubMed]

Qian et al. (8) reported the preparation MGITC-AuNPs-scFvB10. A solution of 3–4 µM of MGITC was added slowly to a rapidly stirred gold colloid solution. After 10 min, a solution of thio-PEG (10 µM) was added dropwise to the MGITC-AuNP solution. The MGITC-AuNP solution was washed and isolated by centrifugation. The core size of each MGITC-AuNP was ~60 nm with >30,000 copies of thio-PEG (5 kDa). For protein conjugation, a solution of thio-PEG-COOH (1 µM) was added slowly to another freshly prepared MGITC-AuNP solution. After 15 min, a large excess of thio-PEG was added to coat the area not covered by the thio-PEG-COOH. A solution of ethyl dimethylaminopropyl carbodiimide and sulfo-N-hydroxysuccinimide was added to activate the carboxyl groups of PEG-COOH on the MGITC-AuNPs. The activated nanoparticles were incubated with scFv B10 antibody (11.2 nmol) for 20 h. MGITC-AuNPs-scFvB10 was isolated and purified by centrifugation and membrane filtration to remove the unreacted antibody. There were ~600 copies of scFv B10 antibody/nanoparticle; there were 1.4–1.5 × 10⁴ MGITC molecules/nanoparticle. MGITC-AuNPs-scFvB10 had a hydrodynamic diameter of ~80 nm. MGITC-AuNPs-scFvB10 has electronic transitions at 633 nm for NIRF imaging and 785 nm for NIR SERS imaging; therefore, MGITC-AuNPs-scFvB10 is a multimodal agent. Another reporter dye (diethylthiatricarbocyanine (DTTC)) conjugated with AuNPs-scFvB10 (DTTC-AuNPs-scFvB10) was also prepared similarly.

In Vitro Studies: Testing in Cells and Tissues

[PubMed]

Qian et al. (8) performed cell-binding assays with DTTC-AuNPs-scFvB10 using human head-and-neck carcinoma Tu866 (HER1-positive) and non-small cell lung carcinoma NCI-H520 (HER1-negative) cell lines. SERS measurements at 30 min of incubation with DTTC-AuNPs-scFvB10 (15 pM) showed that Tu866 cells exhibited strong SERS signals, whereas H520 cells showed little or no signal. Pretreatment of Tu866 cells with a ten-fold excess of scFvB10 abrogated the binding of DTTC-AuNPs-scFvB10 to the cells. No signal was obtained after incubation of Tu866 cells with DTTC-AuNPs-IgG (nonspecific antibody) or DTTC-AuNPs (control).

Animal Studies

Human Studies

[PubMed]

No publication is currently available.

NIH Support

U54 CA119338, P50 CA128613

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