[PubMed]

Epidermal growth factor (EGF) is a 53-amino acid cytokine (6.2 kDa) secreted by ectodermic cells, monocytes, kidneys and duodenal glands (1). EGF stimulates growth of epidermal and epithelial cells. EGF with at least seven other growth factors and their transmembrane receptor kinases play important roles in cell proliferation, survival, adhesion, migration and differentiation. The EGF receptor (EGFR) family consists of four transmembrane receptors, including EGFR (HER1/erbB-1), HER2 (erbB-2/neu), HER3 (erbB-3) and HER4 (erbB-4) (2). HER1, HER3 and HER4 comprise three major functional domains: an extracellular ligand-binding domain, a hydrophobic transmembrane domain and a cytoplasmic tyrosine kinase domain. No ligand has been clearly identified for HER2. However, HER2 can be activated as a result of ligand binding to other HER receptors with the formation of receptor homodimers and/or heterodimers (3). HER1 as well as HER2 are overexpressed on many solid tumor cells such as breast, non-small-cell lung, head and neck, and colon cancer (4-6). The high levels of HER1 and HER2 expression on cancer cells are associated with a poor prognosis (7-10).

Optical fluorescence imaging is increasingly used to monitor biological functions of specific targets (11-13). 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 imaging is becoming a non-invasive alternative to radionuclide imaging in small animals (14, 15).

Fluorescent semiconductor quantum dots (QDs) are nanocrystals made of CdSe/CdTe-ZnS with radii of 1-10 nm (16-18). They can be tuned to emit in a range of wavelengths by changing their sizes and composition, thus providing broad excitation profiles and high absorption coefficients. They have narrow and symmetric emission spectra with long, excited-state lifetimes, 20-50 ns, as compared with 1-10 ns of fluorescent dyes. They process good quantum yields of 40-90% and high extinction coefficients. They are more photo-stable than conventional organic dyes. They can be coated and capped with hydrophilic materials for additional conjugations with biomolecules, such as peptides, antibodies, nucleic acids, and small organic compounds, which were tested in vitro and in vivo (18-22). Although many cells have been labeled with QDs in vitro with little cytotoxicity, there are only limited studies of long-term toxicity of QDs in small animals (23-31). However, little is known about the toxicity and the mechanisms of clearance and metabolism of QDs in humans.

Trastuzumab is a humanized IgG₁ monoclonal antibody (mAb) against the extracellular domain of recombinant HER2 with an affinity constant (K_d) of 0.1 nM (32). Cardiotoxicity is the most serious complication of using trastuzumab in humans with breast cancer (33). One potential application of a radiolabeled anti-HER2 MAb is the pretreatment imaging of breast cancer patients to predict the therapeutic efficacy of trastuzumab. ¹¹¹In-Trastuzumab, Cy5.5-trastuzumab, and ⁶⁸Ga-trastuzumab -F(ab')₂ have been developed for imaging of human breast cancer (34-38). Trastuzumab has also been successfully coupled with quantum dots for optical imaging of HER2 in tumors in mice (39).

[PubMed]

Commercially available QD-antibody labeling kit was used to conjugate trastuzumab with QDs coated with polyethylene glycol to form QD-trastuzumab (QT) nanoparticles (39). In brief, QDs were first activated with the heterobifunctional cross-linker 4-(maleimidomethyl)-1-cyclohexanecarboxylic acid N-hydroxysuccinimide ester (SMCC) to yield a maleimide-nanocrystal surface. Excess SMCC was removed by column chromatography. Trastuzumab was then reduced and fragmented by dithiothreitol (DTT) to expose free sulfhydryl groups, and excess DTT was removed by column chromatography. The activated QDs were covalently coupled with reduced antibody fragments and the reaction was quenched with ß-mercaptoethanol. QT nanoparticles were purified by gel-filtration chromatography. The molar ratio of trastuzumab fragments to QD was estimated to be ~3.0 by spectrometric analyses.

[PubMed]

Li-Shishido et al. (40) and Tada et al. (39) performed cell-binding assays with QT nanoparticles using human KPL-4 breast cancer cells (overexpressing HER2) and MDA-MB-231 cancer cells (low HER2 expression). Incubation of 10 nM QT nanoparticles for 30 min at 37°C showed that higher fluorescence intensity (a.u.) was observed in the KPL-4 cells (7,000 a.u.) than the MDA-MB-231 cells (<1,000 a.u.). Incubation of 30-100 nM QT nanoparticles provided even higher fluorescence intensity in the KPL-4 cells. On the other hand, binding of control QDs in both cell types was low (< 500 a.u.). Similar results were obtained using fluorescence flow cytometry. Pretreatment of KPL-4 cells with 100 nM trastuzumab before incubation with 100 nM QT nanoparticles showed little fluorescence could be detected on the KPL-4 cells.

[PubMed]

Tada et al. (39) studied the accumulation of QT nanoparticles in nude mice bearing KPL-4 tumors using a fluorescence detection system through a dorsal skinfold chamber after injection of QT nanoparticles (200 nmol/mouse). Movement of single QT nanoparticles was imaged at a video rate of 33 ms/frame with a resolution of 30 nm. The speed of movement was calculated from positional changes of the centroid of the QT images with time. The membranes of the KPL-4 cancer cells were clearly delineated with single QT nanoparticle at 6 h after injection. QT nanoparticles were internalized into the perinuclear region of the cancer cells at 24 h after injection. Six stages of movement of QT nanoparticles were detected: (a) blood vessel circulation (100-600 μm/s), (b) extravasation (1-4 μm/s), (c) diffusion movement into the extracellular space (0.0014 μm²/s), (d) binding to HER2 on the cell membrane (200-400 nm/s), (e) movement from the cell membrane to the perinuclear region after endocytosis (100-300 nm/s), and (f) movement to nuclear region (~600 nm/s).

[PubMed]

No publication is currently available.

[PubMed]

No publication is currently available.

[PubMed]

No publication is currently available.