Characterization and Cancer Cell Specific Binding Properties of Anti-EGFR Antibody Conjugated Quantum Dots

Abstract

Synthesis of biologically active antibody conjugated quantum dots (QDs) has been of great importance in cellular imaging and diagnostics. Cetuximab (or Erbitux) is the first monoclonal antibody drug which targets the epidermal growth factor receptor (EGFR) overexpressed in most cancer cells. In the present work, we investigated three different conjugation strategies to obtain the biologically functional QD-cetuximab conjugates for the tumor-specific imaging. Successful conjugation of cetuximab to QDs was achieved using PEG conjugated polymer-coated QDs and two long-chain heterobifunctional linkers, sulfo-LC-SPDP and sulfo-SMCC. The dissociation constant of the QD-cetuximab conjugates to EGFR was determined to be 0.61 +/- 0.28 nM. The cancer cell-specific binding ability of the QD-cetuximab conjugates was evaluated in vitro, and the cellular internalization of the QD-cetuximab conjugates was clearly demonstrated in live cells by confocal microscopy. The cellular imaging experiments using the QD-cetuximab conjugates showed a clear endocytosis pathway, which was evidenced by the colocalization of the QD-cetuximab conjugates with dye-labeled transferrin. These results suggest that the QD-cetuximab conjugates as an imaging modality for tumor EGFR overexpression can be expected to provide important information on the expression levels of EGFR on the cancer cells.

Anti-HER2 antibody and ScFvEGFR-conjugated antifouling magnetic iron oxide nanoparticles for targeting and magnetic resonance imaging of breast cancer

“…31 Strong uptake of anti-HER2-IONPs by HER2-positive cells was demonstrated on Prussian blue staining, as shown in Figure 2A. Inhibiting the HER2 receptor with 100 times excess of free HER2 antibody effectively reduced the amount of blue staining ( Figure 2B), 24 suggesting that uptake of targeted IONPs is specifically mediated by HER-2 receptors on the cancer cells. Additionally, Prussian blue staining was negative in MCF-7 and MDA-MB-231 cancer cell lines after treatment with anti-HER2-IONPs, as shown in Figure 2C Figure 2E and F), suggesting reduced nonspecific binding in the condition when active targeting was taking place.…”

“…As described above, the binding of EGFR-targeted nanoparticles could be extensively inhibited by an excess amount of free ScFvEGFR ( Figure 3B). 24 In comparison, there was no cellular uptake observed in two cell lines (MCF-7 and MDA-MB-453) with no or low EGFR expression ( Figure 3C and D). As a control, the PEO-b-PγMPS copolymer-coated IONPs without targeting ligands showed no uptake in any of the positive or negative cell lines ( Figure 3E and F).…”

“…The conjugation was carried out using a two-step procedure. 24,25 First, 1 mL of aqueous IONPs (1 mg Fe) was washed thoroughly with ethanol to remove any water residue. The particles were dispersed in 0.9 mL of ethanol, and 1.0 µL of APTES in 0.1 mL of ethanol was added dropwise into the suspension.…”

A Review on Aptamer-Conjugated Quantum Dot Nanosystems for Cancer Imaging and Theranostic

“…Therefore, exploiting other set of detecting molecules which show higher binding affinity and more accessibility by chemical synthesis to prepare conjugated quantum dots without aforementioned disadvantages would become a hotspot in the improvement of molecular probes [49]. Aptamers with similar characteristics to antibodies offering additional advantages of small size, easy synthesis and no immunogenicity [50] have been successfully utilized in cancer imaging and cell labeling. For example, Chu et al [21] conjugated the aptamer of prostatespecific membrane antigen (PSMA) with QDs and evaluated the labeling of live cells, fixed cells, and prostate tumor cells (LNCaP) in a simulated tissue [51].…”

“…Aptamers represent the potential applications in medical and pharmaceutical basic research including drug development, analytical science, diagnosis and therapy of disease owing to the specific and unique binding characteristics [55]. The technology to evolve aptamers was discovered in 1990 [50,56] …”