Journal: International Journal of Nanomedicine

Article Title: Cetuximab-conjugated iron oxide nanoparticles for cancer imaging and therapy

doi: 10.2147/IJN.S80134

Figure Lengend Snippet: EGFRvIII-ECD-hFc binding determined by enzyme-linked immunosorbent assay. Notes: Soluble EGFRvIII-ECD-hFc was coated on enzyme-linked immunosorbent assay plates, and binding of cetuximab ( A ) or cetuximab-functionalized and non-functionalized iron oxide nanoparticles ( B ) was detected using a horseradish peroxidase-conjugated goat anti-human kappa light chain antibody. The concentrations tested ranged from 1.34×10 −4 to 10 μg/mL for the anti-EGFR monoclonal antibody cetuximab and from 6.4×10 −4 to 120 μg Fe/mL for cet-PEG-dexSPIONs and PEG-dexSPIONs. The EC 50 was 0.0242 and 0.0646 μg/mL for cetuximab and cet-PEG-dexSPIONs, respectively. Each point represents the mean ± standard deviation of three determinations. Abbreviations: cet, cetuximab; dex, dextran; ECD, extracellular domain; EGFR, epithelial growth factor receptor; OD, optical density; PEG, polyethylene glycol; SPIONs, superparamagnetic iron oxide nanoparticles.

Article Snippet: After acid strip with ice-cold 0.2 M acetic acid and 0.5 M NaCl (pH 2.5), on ice for 5 minutes, the cells were washed once with ice-cold 1× PBS prior to incubation on ice for one hour with phycoerythrin-conjugated mouse anti-human EGFR monoclonal antibody (BD Bio-science Pharmingen) to label EGFR remained on the cell surface.

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: International Journal of Nanomedicine

Article Title: Cetuximab-conjugated iron oxide nanoparticles for cancer imaging and therapy

doi: 10.2147/IJN.S80134

Figure Lengend Snippet: Iron content of differential EGFR-expressing cells after a 2-hour incubation at 37°C with the synthesized iron oxide nanoparticles

Article Snippet: After acid strip with ice-cold 0.2 M acetic acid and 0.5 M NaCl (pH 2.5), on ice for 5 minutes, the cells were washed once with ice-cold 1× PBS prior to incubation on ice for one hour with phycoerythrin-conjugated mouse anti-human EGFR monoclonal antibody (BD Bio-science Pharmingen) to label EGFR remained on the cell surface.

Techniques: Incubation, Synthesized, Concentration Assay

Journal: International Journal of Nanomedicine

Article Title: Cetuximab-conjugated iron oxide nanoparticles for cancer imaging and therapy

doi: 10.2147/IJN.S80134

Figure Lengend Snippet: Iron uptake among the cell lines with differential EGFR expression levels. The A431, 32D, and 32D/EGFR cell lines were incubated with 25 μg Fe/mL of PEG-dexSPIONs or cet-PEG-dexSPIONs at 37°C for 2 hours. The excess iron was removed by washing with 1× phosphate-buffered saline, and the iron concentration in 4×10 6 cells from each cell line was determined using a thiocyanate-based spectrophotometric assay. The results are presented as the mean ± standard deviation of four determinations. Abbreviations: cet, cetuximab; dex, dextran; EGFR, epidermal growth factor receptor; PEG, polyethylene glycol; SPIONs, superparamagnetic iron oxide nanoparticles.

Article Snippet: After acid strip with ice-cold 0.2 M acetic acid and 0.5 M NaCl (pH 2.5), on ice for 5 minutes, the cells were washed once with ice-cold 1× PBS prior to incubation on ice for one hour with phycoerythrin-conjugated mouse anti-human EGFR monoclonal antibody (BD Bio-science Pharmingen) to label EGFR remained on the cell surface.

Techniques: Expressing, Incubation, Concentration Assay, Spectrophotometric Assay, Standard Deviation

Journal: International Journal of Nanomedicine

Article Title: Cetuximab-conjugated iron oxide nanoparticles for cancer imaging and therapy

doi: 10.2147/IJN.S80134

Figure Lengend Snippet: Therapeutic effect of cet-PEG-SPIONs. Notes: ( A ) Inhibition of epidermal growth factor-induced tyrosine phosphorylation of EGFR by cet-PEG-dexSPIONs. A431 cells were serum-starved overnight and then treated with cetuximab, cet-PEG-dexSPIONs, or PEG-dexSPIONs at varying doses for 2 hours at 37°C. A431 cells were further stimulated with 30 ng/mL EGF for 30 minutes at 37°C. Cell lysates were separated by electrophoresis, and immunoblotting was performed to detect the p-EGFR (Tyr1173) and total EGFR levels. β-actin was used as a loading control. A representative of three blots is shown. ( B ) EGFR internalization in response to cet-PEG-dexSPIONs treatment in A431 cells. Cells were incubated for the indicated times in the presence of cetuximab (2.5 μg/mL), cet-PEG-dexSPIONs (50 μg Fe/mL), or PEG-dexSPIONs (50 μg Fe/mL). After stripping off the surface-bound cetuximab or nanoparticles, the cells were stained on ice for one hour with PE-conjugated mouse anti-human EGFR monoclonal antibody to determine the EGFR level remaining on the cell surface by flow cytometry. The expression level of EGFR on untreated A431 cells at 0 hours was set at 100%. ( C ) Apoptosis-inducing activity of cet-PEG-dexSPIONs in EGFR-expressing cell lines analyzed by an Annexin V-fluorescein isothiocyanate/propidium iodide assay. The stacked bar graph shows the proportion of different cell populations among the EGFR-null 32D cells, EGFR-overexpressing 32D/EGFR cells, and A431 cells. Abbreviations: cet, cetuximab; dex, dextran; EGF, epidermal growth factor; PEG, polyethylene glycol; PE, phycoerythrin; SPIONs, superparamagnetic iron oxide nanoparticles; p-EGFR, phosphorylated epidermal growth factor receptor.

Article Snippet: After acid strip with ice-cold 0.2 M acetic acid and 0.5 M NaCl (pH 2.5), on ice for 5 minutes, the cells were washed once with ice-cold 1× PBS prior to incubation on ice for one hour with phycoerythrin-conjugated mouse anti-human EGFR monoclonal antibody (BD Bio-science Pharmingen) to label EGFR remained on the cell surface.

Techniques: Inhibition, Electrophoresis, Western Blot, Incubation, Stripping Membranes, Staining, Flow Cytometry, Expressing, Activity Assay