Synthesis of EGFR-Targeted SPIO Nanoparticles

The EGFR-SPIO nanoparticles are constructed as described previously [31 (link)] in a three steps manner. The lipid-coated SPIO nanoparticles 10 nm in size were purchased from OceanNanoTech Inc. Briefly, 1 mg of amine-functionalized lipid-coated SPIO nanoparticles [concentration: 1 mg/ml (Fe) = 0.86 nmol/ml (nanoparticles)] 10 nm in size were first reacted with 150 μl/ml sulfo-SMCC (10 mg/ml) at room temperature for 1 h to obtain maleimide-functionalized SPIO nanoparticles. Second, 1.29 nmol of the anti-EGFR antibody cetuximab was treated with 35.6 μl iminothiolane (10 mg/ml) at room temperature for 30 min. Third, the antibody and maleimide-lipid-coated SPIO nanoparticles were cleaned up by 10 K Nanosep filter (Pall Corporation; Port Washington, NY, USA), mixed, and reacted at 4 °C overnight. The unused maleimide-functionalized groups were then blocked by excess cystein for 15 min at room temperature. The antibody-conjugated SPIO nanoparticles were separated using an MS column (Miltenyi Biotech, Germany) and washed with sterilized phosphate-buffered saline (PBS) at a volume > 25 times greater than the column bed volume to remove unconjugated antibodies. The number of immobilized EGFR antibody molecules per SPIO nanoparticle was estimated to be 1.5 based on the molarities of components in the reaction.