2 iminothiolane

Pirarubicin (4′-O-tetrahydropyranyldoxorubicin, THP) was purchased from Selleck Biotech (Tokyo, Japan). 6-Maleimidohexanehydrazide trifluoroacetate (EMCH) was purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). 2-iminothiolane was from Thermo Fisher Scientific (Tokyo, Japan). Human serum albumin (HSA) was purchased from the Japan Blood Products Organization (Tokyo, Japan) and defatted by means of a charcoal treatment [35 (link)]. Methanol (Super Dehydrated), diethyl ether, dimethyl sulfoxide (DMSO), acetonitrile, penicillin G, streptomycin, were purchased from Wako Pure Chemical (Osaka, Japan). Dulbecco’s Modified Eagle Medium (DMEM) was purchased from Nissui Seiyaku (Tokyo, Japan). Fetal calf serum was obtained from GIBCO (Grand Island, NY, USA). Diethylenetriaminepentaacetic acid (DTPA) and 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) were purchased from Dojindo Chemical Laboratories (Kumamoto, Japan). A CellTiter 96^® Aqueous One solution containing 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy-methoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) was from Promega (Madison, WI, USA). All chemicals were used without further purification.

Unless otherwise mentioned, all supplies were purchased from Sigma-Aldrich (St. Louis, MO, USA). The antibodies used in this study were the anti-EGFR antibody cetuximab (Merk Serono, Taiwan), the anti-EGFR-AF594 antibody (Cell Signaling Technology, Danvers, MA, USA, #8742), and Alexa-488-conjugated goat anti-rabbit and goat anti-human immunoglobulin (IgG) secondary antibodies (Thermo Fisher Scientific, Waltham, MA USA). The reagents were sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (sulfo-SMCC), 2-iminothiolane (Thermo Fisher Scientific, Waltham, MA, USA), 6–10-nm non-coated amino-terminated SPIOs (Taiwan Advanced Nanotech Inc., Taiwan; TANBead USPIO-101) and 10-nm lipid-based SPIO nanoparticles with amine (Ocean NanoTech, San Diego, CA, USA, #ILA-10).