Imaging specificity of MR-optical imaging agents following the masking of surface charge by poly(ethylene glycol) Shou-Cheng Wu a , Kun-Liang Lin a , Tzu-Pin Wang b , Shey-Cherng Tzou a , Gyan Singh a , Ming-Hung Chen a , Tian-Lu Cheng c , Chiao-Yun Chen d , Gin-Chung Liu d , Te-Wei Lee e , Shao-Hwa Hu f , Yun-Ming Wang a, * a Department of Biological Science and Technology, Institute of Molecular Medicine and Bioengineering, National ChiaoTung University, No. 75 Bo-Ai Street, Hsinchu 300, Taiwan b Department of Medicinal and Applied Chemistry, Kaohsiung Medical University, Kaohsiung 807, Taiwan c Department of Biomedical Science and Environmental Biology, Kaohsiung Medical University, Kaohsiung 807, Taiwan d Department of Medical Imaging, Kaohsiung Medical University Hospital, Kaohsiung 807, Taiwan e Isotope Application Division, Institute of Nuclear Energy Research, Lungtan, Taoyuan 325, Taiwan f Chemical Systems Research Division, Chung Shan Institute of Science and Technology, Lungtan, Taoyuan 325, Taiwan article info Article history: Received 22 January 2013 Accepted 10 February 2013 Available online 1 March 2013 Keywords: EGFR Erbitux MnMEIO MRI Nanocarrier abstract The coupling of specific antibodies to imaging agents often improves imaging specificity. However, free amine groups designed for the coupling can cause nonspecific binding of the imaging agents. We report here development of a nanocarrier, MnMEIO-silane-NH 2 -mPEG nanoparticles (NPs), consisting of a manganese-doped iron oxide nanoparticle core (MnMEIO), a copolymer shell of silane and amine- functionalized poly(ethylene glycol) (silane-EA-mPEG). The key feature in MnMEIO-silane-NH 2 -mPEG is the flexible PEG, which masks the non-conjugated reactive amine groups (eNH 2 4 eNH 3 þ ) and re- duces nonspecific binding of MnMEIO-silane-NH 2 -mPEG to cells. The amine groups on MnMEIO-silane- NH 2 -mPEG were conjugated with the fluorescent dye, Cy777 or antibodies [Erbitux (Erb)] to form a MR- optical imaging contrast agent (MnMEIO-silane-NH 2 -(Erb)-mPEG) for EGFR-expressing tumors. Confocal microscopic and flow cytometric analyses showed that MnMEIO-silane-NH 2 -(Erb)-mPEG displayed low nonspecific binding. Moreover, TEM images showed that MnMEIO-silane-NH 2 -(Erb)-mPEG were endo- cytosed by EGFR-expressing cells. In line with their EGFR expression levels, A431, PC-3, and Colo-205 tumors treated with MnMEIO-silane-NH 2 -(Erb)-mPEG NPs showed 97.1%, 49.7%, and 2.8% contrast enhancement, respectively, in in vitro T 2 -weighted MR imaging. In vivo T 2 -weighted MR imaging and optical images showed that MnMEIO-silane-NH 2 -(Erb)-mPEG could specifically and effectively target to EGFR-expressing tumors in nude mice; the relative contrast enhancements were 7.94 (at 2 h) and 7.59 (at 24 h) fold higher in A431 tumors as compared to the EGFR-negative Colo-205 tumors. On the con- trary, MnMEIO-silane-NH 2 -(Erb) NPs showed only 1.44 (at 2 h) and 1.52 (at 24 h) fold higher in EGFR- positive tumors as compared to the EGFR-negative tumors. Finally, antibodies can be readily changed to allow imaging of other tumors bearing different antigens. These data indicate that masking surface charges on contrast agents is a useful strategy to improve imaging efficacy. Ó 2013 Elsevier Ltd. All rights reserved. 1. Introduction Recently, there has been a considerable interest in developing targeted dual-modality MR-optical imaging agents for early cancer detection [1,2]. The development of these imaging agents often re- quires conjugation of tumor-targeting moieties and near infrared (NIR) molecules onto the surface of superparamagnetic iron oxide (SPIO) nanoparticles (NPs) [3e6]. The surface modification of NPs with linear chains of poly(ethylene glycol) (PEG) derivatives pos- sessing terminal functional groups such as amine or carboxylic acid is the most common strategy employed for conjugation [7e10]. However, non-conjugated reactive functional groups on the surface of NPs can cause severe fouling and nonspecific interaction of NPs to the cell membranes and may lead to inefficient tagging of NPs to the desired targets and ambiguous detection of unintended targets [11e 13]. These inadequacies can severely jeopardize the usefulness of the imaging modality. Consequently, intelligent de- signs of surface structure that achieve excellent stability, multi- functionality and high targeting specificity are urgently needed [14,15]. Previous studies have demonstrated that neutral and * Corresponding author. Tel.: þ886 3 5712121x56972; fax: þ886 3 5729288. E-mail address: ymwang@mail.nctu.edu.tw (Y.-M. Wang). Contents lists available at SciVerse ScienceDirect Biomaterials journal homepage: www.elsevier.com/locate/biomaterials 0142-9612/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biomaterials.2013.02.025 Biomaterials 34 (2013) 4118e4127