Investigating the structural biofunctionality of antibodies conjugated to magnetic nanoparticles† Emanuela Occhipinti,‡ a Paolo Verderio,‡ b Antonino Natalello, a Elisabetta Galbiati, a Miriam Colombo, ab Serena Mazzucchelli, b Agnese Salvad e, b Paolo Tortora, a Silvia Maria Doglia a and Davide Prosperi * ab Received 23rd June 2010, Accepted 3rd September 2010 DOI: 10.1039/c0nr00436g We present the synthesis of trastuzumab-functionalized pegylated iron oxide nanoparticles and provide an FTIR-based approach to gain a direct evidence of the actual conservation of the native structure of conjugated antibody. Their target-selectivity to specific cancer cell receptors has been also assessed. The successful development of targeted nanoprobes for noninvasive medical imaging represents a new frontier in the prevention and treatment of malignant diseases. 1 Among the available choices to obtain an effective targeting action, the conjugation of nanoparticles with monoclonal antibodies (mAbs) is a well-established strategy to deliver the nanoprobe to specific cell types. 2 This approach combines the unique physical properties of nanoparticles, with the specific and selective recognition capability of mAbs to cells and tissues. More- over, the potential improvement in the cellular uptake and the enhanced intracellular stability could be two of the major advantages of using mAb-conjugated nanoparticles. Trastuzumab (TZ) is a recombinant, humanized IgG mAb that selectively binds with high affinity to the extracellular domain of the human epidermal growth factor receptor 2 (HER2), which is expressed in several primary tumors, including breast, ovarian, gastric and salivary cancers, and in metastatic sites. 3 For this reason, TZ has been developed for clinical immunotherapy and the conjugation of TZ with chemotherapeutics may offer an excellent strategy for tar- geted delivery of drugs to malignant cells. 4 The therapeutic action of TZ mainly consists in blocking the dimerization of the two compo- nents of HER2 complex resulting in an interruption of the signal transduction. Colloidal systems, such as those based on inorganic nanomaterials, provide higher drug carrier capacities than individual mAbs, concomitantly acting as signal emitters for medical diagnosis. Therefore, in view of the outstanding potential of mAb-based hybrid nanovehicles, the conjugation of mAbs to nanoparticles is now a widespread practice to gain selective delivery and localization of nanoparticle probes to the desired target site. However, despite the huge interest for mAb nanoconjugates, only poor evidence is presently available on the actual conservation of the protein biofunctionality at the molecular structural level, once mAb has been covalently conjugated to the nanoparticle. Such difficulty mainly resides in the lack of reliable methods capable of providing exhaustive information on structural/conformational features sustaining the protein functionalities of bioconjugate systems. This is indeed a crucial point, which urgently needs to be addressed. Weiss et al. exploited circular dichroism to investigate proteins immobilized on gold nanoparticles. 5 Unfortunately, this technique is expected to fail with magnetic nanoparticles due to the strong absorption of iron oxide in the UV range. The purpose of the present study is to provide a direct evidence on the extent of preservation of the structural bioactivity of IgGs immobilized onto the surface of iron oxide nanoparticles deduced by accurate analysis of the essential folding features obtained by Fourier-transform infrared (FTIR) spectros- copy. As a model for this study, PEG-stabilized, TZ-modified magnetite nanoparticles (TMNP) were developed (Scheme 1). Thanks to the unique magnetic properties of nanoscale iron oxides, this class of nanoparticles could be useful as targeted contrast agents for magnetic resonance imaging of cancer cells. 6–8 Bare monodisperse magnetite nanoparticles (MNP1) were obtained by aqueous alkaline coprecipitation of Fe 2+ and Fe 3+ ions in 1 : 2 molar ratio, as described in a previous work. 9 A bifunctional linker a-u-dicarboxyl-terminated polyethylene glycol (PEG, M w 600 Da) was grafted on the Fe 3 O 4 surface by ultrasound-assisted reaction and the unreacted excess of PEG was removed by dialysis. The synthesized carboxyPEG-functionalized nanoparticles (MNP2) were highly soluble in water, thus the MNP2 suspension was diluted to a final concentration of 1 mg mL 1 , which was stable for several months at room temperature. According to previous reports, PEG- coated nanoparticles are more biodegradable, non-antigenic, non- irritative for tissues and less toxic than unmodified nanoparticles. 10–12 At the same time, the PEG chains are responsible for the so-called ‘‘stealth effect’’, preventing nonspecific adsorption of opsonin proteins. 13 TEM image showed that the core size of MNP2 was unchanged after PEG grafting (10  3 nm, Fig. S2, ESI†). The hydrodynamic diameter of MNP2 was determined to be 82.4  1.0 nm by dynamic light scattering (DLS, Fig. S3a, ESI†). A good contrast power for MNP2 in PBS was deduced by the relaxivity value r 2 , which was calculated to be 131.9 mM 1 s 1 (Fig. S4, ESI†). The pH-dependent stability of MNP2 in water was also investigated by UV-vis absorption spectra (Fig. S5 and Table S1, ESI†). MNP2 were dispersed in deionized water and evaluated in the pH range from 1 to 11, in which they did not display any aggregation up to pH 7–8. A critical formation of large clusters of nanoparticles was instead observed above pH 9 right after 90 min incubation, which resulted in collapsing of the colloidal suspension (Fig. S6, ESI†). Zeta potential (z) of MNP2 in water in a range of pH between 3 and 9 was also a Dipartimento di Biotecnologie e Bioscienze, Universit  a di Milano-Bicocca, P.za della Scienza 2, 20126 Milano, Italy. E-mail: davide.prosperi@ unimib.it; Fax: +39 026448 3565; Tel: +39 026448 3302 b Centro di Microscopia Elettronica per le Nanotecnologie applicate alla medicina, Universit  a di Milano, Via G.B. Grassi 74, 20157 Milano, Italy † Electronic supplementary information (ESI) available: Experimental details, TEM images, DLS and relaxivity plots, additional FT-IR spectra and analyses. See DOI: 10.1039/c0nr00436g ‡ These authors contributed equally to the research. This journal is ª The Royal Society of Chemistry 2011 Nanoscale, 2011, 3, 387–390 | 387 COMMUNICATION www.rsc.org/nanoscale | Nanoscale Downloaded on 11 July 2012 Published on 29 September 2010 on http://pubs.rsc.org | doi:10.1039/C0NR00436G View Online / Journal Homepage / Table of Contents for this issue