Magnetic Nanoparticles with Covalently Bound Self-Assembled Protein Corona for Advanced Biomedical Applications Rina Venerando, † Giovanni Miotto, † Massimiliano Magro, ‡,§ Marco Dallan, ‡ Davide Baratella, ‡ Emanuela Bonaiuto, † Radek Zboril, § and Fabio Vianello* ,‡,§ † Department of Molecular Medicine, University of Padua, 35121 Padua, Italy ‡ Department of Comparative Biomedicine and Food Science, University of Padua , 35020 Padua, Italy § Regional Centre of Advanced Technologies and Materials, Department of Physical Chemistry, Palacky University in Olomouc, 771 47 Olomouc, Czech Republic * S Supporting Information ABSTRACT: Novel surface active maghemite nanoparticles (SAMNs) possess- ing peculiar colloidal properties and surface characteristics are able to covalently bind biomolecules. The interactions of SAMNs and rhodamine derivatized SAMNs (SAMN@RITC) with proteins from cell culture medium were studied by gel electrophoresis and mass spectrometry. Among the 3000 proteins present in fetal calf serum, SAMNs and SAMN@RITC give rise to the formation of a self-assembled corona shell with 22 selected proteins, representing minor plasma proteins, among which α-2-HS- glycoprotein stands out. Bovine serum albumin (BSA), representing 80% of the total serum proteins, shows negligible absorption on the SAMN surface. Nevertheless, SAMNs are able to bind BSA, upon incubation in pure BSA solutions. The interaction between SAMNs and BSA was investigated by optical spectroscopy, circular dichroism, Fourier transform infrared spectroscopy, and transmission electron microscopy. BSA binding resulted a time-consuming process, nevertheless experimental results showed the interaction of 6 ± 2 BSA molecules per nanoparticle, and optical spectra indicate remarkable changes in SAMN optical features, as well as circular dichroism proved secondary structure alteration of bound BSA, suggesting that the protein needs to adapt its structure to adhere to nanoparticle surface. The selectively bound protein corona shell, formed upon SAMNs incubation in calf serum, was responsible for the characteristic behavior when SAMNs were tested for cell internalization and cytotoxicity on HeLa cells. Cytotoxicity of SAMN preparations was extensively studied, and was negligible up to 100 μg mL -1 . Moreover, nanoparticle uptake proceeded for long times, suggesting a correlation between internalization and stability of covalently bound self-assembled protein corona, representing a unique example of magnetic nanoparticle opsonization via covalent binding. We suggest that SAMN based nanobiocomposites can be employed for the preparation of self-assembled opsonized nanoparticles as future candidates for biomedical applications. 1. INTRODUCTION Many different kinds of magnetic nanoparticles have already demonstrated their potential in biomedical applications, 1,2 as these nanostructures properly labeled with bioactive molecules can serve in magnetic separations, 3-5 drug delivery systems, 6 or to generate heat by exposition to an alternating electromagnetic field, thus increasing the temperature of tumor tissues and destroying pathological cells. 7 Moreover, their use in magnetic resonance imaging as contrast agents is common, 8,9 owing to their unique magnetic properties and biocompatibility, 10 and commercial preparations are available (Nanocs Inc., New York, NY; Nanoimmunotech SL, Vigo, Spain; MK Impex Corp., Missisauga, ON, Canada; and many others). However, for most iron oxide nanoparticles, little is known about their potential adverse effects on health due to prolonged exposure in biological systems, representing a hindrance to the develop- ment of novel applications of magnetic nanoparticles in nanobiology, nanomedicine, and nanotoxicology. The meta- bolic and immunological responses induced by these particles have been rarely understood so far. 11 An essential prerequisite for the implementation of bionanotechnological applications is to obtain nanoparticles with a hydrophilic surface able to maintain colloidal stability under physiological conditions. 12 Among magnetic nano- particles, magnetite (Fe 3 O 4 ) appears to be an interesting candidate, owing to its low toxicity, high saturation magnet- ization, and susceptibility. Unfortunately, upon exposure to physiological environments, magnetite nanoparticles exhibit a Received: July 10, 2013 Revised: August 24, 2013 Article pubs.acs.org/JPCC © XXXX American Chemical Society A dx.doi.org/10.1021/jp4068137 | J. Phys. Chem. C XXXX, XXX, XXX-XXX