Covalent functionalization of magnetic nanoparticles for biomedical imaging
Progress in nanotechnology has led to the creation of a new field, namely, nanomedicine.1–3 One fundamental goal of nanomedicine is to develop nanoparticles for diagnosis (through imaging techniques) and treatment, usually termed ‘theranostics’ (therapeutics plus diagnostics). These nanoparticles—specifically, magnetic nanoparticles (MNPs)—should ideally provide a signal in two imaging techniques, typically a functional technique such as fluorescence and a structural technique such as magnetic resonance imaging (MRI).4–6
MNPs can be synthesized in two ways: through coprecipitation or decomposition of organic precursors. The first approach, although widely used for biomedical applications, suffers from several drawbacks, including broad particle size distribution and low reproducibility. The ‘organic approach’ renders MNPs with narrow size distribution, high size control, and high crystallinity. These MNPs are hydrophobic due to the oleic acid surfactant used in the synthesis. This is commonly mentioned as a disadvantage because a second step (phase transfer) is required to disperse the nanoparticles in water. However, we believe the oleic acid presents an opportunity for a new method in building nanoparticles for molecular imaging.7
The micelle approach (using a secondary surfactant) and ligand exchange are two traditional methods for transferring the hydrophobic nanoparticles to water. Both methods are based on weak interactions to camouflage (the micelle approach) or partially remove (ligand exchange) the oleic acid surfactant. These methods have problems, for example, increased MNP size and complicated biofunctionalization. Consequently, we decided to use a method that directly produces water-stable, functionalized MNPs by two different covalent (forming a direct link) techniques.5, 8,9
Our first approach is depicted in Figure 1(A). We started from hydrophobic nanoparticles coated with oleic acid and took advantage of the olefinic functional group. We oxidized this bond with potassium permanganate and trimethylbenzylammonium chloride, in a two-phase reaction.10 We obtained 37nm-diameter MNPs, which can be classified as ultrasmall superparamagnetic iron oxide nanoparticles (USPIO, particles <50nm), and measured their physical properties. Figure 1(B) shows the zeta potential—the charge on the surface of the nanoparticles—with –40mV at physiological pH. We found that the superparamagnetism does not change after the reaction: see Figure 1(C). In addition, a transmission electron microscopy image showed no aggregation in water: see Figure 1(D).10 We also tested these MNPs as liver MRI contrast agents. Figure 2 shows a rat's liver after intravenous injection of the MNPs. Due to the presence of a small charged group on the surface, the particles are rapidly taken up by Kupffer cells in the liver. The liver turned black, due to the presence of the MNPs, a few minutes after the injection.
The direct covalent biofunctionalization of the MNPs can be achieved in two ways: through covalent bonding using the carboxylic acid and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-hydroxysulfosuccinimide chemistry, or ionic bonding due to the intrinsic strong negative charge of the acid.11, 12 We have used the covalent approach for the synthesis of multifunctional nanoparticles functionalized with an allergen and an optical dye. These MNPs provide a signal both in MRI and fluorescence imaging due to the attachment of a dye through a biotin-streptavidin interaction. The main advantage of this technique is the reproducibility (in terms of size, size distribution, and composition), always a problem in the functionalization of MNPs. We have also synthesized MNPs functionalized in such a way that their circulation time in blood is about two hours, suggesting they can be used as blood pool contrast agents and in drug delivery (patent in preparation).
The second technique we have developed allows for the direct functionalization and stabilization of MNPs in water through olefin metathesis. This reaction allows swapping the functional groups between two olefins, one being the oleic acid on the MNPs, and the second any double-bond-containing molecule we are interested in, by the use of a catalyst (see Figure 3).13
In summary, one key objective of nanomedicine is the reproducible synthesis and functionalization of magnetic nanoparticles that allows for diagnosis and treatment of pathologies. Our group has developed new approaches for the covalent biofunctionalization of MNPs. These particles are small with a narrow size distribution, and allow visualization through several imaging techniques. We will attempt to expand the metathesis method to antibodies and enzymes by modifying the reaction conditions.
The authors thank the Spanish Ministry of Science (SAF2008-05412, MAT2008-01489, MAT2010-11349-E, SAF2011-25445) and the European Commission's 7th Framework Programme (FP7-PEOPLE-ITN-264864).
Spanish Cardiovascular Research Centre (CNIC)
Fernando Herranz is a researcher at CNIC. His work focuses on the development of nanoparticles and radiochemicals for cardiovascular molecular imaging.
Juan Pellico is carrying out his PhD in the development of new 68Ga radiochemicals for cardiovascular imaging.
Jesús Ruiz-Cabello is the director of the Advanced Imaging Unit at CNIC. His research extends from nanotechnology and radiochemistry to MRI of the lung and cardiovascular system.