Transmission Electron Microscopy of Lipid Vesicles for Drug Delivery: Comparison between Positive and Negative Staining Valentina Bello, 1, * Giovanni Mattei, 1 Paolo Mazzoldi, 1 Nicoletta Vivenza, 2 Paolo Gasco, 2 Jean Marc Idee, 3 Caroline Robic, 3 and Elisabetta Borsella 4 1 Department of Physics, University of Padova, via Marzolo 8, I-35131 Padova, Italy 2 Nanovector S.r.l., via Livorno, 60, I-10144 Torino, Italy 3 Guerbet, 15 rue des Vanesses, Zone Paris Nord I, 93420 Villepinte (Paris), France 4 ENEA, Dept. FIM, via E. Fermi 45, I-00044 Frascati (Rome), Italy Abstract: Lipid-containing nanostructures, in the form of solid lipid nanoparticles or iron oxide nanoparticles ~NPs! coated with a lipid shell, were used as case studies for assessing and optimizing staining for transmission electron microscopy structural and compositional characterization. These systems are of paramount impor- tance as drug delivery systems or as bio-compatible contrast agents. In particular, we have treated the systems with a negative ~phospshotungstic acid! or with a positive ~osmium tetroxide! staining agent. For iron-oxide NPs coated with the lipid shell, negative staining was more efficient with respect to the positive one. Nevertheless, in particular cases the combination of the two staining procedures provided more complete morphological and compositional characterization of the particles. Key words: lipid nanoparticles, USPIO, staining process, TEM I NTRODUCTION Lipidic systems with size in the nanometer range, such as liposomes, micelles, nanoemulsions, and solid lipid nanopar- ticles ~SLNs!, have been developed as nanovectors for diag- nostics and therapeutic drugs due to their low toxicity, their ability to encapsulate both hydrophilic and lipophilic parti- cles, and their ability to localize and control the release of the active drug ~Morel et al., 1998; Muller et al., 2000; Ugazio et al., 2002; Peira et al., 2003; Muller & Keck, 2004; Gasco, 2007; Weiss et al., 2008!. The development of appro- priate in vitro and in vivo characterization techniques is essential to effectively utilize the potential offered by these systems and to control their quality and physical and chem- ical stability. Concerning physical characterization, photon correla- tion spectroscopy ~PCS! and laser diffraction are the most frequently used techniques for measuring particle size ~Igar- tua et al., 2002; Shah et al., 2007!. Recently, atomic force microscopy has also received increasing attention for deter- mining the mean diameter of lipid particles and for observ- ing the particle surface features through the acquisition of topographic images ~Dubes et al., 2003; Azevedo et al., 2005; Garg & Kokkoli, 2005!. Electron microscopy, and in partic- ular transmission electron microscopy ~TEM!, is also a powerful technique for revealing the ultrastructure of such carriers ~Bargoni et al., 1998; Podio et al., 2000; Dubertret et al., 2002; Jores et al., 2004; Trotta et al., 2005; Cho et al., 2007; Al-Haj & Rasedee, 2009!. Moreover, in combination with X-ray energy dispersive spectroscopy ~EDX!, TEM can provide nanoscale elemental analysis that is fundamental to properly localizing the drug inside the lipid vectors. One of the main limitations for the characterization of lipid systems by TEM or scanning electron microscopy is sample preparation. Fixation and processing can induce artifacts such as lipid particle aggregation, degradation, dehydration, etc., and complicate or alter the interpretation of images. Since lipid carriers are not electron dense enough to be easily visible with electron microscopy, they need to be stained with a heavy metal and images need to be properly interpreted. The purpose of the present study is to characterize by TEM unloaded SLNs and ultrasmall superparamagnetic iron oxide ~USPIO! nanoparticles ~NPs! coated by a lipid shell whose composition is similar to the external layers of the SLNs. The interest in NPs stems from the fact that they can be used as a contrast agent for magnetic resonance imaging ~MRI!. USPIOs are coated nanocrystals of iron oxides, magnetite ~Fe 3 O 4 !, maghemite ~g-Fe 2 O 3 !, or other ferrites, characterized by a large magnetic moment in the Received December 23, 2009; accepted March 20, 2010 *Corresponding author. E-mail: bello@padova.infm.it Microsc. Microanal. 16, 456–461, 2010 doi:10.1017/S1431927610093645 Microscopy AND Microanalysis © MICROSCOPY SOCIETY OF AMERICA 2010