Reagents and equipment

Magnetic fluids used were synthesized by the coprecipitation method of Fe²⁺ and Fe³⁺ ions in alkaline medium and subsequently oxidized by bubbling oxygen. The functionalized fluids of MRC NPs and citrate-loaded maghemite (MC) NPs were obtained by adsorption experiments. MRC with 59.6 µM of γ-Fe₂O₃ and 2.85 µM of RC, MC with 64.7 µM of γ-Fe₂O₃ and 2.55 µM of RC and 2.513 mM Rh₂(H₂Cit)₄ were synthesized at the Institute of Chemistry of Federal University of Goiás (Goiânia, Brazil). For cell-culture maintenance, we used DMEM, RPMI, FBS, and 0.25% trypsin–EDTA (all from Thermo Fisher Scientific, Waltham, MA, USA) and Leibovitz L15 (Sigma-Aldrich, St Louis, MO, USA).

The antibodies used in this study were monoclonal anti-β-actin produced in mice, monoclonal anticlathrin heavy chain produced in mice, polyclonal anti-caveolin 1 produced in rabbits (Sigma-Aldrich), and IgG HRP-conjugated antimouse and antirabbit (Sigma-Aldrich). The reagents used throughout the work were: MTT, BoDipy FL C-lactosylceramide (N-[4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl]), sphingosyl 1-β-d-lactoside and transferrin from human serum, fluorescein conjugate (Thermo Fisher Scientific), methyl-β-cyclodextrin (MβCD; Sigma-Aldrich), Pitstop 2 (Abcam, Cambridge, UK), and bicinchoninic acid (Thermo Fisher Scientific). For Western blot analyses, we used a protease inhibitor (Hoffman-La Roche, Basel, Switzerland). The development was made with Amersham ECL prime Western blot-ting detection reagent, and image acquisition and analyses were performed with an Image Quant LAS 4000 (GE Healthcare, Little Chalfont, UK).

Ultrastructural analyses were performed with transmission electron microscopy (TEM, JEM-1011; JEOL, Tokyo, Japan) operating at 100 kV and field-emission scanning electron microscopy (SEM, JSM-7000F; JEOL). Absorbance reading was performed using SpectraMax M5 equipment, and data were analyzed by SoftMax Pro 5.2, both from Molecular Devices (Sunnyvale, CA, USA). Flow cytometry was performed with a FACSCalibur (BD Biosciences, San Jose, CA, USA), and data analysis was performed with FlowJo 5.2.7 (Tree Star, Ashland, OR, USA). Raman analyses were carried out by confocal Raman microspectroscopy (CRM, Alpha300R⁺; WITec, Ulm, Germany). Statistical analyses and graphs were performed using GraphPad Prism 5 software.