Development of multifunctional liposomes containing magnetic/plasmonic MnFe2O4/Au core/shell nanoparticles

Development of Multifunctional Liposomes Containing Magnetic/Plasmonic MnFe 2 O 4 /Au Core/Shell Nanoparticles Ana Rita O. Rodrigues 1 , Joana O. G. Matos 1 , Armando M. Nova Dias 1 , Bernardo G. Almeida 1 , Ana Pires 2 , André M. Pereira 2 , João P. Araújo 2 , Maria-João R. P. Queiroz 3 , Elisabete M. S. Castanheira 1 and Paulo J. G. Coutinho 1, * 1 Centro de Física da Universidade do Minho (CFUM), Campus de Gualtar, 4710-057 Braga, Portugal; [email protected] (A.R.O.R.); [email protected] (J.O.G.M.); [email protected] (A.M.N.D.); [email protected] (B.G.A.); [email protected] (E.M.S.C.) 2 IFIMUP/IN—Instituto de Nanociência e Nanotecnologia, Universidade do Porto, R. Campo Alegre, 4169-007 Porto, Portugal; [email protected] (A.P.); amper[email protected] (A.M.P.); j[email protected] (J.P.A.) 3 Centro de Química da Universidade do Minho (CQUM), Campus de Gualtar, 4710-057 Braga, Portugal; [email protected]o.pt * Correspondence: [email protected]; Tel.: +351-253-604-321 Received: 20 November 2018; Accepted: 24 December 2018; Published: 31 December 2018 Abstract: Multifunctional liposomes containing manganese ferrite/gold core/shell nanoparticles were developed. These magnetic/plasmonic nanoparticles were covered by a lipid bilayer or entrapped in liposomes, which form solid or aqueous magnetoliposomes as nanocarriers for simultaneous chemotherapy and phototherapy. The core/shell nanoparticles were characterized by UV/Visible absorption, X-Ray Diffraction (XRD), Transmission Electron Microscopy (TEM), and Superconducting Quantum Interference Device (SQUID). The magnetoliposomes were characterized by Dynamic Light Scattering (DLS) and TEM. Fluorescence-based techniques (FRET, steady-state emission, and anisotropy) investigated the incorporation of a potential anti-tumor drug (a thienopyridine derivative) in these nanosystems. The core/shell nanoparticles exhibit sizes of 25 ± 2 nm (from TEM), a plasmonic absorption band (λ max = 550 nm), and keep magnetic character. XRD measurements allowed for the estimation of 13.3 nm diameter for manganese ferrite core and 11.7 nm due to the gold shell. Aqueous magnetoliposomes, with hydrodynamic diameters of 152 ± 18 nm, interact with model membranes by fusion and are able to transport the anti-tumor compound in the lipid membrane, with a high encapsulation efficiency (EE (%) = 98.4 ± 0.8). Solid magnetoliposomes exhibit hydrodynamic diameters around 140 nm and also carry successfully the anticancer drug (with EE (%) = 91.2 ± 5.2), while also being promising as agents for phototherapy. The developed multifunctional liposomes can be promising as therapeutic agents for combined chemo/phototherapy. Keywords: magnetic/plasmonic nanoparticles; multifunctional liposomes; manganese ferrite; gold shell; anti-tumor drugs; cancer therapy 1. Introduction In recent years, a revolution in cancer therapy has taken place due to the development of multi-tasked nanostructures or materials for applications in oncology [1,2]. In chemotherapy, the ideal nano-encapsulation system should have biophysical properties that favor the passive accumulation in tumors upon intravenous administration, as well as controlled triggered release of the encapsulated active molecules. In this context, magnetic nano-encapsulation systems are promising since they can enable the magnetic drug targeting by static gradient magnetic fields and magnetic hyperthermia, which produce local heat as a trigger for drug release and a synergistic 2 of 18 cytotoxic effect in cancer cells [3–7]. Additionally, systems based on superparamagnetic nanoparticles can generate high-resolution images by T2-weighted magnetic resonance imaging (MRI) for tumor diagnosis [7–9]. Noble metal (Ag, Au) nanoparticles strongly absorb light in the visible region due to coherent oscillations of the metal conduction band electrons in strong resonance with visible frequencies of light. This phenomenon is known as surface plasmon resonance (SPR) [10–13] and is highly dependent on nanoparticles size, shape, surface, and dielectric properties of the surrounding medium [14–16]. Light absorbed by nanoparticles is readily dissipated as heat. Due to their large absorption cross sections, plasmonic nanoparticles can generate a significant amount of heat and increase temperatures in their vicinities [17]. If a sufficient number of nanoparticles are present, the temperature fields overlap and create a substantial global temperature rise [18]. From the point of view of cancer therapeutics, noble metal nanoparticles become very useful as agents for plasmonic photothermal therapy (PTT) on account of their enhanced absorption cross sections, which are four to five orders of magnitude larger than those offered by conventional photo-absorbing dyes [16]. This strong absorption ensures effective laser therapy at relatively lower energies, which render the therapy method minimally invasive. Additionally, metal nanostructures have a higher photo-stability and do not suffer from photo-bleaching [16,19]. Recently, plasmonic nanoparticles have also been used as photoacoustic imaging (PAI) agents to increase tissue penetration, as well as sensitivity and spatial resolution [20]. In nanomedicine, systems with combined magnetic and plasmonic properties are of particular interest for theranostics since they combine simultaneously multiple imaging modalities for diagnosis with complementary synergistic strategies for therapy [21–23]. Gold nanoparticles have been largely used in biomedical applications for their low toxicity, great biocompatibility, easy conjugation with active biomolecules, and their remarkable optical properties, which enable their use as diagnostic and therapeutic agents [19,24]. However, recent works have shown that the conjugation of gold nanoparticles with magnetic ones may decrease the overall magnetization of the nanostructure [25]. Thus, coating magnetic nanoparticles with gold should be carefully considered in order to ensure proper magnetic capabilities for their application. Among all magnetic nanoparticles, those of manganese ferrite have recently received great attention for their high magnetic susceptibility, which suggests that they may be promising as hyperthermia and magnetic drug targeting agents [26,27]. In this study, magnetic/plasmonic nanoparticles possessing a manganese ferrite core and a gold shell were prepared. In order to develop applications in cancer therapy, the prepared nanoparticles were entrapped in liposomes (aqueous magnetoliposomes, AMLs) or covered with a lipid bilayer (solid magnetoliposomes, SMLs). These new nanosystems were tested in this scenario as nanocarriers for a potential anticancer drug, especially active against melanoma, breast adenocarcinoma, and non-small cell lung cancer [28]. In addition, the local heating capability of the developed systems was monitored through the fluorescence quenching of rhodamine B incorporated in the lipid layer when excited with a light source. Considering their potentialities, the new nanosystems developed in this study can be promising for future applications in cancer therapy. 2. Materials and Methods All the solutions were prepared using spectroscopic grade solvents and ultrapure water of Milli-Q grade (MilliporeSigma, St. Louis, MO, USA). 2.1. Preparation of Manganese Ferrite/Gold Core/Shell Nanoparticles Manganese ferrite nanoparticles (NPs) were synthesized in 5 mL aqueous solution, by the co-precipitation method, as previously described [26]. First, an aqueous solution containing 612 μL of 50% NaOH solution was heated to 90 °C. Then, a mixture containing 500 μL of 0.5 M MnSO 4 ·H 2 O solution and 500 μL of 1 M FeCl 3 ·6H 2 O solution was added, drop by drop, to the previously warmed basic solution under magnetic stirring. After two hours at 90 °C, manganese ferrite nanoparticles 3 of 18 were formed. For purification, the obtained sample was washed several times with ethanol, by centrifugation (14,000 g) and magnetic decantation. For growth of the gold shell, a method adapted from a previously described procedure was used [19]. In addition, 5 mL of an aqueous dispersion of the synthesized MnFe 2 O 4 nanoparticles (with concentration of 4 mg/mL) were added to 25 mL of glycerol and heated up to 200 °C, under vigorous stirring. Then, 2 mL of 0.02 M solution of gold(III) chloride hydrate (HAuCl 4 ), from Sigma-Aldrich (St. Louis, MO, USA), were added dropwise. After 15 minutes under continuous stirring at 200 °C, the gold shell was formed around the MnFe 2 O 4 core NPs. To remove glycerol residues, the synthesized NPs were washed by centrifugation (14,000 g) with ethanol. 2.2. Preparation of Magnetoliposomes For magnetoliposomes preparation, the lipids L -α-phosphatidylcholine from egg yolk (Egg-PC), and 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), from Sigma-Aldrich (St. Louis, MO, USA), were used in a final concentration of 1 mM. The ethanol injection method was employed to obtain aqueous magnetoliposomes (AMLs) [29]. Accordingly, a 20 mM lipid solution in ethanol was injected, under vigorous vortexing, to an aqueous dispersion of manganese ferrite/gold nanoparticles (with 4 mg/mL concentration). After encapsulation, the ferrofluid was washed with water and purified by magnetic decantation to remove all the non-encapsulated NPs. For the preparation of solid magnetoliposomes (SMLs), a method previously described was used [30]. First, 10 μL of a solution of the synthesized MnFe 2 O 4 /Au core/shell nanoparticles (0.02 mg/mL) were ultra-sonicated for one minute at 189 W, and 3 mL of chloroform were added to the solution. Then, immediately after vigorous agitation, 150 μL of a 20 mM methanolic solution of the lipid DOPG (1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt) were injected under vortexing to form the first lipid layer of the SMLs. To remove the lipid that was not attached to the nanoparticles surface, the particles were washed twice by magnetic decantation with ultrapure water. The lipid bilayer was completed by a new injection of 150 μL of 20 mM lipid methanolic solution, under vortexing, in 3 mL of aqueous dispersion of the particles with the first lipid layer. The SMLs obtained were then washed and purified with ultrapure water by magnetic decantation. The anti-tumor compound methyl 3-amino-6-(benzo[d]thiazol-2-ylamino)thieno[3,2-b]pyridine- -2-carboxylate was incorporated into aqueous magnetoliposomes by the co-injection method (simultaneous injection of compound and lipid) in a final compound concentration of 2 μM. In solid magnetoliposomes, the compound was incorporated by injection of an ethanolic solution (0.2 mM) immediately before the formation of the second lipid layer. 2.3. Preparation of Giant Unilamellar Vesicles (GUVs) GUVs of soybean lecithin ( L -α-phosphatidylcholine from soybean), from Sigma-Aldrich (St. Louis, MO, USA), were obtained by the thin film hydration method [31,32]. For that, a lipid film of 100 μL of soybean lecithin solution (1 mM) was obtained by solvent evaporation under an argon stream, and 40 μL of water were added, followed by incubation at 45 °C for 30 minutes. Then, 3 mL of glucose aqueous solution (0.1 M) were added and the resulting solution was again incubated at 37 °C for 2 hours. After incubation, the GUVs suspension was centrifuged at 14,000 g for 30 minutes at 20 °C, to remove multi-lamellar vesicles and lipid aggregates. 2.4. Spectroscopic Measurements 2.4.1. General Methods Absorption spectra were performed in a Shimadzu UV-3600 Plus UV-vis-NIR (Shimadzu Corporation, Kyoto, Japan) spectrophotometer. Fluorescence measurements were recorded using a Horiba Fluorolog 3 spectrofluorimeter (HORIBA Jobin Yvon IBH Ltd., Glasgow, UK), equipped with double mono-chromators in both excitation and emission, Glan-Thompson polarizers, and a 4 of 18 temperature controlled cuvette holder. Fluorescence spectra were corrected for the instrumental response of the system. 2.4.2. FRET Measurements Förster Resonance Energy Transfer (FRET) assays were employed to confirm the formation of the lipid bilayer in the solid magnetoliposomes (SMLs). For that purpose, the nitrobenzoxazole labeled lipid NBD-C 6 -HPC (1-palmitoyl-2-{6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl} -sn-glycero-3-phosphocholine) (from Avanti Polar Lipids, Alabaster, AL, USA) was included in the first lipid layer, while the rhodamine B labeled lipid Rhodamine B-DHPE (1,2-dipalmitoyl-sn-glycero-3-phospho-ethanolamine-N-lissamine rhodamine B sulfonyl (ammonium salt)) (from Avanti Polar Lipids, Alabaster, AL, USA) was included in the second lipid layer. FRET efficiency, Φ RET , defined as the proportion of donor molecules that have transferred their excess energy to the acceptor molecules, was calculated through donor emission quenching, by taking the ratio of the donor integrated fluorescence intensities in the presence of acceptor (F DA ) and in the absence of acceptor (F D ) (Equation 1) [33]. Φ RET = 1 −     (1) The distance between the donor and acceptor molecules was determined through the FRET efficiency (Equation 2).  =    1 − Φ  Φ      (2) where R 0 is the Förster radius (critical distance), that can be obtained by the spectral overlap, J(λ), between the donor emission and the acceptor absorption, according to Equations (3) and (4) (with R 0 in Å, λ in nm, ε A ( λ ) in M −1 cm −1 ) [33].   = 0 . 2108           ( ! ) #    (3)  ( ! ) = $ %  ( ! &  ) ' ( ( ! ) !  )! (4) where   =23  is the orientational factor assuming random orientation of the dyes, n is the refraction index of the medium, I D (λ) is the fluorescence spectrum of the donor normalized so that +%  &  (!))!=1, and ε A (λ) is the molar absorption coefficient of the acceptor. Φ   , the fluorescence quantum yield of the donor in the absence of energy transfer, was determined by the standard method (Equation 5) [34,35].    = , - .     ,  . -  -   - (5) where A is the absorbance at the excitation wavelength, F is the integrated emission area, and n is the refraction index of the solvents used. Subscripts refer to the reference (r) or donor (D). The absorbance at the excitation wavelength was always lower than 0.1 to avoid the inner filter effects. The NBD-C 6 -HPC molecule intercalated in lipid membranes was used as a reference, Φ r = 0.32 at 25 °C, as reported by Invitrogen [36]. The hydrophobic dye Nile Red (energy acceptor) was also incorporated in magnetoliposomes labelled with NBD-C 6 -HPC (NBD as energy donor) for monitoring the interaction of magnetoliposomes with GUVs by FRET. 2.4.3. Fluorescence Anisotropy Measurements The steady-state fluorescence anisotropy, r, is calculated by the equation below. 5 of 18  = % // − 0 % /1 % // + 2 0% /1 (6) where I VV and I VH are the intensities of the emission spectra obtained with vertical and horizontal polarization, respectively (for vertically polarized excitation light), and 0 =% 1/ /% 11 is the instrument correction factor, where I HV and I HH are the emission intensities obtained with vertical and horizontal polarization (for horizontally polarized excitation light). 2.4.4. Drug Encapsulation Efficiency The encapsulation efficiency, EE (%), of the potential anti-tumor drug in magnetoliposomes, was determined through fluorescence emission measurements. Therefore, drug loaded magnetoliposomes were subjected to centrifugation at 11,000 rpm for 60 min using Amicon  Ultra centrifugal filter units 100 kDa (Merck Millipore, Darmstadt, Germany). Then, the filtrate (containing the non-encapsulated drug) was pipetted out, the water was evaporated, and the same amount of ethanol was added. After vigorous agitation, its fluorescence was measured, which allowed it to determine the drug concentration using a calibration curve (fluorescence intensity vs. concentration) previously obtained in the same solvent. Three independent measurements were performed for each system and standard deviations (SD) were calculated. The encapsulation efficiency was determined using Equation (7). 44 ( % ) = ( 67689 8:7;6 − 8:7;6 7< 7 =>8?@;98 6 =) >7:?7;) ) 67689 8:7;6 × 100 (7) 2.5. Structural Characterization 2.5.1. Transmission Electron Microscopy (TEM) TEM images of nanoparticles and solid magnetoliposomes were acquired using a Transmission Electron Microscope Leica LEO 906E (Leica Microsystems, Wetzlar, Germany) operating at 120 kV, at UME (Electron Microscopy Unit), University of Trás-os-Montes and Alto Douro (Vila Real, Portugal). For SMLs, a negative staining was employed, using a 2% aqueous solution of ammonium molybdate tetrahydrate. In addition, 20 μL of the sample and 20 μL of the staining solution were mixed and a drop of the mixture was placed onto a Formvar grid (Agar Scientific Ltd., Essex, UK), held by tweezers. After 20 s, almost all the solution was removed with filter paper and left to dry. TEM images were processed using ImageJ software (National Institutes of Health (NIH), Bethesda, MD, USA) with the addition of a value to all pixels so that a white background resulted, which was followed by inversion and enhanced local contrast. Subsequently, the ParticleSizer plugin [37] was used and was followed by particle analysis. The area of each particle allowed an estimation of the particle diameter. The resulting histogram was fitted to a bimodal Gaussian distribution. 2.5.2. X-Ray Diffraction (XRD) X-Ray Diffraction (XRD) analyses were performed using a conventional Philips PW 1710 (Royal Philips, Amsterdam, The Netherlands) diffractometer, operating with CuK α radiation, in a Bragg-Brentano configuration. 2.5.3. Dynamic Light Scattering (DLS) The mean diameter and size distribution (polydispersity index) of aqueous and solid magnetoliposomes (1 mM lipid concentration) were measured using Dynamic Light Scattering (DLS) equipment NANO ZS Malvern Zetasizer (Malvern Panalytical Ltd., Malvern, UK) at 25 °C, using an He-Ne laser of λ = 632.8 nm and a detector angle of 173°. The measurements were also carried out for the magnetoliposomes in a solution of human serum albumin (35 mg/mL) in PBS buffer (pH = 7.4). Five independent measurements were performed for each sample. 6 of 18 2.6. Magnetic Measurements Magnetic measurements of the dry core/shell nanoparticles were performed at room temperature in a Superconducting Quantum Interference Device (SQUID) magnetometer Quantum Design MPMS5XL (Quantum Design Inc., San Diego, CA, USA) using applied magnetic fields up to 5.5 T. 2.7. Measurement of the Photothermal Effect Solid magnetoliposomes incorporating the labelled lipid Rhodamine B-DHPE were irradiated and Rhodamine B emission was monitored by a function of time, using the detection system of a SPEX Fluorolog 2 spectrofluorimeter (HORIBA Jobin Yvon IBH Ltd., Glasgow, UK). The irradiation setup consisted in a Xenon arc lamp (200 W) and an optical fiber, using a Thorlabs FEL0600 (Thorlabs Inc., Newton, NJ, USA) long pass filter with cut-on wavelength at 600 nm, to ensure the excitation of only the gold nanoparticles (not exciting Rhodamine B dye). 3. Results and Discussion 3.1. Nanoparticles Characterization 3.1.1. Absorption Spectra Figure 1 displays the UV-Visible absorption spectrum of the synthesized manganese ferrite/gold core/shell nanoparticles. The absorption spectra of net gold nanoparticles and net manganese ferrite nanoparticles are also shown for comparison. Figure 1. UV-Visible absorption spectra of aqueous dispersions of manganese ferrite nanoparticles, gold nanoparticles, and MnFe 2 O 4 /Au core/shell nanoparticles. The spectrum of manganese ferrite NPs is typical of an indirect semiconductor, as reported earlier [26], while the spectrum of gold nanoparticles obtained by the standard Turkevish method [38] reveals a characteristic local surface plasmon resonance (LSPR) band, with a maximum around 530 nm. In comparison, the absorption spectrum of manganese ferrite/gold core/shell NPs exhibits a broader and red shifted plasmon band (maximum at 550 nm). The absorption spectrum of gold nanoshells depends on their thickness, as well as on the refraction index of both core and surrounding media [39]. Theoretical studies have shown that, for an air filled core of 10 nm size and a gold shell of 5 nm in water medium, the resonance peak is expected to occur at 538 nm, while, for a 10-nm shell, it should appear at 552 nm [40]. The increase of the core refraction index results in a red 7 of 18 shift of the plasmon resonance peak [39]. For a 3-nm gold shell thickness on a 10-nm magnetite core, a resonance peak at 560 nm was found [41]. 3.1.2. X-Ray Diffraction (XRD) Measurements XRD analysis confirmed the synthesis of a pure crystalline phase of manganese ferrite/gold nanoparticles, since all their characteristic peaks (CIF 2300618 for manganese ferrite and CIF 9013035 for gold), marked by their indices, were observed (Figure 2b). The percentage amounts obtained for MnFe 2 O 4 and Au were 59.1% and 40.9%, respectively. Mean sizes of 13.3 nm for manganese ferrite and 11.7 nm for gold, were estimated through a Rietveld analysis using Fullprof software [42]. Table 1 summarizes the main results of the Rietveld analysis. For the net manganese ferrite powdered sample, it resulted in a poor R F factor of 9.0. It was possible to improve it by optimizing the overall isothermal factor, B over , but an unreasonable value of −2.79 was obtained. A similar improvement was possible by accounting for the effect of sample microstructure (Figure 2a), according to Equation (8) [43], which gives the micro-absorption correction term, P. B = B  + C  sin  G 1 −  sin  H (8) where P 0 is the bulk contribution to the micro-absorption effect and τ is the normalized surface roughness parameter [43]. Considering I9J= 0.01 (μ is the linear absorption coefficient and 9J is the mean chord length of the powder particle) and the values in Table 1 for Equation (8) parameters, a degree of inversion of i = 0.60 is obtained for manganese ferrite. This value is close to the one reported by Chen et al. (i = 0.67) [44], using a similar preparation method for MnFe 2 O 4 . The Rietveld analysis of gold phase was not optimal, as the intensity of peak (1 1 1) is lower and that of peak (2 0 0) is higher than the experimental ones. In addition, the analysis of MnFe 2 O 4 phase decreased its quality, as the R F factor increased from 3.18 to 4.70 and the peak (3 1 1) got much lower than the experimental one. This could be due to the expected shell morphology of gold in the prepared MnFe 2 O 4 /Au nanocomposites in which a layer of gold grows on the MnFe 2 O 4 surface. This morphology, through lattice mismatch induced stress, is predictable to change the intensity of the diffraction peaks [45]. An atomistic modelling of the core/shell nanoparticle is anticipated to yield a better description of the XRD diffractogram [46] and this will be addressed in a future study. The obtained weight fraction of gold was 40.9%, but this is calculated using proportionality factors between diffraction intensity and mass, ATZs [42], that do not take into account the dependence of diffraction intensity on particle size. Since the lattice constant of Au is less than half that of MnFe 2 O 4 , the reduction of diffraction intensity with particle size is much more pronounced for Au than for MnFe 2 O 4 . This means that the value given by Fullprof software is expected to be much lower than the real one. Considering the obtained size of manganese ferrite of 13.3 nm and using the phase densities that resulted from the XRD analysis (5.04 g cm −3 for MnFe 2 O 4 and 19.4 g cm −3 for Au), the mass percentage of gold would be 98.7% if the obtained size of gold phase (11.7 nm) corresponds to the shell thickness. This percentage would change to 95.6% if the obtained size value of 11.7 nm corresponds to the double of the shell thickness. This would be true if the effect of the two gold layers in given X-ray crosses contributes equally to the broadening of the diffraction peak. Thus, from XRD data analysis, the thickness of the gold shell is expected to be 5.85 nm. This value is compatible with the observed position of the surface plasmon resonance peak, as discussed in the previous section. Additionally, in magnetite/gold core/shell nanoparticles, reported in Reference [41], the diffraction peak widths were identical for the 10 nm magnetite core and for the 2 nm gold shell (where its dimensions were obtained from HR-TEM measurements). Therefore, the actual value of the gold shell thickness in MnFe 2 O 4 /Au NPs could be even lower. A nanostructure consisting of a 5.85 nm gold shell surrounding a cluster of MnFe 2 O 4 nanoparticles cannot be ruled out. In that case, the calculated mass percentage of gold changes to 91% for a compact over-coating layer of 12 spheres. Nevertheless, in the case of gold shell growth, the magnetic nanoparticles are dispersed in glycerol at a high temperature, before the addition of HAuCl 4 solution. This means the magnetic nanoparticles can effectively be dispersed with their 8 of 18 surface well stabilized and passivated by the abundant OH groups of glycerol. Furthermore, the formation of the gold shell occurs through oxidation of glycerol. This process originates in other molecules, such as glyceric acid or tartronic acid, which can act as gold surface stabilizers. This is the case in terms of the citric acid in the Turkevish gold nanoparticles synthesis procedure [38]. Therefore, the prepared MnFe 2 O 4 /Au core/shell nanoparticles are expected to be surface passivated by hydroxyacids and, as such, well dispersible in aqueous media. Figure 2. XRD diffractogram of manganese ferrite (a) and manganese ferrite/gold core/shell nanoparticles (b). Gold diffraction peaks are marked by a filled triangle. Table 1. Selected Rietveld analysis parameters. Sample O x,y,z (*) i (*) Micro Absorption Correction Overall Temperature F actor , B over Lattice Constant (nm) Size (nm) R f χ 2 MnFe 2 O 4 0.251 0.928 No 0 ( + ) 0.84693 13.8 9.03 1.33 MnFe 2 O 4 0.251 0.898 No −2.79 0.84684 13.2 4.45 1.18 MnFe 2 O 4 0.257 0.60 Yes (#) 0 ( + ) 0.84685 13.3 3.18 1.18 MnFe 2 O 4 /Au 0.257 ( + ) 0.60 ( + ) Yes (##) 0 ( + ) 0.84685 ( + ) 13.3 4.70 1.56 --- --- 0 ( + ) 0.406945 11.7 0.68 (#) P 0 = 0.629, C = 1.31, τ = 0.055. (##) P 0 = 0.607, C = 1.15, τ = 0.084. ( + ) fixed values. (*) Values in CIF file 2360018 are O x,y,z = 0.25053 and i = 0.33. 3.1.3. Transmission Electron Microscopy (TEM) TEM images (Figure 3a) of the MnFe 2 O 4 /Au prepared nanoparticles and the corresponding image after processing by ImageJ (Figure 3b) revealed a generally spherical shape with the presence of some aggregation. These aggregates probably arise from the sample preparation on TEM grids (slow evaporation of a drop of an aqueous dispersion of nanoparticles). The size histogram that results from the area of the highlighted particles was fitted to a bimodal Gaussian distribution in order to better separate the presence of aggregates from the individual nanoparticles (Figure 3c). A bimodal size distribution of 25 ± 2 nm and 32 ± 6 nm was obtained. The former population is in accordance with the size estimated from XRD measurements, which is 25 nm when the gold shell thickness is 5.85 nm. 9 of 18 (a) (b) (c) Figure 3. (a) TEM image of the synthesized MnFe 2 O 4 /Au core/shell nanoparticles. (b) TEM image processed by ImageJ (same scale of image a). (c) Particles size histogram and fitting to a bimodal Gaussian distribution (total number of 141 particles). Below is a critical diameter of 42.9 nm. MnFe 2 O 4 nanoparticles possess a superparamagnetic behavior [47,48], losing at least 90% of the magnetization when an applied magnetic field is removed, which is important for biomedical applications. The size of the nanoparticles obtained in this study is within this limit, and, therefore, these NPs are suitable for applications in biomedicine. 3.2. Magnetic Properties The magnetic properties of MnFe 2 O 4 /Au core/shell nanoparticles (Figure 4) were characterized by measuring their magnetic hysteresis loop, which shows the relationship between the induced magnetic moment and the applied magnetic field (H). The core/shell nanoparticles present a superparamagnetic behavior since the ratio between remnant magnetization (M r ) and saturation magnetization (M s ) is below 0.1 [49] (Table 2). Figure 4. Magnetization hysteresis loop of MnFe 2 O 4 /Au core/shell nanoparticles measured at room temperature. Inset: Enlargement of the loop in the low field region. Table 2. Coercive field (H c ), saturation magnetization (M s ), remnant magnetization (M r ), and ratio M r /M s for MnFe 2 O 4 /Au core/shell nanoparticles at room temperature. 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