Shaping Up Zn-Doped Magnetite Nanoparticles from Mono- and Bimetallic Oleates: The Impact of Zn Content, Fe Vacancies, and Morphology on Magnetic Hyperthermia Performance

Shaping Up Zn-Doped Magnetite Nanoparticles from Monoand Bimetallic Oleates: The Impact of Zn Content, Fe Vacancies, and Morphology on Magnetic Hyperthermia Performance Idoia Castellanos-Rubio,*Oihane Arriortua, Lourdes Marcano, Irati Rodrigo, Daniela Iglesias-Rojas, Ander Barón, Ane Olazagoitia-Garmendia, Luca Olivi, Fernando Plazaola, M. Luisa Fdez-Gubieda, Ainara Castellanos-Rubio, JoséS. Garitaonandia, Inaki Orue, and Maite Insausti* Cite This: Chem. Mater. 2021, 33, 3139−3154 Read Online ACCESS Metrics & More Article Recommendations * sıSupporting Information ABSTRACT: The currently existing magnetic hyperthermia treatments usually need to employ very large doses of magnetic nanoparticles (MNPs) and/or excessively high excitation conditions (H×f>10 10 A/m s) to reach the therapeutic temperature range that triggers cancer cell death. To make this anticancer therapy truly minimally invasive, it is crucial the development of improved chemical routes that give rise to monodisperse MNPs with high saturation magnetization and negligible dipolar interactions. Herein, we present an innovative chemical route to synthesize Zn-doped magnetite NPs based on the thermolysis of two kinds of organometallic precursors: (i) a mixture of two monometallic oleates (FeOl + ZnOl), and (ii) a bimetallic ironzinc oleate (Fe3−yZnyOl). These approaches have allowed tailoring the size (10−50 nm), morphology (spherical, cubic, and cuboctahedral), and zinc content (ZnxFe3−xO4, 0.05 < x< 0.25) of MNPs with high saturation magnetization (≥90 Am2/kg at RT). The oxidation state and the local symmetry of Zn2+ and Fe2+/3+ cations have been investigated by means of X-ray absorption near-edge structure (XANES) spectroscopy, while the Fe center distribution and vacancies within the ferrite lattice have been examined in detail through Mossbauer spectroscopy, which has led to an accurate determination of the stoichiometry in each sample. To achieve good biocompatibility and colloidal stability in physiological conditions, the ZnxFe3−xO4NPs have been coated with high-molecular-weight poly(ethylene glycol) (PEG). The magnetothermal efficiency of ZnxFe3−xO4@PEG samples has been systematically analyzed in terms of composition, size, and morphology, making use of the latest-generation AC magnetometer that is able to reach 90 mT. The heating capacity of Zn0.06Fe2.94O4cuboctahedrons of 25 nm reaches a maximum value of 3652 W/g (at 40 kA/m and 605 kHz), but most importantly, they reach a highly satisfactory value (600 W/g) under strict safety excitation conditions (at 36 kA/m and 125 kHz). Additionally, the excellent heating power of the system is kept identical both immobilized in agar and in the cellular environment, proving the great potential and reliability of this platform for magnetic hyperthermia therapies. 1. INTRODUCTION The success of magnetic hyperthermia therapies depends on the heating capacity or specific absorption rate (SAR) of the magnetic nanoparticles (MNPs) when they are exposed to an alternating magnetic field (AMF). 1−3 As a matter of course, to achieve the desired therapeutic effect under a safe frequency field product (H×f<1010 A/m s), the MNP heating power has to be optimized. 4,5 The design of new MNPs with improved magnetothermal efficiency is a challenging task due to the difficulty in controlling and predicting the complex colloidal synthesis of inorganic nanocrystals. 6−8 The preparation of nanostructures with very specific sets of characteristics (size, morphology, homogeneous chemical composition, high purity, and low size/shape dispersity) requires an extremely fine control over the synthetic protocol. 9 In this sense, the thermal decomposition of organometallic precursors opened a new avenue for synthesizing novel iron oxide-based MNPs with a well-defined size and morphology. 10,11 The most commonly used iron oxide MNPs for biomedical applications are of magnetite (Fe3O4) due to their high magnetic response, good biocompatibility, chemical stability, and simple compoReceived: December 16, 2020 Revised: April 2, 2021 Published: April 19, 2021 Articlepubs.acs.org/cm © 2021 American Chemical Society 3139 https://doi.org/10.1021/acs.chemmater.0c04794 Chem. Mater. 2021, 33, 3139−3154 Downloaded via 62.99.67.15 on January 31, 2024 at 17:59:32 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles. sition. 12,13 But interestingly, the introduction of a low quantity of divalent transition-metal ions (MxFe3−xO4, M = Zn, Co, Mn, Ni, etc.) within the spinel structure of magnetite NPs has proven to be a good strategy to obtain mixed ferrites with tuned magnetic performances, 14−17 although in some cases, there are concerns about their dubious biocompatibility. Particularly, Zn-containing ferrite NPs are considered quite biocompatible because zinc is an essential trace element of the human body that has a relatively high toxic dose, up to 450 mg day−1. 18 Currently, zinc ferrites are also being explored for biomedical applications due to their higher stability against oxidation. 19,20 Additionally, as it is well-known, the introduction of diamagnetic Zn2+ ions in the magnetite lattice ([Fe3+]A[Fe2+Fe3+]BO4) can produce significant enhancement of the particle′s magnetic moment, compared to pure magnetite. 21 This is because Zn2+ ions tend to replace Fe3+ in A sites, reinforcing the already existing unbalance between antiferromagnetically coupled A and B sublattices. Unfortunately, this mechanism holds up, in the bulk state, just until a Zn content of x≈0.4 (ZnxFe3−xO4), above which the lack of magnetic moments located at A sites strongly disrupts the exchange interaction between both sublattices, causing a decrease of the total magnetic moment. 22 Another drawback in the preparation of doped ferrite NPs is that the dopant is often assimilated in different positions of the crystal lattice, 23−27 which typically happens due to the nonequilibrium nature of these chemical reactions, making it difficult to achieve the intended theoretical results and running the risk of deriving mistaken conclusions. Thus, the first step in the development of doped ferrite nanomaterials should be the accurate determination of the local geometry of the dopant atoms to ensure reliable properties and trustful potential applications. It is clear, then, that a priori a remarkable improvement of the SAR can be achieved if high-grade Fe3O4NPs are doped with a suitable amount of Zn2+ in the proper lattice position. In addition, as has been reported recently, the heating power of magnetite NPs with nonfluctuating magnetic moment (FMNP), whose average size is over 20 nm, can be significantly greater than that of the superparamagnetic NPs (SP-NPs < 20 nm) if high-enough fields (H>15−20 mT) are used and dipolar magnetic interactions are minimized. 28,29 However, as one would expect, the synthesis of high-quality Zn-doped magnetite FM-NPs is far more challenging than that of undoped magnetite. In recent years, it has been common to synthesize Zn-doped ferrite NPs by thermal decomposition of metal acetylacetonates 14,25,30 and by coprecipitation of corresponding metal chloride or nitrate salts, 25,31−33 which in most of the cases gave rise to polydisperse NPs in size and shape. The decomposition of metal oleates has also been explored in some studies on Zn-ferrites, but it usually has the downside of having to deal with the formation of the wustite (FeO)-phase byproduct. 14,27 Certainly, works describing the synthesis of Zn-doped magnetite NPs with a well-defined morphology, average size larger than 20 nm, and devoid of secondary phases are rather scarce and mostly focused on NPs with a cubic morphology. 20,26,34 There is no doubt that the development of new strategies to prepare Zn-doped ferrites of different sizes and shapes would stimulate the progress of nanoparticle-based platforms for theranostics. Herein, we present an improved protocol to synthesize highly crystalline ZnxFe3−xO4NPs with a low size/shape dispersity and enhanced saturation magnetization. We have explored a new synthetic route based on the decomposition of bimetallic iron-zinc oleates, which has been compared to a more common route employing a mixture of monometallic oleates (iron oleate + zinc oleate). To the best of our knowledge, this is the first time that these two approaches are carefully analyzed and compared. By finely modifying the synthesis conditions of both routes, NPs of different sizes (10− 50 nm), shapes (spheres, cubes, and cuboctahedrons), and zinc contents (0.05 < x< 0.25) have been obtained. These samples have been chemically, structurally, and magnetically analyzed with great accuracy making use of X-ray absorption near-edge structure (XANES), DC magnetometry, and Mossbauer spectroscopy. The study has allowed one to determine reliably both the Zn position/content and the Fe distribution/ vacancies, a subject that has not been sufficiently explored even in the most recent works on this topic. 35,36 Additionally, ZnxFe3−xO4NPs have been specifically PEGylated to avoid NP aggregation in cell environments, and viability assays have been carried out to prove their good biocompatibility. Finally, the heating efficiency of ZnxFe3−xO4@PEG NPs has been studied in detail by measuring the dynamical hysteresis loops at different frequencies (up to a field intensity of 90 mT) and in several dispersion environments (distilled water, agar, and cell culture). The optimal excitation parameters to maximize the heating production under clinical safety limits have been determined for each sample. 2. RESULTS AND DISCUSSION 2.1. Role of Chemical Synthesis on the Size, Shape, Crystalline Structure, and Composition. By carrying out thermolysis of different iron and zinc oleates, ZnxFe3−xO4NPs of different sizes, shapes, and compositions were obtained. Since our goal is to focus on zinc contents x< 0.4 and the dopant has typically to be added in excess to reach the intended compostion, 23,37,38 Fe/Zn oleates with 5:1 and 2:1 ratios have been used in the preparations, employing both Table 1. Summary of Synthesis Conditions and Samples Features Obtained by Mixture of Monometallic Oleates (Gray Shade Rows) And by Bimetallic Oleates: Metal Oleate Used in the Synthesis, Znxfe3−xO4NPs Composition Determined By ICP-MS, Final Temperature, Annealing Time, Particle Mean Dimension Obtained By TEM, Average Crystallite Size Obtained from (311) And (400) Diffraction Peaks, Peak Position of (311) And Lattice Parameter a sample metal oleate used in the synthesis sample composition ICP-MS final T (°C) annealing t (min) DTEM (nm) DXRD (nm) 311 peak position 2θ (deg) lattice parameter a (Å) Zn0.15-10 2.5 FeOl + 0.5 ZnOl Zn0.15Fe2.85O4320 30 10 (1) 8.8 (4) 35.592 8.3910(1) Zn0.1-48 2.5 FeOl + 0.5 ZnOl Zn0.1Fe2.9O4320 80 48 (4) 53 (5) 35.566 8.3940(5) Zn0.1-24 Fe2.5Zn0.5Ol Zn0.1Fe2.9O4320 30 24 (2) 22 (2) 35.581 8.3913(3) Zn0.1-34 Fe2.5Zn0.5Ol Zn0.1Fe2.9O4330 30 34 (3) 34 (3) 35.532 8.3961(4) Zn0.25-39 Fe2Zn1Ol Zn0.25Fe2.75O4320 60 39 (3) 29 (3) 35.530 8.4016(4) Chemistry of Materials pubs.acs.org/cm Article https://doi.org/10.1021/acs.chemmater.0c04794 Chem. Mater. 2021, 33, 3139−3154 3140 monometallic oleates (FeOl + ZnOl) and bimetallic oleates (Fe3−yZnyOl) (y= 0.5 and 1) (see Section 4 and Figure S1, Table S1 in the Supporting Information). The main structural difference between the mixture of monometallic oleates and bimetallic oleates is the presence of heterometallic bridging coordination in the latter (see Figure S2 and Table S2 in the Supporting Information), which reduces the diffusion distance between Zn−Fe centers, affecting the growth dynamics of the ZnxFe3−xO4NPs, as will be shown in the following. The ZnxFe3−xO4samples present a zinc content (measured by inductively coupled plasma mass spectrometry (ICP-MS)) ranging from x= 0.1 to 0.25 and an average dimension from 10 to 50 nm. With the aim of providing a clear picture of the main synthesis parameters affecting the properties of the NPs, five representative samples have been chosen (see Table 1). Samples have been named according to the composition and size as follows: Znx-DTEM, where xis the zinc content in the NPs determined by ICP-MS and DTEM is the average dimension obtained by transmission electron microscopy (TEM) analysis. TEM micrographs in Figure 1 show monodisperse samples with spherical, cubic, and cuboctahedral shapes. When FeOl and ZnOl are reacted together at an Fe/Zn ratio equal to 5:1, using a final Tof 320 °C and 30 min of annealing, spherical particles of 10 nm diameter (sample Zn0.15-10 in Figure 1a) are obtained. By increasing the annealing time to 80 min, the shape of the nanocrystals changes from spheres to cubes and the average dimension increases considerably, from 10 to 48 nm (sample Zn0.1-48 in Figure 1b). Samples Zn0.15-10 and Zn0.1-48 seem to have followed the reaction profile described by Hyeon et al., in which the nucleation process starts between 310 and 320 °C and growth takes places gradually from 1 to 20 min of aging, proceeding rapidly afterward and causing the morphology to evolve to the cubic type. 39 On the other hand, when bimetallic Fe2.5Zn0.5Ol (Fe/Zn = 5:1) is reacted at the same synthesis conditions as those used in sample Zn0.15-10 (see sample Zn0.1-24 in Table 1), the NPs grow further and present a well-faceted octahedral shape with slight truncation (cuboctahedrons) (Figure 1c). The larger size obtained when the bimetallic Fe2.5Zn0.5Ol is used appears to be due to the shorter distance between Zn2+ and Fe3+ cations within the metallo-organic complex. The Zn2+ ions accelerate the transformation of the bimetallic oleate precursor, shifting its decomposition temperature to lower values. 27 Therefore, as could be expected, the reaction of Fe2.5Zn0.5Ol at higher final T (330 °C) produces even larger cuboctahedral nanoparticles (see sample Zn0.1-34 Figure 1d). Thus, it also seems reasonable to postulate that the changes in the decomposition profile of the bimetallic Fe2.5Zn0.5Ol not only speed up the growing stage but also modify the morphology of resulting nanocrystals. On the contrary, the metal oleate type used in the synthesis (monometallic or bimetallic) does not affect the amount of Figure 1. TEM micrographs and corresponding size distributions of samples (a) Zn0.15-10, (b) Zn0.1-48, (c) Zn0.1-24, (d) Zn0.1‑34, and (e) Zn0.2539. (a) and (b) have been obtained from a mixture of monometallic oleates (FeOl + ZnOl). (c)−(e) have been obtained from a bimetallic iron-zinc oleate (Fe3−yZnyOl). White scale bars are 100 nm. Black scale bars are 10 nm. Chemistry of Materials pubs.acs.org/cm Article https://doi.org/10.1021/acs.chemmater.0c04794 Chem. Mater. 2021, 33, 3139−3154 3141 zinc incorporated in the NPs. All of the samples synthesized using an initial Fe/Zn ratio of 5:1 give rise to NPs with a similar zinc content (x≈0.1) regardless of the synthetic route employed. The slightly higher zinc content in Zn0.15-10 is likely due to its larger surface-to-volume ratio, assuming that the dopant concentration tends to be somewhat higher on the NP surface because of internal diffusion constraints. 27,40 With the aim of increasing the zinc content within the NP lattice while facilitating its diffusion, a bimetallic Fe2Zn1Ol with a higher zinc concentration has been used (Fe/Zn = 2:1), expanding the annealing time to 60 min. In this way, the resulting NPs have a higher zinc concentration (x= 0.25), but they present an irregular prismatic shape with twinning planes (sample Zn0.25-39), as can be seen in Figure 1e. It seems feasible that an increase in Zn2+ substitution causes lattice strains and, thus, some kind of crystal distortion. To gain further information on the structural characteristics of the nanoparticles, X-ray diffraction (XRD) has been performed in powder samples. The whole set of ZnxFe3−xO4 samples (see Figure 2a) shows an inverse spinel structure with the space group Fd3m, compatible with the magnetite phase (PDF #880866) and without any trace of the wustite phase, which is a very common byproduct in this kind of iron oxide nanoparticles. 25 After Rietveld refinement, no additional impurity phase has been detected and the observed peaks have been indexed as (111), (220), (311), (400), (422), (511), (440), (620), and (533). The summary of Rietveld refined structural data is displayed in the Supporting Information (Figure S3 and Table S3), and the estimated lattice parameters (a) have been included in Table 1. The diffractions peaks of Zn0.25-39 are the most shifted toward lower angles (see Figure 2b), which give rise to the largest lattice parameter among the samples, i.e., 8.4016 Å, in accordance with its higher zinc content. When the x determined by ICP-MS is taken into account, there is no clear correlation between the lattice parameter and the zinc concentration (see Figure 2c). This seems to suggest that a fraction of zinc may not be within the ferrite lattice. As will be proved in the following (by XANES, magnetometry, and Mossbauer techniques), there are Zn2+ ions on the NP surface; thus, the Zn content in the ferrite lattice is lower than the total zinc amount determined by ICP-MS. If the corrected zinc content within the ferrite is plotted versus the lattice parameter, a linear-like dependence can be observed (see Figure 2d). In any case, an in-depth study of the physical dimension of unit cells should also account for the possible iron vacancies in the crystal lattice, a matter that will be discussed in more detail later. In relation to the average crystallite sizes, they have been calculated from (311) (Figure 2b) and (400) diffraction peaks and are listed in Table 1 (see also Tables S4−S6 in the Supporting Information). In all of the cases, except for Zn0.2539, the average dimensions calculated from TEM measurements match very well with the crystallite sizes, meaning that samples Zn0.15-10, Zn0.1-48, Zn0.1-24, and Zn0.1-34 are composed of single crystals. Nevertheless, the crystallite size of Zn0.25-39 is smaller than the physical average size determined by TEM, which implies that the NPs of this sample are twinned crystals, in agreement with what is seen in Figure 1e. 2.1.1. X-ray Absorption Near-Edge Structure (XANES). XANES is an element-specific technique that allows one to gain information about the oxidation state and the local symmetry of the absorbing element and can be used to identify and quantify inorganic phases and coordination compounds. 41,42 In the present case, X-ray absorption near-edge structure (XANES) has been performed at both Fe K-edge and Zn K-edge to investigate iron and zinc arrangements within the ZnxFe3−xO4NPs. Figure 3a shows the Fe K-edge XANES spectra of the set of ZnxFe3−xO4NPs (x= 0.1, 0.15, and 0.25) together with stoichiometric magnetite (Fe3O4), Zn-ferrite (ZnFe2O4), and wustite (FeO) as references. The comparison of the Fe K-edge XANES spectra of Fe3O4and ZnFe2O4shows that, apart from certain differences in the intensity below and above the edge region, the main changes expected from Zn doping should Figure 2. (a) X-ray powder diffraction patterns of samples Zn0.15-10, Zn0.1-48, Zn0.1-24, Zn0.1-34, and Zn0.25-39. (b) Zoom-in of the (311) diffraction peak and the lattice parameter (a) obtained by Rietveld refinement versus Zn content (x) estimated by (c) ICP-MS and (d) Mossbauer spectroscopy, respectively. Chemistry of Materials pubs.acs.org/cm Article https://doi.org/10.1021/acs.chemmater.0c04794 Chem. Mater. 2021, 33, 3139−3154 3142 appear at the edge energy (see the zoom-in of Figure 3a). In fact, the edge position is a clear-cut indicator of the oxidation state of the absorbing atom. Note that while magnetite is an inverse spinel where the Fe2+ ions occupy octahedral (B) sites and Fe3+ ions occupy both octahedral (B) and tetrahedral (A) sites, ZnFe2O4is a normal spinel in which the Zn2+ cations occupy the A sites and the Fe3+ are located in the B ones. Thus, the oxidation state of the Fe ions in magnetite (Fe2+/ Fe3+ ratio of 1:2) is lower than that in ZnFe2O4(exclusively Fe3+) and, consequently, the edge position appears ∼2eV shifted to lower energies in comparison with the ZnFe2O4 XANES spectrum. 41 In the case of the ZnxFe3−xO4NPs, all samples except for Zn0.15-10 display very similar spectra to the one of magnetite. In samples Zn0.1-48, Zn0.1-24, Zn0.1-34, and Zn0.25-39, the observed variations in the edge positions with respect to magnetite are within the error (0.2 eV) (see the inset of Figure 3a), suggesting that the Zn concentration in the ferrite lattice must be somewhat lower than the Zn content determined by ICP-MS. In contrast, the Zn0.15-10 sample shows a larger shift in the edge position toward higher energies (≈0.6 eV), which can be explained by the presence of the maghemite (Fe2O3) phase on the surface (see Figure S4 in the Supporting Information). A partial oxidation from magnetite to maghemite is not surprising in the Zn0.15-10 sample considering the high surface-area-to-volume ratio in NPs with an average dimension of 10 nm. Additionally, the presence of the maghemite phase in this sample is in accordance with the lower lattice parameter obtained from the Rietveld refinement (see Table 1). Figure 3b displays the Zn K-edge XANES spectra of ZnxFe3−xO4NPs compared to the ZnFe2O4reference. The edge position of the synthesized nanoparticles is coincident with that observed in the ZnFe2O4XANES spectrum, revealing aZn 2+ oxidation state in the sample. Above the edge position, all spectra present three main peaks. Although the positions of those peaks are comparable with that observed in the ZnFe2O4 XANES spectrum, the relative intensity of the peaks varies among the samples. Indeed, while certain similarities are observed between the Zn K-edge XANES spectra of the ZnxFe3−xO4samples and ZnFe2O4, confirming the incorporation of Zn cations in the inner structure of the magnetite in lattice A, an additional contribution is necessary to reproduce the experimental spectra. Therefore, the Zn K-edge XANES spectra of the ZnxFe3−xO4samples were fitted to a linear combination of ZnFe2O4and the available standards. The best linear combination fit was found considering the coexistence of Figure 3. (a) Normalized XANES spectra at the Fe K-edge of ZnxFe3−xO4(x= 0.1, 0.15, 0.25) NPs compared to reference compounds: magnetite (Fe3O4), Zn-ferrite (ZnFe2O4), and wustite (FeO). Zoom: detail of the pre-edge region. (b) Zn K-edge XANES spectra of ZnxFe3−xO4NPs compared to ZnFe2O4. Zoom: detail of the white line. (c) Linear combination fit (l.c.) of the Zn K-edge XANES spectra of sample Zn0.1-34 with 57(2)% ZnFe2O4and 43(2)% Zn2+ Td-complex. (d) Linear combination fit (l.c.) of the Zn K-edge XANES spectra of sample Zn0.25-39 with 79(1)% ZnFe2O4and 21(1)% Zn2+ Td-complex. The linear combination fits for the rest of the samples are in Figure S5 in the Supporting Information. Chemistry of Materials pubs.acs.org/cm Article https://doi.org/10.1021/acs.chemmater.0c04794 Chem. Mater. 2021, 33, 3139−3154 3143 ZnFe2O4and Zn2+ adsorbed onto a hydroxyapatite-like structure (see Figure 3c,d), a partially distorted phase in which Zn2+ favors the tetrahedral coordination. 43,44 The presence of this tetrahedral molecular geometry phase suggests that a fraction of Zn cations are located out of the inorganic core, probably on the surface of the NPs as zinc oleate that have not yielded decomposition. The higher decomposition temperature of ZnOl seems to be the reason why at the final T of the synthesis (320−330 °C), there are still some zinc centers that have not been completely detached from the oleate ligands (see Figure S6 in the Supporting Information). Since this secondary Zn phase is located at the organic surface coating, it will not affect the magnetic properties of the inorganic ferrite core. The linear combination fits presented in Figures 3c,d and S5 provide the percentage of zinc in the inorganic core (as ZnxFe3−xO4) and on the surface (as a metallo-organic structure with tetrahedral coordination); see Table 2. It becomes apparent that the zinc content determined by ICP-MS reflects the total zinc amount in the NP system (ferrite core + organic surface). Thus, to know the real Zn content in the ferrite lattice, the corresponding percentage (second column in Table 2) must be applied to the total zinc amount (ICP-MS data presented in Table 1). The recalculated ZnxFe3−xO4 compositions are listed in Table 2. These corrected xvalues are in agreement with the slight edge-position variations observed at the Fe K-edge (commented above) and the stoichiometries determined by Mossbauer that will be discussed in the following (and are presented in Figure 2d). 2.2. Magnetic Characterization. 2.2.1. DC Magnetometry. The magnetic field (M(H)) and thermal (M(T)) dependence of the magnetization between 5 and 300 K were obtained in the whole set of samples and are presented in Figures 4 and 5. The main properties of the hysteresis loops (saturation magnetization, Ms, coercive field, Hc, and reduced remanent magnetization, Mr/Ms) are summarized in Table 3. As would be expected, Msvalues at 5 K reflect the increase of the net magnetic moment of the lattice as the Zn content increases due to the tetrahedral Fe3+ substitution. As shown in Table 3,Msranges from 122 Am2/kg in the Zn-richest sample (for x≈0.25) to 105−108 Am2/kg in the samples with the lowest Zn content (for x≈0.1). Note that these values significantly exceed the saturation magnetization of pure bulk magnetite (98 Am2/kg at 5 K). However, another direct consequence of the Zn content increase is the concomitant decrease of the Curie temperature, originated by the weakening of the superexchange interaction between A and B sublattices. Such a temperature reduction may be on the order of 200 K for a Zn content of 0.4 relative to pure magnetite (∼950 K) and affects strongly the room-temperature magnetization values. 45 This effect can be observed by plotting Msas a function of temperature (Figure S7, Supporting Information). The curve for sample Zn0.25-39 shows a strong thermal dependence in which the ratio Ms(300 K)/Ms(5 K) becomes much smaller (0.75) than for samples Zn0.1-24 (0.89) or Zn0.1-48 (0.9). As a consequence, the room-temperature Ms of ZnxFe3−xO4samples with x> 0.1 can differ little from that of pure bulk magnetite. Conversely, moderate doping levels (x< 0.1) can provide more benefit at RT; e.g., the Msvalues of samples Zn0.1-48, Zn0.1-24, and Zn0.1-34 (96−97 Am2/kg at RT; see Table 3) notably improved when compared with pure magnetite (92 Am2/kg at RT). Additionally, from the hysteresis loops at 5 K shown in Figure 4, it can be stated that the whole set of NPs is basically single magnetic-phase objects whose magnetization reaches saturation at fields smaller than 0.5 T. This is because the curves do not present kinks and/or linear contributions to the total magnetization in the high-field region, which would be expected if paramagnetic and/or different ferro-/ferri-magnetic phases were significant. The shape of the hysteresis loops at 5 K clearly fits with the Stoner−Wohlfart model of uniaxial single domains for all ZnxFe3−xO4samples (except for Zn0.25-39 composed of twinned NPs), as confirmed by simulations of direct hysteresis loops performed with this model (see Model S1 in the Supporting Information). Note that the model predicts a reduced remanence (Mr/Ms) of about 0.5 (Table 3). In brief, it suggests that interparticle interactions play a minor role at 5 K, so that the magnetization process results from the addition of randomly distributed rotations of noninteracting superspins located at each individual particle. Below the Verwey transition (VT) (T< 100 K), the effective magnetic Table 2. Atomic Percentage of Zn as Zinc Ferrite (in the Inorganic Core) and as Zinc Metallo-Organic Complex (on the Organic Surface) Estimated from the Linear Combination Fit of the Zn K-Edge XANES Spectra of ZnxFe3−xO4Samples (Figures 3c,d and S5) a sample Zn in the core (%) Zn on the surface (%) composition in the core Zn0.15-10 64(1) 36(1) Zn0.1Fe2.9O4 Zn0.1-48 66(2) 34(2) Zn0.07Fe2.93O4 Zn0.1-24 59(2) 41(2) Zn0.06Fe2.94O4 Zn0.1-34 57(2) 43(2) Zn0.06Fe2.94O4 Zn0.25-39 79(1) 21(1) Zn0.2Fe2.8O4 a The ZnxFe3−xO4compositions have been obtained by applying the Zn % in the core to the total zinc amount determined by ICP-MS (Table 1). Figure 4. M(H) curves of ZnxFe3−xO4samples at (a) 300 K and (b) 5 K in the low field region. Chemistry of Materials pubs.acs.org/cm Article https://doi.org/10.1021/acs.chemmater.0c04794 Chem. Mater. 2021, 33, 3139−3154 3144 anisotropy (Keff) arises from the competition of the uniaxial magnetocrystalline effect, originating from the monoclinic distortion of the magnetite lattice 46 and the shape anisotropy. In this case, the differences in the value of the coercive field (Hc) (or effective magnetic anisotropy, Keff) seem to be driven by the shape anisotropy contribution, which depends on the particle’s morphology. Note that the Zn content, and therefore its possible impact on monoclinic distortion, should be quite similar in samples Zn0.1-48, Zn0.1-24, and Zn0.1-34. Otherwise, the lower Hcvalue of Zn0.15-10 at 5 K is due to the nonnegligible thermal fluctuation effects in NPs of 10 nm, which are in the SPM regime at RT. The thermal effects are also visible in the rest of the samples at RT given that the hysterical properties (Hcand Mr/Ms) are considerably reduced. This happens when the total anisotropy energy Keffvis comparable to the thermal energy kBTand/or the dipolar interaction energy. M(T) curves obtained upon zero-field cooling and field cooling (ZFC and FC) conditions are presented in Figure 5. The most evident shared feature is the large magnetization step observed in the vicinity of 100 K when the sample is warmed up (ZFC) as well as cooled down (FC) across it. This step is usually the fingerprint of the pure magnetite phase and originates from the Verwey transition (VT). In Figure 5,itis also observed that this transition slightly moves up and down in temperature from sample to sample (86 K in sample Zn0.134 and around 100 K in samples Zn0.1-24, Zn0.1-48, and Zn0.2539; see Table 3). Even in sample Zn0.15-10 composed of smaller NPs (whose blocking Tis around 55 K), a bump between 80 and 100 is observed (marked in Figure 5a). According to the literature, the lowering of the VT point in bulk magnetite is usually considered as a consequence of either Fe deficit (Fe3(1−δ)O4) in undoped magnetite and/or 3d transition-metal substitution of Fe2+ cations (MxFe3−xO4, with M = Zn, Mn, Co, etc.). 47 Besides, the shifting effect also involves the softening of the transition that becomes gradually one of a second order instead of a first order. 48 The point is that values of δ> 0.03 or x=3δ> 0.01 are sufficient to remove the Verwey transition in bulk single crystals, from which it would be expected that in our ZnxFe3−xO4samples, characterized by nominal x≥0.1, the magnetization step should be no longer observed. However, there is clear experimental evidence that the Verwey transition is strongly affected by surface properties, particularly significant at the nanometer scale. 49 In the work of Guigue-Millot et al., the VT shifted toward higher temperatures and did not fit the relation that exists for bulk single crystals. The authors proposed that the number of Fe2+/Fe3+ pairs per formula unit is the driving force that determines the VT in nanometric grains. The number of Fe2+/Fe3+ pairs in our ZnxFe3−xO4samples will be estimated in the following by gathering together the analysis of Figure 5. Zero-field cooling and field cooling (ZFC-FC) curves together with derivatives of ZFC magnetization (black line) of samples: (a) Zn0.1510, (b) Zn0.1-48, (c) Zn0.1-24, (d) Zn0.1-34, and (e) Zn0.25-39. (f) Verwey transition temperature (Tv) versus Fe2+/Fe3+ pairs obtained by the analysis of Mossbauer spectra. Table 3. Summary of Saturation Magnetization (Ms), Coercivity (Hc), and Reduced Remanence (Mr/Ms) Obtained from the Hysteresis Loops at 300 and 5 K and the Verwey Transition T (Tv) sample Msat RT (Am2/kg) Msat 5 K (Am2/kg) Hc(mT) at RT Hc(mT) 5 K Mr/Msat RT Mr/Ms5 K Tv (K) Zn0.15-10 92 (2) 111 (2) 0.6 (1) 33.5 (1) 0.01 (2) 0.47 (2) 80−100 (1) Zn0.1-48 97 (2) 108 (2) 6.7 (1) 61.0 (1) 0.28 (2) 0.44 (2) 103 (1) Zn0.1-24 96 (2) 105 (2) 7.4 (1) 56.7 (1) 0.30 (2) 0.47 (2) 98 (1) Zn0.1-34 97 (2) 106 (2) 3.4 (1) 50.8 (1) 0.19 (2) 0.48 (2) 86 (1) Zn0.25-39 90 (2) 122 (3) 13.1 (1) 44.8 (1) 0.21 (2) 0.25 (2) 101 (1) Chemistry of Materials pubs.acs.org/cm Article https://doi.org/10.1021/acs.chemmater.0c04794 Chem. Mater. 2021, 33, 3139−3154 3145 Mossbauer spectra and the available magnetization data obtained at 5 K. 2.2.2. Mossbauer Spectroscopy. Mossbauer spectroscopy can help determine the number of Fe2+/Fe3+ pairs by computing the relative occupancy of Fe ions in the inequivalent sites characteristic of the spinel lattice of ferrites. This can be easily achieved from room-temperature spectra in samples above the SPM limit (D> 20 nm), i.e., in the whole set of ZnxFe3−xO4samples except in Zn0.15-10. Figure 6 shows the Mossbauer spectra of Zn0.1-48, Zn0.1-24, Zn0.1‑34, and Zn0.25-39 NPs collected at room temperature together with the Mossbauer fitting parameters. For a better comparison, the area of the spectra has been normalized to the Fe content. If the thermal fluctuation effectissufficiently small, Mossbauer spectra of the stoichiometric Fe3O4magnetite results from the superposition of two well-resolved sextets. The sextet with the higher hyperfine field (I) is ∼49 T, and it is associated with Fe3+ ions in tetrahedral sites (A), while the component with a lower hyperfine field (II) (∼46 T) is assigned to Fe2+Fe3+ atoms in the octahedral (B) ones. 50 The electron hoping among the Fe2+ and Fe3+ atoms in the octahedral (B) position is much faster than the resolution time of the Mossbauer spectroscopy, and the hyperfine values of both Fe2+ and Fe3+ cannot be independently determined by this technique. The low hyperfine field sextet is normally considered to be associated with an only component with an Fe2.5+ intermediate valence representing an Fe2+Fe3+ pair of hopped atoms. The relative resonant area ratio among two components is, thus, SI/SII = 0.5, in accordance with the population of both crystallographic positions A and B in a stoichiometric magnetite. Spectra of the studied ZnxFe3−xO4samples have been properly fitted by superposition of two sextets with hyperfine parameters compatible with those expected from a magnetite phase (Figure 6). The spin relaxation superparamagnetic effects due to reduced sizes of the NPs are not manifested in any spectra. The slightly lower hyperfine fields observed in the spectral components of Zn0.1-24, Zn0.1‑34 and Zn0.25-39 NPs can be attributed to their relatively smaller sizes and their topological cuboctahedral shapes with lower core/surface Fe atoms in comparison to the cubic-shaped Zn0.1-48 sample. The SI/SII (Fe) relative resonant area ratio of ZnxFe3−xO4studied NPs can be found in Table 4. Assuming that Zn ions are located in A sites, as inferred from XANES, the normalized atomic SI/SII (at.) ratios differ from the 0.5 value in all of the samples, evidencing the different stoichiometric compositions of the NPs. Fe3+ ions are substituted by Zn2+ ones in A tetrahedral sites and, to conserve the charge neutrality, a conversion of some Fe2+ ions in Fe3+ and/or generation of Fe2+ vacancies in B octahedral position is provoked. Formally, the nonstoichiometric Zn-doped magnetite NPs can be represented by the following formula [ ][ ] δδδ + − + −− + −− + + + ZnFe(FeFe)Fe O xx x x x 2 1 3 A13 2 13 3 52 3 B 4 (1) where δand xsymbolize the vacancies (0 ≤δ≤0.33) and zinc concentration, respectively. Following expression 1, there are 5δ+ 2x unbalanced Fe3+ ions in the B octahedral position that, as suggested by some authors, do not contribute to Fe2+−Fe3+ electronic hopping but instead to a high hyperfine I sextet. 51 This fact must be reflected on the relative resonant area ratio, being projected by eq 2 δδ=++ −− S Sxx/(Fe) (1 5 )/(2 6 2) III (2) Based on eq 2, the number of Fe2+/Fe3+ pairs per formula unit is given by δ=− −px26 2 (3) Figure 6. Mossbauer spectra of the (a) Zn0.1-48, (b) Zn0.1-24, (c) Zn0.1‑34, and (d) Zn0.25-39 NPs collected at room temperature together with hyperfine parameters obtained from the fittings of the spectra. *IS relative to bcc-Fe. Table 4. Summary of Experimental SI/SII,Ms, and μand the Calculated Fe Vacancies (δ), Zn Content (x), Fe2+/Fe3+ Pairs, and Stoichiometry Using Equations 3 and 4for Zn0.148, Zn0.1-24, Zn0.1-34, and Zn0.25-39 Samples sample SI/SII (Fe) μ (μB)δxFe2+/Fe3+ pairs stoichiometry Zn0.1-48 0.52 4.41 ∼0 0.07 1.86 Zn0.07Fe2.93O4 Zn0.1-24 0.75 4.29 0,04 0.06 1.64 Zn0.06Fe2.9O4 Zn0.1-34 1.17 4.33 0,09 0.08 1.3 Zn0.08Fe2.83O4 Zn0.2539 0.43 4.96 ∼0 0.16 1.68 Zn0.16Fe2.84O4 Chemistry of Materials pubs.acs.org/cm Article https://doi.org/10.1021/acs.chemmater.0c04794 Chem. Mater. 2021, 33, 3139−3154 3146 In addition, the net magnetic moment in the framework of eq 1 can be calculated as μ δδ=−+xx(, ) 6 2 4 (4) Note that an increase in the Zn content (x) reinforces the net magnetic moment, while octahedral vacancies (δ) tend to reduce it. In such a context, eqs 2 and 4allow the calculation of xand δfrom experimental SI/SII (Fe) and μvalues (obtained from the Msdata at 5 K; see Table 3). Table 4 summarizes the experimental SI/SII and μ, the obtained δand xvalues, the pair number (p), and the corresponding stoichiometry for each sample. Samples Zn0.1-24 and Zn0.1‑34 present vacancy concentrations of δ= 0.04 and δ= 0.09, respectively. In contrast, Zn0.1-48 and Zn0.25-39 give rise to slightly negative numbers, leading us to conclude that in these samples there is an apparent absence of Fe2+ vacancies, which seems chemically plausible given that Zn0.1-48 and Zn0.25-39 were synthesized using quite larger annealing times (see Table 1). However, it should be noted that Zn0.25-39 NPs present crystal distortions (see Figure 1), so a precise interpretation of SI/SII (Fe) could require a more specific formulation-frame. It is noteworthy to mention that the Verwey transition temperature presents approximately a linear relation with Fe2+/Fe3+ pairs (see Figure 5f), which is in agreement with the hypothesis proposed by Guigue-Millot et al. 49 commented in the previous section. Regarding the xvalues, they are quite compatible with the ones obtained from the XANES study (see Tables 2 and 4), which supports the conclusion drawn previously about being a minor fraction of Zn (out of the ferrite inorganic core) forming part of the organic coating. Additionally, the stoichiometries determined by Mossbauer spectroscopy and listed in Table 4 are highly consistent with the lattice parameters (a) estimated by Rietveld refinement in the foregoing section (see Figure 2c). Given that vacancies generate local electrostatic repulsion among the remaining ions, which in turn induces an increment of the lattice parameter, 52,53 it seems logical to suppose that sample Zn0.1-34 (with a larger number of vacancies, δ= 0.09) presents a larger lattice parameter among the samples with similar Zn contents (Zn0.1-48, Zn0.1-24 and Zn0.1-34). 2.3. Biomedical Potential of ZnxFe3−xO4@PEG NPs. After having performed a comprehensive physicochemical study and a thorough composition determination of the ZnxFe3−xO4NPs, the work will be completed with a detailed discussion about the biomedical potential of the samples. 2.3.1. Viability of ZnxFe3xO4@PEG Formulations on Cells. First, to make the ZnxFe3−xO4samples hydrophilic and colloidally stable in physiological solutions, samples were coated using the PMAO-PEG copolymer (see Section 4 and Table S7 in the Supporting Information) following a previously published protocol that minimizes collective coatings. 29 Sample Zn0.1-24 was functionalized using 10 kDa PEG, and samples Zn0.1-34, Zn0.1-48, and Zn0.25-39, which are composed of larger NPs, were coated using longer PEG molecules (20 kDa) to better counterbalance the dipolar interaction among NPs. Due to the small size of the NPs forming the Zn0.15-10 sample and, thus, its low potential as a magnetothermal actuator, from here on, this sample will no longer be a part of the discussion. The cytotoxicities of Zn0.1-48@PEG, Zn0.1-24@PEG, Zn0.134@PEG, and Zn0.25-39@PEG after 96 hours have been studied. Figure 7 shows that human colorectal cancer cells (HCT116) incubated with ZnxFe3−xO4@PEG NPs grow at the same rate as cells without NPs (white bar), with no significant differences between the two NP concentrations (C1and C2, grey and black bars, respectively). The zinc content (0.05 < x< 0.25), size (24−48 nm), and morphology (cuboctahedral, cubic, or prismatic) of the samples did not affect the viability, concluding that these ZnxFe3−xO4@PEG formulations are not toxic for the cells. 2.3.2. Magnetic Hyperthermia Efficiency of ZnxFe3−xO4@ PEG Formulations. The potential of MNPs to produce heat depends critically on a number of factors, such as intrinsic properties (morphology, size distribution, magnetization, effective magnetic anisotropy, etc.), as well as “extrinsic” ones (collective assemblies, viscosity of the medium, etc.). Additionally, it is well-known that any potentially efficient magnetic colloid can produce poor results if the radiofrequency excitation is far from certain optimal conditions. 54 The hyperthermia study developed in this work takes into account most of these issues, with the aim of analyzing the impact of zinc doping and the NP shape on the performance of magnetite-based NPs. The specific absorption rate (SAR) was extracted from the analysis of the hysteresis loops obtained at three different frequencies (133, 305, and 605 kHz), which are in the range usually employed in the hyperthermia technique. The data are summarized in Figure 8 and Table 5. AC hysteresis loops of samples composed of single crystals (Zn0.1-48@PEG, Zn0.1-24@PEG, Zn0.1-34@PEG), in Figure 8, are similar to those obtained in pure magnetite FM-NPs prepared following a similar synthetic route. 29,55,56 Importantly, these loops are typical of nearly isolated magnetic single domains whose easy axes are oriented at random relative to the externally applied AC magnetic field. In consequence, the Stoner−Wohlfarth-based approach 57 fits reasonably with most of the hysteresis loops presented in Figure 8d(123). The simulations (see Model S1 in the Supporting Information) have been obtained by assuming a Gaussian distribution of the uniaxial effective anisotropy constants, following the same line of thought as that used in the literature 29,55 for comparable magnetite particles. In this approach, the magnetic anisotropy standard deviation is understood as reflecting a morphological disorder, that is to say, irregularities originated by crystallographic directions growing at different rates. Note that hysteresis loop areas of samples Zn0.1-48@PEG and Zn0.134@PEG, which are composed of comparatively large particles, are frequency-independent (as predicted by the model), while in sample Zn0.1-24, with smaller particles of 24 nm, the area Figure 7. Proliferation assay of cells incubated with ZnxFe3−xO4@ PEG NPs for 96 hours using two different concentrations of NPs (C1 = 0.1 ngNP/cell and C2=1ng NP/cell). Growth rates were plotted as relative increase compared to 0 h. Values are represented as the mean and standard error of three independent experiments. Chemistry of Materials pubs.acs.org/cm Article https://doi.org/10.1021/acs.chemmater.0c04794 Chem. Mater. 2021, 33, 3139−3154 3147 (57) Carrey, J.; Mehdaoui, B.; Respaud, M. Simple Models for Dynamic Hysteresis Loop Calculations of Magnetic Single-Domain Nanoparticles: Application to Magnetic Hyperthermia Optimization. J. Appl. Phys. 2011,109, No. 083921. (58) Gandia, D.; Gandarias, L.; Marcano, L.; Orue, I.; Gil-Cartón, D.; Alonso, J.; García-Arribas, A.; Muela, A.; Fdez-Gubieda, M. L. Elucidating the Role of Shape Anisotropy in Faceted Magnetic Nanoparticles Using Biogenic Magnetosomes as a Model. Nanoscale 2020,12, 16081−16090. (59) Atkinson, W. J.; Brezovich, I. A.; Chakraborty, D. P. Usable Frequencies in Hyperthermia with Thermal Seeds. IEEE Trans. Biomed. Eng. 1984,31,70−75. (60) Hergt, R.; Dutz, S.; Muller, R.; Zeisberger, M. Magnetic Particle Hyperthermia: Nanoparticle Magnetism and Materials Development for Cancer Therapy. J. Phys.: Condens. Matter 2006,18, S2919− S2934. (61) Kossatz, S.; Ludwig, R.; Dahring, H.; Ettelt, V.; Rimkus, G.; Marciello, M.; Salas, G.; Patel, V.; Teran, F. J.; Hilger, I. High Therapeutic Efficiency of Magnetic Hyperthermia in Xenograft Models Achieved with Moderate Temperature Dosages in the Tumor Area. Pharm. Res. 2014,31, 3274−3288. (62) Mamiya, H. Recent Advances in Understanding Magnetic Nanoparticles in AC Magnetic Fields and Optimal Design for Targeted Hyperthermia. J. Nanomater. 2013, No. 752973. (63) Muela, A.; Munoz, D.; Martín-Rodríguez, R.; Orue, I.; Garaio, E.; Abad Díaz de Cerio, A.; Alonso, J.; García, J. A.; Fdez-Gubieda, M. L. Optimal Parameters for Hyperthermia Treatment Using Biomineralized Magnetite Nanoparticles: Theoretical and Experimental Approach. J. Phys. Chem. C 2016,120, 24437−24448. (64) Ravel, B.; Newville, M. ATHENA and ARTEMIS: Interactive Graphical Data Analysis Using IFEFFIT. Phys. Scr., T 2005,T115, 1007−1010. Chemistry of Materials pubs.acs.org/cm Article https://doi.org/10.1021/acs.chemmater.0c04794 Chem. Mater. 2021, 33, 3139−3154 3154