Magnetic hyperthermia with ε-Fe2O3 nanoparticles

Magnetic hyperthermia with 3-Fe 2 O 3 nanoparticles† Yuanyu Gu, ab Marie Yoshikiyo, c Asuka Namai, c Debora Bonvin, d Abelardo Martinez, e Rafael Pi˜ nol, b Pedro T´ ellez, f Nuno J. O. Silva, g Fredrik Ahrentorp, h Christer Johansson, h Joaqu´ ın Marco-Brualla, i Raquel MorenoLoshuertos, i Patricio Fern´ andez-Silva, i Yuwen Cui, a Shin-ichi Ohkoshi c and Angel Mill´ an * b Biocompatibility restrictions have limited the use of magnetic nanoparticles for magnetic hyperthermia therapy to iron oxides, namely magnetite (Fe 3 O 4 ) and maghemite (g-Fe 2 O 3 ). However, there is yet another magnetic iron oxide phase that has not been considered so far, in spite of its unique magnetic properties: 3-Fe 2 O 3 . Indeed, whereas Fe 3 O 4 and g-Fe 2 O 3 have a relatively low magnetic coercivity, 3Fe 2 O 3 exhibits a giant coercivity. In this report, the heating power of 3-Fe 2 O 3 nanoparticles in comparison with g-Fe 2 O 3 nanoparticles of similar size (20 nm) was measured in a wide range of field frequencies and amplitudes, in uncoated and polymer-coated samples. It was found that 3-Fe 2 O 3 nanoparticles primarily heat in the low-frequency regime (20–100 kHz) in media whose viscosity is similar to that of cell cytoplasm. In contrast, g-Fe 2 O 3 nanoparticles heat more effectively in the high frequency range (400–900 kHz). Cell culture experiments exhibited no toxicity in a wide range of nanoparticle concentrations and a high internalization rate. In conclusion, the performance of 3-Fe 2 O 3 nanoparticles is slightly inferior to that of g-Fe 2 O 3 nanoparticles in human magnetic hyperthermia applications. However, these 3-Fe 2 O 3 nanoparticles open the way for switchable magnetic heating owing to their distinct response to frequency. Introduction Magnetic heating with magnetic nanoparticles (MNPs) is an elegant method for non-contact heating that has been implemented in industrial 1 and clinical applications. 2 The generation of heat by MNPs in internal parts of the body (mainly tumors) by application of an alternating current (AC) magnetic eld in the radio-frequency-range was proposed as a hyperthermia therapy decades ago, 3 and it is now in clinical practice 4,5 in a limited number of European hospitals. Unfortunately, the low heating power achievable is still a strongly limiting factor in these 6–10 and other similar applications. 11 Few nanoparticle (NP) magnetic materials are trusted by health authorities for in-body use, where only iron oxides, and particularly maghemite (g-Fe 2 O 3 ), are generally accepted for hyperthermia cancer therapy. Although many other materials present advantages in terms of heating power, their safety is not yet guaranteed. Thus, the only viable opportunity to enhance clinical hyperthermia performance has been the optimization of g-Fe 2 O 3 NP structural features, such as crystallinity, size, shape and state of aggregation. 14–16 Another magnetic iron(III) oxide, 3-Fe 2 O 3 , that could open new perspectives in the eld was rst prepared in the lab in 1934 by Forestier and Guiot-Guillain. 17 But its exceptional magnetic properties remained unexplored until 2004 when it was prepared as a pure phase by Jin et al., 18 who showed that the magnetic behaviour of this phase differs drastically from the rest of iron oxides, in that it has a gigantic coercivity. Based on the development of reliable synthesis methods for the fabrication of pure 3-Fe 2 O 3 NPs by a School of Materials Science and Engineering, Nanjing Tech University, 210009, Nanjing, PR China b Instituto de Ciencia de Materiales de Arag´ on, ICMA-CSIC University of Zaragoza, C/ Pedro Cerbuna 10, 50006, Zaragoza, Spain. E-mail: [email protected] c Department of Chemistry, School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan d Powder Technology Laboratory, Institute of Materials, Ecole Polytechnique F´ ed´ erale de Lausanne, 1015 Lausanne, Switzerland e Departamento de Electr´ onica de Potencia, I3A Universidad de Zaragoza, 50018 Zaragoza, Spain f Servicio de Apoyo a la Investigaci´ on, University of Zaragoza, C/ Pedro Cerbuna 10, 50006 Zaragoza, Spain g Departamento de F´ ısica, CICECO-Aveiro Institute of Materials, Universidade de Aveiro, 3810-193 Aveiro, Portugal h RISE Research Institutes of Sweden, 411 33 G¨ oteborg, Sweden i Departamento de Bioqu´ ımica, Biolog´ ıa Molecular y Celular, Instituto de Biocomputaci´ on y F´ ısica de Sistemas Complejos, University of Zaragoza, C/ Pedro Cerbuna 10, 50006 Zaragoza, Spain †Electronic supplementary information (ESI) available. See DOI: 10.1039/d0ra04361c Cite this: RSC Adv., 2020, 10, 28786 Received 15th May 2020 Accepted 27th July 2020 DOI: 10.1039/d0ra04361c rsc.li/rsc-advances 28786 |RSC Adv.,2020,10,28786–28797 This journal is © The Royal Society of Chemistry 2020 RSC Advances PAPER Open Access Article. Published on 04 August 2020. Downloaded on 1/20/2021 11:07:51 AM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online View Journal | View Issue some of the authors of this report, 18–23 we have investigated for the rst time the utility of these NPs in hyperthermia therapy. The thermal power density of MNPs under an applied AC magnetic eld is the energy dissipated in a hysteresis cycle multiplied by the frequency. Depending on the intensity of the applied magnetic eld, H, and the coercivity, H c , of the NPs, the magnetization will revert by rotation of magnetic moment (i.e., N´ eel relaxation 24 ) or by rotation of the entire NP (i.e., Brownian relaxation 25 ). Each type of magnetization reversal has a different response to the frequency. Moreover, the choice of frequencies, f, and eld intensities, Hfor human use are limited owing to safety considerations, and therefore hyperthermia studies should cover a wide range of frequencies. The acceptable eld limits vary with the magnet coil diameter and congurations. Estimates resulting from human subjective impressions using a one-turn coil around the chest with a diameter of 30 cm xed the limit for that conguration at Hf ¼510 8 Am 1 s 1 . 12 Later, a limit of Hf ¼510 9 Am 1 s 1 was established for smaller loops, 13 and a limit of Hf ¼18 10 8 Am 1 s 1 was proposed for gap magnets. 4 The question then arises regarding the best strategy to improve the performance of clinical magnetic hyperthermia: using high frequencies and low elds, or using low frequencies and high elds. In this report we measured and compared the heating power of 3-Fe 2 O 3 and g-Fe 2 O 3 NPs with a similar particle size (20 nm). A wide range of frequencies (20–900 kHz) and eld amplitudes (4–95 kA m 1 ) were used to determine the optimal eld conditions for the application. The measurements were carried out at various NP mobility conditions: in pure water, under cell cytoplasm viscosity and at complete mobility restriction. We also studied the inuence of a particle polymer coating on the heating power. Finally, some conclusions about the utility of 3-Fe 2 O 3 NPs, and the optimal frequency range for clinical hyperthermia therapy were drawn. Experimental Synthesis Block copolymer P4VP-b-P(MPEGA-co-RhodPEGMA-cocarboxylicPEGMA) (poly(4-vinylpyridine)-block-poly(methoxypoly ethylenglycolacrylate-co-Rhodamine polyethylenglycolmethacry late-co-carboxylic polyethylenglycolmethacrylate)), used for the coating of iron oxide nanoparticles, was prepared by atom transfer radical polymerization (ATRP) according to methods described elsewhere. 26 Details on materials and synthesis procedures are given in ESI.†A scheme of the synthesis route is shown in ESI Fig S1.† 3-Fe 2 O 3 nanoparticles were synthesized by partially arranging the ferrihydrite seed sol–gel method. 20 Basically, a precursor of iron oxide hydroxide nanoparticles embedded in silica matrix was prepared by the sol–gel technique; tetraethyl orthosilicate (TEOS) was added to aqueous dispersion of iron oxide hydroxide nanoparticles, to form silica by the hydrolysis process. Then, the precursor was sintered in air to form 3-Fe 2 O 3 nanoparticles embedded in silica matrix. Finally the silica matrix was etched by NaOH treatment and washed with water several times, and the obtained NPs were dispersed in tetramethylammonium aqueous solution using supersonic waves to obtain a stable basic NP suspension. g-Fe 2 O 3 nanoparticles were synthesized following a protocol modied from Bonvin et al. described previously. 27 Briey, gFe 2 O 3 nanoparticles were synthesized by co-precipitation in combination with a hydrothermal treatment performed at 120 C for 15 h. The NPs were coated with the P4VP-b-P(MPEGA-coRhodPEGMA-co-carboxylicPEGMA) copolymer as described previosly. 26 Briey, the uncoated iron oxide nanoparticles are dispersed in slightly acidic medium (pH ¼2) and mixed with a polymer solution at the same pH. At this pH, the P4VP block is hydrophilic, and, as the pH is increased to 7.4, it becomes hydrophobic encapsulating the nanoparticles. Finally, the suspension is ltered through a 0.22 mm membrane lter to obtain the nal ferrouid. The nal iron oxide concentrations in the 3-Fe 2 O 3 and g-Fe 2 O 3 NP suspension samples were 4.0 g(Fe 2 O 3 per l) and 3.86 g(Fe 2 O 3 per l), respectively. Media for hyperthermia experiments emulating cell cytoplasm viscosity consisted on polyethylene glycol (PEG 8000) aqueous solution. Most of reports on cell cytoplasm viscosity indicate values from 1.2 to 1.5 times that of water. 28–32 However, some authors report values as high as 10 fold. 33 Considering that the viscosity of water is 0.89 mPa s, at 25 C, media with a viscosity 1.9 and 10 times that of water have been obtained from 3 wt% and 10 wt% PEG solutions in water 34,35 with a viscosities of 1.68 mPa s and 8.9 mPa s, respectively. NPs dispersions in these media were prepared by dissolving under sonication 30 mg of PEG8000 in 970 mlof3-Fe 2 O 3 NP suspension (7.0 mg(Fe 2 O 3 ) per ml), and by dissolving 100 mg of PEG8000 in 900 ml3-Fe 2 O 3 (c¼7.0 mg ml 1 ). SAR calculations Specic absorption rate (SAR) values of NPs' suspensions were measured on self-designed and self-made equipment. 2 ml of sample and 2 ml of water were placed in the magnet gap, and their corresponding temperatures were measured by two GaAs ber optic temperature sensors (OptoCon), which were inserted into the liquids and connected to a ber optic temperature monitoring system. When the temperatures of the sample and the water were stable, they started to be recorded: (i) 300 s with the eld off, (ii) 30 s with the eld on, and (iii) 300 s with the eld off. The SAR values were extracted from the T(t) curves (Fig. 1), by using the equation: SAR ¼CpðH2OÞ cðFe2O3Þ dðDTÞ dt(1) where C p (H 2 O) is the heat capacity of water, c(Fe 2 O 3 ) is the concentration of iron oxide NPs, DTrepresents the temperature difference between suspension of iron oxide NPs and water reference, and dðDTÞ dtstands for the initial slop of heating curves obtained by tting the experimental measurements to a second order polynomial (see Fig. 1). Transmission electron microscope (TEM) observations were carried in a JEOL 2000-FXII microscope on carbon coated This journal is © The Royal Society of Chemistry 2020 RSC Adv.,2020,10,28786–28797 | 28787 Paper RSC Advances Open Access Article. Published on 04 August 2020. Downloaded on 1/20/2021 11:07:51 AM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online copper grids aer dip coating of the grids in the ferrouid samples. Dynamic light scattering (DLS) measurements were performed in Zetasizer Nano ZS from Malvern Laser. Chemical analysis of iron content in the samples was carried out in by coupled plasma atomic emission spectrometry (ICP-AES). The 2q–qscan X-ray powder diffraction (XRD) measurements were performed using Rigaku Ultima IV with Cu Karadiation (l ¼1.5418 ˚ A). Rietveld analyses were performed using the PDXL program of RIGAKU. Magnetic measurements of samples were carried out on a superconducting quantum interference device (SQUID)-based magnetometer MPMS-XL5 from Quantum Design. AC magnetic susceptibility measurements versus temperature were carried out in a 10 K to 300 K temperature range, and magnetization vs. eld measurements at 5 K and 300 K were carried out in a 500 Oe to 50 000 Oe eld range. AC magnetic susceptibility vs. frequency measurements at 300 K were carried out in a 1 Hz to 500 kHz frequency range, using the DynoMag AC susceptometer (RISE Research Institutes of Sweden). SAR measurements at several amplitudes and frequencies of the magnetic eld were carried out in a homemade magnetic heating source 26 consisting of a signal generator, a high power amplier and a matching transformer connected a RCL circuit. The magnetic eld was produced in between the gap of a ferrite nucleus with Litz wires windings. The eld intensity and frequency during the measurements were varied in the ranges 8–92 kA m 1 and 25 to 100 kHz respectively. The measurements were performed in aqueous suspensions of uncoated and coated 3-Fe 2 O 3 NPs, uncoated and coated g-Fe 2 O 3 NPs and agar– agar gels of these suspensions. Cell experiments For these experiments, MDA-MB468 breast cancer cell line was purchased from Leibniz Institute and grown in DMEM medium (GIBCO) supplemented with 10% FBS (GIBCO) and with penicillin/streptomycin (GIBCO). About 10 000 cells were seeded in 96-well plates 24 hours prior to the treatments with ferrouids. Then, the cells were incubated with TNPs for 24 hours, with different concentrations of 3-Fe 2 O 3 and g-Fe 2 O 3 (0.1, 0.2, 0.5 and 1.0 mg Fe 2 O 3 per ml). As a control, MDA-MB-468 cells were cultured in the absence of NPs and prepared under the same conditions. Cells were collected by trypsinization and NPs incorporation and cytotoxicity were analyzed by ow cytometry in a FACSCalibur ow cytometer (BD Biosciences). NPs cytotoxicity was evaluated by Annexin-V binding assay. Briey, NPs treated cells were stained for 20 min at room temperature in the darkness with Anexin-Dy634, which binds to the phosphatidylserine exposed in the cell surface, in annexin-binding buffer (140 mM NaCl, 2.5 mM CaCl 2 , 10 mM HEPES/NaOH, pH 7.4). Cell suspension was diluted to 200 ml with the corresponding bufferandanalyzedbyow cytometry. In order to evaluate the internalization capacity of the NPS, rhodamine uorescence was measured by cytometry in the same samples. The effect of NPs on the viability and cell growth of cells was evaluated using the MTT reduction assay according to Mosmann et al. 36 Briey, cells were seeded and treated with ferro- uids in the same way as explained above. Aer 24 hours of culture with NPs, the medium is removed and the cells are washed to eliminate NPs in suspension. Then each well was mixed with 10 ml of a MTT dye solution (3-(4,5-dimethylthiazol2-yl)-2,5 diphenyltetrazolium bromide, 5 mg ml 1 in PBS). Intact cells contain mitochondrial dehydrogenases that can reduce the yellowish water-soluble MTT to insoluble purple formazan crystals, while dead cells do not produce this reduction. Aer 2–3 h of incubation, all formed crystals were centrifuged and solubilized in isopropanol. Finally, the absorbance of each well was measured in a microplate reader at 550 nm and compared to that of untreated cells. A reduction in absorbance reveals a reduced number of living cells. Results and discussion Structural characterization of the nanoparticles The 3-Fe 2 O 3 and g-Fe 2 O 3 NPs were characterized by DLS, TEM and XRD (Fig. 2). Rietveld analysis of the XRD pattern indicates a pure 3-Fe 2 O 3 phase (orthorhombic, space group Pna2 1 ) (Fig. 2(a) and (b)). A comparison of the sample pattern with those of a-Fe 2 O 3 and g-Fe 2 O 3 conrms the absence of these phases in the sample (ESI Fig. S2†). The crystallite sizes calculated by the Scherrer formula were 20.5 and 17.4 nm, for the 3Fe 2 O 3 NPs and g-Fe 2 O 3 NPs, respectively. The DLS results (Fig. 2(c) and (d)) indicated that the hydrodynamic diameters, D H , of the uncoated NPs were 18 and 27 nm for 3-Fe 2 O 3 and gFe 2 O 3 NPs, respectively, which increased aer coating to 29 and 36 nm, respectively. The TEM images of the 3-Fe 2 O 3 and g-Fe 2 O 3 iron oxide-copolymer NPs (Fig. 2(e) and (f), respectively) exhibited a mix of rectangular and hexagonal NPs with a mean size D p (standard deviation: SD) of 19.1 (5.3) nm for 3-Fe 2 O 3 NPs and D p (SD) ¼18.3 (7.3) nm g-Fe 2 O 3 NPs, respectively. Histograms of the particle size distributions derived from the TEM Fig. 1 Heating curves of 3-Fe 2 O 3 in aqueous suspension exposed to an AC field (101 kHz, 51 kA m 1 ). The line corresponds to a fitting to a second order polynomial. The y-axis corresponds to the temperature difference between the NPs suspension sample and the control pure water sample placed in the ferrite magnet gap. 28788 |RSC Adv.,2020,10,28786–28797 This journal is © The Royal Society of Chemistry 2020 RSC Advances Paper Open Access Article. Published on 04 August 2020. Downloaded on 1/20/2021 11:07:51 AM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online images of the 3-Fe 2 O 3 and g-Fe 2 O 3 iron oxide-copolymer NPs are shown in Fig. 2(h) and (j), respectively. The crystalline structure of the 3-Fe 2 O 3 and g-Fe 2 O 3 NP samples was also established from electron diffraction (ED) patterns (Fig. 2(g) and (i), respectively). Finally, a detailed structural characterization of these two NP types can be found in Ohkoshi et al. 18–23 in the case Fig. 2 (a) XRD pattern with Rietveld analysis of 3-Fe 2 O 3 NPs. Red dots, black lines, and grey lines are the observed patterns, calculated patterns, and their differences, respectively. Red bars represent the calculated positions of the Bragg reflections of the 3-Fe 2 O 3 phase (orthorhombic, Pna2 1 ). (b) Crystal structure of 3-Fe 2 O 3 . Blue and red balls indicate Fe and O atoms, respectively. (c) Distribution of hydrodynamic diameters, D H ,fromdynamiclight scattering (DLS) measurements of coated and uncoated 3-Fe 2 O 3 NPs and (d) g-Fe 2 O 3 NPs. (e) TEM images of 3-Fe 2 O 3 NPs and (f) g-Fe 2 O 3 NPs. (g) ED patterns of 3-Fe 2 O 3 NPs. (h) Particle size histograms from TEM images of 3-Fe 2 O 3 NPs, in the inset HRTEM image of a single NP. (i) ED patterns of gFe 2 O 3 NPs.(j)Particlesizehistograms from TEM images of g-Fe 2 O 3 NPs.Intheinset,HRTEMimagesofasingleNP. This journal is © The Royal Society of Chemistry 2020 RSC Adv.,2020,10,28786–28797 | 28789 Paper RSC Advances Open Access Article. Published on 04 August 2020. Downloaded on 1/20/2021 11:07:51 AM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online of 3-Fe 2 O 3 NPs, and in Bonvin et al. 14,27 in the case of g-Fe 2 O 3 NPs. Magnetic properties of the nanoparticles The different magnetic characters of the 3-Fe 2 O 3 and g-Fe 2 O 3 NPs were clearly reected in the M(H) measurements at different temperatures. The hysteresis cycles of 3-Fe 2 O 3 NPs suspensions at a temperature well below the freezing point (i.e., 200 K) exhibited a huge coercivity (Fig. 3(a), Table 1). The coercivity was considerable reduced when the temperature approached the water melting point, although still well above the higher eld amplitude used in SAR measurements. On the contrary, the coercivity in g-Fe 2 O 3 NPs hysteresis cycles (Fig. 3(b), Table 1) was consistently below the SAR eld amplitudes (inset in Fig. 3(b)). At 300 K, when the NPs are free to rotate, the coercivity of the suspensions decreased to 0 in both types of NPs. The AC magnetic susceptibility vs. temperature measurements (ESI Fig. S4†) of uncoated 3-Fe 2 O 3 NPs suspension exhibit a peak in both c0(T) and c00(T) between 80 and 120 K. Both of the c0(T) and c00(T) peaks do not exhibit the typical frequency dependence of the N´ eel relaxation process. An anomaly in this temperature range was also observed previously in M¨ ossbauer measurements, and was attributed to a structural transformation and possible spin reorientation effects. 12 Apart from this peak, c00(T) remains zero in the entire 10–260 K temperature range, implying that no N´ eel relaxation processes are present in this range. At around 260 K the liquid begins to thaw, allowing free rotation of the NPs. Simultaneously, the c00 peak increases abruptly in a frequency-dependent manner, which can be attributed to Brownian relaxation. SAR measurements The SAR values of the 3-Fe 2 O 3 and g-Fe 2 O 3 NPs were measured in a wide range of eld frequencies using two different sets of equipment. Fig. 4(a) and (b) show the variation of SAR value with the safety parameter (Hf) for the 3-Fe 2 O 3 and g-Fe 2 O 3 NPs, respectively. Whereas for g-Fe 2 O 3 NPs all of the SAR values tto a single straight line; those of 3-Fe 2 O 3 NPs were higher at frequencies from 25–61 kHz, being optimal at 45 kHz. It is evident that the heating power is very low at the safety limits proposed for wide coils (i.e.,Hf ¼510 8 Am 1 s 1 ) 12 for both types of NPs herein. Extrapolating to the limit proposed for small coils (i.e.,Hf ¼510 9 Am 1 s 1 ) 13 the SAR of 3-Fe 2 O 3 NPs was 80 W g 1 Fe 2 O 3 (at 75 kHz), and therefore inferior to the value obtained for g-Fe 2 O 3 NPs (160 W g 1 Fe 2 O 3 ). In the highfrequency regime the different magnetic characters of the two types of NPs were evident (Fig. 4(c) and (d)). Within the safety limit, the performance of both types of NPs diminished with respect to that observed in the low-frequency regime. However, the diminished performance was especially acute for 3-Fe 2 O 3 NPs, whose SAR values dropped one order of magnitude. This performance disparity caused the difference in SAR value between the two phases to be about 15-fold. Conversely, although the highest absolute value of SAR in both cases was obtained at f¼710 kHz, the best performance in terms of the safety parameter was obtained at the lowest frequency (419 kHz). It should be noted that most of the experimental points in the high-frequency region lay outside the health safety limit adopted in this report (i.e.,Hf ¼510 9 Am 1 s 1 ). An interesting fact derived from these experiments is that, in the case of a suspension containing a mixture of 3-Fe 2 O 3 and g-Fe 2 O 3 NPs, a change from low to high frequency could switch offthe heating via the 3-Fe 2 O 3 NPs while increasing that of the g-Fe 2 O 3 NPs. Such a switchable system could be useful, for instance, in catalytic cascade reactions. Fig. 4(e) and (f) show the variation of SAR with the eld amplitude, H,ataxed f,atdifferent mobility conditions for the 3-Fe 2 O 3 and g-Fe 2 O 3 NPs, respectively. During complete immobilization of the NPs, the SAR value of 3-Fe 2 O 3 NPs falls to 0 in the entire range of eld amplitudes (Fig. 4(e)), indicating no heating whatsoever by Brownian relaxation. This should be expected from the high coercivity of these NPs (Fig. 3 and Table 1) that results in a very high degree of thermally-blocked MNPs. In fact, the characteristic N´ eel relaxation time for 20 nm-diameter 3-Fe 2 O 3 NPs with an anisotropy constant K¼10 5 –10 6 Jm 3 would be around 1000 years (using s 0 ¼10 10 s). 18,20 Nevertheless, in media with a viscosity in the range of the reported cell cytoplasm values, the SAR values of the 3-Fe 2 O 3 NPs were similar to that in water, indicating that 3-Fe 2 O 3 NPs are fully useful for hyperthermia therapy. NP mobility in the interior of cells can also be restrained by membrane binding or cell-mediated aggregation. However, a dramatic fall of SAR has only been observed in NPs with a D H of several hundreds of nm whereas in our case they are around 30 nm. 37,38 On the other hand, it has been reported that pure magnetomechanical forces induced cell apoptosis and tumor reduction, 39 and in this case 3-Fe 2 O 3 NPs could kill cancer cells both by heating and mechanical stress. Experiments on life cells are currently in their way in our lab to sort out this matter. In the case of g-Fe 2 O 3 NPs, the heating was still appreciable aer gelication (Fig. 4(f)), although the SAR values dropped to half those obtained in the liquid state. This indicates, therefore, a contribution from both N´ eel and Brownian relaxation to the heating via g-Fe 2 O 3 NPs. In fact, considering K¼10 4 Jm 3 for gFe 2 O 3 (one order of magnitude lower than the Kof 3-Fe 2 O 3 ), NPs with a size of 16 nm would have a N´ eel relaxation time of 10 7 s, while NPs with a size of 35 nm would have a N´ eel relaxation time of 1000 s. This is indeed in the range of NP sizes of this sample as measured by TEM. The smaller-sized NPs are therefore expected to have no contribution to heating. At intermediate sizes, heating will arise from the N´ eel relaxation; while larger NPs may only dissipate heat by Brownian relaxation. NP polymer coating To be useful in biological applications, MNPs must be endowed with certain biological functionalities; i.e., stability in biological media, hemocompatibility, long blood circulation times or imaging tags. This is realized herein by covering the NPs with adequate biopolymer coatings, which in turn can affect the hyperthermia performance of the NPs. Consequently, we studied the effect of polymer coating on the magnetic heating 28790 |RSC Adv.,2020,10,28786–28797 This journal is © The Royal Society of Chemistry 2020 RSC Advances Paper Open Access Article. Published on 04 August 2020. Downloaded on 1/20/2021 11:07:51 AM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online properties of both types of NPs. The coating polymer used herein was the P4VP-b-P(MPEGA-co-RhodPEGMA-cocarboxylicPEGMA) copolymer functionalized with a uorescent tag (Rhodamine), in anticipation of cellular hyperthermia experiments. Moreover, this polymer possesses carboxylate residues at the ends of some of the polyethylene glycol (PEG) side chains that are suitable for conjugation to antibodies with specic binding properties to targeted body tissues (e.g., cancer tumors). Details on the polymer preparation and coating procedures are given in the ESI.†Aer being coated with the copolymer, both the 3-Fe 2 O 3 and g-Fe 2 O 3 NPs were very stable in water suspensions at the physiological pH (7.4). It is worth noting that this type of copolymer coating has been developed in our lab for the last two decades, and it provides excellent biodistribution capacity 40 and cell compatibility. 41–45 As respectively shown in Fig. 5(a) and (b), the polymer coatings reduced the SAR values of the 3-Fe 2 O 3 and g-Fe 2 O 3 NPs at low frequencies, and especially in the case of g-Fe 2 O 3 NPs. At Fig. 3 Magnetization vs. field of water suspension of (a) uncoated and (c) coated 3-Fe 2 O 3 NP, and (b) uncoated and (d) coated g-Fe 2 O 3 NPs at different temperatures. In the insets, details of the samples magnetization within the range of field amplitudes used in SAR experiments. This journal is © The Royal Society of Chemistry 2020 RSC Adv.,2020,10,28786–28797 | 28791 Paper RSC Advances Open Access Article. Published on 04 August 2020. Downloaded on 1/20/2021 11:07:51 AM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online high frequencies, however, the SAR values of the 3-Fe 2 O 3 NPs dropped drastically aer coating (Fig. 5(c)). Specically, the SAR was practically 0 within the Hf safety range. Conversely, the SAR values of the coated g-Fe 2 O 3 NPs showed only a moderate decrease in SAR value (Fig. 5(d)). Frequency dependent AC magnetic susceptibility The power density dissipation assuming linear response theory (i.e., low eld amplitudes) is directly related to the out-of-phase susceptibility (i.e., imaginary part), c00, by the relation SAR(f)¼pm 0 H 2 0 fc00(f)(2) where m 0 is the permeability of free space and H 0 is the eld amplitude. Thus, we measured the AC susceptibility (ACS; low eld amplitude) of 3-Fe 2 O 3 NPs and g-Fe 2 O 3 (uncoated and coated) MNPs as a function of the frequency in a range from 10 Hz to 500 kHz (Fig. 6). The c00 vs. log(f) curves for the uncoated 3-Fe 2 O 3 NPs exhibited a maximum in c00 at 15.8 kHz (i.e., Brownian relaxation frequency). In the case of uncoated gFe 2 O 3 NPs, the c00 maximum appeared at about 8 kHz. Obviously, the power density will diminish when shiing the frequency up from this maximum, but an increase in the number of eld loop cycles (frequency) may compensate for that loss. Thus, the optimal frequency for heating may deviate from this maximum and the heating power will be favored by the frequency. The absolute values of ACS (both the in-phase and out-of-phase components) were larger for the g-Fe 2 O 3 MNPs than the 3-Fe 2 O 3 MNPs. Because the particle sizes were almost identical for both samples, the difference in ACS value is likely owing to differences in the intrinsic saturation magnetization between the two samples. The different values of the out-ofphase component will affect the heating properties. All samples exhibited Brownian relaxation as determined by low- eld ACS analysis. The shiof the c00 peak in the polymer-coated sample with respect to the uncoated one was remarkable in 3-Fe 2 O 3 NPs (from 15.8 to 1.0 kHz). The consequence is a decrease of the SAR value in the coated sample compared with the uncoated one, as observed in Fig. 5, which is particularly noticeable at high frequencies. However, at the lowest experimental frequencies the ACS of the coated sample is maximal while that of the uncoated sample is very low, and then the situation may be reversed. Thus, we could expect an enhancement of hyperthermia performance for coated 3-Fe 2 O 3 samples at frequencies well below the range used in SAR experiments. This large shi in frequency aer coating was not observed in the g-Fe 2 O 3 NPs. From eqn (2) it is possible to estimate SAR from the imaginary part of the AC susceptibility at a given frequency, if the imaginary part of the dynamic magnetization is linear to the applied AC eld (i.e. low eld amplitudes). The low eld range also implies that SAR will almost vary with a quadratic Hf behaviour at a constant frequency when assuming small eld dependence in the imaginary part of the AC susceptibility. From the SAR results shown in Fig. 4(c) and (d) we have low eld amplitudes and the almost quadratic behaviour can be seen, and therefore we can apply eqn (2). If we take the uncoated 3Fe 2 O 3 and g-Fe 2 O 3 particles as an example we get for the cases Hf ¼410 9 Am 1 s 1 and 12 10 9 Am 1 s 1 at f¼542 kHz (that give elds of 7380 A m 1 and 22 000 A m 1 ), we obtain SAR ¼4Wg 1 and 35 W g 1 for 3-Fe 2 O 3 , and 35 W g 1 and 315 W g 1 for g-Fe 2 O 3 as estimated from the ACS response. The corresponding measured SAR values given in Fig. 4(c) and (d) (for Hf ¼410 9 Am 1 s 1 and 12 10 9 Am 1 s 1 at f¼542 kHz) is 4Wg 1 and 33 W g 1 for 3-Fe 2 O 3 and 30 W g 1 and 300 W g 1 for g-Fe 2 O 3 , which is quite in accordance with the SAR estimations from the ACS results. Cell experiments Annexin-V cytotoxicity assays on 3-Fe 2 O 3 and g-Fe 2 O 3 NPs coated with the rhodamine functionalized copolymer revealed that none of the NPs had an appreciable effect on the viability of MDA-MB468 cells in the entire range of concentrations used in the incubation stage (0.1 to 1 mg ml 1 ) (Fig. 7(a)). The measurements were single-point, and therefore the differences between the control and NP seeded samples have no statistical signicance. As shown in Fig. 7(b) and (c), MDA-MB-468 cells present high rhodamine uorescence intensity, indicating a high internalization rate for the 3-Fe 2 O 3 and g-Fe 2 O 3 NPs, respectively. As observed in Fig. 7(c), cells treated with the gFe 2 O 3 complex exhibited higher uorescence intensity than those treated with the 3-Fe 2 O 3 complex. Specically, the Rhodamine median uorescence intensity of g-Fe 2 O 3 was 1.43-, 1.29-, 1.55and 1.65-fold higher than that of 3-Fe 2 O 3 for 0.1, 0.2, 0.5 and 1 mg ml 1 concentrations, respectively), indicating greater incorporation of the g-Fe 2 O 3 complex. Although ferrouids do not seem to induce apoptosis in cells, MTT reduction assay analysis of cell growth inhibition via NPs indicated a similar reduction in the growth rate of cells treated with both complexes (Fig. 7(d)). Further, the cell growth rate reduction was concentration-dependent and reached values of around 40% at 0.5 and 1.0 mg ml 1 concentration. Brownian versus N´ eel heating It is clear herein that MNPs with a high coercivity such as 3Fe 2 O 3 NPs can only heat by the Brownian mechanism. It is also clear that the Brownian and N´ eel mechanisms operate in different frequency ranges. While the Brownian mechanism Table 1 Coercivity Fields, H c , and saturation magnetization, M s ,of3Fe 2 O 3 and g-Fe 2 O 3 NPs suspensions, before and after polymer coating, at different temperatures Phase Coating H c (Oe) Ms (emu per g Fe 2 O 3 ) 200 K 250 K 300 K 200 K 250 K 300 K 3-Fe 2 O 3 No 20 500 1900 0 9 10 10 Yes 20 000 3300 0 10 15 15 g-Fe 2 O 3 No 33 23 0 76 74 74 Yes 36 19 0 72 69 72 28792 |RSC Adv.,2020,10,28786–28797 This journal is © The Royal Society of Chemistry 2020 RSC Advances Paper Open Access Article. Published on 04 August 2020. Downloaded on 1/20/2021 11:07:51 AM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online operates below 100 kHz, the N´ eel mechanism is mostly effective above 400 kHz. Consequently, 3-Fe 2 O 3 NPs can be efficient in magnetic hyperthermia only at low frequencies. However, Brownian NPs can be advantageous when the pursued effect is a mechanical one instead of heating. Indeed, it has been suggested in the literature that cell death might also be caused by Fig. 4 SAR values in the low frequency range of aqueous suspension of (a) 3-Fe 2 O 3 NPs and (b) g-Fe 2 O 3 NPs in as a function of health parameter Hf.SAR values in the high frequency range of aqueous suspensions of (c) 3-Fe 2 O 3 NPs and (d) g-Fe 2 O 3 NPs. In the insets, details of the SAR in the health safety range of Hf. Shaded areas mark the health safety region in a–d. SAR vs. Hf at different NP mobility conditions of (e) 3-Fe 2 O 3 NPs and (f) g-Fe 2 O 3 NPs. This journal is © The Royal Society of Chemistry 2020 RSC Adv.,2020,10,28786–28797 | 28793 Paper RSC Advances Open Access Article. Published on 04 August 2020. Downloaded on 1/20/2021 11:07:51 AM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online mechanical effects. 46–50 Moreover, there is increasing interest in using magnetic forces to activate transport through cell membranes. 51,52 Moreover the strong dependence of magnetic heating on particle mobility can be used as an advantage to heat selectively tumors with a sotexture in a hard healthy tissue environment. It was observed that the working frequency range is shied downwards aer coating the NPs owing to their increased size. However, the large frequencies shiof the c00 peak of 3-Fe 2 O 3 NPs aer coating cannot be fully ascribed to the size increase because the size change is relatively small. Another determinant, at least in part, is the strong interactions of the PEG chains with water molecules and the exibility of these chains. Owing to the frequent use of a PEG coating of NPs for in vivo applications, this subject deserves a deeper insight. The distinct behavior of Brownian and N´ eel NPs can be usefully applied toward switchable NP heating systems. For instance, consider a mixture of 3-Fe 2 O 3 and g-Fe 2 O 3 NPs hosting different substances (i.e., two different catalysts) activated at a distance by the heat generated in the NPs. One could switchoffthe activity of the substance hosted by the 3-Fe 2 O 3 NPs while simultaneously increasing the activity of the substance hosted by the g-Fe 2 O 3 NPs by turning the eld from the low-frequency range to the high-frequency range. This is probably the most important outcome of this report, as it opens a new tool in Fig. 5 SAR values in the low frequency range of aqueous suspension of (a) 3-Fe 2 O 3 polymer coated NPs and (b) g-Fe 2 O 3 polymer coated NPs in as a function of health parameter Hf. SAR values in the high frequency range of aqueous suspensions of (c) 3-Fe 2 O 3 polymer coated NPs and (d) g-Fe 2 O 3 polymer coated NPs. In the insets, details of the SAR in the health safety range of Hf. Shaded areas mark the health safety region in (a–d). 28794 |RSC Adv.,2020,10,28786–28797 This journal is © The Royal Society of Chemistry 2020 RSC Advances Paper Open Access Article. Published on 04 August 2020. Downloaded on 1/20/2021 11:07:51 AM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online