Georgia
Basina
*ab,
George
Diamantopoulos
b,
Eamonn
Devlin
b,
Vassilis
Psycharis
b,
Saeed M.
Alhassan
c,
Michael
Pissas
b,
George
Hadjipanayis
a,
Aphrodite
Tomou
bd,
Alexandros
Bouras
e,
Constantinos
Hadjipanayis
*e and
Vasileios
Tzitzios
*bc
aDepartment of Physics and Astronomy, University of Delaware, Newark, DE 19711, USA. E-mail: g.basina@inn.demokritos.gr
bInstitute of Nanoscience and Nanotechnology, NCSR Demokritos, 15310, Athens, Greece. E-mail: v.tzitzios@inn.demokritos.gr
cDepartment of Chemical Engineering, Khalifa University, P.O. Box 127788, Abu Dhabi, United Arab Emirates
dGoodfellow Cambridge Ltd., Ermine Business Park, Huntingdon PE29 6WR, Cambridge, UK
eBrain Tumor Nanotechnology Laboratory, Department of Neurosurgery, Icahn School of Medicine at Mount Sinai, New York, NY, USA. E-mail: Constantinos.Hadjipanayis@mountsinai.org
First published on 25th April 2022
Magnetic Fe3O4 nanoparticles “decorated” by LAPONITE® nanodisks have been materialized utilizing the Schikorr reaction following a facile approach and tested as mediators of heat for localized magnetic hyperthermia (MH) and as magnetic resonance imaging (MRI) agents. The synthetic protocol involves the interaction between two layered inorganic compounds, ferrous hydroxide, Fe(OH)2, and the synthetic smectite LAPONITE® clay Na0.7+[(Si8Mg5.5Li0.3)O20(OH)4]0.7−, towards the formation of superparamagnetic Fe3O4 nanoparticles, which are well decorated by the diamagnetic clay nanodisks. The latter imparts high negative ζ-potential values (up to −34.1 mV) to the particles, which provide stability against flocculation and precipitation, resulting in stable water dispersions. The obtained LAPONITE®-“decorated” Fe3O4 nanohybrids were characterized by powder X-ray diffraction (XRD), transmission electron microscopy (TEM), Mössbauer spectroscopy, dynamic light scattering (DLS) and vibrating sample magnetometry (VSM) at room temperature, revealing superior magnetic hyperthermia performance with specific absorption rate (SAR) values reaching 540 W gFe−1 (28 kA m−1, 150 kHz) for the hybrid material with a magnetic loading of 50 wt% Fe3O4/LAPONITE®. Toxicity studies were also performed with human glioblastoma (GBM) cells and human foreskin fibroblasts (HFF), which show negligible to no toxicity. Furthermore, T2-weighted MR imaging of rodent brain shows that the LAPONITE®-“decorated” Fe3O4 nanohybrids predominantly affected the transverse T2 relaxation time of tissue water, which resulted in a signal drop on the MRI T2-weighted imaging, allowing for imaging of the magnetic nanoparticles.
Amongst the various magnetic materials, the ferrimagnetic magnetite (Fe3O4) is the most extensively studied material in the field of biomedicine,6,8–12 involving magnetic resonance imaging (MRI),13–18 magnetic hyperthermia,19–24 drug delivery25–28 and biomolecular separation.9,29 Magnetite has been involved in clinical use for many decades, demonstrating its safety, utility and versatility and is among the few, if not the only, nanomaterials that are FDA approved for in vivo applications.30 It also continues to emerge as one of the most powerful nanomaterials for other technological10 and environmental applications,31 including arsenic removal from drinking water in both arsenate and arsenite forms32,33 or other magnetic separation technologies34–37 and catalysis.38–40
In the last few decades, a plethora of works have been published on the development of protocols, following both physical and chemical approaches for the synthesis of magnetite (Fe3O4) nanoparticles.41 The most popular are those involving chemical synthesis in solutions due to their ability to precisely control the size and the shape of the particles at the nanometer scale. These include the traditional co-precipitation, polyol, thermal decomposition and organometallic approaches.10,12,13,17,26,41–44
The application of heat as a theranostic methodology has a long history. It firstly appeared in ancient Greeks’, Romans’, and Egyptians’ findings who utilized heat to treat breast masses.45 Afterwards, magnetic hyperthermia, again, was at the forefront of the research in the 1950's when it was applied for the selective inductive heating of the lymph nodes,46 nevertheless it was only approved for phase II clinical trials in the treatment of human cancer patients in 2011.24 Magnetic hyperthermia studies the rate at which the energy is absorbed per unit mass by a human body when it is exposed to a radio frequency (RF) electromagnetic field. This is achieved by the specific absorption rate (SAR); a quantity that is highly dependent on the applied magnetic field frequency and strength, as well as the type and the concentration of magnetic nanoparticles (MNPs). The use of MNPs has been proven to be extremely efficient in achieving high SAR values. In the case of magnetite, the vast majority of the literature reports that there is an optimal size in the range of 15 to 20 nm for individual Fe3O4 nanoparticles in order to achieve the maximum SAR value.8,21,47 Recently, it has been shown that the formation of nano-assemblies using individual superparamagnetic Fe3O4 nanoparticles as building blocks significantly increases the heating performance.14,22,23,48 It is also worth mentioning that the morphology in terms of particle shape is very influential and can significantly affect hyperthermia efficacy.23,49–51 Fe3O4 nanocubes and nanorods are among the most efficient morphologies for magnetic hyperthermia, revealing SAR values up to 1045 W gFe−1.51 In addition, the heating efficacy depends to a large extent on the frequency and amplitude of the magnetic field. The frequency dependence on the produced power is almost linear in a wide frequency range.52 However, there are limits of human exposure for both, with the recommended frequency proposed to be about 200 kHz or lower.53 Selected data associated with the morphology and size effects combined with hyperthermia conditions (magnetic field and frequency) in relation to the SAR values, and therefore magnetic hyperthermia performance, are summarized in Table S1 in the ESI.†
Fe3O4 hybrid platforms, in terms of surface functionalization and fabrication of composite materials, have been extensively studied for both imaging and therapy biomedical applications.12 Lately, two-dimensional materials and their corresponding magnetic composites have attracted enormous interest due to their unique morphological, electronic, and physicochemical properties.54,55 Fe3O4 graphene oxide hybrids are among the most studied for magnetic hyperthermia, with a high SAR value of 543 W g−1 at 325 kHz and an amplified magnitude field (AMF) value of 16.72 kA m−1,56 as well as drug delivery and imaging.57 Furthermore, layered double hydroxide, (LDH), Fe3O4 nanohybrids were also reported due to their negligible toxicity as excellent candidates for biomedical applications, including drug delivery and therapy.58,59 The LAPONITE®/iron oxide nanocomposites have been studied for bioseparation,60 pH, and thermosensitive drug release,61–63 and T2-weighted MR contrast agents.64,65
In this study, we report a large scale (up to 1 L, 10 mg mL−1) ferrofluid synthesis based on LAPONITE®-“decorated” Fe3O4 hybrid nanomaterials, which demonstrate outstanding stability in water, and superparamagnetic behavior, together with excellent cytotoxicity behavior and magnetic hyperthermia performance at a very low frequency (150 kHz). MR imaging of the LAPONITE®-“decorated” Fe3O4 nanoparticles was performed after convection-enhanced delivery (CED) of the nanoparticles into the rodent brain.
The structural, morphological, and physicochemical characteristics of the obtained nanohybrids with tunable magnetic loadings that varied from 25 to 95 wt% are given in the (ESI†). In particular, the overall properties concerning the material with 50 wt% Fe3O4 composition has been selectively shown in Fig. 1. The crystal structure of the LAPONITE®-“decorated” Fe3O4 nanoparticles was evaluated by combining the powder X-Ray diffraction pattern (XRD) and Mössbauer spectroscopy. The XRD pattern in Fig. 1(a) corresponds well to the FeFe2O4 pattern (JCPDS card no. #19-0629) for bulk magnetite, confirming the high crystalline nature of the material. The crystallite size of pure Fe3O4 nanoparticles estimated by the Scherrer equation on the highest intension diffraction peak (311) is about 19.3 nm. At the low angle area (Fig. 1(b)), the absence of the diffraction at 2θ = 5.73°, which corresponds to (001) crystal planes with a d spacing of 15.402 Å and refers to the layer spacing between the LAPONITE® platelets, indicates their successful exfoliation. However, with a magnification of over a hundred times (Fig. 1(b)/inset), the pattern of the hybrid nanomaterial shows the existence of very broad diffraction peaks at 2-theta degrees of about 6°, which corresponds to the interatomic space of dspacing = 14.578 Å and 12° with dspacing = 7.295 Å, respectively. This periodic pattern, which is obviously negligible, is probably due to the (001) and (002) lamellar LAPONITE® structure. Furthermore, XRD patterns of similar materials with 25, 75, 90, and 95 wt.% Fe3O4 nominal compositions are presented in the ESI† (Fig. S1(I)), which reveal similar structures regarding the iron oxide phase. The broad diffraction peaks, which are due to the LAPONITE® presence, are absent in samples with very high Fe3O4 content, as indicated by the low angle XRD patterns in Fig. S1(II), ESI† and are probably due to the composition effects and/or full exfoliation.
The 57Fe Mössbauer spectrum of the frozen solution (colloid) of 50 wt% Fe3O4/LAPONITE® nanohybrid obtained at 130 K is given in Fig. 1(c). The spectrum shows two components with distinct peaks (sextets) and is very similar to the characteristic Mössbauer spectrum of pure magnetite with an inversed spinel structure. The outer sextet (pink in color) corresponds to the A-sites of Fe3+ ions in tetrahedral sites and the inner sextet (violet in color) correlates with the B-sites containing both Fe2+ and Fe3+ ions in octahedral sites. In the 57Fe Mössbauer spectrum of Fig. 1(c), the fitted isomer shift for the A-sites was found to be equal to 0.33 mm s−1, while that of the octahedral B-sites was 0.75 mm s−1, the latter being an intermediate value resulting in electron hopping between the ferrous and ferric cations. The corresponding average hyperfine field, Bhf, was 495 KG for the A-sites and 482 KG for the B-sites. The latter value, combined with the characteristic isomer shift associated with the presence of divalent iron (Fe2+) in the B-sites, matches the expected values for magnetite. In the case of pure magnetite, a relative A:
B sextet area ratio of 1
:
2 is expected, reflecting the A
:
B site occupancy. For the 57Fe Mössbauer spectrum, the fitted A
:
B area ratio obtained is 43
:
57. Assuming that the B component (57% of Fe) derives only from the B site, it indicates that an upper limit of 28% of the Fe of the A component can be attributed to magnetite. Thus, we calculate that 15% of the total Fe may be attributed to non-magnetite trivalent Fe oxides. This gives us an estimation of at least 85% of iron to be present in the form of magnetite. The higher A
:
B ratio value of 0.75 (= 43
:
57) compared to 0.50 (= 1/2) for the bulk inversed spinel ferrite, Fe3O4 structure is probably due to the superstoichiometry in oxygen or cationic vacancies.66
The magnetic properties of various Fe3O4/LAPONITE® hybrids were studied by a Vibrating Sample Magnetometer (VSM) at room temperature. The 50 wt% Fe3O4/LAPONITE® hybrid shows superparamagnetic behavior10 with 45.4 emu g−1 magnetization at 2 Tesla, as evidenced from the room temperature magnetic hysteresis loop in Fig. 1(d). Similar magnetic behavior, with the saturation magnetization values ranging from 18 emu g−1 to 62 emu g−1 for various magnetic contents of 25 to 95 wt% Fe3O4/LAPONITE®; appeared in the whole studied Fe3O4/LAPONITE® powders regardless of the hybrid material composition. In particular, the saturation magnetization values of 25, 75, 90, and 95 wt% Fe3O4 contents are 18, 59.1, 61.1, and 62.2 emu g−1, respectively, as indicated by the relative magnetization curve in Fig. S2 (ESI†). Considering the weight percentages of the diamagnetic clay matrix of LAPONITE® RD in each composite hybrid material, the saturation magnetization values, in particular, correspond to 72 emu g−1 (25 wt% Fe3O4), 90.8 emu g−1 (50 wt% Fe3O4), 78.8 emu g−1 (75 wt% Fe3O4), 67.9 emu g−1 (90 wt% Fe3O4), and 65.5 emu g−1 (95 wt% Fe3O4), respectively. The results show that the magnetization maximized in the sample with 50 wt% Fe3O4 content and reached a very high value, which is approaching the saturation magnetization of bulk magnetite (92 emu g−1).67,68
The determination of the physicochemical properties of such materials is presented in Fig. 1(e) and (f) and in the ESI† (Fig. S3 and S4). The hydrodynamic diameter distribution given in Fig. 1(e) reveals a polydispersity with a mean diameter of 202.6 nm for the Fe3O4/LAPONITE® hybrids with 50 wt% content. This value is quite higher than the one corresponding to the pure LAPONITE® (80 nm in distilled water),69 which is probably attributed to the formation of bridges between the LAPONITE® platelets. Furthermore, the electrokinetic ζ-potential at the slipping plane of 50 wt% Fe3O4/LAPONITE®, shown in Fig. 1(f), indicates a high negative ζ-potential value of −31.6 mV, confirming the stability of the hybrids. Generally, ζ-potential values >|30| mV denote adequate electrostatic repulsion to provide colloidal stability.70 By varying the Fe3O4 content, the hydrodynamic mean diameter ranges from 136, 202.6, and 174 nm for the 25, 50, and 75 wt%, respectively, while the hybrid materials with higher Fe3O4 content (90 and 95 wt%) reveal multimodal size distribution as presented in Fig. S3 (ESI†). On the other hand, the ζ-potential values decrease as the Fe3O4 content increases. The corresponding values are −34.1 mV and −31.6 mV for compositions with 25 wt% and 50 wt% Fe3O4 and −22.3, −17.6, and −11.4 mV for those with 75, 90, and 95 wt% Fe3O4/LAPONITE®, as given in Fig. S4 (ESI†). This behavior is expected since the negative surface charge is originating from the nature of the LAPONITE® platelets, which shows that the hybrid materials with even a high magnetic loading up to 75% Fe3O4 content are stable and with excellent stability for the hybrids of 25 and 50 wt.% Fe3O4 composition.
The precise morphology of the nanomaterials was identified using the TEM studies. Representative TEM images at varied magnifications received from the 50 wt% Fe3O4 content hybrid material are selectively presented in Fig. 2, while the TEM images of the remaining compositions (25, 75, 90, and 95 wt.% Fe3O4) are shown in the ESI† (Fig. S5). At the lower Fe3O4 content (25 and 50 wt%), the nanoparticles possess a cuboidal shape, while by increasing the Fe3O4 content, the shape of the particles turns to multicore like (75 and 90 wt%, Fig. S5(d)–(f) and (g)–(i), ESI† respectively) and mostly irregular for the sample with the higher Fe3O4 content (95 wt%, Fig. S5(j) and (k)ESI†). Concerning the size of the cuboidal particles in the lowest Fe3O4 content sample (25 wt% Fe3O4) varying between 10–20 nm, the latter of which is slightly increased for the 50 wt% Fe3O4 content. Further increase in the Fe3O4 content seems to affect the size significantly and for the sample with higher than 75% Fe3O4 content in which the particles size is in the 60–80 nm range. Additional morphological characterization is also supported by atomic force microscopy images, presented in Fig. S6 (ESI†). The images originate from the 50 wt% Fe3O4 hybrid, and as it turns out, the material maintains the ability to create uniform films due to the layered nano-clay counterpart presence.
The temperature profiles plotted as a function of time are presented in Fig. 3(a) and Fig. S7 in the ESI.† The best performance appeared for the case of 50 wt% Fe3O4 content (Fig. 3(b)). The colloidal solution temperature increased from 297.4 to 350.4 K (ΔT = 53 K) after 45 s of field exposure, displaying a rate of 1.18 °C s−1, which is similar to the work of Lartigue et al.71 in which the heating rate was 1.04 °C s−1, under 29 kA m−1 and 520 kHz. To the best of our knowledge, these heating rates are the highest reported in the literature. Furthermore, we have to notice that in the present work, the heating rate is achieved by the use of an almost similar field (28 kA m−1) but at a much lower frequency (150 kHz). As mentioned above, the most efficient sample is the 50 wt% Fe3O4 as shown in Fig. 3(b). Its SAR values under different concentrations ranging from 503 W g−1 (5.5. mg mL−1) to 520 W g−1 (11.5. mg mL−1) maximize and reach 540 W g−1 under concentrated conditions (22 mg mL−1), while the magnetic content affects significantly the SAR values, which are in the range of 100–250 W g−1 from the lower to the higher magnetic content (Fig. 3(b)) and slightly varied with the colloid concentration. The maximization of the SAR values in the 50 wt% regime is connected with stability issues as well as size, morphology, and magnetic property issues.72 In particular, the cuboidal nature of the Fe3O4 particles, which is obvious in the high resolution TEM images (Fig. 2(h) and (i)), together with the high net magnetization value (90.8 emu g−1 at 2 T), which is close to the bulk magnetite properties, and the successful decoration with the diamagnetic clay, which favors the suppression of the interparticle magnetic interactions is well known to enhance the T2-weighted MRI contrast ability69,73 and is probably responsible for the superior magnetic hyperthermia performance.74
Regarding iron oxide nanomaterials, the SAR values presented here are among the highest compared to the literature (Table S1, ESI†) in terms of magnetic field and frequency conditions and are comparable or even better than the iron oxide cubes,50,75 rods,20 tubes, rings/hollow,49 multicores,71 assemblies,76,77 branched,72 and cube assemblies,23 which have been recently reported. Among them, some materials reveal extremely high SAR values, in the order of a few thousand W g−1, but we have to notice, taking into consideration the linear dependence with the frequency52,78 that this is mainly due to the very high frequency up to 700 kHz. In addition, it should be noted that these frequencies are much higher than the recommended frequency for human exposure (lower than 200 kHz).53
Cell toxicity studies (no alternating magnetic field (AMF)) revealed no difference in toxicity in human GBM and fibroblast cell survival and proliferation 48 h after treatment with the LAPONITE®-“decorated” Fe3O4 nanoparticles (Fig. 4) in comparison to the control untreated cells. A large drop in GBM cell survival and proliferation was found in both multiple epidermal growth factor receptor (EGFR) – expressing and therapy-resistant GBM cell lines after application of AMFs and thermotherapy for 10 min at a low frequency (Fig. 5).
![]() | ||
Fig. 4 Cell toxicity (without AFM field) in GBM cells (U87-EGFRvIII) (a) and human fibroblasts (HFF1) (b). The Fe3O4/LAPONITE® hybrid concentration is 3 mg mL−1. |
The LAPONITE®-“decorated” Fe3O4 nanoparticles predominantly affected the transverse T2 relaxation time of tissue water that resulted in a signal drop on MRI T2-weighted imaging, allowing for imaging of the MNPs (Fig. 6). Deposition of the nanoparticles was found in the brain after CED on day 0 that persisted 7 days after CED.
This nontoxic and facile magnetic platform can be additionally an excellent candidate for various practical fields, including ferrofluid technologies and magnetic sorbents.
The reaction was carried out under as much anaerobic conditions as possible, titrating Fe(OH)2 by exfoliated LAPONITE® platelets followed by oxidation according to Schikorr reaction (reaction (1))79,80 in contrast to our previous work69 in which exfoliated LAPONITE® platelets were decorated with Fe(OH)2 followed by dehydration and condensation reactions. Briefly, the procedure is based on the oxidation of Fe(OH)2 by the water protons under anaerobic conditions according to the following reaction.
3Fe(OH)2 → Fe3O4 + H2 + 2H2O | (1) |
During the synthesis process, the formation of Fe(OH)2 was achieved by ammonia precipitation of ferrous ions (Ferrous acetate) in a well degassed (applying a high vacuum) aqueous solution under continuous nitrogen gas bubbling at 60–80 °C. Subsequently, the deep olive-green Fe(OH)2 dispersion was titrated by the dropwise addition of a well exfoliated, 1 wt% LAPONITE® RD water solution when the anionic LAPONITE® species were readily adsorbed on the positively charged surface of Fe(OH)2, resulting in the formation of a layered composite. Finally, the olive-green composite material of layered LAPONITE®/Fe(OH)2 was turned into a crude black hybrid material consisting of LAPONITE® platelets “decorated” by Fe3O4 nanoparticles. It is worth mentioning that the dark olive color is due to the partial oxidation of Fe2+ ions owing to the presence of the low amount of dissolved oxygen, which still remains even after extensive degassing with nitrogen bubbling.
Footnote |
† Electronic supplementary information (ESI) available: Structural, magnetic, and morphological characterization data based on XRD, VSM, TEM analysis of various LAPONITE®-“decorated” Fe3O4 nanoparticle hybrids with 25 to 95 wt% magnetic content. Summarized hydrodynamic diameter and ζ-potential distribution histograms are also provided for the physicochemical characterization analysis of the prepared Fe3O4/LAPONITE® hybrids. AFM images of the selected 50 wt% Fe3O4/LAPONITE® are presented. Finally, the temperature profile as a function of the field exposure time from various concentrations (5.5–22 mg mL−1) of colloidal solutions of 25, 50, and 75 wt% Fe3O4/LAPONITE® hybrids and a literature review on SAR values as a function of the applied field frequency and strength of Fe-oxide based nanomaterials with various sizes and morphologies are given for comparison. See DOI: https://doi.org/10.1039/d2tb00139j |
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