Mesoscale Assemblies of Iron Oxide Nanocubes ... - ACS Publications

Alessandra QuartaMarina RodioMarco CassaniGiuseppe GigliTeresa PellegrinoLoretta L. del Mercato. ACS Applied Materials & Interfaces 2017 9 (40), 35095...
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Mesoscale Assemblies of Iron Oxide Nanocubes as Heat Mediators and Image Contrast Agents Maria Elena Materia,† Pablo Guardia,† Ayyappan Sathya,† Manuel Pernia Leal,† Roberto Marotta,† Riccardo Di Corato,†,‡ and Teresa Pellegrino*,† †

Istituto Italiano di Tecnologia, Via Morego 30, 16163 Genova, Italy National Nanotechnology Laboratory of CNR-NANO, via per Arnesano km 5, 73100 Lecce, Italy

‡

S Supporting Information *

ABSTRACT: Iron oxide nanocubes (IONCs) represent one of the most promising iron-based nanoparticles for both magnetic resonance image (MRI) and magnetically mediated hyperthermia (MMH). Here, we have set a protocol to control the aggregation of magnetically interacting IONCs within a polymeric matrix in a so-called magnetic nanobead (MNB) having mesoscale size (200 nm). By the comparison with individual coated nanocubes, we elucidate the effect of the aggregation on the specific adsorption rates (SAR) and on the T1 and T2 relaxation times. We found that while SAR values decrease as IONCs are aggregated into MNBs but still keeping significant SAR values (200 W/g at 300 kHz), relaxation times show very interesting properties with outstanding values of r2/r1 ratio for the MNBs with respect to single IONCs. heterostructures made of different magnetic materials.11 The manipulation of MNPs and their aggregation into controlled clusters could play a significant role on the heating performance of nanoparticles. The relation between the heating performance and the aggregation state of MNPs remains an open question. An improvement in the heating performance while aggregating MNPs has been recently reported.12 Nonetheless, an opposite trend has been observed when increasing the magnetic interaction between nanoparticles by increasing the concentration.13 A similar disagreement can be also found in the reported theoretical studies.14 Moreover, this also depends if superparamagnetic, ferromagnetic or nanoparticles at the interface between superpara- and ferromagnetism are taken into consideration. Such a controversy calls for a direct comparison between a single dispersed and a controlled aggregated system made of the same MNPs, especially if the individual nanoparticles have high SAR values. Indeed, this has been one of the purposes of this work. On the other hand, MNPs are also exploited as contrast agents for MRI diagnosis. Owing to their intrinsic properties, they are able to induce strong magnetic field inhomogeneities, which translate into a higher contrast wherever they accumulate, for instance to the tumor areas.15,16 The MRI signal intensity does solely depend on the relaxation of the net magnetization of a proton under a static magnetic field. Under the influence of an external magnetic field an excited proton can relax along longitudinal (T1 or spin−lattice) or trasversal

1. INTRODUCTION Inorganic nanocrystals provide novel tools to address the challenges of current medicine1 including drug delivery,2 detection,3 imaging,4 and hyperthermia applications.5 The concept of magnetic mediated hyperthermia (MMH) relies on the generation of heat via an oscillating magnetic field exploiting magnetic nanoparticles (MNPs) as heat mediators. Under heat, damages are then induced in cancer cells, these being more sensitive than healthy cells to temperatures higher than 41 °C.6 The combination of MMH with conventional therapies, such as chemotherapy or radiotherapy, has proven to be more effective in the treatment of glyoblastoma, a very malignant brain tumor.7 The heating efficiency of a heat probe is evaluated by its specific absorption rate (SAR) value, which provides the power absorbed per unit mass of magnetic material (W/g) when exposed to an alternating magnetic field (AMF). The SAR value of a given MNP does strongly depend on the size, shape, structure of the nanoparticle as well as the frequency ( f) and the amplitude of the magnetic field (H) applied.5a,8 Some medical constraints are imposed to the physical parameters of the AMF for a safe application of hyperthermia to patients as indeed the product of the frequency and the magnetic field amplitude (Hf) cannot exceed a certain threshold (5 × 109 Am−1s−1).9 Clinical experiments on patients have been carried out without any harm at 110 kHz and 10−20 kAm−1. This issue imposes some limitations to the hyperthermia treatment and at the same time pushes toward the development of more suitable and efficient MNPs under these physical conditions. Outstanding SAR values have been achieved by synthesizing cubic-shaped nanocrystals of iron oxides, like the ones used in this study,10 or by preparing © XXXX American Chemical Society

Received: October 6, 2014 Revised: December 13, 2014

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Langmuir (T2 or spin−spin) directions.17 Superparamagnetic iron oxide nanoparticles are able to shorten the T2 relaxation time (or increase r2), resulting in a reduction in MRI signal intensity (negative contrast).18 The efficiency of a contrast agent does also depend on the surface chemistry, size, shape, and aggregation state of the magnetic nanoparticles. The influence on r2 relaxivity of magnetic clusters made up of spherical and superparamagnetic nanoparticles with different size was studied systematically by several groups.19 Meso- or submicrometer three-dimensional structures of multiple magnetic nanoparticles could be obtained by disparate procedures.20 Spherical nanoclusters of MNPs embedded in polymer matrix, also known as magnetic nanobeads (MNBs), are among the most common clusters that can be produced,20 and their surface can be functionalized toward cancer targeting.20d Also, the easy synthesis allows to include fluorescent nanoparticles or dye molecules within the same bead, making the MNBs promising as multifunctional platforms for biomedical applications.20b,21 Also, the outstanding performances of ferromagnetic IONCs of 30 nm as T2 contrast agent22 were recently reported. However, less is known about the r1 and r2 values of clusters of magnetic nanocubes which show magnetic properties at the transition between superpara- and ferromagnetic behavior. For performing our study, the key was to set a protocol to obtain nanobeads using highly interacting magnetic nanocubes. We did succeed, and we found that the aggregation of IONCs into MNBs of 200 nm in size decrease the SAR values, but still keeping significant heat performances for hyperthermia treatment. The absolute values of r1 and r2 relaxation times decrease for the MNBs; however, the r2/r1 ratios are higher compared with those of IONCs.

Scheme 1. Preparation of Water Soluble IONCs (a) and MNBs (b)a

Starting from the same hydrophobic IONCs (23 ± 3 nm), IONCs are transferred in water by mixing the hydrophobic IONCs in toluene with the gallol-bearing PEG ligand (GA-PEG-OH) in the presence of a base (1a). The solution is shaken for a few seconds, and after acetone addition, the PEG-IONCs are extracted in water (2a). Finally, after organic solvent evaporation at reduced pressure, the PEG-IONCs solution is dialyzed to remove the excess of GA-PEG-OH (3a). This protocol provides the single coated nanocubes in water. The MNBs instead are obtained by mixing the hydrophobic IONCs with a poly(maleic anhydride-alt-1-octadecene) polymer (PC18), in CHCl3 (1b). The solution is shaken for few seconds, and then 1 mL of acetonitrile is added at a flow rate of 2 mL min−1 (2b). The MNBs are collected by magnetic sorting and redissolved in water (3b). a

were injected. The mixture was diluted with 50 mL of toluene, shaken, and transferred in a separating funnel. Then, 250 mL of deionized water was added, resulting in a two-phase mixture that was gently shaken. After the ligand exchange took placed, the IONCs were laid at the toluene−water interface. To drive the nanoparticles into the aqueous phase, 50 mL of acetone was added in order to destabilize the particles from the organic phase and drive them quantitatively into the aqueous phase. After emulsification by means of shaking, the phases were allowed to separate and the aqueous phase containing the IONCs bearing GA-PEG-OH was collected. This step was repeated until all the IONCs were transferred into water, and the organic phase was completely transparent. After concentrating them into a total volume of 10 mL under reduced pressure (300 mbar for 30 min, 200 mbar for 30 min, 77 mbar for 30 min, and 10 mbar for 10 min) at 40 °C, the excess of GA-PEG-OH was removed by dialysis versus deionized water using membrane filters with molecular cutoff point of 50 kDa. The sample was left in dialysis for 2 days at room temperature. Finally, the IONCs solution was concentrated by centrifugation (6.6 g/L of Fe) in a centrifuge filter (molecular cutoff point 100 kDa), and the recovered solution of IONCs was analyzed by DLS and TEM. 2.4. Synthesis of MNBs. The synthesis of MNBs used is based on a previous reported work with substantial modifications.20d Briefly, to prepare MNBs of IONCs, in an 8 mL glass vial, 36.5 μL of IONCs (0.12 μM nanoparticles concentration, d = 23 ± 3 nm) in chloroform was sonicated for few minutes. Soon after, 78.5 μL of chloroform and then 10 μL of a stock solution of poly(maleic anhydride-alt-1octadecene) (PC18) in CHCl3 (50 mM, this concentration refers to the polymer monomer units) were added. The mixture was sonicated again for 1 min and then shaken in an orbital shaker (Multi Reax, Heidolph) at 1250 rpm for 30 s at 20 °C. Subsequently, 1 mL of acetonitrile (ACN) was added at a flow rate of 2 mL min−1. To transfer the MNBs in water, the MNBs were quantitatively collected by keeping them on top of an external magnet (0.3 T) for about 10

2. EXPERIMENTAL SECTION 2.1. Materials. Poly(maleic anhydride-alt-1-octadecene), PC18, Mn 30 000−50 000 (Aldrich), Milli-Q water (18.2 MΩ, filtered with filter pore size 0.22 μM) from Millipore, boric acid (Sigma-Aldrich, 99%), sodium tetraborate decahydrate (Sigma-Aldrich, ≥99.5%), acetonitrile (HPLC grade, J.T. Baker), chloroform (Sigma-Aldrich, 99%), iron(III) acetylacetonate (Acros Organics, 99%), decanoic acid (Acros Organics, 99%), dibenzyl ether (Acros Organic, 99%), squalane (Alfa Aesar, 98%), diethylene glycol Reagent Plus (Sigma-Aldrich, 99%), and poly(ethylene glycol) Mn 380−420 (Sigma-Aldrich). All chemicals were used without any further purification. 2.2. Synthesis of Iron Oxide Nanocubes (IONCs) and Magnetic Nanobeads (MNBs). Synthesis of 23 ± 3 nm IONCs. IONCs were synthesized by using a previous reported procedure.23 Briefly, in a 50 mL three-neck flask 0.353 g (1 mmol) of iron(III) acetylacetonate with 0.69 g (4 mmol) of decanoic acid and 18 mL of dibenzyl ether were dissolved in 7 mL of squalane. After degassing for 120 min at 65 °C, the mixture was heated up to 200 °C (3 °C/min) and kept at this value for 2.5 h. Finally, the temperature was increased at a heating rate of 7 °C/min up to 310 °C or reflux temperature and maintained at this value for 1 h. After cooling down to room temperature, 60 mL of acetone was added, and the whole solution was centrifuged at 8500 rpm. After removing the supernatant, the black precipitate was dispersed in 2−3 mL of chloroform, and the washing procedure was repeated at least two more times. Finally, the collected particles were dispersed in 15 mL of chloroform. 2.3. Transfer in Water of IONCs. To transfer IONCs into water, a ligand exchange procedure was used by using a gallic-functionalized poly(ethylene glycol) (GA-PEG-OH) molecule whose synthesis was previously reported (Scheme 1).23,24 To a 5 mL stock solution of IONCs (concentration 1.9 g/L of Fe in CHCl3), 10 mL of the ligand GA-PEG-OH solution (0.05 M in CHCl3) and 1 mL of triethylamine B

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CryoEM Analysis. Frozen hydrated samples were prepared by dropcasting a 3 μL of sample aliquot on 200-mesh Quantifoil holey carbon grids previously glow discharged (Ted Pella). Before plunging into liquid ethane, the grids were blotted for 1.5 s in a chamber at 4 °C and 90% humidity using a FEI Vitrobot Mark IV (FEI). The nanoparticles were imaged with a 2k × 2k US 1000 Gatan CCD camera using a Tecnai G2 F20 transmission electron microscope (FEI), equipped with a field emission gun operating at an acceleration voltage of 200 kV. Dynamic Light Scattering (DLS) Characterization. Dynamic light scattering measurements were performed on a Zetasizer Nano ZS90 (Malvern) equipped with a 4.0 mW He−Ne laser operating at 633 nm and an avalanche photodiode detector. Measurements were conducted with a ZEN0112-low volume disposable sizing cuvette, setting 1.330 as the refractive index and 0.8869 cP as the viscosity. The measurements were performed with 173° backscatter (NIBS default) as angle of detection, with an automatic scan time and three scans per measurement. SQUID Characterization. Magnetization curves at 5 and 300 K were measured in the range from −70 to +70 kOe by using a superconducting quantum interference device (SQUID) from Quantum Design MPMS. Thermal dependence of the magnetization was also measured in zero field cooling (ZFC) and field cooling (FC) runs by applying a cooling field of Hcooling = 100 Oe and a magnetic field of Hmeas = 100 Oe during the measurement. For the measurements, 150 μL of a solution of IONCs and MNBs was dried on a Teflon film and measured. The mass normalization was done by elemental analysis of the Teflon film.

min. The supernatant was then removed, and the MNBs were dissolved in 500 μL of borate buffer solution (pH 9). To completely solubilize the MNBs, it was necessary to sonicate and warm up (∼40 °C) the sample solution for a few minutes. The MNBs were collected again to the magnet and then redissolved in 500 μL of Milli-Q water. To have enough materials for SAR measurements, the above-reported procedure was repeated 100 times. The MNBs from each batch were recovered to the magnet and redissolved in the same volume of water. The final concentration of MNBs was adjusted to 5.5 g/L of Fe. 2.5. Hyperthermia Measurements on IONCs and MNBs. All the measurements were carried out in a commercially available DM100 Series (nanoScale Biomagnetics Corp.) setup. For example, to evaluate the SAR of the IONCs in water (6.6 g/L of Fe), 100 μL of sample was introduced into a sample holder and exposed under an ac magnetic field at two different frequencies (110 and 300 kHz) under magnetic field amplitudes up to 24 kA m−1. Afterward, 100 μL of a fresh solution of IONCs was added to a 4 mL vial and placed under a magnet until all MNPs were collected at the bottom of the vial. Then the supernatant was removed, and IONCs were redissolved in 100 μL of diethylene glycol (DEG) obtaining concentrations of 6.6 g/L of Fe. The same procedure was performed for poly(ethylene glycol) 400 (PEG400) and the final concentration of IONCs adjusted to 7.4 g/L of Fe. Following the same procedure, three solutions of 100 μL of MNBs dispersed in water (5.5 g/L of Fe), DEG (6.2 g/L of Fe), and PEG400 (5.7 g/L of Fe) were prepared and used for the SAR measurements. All reported SAR values and error bars were calculated from the mean and standard deviation, respectively, of at least four experimental measurements. SAR values were calculated according to the equation

⎛W⎞ C dT SAR ⎜ ⎟ = m dt ⎝g⎠

3. RESULTS AND DISCUSSION Synthesis of IONCs and MNBs. As reported in Scheme 1, the same batch of hydrophobic IONCs (23 nm, edge length) was used for preparing either the clusters (MNBs) or the watersoluble single IONCs. For the preparation of water-soluble IONCs it is required the use of suitable ligands which allow, in a polar solvent, to replace the hydrophobic surfactant molecules at the nanoparticle surface and introduce a thin coating shell that avoids interparticle coalescence. The synthesis of MNBs is a one-step procedure and is based on the clustering of hydrophobic IONCs within a polymer shell with several entangled variables that control the cluster formation (Scheme 1). For the water transfer of hydrophobic IONCs we applied a protocol reported by us, which makes use of catechol derivative PEG molecules.23,24 For the MNBs our well-established protocol for superparamagnetic nanoparticles had to be substantially modified as here we used strongly interacting magnetic nanoparticles at the interphase between superparaand ferrimagnetism. For example, in our previous reports,20d,21,25 particles were always transferred from toluene to tetrahydrofuran (THF) before adding the polymer. This involves a solvent drying step which for the case of IONCs always let to a strong aggregation. Indeed, following this procedure with the nanocubes, MNBs with size at micrometer scale and a broad size distribution were obtained (data not shown). Moreover the IONCs are not well soluble in THF. For the synthesis of smaller clusters, we avoided this solvent evaporation step by directly adding both IONCs and polymer in the same solvent (both IONCs and the polymer are fully soluble in CHCl3). Besides, we also observed that after the addition of the polymer a sonication step improves the formation of MNBs below 200 nm although never smaller than 150 nm with a relative narrow size distribution. The second step involves the destabilization of IONCs and polymer by the addition of a more polar solvent. We chose acetonitrile (ACN) as destabilizing agent as it is fully

where C is the specific heat capacity of the solvent (Cwater = 4185 JL−1K−1, CDEG = 2584 J L−1 K−1, and CPEG400 = 2171 J L−1 K−1) and m is the concentration (g/L of Fe) of magnetic material in solution. Note that the final values are reported as (W/gFe). The measurements were carried out in nonadiabatic conditions; thus, the slope of the curve dT/ dt was measured by taking into account only the first few seconds of the curve. 2.6. Relaxivity Measurements. Water solutions of IONCs and MNBs solution containing different Fe concentration ranging from 0.001 to 2 mM were prepared. The longitudinal (T1) and transverse (T2) relaxation times were measured at 40 °C using a Minispec spectrometer (Bruker, Germany) mq 20 (0.5 T), mq 40 (1 T), and mq 60 (1.5 T). The T1 relaxation profile was obtained using an inversion− recovery sequence, with 20 data points and four acquisitions for each measurement. T2 relaxation time was measured using a Carr−Purcell− Meiboom−Gill (CPMG) spin-echo pulse sequence with 200 data points with interecho time of 0.5 ms. The relaxivities ri (i = 1, 2) were determined by the equation 1 1 = + rC i Fe Ti(obs) Ti(H2O)

(i = 1, 2)

where CFe is the concentration of Fe ions. The values are reproducible within 5% deviation. 2.7. Structural and Elemental Characterization. Elemental Analysis. An inductively coupled plasma atomic emission spectrometer (ICP-AES, iCAP 6500, Thermo) was used for the elemental analysis and concentration evaluation of IONCs and MNBs. The samples were prepared by overnight digestion of 25 μL of nanoparticles solution in 2.5 mL of aqua regia. Subsequently, the sample was diluted with deionized water to a final volume of 25 mL. TEM Characterization. Transmission electron microscopy was carried out on a JEOL JEM-1011 with an acceleration voltage of 100 kV. The sample preparation was conducted by drop-casting a droplet of the sample solution onto a carbon-coated copper grid with subsequent removal of the solvent by evaporation at room temperature. C

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When looking at the TEM images of nanobeads at lower magnification (Figure 1D), a mixture of spherical and anisotropic nanobeads of nanocubes was obtained. Given the restricted window of conditions that can be modified to obtain beads of nanocubes, control over the thickness of the polymer shell, of the shape of the MNBs, and the distribution of nanocubes within the beads is rather poor. With the aim to obtain more anisotropic structures, we also attempted to perform the bead protocol in the presence of a small magnet (0.2−0.1 T) placed beneath the vial during the bead formation. A similar procedure was previously exploited by us to arrange spherical superparamagnetic nanoparticles into submicrometer magnetic rod assemblies.26 Nonetheless, for nanocubes this resulted in a massive precipitation during the bead formation (data not shown). Likely the magnet attracts nanocubes as well as increases the magnetic interaction between them inducing an uncontrolled aggregation with consequent loss of control over the MNBs formation. Nevertheless, on the obtained samples of MNBs and IONCs as in Figure 1 the comparative magnetic studies could be carried out. Hyperthermia Performance of IONCs and MNBs. In Figure 2, the SAR values measured at two different frequencies (300 and 110 kHz) and magnetic field amplitudes (in the range between 12 and 24 kAm−1) reveal that MNBs always have a lower heating performance compared to IONCs for all the tested conditions. For instance, the maximum SAR value was measured at 300 kHz and 24 kAm−1, and for IONCs it was up to 382 ± 4 W/gFe with respect to 193 ± 9 W/gFe for MNBs. The decrease in SAR values might be likely attributed to the magnetic interaction between very close nanocubes within the clusters. This is in close agreement with the results reported by Martinez-Boubeta et al. in which the SAR value decreases while increasing the nanoparticle concentration and thus the magnetic dipole−dipole interaction.13 Our results also fit the SAR values estimated by simulations performed by different other groups,14a,c,d which also predict that the heating performance decreases as the magnetic dipole−dipole interactions increase. However, the comparison of our MNBs to those might be not completely appropriate. Unfortunately, only few studies have measured the SAR values on nanoparticle clusters. Hyeon and co-workers for instance, when setting protocols to solubilize 30 nm IONCs in water, have synthesized small clusters made of IONCs within a shell of dextran.12a Besides the difference in the DLS diameter between our clusters and their samples which have a diameter around 103 ± 15 nm, the dextran clusters have a rather 2D organization (usually 4−10 particles per bead) compare to our clusters which are made of many more nanocubes arranged in a 3D object (20−30 nanocubes per bead). This might account for the difference in the heating performance between our clusters and their clusters. Indeed, in contrast to our data, significant higher SAR values were measured on Hyeon’s clusters. Moreover, in our case we do work with 23 nm cubes which are at the interface between superparamagnetic and ferrimagnetic nanoparticles while the chitosan clusters were made of ferrimagnetic 30 nm nanocubes. On the other hand, the lack of SAR values for single 30 nm cubes does not allow us to compare our results with these single IONCs. In another SAR study based on chitosan cluster of superparamagnetic iron oxide nanoparticles of about 5 and 9 nm in diameter, the authors reported larger heat dissipation for the more interacting nanoparticles assemblies.27 In comparison to our MNBs, nanoparticles appear to be well separated within the clusters.

miscible with CHCl3 and has a higher polarity than CHCl3, a condition necessary for inducing aggregation (in comparison, the standard method uses a single phase of THF/ACN). As a further modification to the MNB procedure, while in the previous method the addition rate of the ACN/THF solution was 0.25 mL min−1, here we had to increase the rate up to 2 mL min−1. We observed that at slow addition rates IONCs tend to aggregate faster which leads to bigger beads with large size distribution (see Figure S1 in the Supporting Information). In general, one can assume that the bead formation is a two-step process in which first the nanoparticle aggregation occurs followed by a stabilization step which consists of polymer enwrapping of the preformed aggregates. The addition of ACN to CHCl3 changes both the solubility of the IONCs and that of the polymer. However, due to stronger magnetic interactions, IONCs tend to aggregate very quickly upon ACN addition. Thus, a fast injection of ACN is required to induce a quick polymer destabilization over the clusters thus avoiding the massive precipitation of the nanocube clusters in macroscopically aggregates. Upon collection of the MNBs to the magnet and subsequent redispersion in water, the hydrolysis of the anhydride groups provides negative charges to the polymer bead surface and hence colloidal stability in water by charge repulsion. Figure 1 shows the TEM and cryoTEM images of

Figure 1. TEM (A, B) and cryoEM images (C, D) of water-soluble IONCs (average size = 23 ± 3 nm) and IONCs based MNBs taken on an air dried sample deposited on the TEM grid (A, B) and on samples in their frozen hydrated state (C, D). DLS hydrodynamic diameters on IONCs and MNBs (E). Inset: average hydrodynamic sizes; the polydispersion index (PdI) is 0.15 and 0.09 respectively for IONCs and MNBs.

the obtained PEG-coated IONCs of 23 ± 3 nm (as measured by TEM) and polymer-coated MNBs of 173 ± 25 nm in size together with their respective DLS hydrodynamic diameters which have low polydispersion indexes (PDIs) (see also Figure 2S for DLS spectra of the MNBs plotted by intensity and volume). Worthily, cryoTEM images (Figures 1C and 1D) recorded on frozen solutions of IONCs or MNBs capture the images of both samples in their hydrated state and just suggest that both nanocubes and beads were individually coated by the polymer layers and appeared as well distinct entities. D

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Figure 2. Measured SAR values as a function of the magnetic field amplitude H for water-soluble IONCs (A) and MNBs (B) at 300 kHz (black empty circles) and 110 kHz (green empty triangles). SAR values as a function of the product Hf for water-soluble IONCs (C) and MNBs (D) at 12 kA m−1 (red rhombi), 16 kA m−1 (green triangles), 20 kA m−1 (blue squares), and 24 kA m−1 (black circles). Each experimental data point was calculated as the mean value of at least three measurements, and error bars indicate the standard deviation. Dashed lines are drawn to guide the eyes. The vertical dashed line defines the biological limit (5 × 109 A m−1 s−1).

Figure 3. SAR values as a function of the magnetic field amplitude H (kAm−1) and the viscosity (mPa·s) at 300 kHz for IONCs (A) and MNBs (B) and at 110 kHz for IONCs (C) and MNBs (D). Each experimental data point was calculated as the mean value of at least three measurements.

it is worthy to keep in mind that our MNBs are pseudospherical aggregates. Anisotropic aggregates could have completely different behavior.14b For example, the heat performance of magnetosomes, a chain-like structure of iron oxide nanoparticles of about 35−50 nm naturally produced by magnetotactic bacteria, are among the best so far reported.28 However, in a magnetosome chain, each magnetite crystal is enwrapped

In addition, the difference in size definitively plays a role on the strength of the magnetic dipole−dipole interaction. It is worthy to mention that simulations on the hyperthermia performance of magnetic nanoparticles and their clusters have shown a similar disagreement.14 We also underline that while our results clearly point out to a decrease in the heating performance as particles get aggregated, E

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Langmuir Table 1. Relaxivities (r1, r2) and r2/r1 of IONCs and MNBs under Different Frequencies 20 MHz

40 MHz

relaxivity (mM−1 s−1)

60 MHz

relaxivity (mM−1 s−1)

relaxivity (mM−1 s−1)

sample

RH (nm)

r1

r2

r2/r1

r1

r2

r2/r1

r1

r2

r2/r1

IONCs MNBs

47.5 175

39.3 2.25

317 162

8 72

24.2 1.3

398 146

16.4 113

7.7 0.65

260.3 130.3

33.8 200

Figure 4. r1, r2, and r2/r1 ratio as a function of the applied magnetic field for individual IONCs (A) and MNBs (B).

within a lipid bilayer which keep the MNPs well separated.29 Since the magnetic dipole−dipole interaction follows an inverse cubic law with the distance, the distance between crystals in magnetosomes might result in a weak magnetic dipole−dipole interaction and has to be taken into account. On top of it, also the shape and colloidal anisotropy contribution of the chain might be responsible for the higher heating performance of magnetosomes. Efforts need to be devoted to clarify this point, but so far direct comparison between chain and single magnetosomes has not been reported, likely because obtaining single magnetosomes is challenging. The SAR values for our IONCs and MNBs were also measured when dissolving the samples in more viscous solvents (DEG and PEG400). As reported in Figure 3, SAR values decrease while the viscosity (η) increases, showing a much more significant effect on the MNBs than on single IONCs. This trend was observed at both frequencies and magnetic field amplitudes. One could recall that the power dissipation during a hyperthermia experiment results from the contribution of two relaxation processes: Brownian relaxation (PR) and hysteresis losses (PH).6b,8a In this regard, for both samples the SAR values decrease while increasing the viscosity which could be attributed to the suppression of the Brownian relaxation process.8a,30 The absolute decrease in SAR values is however higher for MNBs than for the single IONCs underlining a stronger Brownian contribution for the MNBs. Indeed, the Brownian relaxation time is directly proportional to the hydrodynamic volume of the probe and the DLS diameter of the MNB is bigger than that of a single nanocube. When comparing the SARs obtained in solvents at different viscosity (Figure 3 and Figures S5−S10), it could be also seen that in the case of single nanocubes there is no further decrease in the SAR values when the viscosity increases (from DEG to PEG400). This suggests that the Brownian contribution to the SAR is completely suppressed already in DEG being the nanocubes smaller in hydrodynamic diameters. On the contrary, MNBs showed a progressive drop of the heating performance as viscosity increases. This trend points out to a stronger dependence of the heating performance of MNBs with viscosity and hence to a more significant contribution of the Brownian relaxation. This might be explained by a different

heating process compared with IONCs. Indeed, as above reported, aggregation of IONCs might compromise the contribution of hysteresis losses to the heating performance, thus increasing that of Brownian relaxation. Finally, it is also worthy to underline the different magnetic behavior between IONCs and MNBs (Figures S3 and S4). Saturation magnetization values at 5 and 300 K are lower for MNBs, in addition to a lower initial susceptibility at both temperatures for the MNBs with respect to IONCs. These differences support the lower SAR values of MNBs with respect to IONCs. Relaxivity of IONCs and MNBs. In order to achieve high sensitivity in magnetic resonance imaging, it is crucial to shorten the relaxation time of tissue protons by using proper magnetic contrast agents.31 The T1 (spin−lattice) relaxation process is an outcome of the dissipation of an excited proton to its surrounding environment whereas the T2 (spin−spin) relaxation process involves the interaction between the excited nuclei and those with lower energy level. Increasing r2/r1 ratio plays a key role in improving the darker signals or T2-weighted images in MRI (when the r2/r1 is greater than 2, the nanoparticles are better for T2 contrast agent for MRI.).32 Table 1 and Figure 4 show the measured r1, r2, and r2/r1 ratio (see also Figure S11). The absolute r2 values of IONCs under 0.5−1.5 T field are about 398−260 mM−1s−1. The relaxivity values for IONCs are comparable with the literature report on iron oxide nanocubes.22a Similarly, the r2 values of our MNBs exhibit about 161 and 130 mM−1 s−1, which is higher than the reported values of similar size nanoclusters that are made up of spherical superparamagnetic iron oxide particles.33 We ascribe the high r2 value of IONCs compared to MNBs to the higher saturation magnetization of IONCs (Figure S3). Indeed, spin relaxation of proton along the transverse direction (r2) is much faster when the magnetic nanoparticles possess high saturation magnetization. Further, the hydrodynamic size of the single magnetic nanocubes is about 47 ± 15 nm, which indicates the IONCs are in the static diphase region where the r2 values reaches its highest values. Instead, the aggregation of multiple magnetic nanocubes and further polymer enwrapping, results in MNBs with hydrodynamic size of about 173 ± 25 nm with larger size distribution (Figure 1E). Therefore, MNBs falls in the size F

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Langmuir region of echo limiting region where the r2 value decreases with increase in size and size distribution.19d The measured r1 values of MNBs are lower than IONCs in the entire fields. Such decrease in r1 relaxivity may be due to the different coatings: While the hydrophobic ligands of IONCs are exchanged with hydrophilic GA-PEG-OH molecules, MNBs show a thicker polymer shell formed by the hydrophobic surfactants (decanoic acid) and the amphiphilic polymer. This extra hydrophobic layer creates a barrier to the protons; though the water molecules come near to the nanobeads, the compact hydrophobic decanoic acid restricts the interaction between the water molecules and IONCs which limits the spin−lattice relaxation rate and subsequently lowers the r1 of the MNBs.19d,33,34 Nevertheless, both r1 and r2 relaxivities of MNBs are lower compared to individual IONCs, the r2/r1 ratio of MNBs are higher and increases dramatically from 72 to 200 with increase in magnetic field from 0.5 to 1.5 T. These results are in accordance with the work by Qin et al. 35 A similar hydrophobic/hydrophilic coating made of PF127 triblock polymer and the poly(propylene oxide)/oleic acid structure in their case applied on single superparamagnetic iron oxide nanoparticles has been exploited to suppress the r1 values and enhance the r2/r1 value up to 229.35

IONCs and MNBs. This material is available free of charge via the Internet at http://pubs.acs.org.

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Corresponding Author

*E-mail: [email protected] (T.P.). Present Address

M.P.L.: Diagnostic Unit, Andalusiahn Centre for Nanomedicine and Biotechnology, BIONAND, Parque Tecnológico de Andalucia,́ Málaga, Spain. Author Contributions

M.E.M. and P.G. have contributed equally to this manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are grateful to Simone Nitti and Giammarino Pugliese for helping with sample preparation. This work was supported by the European project Magnifyco (Contract NMP4-SL-2009228622) by the EU-ITN network Mag(net)icFun (PITN-GA2012-290248) and by the Italian FIRB projects (Nanostructured oxides, contract no. 588 BAP115AYN).

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4. CONCLUSIONS By precisely controlling the synthesis parameters, strongly interacting IONCs were used as building blocks for the synthesis of MNBs. Control aggregation into MNBs was achieved by adjusting the solvent mixture and the injection rate of the polar solvent (2 mL/min). With respect to the single IONCs, MNBs show a lower hyperthermia performance due to the aggregation and hence to an increase of the magnetic dipole−dipole interactions. The strong decrease of the SAR values when IONCs were aggregated into a bead structure matches with both simulations and experiments reported on magnetic interacting particles. The behavior of the SAR as a function of the viscosity suggests that the heating processes involved during a hyperthermia experiment on MNBs and IONCs are rather different. While energy dissipation in IONCs is manly governed by hysteresis losses, this could be partially suppressed in MNBs giving rise to a significant Brownian contribution to the heating process. For the relaxivity, although individual IONCs show higher r1 (11−18 times) and r2 (double) values than MNBs, our nanobeads exhibit very small r1 which in turn corresponds to high r2/r1 values. These suggest that by making magnetic nanocubes as clusters, r2/r1 ratio can be increased to a very high value, which is a key parameter for an efficient T2 contrast agent. This comparative study suggests that with such kind of nanocubes single IONCs have always better SAR and r2 performance than MNBs although when aggregated still the high r2/r1 values and the measured heat are still exploitable for theranostic applications.

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AUTHOR INFORMATION

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ASSOCIATED CONTENT

S Supporting Information *

TEM images of MNBs samples obtained with two different acetonitrile addition flow rates; DLS analysis by number, intensity, and volume for MNBs; SQUID measurements for IONCs and MNBs; SAR measurements for IONCs and MNBs in water, DEG, and PEG 400; relaxivities measurements for G

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