Aluminium hydroxide stabilised MnFe2O4 and Fe3O4 nanoparticles as dual-modality contrasts agent for MRI and PET imaging

Magnetic nanoparticles (NPs) MnFe2O4 and Fe3O4 were stabilised by depositing an Al(OH)3 layer via a hydrolysis process. The particles displayed excellent colloidal stability in water and a high affinity to [18F]-fluoride and bisphosphonate groups. A high radiolabeling efficiency, 97% for 18F-fluoride and 100% for 64Cu-bisphosphonate conjugate, was achieved by simply incubating NPs with radioactivity solution at room temperature for 5 min. The properties of particles were strongly dependant on the thickness and hardness of the Al(OH)3 layer which could in turn be controlled by the hydrolysis method. The application of these Al(OH)3 coated magnetic NPs in molecular imaging has been further explored. The results demonstrated that these NPs are potential candidates as dual modal probes for MR and PET. In vivo PET imaging showed a slow release of 18F from NPs, but no sign of efflux of 64Cu.


Introduction
Superparamagnetic nanoparticles (NPs) have been intensively investigated due to their potential applications in biosensors [1e3], targeted drug delivery [4e7], MRI [8,9] and localised hyperthermia induction [10,11]. An obstacle to application of these NPs is that they tend to aggregate and form larger secondary particles, in order to minimise their surface energy. Moreover, magnetic NPs are most often synthesised in organic solvents and coated with an organic layer of oleylamine or oleic acid rendering them soluble only in non-polar solvents. On the other hand, medical or bio-applications require colloidal stability and dispersibility in water and biological environments. Many methods have been developed to obtain stable colloids of magnetic NPs, reviewed by Laurent et al. [12]. Amongst them, coating with polyethyleneglycol (PEG) [8] or Dextran [13] has been widely used, as these hydrophilic and biocompatible materials not only provide a steric barrier against aggregation, but also make them hardly recognised by the macrophage-monocytic system [14]. To avoid desorption of the polymeric coating by heating or dilution, one or more functional groups, such as carbonate or phosphonate, are necessary to bind with the NPs. Such polymers, however, involve a complicated multi-step synthesis approach [8,15]. Therefore the use of an inorganic shell material that introduces stability, functionality and water-solubility is desirable.
Herein, we report a simple approach to stabilise magnetic NPs by coating them with an Al(OH) 3 layer. The aluminium hydroxide coating was selected, due to its high affinity with fluoride anions [16] and bisphosphonate groups [17], which allow easy radiolabelling and functionalisation, and its biocompatibility as shown by its application in vaccine adjuvants [18].

Materials and general characterisation
All chemicals were used as purchased without further purification. Deionised water was obtained from an ELGA PureLab Option Q system. Bisphosphonate polyethyleneglycol (BP-PEG) polymers were synthesised in house according to published methods [8]. X-Ray powder diffraction (XRD) measurements were recorded at room temperature on a PANalytical X'Pert PRO diffractometer using Cu-K a1 radiation (l ¼ 1.540598 Å) at 40 kV, 40 mA, a scan speed of 0.02 /s and a step size of 0.026 in 2q, at Nottingham University. X-Ray photoelectron spectra were recorded using a Thermo Fisher ESCALAB 250 X-ray Photoelectron Spectrometer with a hemispherical sector energy analyser at Aston University. Monochromatic Al K a X-ray source was used at excitation energy of 15 kV and an emission current of 6 mA. The analyser pass energy of 20 eV with step size of 0.1 eV was used throughout the experiment. Transmission electron microscope (TEM) images were taken on a Tecnai FEI T20 at Centre for Ultrastructural Imaging, King's College London. Attenuated total reflectance infrared (ATR-IR or IR) spectra were recorded on a Perkin Elmer spectrum 100. Dynamic light scattering (DLS) experiments were carried out on Zetasizer Nano ZS from Malvern Instruments with a measure angle 175 and a 632.8 nm laser. Zeta potential for all samples was measured in neutral aqueous solution with a pH value z7.

Synthesis of MnFe 2 O 4 and Fe 3 O 4
Magnetic NPs were obtained via a method reported previously [19,20]. Typically, 6 mmol 1,2-hexadecanediol was added to a 100 ml flask containing 20 ml phenyl ether, 5 ml oleylamine and 5 ml oleic acid at 120 C, and the resultant solution was kept at this temperature under vacuum for over 30 min to remove water in the solvent. To this light yellow solution, 1 mmol Mn(acac) 2 and 2 mmol Fe(acac) 3 (or 2 mmol Fe(acac) 3 for Fe 3 O 4 ), was added under N 2 , and then temperature was increased to 270 C at a rate of 10 C/min with magnetic stirring. After 30 min, the flask was cooled to room temperature by removal from the hotplate. To precipitate out the NPs, 40 ml ethanol was added. The particles were collected by centrifugation (Jouan CR312, at a speed of 3000 rpm for 30 min) and washed with ethanol/hexane twice.

Synthesis of MnFe 2 O 4 @Al(OH) 3 (1)
MnFe 2 O 4 (80 mg, 0.33 mmol) was dissolved in 30 ml diethyl ether by sonication for 20 min to form a dark brown solution, and then 10 ml of a diethyl ether solution containing AlCl 3 (144 mg, 1 mmol) was added dropwise. The mixture was sonicated for 2 min before the addition of 500 ml water (27.8 mmol). The subsequent addition of 10 ml acetone led to a brown suspension. The product was collected by centrifugation and then dried in a stream of N 2 to remove ether and acetone, and redispersed in water.

Synthesis of Fe 3 O 4 @Al(OH) 3 samples (2e4)
In the case of Fe 3 O 4 @Al(OH) 3 (with a precursor molar ratio of Fe 3 O 4 to AlCl 3 of 1:3) (4), a faster uncontrolled hydrolysis method was used. Fe 3 O 4 (82 mg, 0.33 mmol) was dissolved in 30 ml diethyl ether after sonication for 20 min to form a dark brown solution, and then 10 ml diethyl ether solution containing AlCl 3 (144 mg, 1 mmol) was added dropwise. The mixture was sonicated for 2 min before the addition of 10 ml acetone leading to a brown suspension. The product was collected by centrifugation and then dried with a stream of N 2 to remove ether and acetone, and re-dispersed in water. Corresponding amounts of AlCl 3 were used with the same volume of Et 2 O to obtain Fe 3 O 4 @Al(OH) 3 (1:1) (2) and Fe 3 O 4 @Al(OH) 3 (1:2) (3) samples with various coreeshell ratios.

Filtration of MFe 2 O 4 @Al(OH) 3 (M ¼ Mn or Fe)
The Al(OH) 3 @MFe 2 O 4 solution prepared as described in Section 2.2.2 (200 ml) was diluted with water (1 ml) to form a transparent brown solution, and then transferred to a 1 ml centrifuge tube with a filter inside (NanoSep, cut-off-molecular size, 30 K). Brown NPs were obtained on the filter by centrifugation at 5000 rpm for 20 min.

Preparation of Fe 3 O 4 @Al(OH) 3 -BP-PEG(5K)
Bisphosphonate polyethyleneglycol (prepared as described elsewhere [8]) (5 mg) was added to the aqueous solution of Fe 3 O 4 @Al(OH) 3 (5 ml, ca. 4 mg/ml), followed by a sonication treatment for 10 min. 18 3 in NanoSep with a cutoff size of 30k. After 10 min incubation with continuous shaking at room temperature, labelled NPs were separated by centrifugation at 5000 rpm (Eppendorf centrifuge 5424) for 20 min. The radioactivity of the supernatant and particles (on the filter) was measured separately using a gamma counter. The labelling efficiency was given by the following Equation (1):

Radiolabelling with
Labelling efficiency ð%Þ ¼ Activity of NPs Activity of NPs þ Activity of supernatant Â 100% (1) Triplicate samples of 18 F labelled NPs were separated as described above. The NPs retained on the filter were re-suspended in deionised water in the inner NanoSep tube and then centrifuged at 5000 rpm for 20 min. This step was repeated three times. The percent binding retained after each washing step was calculated using equation (1). The correction for cumulative loss of label for the second and third washing steps was performed as exemplified by the following equation (2): Triplicate samples of labelled NPs were prepared on a NanoSep membrane as described above. The NPs retained in the filtrate were re-suspended in 25% serum in water (v/v), incubated at 37 C for a period of up to 6 h, and then centrifuged at 10,000 rpm (Eppendorf centrifuge 5424) for 30 min. The cumulative binding was calculated using equation (2) as described previously.

Adsorption of non-radioactive 19 F
5 mg NP 1 were dissolved in 5 ml freshly prepared NaF solution with concentrations of 0.01 mmol/L, 0.1 mmol/L, 1 mmol/L and 10 mmol/L. The suspensions of NPs were sonicated with the laboratory sonicator bath for 1 h, and then left overnight. The samples were centrifuged for 30 min at 3000 rpm (Jouan CR312) and 4 ml of supernatant was then withdrawn from each sample. The concentrations of fluoride anions in supernatant and corresponding particle-free NaF solution were measured with an Orion Star 214 bench-top meter with a fluoride combination electrode (from Fisher Scientific). Duplicate samples were prepared for each concentration. Adsorption percentage was obtained by dividing the concentration difference between the supernatant and the initial particle-free solution by the initial concentration. NPs) was placed in a NanoSep tube with omega membrane (molecular weight cutoff, 30 kDa). The tubes were centrifuged at 5000 rpm (Eppendorf centrifuge 5424) for 20 min, and then these NPs were re-dissolved in 450 ml water. 50 ml [ 18 F]sodium fluoride (ca. 5 MBq) was added to these NPs solutions in the NanoSep tubes. After 10 min incubation by continuous shaking at room temperature, the tubes were centrifuged at 5000 rpm for 20 min. As described before, the activities in the filtrate and remaining on NPs (on the filter) were separately measured with a gamma counter, to produce a labelling efficiency for the 1st washed Fe 3 O 4 @Al(OH) 3 samples. To measure the labelling efficiency for 2nd washed NPs, the washing step was repeat twice before incubation with 18 F-fluoride radioactivity.

Radiolabelling of 1 with 64 Cu
1 mg bis(dithiocarbamate) bisphosphonate (DTCBP) [15] was dissolved in 100 mM Na 2 CO 3 buffer (pH 9). 200 ml of the above solution was added to 200 ml 64 CuCl 2 radioactivity (ca. 20 MBq) solution that was buffered to pH 5 with sodium acetate. It is essential to maintain the solution at neutral pH, since Al(OH) 3 is not stable either in acidic or in basic solution. After 5 min, 200 ml 0.5 mg/ml MnFe 2 O 4 @Al(OH) 3 solution containing 0.2 mg/ml PEG-5K was added and the mixture was incubated at room temperature for another 5 min. The radiolabelled NPs were isolated by filter centrifugation at speed of 5000 rpm for 15 min, using a Nanosep with a cutoff size of 30 K. There was no radioactivity observed in the filtrate, and all radioactivity remained on NPs in the filter. The 64 Cu radiolabelled NPs were re-dissolved in 100 ml saline for injection.

T 1 , T 2 and T 2 * relaxivity measurement
MR imaging of all particles was performed with a standard extremity flex coil on a clinical 3T Philips Achieva MRI scanner (Philips Healthcare, Best, The Netherlands). T 1 mapping was obtained by using a 2D sequence that employed two non-selective inversion pulses with inversion times ranging from 20 to 2000 ms, followed by eight segmented readouts for eight individual images [21]. The two imaging trains resulted in a set of 16 images per slice with increasing inversion times The acquired imaging data were transferred to a computer running Matlab and analysed using an in-house Matlab tool to receive the relaxation times T 1 and T 2 for each NP concentration (in terms of [Fe] or [Fe] þ [Mn]). Excel was used to plot the relaxation rates against concentration and the relaxivity (i.e. gradient of linear fit) determined from a least squares fit.

In vivo PET/MR imaging
A 6e7 weeks old female C57 black mouse with a weight of 20e21 g was used.
Animal experiments were carried out at the Nanobiotechnology & In Vivo Imaging Center, Semmelweis University in Hungary, with permission from the local institutional animal ethics committee and in compliance with the relevant European Union and Hungarian regulations. PET/MRI images were recorded on a nanoScan (r) integrated PET/MRI system (Mediso, Budapest, Hungary), in which the MR is a preclinical 1T MRI scanner (M2, Aspect Imaging) with horizontal bore magnet, solenoid coil (diameter of 35 mm) and 450 mT/m gradients. Mice were anaesthetised with isoflurane and placed in prone position on the MRI bed. After the pre-contrast MR scan, 85 ml 18 F-labelled (as described above, Section 2.3) NPs 3 solution in saline containing 0.95 MBq-fluoride radioactivity and ca. 60 mg Fe was injected via the tail vein. PET scanning was started immediately after injection and continued for 120 min. Acquisition took place in 1e5 coincidence mode with 5 ns coincidence window, 400e600 keV energy window, 94.7 mm scan range. A 3D expectation maximisation (3D EM) PET reconstruction algorithm (Mediso Tera-Tomo TM) was applied to produce PET images including corrections for attenuation and scatter, dead time, decay and randoms. After 8 iterations the reconstruction stopped resulting in images with 0.1 mm voxel size and time frames of 8 Â 15 min. MR scanning was performed immediately after PET. The images of the two modalities were fused automatically. was injected intravenously via the tail vein. PET scanning was commenced immediately after injection of NPs using a NanoPET/CT scanner from Mediso, with PET acquisition time 120 min with a coincidence mode 1e5 and energy window 400e 600 keV. CT scans were performed immediately after PET. Adjoint Monte Carlo was used for reconstruction, while the detector model and the number of iterations/ subsets were LOR filter and 5/6, respectively.

Results and discussion
Typically, MnFe 2 O 4 @Al(OH) 3 NPs (1) were obtained by adding a diethyl ether (Et 2 O) solution of AlCl 3 to a Et 2 O solution of MnFe 2 O 4 NPs, at the selected mole ratios, whilst stirring. After 10 min, the black mixture was treated with water (500 ml) to induce controlled hydrolysis and stirred for a further hour. The particles were precipitated out by the addition of 10 ml acetone, and then isolated by centrifugal filtration, washed with ethanol and re-dispersed in water. Fe 3 O 4 @Al(OH) 3 samples with different Fe:Al ratios (2e4) were obtained via a quick hydrolysis process, where no water was added prior to the addition of acetone and AlCl 3 was hydrolysed rapidly when the NPs were dispersed in water, rather than by addition of a small amount of water in Et 2 O. Two weak peaks around 21 in the XRD pattern appeared after coating and were associated with the nordstrandite phase of Al(OH) 3 (Fig. 1a) [22]. The infrared spectrum of all Al(OH) 3 coated samples showed the disappearance of adsorption peaks of CeH at 2845 cm À1 and 2950 cm À1 after coating with Al(OH) 3 , confirming that oleylamine had been removed, and the appearance of three absorption peaks at 842 cm À1 and 1645 cm À1 and a broad band from 3000 to 3500 cm À1 , corresponding respectively to the AleO stretching [23], the deformation vibration of water, and OeH stretching mode (Fig. S1). Nanoparticulate MnFe 2 O 4 is soluble in hexane but insoluble in water due to the organic layer (oleylamine and oleic acid) on the surface. Once coated with Al(OH) 3 , the NPs become soluble in water but insoluble in hexane (Fig. 1b). All these features suggest a coating of Al(OH) 3 replacing the oleylamine on the iron oxide NPs. Transmission electron microscopy (TEM), however, revealed no obvious difference size or morphology before and after coating with Al(OH) 3 (Fig. 1ced, Fig. S2). This could be attributed to the poorly crystalline and low-density nature of shell, indicated by the weak and broad diffraction peak on XRD pattern in Fig. 1a and Fig. S3.
X-Ray photoelectron spectroscopy (XPS) spectrum and inductively coupled plasma mass spectrometry (ICP-MS) both indicated that the content of Al in the shell increased with increasing ratios of AlCl 3 to magnetic NPs (Table S1, Figs. S4 and 5). NPs with insufficient Al(OH) 3 , for example 2 tended to aggregate strongly in water, as indicated by TEM images (Fig. S2) and exhibited large hydrodynamic size (hydrodynamic diameter, D h ) up to 400 nm as measured by dynamic light scattering (DLS) experiments (Table S2). This suggested the important role of Al(OH) 3 in stabilising iron oxide NPs in water by converting the hydrophobic surface of oleylamine-coated Fe 3 O 4 NPs into a hydrophilic surface, as well as offering a highly positive surface potential to protect them from aggregation. DLS experiments confirmed that NPs 3 exhibited a highly positive zeta potential up to þ70 mV, and a small D h of 21 nm, reduced from 43.8 nm for Fe 3 O 4 in hexane (as measured by DLS). These coated NPs were stable in water with no obvious changes in D h for over 12 months.
Another benefit of the Al(OH) 3 coating is its high affinity to fluoride ions and bisphosphonate groups [16,17], which allows a simple and easy approach for radiolabelling with [ 18 F]-fluoride or metallic radionuclides conjugated with bisphosphonate. Indeed, a nearly 100% labelling efficiency (LE) was achieved by simply mixing a solution of NPs 1 with radioactive 64 Cu(DTCBP) 2 solution (Fig. 4a) [15] at room temperature for no more than 5 min, and no radioactivity was observed in the supernatant. Moreover, NPs 1 exhibited a high labelling efficiency (LE) with no-carrier-added [ 18 F]fluoride of up to 97% using as little as 10 mg NPs (Fig. 2). The adsorption of fluoride ions by Al(OH) 3 -coated NPs was further confirmed using a fluoride selective electrode, with cold NaF instead of tracer level radioactive 18 F (Fig. S6). The binding capacity was measured to be up to 44.45 mg (fluoride)/g (NPs) (10 times higher than 4e7 mg/g observed for hydroxyapatite [16,25]). The kinetic stability of 18 F binding to NPs (0.34 mg and 0.68 mg) was investigated in water and in serum. The results demonstrated that over 99.8% 18 F remained on the NPs even after washing with water three times (Fig. 2b). However, the stability appeared to become diminished with a smaller sample of NPs (0.07 mg). This may be simply a result of mechanical losses due to manipulation of the very small sample. Studies on the dynamic stability in human serum indicated that there was a slow release of 18 F from radiolabelled NPs 1 over a period of 4 h, with ca. 40% 18 F remaining on NPs after 4 h incubation and no obvious further release of 18 F-fluoride afterwards. The release of 18 F into serum could be a combination of the dissociation of loosely bonded 18 F from the surface, the substitution by other anions in serum, interaction with proteins in serum via hydrogen bonding or ion pairing, and the dissolution of a labile fraction of the Al(OH) 3 layer.
Interestingly, initial results suggested that Fe 3 O 4 @Al(OH) 3 samples (2e4), prepared by a fast, uncontrolled hydrolysis process, are much less efficient in radiolabelling with 18 F than their analogues 1 prepared by controlled hydrolysis (Fig. S7). Moreover, NPs coated with a thicker Al(OH) 3 layer, for example NPs 3 and 4,  showed a worse LE, less than 10%, but higher colloidal stability. NPs 2 have a thinner shell and correspondingly lower colloidal stability. These phenomena lead to the hypothesis that a quick hydrolysis with large amount of water resulted in an unstable Al(OH) 3 layer on the NPs (2e4) whereas a slow hydrolysis with a small amount of water in Et 2 O led to a stable layer (1). An external unstable Al(OH) 3 layer would be washed into the supernatant during the separation process, resulting in a low value of LE. By monitoring the Al concentration in the supernatant after washing and comparing to the initial solution by ICP-MS, we found that almost half of the aluminium was washed out at the first wash for samples 3 and 4 which were synthesised by the quick hydrolysis process. The aluminium remaining on the NPs after washing was stable since no Al was detected in the supernatant after further washing (Table S3). Correspondingly, these NPs 2e4 displayed a high affinity to [ 18 F]fluoride after washing, of up to 94.9%. Only trace amounts of Al were detected in the supernatant of 1, which suggested a stable layer of Al(OH) 3 consistent with the excellent radiolabelling results above.
As expected, these Al(OH) 3 -coated NPs displayed essentially the magnetic properties of the cores and were active as contrast agents in MR imaging, showing a darkening contrast on the T 2 or T 2 * weighted MR images of solutions of NPs as a result of shortening transverse relaxation time of water molecules (Fig. 3). The transverse relaxivity property (r 2 ) of the NPs strongly depends on the shell thickness, weakening dramatically as the Al(OH) 3 shell thickness increases (3 and 4), consistent with previous reports that relaxivity is proportional to the volume fraction of magnetic materials [26]. Fe 3 O 4 @Al(OH) 3 samples 3 and 4 displayed higher relaxivities (r 1 and r 2 ) after washing off the unstable layer; for example, r 2 was improved from 81.6 to 121.9 mM À1 s À1 for NPs 3, and from 60.5 to 116.6 mM À1 s À1 for NPs 4 at 3T magnetic field (Fig. 3, Table S2). For the samples with a stable layer (1), no obvious improvement was observed on the relaxivity properties after washing.
In vivo PET/CT and PET/MR imaging results showed that both 1 and 3 labelled with [ 18 F]-fluoride exhibited a quick uptake, seen by PET imaging, in the spleen and liver after intravenous injection via tail vein, despite their small hydrodynamic size of 21 nm in saline solution. Accumulation of NPs in the spleen and liver was evident also by MR, in a significant darkening contrast in the corresponding areas on MR images in Fig. S8. The combined images show that the magnetic cores and the radioactivity colocalise in the early period after injection but separate with time. Due to the unstable aluminium hydroxide shell, [ 18 F]-fluoride radioactivity was gradually released from NPs 3 in vivo, resulting in a significant bone uptake increasing with time. Consistent with in vitro studies presented above, 1 NPs showed a better in vivo stability and slower, but still significant, release of [ 18 F]-fluoride radioactivity (Fig. S9). By contrast, intravenously injected free [ 18 F]-fluoride, without NPs, is immediately accumulated in bone and not in liver and spleen. PET/CT imaging a normal mouse with 1-64 Cu(DTCBP) 2 showed a similar biodistribution to that of 18 F radiolabeled NPs (Fig. 4). All intravenously administered NPs were taken up by the spleen and liver within 2 h post-injection, and showed no sign of efflux of radiolabel from these organs, in contrast to the 18 F-labelled particles. By comparison, PET/CT using ionic 64 Cu ( 64 CuCl 2 , Fig. S10) showed uptake dominated by liver and kidneys but not spleen. This confirms that 64 Cu radioactivity attached to NPs via bisphosphonate groups co-localises with the magnetic cores and is not rapidly detached from the NPs. The quick clearance of 1 NPs by the liver and spleen was not unexpected, as the in vivo behaviour is determined not only by their hydrodynamic size but also by surface properties (surface chemistry and potential) [27,28].
Generally, intravenously administered NPs over 100 nm are readily cleared by the reticuloendothelial system (RES) through opsonisation, whilst small particles (10e100 nm) tend to stay in the blood pool longer [27]. Thus, although their hydrodynamic size as measured in saline or in water was sufficiently small, to achieve stealth features, the Al(OH) 3 -coated NPs needed further surface modification to neutralise the surface potential. We found that in this case, polymers with anionic functional groups bound to the NPs via bisphosphonate groups, such as bisphosphonate polyethyleneglycol (BP-PEG), could be used to modulate the surface potential of particles (Fig. S11), and protect them from opsonisation and aggregation in serum.

Conclusions
In summary, we have presented a simple approach to convert hydrophobic iron oxide-based magnetic NPs into hydrophilic particles stabilised by an Al(OH) 3 shell. The features of this system, including high efficiency on 18 F or 64 Cu labelling, excellent colloidal stability, small hydrodynamic size, good transverse relaxivity and controllable surface potential, suggest that materials based on Fe 3 O 4 @Al(OH) 3 have potential applications as bimodal contrast agents in PET/MRI imaging. A slow release of 18 F from NPs was observed in vivo, whereas PET imaging with 64 Cu radiolabelled NPs showed no loss of radioactivity from the initially targeted organs (liver, spleen). The ability to derivatise the surface with radiolabels and bisphosphonate groups suggests applications in molecular imaging. Barriers to in vivo use due to toxicity should be low, because of the established use of Al(OH) 3 as adjuvants in vaccines. The high affinity to bisphosphonate groups for Al(OH) 3 allows us to conjugate these NPs with a range of imaging and therapeutic radionuclides which may be used in conjunction with magnetic imaging and therapy.