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Nanomaterials 8 (4): 180 (2018)
Ziemian, Sabina
Löwa, Norbert
Kosch, Olaf
Bajj, Daniel
Wiekhorst, Frank
Schütz, Gunnar
The optimization of iron oxide nanoparticles as tracers for magnetic particle imaging (MPI) alongside the development of data acquisition equipment and image reconstruction techniques is crucial for the required improvements in image resolution and sensitivity of MPI scanners. We present a large-scale water-based synthesis of multicore superparamagnetic iron oxide nanoparticles stabilized with dextran (MC-SPIONs). We also demonstrate the preparation of single core superparamagnetic iron oxide nanoparticles in organic media, subsequently coated with a poly(ethylene glycol) gallic acid polymer and phase transferred to water (SC-SPIONs). Our aim was to obtain long-term stable particles in aqueous media with high MPI performance. We found that the amplitude of the third harmonic measured by magnetic particle spectroscopy (MPS) at 10 mT is 2.3- and 5.8-fold higher than Resovist for the MC-SPIONs and SC-SPIONs, respectively, revealing excellent MPI potential as compared to other reported MPI tracer particle preparations. We show that the reconstructed MPI images of phantoms using optimized multicore and specifically single-core particles are superior to that of commercially available Resovist, which we utilize as a reference standard, as predicted by MPS.
nanomaterialsArticleOptimization of Iron Oxide Tracer Synthesis forMagnetic Particle ImagingSabina Ziemian 1,* ID , Norbert Löwa 2 ID , Olaf Kosch 2, Daniel Bajj 3, Frank Wiekhorst 2and Gunnar Schütz 11 MR and CT Contrast Media Research, Bayer AG, D-13353 Berlin, Germany; gunnar.schuetz@bayer.com2 Physikalisch-Technische Bundesanstalt, D-10587 Berlin, Germany; Norbert.Loewa@ptb.de (N.L.);Olaf.Kosch@ptb.de (O.K.); Frank.Wiekhorst@ptb.de (F.W.)3 University of British Columbia, Vancouver, BC V6T 1Z3, Canada; daniel.bajj@alumni.ubc.ca* Correspondence: sabina.ziemian@bayer.com; Tel.: +49-304-681-94232Received: 23 February 2018; Accepted: 19 March 2018; Published: 21 March 2018Abstract: The optimization of iron oxide nanoparticles as tracers for magnetic particle imaging(MPI) alongside the development of data acquisition equipment and image reconstructiontechniques is crucial for the required improvements in image resolution and sensitivity of MPIscanners. We present a large-scale water-based synthesis of multicore superparamagnetic iron oxidenanoparticles stabilized with dextran (MC-SPIONs). We also demonstrate the preparation of singlecore superparamagnetic iron oxide nanoparticles in organic media, subsequently coated with apoly(ethylene glycol) gallic acid polymer and phase transferred to water (SC-SPIONs). Our aim wasto obtain long-term stable particles in aqueous media with high MPI performance. We found thatthe amplitude of the third harmonic measured by magnetic particle spectroscopy (MPS) at 10 mT is2.3- and 5.8-fold higher than Resovist for the MC-SPIONs and SC-SPIONs, respectively, revealingexcellent MPI potential as compared to other reported MPI tracer particle preparations. We show thatthe reconstructed MPI images of phantoms using optimized multicore and specifically single-coreparticles are superior to that of commercially available Resovist, which we utilize as a referencestandard, as predicted by MPS.Keywords: magnetic particle imaging (MPI); magnetic particle spectroscopy (MPS); iron oxide tracer;superparamagnetic nanoparticles1. IntroductionMagnetic particle imaging (MPI) is a new imaging modality, introduced by Gleich andWeizenecker in 2005, with the potential to enrich modern diagnostic imaging [1]. MPI technology isstill under development [2], and significant challenges remain in streamlining the highly complexinstrumentation as well as optimizing iron-oxide-based MPI tracers. The ideal tracer must haveoptimized physicochemical properties; a high magnetization, consistent morphology, and chemicalstability in biological media are all necessary to generate high-quality three-dimensional images.MPI has been suggested as being suitable for a wide range of applications, such as vasculardiagnostic and interventional procedures, gastrointestinal and pulmonary imaging, as well as cellularand targeted imaging [3–5]. Further uses are envisaged with the development of multicolor MPI,implementing a signal separation acquired simultaneously from different tracer types or from tracersin different environments [6,7]. To date, only research scanners and a few preclinical demonstratorsacquiring one-dimensional (1D), 2D, and 3D images are in use, aiming at establishing the relationbetween the tracer properties, scanner parameters, and the resulting sensitivity and spatial/temporalNanomaterials 2018, 8, 180; doi:10.3390/nano8040180 www.mdpi.com/journal/nanomaterialsNanomaterials 2018, 8, 180 2 of 13resolution of the image [8]. The feasibility of MPI for clinical imaging has yet to be demonstrated in awhole-body clinical scanner [9].The first preclinical in vivo MPI experiments utilized Resovist (Bayer Schering Pharma, Berlin,Germany now available from I’rom Pharmaceutical Co Ltd., Tokyo, Japan), a formulation ofsuperparamagnetic iron oxide nanoparticles and a clinically approved liver-specific magnetic resonanceimaging (MRI) contrast agent. In the MPI community, Resovist has become a reference standard forperformance measurements due to its exceptional signal stability and biocompatibility. The goodMPI signal is explained by the presence of small elementary crystallites of 5 nm, forming compactaggregates of about 25 nm [10,11]. Since only a small fraction of the particles in Resovist contribute tothe signal, further performance improvements of up to 2.5-fold are possible by size fractionation ofthis iron oxide tracer [12,13].Aiming at higher MPI signal improvements, synthesis methods of iron oxide nanoparticleshave been optimized by tailoring the properties of the superparamagnetic iron oxide nanoparticles(SPIONs). The particle size and local environment have an impact on the extent to which Néel andBrownian relaxation contributes to the overall magnetic relaxation, which is a key parameter for spatialresolution [14]. In addition to the iron core diameter and its environment, the relaxation mechanism isdependent on the frequency and the strength of the drive field [15]. Optimal iron oxide core diametermay vary for different drive field settings. The higher the frequency of the excitation field, the lowerthe optimum core diameter [16,17]. Another important parameter for SPIONs in MPI is the anisotropyof particles. A small core anisotropy and coercivity may enhance the performance of the MPI tracer [18].When designing the tracer for use in vivo, care should be taken to optimize the surfactant coating toprevent the aggregation of particles and promote biocompatibility. The coating should be thin enoughto minimize viscous relaxation, but sufficiently thick to prevent aggregation [19].A common approach for obtaining improved MPI tracers is to purposely synthesize multicoreclusters consisting of small iron oxide crystallites. A popular method is coprecipitation from ferrousand ferric chloride in aqueous media under basic conditions, in the presence of a stabilizing agent likedextran. Eberbeck et al. [20] reported on Nanomag-MIP NPs consisting of clustered 5 nm crystallitesshowing the third harmonics employing magnetic particle spectroscopy (MPS) two times better thanResovist. Alternative synthesis routes of multicore magnetic nanoparticles with a variety of coatingpossibilities have been reviewed by Gutiérrez et al. [21]. Nevertheless, the water-based syntheses seemto produce the particles with MPI performance improvements below or comparable to the Resovistseparated by a size fractionation [12].Organic-phase iron oxide synthesis generally allows excellent control over particle shape, size,and morphology, and is typically used to produce single-core nanoparticles. Thermal decomposition ofan iron (III) oleate precursor, reported by Ferguson et al. [22], yielded 26 nm iron oxide nanoparticles.Stability was achieved through coating with poly(maleic anhydride-alt-1-octadecene)-poly(ethyleneglycol) (PMAO–PEG). Such a formulation generated up to three times greater signal intensity thanResovist in MPI [23]. Starmans et al. [24] presented studies on the thermal decomposition of ironoxide hydroxide FeO(OH) resulting in monodisperse 25 nm iron oxide nanoparticles, followed by theencapsulation of magnetic cores in phospholipidic micelles. The particles generated a signal four tosix times higher than Resovist at frequencies below 500 kHz. Published experimental data fluctuatearound 25 nm as a favorable core size for the current MPI applications utilizing an excitation fieldfrequency of 25 kHz.Despite a significant effort on the synthesis, multiple independent research groups working onimproving iron-oxide-based MPI tracers have all converged on improvements in the order of two tosix times better than Resovist.Nature gives us an example of biological magnetosomes consisting of large iron oxide particleswhich fulfill the requirements for high MPS amplitudes, having a high saturation magnetization and alow anisotropy constant. Monocrystalline nanoparticles close to 25.7 nm exhibit the third harmonicswith a seven-fold increase in comparison to Resovist, the highest reported values so far [25].Nanomaterials 2018, 8, 180 3 of 13All the experimental values are still far below the initial calculations based on the Langevintheory, which predicts a cubic improvement of spatial resolution with iron oxide core diameter;hence, calculating a much higher potential for the improvement of the tracer, suggesting a signalincrease even by two orders of magnitude for the core size of 40 nm [1]. The Langevin theory predictsthe MPI signal in relation to the particle core size and does not account for the anisotropy of the particlecore; thus, in practice, the expected MPI signal would differ [18]. More importantly, Tay et al. [26]show that increasing the magnetic relaxation with core size eventually opposes the expected Langevinbehavior, and the high spatial resolution of MPI cannot be further achieved by increasing core sizeabove 25 nm.The motivation behind our studies was the optimization of iron oxide particles for MPI in a cost-and time-efficient manner, utilizing MPS, which is a zero-dimensional MPI scanner, as a screening tool.In this work, we employed a screening technique in both a cheap and simple aqueous synthesis method,as well as a more costly and laborious organic synthesis approach for the preparation of magneticparticles, aiming for a scope of improvement. In the first approach, we optimized large-scale ironoxide water-based synthesis by varying reaction parameters (temperature, stirring speed, flow rate ofprecursor addition, dextran molecular size, and type of coordinating agents to iron), resulting in stabledextran-coated nanoparticles (MC-SPIONs). The second approach first yielded hydrophobic highlymagnetic particles, which have to be functionalized with hydrophilic molecules in order to establish aredispersed aqueous suspension. Here, a biocompatible poly(ethylene glycol) gallic acid (GA-PEG)coating polymer was applied. The particles were subsequently dialyzed in dextran to further enhancetheir stability (SC-SPIONs). For our purpose, MPS could be employed as a facile screening techniquewhich allowed for the quantification of particles’ MPI performance regardless of synthesis conditions,thus avoiding the need for the time-consuming recording of an MPI system function. The excellentperformance of the optimized particles in both synthesis routes was demonstrated in MPI.2. Materials and Methods2.1. MaterialsFerrous chloride tetrahydrate, ferric chloride hexahydrate, sodium hydroxide and formic acidwere purchased from Merck (Merck KGaA, Darmstadt, Germany). Dextran 10 (Dex-10, pharmaceuticalgrade) and dextran 40 were acquired from Pharmacosmos (Pharmacosmos A/S, Holbaek, Denmark)and AppliChem (AppliChem GmbH, Darmstadt, Germany), respectively. Ferric acetylacetonateand zinc acetylacetonate were obtained from Acros Organics (Acros Organics N.V., Geel, Belgium).Oleic acid (Ph. Eur standard, technical grade) and benzyl ether (98%) were obtained fromSigma-Aldrich (Sigma-Aldrich Chemie GmbH, Steinheim) under the Fluka brand. Poly(ethyleneglycol) avg. wt. 10,000 g/mol (PEG 10k), gallic acid (97.5–102.5%), N,N-dimethylformamide (DMF,>99.8%), dicyclohexylcarbodiimide (DCC, 99%), and 4-(dimethylamino)pyridine (DMAP, >99%),chloroform (>99.8%), diethyl ether (anhydrous, >99.7%), and acetone (>99.8%) were all purchasedfrom Sigma-Aldrich. 50 kDa Biotech Grade CE membrane was used from Spectrum Labs (SpectrumLaboratories Inc., Rancho Dominguez, CA, USA). Filter units Millex-HA 0.45 µm and Whatman 0.2 µmwere used. Water was purified by a Milli-Q Gradient machine (Merck KGaA, Darmstadt, Germany)equipped with a 0.16 µm filter. All reagents were used without further purification.2.2. Characterization TechniquesThe hydrodynamic size of the iron oxide nanoparticles was determined utilizing dynamic lightscattering (DLS) with a Malvern Zetasizer Nano Series (Malvern Instruments Ltd., Worcestershire,UK). Crystallite size and morphology were investigated employing JEOL JEM-1011 transmissionelectron microscope (TEM) (JEOL Inc., Peabody, MA, USA) at an acceleration voltage of 80 kV.Qualitative X-ray Diffraction (XRD) analysis was performed facilitating PANalytical EMPYREAN X-raydiffractometer (Malvern Panalytical GmbH, Kassel, Germany) applying Cu Kα radiation (1.54056 Å).Nanomaterials 2018, 8, 180 4 of 13The Fe concentrations were measured by Thermo Scientific ICP-OES iCAP 7600 (Thermo FisherScientific, Waltham, USA) with Y as an internal standard. Zn and Fe concentrations were alsodetermined by Agilent Technologies ICP-MS 7900 (Agilent Technologies Inc., Santa Clara, CA, USA),employing Co as an internal standard. Thermogravimetric analysis (TGA) measurements wereconducted by Mettler TGA/DSC thermal analyzer (Mettler Toledo, Columbus, OH, USA) in thetemperature range 303.15–1273.15 K under oxygen flow of 50 mL/min.The MPS signal was analyzed employing a commercial magnetic particle spectrometer MPS-3,manufactured by Bruker BioSpin (Ettlingen, Germany), operating at a fixed excitation frequency of25.25 kHz and an excitation field up to 25 mT. Aqueous samples at different iron concentrations werestudied in order to determine the concentration dependency of the odd harmonics. Stationary phantomswere imaged with a preclinical MPI scanner 25/20 FF (Bruker BioSpin/Phillips), installed at CharitéUniversity Hospital in Berlin, Germany. This system is a field-free-point (FFP) scanner and employsthe system function (SF) approach for image reconstruction [27]. The spatial resolution of MPI is in therange of 1–2 mm and depends on the magnetic properties of a tracer. The temporal resolution of oneframe is 21.5 ms, and circa 100 averages were acquired to reduce the noise. A selection gradient field of2.5 T/m in the z-direction (1.25 T/m in x- and y-directions) is implemented to suppress the responsesignal of the SPIONs in all regions of the field-of-view (FOV), except for a small volume around theFFP. By moving the FFP through the FOV, a spatially encoded signal of the whole imaging volumecan be detected. This movement of the FFP is accomplished by applying three orthogonal drive-fieldsoscillating at slightly different excitation frequencies about 25 kHz with amplitudes of 12 mT. For thephantom, flexible silicon tubing with an inner diameter of 1.0 mm was used (see Figure 1) and filledwith 71 µL iron oxide tracer redispersed in water at concentrations of c(Fe) = 5, 1, or 0.5 mM.Nanomaterials 2018, 8, x FOR PEER REVIEW 4 of 14 employing Co as an internal standard. Thermogravimetric analysis (TGA) measurements were conducted by Mettler TGA/DSC thermal analyzer (Mettler Toledo, Columbus, OH, USA) in the temperature range 303.15–1273.15 K under oxygen flow of 50 mL/min. The MPS signal was analyzed employing a commercial magnetic particle spectrometer MPS-3, manufactured by Bruker BioSpin (Ettlingen, Germany), operating at a fixed excitation frequency of 25.25 kHz and an excitation field up to 25 mT. Aqueous samples at different iron concentrations were studied in order to determine the concentration dependency of the odd harmonics. Stationary phantoms were imaged with a preclinical MPI scanner 25/20 FF (Bruker BioSpin/Phillips), installed at Charité University Hospital in Berlin, Germany. This system is a field-free-point (FFP) scanner and employs the system function (SF) approach for image reconstruction [27]. The spatial resolution of MPI is in the range of 1–2 mm and depends on the magnetic properties of a tracer. The temporal resolution of one frame is 21.5 ms, and circa 100 averages were acquired to reduce the noise. A selection gradient field of 2.5 T/m in the z-direction (1.25 T/m in x- and y-directions) is implemented to suppress the response signal of the SPIONs in all regions of the field-of-view (FOV), except for a small volume around the FFP. By moving the FFP through the FOV, a spatially encoded signal of the whole imaging volume can be detected. This movement of the FFP is accomplished by applying three orthogonal drive-fields oscillating at slightly different excitation frequencies about 25 kHz with amplitudes of 12 mT. For the phantom, flexible silicon tubing with an inner diameter of 1.0 mm was used (see Figure 1) and filled with 71 µL iron oxide tracer redispersed in water at concentrations of c(Fe) = 5, 1, or 0.5 mM. Figure 1. Tube phantom filled with redispersed tracer in water, mounted on the robot arm. Images were captured with a temporal resolution of approx. 1.7 s. The system functions were acquired for a FOV of 26.4 × 26.4 × 13.2 mm3 with 33 × 33 × 33 voxels resulting in a voxel size of 0.8 × 0.8 × 0.4 mm3. The sample size in the system function recordings was 2 × 2 × 1 mm3 (4 µL), with a concentration of 100 mM for Resovist and the MC-SPIONs, and the SC-SPIONs at the original concentration of 50 mM. In the MPI measurements of the phantom, the background was removed from the recorded data. For this purpose, the background signal was recorded at the beginning of the measurement and then the phantom was moved robotically to the center of the FOV. An average rate of 100 is applied, and images of all tracers were reconstructed by selecting 2209 frequency components and applying the Kaczmarz algorithm [28] with 5 iterations and a regularization factor of 1. The reconstructed volumes were rendered with a threshold of 0.4 related to the maximum intensity of the image. 2.3. Water-Based Sythesis of Multicore Superparamagnetic Iron Oxide Nanoparticles (MC-SPIONs) 887 mL of ferrous chloride (192.9 g/L), 579 mL of ferric chloride (378.4 g/L), 6480 mL dextran solution (40000 g/mol, 25 wt %), 218 g of formic acid (98 wt %), and 2054 mL of MilliQ water were introduced into the reactor (30 L Reactor CA 03) while stirring. The mixture was degassed under nitrogen with a flow rate of 100 L/h and heated to 293.15 K. The temperature was maintained at 293.15 K until a completion of the reaction. 9450 mL of NaOH solution (50 g/L) was added within 5 min. The injection was performed between the two lower levels of the Intermig stirrer. The mixture was stirred Figure 1. Tube phanto filled ith redispersed tracer in ater, ounted on the robot ar .I ages ere capture ith a te poral resolution of approx. 1.7 s. he syste functions ereacq ire for a F of 26.4 26.4 × 13.2 m3 with 33 × 33 × 33 voxels resulting in a voxel size of0.8 0.8 × 0.4 mm3. The sample size in the system function recordings was 2 × 2 × 1 mm3 (4 µL),with a concentration of 100 mM for Resovist and the MC-SPIONs, and the SC-SPIONs at the originalconcentration of 50 . In the PI easure ents of the phanto , the background as re ovedfro the recorded data. For this purpose, the background signal as recorded at the beginning of theeasure ent and then the phantom was moved robotically to the center of the FOV. An average rate of100 is applied, and images of all tracers were reconstructed by selecting 2209 frequency components andapplying the Kaczmarz algorithm [28] with 5 iterations and a regularization factor of 1. The reconstructedvolumes were rendered with a threshold of 0.4 related to the maximum intensity of the image.2.3. Water-Based Sythesis of Multicore Superparamagnetic Iron Oxide Nanoparticles (MC-SPIONs)887 mL of ferrous chloride (192.9 g/L), 579 mL of ferric chloride (378.4 g/L), 6480 mL dextransolution (40,000 g/mol, 25 wt %), 18 g of formic acid (98 wt %), and 2054 mL of MilliQ waterNanomaterials 2018, 8, 180 5 of 13were introduced into the reactor (30 L Reactor CA 03) while stirring. The mixture was degassedunder nitrogen with a flow rate of 100 L/h and heated to 293.15 K. The temperature was maintainedat 293.15 K until a completion of the reaction. 9450 mL of NaOH solution (50 g/L) was addedwithin 5 min. The injection was performed between the two lower levels of the Intermig stirrer.The mixture was stirred at 1400 rpm for an additional 120 min with a nitrogen flow of 100 L/h.In the screening experiments, optimizing the synthesis conditions for the following parameters wasexamined: temperature, stirring speed, flow rate of precursor addition, dextran molecular size,and type of coordinating agent to iron.2.4. Organic Synthesis of Coated Single-Core Superparamagnetic Iron Oxide Nanoparticles Doped withZinc (SC-SPIONs)2.4.1. Organic Synthesis of NanoparticlesThe synthesis was performed based on the reaction of iron oxide nanocubes described byNoh et al. [29]. 0.180 g of ferric acetylacetonate and 0.053 g of zinc acetylacetonate were combinedin a three-neck 100 mL RBF with 1.2 mL of oleic acid and 10 mL of benzyl ether. The mixture wasstirred at 420 rpm. Nitrogen was vigorously bubbled through the solution for 20 min, then the needlewas removed from the reaction solution and the flow reduced to a trickle. The flask was insertedinto a preheated mantle and the temperature rose to 571.15 K at an average rate of 313.15 K/min.The reaction was maintained at the reflux temperature for 30 min, and then cooled to room temperature.The particles were precipitated using 2:1 ethanol by volume and centrifuged at 3220 rcf for 30 min.The supernatant was discarded. The particles were rinsed twice with ethanol and stored for furtheruse. In the screening experiments aiming at the optimization of the nanoparticles, the followingparameters were investigated: zinc concentration, oleic acid content, stirring speed, anhydrousconditions, temperature, and reaction duration.2.4.2. Gallic Acid–PEG Ligand SynthesisUsing a DCC-mediated coupling, one molecule of PEG 10k was coupled with one molecule ofgallic acid in a modified synthesis based on previously published work [30,31]. In brief, 100 g of PEG10k, 1.7 g of gallic acid, and 0.3 g of DMAP were combined in a one-neck 2 L round bottom flask in1 L of DMF under gentle heating and nitrogen flow. The reaction was then stirred vigorously at roomtemperature while 2.48 g of DCC dissolved in 100 mL of DMF was added dropwise over one hour.Once addition was completed, the reaction was stirred for an additional 3 h.Following the completion of the reaction, the solvent was removed by vacuum and the productwas dissolved in 1 L of acetone with heat and vigorous shaking. The acetone was cooled in an ice-bathto precipitate dicyclohexylurea by-product, which was removed by filtration. The remaining acetonewas concentrated to 100 mL and poured into 1 L of cold stirred diethyl ether. The precipitated whiteproduct was then isolated by a vacuum filtration, and after that dissolved into chloroform followed bya subsequent precipitation. The off-white solid was dried under high vacuum for 24 h. The yield wasdetermined at approx. 85% w/w.2.4.3. Phase Transfer and Purification of ParticlesIn a typical phase transfer, 50 mg of dried nanoparticles were added to 6.4 mL CHCl3 andsonicated for one minute to disperse aggregates. 3.2 mL 0.0625 M GA-PEG ligand in chloroform and1.6 mL of triethylamine were mixed and shaken vigorously for 12–15 h in a ligand exchange procedure.After shaking, the solution was transferred into a round-bottom flask and MilliQ water was added at avolume twice that of the coating solution. The chloroform was then slowly removed at 100 mbar whilevigorously mixing the solution. This resulted in a homogenous aqueous solution of nanoparticles atpH 12. Following the phase transfer, the solution was placed into a 50 kDa dialysis membrane anddialyzed against dextran 10 (12 g/L) for 7 days and stored at 278.15 K until further use.Nanomaterials 2018, 8, 180 6 of 133. Results and Discussion3.1. Nanoparticles SynthesisMC- and SC-SPIONs were optimized via water-based and organic synthesis, respectively.The optimization procedure was performed by means of MPS; the straightforward technique basedon the same physical principle as MPI waiving of any spatial encoding [32]. Only homogeneouslyredispersed samples in water were selected for the MPS screening.The water-based synthesis of MC-SPIONs (schematic overview in Figure 2) was performeddirectly in a 30 L reactor, offering high-level control over reaction parameters. The well-establishedmethod of chemical coprecipitation from ferrous and ferric chloride in basic conditions was explored.Various carboxylic acids were studied as potential coordinating agents for iron. Formic acid wasfound to have the most positive impact on the crystal growth and phase purity. It was established thatdextran had to be added at the beginning of the reaction in order to realize the optimal precipitationtemperature. Dextran with MW 40,000 g/mol yielded the best combination of particle stability andmagnetic properties. Temperatures in the range of 293.15 to 333.15 K were studied, but no MPSimprovement was found with a temperature change. Sodium hydroxide addition was evaluatedin terms of agglomeration homogeneity. The best control over particle size and distribution wasaccomplished with the base added between the two lower levels of the Intermig stirrer and additionalduration of 5 min. The optimized product is colloidally stable for years at room temperature,and shows consistent MPS performance across multiple dilutions. The maghemite/magnetite phasewas determined by X-ray Diffraction (XRD) analysis. The brown color of the product suggests thepresence of maghemite rather than magnetite.Nanomaterials 2018, 8, x FOR PEER REVIEW 6 of 14 3. Results and Discu sion 3.1. Nanoparticles Synthesis MC- and SC-SPIONs were optimized via water-based and organic synthesis, respectively. The optimization procedure was performed by means of MPS; the straightforward technique based on the same physical principle as MPI waiving of any spatial encoding [32]. Only homogeneously redispersed samples in water were selected for the MPS screening. The water-based synthesis of MC-SPIONs (schematic overview in Figure 2) was performed directly in a 30 L reactor, offering high-l vel control ove reaction parameters. The well-established method of chemical coprecipitation from ferrous and ferric chloride in basic cond ti s was explored. Various carboxylic acids were studied as potential coordinating agents for iron. Formic acid was found to have the most positive impact on the crystal growth and phase purity. It was established that dextran had to be added at the beginning of the reaction in order to realize the optimal precipitation temperature. Dextran with MW 40,000 g/mol yielded the best combination of particle stability and magnetic properties. Temperatures in the range of 293.15 to 333.15 K were studied, but no MPS improvement was found with a temperature change. Sodium hydroxide addition was evaluated in terms of agglomeration homogeneity. The best control over particle size and distribution was accomplished with the base added between the two lower levels of the Intermig stirrer and additional duration of 5 min. The optimized product is colloidally stable for years at room temperature, and shows consistent MPS performance across ultiple dilutions. The maghemite/magnetite phase was determined by X-ray Diffraction (XRD) analysis. The brown color of the product suggests the presence of maghemite rather than magnetite. Figure 2. Schematic overview of multicore superparamagnetic iron oxide nanoparticle (MC-SPION) synthesis in aqueous media. The organic synthesis of SC-SPIONs was conducted by a thermal decomposition of ferric acetylacetonate in benzyl ether [33]. A schematic overview, including a surface functionalization and a phase transfer to water, followed by a dialysis, is displayed in Figure 3. Figure 3. Schematic overview of single-core (SC)-SPIONs synthesis in organic media, subsequent PEG functionalization and a phase transfer to water, followed by a dialysis in a dextran solution. Figure 2. Schematic over iew of multicore super r agnetic iron oxide anoparticle (MC-SPION)synthesis in aqueo s media.The organic synthesis of SC-SPIONs was conducted by a thermal decomposition of ferricacetylacetonate in benzyl ether [33]. A schematic overview, including a surface functionalizationand a phase transfer to water, followed by a dialysis, is displayed in Figure 3.Nanomaterials 2018, 8, x FOR PEER REVIEW 6 of 14 3. Results and Discussion 3.1. Nanoparticles Synthesis MC- a d SC-SPIONs wer optimized via water-based and o ganic synthesis, respectively. Theoptimization procedure was performed by means of MPS; the straightforward technique based onthe ame physical principl as MPI waiving of any spatial e coding [32]. Only homogeneously redispersed samples in water were selected for the MPS screening. The water-based synth s s of MC-SPIONs (schematic ove vi w in Figure 2) was performdire tly in a 30 L reacto , offering high-level control ove reaction p rameters. The well-estab ishemethod of che ical coprecipitation from ferr us and ferric c lori e in ba ic conditions was explored.Various carboxylic acids were studied as potential coo dinating agents for iron. Formic acid wasfoun to have the most positive impact on the crystal growth and phase purity. It was establishedthat dex ra had to be add d t the beginning of the reaction in order to realize the optimalprecipitation temp ratu e. Dextran with MW 40,000 g/mol yielded the best combination of particlestability and magnetic properties. Temper tures in the range of 293.15 to 333.15 K w re studied, butno MPS improvement was found with a temperature change. Sodium hydroxide a di ion wasevaluated in terms of agglomeration homogeneity. T e best c ntrol o r particle siz and distributionwas accomplished with the bas added between the two lower leve s of the Intermig tirre andadditional dur tion of 5 min. The optimized roduct is c lloidally st ble for years at roomtemperature, a d shows consis nt MPS performance across multiple diluti ns. Themaghemite/magnetite phase was d ter ined by X- y Diffr ction (XRD) analysis. The brown color of the product suggests the presence of maghemite rather than magnetite. Figur 2. Schematic ov rview of multicore superparamagnetic iron oxide nanoparticle (MC-SPION) synthesis in aqueous media. The rganic sy thesis of SC-SPIONs was conducted by a thermal d composition of ferriccetylaceto ate in benzyl ether [33]. A schematic overview, including a surface functionalization and a phase transfer to water, followed by a dialysis, is displayed in Figure 3. Figure 3. Schematic overview of single-core (SC)-SPIONs synthesis in organic media, subsequent PEG functionalization and a phase transfer to water, followed by a dialysis in a dextran solution. Figure 3. Schematic overview of single-core (SC)-SPIONs synthesis in organic media, subsequent PEGfunctionalization and a phase transfer to water, followed by a dialysis in a dextran solution.Nanomaterials 2018, 8, 180 7 of 13The synthesis process was tailored to provide particles large enough for superior MPSperformance, but at the same time still stable for months in aqueous media. A cubic shape wasaimed for, expecting a higher magnetisation value than for spheres resulting from a smaller surfaceanisotropy [29]. In addition, it was decided to dope with zinc in order to enhance magnetic properties.According to theoretical considerations, the net magnetic moment of ferrite per unit cell is determinedby the magnetic moment of divalent ions, while the magnetic spins at the octahedral sites are parallelto each other and antiparallel to the spins at tetrahedral sites. Zinc, which has a magnetic moment ofzero, has a preference to occupy tetrahedral sites, leading to a decrease in the amount of antiparallelinteractions and an increase in magnetism [34]. Here, the optimal zinc content in the synthesis wasbased on empirical measures by MPS. After thorough screening, the best-performing synthesis utilizedan iron/zinc molar ratio of 2.5, which resulted in a ratio of 16.3 in the particles.It was found that the degassing step under vacuum typically led to a more monodisperse particledistribution after 30 min of heating, but with a much smaller particle size than expected. Additionalheating to achieve an acceptable size would bring the size distribution back to the level of the regularsynthesis. A vigorous nitrogen bubbling instead of the degassing step under vacuum, the use ofanhydrous reagents, and the high-temperature heating of glassware prior to use were implemented toprevent water from entering the reaction system.In order to transfer the SC-SPIONs into aqueous media, a polyethylene glycol polymer was utilized.PEG is one of the most thoroughly investigated polymers for nanoparticle encapsulation, owing to afavorable biocompatibility profile, the ability to increase blood circulation time, and high solubility inaqueous media [35]. In addition to this, polyethylene glycols are inexpensive, offer functional groupsfor further functionalization, are linear, and readily available in a variety of sizes, facilitating the abilityto tune the thickness of the particle coating in order to optimize magnetic properties.PEG 10,000 provides a coating thick enough to prevent interactions with neighboring particles,but still sufficient to preserve the Brownian motion of the particles. The linking group, gallic acid,has a high tolerability (a median lethal dose administered intravenously LD50 i.v. is 320 mg/kg inmice), and has been shown to have a high affinity for metal oxides [36]. The ideal scenario would be acomplete exchange of the oleic acid carboxyl groups with the trihydroxyl groups of the gallic acid linker.A study of such a ligand exchange, using 3,4-dihydroxy-phenylalanine functionalized PEG, indicateda substantial removal of oleic functionalities from the particle surface [37]. We found that comparedto conventional catechol linkers, using gallic acid–PEG resulted in fewer large aggregates during thephase transfer step and increased stability after the addition of dextran. It has been suggested thatgallic acid coordinates via two phenoxy groups, with the third remaining unattached [36]. This maylead to a more irregular packing of PEG chains on the nanoparticles, facilitating the incorporation ofdextran into the coating matrix.3.2. Particle SizeThe TEM images shown in Figure 4 depict the differences in the particle shape and size in thetwo studied approaches. MC-SPIONs obtained by aqueous synthesis consist of small nanoparticles inthe 5-nm region (Figure 4a). Dextran appears to form patches of iron oxide-containing agglomerates.In contrast, Figure 4b displays well-redispersed SC-SPIONs of 27 ± 5 nm. The particles tend to beof a hexagonal and cubic shape, which are coated with a very thin shell having a spherical shape.The particle size is within the range of ideal size for MPI, as argued by the research community [22,25].It seems that dextran is well-incorporated into the coating matrix. The large dextran polymerswere intended to function as spacer groups and prevent the formation of particle aggregates overlong storage times. This modification allowed the production of a highly stable PEG dextranSC-SPIONs suspension.The DLS hydrodynamic diameter (Figure 5b) for SC-SPIONs was 38 nm, as determined bynumber-weighted analysis, which represents the iron core plus the coating consisting of PEG anddextran. The dominant peak was at 68 nm for MC-SPIONs (Figure 5a), indicating multiple iron oxideNanomaterials 2018, 8, 180 8 of 13cores embedded in dextran. Hydrodynamic particle size distribution in the volume-weighted analysisis shifted towards higher numbers; 51 nm and 79 nm for SC-SPIONs and MC-SPIONs, respectively.The peak for SC-SPIONs has an extended right shoulder, indicative of a small fraction of aggregates.The average hydrodynamic diameter (Zav) was determined to be larger for both studied samples;96 nm (Polydispersity Index (PDI) 0.24) and 104 nm (PDI 0.11) for SC-SPIONs and MC-SPIONs,respectively, compared to 62 nm for Resovist [38].Nanomaterials 2018, 8, x FOR PEER REVIEW 8 of 14 (a) (b) Figure 4. Transmission electron microscope (TEM) images of (a) MC-SPIONs and (b) SC-SPIONs, with a 100-nm scale bar. The DLS hydrodynamic diameter (Figure 5b) for SC-SPIONs was 38 nm, as determined by number-weighted analysis, which represents the iron core plus the coating consisting of PEG and dextran. The dominant peak was at 68 nm for MC-SPIONs (Figure 5a), indicating multiple iron oxide cores embedded in dextran. Hydrodynamic particle size distribution in the volume-weighted analysis is shifted towards higher numbers; 51 nm and 79 nm for SC-SPIONs and MC-SPIONs, respectively. The peak for SC-SPIONs has an extended right shoulder, indicative of a small fraction of aggregates. The average hydrodynamic diameter (Zav) was determined to be larger for both studied samples; 96 nm (Polydispersity Index (PDI) 0.24) and 104 nm (PDI 0.11) for SC-SPIONs and MC-SPIONs, respectively, compared to 62 nm for Resovist [38]. (a) (b) Figure 5. Hydrodynamic particle diameter distribution weighted by number (solid lines) and volume (dashed lines) measured by dynamic light scattering (DLS) for (a) MC-SPIONs and (b) SC-SPIONs. dmax is the diameter corresponding to the maximum of the peak. FWHM stands for the full width at half maximum of the peak. 3.3. MPS Analysis and MPI Imaging The MPI performance was routinely investigated by MPS at an excitation frequency of 25.25 kHz. The MPS response in all cases was tested at pH 6–8 and across a range of iron concentrations from 1 mM to 50 mM, to confirm the independence of tracer performance from particle concentration (as estimated by the iron concentration). The full curves for MPS data at 5 mT, 10 mT, and 25 mT are displayed in Figure 6 (left column). Data points are presented only to a relative uncertainty of 50%. An increase in the magnitude of the harmonics with increasing field strength coincides with theoretical models [39]. Improvement factors obtained at excitation amplitudes of 5 mT, 10 mT, and 25 mT are presented up to 1200 kHz in Figure 6 (right column). As can be seen, the improvement factors increase with decreasing field strengths. Figure 4. Transmission electron microscope (TEM) images of (a) MC-SPIONs and (b) SC-SPIONs,with a 100-nm scale bar.Nanomaterials 2018, 8, x FOR PEER REVIEW 8 of 14 (a) (b) Figure 4. Transmission electron microscope (TEM) images of (a) MC-SPIONs and (b) SC-SPIONs, with a 100-nm scale bar. The DLS hydrodynamic diameter (Figure 5b) for SC-SPIONs was 38 nm, as determined by number-weighted analysis, which represents the iron core plus the coating consisting of PEG and dextran. The dominant peak was at 68 nm for MC-SPIONs (Figure 5a), indicating multiple iron oxide cores embedded in dextran. Hydrodynamic particle size distribution in the volume-weighted analysis is shifted towards higher numbers; 51 nm and 79 nm for SC-SPIONs and MC-SPIONs, respectively. The peak for SC-SPIONs has an extended right shoulder, indicative of a small fraction of aggregates. The average hydrodynamic diameter (Zav) was determined to be larger for both studied samples; 96 nm (Polydispersity Index (PDI) 0.24) and 104 nm (PDI 0.11) for SC-SPIONs and MC-SPIONs, respectively, compared to 62 nm for Resovist [38]. (a) (b) Figure 5. Hydrodynamic particle diameter distribution weighted by number (solid lines) and volume (dashed lines) measured by dynamic light scattering (DLS) for (a) MC-SPIONs and (b) SC-SPIONs. dmax is the diameter corresponding to the maximum of the peak. FWHM stands for the full width at half maximum of the peak. 3.3. MPS Analysis and MPI Imaging The MPI performance was routinely investigated by MPS at an excitation frequency of 25.25 kHz. The MPS response in all cases was tested at pH 6–8 and across a range of iron concentrations from 1 mM to 50 mM, to confirm the independence of tracer performance from particle concentration (as estimated by the iron concentration). The full curves for MPS data at 5 mT, 10 mT, and 25 mT are displayed in Figure 6 (left colum ). Data points are presented only to a relative uncertainty of 50%. An increase in th magnitude of the harmonics with increasing field trength coincides with theoretical models [39]. Improvement factors obtained at excitation amplitudes of 5 mT, 10 mT, and 25 mT are presented up to 1200 kHz in Figure 6 (right column). As can be seen, the improvement factors incre se with decreasing field strengths. Figure 5. Hydrodynamic particle diameter distribution weighted by number (solid lines) and volume(dashed lines) measured by dynamic light scattering (DLS) for (a) MC-SPIONs and (b) SC-SPIONs.dmax is the diameter corresponding to the maximum of the peak. FWHM stands for the full width athalf maximum of the peak.3.3. MPS Analysis and MPI ImagingThe MPI performance was routinely investigated by MPS at an excitation frequency of 25.25 kHz.The MPS response in all cases was tested at pH 6–8 and across a range of iron concentrations from1 mM to 50 mM, to confirm the independence of tracer performance from particle concentration(as estimated by the iron concentration). The full curves for MPS data at 5 mT, 10 mT, and 25 mT aredisplayed in Figure 6 (left column). Data points are presented only to a relative uncertainty of 50%.An increase in the magnitude of the harmonics with increasing field strength coincides with theoreticalmodels [39]. Improvement factors obtained at excitation amplitudes of 5 mT, 10 mT, and 25 mT arepresented up to 1200 kHz in Figure 6 (right column). As can be seen, the improvement factors increasewith decreasing field strengths.Nanomaterials 2018, 8, 180 9 of 13Nanomaterials 2018, 8, x FOR PEER REVIEW 9 of 14 The ratios of the amplitudes of the third harmonic against Resovist were determined at 2.4, 2.3, and 2.1 for MC-SPIONs at 5 mT, 10 mT, and 25 mT, respectively. We can speculate that such MPS enhancement has its roots in a larger average hydrodynamic size of MC-SPIONs (104 nm), in comparison to Resovist (62 nm). Although the structure of Resovist is very complex, as revealed by the finding of two populations of sizes [10], the presence of the larger population is of key importance for the good MPI performance of Resovist. This has been demonstrated by the fractionation of Resovist [12], which results in an MPS signal change of up to 2.2 for the isolated larger clusters. Also, another direct synthesis of multicore iron oxide nanoparticles [20] led to an increase in the MPS amplitude by a factor of two for Nanomag-MIP particles with a hydrodynamic diameter of 106 nm. The MPS signal improvement at the third harmonic for SC-SPIONs was found to be 6.6, 5.8, and 4.3 at 5 mT, 10 mT, and 25 mT, respectively. In comparison, single-core UW-2 nanoparticles with a core size of 27 nm and hydrodynamic diameter of 47 nm showed improvement of the third harmonic intensity of only 3.5-fold [22]. Iron oxide nanoparticles encapsulated in lipidic micelles possessing a core diameter of 25 nm and hydrodynamic diameter of 61 nm generated four to six times more signal than Resovist at frequencies below 0.5 MHz [24]. When comparing our SC-SPIONs to these published approaches, it appears that the nanoparticles presented here have a similar size (by TEM), but contain zinc as a dopant known to increase particle magnetism; possess a thin layer of GA-PEG coating (based on TEM, see Figure 4b, and TGA analysis, see Figure A1 in Appendix A); and are additionally stabilized with dextran, resulting in an average hydrodynamic particle size of 96 nm, leading to an increased colloidal stability which can have an impact on the MPS signal. Nanomaterials 2018, 8, x FOR PEER REVIEW 10 of 14 Figure 6. Magnetic particle spectroscopy (MPS) experimental data (odd harmonics only) of SC-SPIONs, MC-SPIONs and Resovist plotted as magnetic moment µk versus frequency f (left column); and magnetic moment ratios of SC-SPIONs and MC-SPIONs versus Resovist defined as µk/µk,Resovist (right column) at an excitation field Bex of 5 mT, 10 mT, and 25 mT. Data points are shown up to a relative uncertainty of 50%. The superior MPS performance of the purposely optimized tracers was confirmed on a preclinical MPI scanner (Bruker BioSpin GmbH, Ettlingen, Germany). Accurately reconstructed images visualizing the geometric shape of the tube phantom containing 71 µL of iron oxide particles were obtained at the concentration of 5 mM of iron for all three investigated tracers (Figure 7), which set a sensitivity baseline for Resovist. A further decrease in the iron concentration to 1 mM led to the loss of a full phantom reconstruction for Resovist. MC-SPIONs resulted in image reconstruction superior to Resovist. However, as predicted by MPS performance, SC-SPIONs outperformed both MC-SPIONs and Resovist. Lowering the iron concentration to 0.5 mM led to a full phantom reconstruction for SC-SPIONs only. Nevertheless, the further reduction of the tracer concentration is required for clinical application. Due to a lack of standardization of MPI acquisition, data from different systems are not directly comparable. Figure 6. Magnetic particle spectroscopy (MPS) experimental data (odd harmonics only) of SC-SPIONs,MC-SPIONs and Resovist plotted as magnetic moment µk versus frequency f (left column); and magneticmoment ratios of SC-SPIONs and MC-SPIONs versus Resovist defined as µk/µk,Resovist (right column)at an excitation field Bex of 5 mT, 10 mT, and 25 mT. Data points are shown up to a relative uncertaintyof 50%.The ratios of the amplitudes of the third harmonic against Resovist were determined at 2.4,2.3, and 2.1 for MC-SPIONs at 5 mT, 10 mT, and 25 mT, respectively. We can speculate that suchMPS enhancement as its roots i a larger average hydrodynamic siz of MC-SPIONs (104 nm),in compari on to Resovist (62 nm). Although the structure of Res vist is very c mplex, as r vealed bythe finding of two populations of sizes [10], th pre ence of the larger population is of key importa cefor the g od MPI performanc of Resovist. This has been demonstrated by the f actionati n ofResovist [12], which results in an MPS signal change of up to 2.2 for the isolated larger clusters.Also, another direct synthesis of multicore iron oxide nanoparticles [20] led to an increase in the MPSamplitude by a factor of two for Nanomag-MIP particles with a hydrodynamic diameter of 106 nm.The MPS signal improvement at the third harmonic for SC-SPIONs was found to be 6.6, 5.8,and 4.3 at 5 mT, 10 mT, and 25 mT, respectively. In comparison, single-core UW-2 nanoparticles with acore size of 27 nm and hydrodynamic diameter of 47 nm showed improvement of the third harmonicNanomaterials 2018, 8, 180 10 of 13intensity of only 3.5-fold [22]. Iron oxide nanoparticles encapsulated in lipidic micelles possessinga core diameter of 25 nm and hydrodynamic diameter of 61 nm generated four to six times moresignal than Resovist at frequencies below 0.5 MHz [24]. When comparing our SC-SPIONs to thesepublished approaches, it appears that the nanoparticles presented here have a similar size (by TEM),but contain zinc as a dopant known to increase particle magnetism; possess a thin layer of GA-PEGcoating (based on TEM, see Figure 4b, and TGA analysis, see Figure A1 in Appendix A); and areadditionally stabilized with dextran, resulting in an average hydrodynamic particle size of 96 nm,leading to an increased colloidal stability which can have an impact on the MPS signal.The superior MPS performance of the purposely optimized tracers was confirmed on a preclinicalMPI scanner (Bruker BioSpin GmbH, Ettlingen, Germany). Accurately reconstructed images visualizingthe geometric shape of the tube phantom containing 71 µL of iron oxide particles were obtained atthe concentration of 5 mM of iron for all three investigated tracers (Figure 7), which set a sensitivitybaseline for Resovist. A further decrease in the iron concentration to 1 mM led to the loss of afull phantom reconstruction for Resovist. MC-SPIONs resulted in image reconstruction superior toResovist. However, as predicted by MPS performance, SC-SPIONs outperformed both MC-SPIONsand Resovist. Lowering the iron concentration to 0.5 mM led to a full phantom reconstruction forSC-SPIONs only. Nevertheless, the further reduction of the tracer concentration is required for clinicalapplication. Due to a lack of standardization of MPI acquisition, data from different systems are notdirectly comparable.Nanomaterials 2018, 8, x FOR PEER REVIEW 10 of 14 Figure 6. Magnetic particle spectroscopy (MPS) experimental data (odd harmonics only) of SC-SPIONs, MC-SPIONs and Resovist plotted as magnetic moment µk versus frequency f (left column); and magnetic moment ratios of SC-SPIONs and MC-SPIONs versus Resovist defined as µk/µk,Resovist (right column) at an excitation field Bex of 5 mT, 10 mT, and 25 mT. Data points are shown up to a relative uncertainty of 50%. The superior MPS performance of the purposely optimized tracers was confirmed on a preclinical MPI scanner (Bruker BioSpin GmbH, Ettlingen, Germany). Accurately reconstructed images visualizing the geometric shape of the tube phantom containing 71 µL of iron oxide particles were obtained at the concentration f 5 mM of iron for all three investigated tracers (Figure 7), which set a sensitivity baseline for Resovist. A further decrease in th iron c centration to 1 mM led t the loss of a full phan om reconstructi n for Resovist. MC-SPIONs resulted in image rec struction superior t R sovist. However, as predicted by MPS performance, SC-SPIONs outperformed both MC-SPIONs and Resovist. L weri g the ir concentration to 0.5 mM led to a full phant m reconstruction for SC-SPIONs only. Nevertheless, the further reduction of the tracer concentration is required for clinical application. Due to a lack of standardization of MPI acquisition, data from different systems are not directly comparable. Nanomaterials 2018, 8, x FOR PEER REVIEW 11 of 14 Figure 7. Reconstructed MPI images of phantoms using 5 mM, 1 mM, and 0.5 mM of Resovist, SC-SPIONs, and MC-SPIONs. 4. Conclusions We demonstrated that the MPS-driven screening of synthesis methods is a facile technique for selecting optimized MPI tracers. Improved MPI performance is already shown in the amplitude of the third harmonic of the MPS, which requires only seconds to be recorded, in comparison to the several hours spent on acquiring system function and then image reconstruction in MPI. Although MPS is a zero-dimensional magnetic particle imaging system, it provides a robust set of characterization data of imaging properties, which can be easily compared between various iron oxide nanoparticles formulations once the same reference standard (e.g., Resovist) is utilized. MPS enhancement of up to 2.5-fold was determined for MC-SPIONs obtained via coprecipitation in aqueous media in a large-scale process. A reconstructed MPI image of a tube phantom demonstrates this improvement. An organic synthesis method generates well-defined SC-SPIONs with even higher MPS signal intensities, which in consequence leads to a 6.6-fold signal increase at the third harmonics at 5 T compared to commercially available Resovist. The superior MPI performance of SC-SPIONs estimated by MPS was reflected by MPI experiments visualizing the geometric shape of the phantom at iron concentrations as low as 0.5 mM. Acknowledgments: This research was supported by the German Ministry of Education and Research under Grant FKZ 13N11092 (MapIT). The authors thank G. Winter from Bayer for the TGA measurements. Author Contributions: S.Z. designed the synthesis and characterization experiments, acquired and interpreted data, and wrote the paper; N.L. designed, collected, and interpreted MPS data; O.K. designed, acquired, and interpreted MPI data; D.B. performed SC-SPIONs synthesis experiments and interpreted data; F.W. contributed to the planning of measurements and MPI phantom design; G.S. contributed to the conception and design of the work. Conflicts of Interest: The authors declare no conflict of interest. Figure 7. Reconstructed PI images of phantoms using 5 mM, 1 mM, and 0.5 mM of Resovist,SC-SPIONs, and MC-SPIONs.4. ConclusionsWe demonstrated that the MPS-driven screening of synthesis methods is a facile technique forselecting optimized MPI tracers. Improved MPI performance is already shown in the amplitude of theNanomaterials 2018, 8, 180 11 of 13third harmonic of the MPS, which requires only seconds to be recorded, in comparison to the severalhours spent on acquiring system function and then image reconstruction in MPI. Although MPS isa zero-dimensional magnetic particle imaging system, it provides a robust set of characterizationdata of imaging properties, which can be easily compared between various iron oxide nanoparticlesformulations once the same reference standard (e.g., Resovist) is utilized.MPS enhancement of up to 2.5-fold was determined for MC-SPIONs obtained via coprecipitationin aqueous media in a large-scale process. A reconstructed MPI image of a tube phantom demonstratesthis improvement. An organic synthesis method generates well-defined SC-SPIONs with even higherMPS signal intensities, which in consequence leads to a 6.6-fold signal increase at the third harmonicsat 5 T compared to commercially available Resovist. The superior MPI performance of SC-SPIONsestimated by MPS was reflected by MPI experiments visualizing the geometric shape of the phantomat iron concentrations as low as 0.5 mM.Acknowledgments: This research was supported by the German Ministry of Education and Research underGrant FKZ 13N11092 (MapIT). The authors thank G. Winter from Bayer for the TGA measurements.Author Contributions: S.Z. designed the synthesis and characterization experiments, acquired and interpreteddata, and wrote the paper; N.L. designed, collected, and interpreted MPS data; O.K. designed, acquired,and interpreted MPI data; D.B. performed SC-SPIONs synthesis experiments and interpreted data; F.W.contributed to the planning of measurements and MPI phantom design; G.S. contributed to the conceptionand design of the work.Conflicts of Interest: The authors declare no conflict of interest.Appendix ANanomaterials 2018, 8, x FOR PEER REVIEW 12 of 14 Appendix A Figure A1. TGA analysis of a dried sample of SC-SPIONs with GA-PEG ligands before dialysis in dextran. The sample was heated up under oxygen atmosphere at 10 K/min and the weight loss was recorded as a function of temperature. The remaining mass of inorganic material was 74.3%. References 1. Gleich, B.; Weizenecker, J. 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Vancouver : University of British Columbia Library
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Faculty
Magnetic particle imaging (MPI)
Magnetic particle spectroscopy (MPS)
Iron oxide tracer
Superparamagnetic nanoparticles
Optimization of Iron Oxide Tracer Synthesis for Magnetic Particle Imaging
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