Nanoparticle encapsulation in red blood cells enables blood-pool magnetic particle imaging

hours after injection

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Phys. Med. Biol. 58 (2013) 3965–3977 doi:10.1088/0031-9155/58/12/3965

Nanoparticle encapsulation in red blood cells enablesblood-pool magnetic particle imaging hours afterinjection

J Rahmer1, A Antonelli2, C Sfara2, B Tiemann3, B Gleich1,M Magnani2, J Weizenecker4 and J Borgert1

1 Philips Technologie GmbH Innovative Technologies, Research Laboratories, Rontgenstraße24-26, D-22315 Hamburg, Germany2 Department of Biomolecular Sciences, University of Urbino ‘Carlo Bo’, Via Saffi 2, I-61029Urbino, Italy3 University Medical Center Hamburg-Eppendorf, Martinistraße 52, D-20246 Hamburg, Germany4 University of Applied Sciences, Moltkestraße 30, D-76133 Karlsruhe, Germany

E-mail: juergen.rahmer@philips.com and antonella.antonelli@uniurb.it

Received 1 March 2013, in final form 17 April 2013Published 17 May 2013Online at stacks.iop.org/PMB/58/3965

AbstractMagnetic particle imaging (MPI) is a new medical imaging approach that isbased on the nonlinear magnetization response of super-paramagnetic ironoxide nanoparticles (SPIOs) injected into the blood stream. To date, real-timeMPI of the bolus passage of an approved MRI SPIO contrast agent injected intothe tail vein of living mice has been demonstrated. However, nanoparticles arerapidly removed from the blood stream by the mononuclear phagocyte system.Therefore, imaging applications for long-term monitoring require the repeatedadministration of bolus injections, which complicates quantitative comparisonsdue to the temporal variations in concentration. Encapsulation of SPIOs into redblood cells (RBCs) has been suggested to increase the blood circulation time ofnanoparticles. This work presents first evidence that SPIO-loaded RBCs can beimaged in the blood pool of mice several hours after injection using MPI. Thisfinding is supported by magnetic particle spectroscopy performed to quantifythe iron concentration in blood samples extracted from the mice 3 and 24 h afterinjection of SPIO-loaded RBCs. Based on these results, new MPI applicationscan be envisioned, such as permanent 3D real-time visualization of the vesseltree during interventional procedures, bleeding monitoring after stroke, or long-term monitoring and treatment control of cardiovascular diseases.

S Online supplementary data available from stacks.iop.org/PMB/58/3965/mmedia

(Some figures may appear in colour only in the online journal)

0031-9155/13/123965+13$33.00 © 2013 Institute of Physics and Engineering in Medicine Printed in the UK & the USA 3965

3966 J Rahmer et al

1. Introduction

Magnetic particle imaging (MPI) is a new medical imaging approach that enables 3Dquantitative imaging of magnetic nanoparticle distributions with high temporal and good spatialresolution (Gleich and Weizenecker 2005). Potential applications are seen in dynamic imagingsuch as coronary angiography, cardiac wall motion assessment, or quantitative myocardialperfusion imaging (Schutz 2012). MPI may also be a safe alternative for CT and MRIangiography in patients with chronic kidney disease, who cannot tolerate iodine or gadoliniumbased contrast agents (Saritas et al 2013). These MPI applications can be realized by imaginga bolus of iron-oxide nanoparticles injected into the blood stream. Resovist R© (Bayer ScheringPharma, Germany), a commercial SPIO-based MRI liver contrast agent (Lawaczeck et al1997), has been successfully used as an in vivo MPI tracer for real-time visualization of thebolus passage through the heart of a mouse (Weizenecker et al 2009). However, from the broadspectrum of particle sizes found in Resovist R© (Eberbeck et al 2011), MPI mainly makes useof particles with a core diameter larger than 15 nm, which are most quickly removed from theblood stream by the mononuclear phagocyte system (Corot et al 2006). Thus, the time windowfor bolus-based measurements is only a few minutes (Lawaczeck et al 1997). Within this timewindow, the particle concentration does not reach a constant level, since during the dilutionof the particle bolus in the blood, there is already substantial removal of particles. Whilethis is acceptable for the above-mentioned dynamic imaging applications, other applicationswould benefit from a constant signal level and a homogeneous particle distribution in thevessel tree over a longer period of time. Among these are comparative measurements, e.g. ofblood volume at different locations in the vessel tree or of vessel lumens at different points intime such as in cardiac rest/stress comparisons. Further applications could be the permanentreal-time visualization of a 3D vessel roadmap for catheter-based interventions, monitoringfor bleeding after stroke, or therapy control in oncology with images taken at intervals ofseveral weeks.

A method that has been suggested for the prolongation of the blood retention time ofSPIOs is their encapsulation in red blood cells (RBCs) (Antonelli et al 2008, Magnani et al2011). Optimally, this extends the blood retention time toward the half-life of RBCs, which isaround 11 days in mice (Magnani et al 1990) and 30 days in humans (Bax et al 1999).

Human RBCs typically are disc-shaped with a diameter of 7–8 μm and a thicknessof 1 μm and 2.5 μm at the center and toward the rim, respectively. For encapsulation ofparticles inside RBCs, a loading procedure is applied. A hypotonic solution containing theSPIOs induces a transient opening of pores with diameters between 50 and 200 nm in theRBC membrane (Ihler and Tsang 1987), so that particles can enter the cells. After resealingthe pores in an isotonic buffer, particles are trapped in the RBCs. For Resovist R© loading,transmission electron microscopy (TEM) revealed that particles indeed go into the cells anddo not attach to the cell membrane (Antonelli et al 2011). TEM images furthermore showeda spatially homogeneous distribution of particles inside the cells, without visible signs ofparticle agglomeration (Markov et al 2010). Nuclear magnetic resonance (NMR) relaxationmeasurements demonstrated that loaded RBCs circulated for more than 12 days in mice beforea noticeable reduction in concentration was observed (Antonelli et al 2008).

Since the distribution of hydrodynamic diameters in Resovist R© extends beyond theachievable pore diameters, a size selection effect occurs during the loading procedure. Withtypical loading parameters, this effect leads to a preferred entrapment of particles witha hydrodynamic diameter smaller than 60 nm. Since the hydrodynamic diameter stronglycorrelates with the diameter of the iron core, the peak of the core size distribution of entrappedparticles is shifted to smaller diameters and also drops much more steeply toward larger

In vivo MPI of nanoparticles encapsulated in RBCs 3967

diameters than for bulk Resovist R©. This change in particle size distribution has been verifiedby modeling the magnetization response obtained from vibrating sample magnetometry andmagnetic particle spectrometry (MPS) of entrapped versus unentrapped particles (Markov et al2010). The size selective effect reduces the relative fraction of particles with a core diameter of30 nm by about an order of magnitude. Since for MPI, particles with core diameters larger thanabout 25 nm deliver the signal components responsible for high spatial resolution (Rahmer et al2012, Weizenecker et al 2012), MPI of loaded RBCs is expected to have reduced sensitivity andresolution compared to the same amount of iron in particles directly injected into the blood.

In this paper, in vivo MPI of mice using Resovist R©-loaded RBCs is demonstrated.Resovist R© has been chosen for its good MPI performance and the fact that its suitability forRBC entrapment has already been established. Despite the reduced sensitivity and resolutionresulting from the size selection effect, and despite the difficulties related to the applicationof the loading procedure to mice instead of humans, the sensitivity of the experimental MPIinstrumentation allows dynamic imaging up to several hours after Resovist R©-loaded RBCinjection.

2. Materials and methods

All in vivo experiments were in accordance with local laws and regulations and were approvedby the local authorities as well as the internal committee on biomedical experiments ofPhilips Research. All animal handling was done by a veterinarian. Blood withdrawal, RBCloading, and injection of Resovist R©-loaded RBCs was performed using Crl:CD1(ICR) miceat the veterinary facility of the University Medical Center Eppendorf. Three mice receivedan injection of RBCs with encapsulated Resovist R©. The mice were then transferred to thePhilips facilities for MPI and MRI experiments. MPI scans were performed 3 and 24 hafter loaded RBC injection, respectively. For the scans, the mice were anesthetized using aketamine/xylazine injection. During anesthesia, blood was withdrawn from the ocular veinfor measuring the iron concentration in the blood using MPS. After the last MPI scan, the micewere sacrificed using embutramide (T61 R©), and for mouse 2 and 3, an anatomical referencescan was performed using MRI.

2.1. Encapsulation of magnetic nanoparticles in RBCs

For all experiments, Resovist R© was used, since it exhibits the best MPI performance of allcommercially available magnetic nanoparticle suspensions. Furthermore, it has already beensuccessfully applied for in vivo MPI mouse imaging (Weizenecker et al 2009) and it can betransferred into RBCs with good efficiency (Antonelli et al 2011). Blood was withdrawn fromseveral mice to load RBCs using the approach published by Antonelli et al (2008).

In brief, 1 ml of blood with a hematocrit of 65 % was dialyzed in a membrane tubewith 12–14 kDa cut-off in the presence of 5.9 mg Fe of Resovist R© for 75 min in 50 ml of adialysis buffer (15 mM NaHCO3, 15 mM NaH2PO4, 20 mM glucose, 4 mM MgCl2 pH 7.4)containing 2 mM ATP and 3 mM reduced glutathione. For the treatment of 3 mice, 4.2 ml(or 117.6 mg Fe) of Resovist R© (0.5 mmol(Fe) l–1) were added to 20 ml of murine RBCsat 65 % hematocrit. Resealing of RBCs was achieved by adding 0.1 vol of PIGPA (5 mMadenine, 100 mM inosine, 2 mM ATP, 100 mM glucose, 100 mM sodium pyruvate, 4 mMMgCl2, 194 mM NaCl, 1.606 M KCl, 35 mM NaH2PO4, pH 7.4) per vol of dialyzed RBCsand by incubating at 37 ◦C for 45 min. The resealed cells were recovered by centrifugationat 400 g and washed four times with Hepes buffer to remove unentrapped magnetic particles.The procedures were performed at 4 ◦C under sterile conditions. For the imaging experiments,

3968 J Rahmer et al

the Resovist R©-loaded RBC suspensions with a hematocrit of 44 % were injected into the tailveins of three mice. The injected volume was 1.0 ml for mice 1 and 2, and 0.5 ml for mouse 3,respectively. The weight of each mouse was around 29 g.

2.2. NMR measurements

NMR spectroscopy was applied to determine the iron content of loaded RBCs. To this end,the T1 and T2 relaxation times were used as a measure for iron content. T1 was measured usingan inversion recovery sequence, whereas T2 was determined using a Carr–Purcell echo train.NMR was performed as a service measurement by the chemistry department of the Universityof Hamburg using a commercial 400 MHz spectrometer (Bruker Biospin, Germany). Forcalibration, a concentration series of Resovist R© diluted in RBC suspensions at 44% hematocritwas measured. The series comprised seven suspensions with iron concentrations ranging from0 to 5 mmol(Fe) l–1. The inverse relaxation times, i.e. the relaxation rates R1 and R2 as afunction of concentration c were fitted to obtain the relaxivities r1 and r2 and base relaxationrates R1,0 and R2,0 according to R1/2 (c) = r1/2 c + R1/2,0. Using these relaxivities and baserelaxation rates, the particle concentration was determined from the measured R1 and R2 valuesof the loaded RBC sample.

2.3. Magnetic particle spectroscopy

MPS was used to determine the iron content and the spectral properties of the particle response(Biederer et al 2009). It was performed using a prototype spectrometer with an excitationfrequency of 25 kHz and an excitation field amplitude of 10 mT. The measurement time was30 s. The detection bandwidth was 2.5 MHz. For data evaluation, only odd harmonics ofthe excitation frequency were considered over a bandwidth of 1 MHz, since other spectralcomponents do not contain relevant information. The measured volume of the individualsamples was 10 μl. Resovist R© was used as a reference at a dilution of 1:10, i.e., with an ironconcentration of 50 mmol(Fe) l–1.

For a given excitation amplitude and frequency, the shape of the spectrum depends onthe steepness of the magnetization curve. The steepness is determined by the field interval inwhich the magnetization flips from its negative to its positive saturation value. It can be usefulto express this interval as the full width at half maximum of the derivative of the magnetizationcurve, which is also a measure of achievable spatial resolution in MPI (Rahmer et al 2009).A steep magnetization curve generates strong higher harmonics of the excitation frequency,whereas for a shallow curve, the signal drops rapidly toward increasing frequencies. Sincethe steepness of the magnetization curve is proportional to the third power of the diameterof the particle core (Weizenecker et al 2007), the shape of the spectrum is very sensitive tothe particle size distribution. Therefore, to assess iron content, the 3rd harmonic is used, as itis rather independent of particle size, whereas the higher harmonics are used to assess MPIperformance and changes in particle size distribution.

MPS measurements were performed on the Resovist R©-loaded RBC sample and on bloodsamples that were drawn from the ocular veins of the mice 3 and 24 h after injection of theloaded RBCs, respectively.

2.4. Magnetic particle imaging

MPI was applied with the aim of visualizing RBC-encapsulated iron particles in the bloodstream. An experimental MPI mouse scanner was used as described in previous publications(Weizenecker et al 2009, Rahmer et al 2012). The mice were inserted on a bed into the

In vivo MPI of nanoparticles encapsulated in RBCs 3969

cylindrical bore of the scanner. The bore had a diameter of 32 mm and its axis definedthe x direction. A static selection field was used to generate rather constant field gradientsand a field-free point (FFP) at the center of the bore. The selection field gradient strengthwas 5.5 T/m/μ0 in the vertical direction (z direction) and half this value in the horizontaldirections. Drive fields were used to rapidly move the FFP in space and thus generate signalfrom the particles that are traversed by the FFP. Three orthogonal drive field coils generatedhomogeneous fields at three different frequencies fx = 24.51 kHz, fy = 26.04 kHz, and fz =25.25 kHz with an amplitude of 18 mT. For this frequency ratio, the trajectory of the FFPrepeats after TR = 21.54 ms. During one TR, the FFP trajectory densely fills a box-shapedvolume of 13.1 × 13.1 × 6.5 mm3. From the acquired signal, a series of images wasreconstructed to a slightly larger field of view (FOV) of 16.8 × 20.4 × 12.0 mm3 with avoxel size of (0.6 mm)3.

For mouse imaging, the anesthetized animals were placed in the supine position on theanimal bed such that in the transverse directions, the FOV covered the central part of the mousebody. To image a larger volume of the mice in axial direction, the mouse bed was manuallymoved in steps along the bore. Each station lasted 10 s and the whole length of the mouse wascovered using this multi-station approach. The mice were imaged 3 and 24 h after injection ofResovist R©-loaded RBCs.

For image reconstruction, the system function describing the relation between particleposition and receive signal was established beforehand in a calibration scan using a voxel-sized sample of undiluted Resovist R© (Weizenecker et al 2009). The inverse imaging problemwas solved iteratively using the Kaczmarz method (Dax 1993). Regularization was used toadjust the balance between resolution and SNR in the images (Weizenecker et al 2007). Sincein the present experiments, only very low concentrations of MPI-active particles were achievedin vivo, rather strong regularization was used to increase image SNR at the cost of resolution.Furthermore, the size selection occurring during Resovist R© encapsulation in RBCs furtherdegrades sensitivity and resolution. Thus, spatial resolution was of the order of 2–3 mm andtherefore lower than in bolus-based experiments where higher particle concentrations wereavailable.

The sensitivity of the system at the temporal resolution of TR = 21.54 ms was determined inprevious quantitative in vivo measurements using bolus-injected Resovist R©, where for similarregularization parameters the noise floor in a voxel was comparable to the signal obtained froma Resovist R© concentration between 20 and 50 μmol(Fe) l–1 in that voxel. The range indicates aspatial dependence of the SNR which results from a signal drop observed toward the edges ofthe FOV. Thus, to be able to image Resovist R© in the blood using this scanner at high temporalresolution, the Resovist R© concentration has to be of the order of 50 μmol(Fe) l–1.

2.5. Reference MR imaging

For obtaining anatomical reference information, MRI was performed on a clinical 3.0 T humanwhole-body scanner (Achieva 3.0 T, Philips, The Netherlands) with a dedicated receive coil formouse measurements. A T1-weighted turbo-spin-echo sequence was applied to acquire sagittalmulti-slice images. A FOV of 79 × 24 × 25 mm3 was covered with an in-plane resolution of0.25 × 0.25 mm2 and a slice thickness of 0.50 mm. Total scan time amounted to 11 min. Aftertri-linear interpolation of the MPI data to the MRI resolution, image overlays were made byapplying the 3D translations derived from the positions of the mice in the two scanners. Thetranslations have an error of a few millimeters, so that in a second step, manual registrationwas performed based on anatomical landmarks such as the diaphragm or the heart. In theoverlay images, the MPI data are displayed using a color map with a color change from red toyellow to allow for easy differentiation from the grayscale MRI data.

3970 J Rahmer et al

(a) (b)

Figure 1. MPS spectra of diluted Resovist R©, loaded RBC suspension, and blood withdrawalsamples for mouse 1 (a) and mouse 3 (b). The sample volume was 10 μl. Only the odd harmonicsof the excitation frequency of 25 kHz are shown. The signal of the Resovist R©-loaded RBC sampledrops faster with frequency than the signal of diluted Resovist R©. This indicates a particle sizeselection effect during RBC loading. For the determination of iron content, the signal in the thirdharmonic (at 75 kHz, leftmost points) was used. For reference, the background noise of the MPSsystem is plotted. Also, the Resovist R© curve is scaled to a concentration of 50 μmol(Fe) l–1 inorder to indicate the sensitivity limit of the MPI scanner used for imaging.

Table 1. Iron concentration in loaded RBC suspension determined using different methods.

NMR T1 NMR T2 MPS (third harm.) MPS (seventh harm.) MPI

Iron concentration 2.1 3.1 1.5 0.93 2.0(mmol(Fe) l–1)

3. Results

In the evaluation of the MPI measurements of the three mice, it was found that a substantialfraction of the signal obtained from mouse 2 was corrupted by an oscillation built-up in thelow noise amplifiers. This was probably due to a high integral iron concentration present inthe FOV. Therefore, mouse 2 was removed from the evaluation and results are only reportedfor mouse 1 and 3. As mentioned above, mouse 1 was injected with 1 ml of the loaded RBCsuspension, whereas mouse 3 was injected with only 0.5 ml.

The iron concentration achieved in Resovist R©-loaded RBCs by the loading procedurewas determined using NMR relaxometry. For NMR evaluation, a volume of about 160 μlwas removed from the loaded RBC suspension later to be injected into the three mice. Themeasured longitudinal relaxation time was T1 = 0.50 s and the transverse relaxation time wasT2 = 3.5 ms. Using the calibration curve derived from a dilution series, iron concentrations of2.1 mmol(Fe) l–1 and 3.1 mmol(Fe) l–1 were calculated from T1 and T2, respectively.

MPS was used to determine iron concentrations in the loaded RBC suspension and inthe blood, as shown in figure 1 for blood withdrawn from mouse 1 (a) and mouse 3 (b). TheResovist R©-loaded RBC suspension has a significantly lower iron concentration compared to50 mmol(Fe) l–1 Resovist R© in aqueous solution. From the evaluation of the third harmonic, aniron concentration of 1.5 mmol(Fe) l–1 was determined for the loaded RBCs. Table 1 comparesthe iron concentrations obtained for the loaded RBC suspension using different methods.The injection into the blood circulation further dilutes the RBCs, so that a sample of bloodwithdrawn 3 h after injection shows a reduction by a factor of 8.5 and 17.8 compared to theinjected suspension for mouse 1 and 3, respectively. The corresponding iron concentrations inthe blood are 179 and 85 μmol(Fe) l–1. After 24 h, a further reduction by a factor of 2.6 and

In vivo MPI of nanoparticles encapsulated in RBCs 3971

1.6 is observed, leading to 70 and 53 μmol(Fe) l–1 for mouse 1 and 3, respectively. The signallevels are still clearly above the instrumentation noise of the MPS, but the concentrationsapproach the sensitivity limit of 50 μmol(Fe) l–1 of the MPI scanner, which is indicated by thedashed lines in figure 1.

For the loaded RBC samples, the higher harmonics in the MPS spectra exhibit a more rapiddrop in signal intensity than for Resovist R©. If the above concentrations had been determinedusing the seventh harmonic, only a concentration of 0.93 mmol(Fe) l–1 would have been foundfor the loaded RBCs, whereas evaluation of the 3rd harmonic yielded 1.5 mmol(Fe) l–1. Thisdrop was expected from the size selection effect when the particles diffuse into the RBCsthrough the opened membrane pores during the loading process. Since for imaging, all signal-containing harmonics are used and higher harmonics encode fine spatial detail (Rahmer et al2012), imaging performance of Resovist R© inside the RBCs may be more strongly reducedthan expected from the reduction in iron content derived from the third harmonic.

To assess the imaging performance, a vial containing loaded RBCs from a differentloading experiment performed under the same conditions was imaged in the MPI scannertogether with three vials containing Resovist R© at dilutions of 1:10, 1:100, and 1:1000 (notshown). Since MPI images are quantitative, a voxel based comparison between the obtainedsignal levels can be made. This comparison shows that the loaded RBC suspension performsas if it contained 611 μmol(Fe) l–1 of Resovist R©. MPS measurements showed that this RBCsuspension had a 3.3 times lower iron concentration than the suspension used in the currentloading experiment, thus the MPI signal of the actually used suspension would correspond toroughly 2 mmol(Fe) l–1. Using this relation, the MPI signal levels have been transformed intoconcentrations in the evaluations following below.

For MPI, since the FOV is much smaller than the mouse body, the mice were shifted step-wise along through bore to cover both the thoracic region and the abdomen. The followinganalysis only concentrates on the volume covering the heart and liver, since in this region,additional dynamic information can be extracted.

Figure 2 shows MPI data acquired on mouse 1 3 h (a–c) and 24 h (d–f) after injectionof Resovist R©-loaded RBCs, respectively. A sagittal slice has been extracted from the 3DMPI data at a point in time where the heart and liver were positioned in the FOV (a, d).To get an impression of the real-time signal variation, movies showing the slice depicted infigure 2(a) over a couple of seconds can be found in the supplemental materials, available fromstacks.iop.org/PMB/58/3965/mmedia. In the upper part of figure 2(a), a structure is visible,whose position and dynamic behavior matches the heart. The signal farther below originatesfrom the liver region. In figure 2(d), i.e. on the next day, the heart signal is much weaker incomparison to the liver signal. Since spatial resolution is limited, dynamic information hasbeen used to verify the above signal assignment. Due to the motion of the upper structure,signal at its edge is modulated as visible in the time trace of the single voxel signals shownin the upper graphs of figures 2(b) and (e). A periodic modulation is visible, whose frequencymatches the heart rate of a sedated mouse, as shown in the Fourier transformed time tracesin figures 2(c) and (f). The edge of the structure assigned to the liver gives a slower signalvariation (center rows in figures 2(b) and (e)), whose frequency matches the breathing rate ofa mouse, cf figures 2(c) and (f). The signal at the bottom of the image only has small dynamiccontributions (bottom rows in figures 2(b), (e), (c) and (f)), which can be explained by its largedistance to the heart and the diaphragm.

Figure 3 shows the same evaluation for mouse 3 together with sagittal MRI slices acquiredon this mouse after MPI. On the MR image, the liver is black, indicating that a substantialamount of the loaded RBCs ended up in the liver. In figures 3(b) and (g), the MPI images havebeen interpolated to the MRI grid to be displayed as an overlay in orange color. Clearly, the

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(a) (b) (c)

(d) (e) (f)

Figure 2. MPI and single voxel dynamic evaluation of mouse 1, 3 h (top row) and 24 h (bottom row)after injection of Resovist R©-loaded RBCs into the tail vein. A sagittal slice through the position ofthe heart has been selected for display (a), (d). Letters AP indicate the anterior–posterior direction(front-back), whereas SI indicates the superior–inferior direction (head–foot). The signal variationfor three selected voxels has been extracted from each slice (b), (e). The right ordinate axes show theiron concentrations derived from the signal level as described in the text. The temporal resolutioncorresponds to the repetition time of the FFP trajectory, which is TR = 21.5 ms. The voxels havebeen chosen at the edge of the suspected heart, the edge of the suspected liver, and at a positiondeeper in the abdomen (from top to bottom). The edges have been selected, since in these voxelsthe signal variation due to the heart beat and breathing is maximal. The selected time series havebeen Fourier transformed (c), (f) to show that two distinct peaks can be observed. The frequency isgiven in beats per minute (bpm). From the origin and frequency spectrum of the observed signals,it is inferred, that the heart beat and breathing motion is observed. Movies directly showing thetemporal variation in the sagittal slices can be found in the supplemental materials, available fromstacks.iop.org/PMB/58/3965/mmedia.

upper structure in the MPI image matches the heart, whereas the lower structures are foundin the region of the liver and further down in the abdomen. Again, the single voxel temporalsignal evolution supports the anatomical assignment. After 24 h, the signal in the heart regionis strongly reduced in comparison to the liver signal. Nonetheless, the available signal stillcontains the temporal pattern of the beating heart (top row of figures 3(i) and (j)).

To further support the anatomical assignment of the MPI signal, the Fourier transformof the signal evolution has been calculated for all voxels of the MPI data acquired 3 h afterinjection on mouse 3. Figures 4(b) and (c) displays overlay images of the magnitude of the threestrongest spectral MPI signal components on the MR reference image shown in figure 4(a).The frequencies correspond to the constant (dc) Fourier component (b) and the breathing (c)and heart rate (d), i.e. the peaks found in the spectra of figure 3.

While the dc component delineates all regions from which MPI signal arises, signal atthe breathing frequency is mainly found close to the diaphragm and in the heart, which sits onthe diaphragm and thus follows the breathing motion. In figure 4(c), there is a signal-void linewhich roughly follows the diaphragm. It separates regions whose signal changes with oppositephase, as can be verified by inspecting the complex data (not shown). When the diaphragm

In vivo MPI of nanoparticles encapsulated in RBCs 3973

(a) (b) (c) (d) (e)

(j)(f) (g) (h) (i)

Figure 3. MRI reference images, MPI and single voxel dynamic evaluation of mouse 3. Fromthe 3D MPI data acquired 3 h after injection of Resovist R©-loaded RBCs (top row) and 24 hafter injection (bottom row), a sagittal slice through the position of the heart has been selectedfor display (c), (h). Letters AP indicate the anterior–posterior direction whereas SI indicates thesuperior–inferior direction. For reference the corresponding slices of the MRI data set are shown(a), (f) as well as the overlays of the interpolated MPI signal (orange) on the MRI data (b), (g).The signal variation for three selected voxels has been extracted from each slice (d), (i). The rightordinate axes show the iron concentrations derived from the signal level as described in the text.The voxels have been chosen at the edge of the suspected heart, the edge of the suspected liver,and at a position deeper in the abdomen (from top to bottom). The selected time series have beenFourier transformed (e), (j) to show that distinct peaks for the heart beat and breathing motion canbe observed. Movies directly showing the temporal variation in the sagittal slices can be found inthe supplemental materials, available from stacks.iop.org/PMB/58/3965/mmedia.

moves upward, the bright signal areas in the heart and liver also move upward, thus signalincreases in the areas above the diaphragm but decreases in the area below. Thus, an oppositephase behavior results. In figure 4(d), one sees that at the heart frequency, signal is mainlyconcentrated on the heart region. Here, an opposite phase is observed between the atria andthe ventricles.

4. Discussion and outlook

The iron concentration of the loaded RBC suspension exhibits discrepancies between thevalues determined by T1 and T2 NMR relaxometry, MPS, and MPI.

The discrepancy between the concentrations determined from T1 and T2 in the NMRmeasurement, namely 2.1 and 3.1 mmol(Fe) l–1, results from two effects. The first is thesize selection effect occurring during RBC loading. A different size distribution of particlesleads to different relaxation rates R1 and R2, so that the calibration curves acquired withResovist R© diluted in murine RBC suspension are no longer the correct reference. The secondeffect is that the particles experience a different environment, i.e. exhibit a different diffusionbehavior, inside the cells as compared to the extracellular compartment again with differenteffects on T1 and T2.

The concentration obtained from MPS, 1.5 mmol(Fe) l–1, is lower than both valuesobtained from NMR. This is due to the fact that very small iron oxide particles hardly

3974 J Rahmer et al

(a) (b) (c) (d)

Figure 4. MPI/MRI overlay for different frequency components of data acquired on mouse 3. Theoverlay data were obtained by Fourier transformation of the evolution of the image intensity inevery single voxel. The static frequency component (b), the frequency component correspondingto the breathing frequency of 123 bpm (c), and the frequency component corresponding to theheart beat frequency of 209 bpm (d) are shown. The gray frame in (a) indicates the region enlargedin (b)–(d), whereas the orange frames indicate the MPI FOV in the displayed plane. Letters APindicate the anterior–posterior direction whereas SI indicates the superior–inferior direction.

generate any harmonics at the MPS excitation amplitude of 10 mT, while NMR relaxometryis susceptible to small particles as well. Since the size selection effect during loading of theRBCs increases the fraction of smaller particles, the concentration determined from the 3rdharmonic of the MPS spectrum underestimates the total iron content.

The concentration obtained by comparing vials in MPI, 2.0 mmol(Fe) l–1, is close to thevalue obtained by T1 NMR, but higher than the value obtained from MPS and lower thanthe value determined by T2 NMR. The discrepancy with the MPS result can be explained bythe fact that the drive amplitude in the MPI scanner was larger than in the MPS, namely 18 mTversus 10 mT. Given the larger fraction of smaller particles inside the RBCs (Markov et al2010), their contribution to the signal in higher harmonics grows stronger with drive amplitudethan for the larger particles in Resovist R©. Thus, the higher amplitude used in MPI generatesmore signal from the encapsulated particles and therefore a higher iron concentration results.

In view of the fact, that both the NMR relaxation times and the MPI/MPS signals dependon the size distribution of particles, a rather large error of about 50% has to be assumed in theconcentrations obtained for the loaded RBC material.

Despite the low MPI active iron concentrations, MPI of RBCs flowing in the blood seemsfeasible. After injection of Resovist R©-loaded RBCs into the tail vein of the mouse, the loadedRBCs are diluted in the blood stream. Assuming a blood volume of 2.5 ml, the 1 ml injectionin mouse 1 leads to a dilution by a factor of 3.5, while the 0.5 ml injection in mouse 3 leads toa dilution by a factor of 6. Expected MPS concentrations in the blood would thus be 430 and250 μmol(Fe) l–1 for mouse 1 and 3, respectively. The values are between a factor of two andthree higher than the concentrations actually measured on blood extracted 3 h after injection,namely 179 and 85 μmol(Fe) l–1. This indicates that by this time, more than half of the iron hasbeen removed from the blood. It is assumed, that during the loading procedure, a substantialamount of RBCs has been slightly damaged, which is then rather rapidly removed by the liverand spleen (Muzykantov 2010). In loading of human RBCs, a cell recovery rate of 60 to 70%

In vivo MPI of nanoparticles encapsulated in RBCs 3975

is achieved (Markov et al 2010). Due to the higher membrane fragility, the loading processis slightly different for murine RBCs (DeLoach 1987) and cell recovery rates are typicallyonly in the range between 25 and 30%. This is confirmed by the high MPI signal observedin the liver. A smaller fraction of the loaded RBCs is still present in the blood. Due to theproximity to the detection limit of the MPI scanner, their signal can only be discerned in areaswith a large blood volume, i.e. the heart. A dynamic data evaluation was used to discern heartmotion from the signal in the liver, which is mainly subject to breathing motion. From thepumping motion visible in the dynamic MPI data and the iron concentration determined fromthe extracted blood, one can infer that Resovist R©-loaded RBCs indeed still flow in the blood3 h after injection.

According to the MPS measurements, 24 h after injection, the iron particle concentrationin the blood has further decreased by a factor of 2.6 and 1.6 for mouse 1 and 3, respectively.Again, this decrease is more rapid than expected for healthy RBCs. Aside from the short life-time of RBCs damaged during the loading procedure, the large relative volume of the injectedblood may also be detrimental to the RBC lifetime. It is assumed that these issues would beless severe in human applications, where rather large blood volumes can be extracted, thusfacilitating the loading procedure so that a higher iron concentration can be achieved whileat the same time the amount of damaged RBCs is reduced. For comparison, NMR evaluationshowed iron concentrations as high as 13.9 mmol(Fe) l–1 in Resovist R©-loaded human RBCswithout negative effects on cell recovery (Markov et al 2010), whereas in this work, onlyaround 2 mmol(Fe) l–1 were achieved in murine RBCs. With higher concentrations, smallerrelative amounts of blood have to be injected, thus putting less strain on the organism. It wouldthen be possible to extend the lifetime of the loaded RBCs toward that of healthy RBCs. Healthyreinjected human RBCs have shown a half-life of more than three weeks (Magnani et al 1998).

For loading of human RBCs, an automated procedure is available, that performs allprocessing steps under sterile conditions within 2 h (Magnani et al 1998). For this, a minimumblood volume of 50 ml is necessary. The method has been routinely applied to load RBCswith drug precursors for the treatment of inflammatory diseases in clinical studies (Castro et al2007, Bossa et al 2008). For MPI, the necessary blood volume to be extracted for RBC loadingis calculated in the following. It is assumed that an iron concentration of 10 mmol(Fe)/l can beeasily reached in loading human RBCs with Resovist R© and that a clinical MPI system reachesa similar sensitivity as the mouse scanner, i.e. 50 μmol(Fe) l–1. Then, with a total bloodvolume of 5 l, a volume of 25 ml would have to be loaded. Since it is not a problem to collect100–200 ml of blood, there is enough room to compensate for the loss in MPI performanceby particle encapsulation. Thus, one can envision a clinical scenario where blood extraction,RBC loading, blood reinjection, and subsequent MPI are performed in a single session.

Aside from the better performance of the loading procedure, major improvements arealso expected on the particle side (Ferguson et al 2012). With a better starting material, animproved MPI signal can be obtained at the same iron concentrations.

Slight image distortions and blurring, which result from the use of a system functionacquired on pure Resovist R© for the reconstruction of signal generated by particles entrappedin RBCs, make assignment of some MPI signal contributions difficult. Pure Resovist R© hadto be used to achieve a high SNR despite the limited scanner sensitivity and the requirementthat the calibration sample does not exceed the dimensions of a voxel. Here, the new andmore sensitive calibration approach using a ‘system calibration unit’ (Halkola et al 2012) incombination with better particles will allow the acquisition of a system function directly onloaded RBC samples. This will remove all effects related to the mismatch in particle sizedistribution and dynamics between aqueous solution and inner RBC volume. It would also bepossible to acquire a system function of particles accumulated in the liver, so that the liver signal

3976 J Rahmer et al

can be reconstructed more accurately. In the presented measurements, a discrepancy betweenthe homogeneously blackened liver in MRI and the rather inhomogeneously distributed MPIsignal in the liver is observed.

Future clinical MPI scanners are expected to deliver a spatial resolution quite similarto the one demonstrated on the mouse scanner (Borgert et al 2012). In view of the largerhuman anatomy, it can be expected that not only the heart chambers and the major vesselslike aorta and pulmonary veins, but also smaller vessels like the coronaries can be easilyresolved. Furthermore, the signal obtained from perfused tissue can deliver quantitative bloodvolume maps. MPI with loaded RBCs may therefore be a valuable tool for monitoring therapyresponse of cardiovascular interventions or medication over the course of time periods rangingfrom days to weeks.

Furthermore, newer scanners implement additional fields that increase spatial coverageby shifting the focus of the FOV over a larger region in space (Gleich et al 2010) or can evendecrease the selection field gradient to enable overview imaging of a large volume at reducedspatial resolution. These abilities will ease anatomical assignment of signal and will removethe limitations given by the fixed position of the FOV in the mouse scanner.

The rather constant signal level provided by iron-oxide loaded RBCs also simplifiesthe comparison of exams taken at different point in times. It furthermore opens the doorfor averaging of data by the use of gating or triggering techniques to further increase MPIsensitivity. In conventional bolus-based exams, these approaches are difficult to implementdue to the rapidly changing particle concentrations in the blood.

5. Conclusion

By loading RBCs with Resovist R©, MPI of the blood pool of living mice is feasible severalhours after injection with current MPI equipment. This has been demonstrated by visualizationof cardiac motion in MPI and by determination of the concentration of iron loaded RBCs inthe blood using MPS. These results prove the efficacy of the RBCs loading method forincreasing the blood retention time of iron oxide nanoparticles. A prolonged blood retentiontime enables therapy monitoring, but also more reliable concentration and blood volumeevaluations as compared to the dynamic concentration variations occurring in bolus exams.The achieved iron concentrations in the blood are close to the sensitivity limit of the currentequipment, but the real-time information delivered by the rapid 3D MPI acquisition givesenough additional information to allow assignment of signal to certain anatomies. Current andfuture improvements in particle performance, loading procedure, MPI hardware and calibrationas well as the transfer to human anatomy are expected to increase the signal levels and thusimprove image quality strongly.

Acknowledgment

The authors from Philips acknowledge funding by the German Federal Ministry of Educationand Research (BMBF) under the grant numbers 13N9079 and 13N11086.

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