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Biomaterials 35 (2014) 1627e1635

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Biomaterials

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Sensitive in vivo cell detection using size-optimizedsuperparamagnetic nanoparticles

Jesse Trekker a,b,*, Cindy Leten b, Tom Struys b,c, Vera V. Lazenka d,1, Barbara Argibay e,Liesbeth Micholt a, Ivo Lambrichts c, Willem Van Roy a, Liesbet Lagae a,d,Uwe Himmelreich b

a IMEC, Leuven, BelgiumbDepartment of Imaging and Pathology, Biomedical MRI/Mosaic, KU Leuven, Leuven, Belgiumc Lab of Histology, Biomedical Research Institute, Hasselt University, Diepenbeek, BelgiumdDepartment of Physics, Solid State Physics and Magnetism, KU Leuven, Leuven, BelgiumeDepartment of Neurology, Clinical Neuroscience Research Lab, Hospital Clinico Universitario, University of Santiago de Compostela, IDIS, Santiago deCompostela, Spain

a r t i c l e i n f o

Article history:Received 4 October 2013Accepted 1 November 2013Available online 15 November 2013

Keywords:MRIMagnetic nanoparticleCell labelingCell trackingMesenchymal stem cells

* Corresponding author. Tel.: þ32 16 281057.E-mail address: trekker@imec.be (J. Trekker).

1 Current address: Nuclear and Radiation PhysicsGroup, KU Leuven, Belgium.

0142-9612/$ e see front matter � 2013 Elsevier Ltd.http://dx.doi.org/10.1016/j.biomaterials.2013.11.006

a b s t r a c t

Magnetic nanoparticle (MNP) enabled cell visualization with magnetic resonance imaging (MRI) iscurrently an intensively studied area of research. In the present study, we have synthesized polyethyleneglycolated (PEG) MNPs and validated their suitability as MR cell labeling agents in in vitro and in vivoexperiments. The labeling of therapeutic potent mesenchymal stem cells (MSCs) with small core andlarge core MNPs was evaluated. Both MNPs were, in combination with a transfection agent, stablyinternalized into the MSCs and didn’t show an effect on cell metabolism. The labeled cells showed highcontrast in MRI phantom studies. For quantification purposes, the MRI contrast generating properties ofcells labeled with small core MNPs were compared with large core MNPs and with the commercialcontrast agent Endorem. MSCs labeled with the large core MNPs showed the highest contrast generatingproperties in in vitro phantom studies and in in vivo intracranial stereotactic injection experiments,confirming the sizeerelaxivity relationship in biological systems. Finally, the distribution of MSCs pre-labeled with large core PEGylated MNPs was visualized non-invasively with MRI in a glioma model.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Non-invasive imaging has greatly improved biomedical researchand has proven its value in diagnostic and therapeutic de-velopments [1e5]. Especially in the field of cell based treatments, inwhich assessing the fate of the administered cells is crucial, theseimaging techniques have contributed to the success of therapy [6,7].Magnetic resonance imaging (MRI) is one of themost powerful non-invasive imaging techniques, due to its safety, its excellent soft-tissue contrast and its high spatial resolution [8]. One of the maindrawbacks however, is its lowsensitivity compared to other imagingmodalities, hampering its direct applicability to the previouslymentioned advanced application for cell tracking. Ex vivo labeling ofthese cells with MR contrast agents partially overcomes this

Section, Nuclear Solid State

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problem [9]. Routinely used MR cell labeling agents are based onmagnetic nanoparticles (MNPs). The magnetic strayfields of theMNPs create local magnetic field inhomogeneities, acceleratingproton spin dephasing. This enhanced relaxation phenomenon in-duces hypointense signals in T2=T*

2 weighted MR images [10].Several MNPs have been designed and proposed as MR cell la-

beling agents. In general, they are built up of a magnetic iron oxidecore (IOC), which is surrounded by a stabilizing coating [11]. Forproducing high MRI contrast, highly crystalline IOCs with highmagnetization saturation values are primordial. A tight IOC sizecontrol is needed as this is strongly related to the magnetic prop-erties. Commonly used IOC synthesis methods are based on theprecipitation of iron salts in an aqueous environment [12], but lacka good size control. Organic solvent based MNP synthesis routes,such as the thermal decomposition method, are preferred forproducing monodisperse MNPs with tight size control [13], buthave only recently been used for MNP cell labeling purposes [14].This is partially due to the non-polar surface properties of thesynthesized IOCs, which demands a modification of the coating.

J. Trekker et al. / Biomaterials 35 (2014) 1627e16351628

Various molecules, such as lipids [15], sugars [16], blockpolymers[17] and surfactants [18] have been applied to the MNPs for thispurpose. This coating must provide a good stability to the MNPdispersion and must therefore be strongly anchored to the MNPs.Loosely bound coatings such as dextrans have been reported todegrade in the presence of an acidic environment such as the cellsendosomal compartments [19]. This creates a rapid modification ofthe magnetic properties and thus hampers long-term follow up byMRI. Additionally, the leached iron atoms have been reported toinduce reactive oxygen species and hence cytotoxicity.

The mechanism of MR contrast generation with MNPs is com-plex and is influenced by many variables. Many studies havefocused on understanding and modeling the effect of MNPs on theproton relaxation time. In the case of uniform distributions of ultra-small MNPs a complete description of this phenomenon has beenobtained [20]. For larger diameters, computational and analyticalmodels have been developed explaining the transverse relaxationprocess at high fields. Recently a newapproach has emerged, whereresearchers focus on the synthesis of clustered or aggregated MNPsystems [21e23]. It indeed has been shown that densely packedMNPs have relaxation rates and mechanisms different from evenlyspaced MNPs. To describe this behavior, studies have turned toMonte Carlo simulations to gain more insights in the relaxationprocesses [24]. From this literature, it is obvious that the develop-ment of MNPs as MR cell labeling agents requires control of severalparameters to be able to draw reliable conclusions from experi-ments. One can understand that the accumulation of MNPs incellular structures, such as endosomes, leads to a virtual aggrega-tion of the MNPs and thus to a changing in the induced relaxationrates [25,26]. It is therefore crucial to develop labeling protocolsthat result in a complete internalization of the MNPs. Also over-loading the cells with MNPs should be avoided, as it could formMNP deposits on the surface of saturated cells, inflicting cytotoxiceffects and hampering complete MNP internalization [25]. Inaddition, cell surface bound MNPs could be taken up by cell scav-engers such as macrophages after in vivo engraftment, leading tobiased results when determining cell biodistribution [1].

Mesenchymal stem cells (MSCs) are multipotent stem cells withself-renewal capacity that have been harvested from several tissuessuch as bone marrow, muscle and dental pulp [27]. Their stem cellnature allows them to differentiate in several cell types such asbone, cartilage and adipose tissue. They have also been shown toexhibit tumor-tropic homing and migration capacities, infiltratingtumor extensions and tracking distal tumor micro satellites [28].MSCs are therefore of high interest as a cytotherapeutic withinregenerative medicine and cancer therapy [29].

Keeping in mind the above mentioned concerns regarding celllabeling, we selected a small and a large IOC MNP to labeled MSCs.Previously, we have developed a procedure for the production ofPEGylated iron oxide MNPs with various core sizes [30]. TheseMNPs had tunable magnetic properties, had a narrow size distri-bution and were stable in biological fluids. It is the main aim of thisstudy to evaluate their use as cell labeling agents for in vivoMR cellvisualization. First, the extent of MNP cell uptake and the effect oftheMNPs on cell metabolismwere assessed. Based onMR phantomstudies, we selected the best performingMNP for further in vivoMRcontrast generation evaluation. Finally, we assessed with MRI andsubsequent histological validation the distribution of the labeledcells after intracranial injection in a glioma mouse model.

2. Materials and methods

2.1. Cell lines/animals

GL261 cells were obtained from Dr. S. Van Gool, KU Leuven and werecultured in DMEM þ glutamax medium (Gibco Invitrogen, Paisly, UK) supple-mented with 10% fetal bovine serum (FBS, Gibco Invitrogen, Paisly, UK) and 1%

penicillin/streptomycin (10,000 unit/mle10,000 mg/ml, Gibco Invitrogen, Paisly,UK). Mouse mesenchymal stem cells (mMSCs) were obtained from the Stem CellInstitute Leuven at KU Leuven and were further handled as previously described[31]. C57Bl6/j mice (Janvier labs, Le Genest-Saint-Isle, France) were used for thein vivo experiments. All animal experiments were conducted according to theEuropean Union Community Council guidelines and were approved by the localanimal ethics committee (KU Leuven).

2.2. Production of Seeds-mPEGSi and second seed mediated growth-mPEGSi

Seeds-mPEGSi and second seed mediated growth (SMG2-mPEGSi) were pre-pared in two steps. First the monodisperse MNPs (Seeds and SMG2 MNPs) weresynthesized and in a next step coated with triethoxy (methoxypolyethyleneoxy)silane, 5K (mPEGSi) (Laysan Bio, Inc, Arbab, AL, USA).

2.2.1. Synthesis of seeds and SMG2 MNPsSmall “Seeds” MNPs and larger “SMG2” MNPs were synthesized based on the

thermal decomposition and seed mediated growth method as previously reported[18,30].

2.2.2. PEGylated silane coatingThe silane functionalization of theMNPswas performed as described in Ref. [30].

In case of fluorescently taggedMNPs,10mg of FITC-PEGSi, 5K (Nanocs, Inc, New YorkCity, USA)was added to the functionalizationmixture. Prior to cell labeling, theMNPswere washed in a magnetic column (MACS, Miltenyi Biotec B.V., Leiden, TheNetherlands) with 10 ml of 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid, N-(2-Hydroxyethyl)piperazine-N0-(2-ethanesulfonic acid) (HEPES) buffer (10 mM, pH7.4, Sigma, Bornem, Belgium) solution andfinally dispersed in 1ml of the samebuffer.

2.3. Preparation of PLL-coated-mPEGSi-MNPs

One hour prior to cell incubation, the mPEGSi-MNPs were mixed with mMSCmedium and with 0.75 or 1.5 mg/ml PLL (388 kDa, Sigma, Bornem, Belgium) andvigorously shaken.

2.4. Cell labeling with PLL-coated-mPEGSi-MNPs

mMSCs were incubated with different concentrations of PLL-coated-mPEGSi-MNPs (200, 100, 50, 20, 10 mg Fe/ml medium) for 24 h. The MNP containing me-diumwas removed and the cells were washed three times with 1.5 ml of PhosphateBuffered Saline (PBS without Mg2þ and Ca2þ, Gibco Invitrogen, Paisly, UK) to removenon-attached MNPs. After washing, the cells were left overnight in 1.5 ml of freshmedium. The following day, the medium was removed, the cells were washed oncewith 1.5 ml of PBS and 0.5 ml of EDTA-trypsin (0.05%, Gibco Invitrogen, Paisly, UK)was applied to detach the cells from the well. The trypsinwas neutralized with freshmedium and the detached cells were collected in an Eppendorf tube. After a mildcentrifugation (1500 rpm for 5 min), the supernatant was discarded by decantationand the cells were resuspended in 1 ml of fresh medium. Total cell count was per-formed with an automatic cell counter (Chemometec, Lillerod, Denmark). For allfurther cell characterization steps, the just described labeling techniquewas applied.

2.5. Intracellular iron determination

For Seeds-mPEGSi and SMG2-mPEGSi, uptake the amount of iron per cell wasquantified by dissolving 50,000 labeled cells in 0.5 ml of 36% HCl (Honeywell, Sleeze,Germany). Distilled water was added to exactly match a volume of 5 ml. The ironconcentration was determined by inductive coupled plasma optical emissionspectroscopy (ICP-OES) (Varian Inc., Palo Alto, USA). The intensity of the emissionline at 238.204 nm was measured for iron and compared to a standard solution.

2.6. Microscopic procedures

2.6.1. Prussian blue stainingTo confirm the uptake of the MNPs by the cells, the iron in the labeled cells was

stained by Prussian blue. Hereto, adherent cells were washed with PBS and subse-quently incubated for 20 min in a 2% aqueous solution of HCl and a 2% aqueoussolution of potassium ferrocyanide (Sigma, Bornem, Belgium).

2.6.2. Confocal laser microscopy (CFLM)Cells (50,000) were plated on a glass cover slide and fixed in paraformaldehyde

(4% PFA and 4% Sucrose in PBS) at 37 �C for 10 min. Next, they were permeabilizedfor 5 min in 0.1% Triton X-100/PBS. Actin was visualized with Phalloidin coupled tofluorophore Alexa-568 (Invitrogen, Paisly, UK) and nuclei were stained with theHoechst compound (Hoechst 33342, Invitrogen, Paisly, UK). CFLM images were ac-quired with a Zeiss LSM 780 (Zeiss, Jena, Germany) confocal microscope equippedwith a Blue Diode 430 nm laser, an Argon 488 nm laser and a DPSS 561 nm laser.

2.6.3. Transmission electron microscopy (TEM)For TEM analysis, labeled cells were seeded at a density of 5 � 104 cells/cm2 on

plastic Thermanox� coverslips. After adherence, the cells were fixed with 2%glutaraldehyde (Laborimpex, Brussels, Belgium) in 0.05 M sodium cacodylate buffer

J. Trekker et al. / Biomaterials 35 (2014) 1627e1635 1629

(Aurion, Wageningen, The Netherlands) (pH ¼ 7,3) at 4 �C. Following fixation, thesamples were washed twice for 5 min with 0.05 M sodium cacodylate (pH ¼ 7.3)(Sigma Chemical Co, St. Louis, USA) and 0.15 M saccharose (Sigma Chemical Co, St.Louis, USA) at 4 �C. Postfixation was achieved by treating the samples with 2%osmiumtetroxide (Aurion, Wageningen, The Netherlands) in 0.05 M sodium caco-dylate buffer (pH ¼ 7.3) for 1 h at 4 �C. The samples were dehydrated by exposingthem to increasing concentrations of acetone (Honeywell, Sleeze, Germany). Thesamples were impregnated overnight in a 1:1 mixture of acetone and araldite epoxyresin (Aurion, Wageningen, The Netherlands) at room temperature. In a followingstep, the samples were embedded in araldite epoxy resin at 60 �C. After applying thepop-off method, the embedded samples were cut in sections of 40e60 nm, using aLeica EM UC6 microtome (Leica, Groot Bijgaarden, Belgium). Sections were thentransferred to 50 mm mesh copper grids (Aurion, Wageningen, The Netherlands)coated with 0.7% formvar (Sigma Chemical Co, St. Louis, USA). The samples wereautomatically stained using a Leica EM AC20 (Leica, Groot Bijgaarden, Belgium) with0.5% uranyl acetate and a stabilized solution of lead citrate (both from Laurylab, SaintFons, France). TEM analysis was performed with a Philips EM208 S electron mi-croscope (Philips, Eindhoven, The Netherlands) operated at 80 kV. The microscopewas provided with a Morada Soft Imaging System camera to acquire high resolutionimages of the evaluated samples. Digital processing of the images was performedwith the iTEM-FEI software (Olympus SIS, Munster, Germany).

2.7. Magnetic characterization

Hysteresis magnetization measurements were performed to study the magneticproperties of the MNPs after cell internalization using a SQUID magnetometer(MPMS XL-5, Quantum Design inc, San Diego, USA) in RSO mode. Labeled cells(400,000) were resuspended in 100 ml of PBS and dispersed on a cotton ball andmounted in the SQUID magnetometer using a plastic straw. The hysteresis curveswere measured at 300 K and fitted with a Langevin function using Origin 8.1 (Ori-ginLab Co., Northampton, USA).

2.8. Cell viability assay

For assessing the viability of the cells after labeling, 100,000 cells were plated in96 well plates and allowed to adhere. After adherence, the medium was replacedwith 90 ml of freshmedium and 10 ml of PrestoBlue� reagent (Invitrogen, Paisly, UK).After 90 min, the top-read fluorescence of labeled cells was determined on aninfinite M1000 microplate reader (Tecan, Männedorf, Switzerland) at excitation560 nm and emission 590 nm and compared to non-labeled cells.

2.9. In vitro magnetic resonance imaging of labeled cells

In preparing a phantom background, 60 ml of liquid 1.5% agar gel (microbiologygrade, Sigma Aldrich, Bornem, Belgium) was put into a cylindrical custom builtTeflon holder (diameter 7 cm). Next, freely suspended Eppendorf tubes were placedinto the liquid agar gel and the whole phantom was placed at 4 �C. After solidifi-cation the Eppendorf tubes were carefully removed, leaving an Eppendorf tubeimprint in the agar phantom. In the following step, 100,000 cells labeled withdifferent concentrations of Seeds-mPEGSi or SMG2-mPEGSi or Endorem were sus-pended in 100 ml of PBS andmixed homogenously with 100 ml of 2% agar gel. The cellsuspension was immediately transferred to the different imprints in the agarphantom at a final cell density of 500 cells/ml. As a control 100,000 non-labeled cellswere used. The phantomwas topped up with 10 ml of 1.5% agar gel and placed in aBruker Biospec 9.4 T small animal MR scanner (Bruker Biospin, Ettlingen, Germany;horizontal bore, 20 cm) equipped with actively shielded gradients (600 mT m�1). Aquadrature radio-frequency transmit/receive resonator (inner diameter 7 cm,Bruker Biospin) was used for data acquisition. Measurements of the T*2 relaxationtimes were performed using a multigradient echo pulse sequence with 8 TE in-crements (TR ¼ 1500 ms, first TE ¼ 4.44 ms with increments of 6.75 ms, 400 � 400matrix, 187.5 � 187.5 mm in plane resolution, 0.35 mm slice thickness, 12 slices).ImageJ (NIH, USA) was used for further image processing. Signal intensities overtime were determined as mean values of one slice of a homogenous section of thecell loaded areas in the agar phantoms. The relaxation time was drawn from thebest-fit least square first order exponential decay line with variable offset of themeasured values. For these fittings, Origin 8.1 was used. The T*2 relaxation times oflabeled cells versus intracellular iron uptake were evaluated.

2.10. Stereotactical injections

Animals were anaesthetized by an intraperitoneal injection with a mixture ofKetamine (4.5 mg/kg)/medetomidin (0.6 mg/kg). Local analgesia (2% xylocain) andantibiotics (ampiciline) were administered. After fixation of the animals in a ste-reotactic device (Stoelting Europe, Dublin, Ireland) cells were injected with a 10 mlHamilton syringe, equipped with a 22 G needle, into the right and left striatum ofC57Bl6/j mice at following coordinates: 0.5 mm anterior and 2.0 mm lateral tobregma and 3.0 mm from the dura.

2.10.1. Control injectionsFor the control injections, 9 animals were used. In total 18 injections were

performed of which 1 with PBS and 1 with 300,000 non-labeled cells. Eight

injections occurred with cells labeled with SMG2-mPEGSi and 8 with cells labeledwith Endorem. Of the eight injections, 4 were with 100,000 cells and 4 with300,000 cells. Two of the 4 injections were labeled with a low concentration ofMNPs (w5 pg Fe/cell) and 2 were labeled with a relative high concentration of MNPs(w10 pg Fe/cell).

2.10.2. Injections in a glioma modelFor the glioma tumor model, 2.5 �105 GL261 cells were injected in one C57Bl6/j

mouse two weeks prior to stem cells injection. 500,000 stem cells of which 1% werelabeled with SMG2-mPEGSi were injected intracranial (w10 pg Fe/cell). MRI wasperformed 3 days post injection to follow-up stem cell location.

2.11. In vivo magnetic resonance imaging of labeled cells

All MR images were acquired with a 9.4 T Biospec small animal MR scanner(Bruker Biospin, Ettlingen, Germany, horizontal magnet) equipped with activelyshielded gradients of 600 mT m�1 and using a 7 cm linearly polarized resonator fortransmission and an actively-decoupled dedicated mouse surface coil for receiving(Rapid Biomedical, Rimpar, Germany). Prior to scanning, mice were anaesthetizedwith 2% isofluorane for induction and 1.5% isofluorane for maintenance. Tempera-ture and respiration were monitored throughout the experiment and maintained at37 �C and 100 � 20 breaths min�1. Furthermore, 3D T*2 weighted gradient echoimages (FLASH, TR: 100 ms, TE: 12ms, flip angle: 20� , 78 mm isotropic, field-of-view:2.0� 1.5� 0.75 cm) were acquired to visualize the labeled stem cells. The volume ofthe signal void was determined by outlining it manually on all slices using the 5.1Paravision software (Bruker Biospin, Ettlingen, Germany). Furthermore, 2D T2weighted multi-slice-multi-echo scans (coronal orientation, TR: 3000 ms; TE:50.2 ms; in plane resolution: 78 mm/pixel; field-of-view: 2.0 � 2.0 cm; 16 slices of0.05 cm) were acquired for visualization of the tumor.

2.12. Histological assessment of in vivo cell engraftment

For performing a Trichome/Prussian Blue staining, animals were sacrificed by anintraperitoneal overdose of Nembutal (300 ml; Ceva) and subsequently perfusedwith 4% ice-cold paraformaldehyde (PFA) solution (Sigma, Bornem, Belgium). Afterovernight postfixation in 4% PFA, the brain tissue was stored in a 0.1% sodium azidesolution (Sigma, Bornem, Belgium) at 4� C. Brains were embedded in paraffin, 5 mmparaffin sections were sliced and a Masson Trichome staining was performed. Sec-tions were deparaffinized in xylol, followed by rehydration through ethanol. Sub-sequently, sections were stained with hematoxyline, ponceau/fuchine and anilineblue. Finally, sections were dehydrated and mounted with di-n-butyl phthalate inXylene (Sigma, Bornem, Belgium). Furthermore, Prussian blue staining was per-formed by combining a 20% aqueous solution of hydrochloric acid and a 10% aqueoussolution of potassium ferrocyanide and counterstained with nuclear fast red stain-ing. Finally, slices were scanned with a Mirax desk scanner (Zeiss, Jena, Germany)and pictures were taken with the Mirax viewer software (Zeiss, Jena, Germany).

2.13. Statistical analysis

Values are represented as the mean � standard deviation (SD). Significant dif-ferences between the slopes of a linear regression of the experimental data weredetermined using Graphpad Prism 5 software (Graphpad, California, USA). The de-gree of significance is indicated when appropriate (*: p < 0.05; **: p < 0.01; ***:p < 0.001).

3. Results

By following the procedure described in Ref. [30], narrow sizedistributed MNPs were obtained with hydrophilic surface proper-ties. Two batches were selected for further use. The first batch,Seeds-mPEGSi, had a mean core size of w7 nm and the secondbatch, SMG2-mPEGSi, was on average w13 nm in diameter. Mousemesenchymal stem cells (mMSCs) were further incubated withboth MNP batches.

3.1. Cellular internalization of NPs

To quantify the efficiency of uptake during the pulse period, themMSCs were exposed to different concentrations of Seeds-mPEGSiand SMG2-mPEGSi MNPs. If no transfection agent was used incombination with the MNPs, no iron could be detected in the cells(data not shown). As previously reported, poly-L-lysine (PLL) can beused as a transfection agent to enhance the uptake of MNPs in stemcells [32]. Two concentrations of PLL in combination with 5 expo-sure amounts of MNPs were used, revealing a concentrationdependent uptake mechanism (Fig. 1). At the maximum exposure

Fig. 1. Intracellular iron per exposure concentration determined by ICP-OES.

J. Trekker et al. / Biomaterials 35 (2014) 1627e16351630

of 200 mg Fe/ml, an intracellular iron amount of Seeds-mPEGSi wasmeasured of 6.5 � 0.2 pg Fe per cell and 15.6 � 2.9 pg Fe per cellwith 0.75 mg and 1.5 mg PLL per ml, respectively. At the sameexposure concentration, SMG2-mPEGSi were internalized atamounts of 8.0 � 0.4 pg Fe per cell and 16.5 � 1.3 pg Fe per cell atPLL concentrations of 0.75 mg/ml and 1.5 mg/ml, respectively.

When labeling mMSCs with MNPs, an incubation pulse of 24 hand a chase of 12 h was followed to ensure full cell internalization,thereby limiting cell surface bound NPs. Several microscopictechniques were used to confirm the MNP internalization. First ofall, staining of the iron of the MNPs with Prussian blue revealedthe typical blue color in the cells for both the Seeds-mPEGSi andSMG2-mPEGSi labeled cells. As can be seen in Sup Fig. 1, the bluecolor is homogenously distributed amongst the cells and almostevery cell showed blue staining deposits. For further detail,confocal microscopy was used on cells incubated with fluo-rescently tagged MNPs. Z-stacking of the confocal images ensuredvisualization of the cell internals and not of the cell surface. Fig. 2Adepicts control cells devoid of MNPs and Fig. 2B clearly visualizesthe internalized MNPs. By applying an F-actin staining and a nu-cleus staining, the localization of the MNPs can be further inves-tigated, confirming that MNPs were confined to a perinuclear ringin the cells. In a next step, high resolution TEM images were takenfrom labeled cells.

Fig. 2. Confocal fluorescent microscopy images of fluorescently stained mMSCs. (A) ContromMSCs labeled with (B) FITC-PEGSi-MNPs (white arrows) (N ¼ nucleus). The scale bar is 5

As can be seen in Fig. 3, the MNPs are found in small vesiclesinside of the cells. These images also reveal that the cellular outermembrane is devoid of MNPs and that all surface boundMNPswereinternalized during the chase period.

3.2. Effect of internalization on the MNPs and on the cells

Applying MNPs as MR cell labeling probes requires robustmagnetic properties of the probes. Therefore in a following step,the effect of the internalization on the structural and magneticproperties of the Seeds-mPEGSi and SMG2-mPEGSi was investi-gated. The diameter of the MNPs was determined by TEM (SupFig. 2AeD). The internalized Seeds-mPEGSi measured7.5�1.0 nm and the SMG2-mPEGSi were 12.2� 1.5 nm in diameter.A SQUID study was used to determine the magnetic properties.Cells labeled with Seeds-mPEGSi or SMG2-mPEGSi portrayed noremnant magnetization. SMG2-mPEGSi labeled cells attained fastermagnetization saturation than cells labeled with Seeds-mPEGSi.When determining the magnetic diameter of the MNPs based onthe Langevin fitting of the hysteresis curves, a value of 6.7e7.0 nmwas found for the Seeds-mPEGSi and a magnetic diameter of 10.2e10.7 nm was derived for the SMG2-mPEGSi. The lower value wasobtained when using the parameters for magnetite and the highervalue of the range was calculated based on maghemite parameters.

The effect of the MNPs on the cell viability was evaluated next.The cell count and metabolic activity of labeled versus non-labeledcontrol cells were determined 36 h post initial MNP exposure.Possible acute cytotoxicity was investigated on cells, which hadinternalized different amounts of MNPs. Based on Fig. 4, no sig-nificant differences in cell count or metabolic activity could be seenin the labeled cells versus the unlabeled control cells. This was thecase for cells labeled with Seeds-mPEGSi and for cells labeled withSMG2-mPEGSi. As a positive control for hampered metabolic ac-tivity, 2% Triton-X was added to a set of cells.

3.3. Transverse relaxivity ðR*2Þ of labeled mMSCs in vitro

To assess the transverse relaxivity, mMSCs were labeled withdifferent amounts of Seeds-mPEGSi, SMG2-mPEGSi and Endoremand homogeneously suspended in an agar phantom at 500 cells/ml(Fig. 5A). Multi gradient echo pulse sequences were used todetermine the R*2 relaxation rate of the labeled cells. The relaxationrate was plotted versus the concentration of iron, thereby enablinga comparison of the contrast generation of the different cell labels(Fig. 5B). A linear relationship was found between the R*2 and the

l cells didn’t show green fluorescence, which in contrary could clearly be detected in0 mm.

Fig. 3. Bright field TEM images of mMSCs labeled SMG2-mPEGSi. (A) The outer cell membrane is clearly devoid of MNPs. (B) The MNPs are packed together in endomembranestructures (black arrow). Scale bar in image A is 5 mm and in image B is 2 mm.

J. Trekker et al. / Biomaterials 35 (2014) 1627e1635 1631

MNP concentration, consistent with published studies [26,33], andenabling the calculation of the transverse relaxivity (R*2�slope ofthe linear regression curve). For mMSCs labeled with Seeds-mPEGSi, a value of 631 � 35 mM

�1 s�1 was calculated. SMG2-mPEGSi labeled cells resulted in 972 � 46 mM

�1 s�1 and cellslabeled with Endorem showed a value of 655 � 22 mM

�1 s�1.

3.4. Assessment of engrafted labeled cells in vivo

For further in vivo applications, the engraftment site of stereo-tactically injected cells was investigated with histology and TEM.

Fig. 4. Cell viability tests for mMSCs labeled with different concentrations of mPEGSi-MNPs. Upper graph shows cell count (percentage compared to unlabeled control cells)and lower graph shows their metabolic activity after labeling (percentage compared tounlabeled control cells) as determined by a Presto Blue metabolic assay (n � 3).

Labeled and non-labeled cells were injected in the left and rightstriatum of mouse brains. Fig. 6A1 shows a full brain slice in whichthe graft of cells labeled with SMG2-mPEGSi can be seen as indi-cated by the white arrow. When looking closer (Fig. 6A2) at theengraftment site, the MNPs can be seen as black deposits in the cell(white arrows). In more detail, the TEM analysis of the brain tissuewith labeled cells shows the MNPs inside the cells (Fig. 6A3)enwrapped by a membrane (Fig. 6A4). In Fig. 6B1e2, the injectionsite of non-labeled cells can be seen, which is clearly devoid of anyMNP deposits. Determined by visual inspection no obvious differ-ences in cell distribution or volumes for the various injections wereobserved.

3.5. In vivo contrast generation of injected labeled cells

For determining the efficiency of contrast generation of thelabeled cells in vivo, cells were labeled with different concentra-tions of SMG2-mPEGSi and Endorem. A set number of these cells(100,000 or 300,000) were engrafted in the mouse brain. Fig. 7Adepicts the signal void which is created by the labeled cells in T*

2weighted MR images as indicated by the black arrow. Almost nosignal void could be observed in animals with control PBS in-jections or injection of non-labeled cells. The total hypointensevolume was calculated for each 3D brain image and compared tothe total amount of iron that had been injected. As can be seen inFig. 7B, hypointense volumes of cells labeled with SMG2-mPEGSiwere on average 4e5 times larger than for Endorem labeled cellsfor similar amounts of total internalized iron mass. A maximum ofhypointense volume of 33 mm3 was obtained for cells labeled withSMG2-mPEGSi. Only slight differences were observed when im-aging 2 days post injection or 8 days post injection (data notshown).

3.6. Visualization of cell distribution

The in vivo cell distribution in a tumor model was assessedafter stereotactic injection of SMG2-mPEGSi labeled cells in thebrain of glioma bearing C57Bl6/j mice. Labeling only 1% of theinjected cells allowed sufficient visualization of the labeled stemcells relative to the tumor lesion. Fig. 8A shows T2 and T*

2weighted brain images prior and after cell injection. The T2weighted images revealed the brain tumor as a hyperintensespot, indicated by the black arrow. Only after injection of thelabeled cells, a hypointense spot was generated by the engraftedcells, which surrounded the tumor as specified by the white ar-row and which seemed more pronounced in the T*2 weighted MRimages versus the T2 weighted MR images. Prussian blue stainingof the brain slices indicated several blue iron deposits at theoutskirts of the glioma (Fig. 8B).

Fig. 5. (A) T*2 weighted MR images of agar phantoms loaded with labeled mMSCs. The numbers indicate the initial incubation concentration in mg Fe/ml. (B) Transverse relaxation

rate ðR*2Þ plotted versus intracellular iron amount (Iron concentration (mM) or pg iron per cell at 500 cells/ml) of mMSCs labeled with different amounts of Seeds-mPEGSi, SMG2-mPEGSi or Endorem (n � 3).

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4. Discussion

The non-invasive visualization of the fate and distribution oftransplanted cells by MRI becomes increasingly custom in the fieldof biomedical research. MRI has in this regard outperformed othernon-invasive imaging techniques such as SPECT and PET for itssafety, its high soft tissue contrast and high anatomical informationprovided by those images. This technique does require the cells tobe pre-labeled with MRI contrast agents such as magnetic nano-particles [7]. Several reports have developed and proposedMNPs asMR cell labeling agents, however, these studies have limited theirassessment to the contrast generating properties of the MNPs assuch. Indeed, it has been reported that MNP clustering [21,24] andcell compartmentalization [26] drastically changes the contrastgenerating mechanisms of the MNPs.

In the present study, we have evaluated a MNP based system,which enables efficient and non-invasive imaging of the

Fig. 6. (A1, A2 and B) Trichrome staining of brain sections with injected labeled mMSCs (Ainjected cells. Deposits of SMG2-mPEGSi are visible in A2 (white arrows). (A3, A4) Bright fieldare indicated with the black arrow. (N ¼ nucleus). Scale bar in A1 and B1 is 1000 mm, in A2

distribution of therapeutic cells after administration. To reach this,several MNP characterization techniques and MNP cell interactionstudies were executed both in vitro and in vivo, which allowedselecting the better performing MNP system. Previously, we haveshown that by applying the thermal decomposition method and aseed mediated growth method, MNPs could be synthesized withdifferent well-defined core sizes of 7 and 13 nm and a narrow sizedistribution. After coating these MNPs with mPEGSi, stable MNPdispersions could be obtained in biological solutions [30]. Wetherefore aimed to select the most efficient MNP system to be usedas a cell labeling agent for in vivo MRI. Two MNPs, one with a coresize of 7 nm (Seeds-mPEGSi) and one with a core size of 13 nm(SMG2-mPEGSi) were assessed. The contrast generating propertiesin vitro and in vivo were compared to cells labeled with the com-mercial contrast agent Endorem.

Labeling with Endoremwas not shown as this has already beenpreviously reported on [32]. Cellular uptake was mainly governed

1, A2) and non-labeled mMSCs (B). The white arrows in panel A1 and B1 indicate theTEM images of brain section of injected labeled mMSCs. Endosomes with SMG2-MNPsand B2 is 50 mm, in A3 is 2 mm and in A4 is 200 nm.

Fig. 7. (A) T*2 weighted MR images of mice brain stereotactily injected with PBS or with 100,000 mMSC, which were labeled with SMG2-mPEGSi or Endorem. The black arrow

indicates the injection site. (B) Hypointense voxel volume determined based on T*2 weighted images plotted versus the total mass of iron injected with the cells. (B0) Zoom of low end

part of graph B. The dotted lines are for visual clarity and do not indicate a linear dependency of the variables.

J. Trekker et al. / Biomaterials 35 (2014) 1627e1635 1633

by the coating of the MNPs. In general, incorporation of poly-ethylene oxide (PEO) units stabilizes the MNPs and limits biologicalinteractions [34]. Consequently, limited ironwas found in cells onlyincubated with the mPEGSi-MNPs (Fig. 1). In combination with atransfection agent, MNP uptake occurredwhich apparently reacheda saturation level of internalized iron. The Seeds-mPEGSi andSMG2-mPEGSi were internalized in similar amounts, which is ex-pected for similarly coated MNPs within the same size range. Thestabilizing effect of the mPEGSi coating may on one hand limit highamounts of MNP internalization, but it is also important for in vivoapplications. MNPs with limited stability due to a rapidly degradingcoating, like citrate, have shown to remain deposited on the surfaceof cell membranes, hampering their efficiency as a cell label andmay induce oxidative stress due to the formation of reactive oxygenspecies [25]. Prussian blue staining and CLSM images confirmed theuptake of both Seeds-mPEGSi and SMG2-mPEGSi and showed theMNPs to be located in the perinuclear area. No blue deposits orMNP related fluorescence were seen at the cell surface. In evenmore detail, TEM images of the labeled cells clearly indicated MNPsinternalized in vesicular structures and a cell membrane devoid ofMNPs. It was thus confirmed that with the used protocol a com-plete internalization of the MNPs could be achieved. Cell surfaceassociated MNPs could result in label transfer to cells of the host,leading to unspecific contrast and possible misinterpretation of theMR results.

After cellular internalization, the MNPs can be transformed bytheir incorporation into acidic vesicles like lysosomes [35e37].Consequently, it is likely that this affects their magnetic propertiesand thus their efficiency to be used as MRI contrast agents. Withinour time course, the SQUIDmeasurements didn’t show any effect ofthe cellular uptake on the structural and magnetic properties of theMNPs. Size determination based on the TEM images (structural)and Langevin fitting (magnetic) revealed similar sizes for the syn-thesizedMNPs [30] compared to the internalizedMNPs (Sup Fig. 2).This was the case for both Seeds-mPEGSi and SMG2-mPEGSi and isin concert with a previous report [38]. MNP internalization by thestem cells also did not lead to a cytotoxic effect at the concentra-tions used in our experiments as indicated by the unchangedrelative cell numbers and unchanged relative metabolic activity atdifferent intracellular iron concentrations. Cytotoxicity is mainlyinflicted by the amount of iron released after intracellular

degradation [35], which depends on the total internalized amountof iron per cell and MNP stability. In general, the maximal ironuptake we obtained for Seeds-mPEGSi and SMG2-mPEGSi wasabout 15 pg per cell. As this value is at the low to medium endcompared to other uptake studies [35], a clear cytotoxicity was notexpected.

Nonetheless these amounts were sufficient to induce a contrastenhancement in MRI phantom studies [32,39]. Moreover a quan-titative assessment of the efficiency of contrast generation waspossible for the two newly developed MNPs, Seeds-mPEGSi andSMG2-mPEGSi and compared to a commercially available contrastagent, Endorem. Previous studies evaluating the relaxivity of MNPshave shown a higher effect on T*

2 versus T2 weighted images[26,33], which was linked to the cell internalization of the MNPs.We therefore focused on T*

2 measurements in phantom studies tocompare relaxation rates (Fig. 5). Based upon these measurements,the SMG2-mPEGSi outperformed both Seeds-mPEGSi and Endoremin their MRI contrast enhancing efficiency. This is consistent withthe finding that larger magnetic cores (SMG2-mPEGSi, 13 nm) havea larger effect on the dephasing of diffusing protons compared tothe smaller Seeds-mPEGSi (7 nm) [21,24,40] or compared toEndorem, which consists of clustered cores in the size range of 5e6 nm [41]. Hereby, several parameters need to be considered. Firstof all, nanoscale surface phenomena decrease the magnetizationsaturation values of smaller cores [42]. The related decreasedstrength of the stray field affects the proton dephasing to a lesserextent. Both, Seeds-mPEGSi and SMG2-mPEGSi have a similarcoating made up of a PEG polymer of molecular weight 5000. Astudy by Tong et al. showed a dependency of R2 relaxivity on thecore size and the PEG chain length, where the relaxivity of a core of5 nm was highly diminished by a PEG of 5000 MW. This dimin-ishing effect of the PEG chain length was not that large for a largercore of 14 nm [40], providing an additional explanation why theoverall volume magnetization (core and PEG coating) of the Seeds-mPEGSi MNPs was smaller than the volume magnetization of theSMG2-mPEGSi MNPs.

The in vitro evaluation allowed selecting the SMG2-mPEGSi forfurther in vivoMRI assessment. It must be noted though, that in vivoimaging is profoundly different from imaging in phantom set-ups.Parameters that influence the in vivo imaging, such as correlationand diffusion time, depend strongly on the micro-environment of

Fig. 8. (A) T2 weighted and T*2 weighted MR images of mice brain with a glioma (black arrow). Images were taken pre and post injection of mMSCs labeled with SMG2-mPEGSi. (B)

H1 and Prussian blue staining of a mouse brain section with a glioma. Blue iron deposits of the SMG2-MNPs can be seen in the periphery of the tumor. Scale bar in B1 is 2000 mm andin B2 is 100 mm (n ¼ 1). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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the protons, which, in the case of MNP enhanced MR imaging, isinfluenced by iron distribution, cell water content, surroundingmacromolecules etc. These conditions are difficult to mimic in vitro.Still, phantom studies remain of interest for MNP enhanced cellimaging as their value lies in the ability to quantitatively estimateunder well-defined conditions the influence of NP concentrationand the type of NP. The in vivo assessment was as much as possibledefined by injecting distinct cell numbers loaded with definedconcentrations of SMG2-mPEGSi or Endorem in the mice striata.The injection sitewith the labeled cells was clearly visible in theMRimages (Fig. 6), in histology and in more detail in TEM, whichrevealed intracellular SMG2-mPEGSi MNPs, pointing towardsretention of the MNPs in the cells (Fig. 6). This is, similarly to thein vitro study, important when further in vivo validating the SMG2-mPEGSi MNPs. Although volume calculations of the signal voids inT*2 weighted MRI has been shown to be prone to variations [43], itdid allow us to compare the signal voids created by cells labeledwith SMG2-mPEGSi MNPs versus cells labeled with Endorem. Thecalculations per amount of iron revealed larger volumes for SMG2-mPEGSi MNPs versus Endorem, which were on average 1.5e2 timeslarger.

In a last step, the labeling of the cells with SMG2-mPEGSi MNPsallowed to visualize the cell distribution after injection in a gliomamodel. This was further confirmed by histology. Being able tofollow this distribution has been shown inevitable to determine theonset of therapy in a bystander killing treatment of brain gliomasusing the therapeutic cells as transport agents for chemothera-peutics [44]. Finally, it must be noted thatMNP visualization byMRIremains an indirect technique and is always related to the totalamount of MNPs and not to the amount of cells. Cell proliferation,cell death or lysis will hamper exact and reproducible MRI quan-tification especially over longer time points. As the contrast gen-eration of the MNPs is an inherent property of the MNP, it is notrelated to the cell’s viability and cannot be used as a cell functionimaging probe. Combination with optical imaging, in particularwith bioluminescence, can aid in this regard.

5. Conclusion

The results of our study showed that the relaxivity of labeledcells depends on the core size of the internalized MNPs. Althoughdifficult to quantify, a similar effect was seenwith the volume of thesignal voids created in in vivo injected labeled cells. Furthermore,mPEGSi coated MNPs in combination with a transfection agent can

be sufficiently taken up by therapeutic cells, such as mMSCs, forfurther use in MRI cell distribution studies. The labeling protocolensured a full internalization of theMNPswith a plasmamembranedevoid of MNPs, which is a prerequisite for a successful MRIcontrast enhanced efficiency assessment. Finally, evidence has beenset forth that these MNPs can be used to assess the distribution oftherapeutic cells after engraftment in a tumor. This can providecrucial information for the timely administration of co-drugs incell-delivered chemotherapy.

Acknowledgments

JT is the recipient of a research grant from the IWT-Vlaanderen.LL, IL and UH are recipients of an IWT grant-sponsoring projectSBO80017 (‘Imagine’). UH received financial support from the KULeuven Program financing ‘IMIR’, from the European Commissionfor EC-FP7 network ‘ENCITE’ (2008-201842), and from the ‘Vibrant’project (FP7-NMP-2008-228933). The valuable help of Tim Stey-laerts with the MNP synthesis is highly appreciated. Ashwini AtreKetkar is thanked for her support with the cell culture. The excel-lent technical assistance of Marc Jans in performing the TEM ex-periments is gratefully acknowledged. Gratitude goes to dr. TomDresselaers for his help with the MRI measurements and BrittVandenbroeck from the Material Science Institute (MTM) of the KULeuven is acknowledged for her help with the ICP-OESmeasurements.

Appendix A. Supplementary data

Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.biomaterials.2013.11.006.

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