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Biomaterials xxx (2014) 1e16

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Biomaterials

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Ultrasmall superparamagnetic iron oxide nanoparticle prelabelling ofhuman neural precursor cells

Steven S. Eamegdool a, Michael W. Weible II a,b, Binh T.T. Pham c, Brian S. Hawkett c,Stuart M. Grieve d, Tailoi Chan-ling a,*

aDepartment of Anatomy and Histology, Bosch Institute, Sydney Medical School, University of Sydney, NSW 2006, AustraliabBiomolecular and Physical Sciences, Griffith University, QLD 4111, Australiac School of Chemistry, Key Centre for Polymers and Colloids, University of Sydney, NSW 2006, Australiad Sydney Translational Imaging Laboratory, Sydney Medical School, University of Sydney, NSW 2006, Australia

a r t i c l e i n f o

Article history:Received 26 February 2014Accepted 21 March 2014Available online xxx

Keywords:MRI (magnetic resonance imaging)BiocompatibilityIron oxide nanoparticlesNeural cellStem cellsRegenerative medicine

* Corresponding author. Discipline of Anatomy anSchool, Anderson Stuart Building F13, The University oAustralia. Tel.: þ61 2 9351 2596; fax: þ61 2 9351 655

E-mail address: tailoi@anatomy.usyd.edu.au (T. Ch

http://dx.doi.org/10.1016/j.biomaterials.2014.03.0610142-9612/Crown Copyright � 2014 Published by Els

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a b s t r a c t

Stem cells prelabelled with iron oxide nanoparticles can be visualised using magnetic resonance imaging(MRI). This technique allows for noninvasive long-term monitoring of migration, integration and stemcell fate following transplantation into living animals. In order to determine biocompatibility, the presentstudy investigated the biological impact of introducing ultrasmall superparamagnetic iron oxide nano-particles (USPIOs) into primary human fetal neural precursor cells (hNPCs) in vitro. USPIOs with a meandiameter of 10e15 nm maghemite iron oxide core were sterically stabilised by 95% methoxy-poly(ethylene glycol) (MPEG) and either 5% cationic (NH2) end-functionalised, or 5% Rhodamine Bend-functionalised, polyacrylamide. The stabilising polymer diblocks were synthesised by reversibleaddition-fragmentation chain transfer (RAFT) polymerisation. Upon loading, cellular viability, total ironcapacity, differentiation, average distance of migration and changes in intracellular calcium ion con-centration were measured to determine optimal loading conditions. Taken together we demonstrate thatprelabelling of hNPCs with USPIOs has no significant detrimental effect on cell biology and that USPIOs,when utilised at an optimised dosage, are an effective means of noninvasively tracking prelabelledhNPCs.

Crown Copyright � 2014 Published by Elsevier Ltd. All rights reserved.

1. Introduction

Neural stem cells (NSCs) in the mammalian brain can give rise toastrocytes, neurons and oligodendrocytes and functionallycontribute to cognition and repair processes after nervous tissueinjury. Transplanted NSCs are promising candidates for providingbeneficial effects on recovery from central nervous system (CNS)trauma and neurodegenerative disorders. However, clinical appli-cations remain elusive, positive-outcomes limited and significantroadblocks to clinical translation remain [1]. Critical shortcomingsof current transplantation methodologies are low cellular survival,limited migration through host parenchyma and limited functionalintegration with the host neuronal circuitry [2]. Typical survivalranges recorded fall between 10 and 20% of engrafted cells [3e5],

d Histology, Sydney Medicalf Sydney, Sydney, NSW 2006,6.an-ling).

evier Ltd. All rights reserved.

ol SS, et al., Ultrasmall superx.doi.org/10.1016/j.biomateri

and of these,migration from the initial site of engraftment generallyreaches amaximumdistance of between 700 and 800 mm [6]. Thesefactors mean that only a small number of cells survive trans-plantation, and that an even fewer number actually migrate farenough to functionally integrate into the host neuronal circuitry [7].Traditional histopathological methods are routinely employed tovisualise cell integration post-transplantation surgery, howeverthese methods rely heavily on processing post-fixed tissue ex vivo.The ability to track endogenous precursors under pathophysiolog-ical conditions is therefore restricted and it is not possible to eval-uate in vivo changes at the site of engraftment or longitudinallypatterns of survival andmigration in live animals [8,9]. To accuratelyassess the long-term efficacy of cell-replacement therapy in thenervous system, reliable methods for quantification of engraftmentand visualisation technologies therefore need to be developed [10].

At present, long-term noninvasive imaging techniques fortracking donor cells include ultrasonography, radionuclide to-mography, two-photon fluorescent optical imaging, and magneticresonance [11]. MRI permits effective detection of transplanted

paramagnetic iron oxide nanoparticle prelabelling of human neuralals.2014.03.061

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stem cells following prelabelling with iron oxide nanoparticles (NP)serving as T1 and T2 contrast agents [12]. Hawrylak et al. were thefirst to use MRI to track iron oxide labelled fetal neural tissue afterinjection into the brain of live rats [13]. Since then, numerouslaboratories have utilised contrast agents to track stem cell trans-plants prelabelled in vitro as well as endogenous adult stem cellslabelled in vivo in order to assess migration and integration into thenervous system via quantitative MRI [14,15].

One of the advantages of using MRI to investigate long-termstem cell engraftment is its ability to map positional data directlyonto images of detailed neuroanatomy made during the sameacquisition [16]. The combination of NP contrast agents withanatomical information has the potential to be extended furtherthrough the use of connectivity measurement techniques such asresting state functional MRI (rs-fMRI), diffusion tensor imaging(DTI) and manganese-enhanced axonal tract-tracing [17].

NPs can be created from solid lipids, polymers or hybrids usingmetal cores of gold, gadolinium, manganese or iron [11]. Super-paramagnetic iron-oxide nanoparticles (SPIOs), with their smallsize and coating versatility, have been shown to increase iron-loading efficiency and hence confer the needed MRI signalchanges required for live-cell tracking [18]. SPIO polymer coatingscan be further modified and attached to functional groups,including growth factors, fluorescent dyes, and small molecules toenhance biocompatibility [14]. Depending on the desired applica-tions, NP size, stabiliser end group charge and functionalisation,and toxicity, can be customised and finely tuned dependent onexperimental needs or clinical application.

Once altered, the accompanying changes in biocompatibility dueto the emergent properties of the NP must then be determinedthrough in vitro experimentation. This is because any changesmadeto NP composition may significantly impact cellular biologicalproperties such as uptake, viability and/or prelabelling efficiency[19]. Furthermore, earlier available USPIOs suffered from tendencyto coagulate when internalised within cells and become unstable inphysiological conditions [19,20]. The coating is therefore of criticalimportancewhen determining NP physical characteristics, which inturn affect their use [21]. Dextran is the most common coatingpolymer for SPIOs, which has FDA approval for use in patients [22].However, its highmolecular weight, which contributes significantlyto the hydrodynamic size, results in relatively lower total metalcontent of the final coated USPIOs and reduction in NP-labellingefficiency [23]. To overcome these limitations we have previouslysynthesised USPIOs, utilising RAFT diblocks to stabilise the ironoxide core, which exhibit long-term stability in water and physio-logical solutions [23]. Advantages of RAFT polymerisation are thatthe diblocks for USPIOs, with an anchoring and stabilising block, areshort chains and narrow in molecular weight distribution, andtherefore have a minimal addition to the overall hydrodynamicdiameter of the coated particle. More importantly, with a functionalgroup at the end of each stabilising block, these RAFT diblock co-polymers can be modified by attaching targeting agents such asfluorescent molecules, antibody or receptor binding molecules(Fig. 1AeE). The stabilising method is also flexible, providing thepossibility tomix andmatch different types of stabilisingmolecules,which allows the USPIOs to be fine-tuned to their physiologicalenvironment aswell as to act as effective drug delivery vehicles [23].Moreover, the anchored stabilisers remain attached to the particlesand stabilise them after they are taken up within the target cells,making them ideal candidates for use in cellular labelling and MRIvisualisation. The biological consequences of pre-labelling hNPCswith such USPIOs have not been thoroughly characterised previ-ously. Given the robust nature of our sterically stabilised NPs, withfunctional end groups on the stabiliser, NPs synthesised with thistechnique have immense potential for stem cell based clinical

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applications. Such NPs could be modified to optimise tracking ofhuman neural stem cells using MRI to simultaneously monitormigration as well as changes in the surrounding parenchyma.

This study setout todetermine thebiological impactofUSPIOpre-labelling on neural stem cell viability, cell cycling, proliferation,apoptosis, migration, lineage potential and intracellular calciumconcentration, utilising our previously characterised population ofhNPCs [24]. The uptake of USPIOs into hNPCs, at various concentra-tions and incubation times, was investigated quantitatively bygraphite furnace atomic absorption spectrometry, and by trans-mission electron microscopy and super-resolution laser microscopy.Furthermore, in vitro visualisation of USPIO-labelled hNPCs wasperformedusingMRI. Future studieswill investigate the applicabilityof USPIO-labelled hNPCs for in vivo tracking of transplanted cells.

2. Materials and methods

2.1. Synthesis of iron oxide nanoparticles

Unless otherwise indicated, all materials were obtained from SigmaeAldrich.RAFTagents 2-{[(butylsulfanyl)carbonothioyl]sulfanyl}propanoic acid andmethoxy-polyethylene glycol modified 2 [(butylsulfanyl)carbonothioyl] sulfanyl}propanoicacid were kindly provided by Dr Algi Serelis (DuluxGroup). 1,4-Dioxane (Fluka) wasdistilled under reduced pressure. Monoacryloxyethyl phosphate (MAEP) was passedthrough an inhibitor removal column. Acrylamide (AAm), 4,40-azobis(4-cyanovalericacid) (V-501, Wako), sodium hydroxide (NaOH), N-hydroxysuccinimide (NHS), 1-ethyl-3-(3-dimethylamino-propyl)- carbodiimide (EDC) and 2,20-(ethylenedioxy)bis-(ethylamine) were used as received. Maghemite iron oxide cores (g-Fe2O3) wereproduced, as previously described, using the Massart method [25].

RAFT diblocks for coating iron oxide cores were synthesised and prepared ac-cording to Bryce et al. [23]. Magnetic iron oxide nanoparticles were prepared bycoating the iron oxide cores with the desired combination of the steric stabilisers of95%MPEG and 5% NH2 end functionalised RAFT diblocks (Fig. 1AeE). For Rhodaminelabelled particles, a desired amount of rhodamine B isothiocyanate was added to thecoated USPIO in water, pH 7e8, protected from light by aluminium cover and mixedfor 2 h at room temperature. The free rhodamine was washed with water andremoved via centrifugation.

2.2. Characterisation of USPIOs

Particles size distribution, zeta potential and transmission electron microscopy(TEM) of the iron oxide cores and coated nanoparticles have been characterised byBryce et al. [23]. Briefly, nanoparticle morphology was visualised by TEM before andafter steric stabilisation. One drop of particle dispersion in water (0.001% (w/v)) wasplaced on a carbon coated copper grid and left to dry at room temperature. Speci-mens were imaged on a Phillips CM120 Biofilter transmission electron microscope(Philips, The Netherlands) at 120 kV. Particle size distribution and zeta potential ofparticles in water was measured with a dynamic light scattering (DLS) instrument(Malvern, Zetasizer nano series) with a detection angle of 173� , 25 �C. The iron oxidecores had diameters of 10e15 nm and zeta potential of 55 mV. The coated nano-particles had an average hydrodynamic diameter Z-average of 60 nm (PDI ¼ 0.15)and zeta potential of �34 mV and �18 mV after Rhodamine labelling.

The stability of coated NPs in physiological conditions was studied by using adispersion of the particles in phosphate-buffered saline (PBS). Visual observationand DLS measurements (data not shown) showed no aggregation/settling of parti-cles, and a constant hydrodynamic diameter was observed.

2.3. MR relaxation rate

The T2 relaxation rate of the USPIOs was measured using a Bruker 9.4 T BiospecNMR (Bruker, Ettlingen, Germany) at iron concentrations of 1, 2, 5, 10, 20 and 50 mg/mL in water solution at 25 �C. T2 measurements were made by spin-echo sequenceand T2 were calculated by a mono-exponential fit. The transverse relaxivity (R2) wasthen determined by linear regression of R2 versus iron concentration. The R2relaxivity of the USPIOs was 368.2 mM�1 s�1 (Fig. 1F).

2.4. Cell cultures

Primary cultures of hNPCs were generated from fetal cortices between the agesof 16e19 weeks gestation obtained under approval from the Human Ethics Com-mittee of the University of Sydney with informed consent, as previously described[24]. Briefly, cortical dissections were processed by mincing tissue (twice at 90�

angle for 30 s) followed by trypsinisation (0.25% Trypsin/EDTA, 37 �C for 30 min),triturationwith a flame polished Pasteur pipette, pelleted (centrifugation at 300� g)andwashed in PBS to create a single cell suspension. Using this methodology a singlecell suspension with >90% viability was produced when assayed using trypan-blue.Cells were then plated in a medium composed of Neurobasal medium enriched(eNB) with B27 supplement, GlutaMax, epidermal growth factor (EGF, 20 ng/mL),

paramagnetic iron oxide nanoparticle prelabelling of human neuralals.2014.03.061

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and basic fibroblast growth factor (bFGF, 20 ng/mL) (Invitrogen) as previouslydescribed [24]. Viable cells were maintained as neurospheres in eNB at 37 �C in 5%CO2, with 50% media changes performed biweekly. Cultures were passaged (P) withTrypLE (Invitrogen) every 2 weeks and experiments conducted on P4 e 6 cultures.

2.5. Prelabelling of NPCs with USPIOs

NPCs were seeded into glass bottom wells or glass coverslips at 7.5 � 104 cells/mL sequentially coated with poly-L-ornithine (10 mg/mL), human placental laminin(5 mg/mL) and human plasma fibronectin (2.5 mg/mL) as single-cell monolayers andincubated for 24 h under standard culturing conditions as previously described [24].The cells were then labelled with USPIOs (1e100 mg/mL) by incubation for 10 min to48 h in eNB medium. Following incubation cells were washed with PBS to removeunattached USPIOs and cultured in eNB.

2.6. Viability proliferation and apoptosis

The impact USPIOs prelabelling had on NPCs in culture was measured using thetetrazolium dye (MTT) colorimetric assay for cellular growth and viability. Briefly,labelled cells (2 � 105 cells/mL) were incubated with 1 mg/mL 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide for 4 h at 37 �C, 5% CO2.Media containing MTTwas then removed and the dyewas solubilised with dimethylsulfoxide for 10 min after which the absorbance (590 nm) was read using aPOLARstar Galaxy microplate reader (BMG Labtech, Germany). All data was nor-malised against control (0 mg/mL) conditions at each time-point. Using the absor-bances, percentage viability was calculated using blank/no cell absorbances (AB),control (0 mg/mL) absorbances (Acontrol) and sample absorbances (Asample):(Asample � AB)/(Acontrol � AB) � 100.

2.7. Proliferation assay

Proliferation was assessed using the Click-iT� EdU Cell Proliferation Assay(Invitrogen) according to the manufacturers’ protocol. USPIO prelabelled cells(10 mg/mL, 2 h) were incubated for 1, 3, or 7 days in vitro (DIV), pulsed with EdU(1 mM) for 1 h and then postfixed in 4% paraformaldehyde (10 min). Cells were thenpermeabilised with 0.5% Triton X-100 and incubated with the Alexa Fluor 555 re-action cocktail (Invitrogen) according to the manufacturers’ protocol. Nuclei werecounterstainedwith 40 ,6-diamidino-2-phenylindole (DAPI) (1.25 mg/mL) and imagedusing a LSM 510 Meta Confocal microscope (Carl Zeiss, Germany). The mean per-centage of cells positive for EdU was determined from 10 fields-of-view (FOV) foreach triplicate condition and was measured for cells double positive for EdU andDAPI against total number of nuclei.

2.8. Apoptosis assay

Apoptosis was assessed using Muse� Caspase-3/7 kit and Muse� Cell Analyzeraccording to themanufacturers’protocol. Briefly, labelled cells (1e100mg/mL; 10min,2 h, or 24 h) were incubated for 1, 3, or 7 DIV at 37 �C, 5% CO2, and the monolayersdetached for cell retrieval using TrypLE� (Invitrogen). These were pelleted, washed,and resuspended in 5% bovine serum albumin (BSA) in minimal essential medium(MEM) (Invitrogen). Cell samples were incubated with Muse� Caspase-3/7 reagentfor 30 min at 37 �C, 5% CO2, and with Muse� Caspase 7-AAD solution for 5 min atroom temperature prior to analysing using the Muse� Cell Analyzer. Apoptosis wasinduced in retrieved cell samples using heat-shock treatment, by incubating the cellsfor 10 min, at 56 �C. The mean percentage of cells positive for Caspase-3/7 wasmeasured against total 7-AAD positive cells for each triplicate condition.

2.9. Cell cycle analyses

Cell cycle was assessed using Muse� Cell Cycle kit and Muse� Cell Analyzeraccording to the manufacturers’ protocol. Briefly, labelled cells (10 mg/mL) wereincubated for 10 min, 2 h, or 24 h, at 37 �C, 5% CO2 and retrieved using TrypLE,washed and pelleted and then resuspended in PBS prior to post-fixation in ice-coldethanol (70%) for 24 h. Cells were then incubated with Muse� Cell Cycle reagent for30min at room temperature prior to analysing using theMuse� Cell Analyzer. G2/Mcell cycle accumulation and G0/G1 reductionwas induced in cells by incubationwith660 nM Nocodazole for 24 h prior to cell retrieval. The mean percentage of cells, foreach triplicate condition, at different stages of the cell cycle was measured based ondifferential propidium iodide nuclei staining.

2.10. Iron quantification

NPCs (2 � 105 cells/mL) were plated in triplicate in coated 24-well plates andlabelled with USPIOs (1e100 mg/mL, 10 min to 24 h). Cells were retrieved with TrypLEwashed and pelleted in PBS prior to cellular digestion with 69% ultra-pure Nitric acid(HNO3) for 24 h, at 60 �C. Once the cellswere fully digested, 2% ultra-pure hydrochloricacid (HCl) was added (9 parts HCl to 1 part HNO3) to the samples, and analysed usingGraphite Furnace Atomic Absorption Spectrometry (GFAAS) (Agilent Technologies).

2.11. USPIO intracellular location

NPCs prelabelled with USPIOs conjugated with Rhodamine B (USPIO-R) at 5e20 mg/mL for 10 min to 24 h prior to cellular fixation (Section 2.5). Epi-fluorescent

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images were captured with an EM-CCD Camera iXon DU897 mounted on anEclipse Ti inverted Nikon N-SIM Super Resolution microscope system (Nikon,Japan).with Apo TIRF 100x 1.49 (Oil) objective. Super-resolution images were con-structed using the 561 nm laser line. A minimum of 10 FOV for each sample wererandomly selected for analysis.

2.12. Cellular TEM

NPCs (1 �106 cells) prelabelled with USPIOs at 10 mg/mL for 2 h were incubatedin vitro for a further 6 h and then fixed in 2% glutaraldehyde for 20 min before beingwashed in four changes of 0.1 M sodium cacodylate buffer (pH 7.4). Samples werethen post-fixed in osmium tetroxide for 1 h before being washed with distilledwater, dehydrated through a series of graded ethanol dilutions (50e100%), resinembedded, cut in 100 nm sections, and stained with lead citrate on a copper grid. AJEOL1011 transmission electron microscope (JEOL, Japna) operating at 60 keVcoupled with a MegaView G2 camera system (Olympus, Germany) and iTEM soft-ware (Olympus, Germany) was used for image analysis.

2.13. hNPC differentiation

The effect of USPIO prelabelling on hNPC differentiationwas assessed usingmulti-marker immunocytochemical staining. Labelled cells (10 mg/mL, 2 h)were incubated ineNB for 1, 3, or 7 days at 37 �C, 5% CO2, prior to cellular fixation (Section 2.5). Fordetection of cytoplasmic antigens, cells were permeabilisedwith 0.5% Triton X-100 for30 min, prior to incubation with primary antibodies, made up with 1% bovine serumalbumin in PBS, for 16 h at 4 �C. Antibodies used included those specific for; neuralstem/precursor cells: nestin (mouse IgG1; 1:500; R&D Systems) and sox2 (rabbit IgG;1:500; Cell Signalling), astrocytes: GFAP (chicken IgG; 1:2000; Abcam) and s100b(mouse IgG1; 1:1000), neurons: bIII-tubulin (chicken IgG; 1:2000; Chemicon) anddouble cortin (DCX, rabbit IgG; 1:500; Cell Signaling), and oligodendrocytes: GalC(mouse IgG3 hybridoma; 1:50; European Collection of Cell Cultures) and the oligo-dendrocyte precursors marker O4 (mouse IgM hybridoma; 1:4; ECACC) [24,26e28].Immune complexes were detected with goat antibodies (1:200) (2 h, room tempera-ture) to chicken-, mouse-IgG1 or IgG3, or rabbit-IgG; secondary antibodies werelabelledwithAlexa-Fluor488, 555 (Invitrogen),Cy3orCy5 (Jackson ImmunoResearch).Nucleiwere counterstainedwithDAPI prior tomountingonto glass slideswith glycerolin PBS (1:1) and sealed with nail varnish. All images were captured using a LSM 510Meta Confocal microscope (Carl Zeiss, Germany). The mean percentage of each celltypesweredetermined from10FOV for each triplicate condition, andwasmeasured forcells double positive for each lineage marker against total number of nuclei.

2.14. Cellular migration

For migration analyses, hNPCs were grown as free-floating neurospheres (200e400 mm) and plated (n ¼ 10) in triplicate in coated 24-well plates and labelled withUSPIOs (10 mg/mL, 10 min to 6 days). Images of each sphere were taken over thesubsequent 6 DIV using an Olympus CKX31/CKX41 Phase Contrast and FluorescenceInverted microscope with ProgRes C5 Cooled Colour CCD Camera (Olympus, USA).Mean distance from the edge of sphere to the furthest distal cell was measured at 0,90, 180 and 270� to the sphere, using ImageJ (NIH, USA), and was averaged betweeneach neurosphere for each triplicate conditions.

2.15. Calcium physiology

Data were collected on dynamic changes in the intracellular concentration of cal-cium ions [Ca2þ]I [29] through indirect measurement using the calcium indicator dyeFluo-4 AM (Molecular Probes). Cells were loadedwith 1 mM dye in 0.05% (w/v) pluronicacid F-127, for 40 min at 37 �C, 5% CO2. Following incubation, cells were washed andcultured an additional 30 min to ensure ester hydrolysis. Prelabelled cells, (10 mg/mL,2h),were seeded intowells ineNBmedium for 2hprior to adding thedye. Prior to Ca2þ

imaging, cellswere left in either eNB (containing Ca2þ) or calciumandmagnesium freeHanks balanced salt solution (HBSS) supplemented with 2 mM 1,2-bis(o-amino-phenoxy)ethane-N,N,N0 ,N0-tetraacetic acid (BAPTA) to chelate residual Ca2þ (Invi-trogen). Cells were imaged for 30 s prior to addition of 100 mM adenosine triphosphate(ATP) at which point the cells were imaged for a further 2 min. For inhibiting endo-plasmic reticulum ATPase-mediated refilling of the Ca2þ store, cells were incubatedwith 1 mM thapsigargin (Invitrogen) for 10 min at 37 �C, 5% CO2, prior to beingwashedwithMEMand imaged in HBSS/BAPTA.Micrographswere captured on a LSM510Metaconfocalmicroscope equippedwith heated stage and 488 nm laser line. Aminimumof10 FOV were randomly selected from monolayers (>40 representative cells per field)and analysed. LSMMeta 4.2 (Carl Zeiss, Germany) and ImageJ software were used forimage acquisition and analysis. For calcium imaging data analysis, Fluo-4 fluorescenceintensity was expressed as the change in fluorescence intensity relative to baselineintensity (DF/F0), where DF is the background subtracted fluorescence intensity sub-tracted to F0, and F0 was the background subtracted fluorescence fromeach cell at rest.

2.16. In vitro MRI

The ex vivoMR images were acquired using a vertical 9.4 T Biospec MRI (Bruker,Ettlingen, Germany). Total time for imaging was 4 h for each sample. To test thefeasibility of imaging labelled cells, hNPCs neurospheres were labelled with USPIOs

paramagnetic iron oxide nanoparticle prelabelling of human neuralals.2014.03.061

0.0 0.1 0.2 0.3 0.4 0.50

50

100

150

200

[Fe] (mM)

R2

Rel

axat

ion

Rat

e (s

-1)

F

Anchoring block

Steric stabilizing block

RAFT end group

Functional end group, e.g., -CH3 or -COOH or -NH2

Rhodamine

A B

C

D E

Fig. 1. Properties of our custom designed and manufactured USPIOs. Schematic of the iron oxide particles sterically stabilised by the RAFT diblocks (A) without and (B) withrhodamine conjugation. (C) Schematic representation of a RAFT diblock. (D) Chemical structure of 95% MPEG steric stabilising block (blue). (E) Chemical structure of 5% NH2 endpolymer. (F) R2 Relaxation rates (1/T2, s�1) as a function of iron concentration (mM) of USPIOs in water. The R2 relaxivity rate was 368.2 mM�1 s�1 (r2 ¼ 0.99, p ¼ 0.001). (Forinterpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

S.S. Eamegdool et al. / Biomaterials xxx (2014) 1e164

over a range of concentrations from 1 to 100 mg/mL for 24 h. Following incubationcells were washed with water to remove unbound USPIOs, then embedded in 1%agarose to set at room temperature. The agar samples were imaged using T2*-weighed gradient echo imaging and T2 spin-echo techniques. High resolution 3Dgradient echo sequences (3DGE) were acquired using the following parameters:

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NA ¼ 1; TE (echo time) ¼ 3.5 ms; TR ¼ 60 ms; FOV, field of view:19.4�19.4�19.4mm;matrix: 256� 256� 256; resolution: 35� 35� 35 mm (zero-filled to 15 � 15 � 15 mm). Lower resolution images with variable TE were acquiredusing a TE from 3.5 to 8 ms and a resolution of 70 � 70 � 140. 3D turbo spin echosequences (3DTSE) were acquired using the following parameters: NA ¼ 1;

paramagnetic iron oxide nanoparticle prelabelling of human neuralals.2014.03.061

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TE ¼ 40 ms; TR ¼ 200 ms; FOV (field of view): 19.4 � 19.4 � 19.4 mm; matrix:256 � 256 � 256; resolution: 35� 35� 70 mm (zero-filled to 37.5 � 37.5 � 37.5 mm).Conditions with agarose only, cells only, and USPIOs only controls were alsoincluded for imaging.

2.17. Statistical analysis

Data was expressed as mean � standard error of the mean (SEM) with samplesize (n). Significance was determined between the average of two groups using theindependent samples t-test and for groups of three or more by two-way analysis ofvariance (ANOVA). When two-way ANOVA was utilised, the post-hoc analysis forpairwise comparison was the Bonferroni test. An alpha value of 5% (P < 0.05) wasconsidered statistically significant and indicated in the figures as *(p < 0.05) or**(p < 0.01) where appropriate. Statistical analysis was performed using GraphPadPrism 5.0 (GraphPad software, USA) for Mac.

3. Results

3.1. Viability of USPIO-labelled hNPCs

TEM imaging of NP-labelled hNPCs provided ultrastructuralevidence for NP internalisation by hNPCs (Fig. 2A, B) and a lack ofNP coalescence. The effect of NP load on hNPCs viability wascolorimetrically assessed via the formation of MTT formozan

Fig. 2. Screening and optimisation of NPs labelling time and concentration. Transmission eleNPs remain dispersed as single NPs and can be localised within intra-cellular vesicles (red aconditions on hNPCs (C) viability and D) intracellular iron content. Cell viability remainedsignificantly decreasing for NP concentrations >20 mg/mL, 24 h. Significant iron uptake watimes). Significance is relative to (C) control (0 mg/mL) and (D) the respective values. (For inteweb version of this article.)

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(Fig. 2C). From 1 to 10 mg/ml, it was found that NP concentrationsfor all incubation time points (1e48 h) had no significant effect oncell viability, which was greater than 90%, when compared andnormalised against unlabelled cells in control conditions (0 mg/mLin carrier). However our post-hoc analysis showed all concentra-tions higher than 20 mg/mL resulted in a significant decrease(p < 0.01) in cell viability after a 24 h incubation period: (20 mg/mL(82.63 � 5.83%); 50 mg/mL (73.05 � 2.74%); 100 mg/mL(63.47 � 11.96%) (Fig. 2C).

The quantification of intracellular NPs using GFAAS confirmedthe dose-dependent uptake of NPs by hNPCs is a function of NPsconcentration and incubation time (Fig. 2D). After 10 min, wemeasured a ten-fold increase in intracellular iron content overcontrol when hNPCs were incubated at 100 mg/mL NPs concentra-tion (1.17 � 0.19 pg/cell; p < 0.01). Post-hoc analysis revealed thatintracellular iron content was significantly higher for both 10 mg/mLand 100 mg/mL NP concentrations when incubated for 2 h (10 mg/mL: 0.58 � 0.08 pg/cell (p < 0.05); 100 mg/mL: 1.68 � 0.14 pg/cell(p < 0.01)) and 24 h (10 mg/mL: 0.91 � 0.04 pg/cell (p < 0.05);100 mg/mL: 2.9 � 0.6 pg/cell (p < 0.01)) (Fig. 2D). No significantdifference in iron content was detected when cells were incubated

ctron microscopy images of hNPCs (A) without and (B) with NP exposure showed thatrrows) 6 h post-incubation at 10 mg/mL. Scale bar ¼ 2 mm. Optimisation of NP-labelling>90% (dotted line), for NP concentrations 1e10 mg/mL for all exposure times, befores achieved at NP concentrations 10 mg/mL (2 and 24 h), and 100 mg/mL (all exposurerpretation of the references to colour in this figure legend, the reader is referred to the

paramagnetic iron oxide nanoparticle prelabelling of human neuralals.2014.03.061

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with 1 mg/mL NPs, for all incubation time-points. By using bothMTTassay and GFASS, we were able to systematically screen for theoptimum NP-labelling concentration (10 mg/mL) and incubationtimes (2 he24 h) which had an insignificant effect on the cellviability (greater than 90%) over the long-term treatment, andshowed an increasing amount of intracellular iron with time. Thisconcentration and time point was used in the subsequentexperiments.

3.2. Effect of USPIO-labelling on apoptosis, proliferation index, andcell cycle progression

The rate of cellular apoptosis, proliferation, and cell cycle pro-gression, were quantified as indicators of cell survivabilityfollowing a time dependant USPIOs prelabelling treatment.Apoptosis analysis, via caspase-3/7 activity, showed that NP loaddid not induce cell apoptosis at concentrations between 1 and10 mg/mL, for 10 min, 2 h, and 24 h incubation time periods(Fig. 3AeC). At 100 mg/mL concentration, 24 h incubation time,significant cellular apoptosis was measured (Live: 84.95 � 2.5%(p< 0.05); Early Apoptosis: 25.60� 2.8% (p< 0.01); Late Apoptosis/Dead: 5.91 � 0.45% (p < 0.01)) compared to unlabelled hNPCs(Fig. 3AeC). Heat-shocked induced apoptosis was used as a positivecontrol, to confirm that hNPCs undergo caspase-3/7-dependentcellular apoptosis, and resulted in a drastic and significant

Fig. 3. Effects of NPs on cellular apoptosis. No significant difference in the percentage of (labelled (1, 10 mg/mL; 10 min, 2 h, 24 h) hNPCs. NP-labelled cells labelled at 100 mg/mL,cells and an increase cells undergoing (B) early- and (C) late-apoptosis. Control and NP-labeincubation. Significance is relative to control.

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increase (p < 0.01) in early- (40.5 � 4.9%) and late-apoptosis/death(45.5� 5.4%) (Fig. 3AeC). Following NPs incubation at 10 mg/mL, for2 h, cellular apoptosis was analysed 1, 3, and 7 days post-incubationin order to investigate the long-term survivability of NP-labelledhNPCs, and for each of the time periods NP load did not cause asignificant change in cellular apoptosis in any of the samples tested(Fig. 3D).

The proliferation capacity of hNPCs following NP load wasquantitatively determined with EdU incorporation, indicative ofDNA synthesis, and was measured against total DAPI counter-stained cells (Fig. 4AeD). Cells incubated with 10 mg/mL NP-Rhodamine (NP-R) for 2 h (Fig 4A), retained their ability to prolif-erate (Fig. 4D, left arrow). Between 1 and 7 DIV, there was no sig-nificant difference in the average numbers of NP-labelled cellsundergoing proliferation compared against control (Fig. 4E).

Flow cytometric cell cycle analysis was used to determine ifprelabelling hNPCs with NPs had significantly altered cell cyclephases. Incubating hNPCs with 10 mg/mL NPs for 10 min, 2 h, and24 h did not result in significant changes in the average cell cycledistribution when compared against unlabelled control pop-ulations (G0/G1: 77.3 � 4.2%; S: 6.1 � 2.2%; G2/M: 12.2 � 3.1%)(Fig. 5A). Importantly, this data provides good evidence that hNPCpopulations maintain a consistent cell cycle distribution, for allconditions, when exposed to our custom USPIOs e which was apotential concern as researchers have previously observed a

A) live, (B) early apoptotic, and (C) late apoptotic/dead cells between control and NP-24 h, or induced with heat-shock treatment, led to a significant (A) reduction in livelled (10 mg/mL, 2 h) hNPCs displayed similar apoptotic profiles (D) 1, 3 and 7 days post-

paramagnetic iron oxide nanoparticle prelabelling of human neuralals.2014.03.061

Fig. 4. Proliferation of NP-labelled hNPCs. Cellular proliferation of unlabelled and NP-labelled hNPCs were (A, B, C, D) visualised by confocal microscopy and (E) quantified by EdUstaining. Cells were labelled with (A) NP-R (10 mg/mL, 2 h) (red), and colocalisation between (B) EdU (green) and (C) DAPI (blue) was indicative of cellular proliferation. (D) NP-labelled hNPCs undergoing proliferation (left arrow) is displayed. Scale bar ¼ 50 mm. No significant difference in proliferation between control and NP-treated hNPCs wereobserved (E) 1, 3 and 7 days post-incubation. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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significant change in the rate at which cells incorporate foreignmaterial is dependent on the phase of the cell cycle [30]. There wasalso no effect of NP load on hNPCs long-term cell cycling whenobserved at 1, 3, and 7 days post-incubation (Fig. 5B).

To induce G2/M cell cycle arrest and synchronise cell cycles themicrotubule inhibitor nocodazole was used. No changes in cellcycle phase patterns were observed in NP prelabelled hNPCscompared to control populations. Both were found to undergo atwo-fold increase (p < 0.01) in cells entering G0/G1 phase (control:73.0 � 1.5%; NPs: 74.2 � 2.3%; nocodazole: 47.0 � 0.7%) (Fig. 5C)followed by a three-fold decrease (p < 0.01) in the total numbers ofcells in the G2/M phase (control: 16.7 � 1.3%; NPs: 13.6 � 1.4%;nocodazole: 38.7 � 0.4%) (Fig. 5C). There were no significant dif-ferences in the proportion of cells entering the S phase betweencontrol, prelabelled and nocodazole treated cells (Fig. 5C).

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3.3. Intracellular localisation of NPs using super-resolutionmicroscopy

Following incubationwith NP-R intracellular ironwas visualisedby epi-fluorescence microscopy and images reconstructed usingsuperresolution image analysis. At 5, 10 and 20 mg/ml concentra-tions, 10 min incubation time, intracellular USPIOs mainly localisedto the cytoplasm (Fig. 6A1, E1, I1). Prolonged incubation, for 2 h,resulted in a limited amount of USPIOs observed inside the nucleus(Fig. 6B1, F1, J1). After 12 h incubation period a significant propor-tion was also observed in the perinuclear region with numerousNPs found clustering around the outer nuclear membrane (Fig. 6C1,G1, K1) and at 24 h most NPs had passed through the nuclear en-velope, possibly via nuclear pore complexes, and concentrated inthe nucleus (Fig. 6D1, H1, L1).

paramagnetic iron oxide nanoparticle prelabelling of human neuralals.2014.03.061

Fig. 5. Cell-cycle assessment of NP-labelled hNPCs. No significant difference in the percentage of cells at G0/G1, S, or G2/M phase between control and (A) NP-labelled hNPCs at10 mg/mL, for 10 min, 2 h, and 24 h, and (B) NP-labelled hNPCs (10 mg/mL, 2 h) at 1, 3, and 7 days post-incubation. (C) Nocodazole treatment of hNPCs caused a significant increase inthe percentage of cells in the G2/M phase (double nuclei), as well as decrease in the G0/G1 phase (single nucleus) compared to control and NP-treated hNPCs. Significance is relativeto control and NP-labelled hNPCs.

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3.4. Multipotency of USPIO-labelled hNPCs

To examine the effects of NP load on hNPC lineage elaborationwe phenotypically characterised and quantified the proportion ofeach cell type by morphology and multiple-marker immunocyto-chemistry. Due to temporal-, and spatial-specific heterogeneity,with regards to protein expression patterns [24,26e28], cell typewas determined by using multiple lineage-specific markers. Allexperiments were conducted in the presence of growth factors(eNB) in order to specifically discern the effect of NPs on hNPCsdifferentiation. Under this culture system, hNPCs were previouslyshown to be capable of undergoing trilineage differentiation overtime [24]. At 7 DIV, cells were identified as 63.7 � 1.3% nestinþ/SOX2þ NPCs (Fig. 7DeG); 34.1 � 3.3% GFAPþ/s100bþ astrocytes(Fig. 7KeN); 19.5 � 1.7% bIII-tubulin/DCX neurons (Fig. 7ReU) and0.9 � 0.008% GalCþ/O4þ oligodendrocyte precursors (Fig. 7YeB1)with no significant differences when compared against control.Similar results were found at 1 DIV (Fig. 7C1), and 3 DIV (Fig. 7D1).To visually confirm each lineage could be labelled with NPs (10 mg/mL, 2 h), we also labelled the cells with NP-R under the sameconditions. It was found that NPCs, astrocytes, neurons, and oli-godendrocytes were equally labelled with NP-R, and that the cellsexhibited long-term retention of the NPs, (Fig. 7E, L, S, Z) at 7 DIVpost-NP loading.

Comparisons between control and NP-prelabelled cells showedno morphological change to cells of each lineage (Fig. 7AeA1),which were consistent with our previous findings: NPCs (largenuclei with an epithelial-like cell body), astrocytes (flat, epithelial-like), neurons (compact nuclei, elongated, uni-, bi-polar), and oli-godendrocytes (compact nuclei with multiple branched processes)

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[24]. There was no significant difference in the population of NPCs,astrocytes, neurons, oligodendrocytes, following NP-labelling, afterquantification at 1, 3, and 7 DIV post-NP labelling (Fig. 7C1eE1).These data provide evidence that NP prelabelled hNPCs retainmultipotentiality and are able to continuously give rise to each ofthe main neural cell types following NP load (Fig. 7E1).

3.5. Influence of USPIOs on the migratory capability of hNPCs

To determine the effects of NPs on hNPCsmigration, hNPCswereplated as neurospheres and monolayers allowed to form. Cellularmigration was quantified via serial imaging at 24 h periods, withthe distance between the furthest distal cell and the edge of theneurosphere measured at four distinct locations per sphere. As wehave previously shown that there was no significant difference incellular apoptosis (Fig. 3) and proliferation rate (Fig. 4) betweenunlabelled and NP-labelled hNPCs, we were able to determine thatany changes in migration distance could be attributed specificallyto the effect of NPs on cell motility. Cells incubated with 10 mg/mlNPs for 10min, 2 h, and 24 h showed no significant difference in themigration distance at 6 DIV (Fig. 8AeC). However, when hNPCswere incubated with NPs at non-optimal conditions (Fig. 2CeD), at100 mg/mL for the entire 6 DIV, at 3 DIV NP-labelled hNPCs had areduced migration when compared to optimal label and control(Control: 580.5 � 47.3 mm; NP-labelled: 415.4� 82.2 mm) (p< 0.01)(data not shown). This effect was persistent up until post-labelling6 DIV (Control: 1004.08 � 39.5 mm; NP-labelled: 506.8 � 39.5 mm)(p < 0.01) (Fig. 8D), indicating that prolonged exposure to NPs(100 mg/mL, 6 days) resulted in a substantial reduction in: total

paramagnetic iron oxide nanoparticle prelabelling of human neuralals.2014.03.061

Fig. 6. Localisation of USPIOs within hNPCs. Rhodamine-tagged USPIOs were visualised in labelled hNPCs by (AeL) epi-fluorescence and using a (A1eL1) Nikon N-SIM SuperResolution microscope system. Cells were exposed to NP-R (5, 10, 20 mg/mL) for 10 min, 2 h, 12 h, 24 h, and, (A, E, I, A1, E1, I1) after entry into the cell, USPIOs were found to localisein the (B, F, J, B1, F1, J1) cytoplasm after entry into the cell, moving to the (C, G, K, C1, G1, K1) peri-nuclear rim and then concentrating in the (D, H, L, D1, H1, L1) nucleus. Scalebar ¼ 5 mm.

S.S. Eamegdool et al. / Biomaterials xxx (2014) 1e16 9

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Fig. 7. Representative multi-marker immunocytochemistry of post-labelled neural lineages at Day 7. Neural stem/precursor cells were identified via (A, D) Nestin and (B, F) Sox2 (C,G) colocalisation. Astrocytes were identified via (H, K) s100b and (I, M) GFAP (J, N) colocalisation. Neurons were identified via (O, R) bIII-tubulin and (P, T) DCX (Q, U) colocalisation.Oligodendrocytes were identified via (V, Y) O4 and (W, A1) GalC (X, B1) colocalisation. Cells were labelled with NP-R (10 mg/mL, 2 h) in order to visualise the presence of USPIOs in(E, L, S, Z) each lineage. Nuclei were counterstained with DAPI (blue). Scale bar ¼ 20 mm. Quantification of each lineage population indicated no significant difference between ofcontrol and NP-labelled cells at (C1) 1, (D1) 3, and (E1) 7 days post-incubation. (For interpretation of the references to colour in this figure legend, the reader is referred to the webversion of this article.)

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migration distance and, increment in migration over 24 hr-intervals, compared to unlabelled cells.

3.6. Calcium signalling in USPIO-labelled hNPCs

We evaluated whether ATP-induced transients in [Ca2þ]I ofhNPCs were perturbed following NP-labelling. In the absence ofextracellular Ca2þ, ATP can activate metabotropic P2Y purinor-eceptors (P2YR), which then induces inositol-1,4,5-trisphosphate(IP3)-mediated [Ca2þ]I and downstream signalling cascades,therefore, Ca2þ imaging of NP-labelled hNPCs was performed inHBSS (no extracellular Ca2þ) [29].

A 100 mm pulse of ATP induced a transient [Ca2þ]I response inNP-labelled (10 mg/mL, 2 h) hNPCs that significantly differed fromunlabelled control cells in measurements taken in both eNB me-dium (contains extracellular Ca2þ), and HBSS. Addition of ATP

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evoked a peak transient rise in [Ca2þ]I in NP-labelled hNPCs (1.18 at61 s), followed by a slow decline to baseline (0.84 at 135 s),compared to control eNB (1.12 at 50 s) and control HBSS (0.80 at59 s) conditions which had relatively faster declination to baseline(eNB: 0.32 at 135 s; HBSS: 0.05 at 135 s) (Fig. 9A). Treatment ofhNPCs with thapsigargin prior to addition of ATP resulted in com-plete abolition of [Ca2þ]I transients, confirming that endoplasmicreticulum-mediated release of Ca2þ was largely responsible for theobserved ATP-induced transients in [Ca2þ]I in the cells [29]. Addi-tionally, the maximal ATP response of NP-labelled hNPCs(105.8 � 7.4%) was similar to eNB-conditions (100%), and wassignificantly greater (p < 0.05) than HBSS-conditions (73.1 � 5.7%)and thapsigargin-treated cells (16.8 � 1.9%) (p < 0.01) (Fig. 9B).However, NP-labelled hNPCs response to ATP recovered back downto similar levels as unlabelled hNPCs in HBSS conditions after oneday post-incubation (Fig. 9CeD). This effect remained at day 3

paramagnetic iron oxide nanoparticle prelabelling of human neuralals.2014.03.061

Fig. 8. Migration of NP-labelled hNPCs neurospheres. Cells grew over 6 days after a 24 h initial attachment period (Day 0), during which the cells were labelled with USPIOs. Nosignificant difference in the migratory capacity was observed between control hNPCs and hNPCs labelled with NPs (10 mg/mL) for (A) 10 min, (B) 2 h, and (C) 24 h. Migrationdistance of hNPCs was reduced when they were labelled with NPs (100 mg/mL) for (D) 7 days. Significance is relative to control.

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(Fig. 9EeF) and 7 (Fig. 9GeH) post-incubation. Taken together, wefound that NP load increases ATP-evoked [Ca2þ]I levels and pro-longs [Ca2þ]I transients in USPIO-labelled hNPCs.

3.7. In vitro MRI visualisation of USPIO-labelled hNPCs

In order to explore the feasibility of utilising these NPs as MRIcontrast agents, hNPCs neurospheres were labelled with NPs over arange of concentrations (1e100 mg/mL, 24 h), dispersed in 1%agarose, and then imaged byMRI (Fig.10AeR). An agarose only, anda non-NP incubated hNPC neurosphere sample were included ascontrols (data not shown). NP-labelled neurospheres showed sig-nificant local signal loss in T2*-weighted images, corresponding tointernalised NPs. Adequate uptake for full penetration of the neu-rospheres occurred between 5 and 10 mg/mL (Fig. 10DeF, GeI).Concentrations above this level at 20 (Fig. 10 JeL), 50 (Fig. 10MeO),and 100 mg/mL (Fig. 10PeR) resulted in more intense signal loss(Fig. 10J), consistent with the quantitative MTT and GFASS datapresented above.

In order to further explore the localisation and effectiveness ofNP uptake we examined the effect of increasing TE on the NP-labelled hNPC neurospheres at 5 mg/mL (Fig. 10 SeU) and 20 mg/mL (Fig. 10VeX) concentrations. There was a marked signal losspresent in the 20 mg/mL sample that was especially prominent at aTE value of 10 ms (Fig. 10X) compared to a TE value of 3.5 (Fig. 10V)and 5 ms (Fig. 10W). The full FOV minimum intensity projections

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(minIPs) (Fig 10SeX) showed that although uniform labelling wasnot achieved using a 5 mg/mL incubation concentration (Fig. 10S),reasonable contrast was still generated at higher TE (Fig. 10T, U).

4. Discussion

Stem cell therapies have shown great potential in providingfunctional regeneration to damaged tissues. However, the inherentlack of knowledge of the interactions between host and trans-planted cells, as well as a paucity of spatial, temporal, andbehavioural information regarding the transplanted cells, haveresulted in significant challenges that have inhibited clinicaltranslation. MRI is an attractive tool for providing spatiotemporallocalisation of transplanted cells, but requires cellular labellingtechniques to enable specific detection of transplanted stem cells.Although NPs are widely used in research and clinical settings,their impact on cellular mechanisms and function is not fullyunderstood, primarily due to discrepancies in NPs design and celltypes [11,12,14].

We have developed sterically stabilised USPIOs which exhibitlong-term stability, with narrow size distribution, and modifiablestabiliser end functionality. The ability to attach functional groupssuch as fluorescent-dyes and pharmacological drugs permit bothcell-labelling and modulation of cellular function. In this study, thebiocompatibility and imaging capability of our USPIOs on primaryhNPCs, feasible candidates for neurological repair, are reported.

paramagnetic iron oxide nanoparticle prelabelling of human neuralals.2014.03.061

Fig. 9. Rise in [Ca2þ]I of NP-labelled hNPCs neurospheres in response to ATP. Cells labelled with USPIOs (10 mg/mL), in HBSS (no extracellular Ca2þ), for (A) 2 h displayed increased[Ca2þ]I levels, similar to (B) eNB conditions (with extracellular Ca2þ), with a slower return to baseline level. After (C, D) 1, (E, F) 3, and (G, H) 7 days post-incubation, NP-labelledhNPCs (10 mg/mL, 2 h) displayed [Ca2þ]I traces, and peak response to ATP, similar to HBSS control. Significance is relative to eNB unless otherwise indicated.

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We have shown that NPs have a negligible effect on cellularviability below a threshold of 10 mg/mL, and that deleterious effectswere dose and time dependent above these parameters. In addi-tion, under optimal incubation conditions, USPIOs have no effect oncellular apoptosis and proliferation as measured by Caspase-3/7

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activity, EdU-labelling and cell cycle analysis. NPs were found tolocalise predominantly in the cytoplasm, but concentrate towardsthe peri-nuclear rim, and into the nucleus with increasing incu-bation time and concentration. NPs did not affect the trilineagepotential of hNPCs in differentiating into astrocytes, neurons, or

paramagnetic iron oxide nanoparticle prelabelling of human neuralals.2014.03.061

Fig. 10. In vitro MRI of NP-labelled hNPCs neurospheres. hNPCs incubated for 24 h with (AeC) 1 mg/mL, (DeF) 5 mg/mL, (GeI) 10 mg/mL, (JeL) 20 mg/mL, (MeO) 50 mg/mL and (PeR)100 mg/mL NP concentrations. (A, D, G, J, M, P) Selected minimum intensity projection images (minIP) of a 20 mm thickness slice over the full field of view. (B, E, H, K, N, Q) Selectedmagnitude images of a smaller neurosphere (approximate size 200 mm). The FOV is 1.5 mm (C, F, I, L, O, R) Selected magnitude images of a larger neurosphere (approximately500 mm). Dark areas indicate hypointensity, corresponding to intracellular NPs. The TE in all images is 3.5 ms. (SeX) The effect of increasing TE on contrast on NP-labelled hNPCsneurospheres at 5 mg/mL versus 20 mg/mL NP concentration. Minimum intensity projections (minIP) images over the full field of view of hNPCs neurosphere labelled with (SeU)5 mg/mL or (VeX) 20 mg/mL, using a 5 mm slice thickness at (S, V) TE ¼ 3.5 ms, (T, W) TE ¼ 5 ms and (U, X) TE ¼ 10 ms Close-ups of a representative labelled neurosphere at (SeU)5 mg/mL or (VeX) 20 mg/mL NP concentration with a diameter of approximately 500 mm are shown below. Scale bar ¼ 500 mm.

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oligodendrocytes. Cellular migratory capacity was not severelyaffected by NP-labelling, unless the NPs were present with the cellsfor extensive periods of time (over 6 days). Finally, NP-exposedhNPCs exhibited increased intracellular calcium fluxes, howeverthis was followed by slow recovery to baseline levels one day post-exposure. We confirmed that the imaging characteristics of NP-loaded neurospheres are favourable for cell-tracking, with pro-found signal changes demonstrated using T2*-weighted imaging.Thus, we were able to extensively characterise the effects of NPs onhNPCs biological function and cellular profile, which is importantfor future application of these cells in in vivo cellular tracking, MRIdetection, and monitoring them in cell transplantation studies.

4.1. Impact of NPs labelling parameters on hNPCs survivability andsubcellular NPs location

In these studies, hNPCs were shown to remain viable and sur-vivable under optimal NP-labelling conditions. This is consistent

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with previous studies, which have found that SPIOs did not affectthe viability of mesenchymal stem cells or neural stem cells [31,32].We found that at lower NP concentrations (1e10 mg/mL) theviability of NP-labelled hNPCs remained >90% over 48 h (Fig. 2).However, at concentrations greater than 20 mg/mL, cell viabilitywas significantly reduced. Taken together, our findings showed thathNPCs are highly responsive to the presence of foreign particulatematter in their environment at high doses. In this context, the highrelaxivity of our USPIOs (T2 relaxivity: 368.2 mM

�1 s�1, versuscommercial NPs, Resovist: 61 mM

�1 s�1; Feridex: 126.0 mM�1 s�1)

represents an important advantage [33,34]. NPs demonstrated noeffect on hNPC apoptosis in the long-term, as measured throughCaspase-3/7 activity (Fig. 3). Meng et al., have previously reportedthat survivability of neural stem cells was compromised at higherNPs concentrations, but not lower NPs concentrations, while Gep-pert et al., showed that viability of rat astrocytes was substantiallyreduced following prolonged incubation with SPIOs [32,35]. It isknown that intracellular iron overload can lead to cytotoxicity and

paramagnetic iron oxide nanoparticle prelabelling of human neuralals.2014.03.061

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functional impairment, through the generation of reactive oxygenspecies (ROS), formation of DNA strand breaks, and impairment tothe mitochondrial function [36].

There have been numerous studies pertaining to the effects ofNPs on cellular proliferation, with findings of NPs either reducing,or promoting cellular proliferation being reported [37,38]. Withregards to their usage in clinical applications, and the potential forNPs to alter the tumorigenesis of the cells, it was important that weshowed that hNPCs proliferation (EdU incorporation and cell cycleanalysis) was not changed as a result of NP-labelling.

Intracellular iron concentration depends on properties of NPs,such as charge, composition and size [12]. We found that labellinghNPCs with NPs resulted in an optimal loading capacity of 0.6 pg/cell, and a maximal loading capacity of 3 pg/cell. Our optimalloading capacity of 0.6 pg/cell was significantly lower than thatreported for one other USPIOs in mesenchymal stem cells (6 pg/cell) [39], and Ferumoxide (Feridex) in human neural stem cells(1 pg/cell) [40]. The lower uptake by NPs in our study may beexplained in part by the limitation in iron content that is associatedwith small core particle size (<15 nm), compared to other studieswhich have used USPIOs, with wide size ranges (50e200 nm), ormicron-size NPs (MPIO >0.9 mm), which imparts higher intracel-lular iron content [19,41]. However, under these labelling condi-tions, the presence of NPs inside the cells were visible anddetectable through TEM (Fig. 2), fluorescence microscopy (Fig. 6),and MRI (Fig. 10). Following internalisation of NPs into the cells, ithas been shown that NPs can reside in the endosome [31], cyto-plasm [35], and/or the perinuclear region [42]. NPs are graduallycleared from the cells either via lysosomic degradation or cell cycledivision [36,43]. As we observed, the location of NPs in hNPCsdepended on the concentration and incubation time, with NPstranslocating into the perinuclear region after initially residing inthe cytoplasm (Fig 6). As such, this may account for the reduction inviability observed following extensive incubation with higher NPconcentrations, as NPs can cause irreversible DNA damage andperturb normal gene expression [36]. It is possible that the mode ofNP entry into the cell is likely via passive diffusion, as cells of thedifferent lineages were shown to contain NPs. Rat astrocytes wereshown to take up NPs via macropinocytosis and clathrin-mediateduptake [44], suggesting that similar mechanisms could be involvedin the uptake of NPs by hNPCs.

4.2. Differentiation potential of UPSIO-labelled hNPCs

Neurogenesis in the mammalian CNS is thought to arise fromnumerous cell types, most of which originate and differentiate fromneural stem cells [1]. In this study we have used multi-markerimmunocytochemistry to phenotypically characterise the propor-tion of NPCs, astrocytes, neurons and oligodendrocytes in vitro, andhave shown that NPs did not significantly favour differentiationtowards either lineage, but rather that hNPCs retained their dif-ferentiation potential in the long-term, and that cells of all lineageswere able to be equally labelled with NPs (Fig. 7). This is consistentwith other studies that have found that mesenchymal and neuralstem cells retained their multipotentiality following NP-labelling[31,32]. Despite the heterogeneous nature of our cell populationin vitro, we have shown that the proportion of cells of each lineageremained constant over the long-term, and that the presence ofeach cell type is likely to be essential in the maintenance of cellphenotype and function. It has been postulated that astroglial cellspossess an active regulatory role in neurogenesis throughinstructing NPCs towards the neuronal lineage [45]. Although NPCshave been widely used in cell transplantation studies [46], othersgroups have shown that astrocytes (spinal cord injury) [47], neu-rons (stroke) [48], and oligodendrocytes (spinal cord injury) [49]

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are capable of providing functional recovery to the CNS. There-fore, it is worth noting that co-transplantation of NP-labelled NPCs,in addition to NP-labelled committed neural cell types, mightprovide the necessary conditions and regulatory cues which allowsfor successful stem cell therapies.

4.3. Role of NPs on functional Ca2þ stores and signalling

Intracellular calcium homoeostasis is critical for cellular func-tion, as calcium signalling is involved in many key biological as-pects such as proliferation, apoptosis, metabolism, and migration[50]. Excess intracellular calcium is detrimental to cells, and hasbeen associated with various pathological conditions such as car-diac arrhythmias [51] and amyotrophic lateral sclerosis [52], likelyas a result of enzymatic cellular apoptosis (such as caspases andcalpain) [53]. Currently, there are a limited number of studieswhich have specifically investigated the effects of iron-oxidenanoparticles on cell calcium signalling, especially in neural stemcells. Titanium-oxide nanoparticles have been shown to increase[Ca2þ]I in human keratinocytes, leading to actin reorganisation, andenhanced differentiation, likely as a result of co-localisation ofnanoparticles to the Golgi apparatus and deregulation of [Ca2þ]Ipools [54]. Arvizo et al., found that charged gold nanoparticles havethe ability to cause mitochondrial membrane potential depolar-isation, which inhibits their ability to buffer [Ca2þ]I overload,resulting in substantial increases in [Ca2þ]I upon activation [55].Weobserved a significant increase in [Ca2þ]I NP-labelled hNPCsfollowing ATP addition, and that there was a delayed reduction tobaseline, compared to unlabelled hNPCs. However, cells returned tobasal calcium levels after 1 day and no changes persisted thereafter.Further studies are required in order to determine the effects ofelevated [Ca2þ]I on hNPCs. Previously, it has been shown that ironmolecules can stimulate Ryanodine receptor-mediated endo-plasmic reticulum calcium-induced calcium release, through thegeneration of reactive oxygen species (ROS) [56]. This in turn leadsto ERK pathways phosphorylation, sustained long-term potentia-tion, and modulation of synaptic plasticity. ROS production hasbeen shown to be elevated in rat astrocytes [35], and neural pro-genitor cells [57], after loading with iron oxide nanoparticles.Bhattacharjee et al., reported that positive-charge silicon nano-particles can uncouple the electron transport chain, which perturbsmitochondrial membrane potential and increases intracellular ROSproduction [58]. Therefore, it is critical that long-term effects of NP-labelling on cell calcium signalling are determined, prior to theirutilisation in cell transplantation studies.

4.4. USPIOs as potential MRI contrast agents for in vivo tracking oftransplanted cells

Cellular migration is essential for the development of the CNS,as it accounts for generating correct cellular compositions, spatialorganisation, and brain function. Marin and Rubenstein havedescribed two different types of migration that occur: 1) radialmigration, which involves neuronal migration along glial fibresfrom the progenitor zone towards the brain surface for formingthe cytoarchitecture, and; 2) tangential migration, where the cellsmigrate orthogonal to radial migration, forming cellular connec-tivity and spatial distribution [59]. Impaired migration is known tocause developmental abnormalities such as lissencephaly and graymatter heterotopia [60,61]. In this study, NPs were shown to haveno effect on migration of hNPCs, except in conditions whereprolonged NPs exposure occurred, for which a substantial reduc-tion in hNPCs migratory capacity was observed (Fig. 8). Soenenet al., have shown that high concentrations of intracellular nano-particles can disrupt cellular actin cytoskeleton and microtubule

paramagnetic iron oxide nanoparticle prelabelling of human neuralals.2014.03.061

S.S. Eamegdool et al. / Biomaterials xxx (2014) 1e16 15

networks, thereby reducing focal adhesion points portending toreduced migratory capacity [57]. Therefore, it is essential that wehave shown NP-labelled hNPCs retained their migratory capacity,as future uses in cell transplantation studies would require thecells to home into the lesion sites and provide functionalintegration.

MRI of engrafted NP-labelled stem cells is crucial for tracking themigration of the cells towards the site of injury following trans-plantation, giving insight into how these cells participate in tissueregeneration. Lee et al., were able to visualise transplanted NP-labelled mesenchymal stem cells utilising MRI to demonstratemigration of transplanted cells towards the thrombotic stroke sitein rats [31]. In this study, our USPIOs demonstrated a high T2relaxivity of 368.2 mM

�1 s�1, which was significantly greater thancommercial iron oxide nanoparticles of similar core size (Resovist:61 mM

�1 s�1; Feridex: 126.0 mM�1 s�1) [33,34], indicating their

superiority as MRI contrast agents. We performed in vitro MRI ofNP-labelled hNPCs neurospheres embedded in agarose and foundthat NP-labelled neurospheres displayed excellent contrast prop-erties, and an enhanced MRI signal-to-noise ratio, compared tounlabelled neurospheres. Optimisation of in vitro MRI provides asolid platform for the utilisation of these cells in future cell trans-plantation studies, as others have reported good correspondencebetween in vitro and in vivo detection [62,63].

5. Conclusions

The biological effects of USPIO nanoparticles on the stem cellpopulation of interest need to be determined prior to their appli-cation in cell transplantation therapies. This study provided athorough investigation into the relationship between iron-oxidenanoparticle labelling conditions and neural stem cell function,and found that hNPCs can be efficiently loaded with NPs at optimalconditions. We also demonstrated that the presence of intracellulariron at optimal concentrations does not affect hNPCs’ viability,survivability, multipotency, and migratory capacity in the long-term. With prolonged NPs exposure, NPs translocated from thecytoplasm into the peri-nucleus region, and hNPC migrationbecame severely limited. We also observed alterations in ATP-evoked intracellular calcium transients, however this phenome-non normalised 1 day post-incubation. We were able to demon-strate that our custom designed and manufactured USPIO-labelledhNPCs can be detected with MRI in vitro, which enables thesefindings to be applied in future work involving experimentalmodels of neurological damage/diseases.

Acknowledgements

This work was supported by grants from the National Healthand Medical Research Council (NHMRC) of Australia (No. 571100and No.1048082) the Baxter Charitable Foundation, the Alma HazelEddy Trust, the Rebecca L. Cooper Medical Research Foundation(Sydney, Australia) and the Brian M. Kirby Foundation Gift of SightInitiative (Sydney, Australia) to TC-L. SE is a recipient of the BrianM.Kirby Foundation Gift of Sight Initiative scholarship. BTTP and BSHacknowledge the financial support from Sirtex Medical Ltd. SMGacknowledges the support of the Sydney Medical Foundation. Theauthors gratefully acknowledge the advice and assistance related tomicroscopy experiments provided by Dr. Louise Cole (Bosch Insti-tute Advanced Microscopy Facility, University of Sydney); the ironcontent measurements by Atomic Absorption Spectroscopy pro-vided by Nguyen Pham (School of Chemistry, University of Sydney).The authors declare no conflict of interest in this work.

Please cite this article in press as: Eamegdool SS, et al., Ultrasmall superprecursor cells, Biomaterials (2014), http://dx.doi.org/10.1016/j.biomateri

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