Labeling Stem Cells with a New Hybrid Bismuth/Carbon ...downloads.hindawi.com/journals/cmmi/2019/2183051.pdf · Research Article Labeling Stem Cells with a New Hybrid Bismuth/Carbon

Research ArticleLabeling Stem Cells with a New Hybrid BismuthCarbonNanotube Contrast Agent for X-Ray Imaging

Mayra Hernandez-Rivera1 Stephen Y Cho1 Sakineh E Moghaddam1

Benjamin Y Cheong2 Maria da Graccedila Cabreira-Hansen23 James T Willerson23

Emerson C Perin23 and Lon J Wilson 1

1Department of Chemistry MS-60 Rice University PO Box 1892 Houston TX 77251 USA2CHI St Lukersquos HealthmdashBaylor St Lukersquos Medical Center 6720 Bertner Ave MC 2-270 Houston TX 77030 USA3Texas Heart Institute 6770 Bertner Ave C350 Houston TX 77030 USA

Correspondence should be addressed to Lon J Wilson durangoriceedu

Received 23 March 2019 Accepted 7 May 2019 Published 11 June 2019

Academic Editor Marıa L Garcıa-Martın

Copyright copy 2019 Mayra Hernandez-Rivera et al is is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in anymedium provided the original work isproperly cited

e poor retention and survival of cells after transplantation to solid tissue represent a major obstacle for the eectiveness of stemcell-based therapies e ability to track stem cells in vivo can lead to a better understanding of the biodistribution of transplantedcells in addition to improving the analysis of stem cell therapiesrsquo outcomes Here we described the use of a carbon nanotube-based contrast agent (CA) for X-ray computed tomography (CT) imaging as an intracellular CA to label bone marrow-derivedmesenchymal stem cells (MSCs) Porcine MSCs were labeled without observed cytotoxicity e CA consists of a hybrid materialcontaining ultra-short single-walled carbon nanotubes (20ndash80 nm in length US-tubes) and Bi(III) oxo-salicylate clusters whichcontain four Bi3+ ions per cluster (Bi4C) e CA is thus abbreviated as Bi4CUS-tubes

1 Introduction

In recent years stem cell research has taken many newdirections In particular adult stem cells are being used asvectors to provide extracellular survival and growth factorsin tissue repair as well as to treat disease by replacingdamaged tissue [1] Clinical trials involving cell-based au-tologous therapies (patients receive their own cells) andallogenic therapies (recipients receive cells from a donor)have been conducted for a plethora of diseases such asdiabetes paraplegia multiple sclerosis critical limb ische-mia cerebrovascular diseases blood-related cancers andsome solid tumors [1ndash3] Among the adult stem cells foundin the human body multipotent mesenchymal stromal cells(MSCs) have been widely used due to their relatively easyisolation and ex vivo expansion In addition MSCs have theadvantage of being less immunogenic than other stem cellsdue to the lack of costimulatory agents of the B7 family that

are required to initiate an immune response [4ndash6] isallows the use of MSCs without concerns about immuno-logical rejection or the need for immunosuppressant drugsmaking MSCs a universal stem cell source With the rapidincrease of reported cases of MSC-based therapies there isan urgent need to track the cells in vivo during preclinicaland clinical trials to further understand and evaluate thebehavior and fate of transplanted MSCs

To address this need various imaging techniques havebeen proposed and investigated including X-ray computedtomography (CT) imaging [7] magnetic resonance imaging(MRI) [8ndash13] optical imaging (such as bioluminescence andyenuorescence) [14ndash16] ultrasound-guided photoacoustic(USPA) imaging [17] single-photon emission computedtomography (SPECT) imaging [18] and positron emissiontomography (PET) imaging [19] Of these techniques op-tical imaging has been shown to be a potent tool in pre-clinical small animal studies but its use alone is not yet

HindawiContrast Media amp Molecular ImagingVolume 2019 Article ID 2183051 11 pageshttpsdoiorg10115520192183051

translatable to clinical practice due to its low tissue pene-tration depth [20 21] -e use of each of these imagingtechniques for stem cell tracking is reviewed elsewhere [20]Currently the most used and preferable imaging modalityfor stem cell tracking is MRI with T2Tlowast2 contrast acquiredwhen stem cells are labeled with superparamagnetic agentssuch as iron oxide nanoparticles [22ndash24] However a sig-nificant clinical limitation of MRI is its incompatibility withvarious medical and life support devices such as pacemakersand defibrillators whose presence can cause serious issues(ie magnetic field interactions heating and other arti-facts) Another disadvantage is the long scan time (20ndash90min) which requires patients to remain still in anenclosed space potentially causing discomfort anxiety andclaustrophobia X-ray CT although it is based on ionizingradiation it provides a number of advantages for example itrequires much less time per acquisition (each scan can beperformed in less than 1min) and does not possess a harmfor patients with transplanted magnetics medical devicesAlso X-ray CT and other X-ray-based imaging modalitiesare often more readily available than other imaging tech-nologies especially in countries that are less industrializedand economically developed [25ndash27]

In a previous publication we reported the use of CT incombination with a carbon nanotube- (CNT-) based con-trast agent (CA) to track stem cells [7] X-ray CAs (orradiocontrast agents) are used to provide transient contrastenhancement in X-ray-based imaging modalities such asradiography CT and fluoroscopy and are currently underinvestigation for cell labeling [28] Contrast enhancementcomes largely from the photoelectron effect due to highatomic numbers As a rule materials possessing a higherdensity (ρ) and high atomic number (Z) absorb X-rays moreeffectively [29 30] -e capability of matter to attenuateX-rays is measured in Hounsfield units (HU) By definitionwater has a HU value of 0 and air has a value of minus1000HUwhile most soft tissues fall within 30ndash100HU -e HU of amaterial with a linear X-ray absorption coefficient (μ) isdefined as [29 30]

HU μminus μwater( 1113857

μwatertimes 1000 (1)

Radiocontrast agents with good X-ray attenuation (highHU values) facilitate the process of distinguishing the regionof interest by increasing the HU value of the tissue of interestrelative to the background Currently there are two types ofCAs approved for human use barium sulfate suspensions(ZBa 56) and small water-soluble iodinated molecules(ZI 53) [29] However both of these types of agents areexclusively for extracellular use More recently research hasbeen focused on the use of small molecules or nanoparticlescontaining atoms with higher atomic numbers For examplegold nanoparticles (ZAu 79) are an ideal alternative sincegold has both a high density and a high atomic number andit provides about 27 times greater contrast per unit weightthan iodine [31ndash35] Another material that has been in-vestigated is tantalum oxide (Ta2O5 ZTa 73) nanoparticlesand its use has been evaluated both in vitro and in vivo[36ndash39] Bismuth-based CAs are considered an excellent

alternative among metal-containing CAs because bismuth isone of the heaviest and most dense metals (ZBi 83) withlow levels of toxicity For decades bismuth has been used incosmetic and medical formulations and its toxicity profilehas been studied extensively [40ndash47] Due to bismuthrsquos highatomic number materials containing bismuth such asbismuth sulfide (Bi2S3) nanoparticles and hybrid nano-crystals of iron oxide and bismuth oxide have demonstratedexcellent in vitro and in vivo contrast enhancement longcirculation times and safety profiles [48ndash53] Since differentmetals and nanoparticles have shown to be good X-rayattenuators the standardization of the quantity of materialsneeded within cells to produce a detectable signal is difficultto determine Combinations of different elements theirratios and the attenuation capability of each element willsignificantly influence how much material is needed thusindependent in vivo studies must be conducted for eachradiocontrast agent to experimentally evaluate how muchmaterial is required per cell to produce a signal in a par-ticular tissue of interest

Here we describe the successful internalization of aCNT-based X-ray CA into MSCs -e CA consists of ahybrid material containing ultra-short single-walled carbonnanotubes (20ndash80 nm US-tubes) and a Bi(III) oxo-salicylatecluster ([Bi4(μ-O)2(HO-2-C6H4CO2)8]middot2MeCN) whichcontains four Bi3+ ions in its core (Bi4C)-is cluster as withmost bismuth compounds is insoluble in aqueous mediawhich limits its use in biological studies Carbon nanotubesalthough insoluble in water in their pristine state can beeasily modified to increase their suspendability in aqueousmedia and CNT-based materials have shown promise asbiocompatible platforms for delivering imaging agents intocells in a safe manner [8 10 54 55] In previous work wedescribed the use of a CNT-based material containing sim 26wt of Bi3+ (BiUS-tubes) as an intracellular CA for X-rayCT [7] Here we report the performance of a new andconsiderably improved material (Bi4CUS-tubes) as anintracellular contrast agent for MSCs which contains 20bismuth by weight

2 Materials and Methods

21 Preparation of Bi4CUS-Tubes Bi4CUS-tubes wereprepared as previously reported [56] Briefly the US-tubesand the Bi(III) oxo-salicylate cluster were prepared sepa-rately and then combined by simply sonicating both togetherfor 1 h in dried tetrahydrofuran (THF) -e US-tubeswere produced by cutting full-length single-walled carbonnanotubes (SWCNTs) by the fluorination method describedelsewhere [57] and the synthesis of the Bi(III) oxo-salicylatecluster ([Bi4(μ3-O)2(HO-2-C6H4CO2)8]middot2MeCN) was per-formed as previously reported [58] and placed under vac-uum for 48 h for solvent removal For in vitro studies alabeling solution was prepared by suspending Bi4CUS-tubes in a 017 (wv) solution of Pluronicreg F-108 anonionic surfactant via probe sonication for 5min -esamples were centrifuged at 3200 rpm for 10min and thesupernatant was used for the in vitro experiments -ebismuth concentration was determined using inductively

2 Contrast Media amp Molecular Imaging

coupled plasma optical emission spectrometry (ICP-OESOptima 4300 from PerkinElmer Inc) and adjusted to 1 μM

22 StabilityChallengeofBi4CUS-Tubes -e stability of theBi4CUS-tube material has been previously reported [56] Inthe present study the stability of the Bi4CUS-tubes sus-pended in 017 Pluronicreg was assessed to determinewhether a rigorous sonication process affects the stability ofthe Bi4CUS-tubes when suspended Aliquots of the labelingsolution were placed in centrifuge filter tubes (50mL tubes10 kDa) kept at 37ndash40degC for 24 h and finally centrifuged(Figure S1) -e supernatant was treated with additions of70 HNO3(aq) trace-metal grade and 26 HClO3(aq) underheat to digest the organic material Samples were then ana-lyzed for bismuth by inductively coupled plasma massspectrometry (ICP-MS Elan 9000 from PerkinElmer Inc)

23 Labeling MSCs with Bi4CUS-Tubes Porcine MSCsisolated from the bone marrow of adult male pigs (threedifferent animals) were intracellularly labeled with Bi4CUS-tubes Prior to labeling the concentration of the labelingsolution was determined by ICP-OES and adjusted to10mM Bi3+ To sterilize the labeling solution the samplewas exposed to UV light for 3 hours with rocking MSCswere grown in 175 cm2 flasks in α-minimal essential medium(α-MEM) supplemented with 10 fetal bovine serum (FBS)and incubated at 37degC in a humidified atmosphere con-taining 5 CO2 in air Prior to labeling cryopreserved cellsat the third passage were thawed and then allowed to growuntil 70ndash80 of confluence -e labeling solution was di-rectly added to the culture medium to obtain the desiredfinal concentration During this process FBS-free α-MEMwas used and FBS was added to the cell cultures 4 hpostlabeling to obtain a 10 FBS final solution Sub-sequently cells were incubated and left undisturbed for 20 hFor US-tubes a 24 h incubation time was found to be op-timal to obtain maximum intracellular labeling when in-cubating MSCs with the material [10] For this reason a 24 hlabeling protocol was also used here for the Bi4CUS-tubes

For cell collection MSCs were trypsinized and passedthrough a 70 μm filter to eliminate large cell aggregates Adensity gradient separation using Histopaquereg 1077 (25degCSigma-Aldrich) was performed as described elsewhere[8 10] to remove free Bi4CUS-tubes from the cell sus-pension Labeled MSCs were isolated from the interface ofthe α-MEM and Histopaquereg layers using a plastic transferpipette and washed with PBS Finally cells were countedusing a particle counter (Beckman Counter MultiSizer 3)Experiments with just the Bi4C cluster as the labeling agentwere not possible to conduct due to the water-insolublenature of the cluster thus unlabeled MSCs were used ascontrol cells Experiments were performed in triplicate usingcells from three different animals unless otherwise specified

24 Elemental Analysis of Bi4CUS-Tube-Labeled MSCsAliquots of cell suspensions were collected in glass scintil-lation vials to determine the Bi3+ ion concentration within

the cells by ICP-MS analysis To prepare the samples foranalysis cells were heated and two alternating aliquots of500 μL 70 HNO3(aq) trace metal grade and 26 HClO3(aq)were added to digest the organic matter -e samples wereallowed to dry between treatments Finally the samples werediluted to 5mL with 2 HNO3(aq) trace metal grade andfiltered through a 022 μm pore size syringe filter

25 Viability of Bi4CUS-Tube-Labeled MSCsBi4CUS-tube-labeled MSCs were prepared as describedabove After a 24 h incubation time with the Bi4CUS-tubeslabeled cells were collected for the viability study Positivecontrol (unlabeled MSCs) and negative control (dead un-labeled MSCs treated with 70 methanol for 20min) werealso studied To determine the viability of the MSCs a LIVEDEAD viabilitycytotoxicity assay kit (Invitrogentrade) wasused -e kit consists of two reagents calcein AM whichfluoresces green when cells are viable and ethidiumhomodimer-1 (EthD-1) which fluoresces red when thecellular membrane is compromised -e reagents wereadded to each sample and cells were incubated in the darkfor 20min at room temperature Fluorescence-activated cellsorting (FACS) was performed using a LSR flow cytometer(Becton Dickinson)

26 Subcellular Localization and Label Retention ElectronMicroscopy TEM analysis was performed to determine thesubcellular localization of the Bi4CUS-tubes LabeledMSCs (300 μM Bi3+ for 24 h) and unlabeled MSCs werecentrifuged separately at 1500 rpm for 10min to form a cellpellet (sim1times 106 cellspellet) -e supernatant was removedwithout disturbing the cell pellet and fixed with 4 glu-taraldehyde Samples were left undisturbed for 2 days at 4degCSubsequently samples were washed with PBS and postfixedwith 1 OsO4 for 1 h followed by dehydration with in-creasing concentrations of ethanol Finally samples wereinfiltrated with acetone and Epon 812 resin and embedded ina mold with 100 Epon 812 Using a Leica EM UC7 ultra-microtome blocks containing the embedded samples werecut in 1mm sections and stained with 1 methylene blueand 1 basic fuchsin and also cut in ultra-thin sections(80 nm) and framed on 100-mesh copper grids Grids werestained with 2 alcoholic uranyl acetate and Reynoldsrsquo leadcitrate -e grids were examined using a JEOL 1230 TEMinstrument equipped with an AMTV 600 digital imagingsystem To verify the clearance of the intracellular Bi4CUS-tube material from the labeled MSC cultures the in-tracellular Bi3+ ion content and the amounts found in theculture medium were quantified for up to 72 h postlabelingMSC cultures from two animals were labeled with Bi4CUS-tubes (300 μM Bi3+) in triplicate as described above Afterlabeling cells were replated in 6-well plates at 20times103 cellswell Cultures were subjected to medium changes (2mL)every 24 hours -e 2mL media collected at 24 48 and 72 hwere centrifuged and the top 1mL was removed and filteredwith 20 μM membrane -e pelleted material (dead floatingcells and cell debris) was resuspended in the remaining 1mLand used to determine the number of dead cells present in

Contrast Media amp Molecular Imaging 3

the medium Elemental analysis was performed by ICP-MSBi3+ ion concentrations were determined in both the filteredcell culture medium and in the adherent cell monolayerDeaddetached cells were counted using the Trypan Blueexclusion method

27 Population Doubling Time (PDT) Assay Cell pro-liferation was measured every 24 hours for up to 144 h usingthe CyQUANTregNFCell Proliferation Assay Kit (Invitrogen)which is based on a DNA fluorescent dye which measures theDNA content MSC cultures previously labeled for 24 hourswith Bi4CUS-tubes (300 μM Bi3+) and unlabeled cells in-cluding cells cultured in regular medium and cells exposed tomedium containing 017 Pluronicreg were subcultured in 96-well plates (sim1times 103 cellswell) -e CyQUANT reagent wasadded to triplicate wells after medium removal and incubatedfor 1 h at 37degC after which the nuclear fluorescence wasmeasured using a TECAN Safire 2trademicroplate reader at 485528 nm (excitationemission) To calculate the cell numbercorresponding to the fluorescence intensities we used astandard curve previously made by plating a known numberof MSCs for all experimental conditions

28 Colony-Forming Unit Fibroblast (CFU-F) Assay -eability of MSCs to self-renew upon Bi4CUS-tube labelingwas also investigated Bi4CUS-tube-labeled MSCs wereplated in triplicate 75 cm2 tissue culture flasks at 1500 cellsflask (20 cellscm2) -e low plating density allowed cells togrow as individualized colonies Cells were incubated for14 days with medium replacement every 3 to 4 days Flaskswere then washed with PBS fixed with 70methanol driedand stained with Giemsa Unlabeled MSCs and MSCstreated with 017 Pluronicreg for 24 h were also plated underthe same conditions and used as controls For the CFU-Fenumeration a stereomicroscope was used

29Aree-LineageDifferentiation To determine whether theBi4CUS-tube material affects the ability of the cells todifferentiate into different progenitor cells unlabeled MSCsand Bi4CUS-tube-labeled MSCs were exposed todifferentiation-inducing media to promote adipogenic os-teogenic and chondrogenic differentiation

For adipogenic and osteogenic differentiation labeledand unlabeled counterpart MSC cultures were initiallyplated and grown in 6-well tissue culture plates atsim20times103 cellswell in α-MEM After 24 h the culture me-dium in the plates prepared for osteogenic differentiationwas replaced with the differentiation medium (α-MEMsupplemented with 10 FBS 50 μgmL ascorbate 2-phos-phate 01 μM dexamethasone and 10mM β-glycerolphosphate) Cultures were maintained in differentiationmedium for 14 days with medium changes every 3 to 4 daysTo demonstrate osteoblastic differentiation cultures werestained with Alizarin Red S to detect extracellular calciumdeposition

Adipogenic differentiation medium (α-MEM supple-mented with 10 FBS 1 insulin-transferrin-selenium (ITS)

1 μM dexamethasone 05mM methyl-isobutylxanthine and100 μM indomethacin) was added to the cultures whenconfluence levels reached 50 Cultures were kept in thedifferentiation medium for 3 days and subsequently in adi-pogenic maintenance medium (α-MEM supplemented with10 FBS and 1 ITS) for the following 48 hours -is pro-cedure was repeated twice To evidence adipogenic differ-entiation cultures were stained with Oil Red O andhematoxylin to reveal lipid vacuoles in red and cell nuclei inblue respectively

For chondrogenic differentiation sim200times103 MSCs weretransferred into a 15mL conical tube and centrifuged at1200 rpm for 5min followed by careful removal of the su-pernatant Cell pellets were then incubated in chondrogenicdifferentiation medium (α-MEM supplemented with 1 ITS40 μgmL proline 100 μgmL sodium pyruvate 01 μMdexamethasone 50 μgmL ascorbate 2 phosphate 10 ngmLTGF-β3 and 02 μgmL BMP6) for 21 days Differentiationmedium was replaced every 3 to 4 days Upon completion ofthe assay cell pellets were fixed with 4 paraformaldehydeembedded in paraffin and stained with Alcian blue to revealaccumulation of glycosaminoglycans an abundant extracel-lular component of cartilaginous matrices

210 X-Ray CTof Bi4CUS-Tube-Labeled MSCs -e abilityof Bi4CUS-tubes to attenuate X-rays once internalized inMSCs was evaluated using a clinical CT scanner (iCT 256Phillips) at St Lukersquos Baylor Hospital Houston TX Bi4CUS-tube-labeled MSCs were prepared as described abovefollowed by centrifugation for 10min at 1200 rpm to form acell pellet (sim200times106 cellspellet) Separately a cell pellet ofunlabeled control MSCs was also prepared in a 15mLEppendorf tube 500 μL of 5 agar was added carefully ontop of the cell pellets and samples were kept refrigerated at4degC Data were acquired using the following scanner pa-rameters tube voltage 120 kV pitch 0664 gantry rota-tion time 033 s mAsmA 150302 and reconstructionslice thickness 06250312 cm For quantitative analysisthree areas per sample were selected as regions of interest(ROI) andmeasured in Hounsfield units (HU)-e reportedvalues are the average measured for axial and coronal viewsAnalysis was performed using OsiriX v 412 32 bit an opensource software

211 Statistical Analysis All experiments were conducted intriplicate and values are reported as meanplusmn standard de-viation All statistical analyses were performed usingGraphPad Prism 700 -e single-factor analysis of variance(ANOVA) test was used to determine statistical significanceand the level of significance alpha was defined at 5 unlessotherwise specified

3 Results and Discussion

31 Bi4CUS-Tube Stability Studies -e preparation of the1mM Bi3+ labeling solution (Bi4CUS-tubes suspended in a017 Pluronicreg solution) requires probe sonication aprocess that might disrupt the interaction between the Bi4C


cluster and the US-tubes To investigate possible detachmentof bismuth from the material after sonication umlltrationchallenge was performed as described above No detectablebismuth was observed in the umlltered solution by ICP-MSis umlnding demonstrated that the Bi4C cluster remainedbound to the US-tubes even after the vigorous sonicationprocess

32 Bi4CUS-TubeMSCLabelingViability and IntracellularRetention MSC cultures isolated from adult male pigs(N 3) were used for the in vitro studies A stock solution(1mM Bi3+) was added to the culture medium directlyFifteen dierent labeling concentrations (10 20 30 40 5060 70 80 100 120 140 160 180 200 and 300 μM Bi3+)were evaluated Cell viability under these dierent con-centrations was determined using yenow cytometry for cellsstained with calcein AM and EthD-1 As seen in Figure 1(a)the viability of theMSCs remained greater than 96 even forthe highest concentration studied (300 μM Bi3+) demon-strating that MSCs tolerate well the incorporation of Bi4CUS-tubes Viability for unlabeled MSCs was 97plusmn 3 positivefor calcein AM Dead MSCs (methanol induced) were usedas a negative control in each experiment and found to be98plusmn 2 positive for EthD-1 More detailed information canbe found in Figure S2

To determine concentrations of intracellular Bi4CUS-tubes MSC cultures were plated and labeled as de-scribed earlier After 24 h incubation cells were collectedcounted and prepared for elemental analysis of Bi3+ ione results of these elemental analyses are depicted inFigure 1(b) e uptake of the Bi4CUS-tubes varied from12 times107 to 16 times109 ions of Bi3+ per cell We observed asigniumlcant correlation (plt 00001 r2 086) between in-tracellular concentrations of Bi3+ and the labeling con-centration in the medium e incubations with lowerconcentrations (10 to 80 μM) produced minimal amountsof bismuth uptake However at labeling concentrationshigher than 100 μM greater uptake of the Bi4CUS-tubematerial was observed

TEM was used to further investigate MSC intracellularlabeling To obtain the greatest Bi3+-ion loading 300 μMBi3+ was selected as the labeling concentration whichwould also result in the greatest X-ray attenuation TEMimages of the labeled MSCs (Figure 2) showed that Bi4CUS-tubes accumulated exclusively in the cytoplasm of thecells with no evidence of translocation into the nucleuse same result has been reported for similar materialssuch as the Gadonanotubes [10] which are US-tubesloaded with Gd3+-ion clusters and other analogous ma-terials [8 10] Importantly the Bi4CUS-tubes are the umlrstof a US-tube-based material to become exclusively en-capsulated within vesicles in the cytoplasm of the MSCshowever the encapsulation of US-tube materials withinvesicles has been previously observed in cancer cells(Hep3B and HepG2) [59] In some of the vesicles thecharacteristic formation of umlber-like structures that CNTmaterials often adopt in aqueous media is also clearlyvisible (Figure 2(c)) e fact that Bi4CUS-tubes are

seemingly being internalized by the MSC via an activetransport mechanism might be due to the surface charge ofthe nanomaterial Bi4CUS-tubes have a slightly morepositive charge compared to US-tubes and Gadonano-tubes as conumlrmed by its zeta potential obtained using aMalvern Zen 3600 Zetasizer (Table S1) e present TEMdata clearly demonstrate that Bi4CUS-tubes do not crossthe nuclear membrane but instead are only enclosed invesicles which suggests an active cellular transportmechanism as found for other CNT-based materials usedto label MSCs [54]

e likely clearance of incorporated Bi4CUS-tubematerial from cells represents a crucial issue that canimpact cell tracking performance To determine whetherintracellular Bi4CUS-tubes are released from the cellsover time we investigated the Bi3+-ion content in theculture medium and within the cells for up to 72 h asdetailed in Section 2 Figure 3(a) shows the average Bi3+concentration detected in the culture medium as wellas in the cell fraction samples collected at 24 48 and72 h after labeling We observed no signiumlcant dier-ence between baseline 24 h Bi3+ concentration in thecellular fraction and the two later time points (48 and72 h) indicating that cells retained incorporated Bi4CUS-tubes e slight decrease in the average of the Bi3+

0

96

98

Via

bilit

y of

cells

()

100

50 100 150Labeling concentration (μM Bi)

200 250 300

(a)

00

Bi(I

II) i

ons

cell

50 times 108

10 times 109

15 times 109

20 times 109


200

8060402000

2 times 107

4 times 107

6 times 107

250 300

(b)

Figure 1 Cell viability and Bi4CUS-tube uptake under dierentlabeling concentration (a) Average cell viability measured byyenuorescence activation cell sorting (FACS) analysis (b) In-tracellular incorporation of Bi4CUS-tubes inferred by the con-centration of Bi3+ ioncell from ICP-MS measurements e insetshows the plot for the lower concentration range


concentration in the cell fraction could be due simply tothe initial removal of residual material from the externalcellular membrane e Bi3+ concentrations in the umllteredculture medium showed signiumlcant dierences among the

three time points which is a result of the daily mediumchange

A detailed representation of the intracellular concen-tration of the Bi4CUS-tube material per viable cell is

(a) (b) (c)

Figure 2 TEM images of Bi4CUS-tube-labeled cells (a and b) Yellow arrows indicate Bi4CUS-tubes encapsulated in vacuoles localizedin the cytoplasm while red arrows show the nucleus (c) An enlarged image of the Bi4CUS-tube material where umlber-like agglomeratescan be seen Scale bar 1 μm

24 48 7200

05

10

15

Time (h)

Bi (μ

gm

L)

CellsCulture medium

(a)

24 48 72

50 times 108

10 times 109

15 times 109

20 times 109

25 times 109

Time (h)

Bi(I

II) i

ons

cell

(b)

24 48 72

10 times 104

50 times 104

90 times 104

13 times 105

17 times 105

Time (h)

Cell

num

ber

Viable cellsDead cells

(c)

Figure 3 (a) Bi3+-ion concentration in cell samples and cell culture medium supernatant over time (b) Bi3+-ion concentration per viablecell over time (c) Cell numbers for the viable and dead cells after plating Some error bars are too small to show Data are presented inmeanplusmn SD


presented in Figure 3(b) An initial concentration of about17times109 ions Bi3+cell was obtained after incubation andreplating (24 h) after which a decrease in the metal con-centration per cell was observed is can be explained bythe expanding cell numbers that is depicted in Figure 3(c)(attached cells) Also Figure 3(c) shows the number of deadcells found yenoating on the medium during the experimentduration Our results indicate that uptake of Bi4CUS-tubesby MSCs is persistent allows cell growth and does not aectcell viability

33 Proliferative Properties of Bi4CUS-Tube Labeled MSCsTo study the eect of Bi4CUS-tubes on MSC proliferativecapacity we determined the population doubling time(PDT) and enumerated colony-forming unit umlbroblast(CFU-F) in control (unlabeled and Pluronicreg-treated cells)and Bi4CUS-tube-labeled MSCs Growth kinetics is il-lustrated in Figure 4(a) No dierence among the threedierent conditions was observed Labeled and unlabeledcells (control and Pluronicreg-treated) reached full conyenuenceat 144 h after plating Also no dierence was found in thePDTvalues obtained for control cells (193plusmn 31) Pluronicreg-treated MSCs (248plusmn 84) and Bi4CUS-tube-labeled MSCs(201plusmn 28) Self-renewal a stem cell hallmark was evaluatedusing the CFU-F assay No statistical dierence in CFU-Fnumbers obtained from plating of Bi4CUS-tube-labeledMSCs and those from unlabeled control and Pluronicreg-treatedMSCs (Figure 4(b)) was observedese results showthat MSC proliferative properties were not altered by Bi4CUS-tube incorporation

34 ree-Lineage Dierentiation As multipotent pro-genitors mesenchymal cells have the ability to dierentiateinto fat bone and cartilage (adipocytes osteocytes andchondrocytes respectively) upon culture in dierentiation-inducing medium As seen in Figure 5 Bi4CUS-tube-labeled MSCs successfully dierentiated in fat bone andcartilage tissues as evidenced by staining of intracellular lipidvacuoles (in red A) calcium deposit (in red B) and gly-cosaminoglycans (in blue C) As seen in Figure 5(c) theBi4CUS-tube material remained in the center of the cellsphere and direct prolonged-exposure (sim3weeks) to theBi4CUS-tube material did not change the normal behaviorof MSCs is conumlrms that the labeling of MSCs with theBi4CUS-tube material did not aect MSC dierentiationsuggesting that Bi4CUS-tube-labeled MSCs may retaintheir therapeutic potential

35 X-Ray CT of Bi4CUS-Tube-Labeled MSCs e po-tential of the Bi4CUS-tube material to function as anintracellular contrast agent for X-ray CTwas evaluated bypreparing cell pellets of unlabeled MSCs and Bi4CUS-tube-labeled MSCs as described above Figure 6(a) showsEppendorf tubes containing 200 times106 cells each Un-labeled MSCs appear white while labeled MSCs are darkdue to cellular internalization of the Bi4CUS-tubematerial Analysis of regions of interest revealed HUvalues which is an indicator of the ability of the materialunder study to attenuate X-rays with respect to water(0 HU) and air (minus1000 HU) HU values obtained for thecontrol (unlabeled) MSCs and for Bi4CUS-tube-labeled

0 50 100 1500

10000

20000

Num

ber o

f cel

ls

30000

Time (h)

ControlPluronicregBi4CUS-tubes

(a)

Cont

rol M

SCs

Plur

onic

-MSC

s

Bi4C

U

S-tu

be-M

SCs

450

500

550

Num

ber o

f col

onie

s

(b)

Figure 4 (a) Proliferation and (b) clonogenic assays in control MSCs MSCs treated with Pluronicreg and Bi4CUS-tube-labeled MSCs


MSCs were 30 plusmn 9 and 214 plusmn 22 respectively us the HUvalue obtained for the Bi4CUS-tube-labeled MSCs isapproximately 2 times greater than those previously re-ported for BiUS-tube-labeled MSCs (1101 plusmn 49 HU)[7] e HU values of the dierent iodinated contrastmedia used in the clinic in soft tissues range from 100 to300 HU [60] hence the performance of Bi4CUS-tube-labeled MSCs is clearly within an acceptable range forX-ray attenuation application of clinical value

4 Conclusion

e work presented here introduces X-ray CT as an alter-native imaging technology for the imaging of MSCs Manyother imaging techniques have been investigated for thispurpose such as MRI and optical imaging modalitieshowever CT oers some advantages over these imagingtechnologies such as fast data collection and high spatialresolution e new CNT-based CA reported here Bi4CUS-tubes is taken up by MSCs without the use of trans-fection agents via an active transport mechanism to formaggregates within vesicles in the cytoplasm After in-ternalization of the Bi4CUS-tube material within the cellsan increase in X-ray attenuation was obtained allowing CTimages of the labeled MSCs to be brighter compared tounlabeled control cells e Bi4CUS-tubes do not alter the

Unlabeled MSCs Bi4CUS-tube-labeled MSCs

(a)

(b)

(c)

Figure 5 Histochemical staining of unlabeled and Bi4CUS-tube-labeled MSCs (a) Adipocytes are evidenced by the red stain of lipids(b) Osteoblast activity is demonstrated by extracellular calcium deposits (c) Hypertrophic chondrocytes are located at the cell pelletperiphery

(a)

(b)

(c)

Hig

hX-

ray

atte

nuat

ion

Low

Figure 6 (a) Photograph of the unlabeled MSCs (left) and Bi4CUS-tube-labeled MSCs (right) (b) Conventional black and whiteCT images (coronal view) and (c) color CT images


viability proliferation or differentiation potential of theMSCs which suggest that the stem cells retain their char-acteristic and therapeutic properties -is new intracellularCA material with 20 bismuth by weight is approximately 2times brighter when localized within MSCs than the first-generation material BiUS-tubes which contained onlysim26 bismuth by weight Taken together these two worksserve as a proof-of-concept example which demonstratesthe feasibility of using an intracellular radiocontrast markerof bismuth to visualize and potentially track stem cells In thepresent work X-ray CTwas the imaging modality of choicehowever other X-ray-based technologies such as conven-tional X-ray and fluoroscopy could also be used to visualizethe labeled cells

To the best of our knowledge BiUS-tubes and Bi4CUS-tubes are the first CNT-based materials containingbismuth to be used as intracellular CAs for the imaging ofcells by X-ray CT A similar work has been reported forMSCs labeled with gold nanoparticles and transplanted in arat model however the cell growth after labeling was notevaluated [61] A similar work with gold nanoparticles wasreported for transplanted MSCs for bone regeneration [62]Other previously reported methods to track cells by CT havenot involved the CA being intracellular in nature For ex-ample alginate-poly-L-lysine-alginate microcapsules con-taining barium sulfate or bismuth sulfate have beeninvestigated as radiopaque capsules to track human ca-daveric islets by encapsulating the cells in the inner space ofthe capsulates [63] Similar microcapsules containing goldnanoparticles [60 64 65] and perfluorocarbons [66] havealso been reported for multimodal imaging including CTand for the encapsulation of different types of cells-erefore the present Bi4CUS-tube formulation (and itsBiUS-tube predecessor) represents significant progress inthe development and implementation of intracellular CNT-based CAs containing bismuth for X-ray-based imaging oflive cells Moreover animal studies still need to be conductedto evaluate the X-ray attenuation capability of the material invivo and its possible eventual elimination from the body aswell as the minimum number of labeled cells needed toproduce a detectable signal

Abbreviations

AFM Atomic force microscopyBi4C Bi(III) oxo-salicylate clusterCA Contrast agentCFU-F Colony-forming unit fibroblastCNT Carbon nanotubesCT Computed tomographyEDS Energy dispersive spectroscopyHU Hounsfield unitsICP-MS Inductively coupled plasma mass spectrometryICP-OES Inductively coupled plasma optical emission

spectroscopyMSCs Mesenchymal stem cellsPDT Population doubling timeRBM Radial breathing modeSTEM Scanning transmission electron microscopy

TEM Transmission electron microscopyTGA -ermogravimetrical analysisXPS X-ray photoelectron spectroscopyUS-tubes Ultra-short carbon nanotubes

Data Availability

-e data used to support the findings of this study are in-cluded within this article and in the supplementary materialfile Any other additional information is available uponrequest

Conflicts of Interest

-e authors declare that they have no conflicts of interest

Acknowledgments

At Rice University this work was supported by the WelchFoundation (Grant C-0627 to L J Wilson) -e authorsthank Alon R Azares (Texas Heart Institute Houston TXUSA) for flow cytometry analysis Dr Deborah Vela (BaylorSt Lukersquos Medical Center Houston TX USA) for thehistological analyses Ralph Nichols (Baylor St LukersquosMedical Center Houston TX USA) for acquiring the TEMimages and Dr Louis Maximilian Buja (University of TexasHealth Science Center of Houston Houston TX USA) forhis assistance with TEM analysis

Supplementary Materials

Additional data (raw data obtained by fluorescence-activated cell sorting (FACS) of unlabeled cells positivecontrol cells negative control cells and Bi4US-tube-labeledMSCs and Z-potential values for Pluronicreg alone SWCNTsUS-tubes Gadonanotubes and Bi4US-tubes) as well as arepresentation of the filtration-challenge process performedin the cell labeling solution can be found in the SupportingMaterial file (Supplementary Materials)

References

[1] D G Phinney and D J Prockop ldquoConcise review mesen-chymal stemmultipotent stromal cells the state of trans-differentiation and modes of tissue repair-current viewsrdquoStem Cells vol 25 no 11 pp 2896ndash2902 2007

[2] A Trounson R G -akar G Lomax and D GibbonsldquoClinical trials for stem cell therapiesrdquo BMC Medicine vol 9no 1 p 52 2011

[3] N Kim and S-G Cho ldquoClinical applications of mesenchymalstem cellsrdquo Korean Journal of Internal Medicine vol 28 no 4pp 387ndash402 2013

[4] L C Amado A P Saliaris K H Schuleri et al ldquoCardiacrepair with intramyocardial injection of allogeneic mesen-chymal stem cells after myocardial infarctionrdquo Proceedings ofthe National Academy of Sciences vol 102 no 32pp 11474ndash11479 2005

[5] S Tipnis C Viswanathan and A S Majumdar ldquoImmuno-suppressive properties of human umbilical cord-derivedmesenchymal stem cells role of B7-H1 and IDOrdquo Immu-nology and Cell Biology vol 88 no 8 pp 795ndash806 2010


[6] J M Hare J E Fishman G Gerstenblith et al ldquoComparisonof allogeneic vs autologous bone marrowmdashderived mesen-chymal stem cells delivered by transendocardial injection inpatients with ischemic cardiomyopathyrdquo JAMA vol 308no 22 pp 2369ndash2379 2012

[7] E J Rivera L A Tran M Hernandez-Rivera et alldquoBismuthUS-tubes as a potential contrast agent for X-rayimaging applicationsrdquo Journal of Materials Chemistry Bvol 1 no 37 pp 4792ndash4800 2013

[8] A Gizzatov M Hernandez-Rivera V Keshishian et alldquoSurfactant-free Gd3+-ion-containing carbon nanotube MRIcontrast agents for stem cell labelingrdquo Nanoscale vol 7no 28 pp 12085ndash12091 2015

[9] T Kim E Momin J Choi et al ldquoMesoporous silica-coatedhollow manganese oxide nanoparticles as positive T1 contrastagents for labeling and MRI tracking of adipose-derivedmesenchymal stem cellsrdquo Journal of the American ChemicalSociety vol 133 no 9 pp 2955ndash2961 2011

[10] L A Tran R Krishnamurthy R Muthupillai et al ldquoGado-nanotubes as magnetic nanolabels for stem cell detectionrdquoBiomaterials vol 31 no 36 pp 9482ndash9491 2010

[11] A M Reddy B K Kwak H J Shim et al ldquoIn vivo tracking ofmesenchymal stem cells labeled with a novel chitosan-coatedsuperparamagnetic iron oxide nanoparticles using 30TMRIrdquoJournal of Korean Medical Science vol 25 no 2 pp 211ndash2192010

[12] D L Kraitchman A W Heldman E Atalar et al ldquoIn vivomagnetic resonance imaging of mesenchymal stem cells inmyocardial infarctionrdquo Circulation vol 107 no 18pp 2290ndash2293 2003

[13] J M Hill A J Dick V K Raman et al ldquoSerial cardiacmagnetic resonance imaging of injected mesenchymal stemcellsrdquo Circulation vol 108 no 8 pp 1009ndash1014 2003

[14] S Kidd E Spaeth J L Dembinski et al ldquoDirect evidence ofmesenchymal stem cell tropism for tumor and woundingmicroenvironments using in vivo bioluminescent imagingrdquoStem Cells vol 27 no 10 pp 2614ndash2623 2009

[15] A B Rosen D J Kelly A J T Schuldt et al ldquoFindingfluorescent needles in the cardiac haystack tracking humanmesenchymal stem cells labeled with quantum dots forquantitative in vivo three-dimensional fluorescence analysisrdquoStem Cells vol 25 no 8 pp 2128ndash2138 2007

[16] X Wang M Rosol S Ge et al ldquoDynamic tracking of humanhematopoietic stem cell engraftment using in vivo bio-luminescence imagingrdquo Blood vol 102 no 10 pp 3478ndash3482 2003

[17] S Y Nam L M Ricles L J Suggs and S Y Emelianov ldquoInvivo ultrasound and photoacoustic monitoring of mesen-chymal stem cells labeled with gold nanotracersrdquo PLoS Onevol 7 no 5 Article ID e37267 2012

[18] K J Blackwood B Lewden R G Wells et al ldquoIn vivo SPECTquantification of transplanted cell survival after engraftmentusing 111In-tropolone in infarcted canine myocardiumrdquoJournal of Nuclear Medicine vol 50 no 6 pp 927ndash935 2009

[19] Z Love F Wang J Dennis et al ldquoImaging of mesenchymalstem cell transplant by bioluminescence and PETrdquo Journal ofNuclear Medicine vol 48 no 12 pp 2011ndash2020 2007

[20] J V Frangioni and R J Hajjar ldquoIn vivo tracking of stem cellsfor clinical trials in cardiovascular diseaserdquo Circulationvol 110 no 21 pp 3378ndash3383 2004

[21] G D Luker and K E Luker ldquoOptical imaging current ap-plications and future directionsrdquo Journal of Nuclear Medicinevol 49 no 1 pp 1ndash4 2008

[22] J W M Bulte and D L Kraitchman ldquoIron oxide MR contrastagents for molecular and cellular imagingrdquo NMR in Bio-medicine vol 17 no 7 pp 484ndash499 2004

[23] Y Amsalem Y Mardor M S Feinberg et al ldquoIron-oxidelabeling and outcome of transplanted mesenchymal stem cellsin the infarcted myocardiumrdquo Circulation vol 116 no 11pp Indash38ndashIndash45 2007

[24] E M Shapiro S Skrtic and A P Koretsky ldquoSizing it upcellular MRI using micron-sized iron oxide particlesrdquo Mag-netic Resonance in Medicine vol 53 no 2 pp 329ndash338 2005

[25] OECD Geographic Variations in Health Care OECDPublishing Paris France 2014 httpwwwoecd-ilibraryorgsocial-issues-migration-healthgeographic-variations-in-health-care_9789264216594-en

[26] OECD Magnetic Resonance Imaging (MRI) Units OECDPublishing Paris France 2015 httpwwwoecd-ilibraryorgsocial-issues-migration-healthmagnetic-resonance-imaging-mri-unitsindicatorenglish_1a72e7d1-en

[27] OECD Computed Tomography (CT) Scanners OECDPublishing Paris France 2015 httpwwwoecd-ilibraryorgsocial-issues-migration-healthcomputed-tomography-ct-scannersindicatorenglish_bedece12-en

[28] J Kim P Chhour J Hsu et al ldquoUse of nanoparticle contrastagents for cell tracking with computed tomographyrdquo Bio-conjugate Chemistry vol 28 no 6 pp 1581ndash1597 2017

[29] S-B Yu and A D Watson ldquoMetal-based X-ray contrastmediardquo Chemical Reviews vol 99 no 9 pp 2353ndash2378 1999

[30] H Lusic and M W Grinstaff ldquoX-ray-computed tomographycontrast agentsrdquo Chemical Reviews vol 113 no 3pp 1641ndash1666 2013

[31] J F Hainfeld D N Slatkin T M Focella andH M Smilowitz ldquoGold nanoparticles a new X-ray contrastagentrdquo British Journal of Radiology vol 79 no 939pp 248ndash253 2006

[32] D Kim S Park J H Lee Y Y Jeong and S Jon ldquoAnti-biofouling polymer-coated gold nanoparticles as a contrastagent for in vivo X-ray computed tomography imagingrdquoJournal of the American Chemical Society vol 129 no 24pp 7661ndash7665 2007

[33] R Guo H Wang C Peng et al ldquoX-ray attenuation propertyof dendrimer-entrapped gold nanoparticlesrdquo Journal ofPhysical Chemistry C vol 114 no 1 pp 50ndash56 2010

[34] R Popovtzer A Agrawal N A Kotov et al ldquoTargeted goldnanoparticles enable molecular CT imaging of cancerrdquo NanoLetters vol 8 no 12 pp 4593ndash4596 2008

[35] Q-Y Cai S H Kim K S Choi et al ldquoColloidal goldnanoparticles as a blood-pool contrast agent for X-raycomputed tomography in micerdquo Investigative Radiologyvol 42 no 12 pp 797ndash806 2007

[36] Y Jin Y Li X Ma et al ldquoEncapsulating tantalum oxide intopolypyrrole nanoparticles for X-ray CTphotoacoustic bi-modal imaging-guided photothermal ablation of cancerrdquoBiomaterials vol 35 no 22 pp 5795ndash5804 2014

[37] A S Torres P J Bonitatibus R E Colborn et al ldquoBiologicalperformance of a size-fractionatedcore-shell tantalum oxidenanoparticle X-ray contrast agentrdquo Investigative Radiologyvol 47 no 10 pp 578ndash587 2012

[38] M H Oh N Lee H Kim et al ldquoLarge-scale synthesis ofbioinert tantalum oxide nanoparticles for X-ray computedtomography imaging and bimodal image-guided sentinellymph node mappingrdquo Journal of the American ChemicalSociety vol 133 no 14 pp 5508ndash5515 2011

[39] P J Bonitatibus A S Torres G D Goddard P F FitzGeraldand A M Kulkarni ldquoSynthesis characterization and


computed tomography imaging of a tantalum oxide nano-particle imaging agentrdquo Chemical Communications vol 46no 47 pp 8956ndash8958 2010

[40] P T Reynolds K C Abalos J Hopp and M E WilliamsldquoBismuth toxicity a rare cause of neurologic dysfunctionrdquoInternational Journal of Clinical Medicine vol 3 no 1pp 46ndash48 2012

[41] M F Gordon R I Abrams D B Rubin W B Barr andD D Correa ldquoBismuth subsalicylate toxicity as a cause ofprolonged encephalopathy with myoclonusrdquo MovementDisorders vol 10 no 2 pp 220ndash222 1995

[42] G G Briand and N Burford ldquoBismuth compounds andpreparations with biological or medicinal relevancerdquoChemical Reviews vol 99 no 9 pp 2601ndash2658 1999

[43] S P Lee T H Lim J Pybus and A C Clarke ldquoTissuedistribution of orally administered bismuth in the ratrdquoClinical and Experimental Pharmacology and Physiologyvol 7 no 3 pp 319ndash324 1980

[44] L Z Benet ldquoSafety and pharmacokinetics colloidal bismuthsubcitraterdquo Scandinavian Journal of Gastroenterology vol 26no 185 pp 29ndash35 1991

[45] A Slikkerveer and F A Wolff ldquoPharmacokinetics and tox-icity of bismuth compoundsrdquoMedical Toxicology and AdverseDrug Experience vol 4 no 5 pp 303ndash323 2012

[46] N Krain and M Y Allain ldquoEnhancement of bismuth toxicityby L-cysteinerdquo Research Communications in Molecular Pa-thology and Pharmacology vol 89 no 3 pp 357ndash364 1995

[47] V Rodilla A T Miles W Jenner and G M HawksworthldquoExposure of cultured human proximal tubular cells tocadmium mercury zinc and bismuth toxicity and metal-lothionein inductionrdquo Chemico-Biological Interactionsvol 115 no 1 pp 71ndash83 1998

[48] S Veintemillas-Verdaguer Y Luengo C J Serna et alldquoBismuth labeling for the CT assessment of local adminis-tration of magnetic nanoparticlesrdquo Nanotechnology vol 26no 13 article 135101 2015

[49] O Rabin J Manuel Perez J Grimm G Wojtkiewicz andR Weissleder ldquoAn X-ray computed tomography imagingagent based on long-circulating bismuth sulphide nano-particlesrdquo Nature Materials vol 5 no 2 pp 118ndash122 2006

[50] J M Kinsella R E Jimenez P P Karmali et al ldquoX-raycomputed tomography imaging of breast cancer by usingtargeted peptide-labeled bismuth sulfide nanoparticlesrdquoAngewandte Chemie International Edition vol 50 no 51pp 12308ndash12311 2011

[51] P C Naha A Al Zaki E Hecht et al ldquoDextran coatedbismuthndashiron oxide nanohybrid contrast agents for computedtomography and magnetic resonance imagingrdquo Journal ofMaterials Chemistry B vol 2 no 46 pp 8239ndash8248 2014

[52] K Ai Y Liu J Liu Q Yuan Y He and L Lu ldquoLarge-scalesynthesis of Bi2S3 Nanodots as a contrast agent for in vivoX-ray computed tomography imagingrdquo Advanced Materialsvol 23 no 42 pp 4886ndash4891 2011

[53] Y Fang C Peng R Guo et al ldquoDendrimer-stabilized bis-muth sulfide nanoparticles synthesis characterization andpotential computed tomography imaging applicationsrdquo AeAnalyst vol 138 no 11 pp 3172ndash3180 2013

[54] E Mooney P Dockery U Greiser M Murphy andV Barron ldquoCarbon nanotubes and mesenchymal stem cellsbiocompatibility proliferation and differentiationrdquo NanoLetters vol 8 no 8 pp 2137ndash2143 2008

[55] M Hernandez-Rivera N G Zaibaq and L J Wilson ldquoTo-ward carbon nanotube-based imaging agents for the clinicrdquoBiomaterials vol 101 pp 229ndash240 2016

[56] M Hernandez-Rivera I Kumar S Y Cho et al ldquoHigh-performance hybrid bismuthndashcarbon nanotube based con-trast agent for X-ray CT imagingrdquo ACS Applied Materials ampInterfaces vol 9 no 7 pp 5709ndash5716 2017

[57] Z Gu H Peng R H Hauge R E Smalley and J L MargraveldquoCutting single-wall carbon nanotubes through fluorinationrdquoNano Letters vol 2 no 9 pp 1009ndash1013 2002

[58] T D Boyd I Kumar E E Wagner and K H WhitmireldquoSynthesis and structural studies of the simplest bismuth(iii)oxo-salicylate complex [Bi4(μ3-O)2(HO-2-C6H4CO2)8]middot2Solv(Solv MeCNorMeNO2)rdquoChemical Communications vol 50no 27 pp 3556ndash3559 2014

[59] S Phounsavath ldquoRF heating of ultra-shortsingle-walledcarbon nanotubes and gadonanotubes for non-invasivecancer hyperthermiardquo -esis Rice University HoustonTX USA 2014 httpsscholarshipriceeduhandle191177425

[60] W De Vos J Casselman and G R J Swennen ldquoCone-beamcomputerized tomography (CBCT) imaging of the oral andmaxillofacial region a systematic review of the literaturerdquoInternational Journal of Oral and Maxillofacial Surgeryvol 38 no 6 pp 609ndash625 2009

[61] O Betzer A Shwartz M Motiei et al ldquoNanoparticle-basedCT imaging technique for longitudinal and quantitative stemcell tracking within the brain application in neuropsychiatricdisordersrdquo ACS Nano vol 8 no 9 pp 9274ndash9285 2014

[62] D Wan D Chen K Li et al ldquoGold nanoparticles as a po-tential cellular probe for tracking of stem cells in bone re-generation using dual-energy computed tomographyrdquo ACSAppliedMaterials amp Interfaces vol 8 no 47 pp 32241ndash322492016

[63] B P Barnett D L Kraitchman C Lauzon et al ldquoRadiopaquealginate microcapsules for X-ray visualization and immu-noprotection of cellular therapeuticsrdquo Molecular Pharma-ceutics vol 3 no 5 pp 531ndash538 2006

[64] D R Arifin C M Long A A Gilad et al ldquoTrimodalgadolinium-gold microcapsules containing pancreatic isletcells restore normoglycemia in diabetic mice and can betracked by using US CT and positive-contrast MR imagingrdquoRadiology vol 260 no 3 pp 790ndash798 2011

[65] J Kim D R Arifin N Muja et al ldquoMultifunctional capsule-in-capsules for immunoprotection and trimodal imagingrdquoAngewandte Chemie International Edition vol 50 no 10pp 2317ndash2321 2011

[66] B P Barnett J Ruiz-Cabello P Hota et al ldquoFluorocapsulesfor improved function immunoprotection and visualizationof cellular therapeutics with MR US and CT imagingrdquoRadiology vol 258 no 1 pp 182ndash191 2011


Stem Cells International

Hindawiwwwhindawicom Volume 2018


MEDIATORSINFLAMMATION

of

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Immunology ResearchHindawiwwwhindawicom Volume 2018

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine


Behavioural Neurology

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwwwhindawicom

Submit your manuscripts atwwwhindawicom

translatable to clinical practice due to its low tissue pene-tration depth [20 21] -e use of each of these imagingtechniques for stem cell tracking is reviewed elsewhere [20]Currently the most used and preferable imaging modalityfor stem cell tracking is MRI with T2Tlowast2 contrast acquiredwhen stem cells are labeled with superparamagnetic agentssuch as iron oxide nanoparticles [22ndash24] However a sig-nificant clinical limitation of MRI is its incompatibility withvarious medical and life support devices such as pacemakersand defibrillators whose presence can cause serious issues(ie magnetic field interactions heating and other arti-facts) Another disadvantage is the long scan time (20ndash90min) which requires patients to remain still in anenclosed space potentially causing discomfort anxiety andclaustrophobia X-ray CT although it is based on ionizingradiation it provides a number of advantages for example itrequires much less time per acquisition (each scan can beperformed in less than 1min) and does not possess a harmfor patients with transplanted magnetics medical devicesAlso X-ray CT and other X-ray-based imaging modalitiesare often more readily available than other imaging tech-nologies especially in countries that are less industrializedand economically developed [25ndash27]

In a previous publication we reported the use of CT incombination with a carbon nanotube- (CNT-) based con-trast agent (CA) to track stem cells [7] X-ray CAs (orradiocontrast agents) are used to provide transient contrastenhancement in X-ray-based imaging modalities such asradiography CT and fluoroscopy and are currently underinvestigation for cell labeling [28] Contrast enhancementcomes largely from the photoelectron effect due to highatomic numbers As a rule materials possessing a higherdensity (ρ) and high atomic number (Z) absorb X-rays moreeffectively [29 30] -e capability of matter to attenuateX-rays is measured in Hounsfield units (HU) By definitionwater has a HU value of 0 and air has a value of minus1000HUwhile most soft tissues fall within 30ndash100HU -e HU of amaterial with a linear X-ray absorption coefficient (μ) isdefined as [29 30]

HU μminus μwater( 1113857

μwatertimes 1000 (1)

Radiocontrast agents with good X-ray attenuation (highHU values) facilitate the process of distinguishing the regionof interest by increasing the HU value of the tissue of interestrelative to the background Currently there are two types ofCAs approved for human use barium sulfate suspensions(ZBa 56) and small water-soluble iodinated molecules(ZI 53) [29] However both of these types of agents areexclusively for extracellular use More recently research hasbeen focused on the use of small molecules or nanoparticlescontaining atoms with higher atomic numbers For examplegold nanoparticles (ZAu 79) are an ideal alternative sincegold has both a high density and a high atomic number andit provides about 27 times greater contrast per unit weightthan iodine [31ndash35] Another material that has been in-vestigated is tantalum oxide (Ta2O5 ZTa 73) nanoparticlesand its use has been evaluated both in vitro and in vivo[36ndash39] Bismuth-based CAs are considered an excellent

alternative among metal-containing CAs because bismuth isone of the heaviest and most dense metals (ZBi 83) withlow levels of toxicity For decades bismuth has been used incosmetic and medical formulations and its toxicity profilehas been studied extensively [40ndash47] Due to bismuthrsquos highatomic number materials containing bismuth such asbismuth sulfide (Bi2S3) nanoparticles and hybrid nano-crystals of iron oxide and bismuth oxide have demonstratedexcellent in vitro and in vivo contrast enhancement longcirculation times and safety profiles [48ndash53] Since differentmetals and nanoparticles have shown to be good X-rayattenuators the standardization of the quantity of materialsneeded within cells to produce a detectable signal is difficultto determine Combinations of different elements theirratios and the attenuation capability of each element willsignificantly influence how much material is needed thusindependent in vivo studies must be conducted for eachradiocontrast agent to experimentally evaluate how muchmaterial is required per cell to produce a signal in a par-ticular tissue of interest

Here we describe the successful internalization of aCNT-based X-ray CA into MSCs -e CA consists of ahybrid material containing ultra-short single-walled carbonnanotubes (20ndash80 nm US-tubes) and a Bi(III) oxo-salicylatecluster ([Bi4(μ-O)2(HO-2-C6H4CO2)8]middot2MeCN) whichcontains four Bi3+ ions in its core (Bi4C)-is cluster as withmost bismuth compounds is insoluble in aqueous mediawhich limits its use in biological studies Carbon nanotubesalthough insoluble in water in their pristine state can beeasily modified to increase their suspendability in aqueousmedia and CNT-based materials have shown promise asbiocompatible platforms for delivering imaging agents intocells in a safe manner [8 10 54 55] In previous work wedescribed the use of a CNT-based material containing sim 26wt of Bi3+ (BiUS-tubes) as an intracellular CA for X-rayCT [7] Here we report the performance of a new andconsiderably improved material (Bi4CUS-tubes) as anintracellular contrast agent for MSCs which contains 20bismuth by weight

2 Materials and Methods

21 Preparation of Bi4CUS-Tubes Bi4CUS-tubes wereprepared as previously reported [56] Briefly the US-tubesand the Bi(III) oxo-salicylate cluster were prepared sepa-rately and then combined by simply sonicating both togetherfor 1 h in dried tetrahydrofuran (THF) -e US-tubeswere produced by cutting full-length single-walled carbonnanotubes (SWCNTs) by the fluorination method describedelsewhere [57] and the synthesis of the Bi(III) oxo-salicylatecluster ([Bi4(μ3-O)2(HO-2-C6H4CO2)8]middot2MeCN) was per-formed as previously reported [58] and placed under vac-uum for 48 h for solvent removal For in vitro studies alabeling solution was prepared by suspending Bi4CUS-tubes in a 017 (wv) solution of Pluronicreg F-108 anonionic surfactant via probe sonication for 5min -esamples were centrifuged at 3200 rpm for 10min and thesupernatant was used for the in vitro experiments -ebismuth concentration was determined using inductively






























0

96

98

Via

bilit

y of

cells

()

100


200 250 300

(a)

00

Bi(I

II) i

ons

cell

50 times 108

10 times 109

15 times 109

20 times 109


200

8060402000

2 times 107

4 times 107

6 times 107

250 300

(b)






(a) (b) (c)


24 48 7200

05

10

15

Time (h)

Bi (μ

gm

L)

CellsCulture medium

(a)

24 48 72

50 times 108

10 times 109

15 times 109

20 times 109

25 times 109

Time (h)

Bi(I

II) i

ons

cell

(b)

24 48 72

10 times 104

50 times 104

90 times 104

13 times 105

17 times 105

Time (h)

Cell

num

ber


(c)







0 50 100 1500

10000

20000

Num

ber o

f cel

ls

30000

Time (h)


(a)

Cont

rol M

SCs

Plur

onic

-MSC

s

Bi4C

U

S-tu

be-M

SCs

450

500

550

Num

ber o

f col

onie

s

(b)




4 Conclusion



(a)

(b)

(c)


(a)

(b)

(c)

Hig

hX-

ray

atte

nuat

ion

Low





Abbreviations




Data Availability




Acknowledgments




References











































































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of















































0

96

98

Via

bilit

y of

cells

()

100


200 250 300

(a)

00

Bi(I

II) i

ons

cell

50 times 108

10 times 109

15 times 109

20 times 109


200

8060402000

2 times 107

4 times 107

6 times 107

250 300

(b)






(a) (b) (c)


24 48 7200

05

10

15

Time (h)

Bi (μ

gm

L)

CellsCulture medium

(a)

24 48 72

50 times 108

10 times 109

15 times 109

20 times 109

25 times 109

Time (h)

Bi(I

II) i

ons

cell

(b)

24 48 72

10 times 104

50 times 104

90 times 104

13 times 105

17 times 105

Time (h)

Cell

num

ber


(c)







0 50 100 1500

10000

20000

Num

ber o

f cel

ls

30000

Time (h)


(a)

Cont

rol M

SCs

Plur

onic

-MSC

s

Bi4C

U

S-tu

be-M

SCs

450

500

550

Num

ber o

f col

onie

s

(b)




4 Conclusion



(a)

(b)

(c)


(a)

(b)

(c)

Hig

hX-

ray

atte

nuat

ion

Low





Abbreviations




Data Availability




Acknowledgments




References











































































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of






































0

96

98

Via

bilit

y of

cells

()

100


200 250 300

(a)

00

Bi(I

II) i

ons

cell

50 times 108

10 times 109

15 times 109

20 times 109


200

8060402000

2 times 107

4 times 107

6 times 107

250 300

(b)






(a) (b) (c)


24 48 7200

05

10

15

Time (h)

Bi (μ

gm

L)

CellsCulture medium

(a)

24 48 72

50 times 108

10 times 109

15 times 109

20 times 109

25 times 109

Time (h)

Bi(I

II) i

ons

cell

(b)

24 48 72

10 times 104

50 times 104

90 times 104

13 times 105

17 times 105

Time (h)

Cell

num

ber


(c)







0 50 100 1500

10000

20000

Num

ber o

f cel

ls

30000

Time (h)


(a)

Cont

rol M

SCs

Plur

onic

-MSC

s

Bi4C

U

S-tu

be-M

SCs

450

500

550

Num

ber o

f col

onie

s

(b)




4 Conclusion



(a)

(b)

(c)


(a)

(b)

(c)

Hig

hX-

ray

atte

nuat

ion

Low





Abbreviations




Data Availability




Acknowledgments




References











































































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of

























0

96

98

Via

bilit

y of

cells

()

100


200 250 300

(a)

00

Bi(I

II) i

ons

cell

50 times 108

10 times 109

15 times 109

20 times 109


200

8060402000

2 times 107

4 times 107

6 times 107

250 300

(b)






(a) (b) (c)


24 48 7200

05

10

15

Time (h)

Bi (μ

gm

L)

CellsCulture medium

(a)

24 48 72

50 times 108

10 times 109

15 times 109

20 times 109

25 times 109

Time (h)

Bi(I

II) i

ons

cell

(b)

24 48 72

10 times 104

50 times 104

90 times 104

13 times 105

17 times 105

Time (h)

Cell

num

ber


(c)







0 50 100 1500

10000

20000

Num

ber o

f cel

ls

30000

Time (h)


(a)

Cont

rol M

SCs

Plur

onic

-MSC

s

Bi4C

U

S-tu

be-M

SCs

450

500

550

Num

ber o

f col

onie

s

(b)




4 Conclusion



(a)

(b)

(c)


(a)

(b)

(c)

Hig

hX-

ray

atte

nuat

ion

Low





Abbreviations




Data Availability




Acknowledgments




References











































































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of






















(a) (b) (c)


24 48 7200

05

10

15

Time (h)

Bi (μ

gm

L)

CellsCulture medium

(a)

24 48 72

50 times 108

10 times 109

15 times 109

20 times 109

25 times 109

Time (h)

Bi(I

II) i

ons

cell

(b)

24 48 72

10 times 104

50 times 104

90 times 104

13 times 105

17 times 105

Time (h)

Cell

num

ber


(c)







0 50 100 1500

10000

20000

Num

ber o

f cel

ls

30000

Time (h)


(a)

Cont

rol M

SCs

Plur

onic

-MSC

s

Bi4C

U

S-tu

be-M

SCs

450

500

550

Num

ber o

f col

onie

s

(b)




4 Conclusion



(a)

(b)

(c)


(a)

(b)

(c)

Hig

hX-

ray

atte

nuat

ion

Low





Abbreviations




Data Availability




Acknowledgments




References











































































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of























0 50 100 1500

10000

20000

Num

ber o

f cel

ls

30000

Time (h)


(a)

Cont

rol M

SCs

Plur

onic

-MSC

s

Bi4C

U

S-tu

be-M

SCs

450

500

550

Num

ber o

f col

onie

s

(b)




4 Conclusion



(a)

(b)

(c)


(a)

(b)

(c)

Hig

hX-

ray

atte

nuat

ion

Low





Abbreviations




Data Availability




Acknowledgments




References











































































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















4 Conclusion



(a)

(b)

(c)


(a)

(b)

(c)

Hig

hX-

ray

atte

nuat

ion

Low





Abbreviations




Data Availability




Acknowledgments




References











































































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of





















Abbreviations




Data Availability




Acknowledgments




References











































































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of























































































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of























of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of



















Documents

Labeling Stem Cells with a New Hybrid Bismuth/Carbon ...downloads.hindawi.com/journals/cmmi/2019/2183051.pdf · Research Article Labeling Stem Cells with a New Hybrid Bismuth/Carbon