Biomaterials 34 (2013) 3837e3845. Contents lists available at SciVerse ScienceDirect. Biomaterials

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1 Biomterils 3 (13) 3837e385 Contents lists ville t SiVerse SieneDiret Biomterils journl homepge: Monodisperse mgnetite nnoprtile trers for in vivo mgneti prtile imging Amit P. Khndhr, R. Mtthew Ferguson, Hmed Armi, Knnn M. Krishnn * University of Wshington, Mterils Siene & Engineering, Settle, WA 98195, USA rtile info strt Artile history: Reeived 9 Jnury 13 Aepted 7 Jnury 13 Aville online 1 Ferury 13 Keywords: Mgneti prtile imging Monodisperse iron oxide nnoprtiles Biodistriution Cirultion time Contrst gents Mgneti Prtile Imging (MPI) is new iomedil imging modlity tht produes rel-time, highresolution tomogrphi imges of superprmgneti iron oxide (SPIO) nnoprtile trer distriutions. In this study, we synthesized monodisperse trers for enhned MPI performne nd investigted oth, their lood lerne time using 5 khz mgneti prtile spetrometer (MPS), nd iodistriution using omintion of quntittive T-weighted MRI nd tissue histology. In vitro nd in vivo MPI performne of our mgneti nnoprtile trers (MNTs), sujet to iologil onstrints, were ompred to ommerilly ville SPIOs (Resovist). Monodisperse MNTs showed -fold greter signl per unit mss, nd % etter sptil resolution. In vitro evlution of trers showed tht MPI performne of our MNTs is preserved in lood, serum-rih ell-ulture medium nd gel; thus independent of hnges in hydrodynmi volume nd fluid visosity e ritil prerequisite for in vivo MPI. In rodent model, our MNTs irulted for 15 min e 3 longer thn Resovist e nd supported our in vitro evlution tht MPI signl is preserved in the physiologil environment. Furthermore, MRI nd histology nlysis showed tht MNTs distriute in the retiuloendothelil system (RES) in mnner similr to linilly pproved SPIO gents. MNTs demonstrting long-irultion times nd optimized MPI performne show potentil s ngiogrphy trers nd lood-pool gents for the emerging MPI imging modlity. Ó 13 Elsevier Ltd. All rights reserved. 1. Introdution Mgneti Prtile Imging (MPI) is n emerging rel-time tomogrphi imging modlity tht quntittively detets nd imges superprmgneti iron oxide (SPIO) nnoprtiles [1,]. In ddition to its intrinsi dvntges in imging, for its first linil pplition MPI is eing developed s sfe nd ompetitive lterntive to CT-ngiogrphy. Presently, CT-ngiogrphy sns use iodinted ontrst medi (ICM) for dignosing rdiovsulr disese. ICM undergo renl lerne nd put ptients with underlying renl dysfuntion t high risk of ontrst-indued nephropthy (CIN); w5% of potentil CT-ngiogrphy ptients lso hve hroni kidney disese (CKD) [3e6]. In ontrst, MPI uses sfe mgneti fields (no ionizing rdition) nd SPIO mgneti nnoprtile trers 1 (MNTs) tht re generlly well tolerted in CKD ptients. * Corresponding uthor. E-mil ddresses: knnnmk@uw.edu, knnnmk@u.wshington.edu (K.M. Krishnn). 1 Sine MPI is truly trer-imging tehnology, we refer to SPIO nnoprtiles ssoited with MPI s mgneti nnoprtile trers (MNTs) rther thn ontrst gents. However, for MPI to e linilly ompetitive, the in vivo performne of MNTs must e optimized. In our previous work we hve modeled [7] nd experimentlly tilored [8,9] MNT size nd size distriution to enhne MPI performne nd demonstrted 3- fold gin in sensitivity nd 37% etter sptil resolution thn the est ville ommeril trers (ResovistÔ) in phntom imging [RM Ferguson et l., in preprtion]. In linil pplitions, however, MPI imging performne depends fundmentlly on the intrinsi properties nd performne of MNTs in omplex iologil environment, nd thus the physil optimiztion of trers is est rried out in onert with pproprite in vitro nd in vivo models. Current SPIO ontrst gents developed for MRI, when used offthe-shelf, re grossly indequte for MPI [1,3,9,1] e mere 3% of nnoprtiles in ResovistÔ ontriute to the MPI signl [1,] e nd simply do not trnslte very well in vivo. Fundmentlly, MNTs re the only soure of signl in MPI, nd s iologil tissue is dimgneti, it leds to ner-infinite imge ontrst. In MPI, the hrteristi nonliner mgnetiztion reversl of SPIO is exited in n AC-field to produe time-vrying indutive signl in the reeive oil; further, signl loliztion is hieved y snning field-free point ross the entire imging volume. In order to optimize MPI 1-961/$ e see front mtter Ó 13 Elsevier Ltd. All rights reserved.

2 3838 A.P. Khndhr et l. / Biomterils 3 (13) 3837e385 signl, the mgnetiztion reversl dynmis, whih re governed y nnoprtile relxtion, must e tuned to the field frequeny [7]. Sine nnoprtile size nd size distriution determine the relxtion mehnism nd distriution of relxtion times [11], respetively, they must e tilored to the exittion frequeny in order to optimize MPI performne. Furthermore, the optimized reversl dynmis, nd thus MNT performne, must e preserved in iologil environment for linil relevne. In this work we desrie in detil the in vitro nd linilly relevnt in vivo MPI performne of our monodisperse MNTs. Speifilly, we investigted the ritil properties e lood irultion time, MPI signl per unit mss nd iodistriution e tht hrterize MNTs suitle for in vivo pplitions in MPI-sed ngiogrphy, nd furthermore, differentite them from ommeril SPIO trers (ResovistÔ). To evlute MPI performne, we used mgneti prtile spetrometer (MPS): rpid nd urte method to ssess the MPI-relevnt imging performne of MNTs, s pre-linil nd linil snners re still under development. In short, we demonstrte tht these MNTs hve pproprite physil nd iologil hrteristis for immedite trnsltionl pplitions of MPI suh s ngiogrphy. To present ontextul frmework for this work, in the following setions we provide rief overview of the dvntges of using MPI over trditionl CT-ngiogrphy sns nd summrize the physil ides underpinning the novel method of MNT detetion using MPI (omprehensive explntions n e found elsewhere [1,1]) MPI nd medil imging SPIOs hve history of regultory pprovl nd linil use; first introdued to the linil mrket in 1995, SPIO nnoprtiles were used for deteting liver lesions using T-weighted mgneti resonne imging (MRI) [13]. In ft, Ferheme (ferumoxytol), dextrn-oted iron oxide nnoprtile formultion ws pproved in 9 for tretment of iron defiieny nemi in CKD ptients [1]. Due to their sfe linil history, SPIOs re lso the mterils of hoie for development of MPI trers. Unlike MRI, where the lrge mgneti moment of SPIO nnoprtiles inreses T-relxivity of nery protons to enhne negtive tissue ontrst, MPI exploits the hrteristi nonliner mgnetiztion of SPIO nnoprtiles to onstrut high temporl (milliseond time-sles) nd sptil resolution (su-mm) imges of nnoprtile distriutions. Physil simultions nd models [7,15,16] suggest the possiility of su-mm resolution nd nnomolr sensitivity in MPI, while experimentl results [8,1] show tht miromolr sensitivity nd w1 mm resolution re lredy ttinle [1]. To pproh the gol of su-mm resolution nd mke MPI ompetitive with other trer-sed linil imging proedures suh s x-ry CT, signifint improvements in trer design nd optimiztion re required. Additionlly, ICM gents re dministered rterilly using invsive/risky theteriztion proedures nd require high doses (3e1 ml of 3 mgi/ ml iodixnol for imging oronry rteries) to delinete nrrow lood vessels from surrounding tissue. Sine there is no osuring tissue noise in MPI, the required dose of MNTs is signifintly lower nd MNTs with suffiient irulting times, s demonstrted here, n e sfely dministered intrvenously. 1.. SPIO nnoprtile detetion in MPI At room temperture nd 1 s mesurement time, mgnetite (Fe 3 O ) MNTs less thn w7 nm (di.) re superprmgneti, i.e. the net mgneti moment (m) of n ensemle of prtiles is thermlly exited nd responds nonlinerly (Fig. 1) to n externl field (H) [17,18]. In MPS, modulting (AC) exittion field, H(t), of fixed drive frequeny (typilly 5 khz) nd suffiient field strength (typilly m H w mt pep ) to sturte MNTs is trnsmitted. The resulting time-vrying mgnetiztion response, m(h(t)), indues voltge in the reeive oil, whih is proportionl to the time derivtive of mgnetiztion, m (H(t)) (Fig. 1) (The onstnts of proportionlity relting m [Am /s] to the mesured voltge re the oil sensitivity, S [1/m], nd m,p 1 7 [Vs/Am]). Fig. 1 is prmetri plot of m (H(t)) vs. H(t); sine this figure is unique to eh trer formultion, we ll it the Prtile Response Funtion (PRF). For given exittion field frequeny there is single prtile dimeter ssoited with resonnt, or perfetly in-phse, mgnetiztion response of mximum signl intensity. Thus, it is ritil tht the size distriution e monodisperse for the entire ensemle of nnoprtiles to mgnetize simultneously. The PRF signl intensity is lso liner with MNT onentrtion; ritil property for in vivo quntifition of MNTs during rel-time MPI sns. In -D or 3-D MPI snner, field grdient (we8 T/m) within the region of interest (ROI) retes single point with zero-field lled the field-free point (FFP) (see Refs. [1e3] for more detils). Due to the field grdient, MNTs loted in the ROI experiene mgneti sturtion everywhere exept the FFP. Sturted MNTs do not respond to the AC-field, while MNTs within the FFP generte time-vrying signl, proportionl to m (H(t)), tht revels their lotion. The volume of the FFP determines the instrument s viewing window within whih MNTs re resolved in spe; lrge field grdients result in smll FFP. Together, the PRF nd the field grdient strength determine the system Point Spred Funtion (PSF); ritilly, the PSF is property of the trer nd hrdwre. The full width hlf mximum (FWHM) of the PSF is the system sptil resolution nd the height is the signl intensity (in V/g Fe). To generte n imge, the FFP is shifted in 3-dimensions using eletromgnets nd sptil distriution of MNTs in the ROI is reveled. In the x-spe pproh [19], imge reonstrution is hieved y onvolution of the trer distriution nd PSF; H(t) m(h(t)) m'(h(t)) m m'(h) H time H(t) Fig. 1. Shemti illustrtion of mgneti nnoprtile detetion in MPS. () The hrteristi non-liner mgnetiztion response of superprmgneti nnoprtiles to n pplied field. () MPS uses n AC-field, H(t), to mgnetize MNTs, m(h(t)), nd indue time-vrying signl, m (H(t)), in the reeive oil. The resulting prtile response funtion (PRF), shown in (), is fundmentl property of MNTs.

3 A.P. Khndhr et l. / Biomterils 3 (13) 3837e TE [se.] normlized intensity Con. [µm Fe] /T [s -1 ] r = 9.8 mm -1 s -1 Con. [mm Fe]..8 TE [se.].1. Con. [mm Fe] Fig.. () T-weighted imges of UW-17 MNTs prepred t 5 onentrtions in 1-wt% gr gel nd () their orresponding intensityedey profiles fit to eqution [1]. () Liner fit to T-relxivity (1/T) s funtion of MNT onentrtion; slope of fit isr[mm 1 s 1 ]. lterntively, in the fst System Mtrix pproh, imge reonstrution is hieved through mtrix inversion of system funtion mesurements []. In priniple, MPS is -dimensionl MPI snner (no field grdients) tht mesures the instrumentindependent MNT performne, or the PRF, nd enles trer development independent of snner development. Without n imging snner, trer development for linil pplitions of MPI is hllenging. In this study, we dopted MPS to study the in vitro MPI performne of MNTs, nd further evlute their in vivo performne nd iovilility (irultion time) in rodent model. Sine ll ommeril snners under development will operte t w5 khz, ll our mesurements were done in 5 khz MPS to lign our optimiztion efforts for future linil snners.. Mterils nd methods.1. Synthesis of mgnetite (Fe 3 O ) MNTs Mgnetite (Fe 3 O ) nnoprtiles were synthesized ording to pulished methods [1,]. The synthesis is sed on pyrolysis of the Fe 3þ olete preursor with exess olei id surftnt in 1-otdeene. Synthesis of 17 nm nnoprtiles, for instne, ws hieved y therml deomposition of.1 mmol Fe 3þ olete nd 1.5 mmol olei id surftnt t reflux temperture of 3 C for h. After synthesis, olei id oted MNTs were trnsferred from orgni to queous phse using PEG-ylted mphiphili polymer [poly(mlei nhydride-lt-1 otdeene)-poly(- ethylene glyol); PEG M w w 5 D]. Detiled synthesis, phse trnsfer methods nd hrteriztion results, inluding invitro ytotoxiity, re disussed inprevious work [3]. ResovistÔ, ommeril MRI-ontrst gent, ws quired from Byer Shering Phrm AG. 3 In ddition to physil size nlysis y TEM, we used the Chntrell method [] to determine the medin mgneti ore dimeter (D C ) of the prtiles nd its stndrd devition (s). The ltter method ssumes lognorml distriution of sizes nd fits the Lngevin funtion to the room temperture mgnetiztion urve s mesured in virting smple mgnetometer [VSM, Lkeshore]. Hydrodynmi size ws mesured, oth in wter nd in RPMIþ1% FBS medium, using dynmi light sttering (DLS). Finlly, the PRF of MNTs ws hrterized in our home-uilt 5 khz mgneti prtile spetrometer (MPS) using field mplitude of 36 mt pep (detils on the design nd onstrution of the spetrometer n e found elsewhere [8]). For injetions in mie, exess polymer nd tre orgni solvents were removed using sephryl S- HR gel olumns [GE Helthre]. Before injeting mie, MNTs were filtered with sterile.-mm syringe filters nd dispersed in USP-grde 1X phosphte uffered sline (PBS) t gfe/l. As-reeived Resovist MRI-ontrst gent (w7.9 gfe/l) ws lso diluted to gfe/l using USP-grde 1X PBS... In vitro nd in vivo studies Femle CD-1 mie [Chrles River Lortories], 5e3 g, were used for ll niml studies. Sixty-five mie were used in totl, forty-five for the irultion The system funtion mesurement mps the prtile response to the frequeny in the field of view. The dt is stored in frequeny-spe, whih is then inverted in rel-spe for imge reonstrution. 3 Prodution of Resovist ws disontinued in 9. study nd twenty for iodistriution. The University of Wshington s Institutionl Animl Cre nd Use Committee pproved ll niml protools...1. In vitro evlution The PRF of trers ws mesured using our 5 khz MPS in iologil environments, inluding whole lood, RPMIþ1% FBS ell-ulture medium nd 1-wt% gr gel, whih physilly immoilized MNTs.... In vivo irultion study Mie were injeted with 1 ml. gfe/l MNTs dispersed in 1X PBS through the til-vein. From eh mouse, pproximtely 1 ml of lood ws drwn retro-oritlly t two time points, fter whih mie were euthnized. Similr dt sets were otined for the ontrol nd Resovist groups, whih were injeted with 1 ml 1X PBS nd. gfe/l Resovist solution, respetively. Blood smples were hrterized in our 5 khz MPS (36 mt pep ), while prllel hrteriztion in VSM ws performed to ompre the stti (VSM) nd dynmi (MPS) mesurements. For MNT quntifition, series of dilutions prepred using Indutively Coupled Plsm- Optil Emission Spetrophotometer [ICP-OES, Perkin Elmer] were used to lirte the MPS nd VSM signls s funtion of onentrtion. Whole lood smples (1 ml), extrted t vrious time points fter til-vein injetions, were trnsferred in.5 ml Eppendorf tues nd mesured diretly in the MPS; no proessing steps were neessry. The sme smples were then trnsferred to polyronte psules for mesurements in the VSM...3. Biodistriution Mie were injeted with either.5 or. gfe/l MNTs through the til-vein. Control mie were injeted with 1 ml 1X PBS. Mie were nesthetized using 5% isoflurne nd mintined t 1e% during the imging period in the 1-T MRI [Bruker Biospin]. Respirtion rte ws monitored nd mintined etween 1 nd 15 reths/min. To quire referene sns, ll mie were imged efore injetion. After injetion, mie were imged t.5-h, 1-h nd -h time points. T-relxivities (R) of liver, spleen nd kidneys were nlyzed to quntify nnoprtile uptke. Following the -h time point, mie were euthnized nd their orgns were hrvested, fixed in 1% formlin nd emedded in prffin for histologil nlysis., spleen, kidneys, hert, lungs nd rin were olleted. Tissue setions were stined for iron using Prussin lue..3. Imging prmeters A 1-T (6 MHz) vertil ore Bruker Mgneti Resonne Spetrometer ws used for imging. To quire T-mp, we used Bruker s MSME-T protool: 1 eho times; TR/TE ¼ / For iron oxide quntifition, the T-relxivity s funtion of onentrtion (r mmfe 1 s 1 ) ws mesured in 1 wt.% gr gel (Fig. ), whih is often used s tissue equivlent phntom mteril for mesuring ontrst gent relxivity [5]. Five onentrtions were used for r quisition: 9., 17.9,.8, 89.5 nd 179. mm Fe, inluding 1 wt.% grose ontrol. For niml imging, 5 slies, eh 1-mm thik, were imged using the sequene desried ove, nd nlyzed using ImgeJ softwre. T-relxtion times were lulted using the following eqution: t IðtÞ ¼I exp T where, I(t) is the MRI signl s funtion of time, I is the initil signl nd T (units: s) is the relxtion time required to reh 37% of the originl signl (I ). The T- relxivity, R (units: s 1 ), is the inverse of T nd is used to estimte MNT onentrtion in liver, spleen nd kidneys ording to the following eqution: (1)

4 38 A.P. Khndhr et l. / Biomterils 3 (13) 3837e385 Tle 1 Chrteriztion dt of UW-17 nd Resovist MNTs. D C [nm] (s) R MNT ¼ R þ r*c () where, R MNT is the T-relxivity fter MNT injetion, R is T-relxivity efore MNT injetion, r is the T-relxivity s funtion of onentrtion (units: mmfe 1 s 1 ) nd C is MNT onentrtion (units: mmfe). Thus, from Eqution (), MNT onentrtion C is given y DR/r, where DR ¼ R MNT e R. The reltive tissue ontrst efore nd fter injetions ws lso used for qulittive nlysis of MNT uptke in liver, spleen nd kidneys. For eh orgn, gry sle intensities from five rndomly seleted regions of interest (ROI) were mesured nd verged. After h, mie were euthnized, their tissues hrvested, nd stined for iron using Prussin lue. 3. Results nd disussion 3.1. Chrteriztion of MNTs MPS VSM TEM D H [nm] M s [ka m -1 ] Signl W FWHM [V mgfe 1 ] UW (.) 19 (.15) (7) Resovist 1 (.7) 5 (.37) 7 3 (3).8 1. TEM hrteriztion dt from [6], whih provides extensive hrteriztion of Resovist. s is the stndrd devition of the lognorml distriution. Chrteriztion dt of the two trers is summrized in Tle 1. TEM nlysis of UW MNTs (leled s UW-17; Fig. 3) yielded medin dimeter (D C )of19nm(s ¼.15), while Chntrell fitting of the sme smple yielded ore size of 17 nm (s ¼.); pproximtely 1% smller. Mgnetiztion mesurements typilly return smller dimeters thn the oserved TEM size, presumly due to mgnetilly ded surfe lyer. Resovist, on the other hnd, showed signifint disrepny (w65%) in the ore size mesurements. Extensive hrteriztion of Resovist suggests tht it is omposed of two size frtions: 5 nm nd nm prtiles [6]. The lrger size is result of ggregted nnoprtiles tht re frequently oserved in TEM imges of Resovist [8]. The overll size distriution of Resovist is thus imodl nd onsiderly roder thn UW-17. Hydrodynmi size mesurements in wter show tht the medin dimeter of UW-17 is 86 nm, while Resovist is 7 nm (Fig. 3). In ddition to the tilored size nd uniform size distriution, UW- 17 MNTs exhiit superior mgneti properties ompred to Resovist (Fig. 3); sturtion mgnetiztion (M s ) of UW-17 is nerly 88% of the ulk sturtion vlue for mgnetite (w7 ka m 1 ) [7], wheres Resovist is signifintly lower t only 6%. Prtiulrly, the MPI performne of UW-17, s dedued from MPS mesurements, is sustntilly etter thn Resovist (Fig. 3d). The PRF pek width (W FWHM ) of UW-17, whih determines the instrument-independent imging resolution nd depends entirely on the trer properties, is w% nrrower nd the signl per unit mss [V mgfe 1 ], ritil property from linil dose perspetive, is higher thn Resovist. In our previous results [RM Ferguson et l., in preprtion], we hve shown tht w5 nm represents the optimum ore dimeter for 5 khz exittion frequeny. In this study, however, we hose 17 nm MNTs, with hydrodynmi dimeter of 86 nm, to hieve lne etween MPI performne nd phrmokinetis. Due to strong inter-prtile mgneti intertions nd potentil lustering of nnoprtiles, hydrodynmi size of our nm (nd lrger) MNTs is >1 nm, wheres optiml size rnge for improving irultion time is typilly etween 15 nd 1 nm [8e3]. The lower nd upper limits on hydrodynmi size re result of physiologil onstrints; the lower limit set y the size of kidney fenestre (w15 nm), nd the upper limit y the size of sinusoidl 15 UW-17 Resovist 1 Di. [nm] Numer [%] nm Dimeter [nm] M [ka/m] [V mgfe-1].1 norm. inten µh [mt] d 1 1 µh [mt] Fig. 3. () TEM imge of UW-17 nd its orresponding histogrm (inset) with lognorml fit. Chrteriztion of UW-17 (solid) nd Resovist (dshed) MNTs in DI wter: () hydrodynmi size mesured using DLS; () stti mgnetiztion response mesured in VSM; (d) intensity (top) nd mss (ottom) normlized PRFs mesured in our 5 khz MPS.

5 A.P. Khndhr et l. / Biomterils 3 (13) 3837e Signl [V mgfe -1 ].1 UW-17 DI Blood Resovist µ H [mt] µ H [mt] RPMI+1%FBS Gel Fig.. In vitro MPS hrteriztion of UW-17 MNTs (left) showed nerly no hnge in PRF in vrious iologil medi unlike Resovist (right). pillries in the liver nd spleen (w5e18 nm) [31], whih omprise the retiuloendothelil system (RES). Thus, prtiles smller thn 15 nm, nd most moleulr ontrst gents suh s ICM nd Gdolinium-heltes re swiftly lered through the kidneys, while prtiles lrger thn w1 nm re rpidly sequestered in the RES. For effetive MPI lood-pool imging tht uses low trer dose nd does not require invsive theteriztion, trers tht demonstrte oth, optiml MPI performne nd lood irultion hrteristis re required. In the urrent study, UW-17.1 MNTs demonstrte signifint improvements in MPI performne over Resovist while lso exhiiting omprle hydrodynmi size (Tle 1). We re urrently optimizing our phse trnsfer proess to minimize nnoprtile lustering; these improvements will enle use of w5 nm ore nd w3e5 nm hydrodynmi dimeter MNTs, whih demonstrte oth, exellent MPI performne nd long irultion times. 3.. In vitro trer evlution In vitro evlution of MPS performne proved ritil to understnding MNT relxtion ehvior in iologil environments, nd thus prediting their in vivo MPI performne. Blood nd ell-ulture medium re rih in serum proteins nd simulte the ngiogrphy environment, while gr gel simultes nnoprtile immoiliztion in tissues. We oserved ler differenes in the PRF of UW-17 nd Resovist MNTs (Fig. ) reltive to their orresponding PRFs in DI wter. UW-17 MNTs showed no loss in MPS signl when dispersed in either lood or RPMIþ1% FBS ell-ulture medium, while in 1-wt% gr gel, the signl deresed mrginlly (w8%). Resovist, on the other hnd, showed n 8% signl loss in lood, nd signifint loss in ell-ulture medium (53%), nd 1-wt% gr gel (7%). The differenes in mgnetiztion reversl, nd thus MPS performne, of UW-17 nd Resovist re governed in generl y two relxtion mehnisms: Néel nd Brownin relxtion; eh defined y time onstnt s N nd s B, respetively. Néel relxtion Signl [V]. Pek signl [V].1 µ H [mt] µgfe/ml] Con. [µgfe/ml] Moment x 1-6 [Am ] m st x 1-6 [Am ] µgfe/ml] 7 9 µ H [mt] d 1 1 Con. [µgfe/ml] Fig. 5. The () MPS nd () VSM signls of UW-17 MNTs re liner s funtion of onentrtion, () nd (d) respetively.

6 38 A.P. Khndhr et l. / Biomterils 3 (13) 3837e385 ours y rottion of the mgnetiztion vetor inside the prtile; thus it is independent of hnges in the surrounding environment. The time onstnt s N is expressed y the Arrhenius eqution: DE s N ¼ s exp k B T where s is the ttempt time typilly in the rnge of 1 11 e1 9 s [3]; k B [ JK 1 ] is Boltzmnn s onstnt; T [K] is the solute temperture nd DE [J], expressed s KV C (1 h), is the energy rrier to hieve mgnetiztion reversl e it depends on the nisotropy onstnt K [Jm 3 ], mteril property of either mgnetorystlline or shpe origin; the ore volume V C [m 3 ]; nd the redued field h ¼ H/H K, where H K is the internl nisotropy field. Thus, Néel relxtion depends primrily on the MNT ore properties nd the field mplitude. Brownin relxtion, on the other hnd, involves physil rottion of the entire prtile; thus it is sensitive to hnges in hydrodynmi size nd fluid visosity tht dd resistne to rottion. The time onstnt s B is given y: s B ¼ 3hV H k B T where h [P s] is the rrier fluid s visosity nd V H [m 3 ] is the hydrodynmi volume. During irultion, nnoprtiles often undergo opsoniztion e protein sorption to surfe e tht slows Brownin relxtion due to inrese in V H. Furthermore, uptke in tissues n immoilize nnoprtiles nd physilly impede Brownin relxtion. Thus, to prevent signl loss, Brownin ontriution (3) () to mgnetiztion reversl must e minimized. Our results indite tht mgnetiztion reversl in UW-17 MNTs is independent of hnges in surrounding medi, nd thus predominntly Néel, wheres Resovist hs signifint ontriution from Brownin relxtion In vivo irultion study To test the in vivo MPI performne of trers, we injeted CD-1 mie with 1 ml-. gfe/l of either UW-17 or Resovist vi the tilvein, nd mesured the MPS signl from lood smples olleted t vrious time intervls. Clirtion urves (Fig. 5) showed tht the signl ws liner with onentrtion in oth MPS nd VSM mesurements. Thus, they were used to quntify the rw MNT signl (Fig. 6), whih ws normlized to mouse ody weight (in kg) nd plotted s funtion of time (Fig. 7). Our results highlight key fetures tht distinguish the in vivo performne of MPI-tilored MNTs (UW-17) from trditionl SPIO ontrst gents (Resovist). First, s UW-17 MNTs re lered from irultion, the MPS signl, defined y the PRF pek-height (Fig. 6), dereses with time; it tkes w5 min for the signl to reh hlf its initil deteted vlue, nd nerly 15 min to reh the noise floor (Fig. 7). On the other hnd, MPS signl from Resovist nnoprtiles is onsiderly low nd nerly sent pst 5 min of irultion (Fig. 7). The inrese in signl deteted t 5 min suggests tht the injeted olus tkes e 5 min for uniform distriution, nd is susequently lered from irultion. Seond, unlike Resovist, UW-17 shows notle greement etween MPS (f ¼ 5 khz) nd VSM (f z khz) mesurements. The ltter leds us to onlude tht the irulting Moment x 1-6 [Am ] 1 1 signl x 1-3 [V] µ H [mt] Time [min] µ H [mt] Moment x 1-6 [Am ].5.5 signl x 1-3 [V]..1.1 d 5 5 µ H [mt] Time [min] µ H [mt] Fig. 6. VSM nd MPS mesurements of lood drwn retro-oritlly, olleted t different time points, from mie injeted with UW-17 ( nd ) nd Resovist ( nd d) MNTs.

7 A.P. Khndhr et l. / Biomterils 3 (13) 3837e norm. on. [ gfe*kg/ml] UW-17 VSM MPS 1 3 Time [min] norm. on. [ gfe*kg/ml] Resovist VSM MPS 6 Time [min] Fig. 7. () UW-17 nd () Resovist onentrtion in mouse lood (verge of three runs) s funtion of time. UW-17 MNTs re superprmgneti, s onfirmed in the stti VSM mesurement, ut more importntly re lso MPI-ple. Resovist MNTs re deteted in VSM, ut not t the sme sle in MPS, whih suggests tht though nnoprtiles irulte, only frtion of Resovist MNTs re MPI-ple; indeed, the inventors of MPI hypothesized tht only 3% of Resovist ontriutes to MPI [1]. There re severl ftors tht ffet irultion time of nnoprtiles, inluding hydrodynmi size, surfe oting nd hrge, nd opsoniztion dynmis [8]. While it is importnt tht the properties ffeting irultion time of MNTs re optimized, it is lso ritil tht the physil properties optimizing MPI response re preserved. Our in vitro study shows tht the MPI response of UW-17 is predominntly governed y Néel relxtion; thus independent of hnges in the environment nd preserved in vivo. We ntiipte further improvement in irultion time will entil simultneous optimiztion of the surfe oting nd the ore. For Reltive ontrst (.u.) Reltive ontrst (.u.) Reltive ontrst (.u.) Pre-inj..5 hr. 1 hr. hr. Pre-inj..5 hr. 1 hr. hr. Pre-inj..5 hr. 1 hr. hr. Inten. [.u.] Inten. [.u.] Inten. [.u.] d Pre-inj..5h h :.5 gfe/l :. gfe/l :.5 gfe/l :. gfe/l :.5 gfe/l :. gfe/l e.5.1 TE [se.].5.1 TE [se.] Fig. 8. Imge nlysis of mie injeted with () 1X PBS, ().5 gfe/l nd (). gfe/l UW-17 MNTs; reltive ontrst in T-weighted imges (TE ¼ 6.8 ms) is the rtio of gry intensity vlue in ROI reltive to the vertere in the sme slie. (d nd e) T-plots for mesuring hnge in relxivity (DR) were used to quntittively determine MNT uptke in liver, spleen nd kidney.

8 38 A.P. Khndhr et l. / Biomterils 3 (13) 3837e385 Tle Relxivities of liver, spleen nd kidney efore nd fter injetion with 1-ml.5 gfe/l UW-17 MNTs. Eqution () ws used to estimte MNT onentrtion. R [s 1 ] R MNT [s 1 ] DR ¼ R MNT e R [s 1 ] C [umfe] Pre-MNT t ¼.5 h t ¼ h t ¼.5 h t ¼ h t ¼.5 h t ¼ h Tle 3 Relxivities of liver, spleen nd kidney efore nd fter injetion with 1-ml. gfe/l UW-17 MNTs. R [s 1 ] R MNT [s 1 ] DR ¼ R MNT e R [s 1 ] C [umfe] Pre-MNT t ¼.5 h t ¼ h t ¼.5 h t ¼ h t ¼.5 h t ¼ h 76. e e e e e e e e T-fit not possile due to quenhed signl (see plot for liver in Fig. 8e). DR vlues negtive. instne, deresing hydrodynmi size to 3e5 nm, while mintining olloidl stility in iologil medi, will prolong irultion time nd enle use of nm (or lrger) MNTs tht show exellent MPI performne. 3.. Biodistriution MNTs designed for MPI re tilored preisely y ontrolling size to respond optimlly to n pplied frequeny. As result, we utilized the well-estlished [1,] high temperture pyrolysis of Fe(III)-olete in orgni solvents pproh; it offers exeptionl ontrol over nnoprtile size nd size distriution. The resulting MNTs re hydrophoi, nd susequently trnsferred to wter phse using PEG-sed mphiphili polymer. Sine there is no linil history of iron oxide nnoprtiles synthesized in orgni solvents, we performed preliminry study to determine the iodistriution of UW-17 MNTs using rodent model. For qulittive iodistriution nlysis, we ompred the reltive MRI ontrst from xil slies ross the domen (Fig. 8e), nd the rte of MRI-signl dey (T-relxivity) ws used for quntittive nlysis (Fig. 8 d nd e). It should e noted tht the MRI signl from mie dosed t. gfe/l UW-17 ws quenhed (Fig. 8e), suggesting high degree of MNT uptke in the liver. Sine the quenhed signl is diffiult to quntify, we inluded n dditionl group of mie tht ws dministered 75% lower dose (.5 gfe/l). At.5 h post-injetion, oth groups (.5 gfe/l: Fig. 8, nd. gfe/ L: Fig. 8) showed n inrese in reltive ontrst in the liver nd spleen, while ontrst in kidneys ws similr to pre-injetion stte. At the -h endpoint, we oserved sustntil signl reovery to pre-injetion levels in the.5 gfe/l group (Fig. 8) e suggesting MNT digestion through metoli pthwys in the liver nd spleen e while the. gfe/l group showed only mrginl reovery (Fig. 8). Quntittive nlysis of the.5 gfe/l-group supported the qulittive ssessment (Tle ): from.5 to h, DR dereses Fig. 9. Tissue setions olleted h post-injetion nd stined with Prussin lue ( mgnifition). Control group ws injeted with 1 PBS. Note the nturlly high onentrtion of ferri iron in the spleen ontrol mkes qulittive omprison etween ontrol nd MNT group diffiult.

9 A.P. Khndhr et l. / Biomterils 3 (13) 3837e % (.3 s 1 e17.5 s 1 ) in the liver nd 5.8% (1. s 1 e.7 s 1 )in the spleen. DR, in omprison, dereses y signifint 8.8% (6.6 s 1 e1. s 1 ) from.5 h to h. After.5 h post-injetion, there ws 1.9% inrese (DR ¼ 6.6 s 1 ) in kidney R vlue, whih indites MNTs re still irulting. However, the differene in R vlues (DR ¼ 1. s 1 ) etween pre-injetion nd h postinjetion is less thn the 5% vriility in signl mesurement, thus negligile. Quntittive nlysis of the. gfe/l group is summrized in Tle 3; liver signl in the. gfe/l group ws quenhed nd thus not quntifile. However, signl in the spleen ws quntified nd showed n inrese in DR from.5 h to h post-injetion, suggesting ontinued nnoprtile uptke till the -h endpoint. Histologil nlysis (Fig. 9) of tissue setions stined with Prussin lue further onfirms MRI nlysis e MNTs umulte primrily in the liver nd spleen. In summry, preliminry sfety ssessment of our trers is promising, sine iodistriution nd lerne routes re nlogous to linilly pproved SPIO ontrst gents (Feridex nd Resovist) tht re lered vi the RES [33] with no long-term renl involvement.. Conlusion MPI is new imging modlity tht is the first to truly exploit nnosle mgneti properties of SPIOs. However, existing ommeril SPIO trers perform su-optimlly nd hinder its linil trnsltion. Our monodisperse MNTs (UW-17) e synthesized in non-queous solvents nd susequently trnsferred to queous phse using PEG-sed mphiphili polymer e showed -fold greter signl per unit mss nd % etter sptil resolution thn ommeril SPIO gent (Resovist) in 5 khz MPS. In vitro trer evlution showed tht the MPI response of UW-17 MNTs, unlike Resovist, ws independent of hnges in hydrodynmi size nd the rrier fluid s visosity e ritil feture tht ensures the MPI performne will e preserved in the physiologil environment. Furthermore, in vivo irultion time of UW-17 in mie, s mesured in our MPS (f ¼ 5 khz), ws 3 longer thn Resovist nd showed exellent greement with the orresponding stti (VSM: f z khz) mesurement. The ltter onfirmed tht the irulting UW-17 MNTs re MPI-ple in vivo, while Resovist MNTs though irulting, re unsuitle for MPI. The iodistriution of UW-17 MNTs in mie ws similr to linilly pproved SPIO gents, primrily distriuting in the liver nd spleen. Further improvements in phse trnsfer nd surfe otings will filitte the synthesis of lrger ore MNTs (> nm di.) with hydrodynmi dimeter elow 1 nm, whih will enle synthesis of long-irulting MNTs tht re truly optimized for MPI. Suh long irulting optimized nnoprtiles show promise s ngiogrphy trers nd lood-pool gents for the emerging MPI imging modlity. Aknowledgments This work ws supported y NIH grnts 1R1EB / NIBIB nd 1R1EB135-1, NSF grnt IIP , UW/CGF ommeriliztion grnt nd Commeriliztion Fellowship (RMF). 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