J0URNAL oF PHyslcs: C0NDENSEDMATTEß

INsrrru'IE oF PHYslcs PUBLISHING

UNCORRECTED PROOF

J. Phys.:Condens.Matter 18 (2006)l-12

rmprovement of the separationof tumour cells from peripheral blood cells using magneticnanoparticles M Schwatbe, K Pachmann, K llöffken and J II Clementl Departmenrof Intemal Medicine II, Friedrich Schiller university Jena,ErlangerAllee l0l, D,07740 tena, Germany med.uni-jena.de E-rnail: joachim-clement@

Received I August 2006 Rrblished 8/l Online at stacks.iop.org/JPhysCtvf/l Abslract Circulating tumour cells are'a key challenge in tumour therapy- Numerous approachesare on *re way to aöhieving the elimination of tlrese potential sources of metastasisformatiOhl Antibody-direcbd magnetic cell sorting is supposed to enrich tumouf cells, with high selectivity, trut low efficiency. The short term application of carboxymethyl dextran (CMD) coated magnetifmaghemit nanopnrticles allorrs dne discrimination of tumour cells q.r frorn leukocytes- In the Fesent work we show that the interaction of CMD nanoparticles is cell-type specific and time dependent. The breast cancer cell tine ucr-z and the cML cell line K-562 are characterizd by a rapid and high interaction rate, whereasleukocytes exhibit adecelerated behaviour- The addition of carboxymethyl dextran or glucosestimulated the magnetic labelling of leukocytes. The variation of the degree of substitution of dextran with carboxymethyl grcups did not affect tlle trabelling profile of leukocytes and MCF-7 cells. In order to verify ttre in vitro results, whole blood samplesfrom I 3 cancer patients were analysedex vivo. Incubation of the purified leukocyte fraction with CMD nanoparticles in the presenceof low amounts of plasma reducedthe overall cell content in the positive fraction. In contrast,the absolute number of residual tumour cells in the positive fraction was 9ovo of the initial amount.

l. Introduction Magnetic nanoparticleswith various shells are widely used for specific labelling and detection of cells. Treatment of solid tumours and hae,rnatologicaldisorders would benefit from an accurateand quantitativediscrimination of tumour cells from healthy cells, e.g. in ttreperipheral blood. During the last decadea couple of nanoparticlebasedtechniqueshave been developed I Autlor to whom any correspondenceshould be addressed. @2006IOPPublishingLtd PrintedintheUK 0953-8984/06/txl{Xl0l+12$30"00

Ascii,/lJord

-

JPC/ cn22997 2./SPE:lFerrof luids I Printed

(ta:

l1/8/2006

Emily)

File nane Date req. Issue no. ArtnuB

CRC data page First Last page Total pages Cover date

I

M Schwalbe er aJ

to target tuürour cells and eventually magnetic assistedcell sorting (MACS) has become a powerfirl tool for detecting and emiching circulating disseminatedtumour cells (Wong er al et al 1995,Berois a al 1q4.7,Eaton et al l99'l, Maparaet aI 1997,Naumeet al l97,Is'datr1iln 1998). Mostly magnetic nanoparticlesrequire covering with a shell which plays an important 6e f6q the interaction with its target. For biomedical applications the biocompatibility of the magnetic nanoparticles, especially the shell, is essential. One of the most popular coatings is dextran and its derivatives. They show a low unspecific reactivity with peripheral blood cells (Pitha 1978) and loaded with specific ligands they could interact with peripheral blood cells specifically (Ivlotday and MacKenzie 1982). The magneticproperties could then be used to emich or deplete cell popnlations from peripheral blood or other complex cell mixtures ex vivo (Trickett et al 1991,Hancockand Kemshead1993,Haukanesand Kvam 1993)- The appLicationof dextran coated rnagnetic nanopartictresin vivo has allowed discrimination of tudlou111 infiltrated lymph nodesfrom lyurph nodesftee of metastasis( Anzxi et al1994)- Several other approachesbasedon magnetresolranceirnaging me usedto detectlyrnph node metastases wirh high precision (Harisinghani and Weissleder 20(X, Wilt er al 2ffi6). Interaction of magnetic nanoparticleswith cells is not only resrtrictedto the cell surface. The particles can be incorporatedinto cells by endocytosisand be accumulatedin endosomes(Jordanet al 1996, 1997, Schoepfet aI l99l,Wagrcr A al2964)r as demonsfratedby electron microscopy. We have shown previously that hrmour cell line cells can be separatedefficiently from a leukocyte--tumowcell suspensionusing magnetic nanoprticles with a caöoxyrnethyl dextran (CMD) sbell (Schwalbe et al 2ffi5, Clement et al 2ffi6} The emichment of the tumour cells wa.sdependent on factoß such as tLreincr{bation time, the osmolality or the plasma concentration during incubation. The majority of the tumour cells could be labelled within 4*8 min whereas only a minority of leukocytes were significantly loaded with magnetic nanoparticles. Theseobservationsshow that tlre dextran shell is an important prerequisite for separatingtumour cells ftom leukocytes. The aim of this study was to ftuther investigate and improve the specif,c interaction of carboxynrethyl dextran coaed magretic nanopmticles witb leukocytes foom peripheral blood and tumour cells. For this purpose we investigated the role of the degree of substitution of dextran with carboxymethyl groups and the incubation conditions with special regard to temperatureand carbohydrate supplementationduring nanopartiale cell interaction. Finally, in vitro observationswere transf,erredto the ex vivo situation and peripheral blood samples from cancerpatients analysedfor tumour cell loading2. Materials and methods 2.1. Cell culture The cell lines used for these investigations were obtained from DSMZ (Braunschweig, Gerrnany) or AICC (Rockville, USA) and are listed in table l. The cell lines were cultivated under standardconditions as indicated. Aürerent cell line cells were harvestedby treatment with Urypsinin order to detachthe cells from the plastic surfaceof the cell culture flask. 2.2. Prcparation oJ leulunytesfrcm peripherel btaod Leukocytes from peripheral blood were prepared by erythocyte lysis (Qiagen, Hilden' Germany) from whole blood samplesof healthy volunteersorpatierts with informed consent. Before erythrocyte lysis the plasma fraction was removed by centrifugation (300 x g, 5 min,

of tumour cell-leDkocyte differcnriation Tabte 1. Origin and cultivation of cell lines- (FCS: foetal calf senrm') Cell line

Origin

Culture medium

BT-20 BT-4'14 Caski CIIRF HBMEC HepG2 K-562 MCF-7 NB-4 SK-BR-3 T-47D

Human breastadenocar€inoma Hümall brast drctal carcinoma Human cervix epidermoidcarcinoma Humm megakarYoblastic Human brain microvassllar endothelium Humanhepatocellularcarcinoma Hüman chmnic myeloid leukemiain blast crisis Human brmst adenocarcinoma Human acutepromyelocyticleukemia

DMEM + 10%FCS RPMII64O+ IO% FCS RPMII64O + IO% FCS RPMtl640 + l07c FCS RPMI164O+ IO% FCS DMEM + 100,6FCS RPMI164O+ IO%FCS DMEM + 10% FCS RPMII640 + l0% Fcs RPMII64O + IO% FCS RPMI!640 + 15%FCS

Humanbreastadenocucinoma Human breast ductal carcinoma, pleural effusion

14"C); the whole supematantwas defined as plasma. The leukocyte pellet was washe.dwith erythrocyte lysis buffer twice and finally resuspendedin PE buffer (phosphate-bufferedsaline (PBS)and2 mmolEDTA). 2.3. Magneti c nanopa rt icles Magnetic nanoparticlescon-sistingof a superpia(ferro)magneticcore made from magnetiie/maghemitewere otrtainedfrom N Buske (Magneticfluids,Berlin' Germany)' The TEM size diameter of the core vmied between 3 and 15 nm. The averagediameter was 5 nm. The nanoparticleswere coated with carboxymethyl dextran as previously described (Wagner er al 2004). The averagedegreeof substitution of the dextran with carboxymethyl groups was 0-8. Repeating of the procedure fesulted in higher DS values (lüagner et al 2U){). The hydrodynamic diameter of the generatednanoparticle clusters was 200-3(D nm. The sahration magnetization ranged from 4.5 to 6.2 mT. Iron oxide nanoparticles coated with citric acid' cmboxymethyl beta-cyclodextrin, bovine ser,umalbumin and L-glutamic acid as well as cobalt q.2 ferrit nanoparticles were kindly provided by N Buske. The average nanoparticle size was (IPHI GerJena' Müller grft R generous from a were nanoparticles ferrit The barium t0 nm. many). Theseparticles with a core size of 7G-100 nm were coated with CMD as previously described(Schwalbeet al 2AJ6)2.4. Incubation af cells with mngneticnanoparticles For kinetic analysiscell line cells (l x lff cells per 500 pl PE) and/orleukocytes(2.5 x 106 cells per 5ü) pl PE) were incubatedin shortterm incubationwith 2.5 U'I/Sffi pl PE magnetic nanoparticlesat 37'C. Precedingexperimentsshowedthat addition of 2.5 p'lof a nanoparticle solution led to an optimal interaction rate independentof the batch. A firther increaseof the nanopafticle concentrationin the reaction did not increasethe interaction behaviour ofthe cells indicating that the nanoparticlesare in far excess. Fot ex vivo investigationsthe white blood cells, containing the putative tumour cell fraction, derived from I ml tumour patient blood were incubatedfor the times indicated in a plasma-PE rnixture supplementedwith different amounts of plasma (0, l, 5%). After the treätment magnetically labelled cells were separatedusing a SuperMACS and MS columns (Miltenyi-Biotec, Bergisch$ladbach, Germany). The separated cells were designed as positive fractinn (retained in colwnn) and negative fraction (effiuent). The number of cells in each fraction was enumerated(Particle Count & Size Analyzer 22, Beckman-Coulter,Krefeld, Germany). For determinationof the tumour cell content in the

M Schwalbe et al

a Er loo

F m Eoo 6zo Ieo

--a--iiEF-7 --t-BT-A) -rBT-474 - -'e -SK€R-3 --{-T-47D

; 5 0

3+o

Peo .c

20

ä( )r o 0 0

o

et*

E g o

tao 5 7 0

S m

* 5 9 0) .a ,!0

'8

4

8 1 2 1 6 incubalionlime [min]

2

0

%,ft* lr'

eo

ao ,o

g r o

8 o 4

I

t2t

t6

incubation rirne [min] labelting of rell line cells cnd teukocytes with CMD coated magnetic Figure l. Time.delmdent nanoparticles. C6lls were indtbated with nanoparticles in PE for the times as indicated and separated by MACS. Cell content in th€ positive änd negative fraction was estimaaed. (a) Breast cancer cell lines and leukocytes; ( ) vadnus cell lines.

ex vivo experimentsan aliquot of the original sarnple,of the positive and the negativefraction was analysed by flow cytometry (FACS Calibur, Becton-Dickinson, Heidelberg, Germany) using epithelium--antigenspecific staining fm the detection of tufilour cells (Anti-tIEA FITC, Miltenyi biotec, Bergisch-Gladbach,Germany) and CDf5 for the estimation of the leukocyte conteot (CD15 PE, Miltenyi Biotec, Bergisch-Gladbach,Germany). 3. Results 3.1. Cell-type specific interaction of cells with magnetic narwparticles Magnetic nanoparticlesprovide a suitable strategy for separatingdistinct subpopulations of cells from cell suspensionswhere the interaction of nanoparticles with the cells is usually directed by antibodiesattachedto the nanoparticles(Kandzia et al 1981,AbLser at 1989). We have, however, shown previor:sly thx there is a diffetentiatrinteraction of only carboxymethyl dextrancoatedmagnetic nanoparticleswith nrmourcells and leukocytes{Schwalbeet a12ffi5). Tbnrour cells showeda rnore ra,pidinteraction than leukooytesduring an 8 min exposure. The difference could be further enhancedby a slightly increasedpH ofthe incubation solution and by the addition of up to 57o plasma. In order to further improve the preferential labelling of nrmourcells we investigatedthe time dependenceof the interaction. Variouscell lines (table l) blood were incubated with CMD coated nanoparticles. At and leukocytes frorn points were magnetically separated(figure l). The breast cancer cell the cells defined time

Improvement of tumour cell-leukocyte differentiation ioo

€

='1oo

:

! o n

o 8 0

o g o

.9 oo o

Eso

6 q n

9 4 0

g,

40

.E

en

tr.20 '-

'-

1n

- n n o

-

g 4

8

incutration time [min]

io

0 4

8

incubation time [min]

Figure 2. The degreeof substitution(DS) of dextran with cü{boxymethylgroupsdoesnot allbct the labelling efficiency. MCF-? celts {black bars) aod leukocytes from peripheral blood (white bars) were incubatedwirh CMD coated nanoparticleswith DS 0.8 and DS 1.6 lbr 8 min in PE. Cells wae separatedby MACS and the cell contentin the positive and negativefraction was estimated. MCF-7: n = 9; leBko!'ytes:n = 14"

lines MCF-7, BT-20, F[474, SK-BR-3 and.T47D showeda continuousincreaseof labelled cells over a time period of 20 min. MCF-? was tlg:.mostactivecell line with a maximum of nearlygOVolabelledcells whereasBT-474 exhibitedonly a low degreeof labelling (307o).In contrast to the breastcancer cell lines the bloodderived leukocytes showeda sigmoidal curve with a sharp increasebetween 4 and 16 min (figiure l(a)). The interaction of CMD coated magnetic nanoparticleswith Caski, HepGZ, CHRF and NB4 was more or less unaltered over the whole incubation period. The interaction proflle of K-562 was similar to that of MCF7 whereasHBMEC cells displayeda steadyincreasedrning the fust 8 min with a flattening of labellingwascell-typespecific.MCFduring the following 12 min (figure l(b)). The degre.e 7, Caski and HBMEC showed a high degree of tabelling. In contrast, NB-4 was nearly not affected by the CMD coated nanoparticles. 3.2. The nanoparticle shell is criticalfor the nanoparticle-{ell interaction Magnetic nanoparticleswithout a cmbohydrate shell interact instantaneouslywith most cell types. This holds rue for magnetit/maghemitnanoparticlesas well as for cobalt ferrite and barium ferrite nanoparticles,which are toxic for the cells. A bmad variety of carbohydratesis, therefore, used to cover magnetic nanoparticles, e.g. for biocompatibility reasons. Such coatings may influence the interaction of the particles with different cell surfaces. We studied several of these coatings in order to maximize differential interaction of magnetic nanoparticles with different cells. In our hands carboxymethyl dextran proved to optimally differentiate tumour cells from leukocytes (Schwalbe et al 2AO6). Carboxyrnethyl groups are negatively charged and therefore the degreeof substitution of dexran with carboxyrnethyl groups alters the overall charge and in consequencemay influence the interaction of the coated nanoparticleswith cells. In order to optimize the chmge of the CMD nanoparticleswe investigatedthe role played by the degreeof substitution (DS) of dextran with carboxymethyl groups. In figure 2 the interaction proflles of CMD nanoparticleswith a DS of 0.8 and I .6 are shown. With both types of CMD nanoparticles leukocytes could clearly be discrirninated f.rom MCF-7 cells after a 4 min as well as 8 min incubation. There was only a minor differcnce between these two preparations. For further analysis we used carboxyrnethyl dextan with DS 0.8.

M Schwalbe el al

€ , o 880 t 7 0 H 6 0 o:

5u

= 4 0 o

Rso .E zo .9. 8 1 0 0

incubationternPerature["C] Figune 3. The labelling of cells with magnetic nenoparticlesis temperatufedependent. cells and incubation medium were a justed to the indicated temperatures for 30 min" AIter 8 min of incubation cells were sparated by MACS. The ceu content of the positive and negativefraction was estimad. MCF-? (black bars): n : 2; Ieukocytes(white bars): z = 2. Table 2. Interactionof nanoparticles.withvarious shellswith cells. Magnetic nanoparticles

Imn oxide Iron oxide Iron oxide Iron oxide Iron oxide Cobalt ferit Barium lbrit

Caf boxv methv I beta-cyclodextnn Citric acid Bovine serum albumin L-glutamic acld Withoüt With0ut rJfithout

Cells in positive tiaction {oÄ)u

MCF-7

Leukocytes

96 92 8l 84 9l 97 9l

94 98 19 82 88 98 93

a Incubationtime: 8 min.

An interesting carbohydrate is carboxymethyl beta-cyclodextrin which forms tube-like strucnrres.Magneticnanoparticlescoatedwith catboxymethylbeta-cyclodexaindid not allow discrimination between the tumour cell lines MCF-7, K-562 and peripheral blood leukocytes (table 2). Imnrcdiately after addition of the nanoparticles more than 85Va of the cells, independentof their origin could be separatedby magnetic force. Interestingly, the maximum amountof labelled MCF-7 and K-562 cells wasreachedalready after 4 min whereasleukocytes required 8 min of interaction to becomeseparableto the sameextent. The coating of magnetic nanoparticleswith various shells, e.g. citric acid, L-glutamic acid or bovine serum albumin did not allow sqrmation of cells in a cell-qpe specific manner after an 8 min incubation (table 2)^ 3.3. Tbmperatureand saluble carbohydratesaffect the nanoparticle-cell interaction Besides core and shell of the nanoparticles the buffer conditions might also modulate the interaction with cells. Usually cell culture cells are cultivated at 3?'C comparable to the mean temperatureconditions of the human body. The temperaturedistribution in the human body however is not homogeneous.The temperatureof the torso is quite uniform but in the limbs for instance the temperanre is reduced. Therefore we performed incubations of CMD 'C (flgure 3). An increase nanoparticleswith leukocytesand MCF-? cells at 4,24,37 and 40 "C during an incubation for eight minutes doubled the amount in temperaturefiom 37 to 40

Improvement of tumour cell-leukocyb qtr".*g"tigl 1nfr

€ o s E 0 e 7 n

.9 i(o5 B o t s 5 0

o)

'E +o o

&30 .g o

20

o 0.8

3.2

1,6

carboxymeüryl dextran {mg/mll

b

5'roo geo {ao 5?s Eoo + 5 0 o .3 40

'B

ro

o - ^ c t u

g t o

8 o

0

0.25

1-25

5.O0

12.50

25.00

glucose [mg/mfl Flgune 4 The addition of soluble carboxymethyl dextrafi aüd glÜcos€ enhances the interacdon the of CMD nanoparticles and leukocytes. MCF-7 cells and leukocytes werc incubated with glucose for nanoparticles in pE and the indicated concentrations of carboxymethyl dextran and g min. CeIs were separatedby MACS. The amonnr of cells in the positive ard the negative fraction was estimated. (a) Addition of corboiymethyl dextran; (b) supplementationof glucose' MCF-7 (black bars): a : 3; leukocytes(white bars): n = 3'

of separableleukocytes, whereasat a temperamreof 24"C the leukocyte pool in the positive "C. MCF-7 cells were also affected by the fraction was reduced to 5OVoas compaed to 37 temperaturebut to a lesserextent. The amountof labelled MCF-7 cells was reducedfrom 857o ar 4b "C to 6A% et 24"C. The interaction of CMD nanoparticles with leukocytes and with "C. MCF-7 is nearly completely abolishedat 4 The carbohydrate shell plays an important role for the cell-type specific and timedepcndentinteraction of the nanoparticleswith cells. The addition of soluble carbohydütes during incubation might influence that interaction. We tested severalcarbohydratesknown to be biocompatible for their ability to interfere with the cell-type specific magnetic labelling of tumour cells and leukocytesduring incubation (figure 4). Addition of cdoxymethyl dextranin a rangefrom 0.8 to 8.4 mg ml-l enhancedthe labelling of leukocyteswith CMD nanoparticles from 30goto 8t% during a incubation of eight minutes. The portion of MCF-7 cells in the positive ftaction was only marginally increased &om 807o to X)Va. Other polysaccharides like methylcellulose or polysucrosedid not elter tbe labelling profiIe of leukocytes or MCF-7 cells. Among the rnonosaccharidesonly glucoseinterfered with the cell-type specific magnetic I labelling of cells. In the rangefrom 0.25 to 25 mg ml- glucosethe portion of leukocytesin the

hI Schwalbe er aI

only positive fraction raised fuom2OVoup to 9ÜVowithin 8 min of incubation' Glucose induced or like fucose monosacchaüdes cells. other MCF-7 labelled of magnetically a minor increase leukocytes' and mannosehad no effect on the cell-typespecificinteractionof tumour cells 3.4. Separationof epithelialtumaur-derivedcellsfrom peripheral hlood These experiments show that the cell-type specific interaction of magnetic nanoparticles well as the with tumour cells can be modulated by the nature of the carbohydrate shell as affects plasma human that demonstrate incubation conditions. In addition, we could recently In brief' et aI 2{fi5)' (Schwalbe cells with the interactionof CMD magneticnanoparticles CMD with incubated cells MCF-7 of alnounts various wittr leukocyte preparations spteO number magneticnanoparticlesand subsequentseparationled to a dramatic reduction of the cell positive the in retained cells of MCF-? number the total Surprisingly, positive fraction. Äe in plasmacontent fraction was almost constantat a plasmacontentof l7o and sliglrtly reducedat a of 5Va. To prove the hypothesisthat our approachis suitablefor the separationof tumour cells from the peripheralblood of cancerpatientswe analysedblood samplesfrom 13 patientssuffering from various solid tumours (e.g. breast cancef, non-srnall cell lung cancer, gastric cancer)' After erytlrocyte lysis of the whole blood' the remaining leukocyte fraction was incubated with CIVID nanoparticles without plasma or in the presenceof l% and 57o plasma' After magnetic separationthe positive and negative fraition was analysedby FACS and compared to a control sample acquired before the incubation with magnetic nanoparticles. Epithelial cells were labelled with a HEA specific antibody,whereasthe leukocytes were detectedusing peripheral anti-C}ls. Thking into accountthat usually no epithelial-like cells are detectablein proportion of average The cells. tumour disseminated as cells HEA-positive we consider blood t O-17 A.46Vo' leukocytes and tumour eells in the untreatedsampleswas 99.83 t 0.467oversus Incubation of the leukocyte fraction with CMD nanoparticles for 8 min in the presenceof plasmaled to a dramaticreductionof cell numberin the positivefraction {figure 5(a))' Without uCaition of plasma the positive fraction contained 8l7o of the initially applied cells- [n the presenceof l% plasma the number of magnetically labelled cells in the positive fraction was in ieduced to5l7o and in the presenceof 5?o plasma to l7?o. Inverselycells accumulated the with threefold the negative fraction. [n contast t]re HEA-positive cells increasedup to increment of plasma in the positive fraction 1figure 5(b)) and declined in the negativefraction (flgure 5(c)). The absolute number of tumour cells in the positive fraction was 907o of the initiatly measgredcontent in the not separatedsample.This was independentof the addition of plasma. 4. Discussion The separation of selected cell populations is a vital problem in biomedicine. Magnetic nanoparticles have become an important tool for meeting this challenge. An essential component of a biocompatible magnetic nanopafücle is the shell and its functional groups. In caseofantibody-directed cell separationantibodiesagainstcell surfacemarkersare attached to the nanoparticles.They allow a preciseandhigh enrichmentof thecellsbearingthe antigenic determinant (Abts er at 1989,Miltenyi et al l99O). However, the separatedfraction has high homogeneity but usually a low recovery with a substantial amount of positive target cells remaining in the flow through. This might contribute to the hitherto disappointing results of clinical trials evaluatingthe effect of in virm purging in the sening of autologoustransplantation for aggressivelymphomas(Jacobsenand Reedman 1004). In attemptsto purify humanblood

Improvementof tumour cell-leukocyte differentiation

100

5 e 0 EE 80 g* 1s eb 60

E s 4s0o äE

. s l 3 0 .!aä zo [) o 1 0 0

0

:.€E

Ann

E E soo o ö .E 3 aoo u'

-B E soo 9 s 200 =l!

8E

+ö 100
o

0

160

(F(|) cD= o l Y

140

a 0 , = 0 O o

100

r D O o c

120 80

. g a 60 o : o.Y o'
S

c\t

(\|

N

(a

o

l" tit

c .9 o .9. E

o o o 6-or E F o o .E (r, v

lr,

F

(Y'

@ s

E N E R

@ r

o o o r

(o

(\ ro

I*

O)

c

o (u o

.g o ü,

ä ;

ää lä

s 8 f,

E . - o Eo

E

EE

bE

ä

blf, ö ö

öro ö

ö u

5

E E t r t rE E r

-

.g o

5_

.s,

_c

9

E g ie F € ö ls o f b E E

E

E E

E

Ü E

0) r

S F

S

cl C\l q)

.o d F

M Schwalbe et dJ

tz

Milreeyi S, MüIer W, Wciehel W and RadbnrohA 199{l t(igh gradi*s magrrericcell sopararis u/ith MACS Cyometry 1l 231-8 furonag:uetic hor}-ddttdn rEageBtsfor the labelling and Molday R S oä MacKcocb D f9S2 hmumryific msgnetic ss4latationof cells J. ImmarcL Methods 3235941 Narlme 3, Boryen E, Beiskc tri, llerstld T K Ravoas C, Renoien A, Ilachsel S, Thrane-Ste€n K Funderud S and Kvdbim G l99l lrnmunomagFtic ledrniryes for th corichmert md detction of isolarcd breast carcinoma cclls in bqp mtrom and peripherat blood f- fiermtaher 6lO3-14 Pith J l97E ReagoE qrcifc fmcell wfrcc conpomtsEnn I. Nxtwm.t2285-$2 R l99Blüuocellularmogrretic labellitrg of lyrnphocytes K andWeissl& Schoepfu, MarscosEM,McldecR.I,IainR fc in vivo taffcftilg sFdics Itrecänrqr€s 24642-1t Schwalbe M, Buske N, Vetterlein M, tlöffken & PachmannK and Ciement I H 2{Xl6 The carboxymeüyl dextran sheU is an important modulator of magnelic nanoparticle uptake in human cells Z Plrys. Chcn- 22O 125'31 Schwalbo M, Xükc C, Bür,k€ N, t{ittrken Il Padrmann K ond Cl€mcnt I H 2{m5 Selective reduction of the interaction of m8gpetic sampsrticles sirh l€DkßTres andtunor cetts by human plasma J. Magn. Magn Maet 293 433-7 boe marmw pttging of common Tlicken A E, Ford D J, I.8m-Fo-Ihng F R.d Vwels M R !991 Immumagoaic acue lympüobla-sticlcdrcmia cclls: srirability of tsioMag parriclqs ßoru Ma rmw Tranrylant 7 199-m3 Wagner K, Kautr A, Röder M, Schwalbe M, Pachmänn K, Clement J H and Schnabelrauch M 2004 Synthesis of oligonücleolide-functionalized magnedc nanqmrlicles and study on tfteir ix vi/m cell v'pttdreAppL Organomct. Cfun lE5l4--9 \l/itl O, Purtayastha S, Cbo[ C, Aüanasim T, Drrzi A W, Hroyc W and Teltis P P 2[D6 Diagnmtic precision of näropaiticle-€nhancodMRI for lymph-nod€ metaßtases:a meta-analysis Lanut. Oncol. T 52"4 Wong L S, Bateman W I, Mcris A 0 ard Fraser I A i995 DetEction of circulating &mour cells with the magnetic ac*ivaredcell sorterBz J" Sr4. &,.1333-7 Yamada K M, Ohanian S H and Pasran I 1976 Celt surface qg*gin decreasesmlrovilli d mfles on traüsfdmed mcuseaodclrick cxlllsCcdg2|l-S

,,;)