A. HALBREICH, J. ROGER, J.N. PONS, D. GELDWERTH, M.F. DA

Bhu'himh, ~1998) 80. 379-390
0 Socidt6 i'ran~aise de biochimie et biologic mo16cukm'c/ EIsc~ IcL Paris
B omed cai @p| cat ons of maghenfi e ferroflu d
A H a l b r e i c D L J R o g e r ~', J N P e n s b, D G e l d w e r t D ,
M F D a S i l v a a, M R o u d i e r ~, J C Bacri a
~aLaboratoire des Milieux D~sordonnt;s et H6tdre:gbnes, case 78:
i,~#~oratom' des Liquhh's hmiques el b~le~.'/~-r'esCha~,~ds. case 63, univer~'il~;Pierre-et-MaHe-Curie, 4. place Jussieu. 75252 Paris c~-'dex 05:
Chtstitut de Bioh~gie Phvsico-Chimique {IBPCL 13. rue Pier,e-el-Marie-Curie. 7,5005 Paris. France:
dDepartameltlo ~h, Fisi('a, I,htiverskk~& ~h, Brasilk~, Com/,,tl.~; Universilario, Asa Norle, Caixa Postal (M455. ('EP-70910-9~Z Brasi/ia DE Brazil.
eService ~h, Gdronloiogie Clinique. H~)pilal CharLes Richel. 95540 Villier~-h,-B,,I. f"mm,'e
b
i Received 8 December 1997, accepted 27 April 1998)
S u m m a r y - - The use of cell-largeted I'errofhlid in the characterization of modil'ic~ltionsof ceil membranes is reviewed. Maghemite ferrofluk! was
synthesized by the Massart nlelhod, complcxcd with dimercaplosuccmic ackl (FF). Cell targeting by Ft- ~,~lsdeve!ol~d by coupling FF to v~u'ious
biological effectors such as antibodies, iectins, etc, which enabled magnetic cell sorting. Modificalk~ns m erythrocyle membranes were sludied using
FF l~und to recombinant human annexin V (AnxFF) which i~, ~ery sensitive, compared to other Anx-based Ivagenls. in the early ddecuon of
phosphatidylserine (PS) exl~)sition on lhe outer leallet of the plasma membnme. Thus PS exD~nitionon I1)t}useRBC was detected already after a 24-h
storage at 4°C and, transiently, 24 il after tlleir infection by Pktsmodiunt pm'asites, at which time the parasites are still confined to tlae tivet; thus leading to
the i~cruimlentof young RBC and tileaccumulationof a species, intermediateI~tween rcticul~ytes and eo'thr~:ytes, and the actual RBC tat'getof pike+me,dial
invasion. AnxFF ~vealed PS exposition on RBC li'om sickle cell anemia patterns, ioilowingv;u'ious it~flammationsand already atier 20 days of human
bltxxl stonlgc under bkx~ ballk conditions. Such a sensitive detection slmuld ix: simil~u"to that of rnacml~hageswhich recognizeextx~sedPS on ceils and
bring al:~)utthe latter's eliminatkm lixm~the circtdation. AnxFF binding detemaination was combined with that of cell eieclxophoretic mobility,glycerol
resistance and filterability to characterize RBC membrane m(~.tificalions in Al~heimer's disease patienls which .suggested a continuous damage and
regeneration in RBC of these patients. A logisticanalysissuggestedthat several tiwee-paramelercombinationscould permitdiagnosisof AlzheJmcr'sdis,.:a~
with up IO 95~'~ accuracy. THPI cells and macrophagcs, derived themselves by incubation with retinoic acid, were l~mnd to ~ and placed in a ntdit)
fi'equency alterllating nlagnelic Iiekl. Magnelt~.'ylolysiswas asso~..'iated will) FF attachnlenl to the cells without damage to non-t'~mndcells and without
healing of the sttiTotmdmgsolution (() S(~-idt6li'an~'aisetie bit~'himieet biologie moldculaire/ Eiseviel; Paris~.
ferroflnid / au|ileXill-|'~rroflllid / llnllttrxi|| V / hhmd stonlge / blood sedimt'nt¢ltion rllle / AIzheimer's dist'~t~t' / malarial / imignetic ~.ell
snriing I nu!gnetocytolysis
Introdn¢!ion
Ferrofhtids Ill or magnetic fluids 12I are colloidal soluo
lions of monodomain magnetic nanoparticles (usually 5.~
15 nm in diameter) in a suitable liquid. The magnetic
particles, most frequently used, are ferrites, having the
general composition MFe204 (with M being a bivalent
metal cation such as Ni, Co, Mg or Zn and including magnetite Fe304) and nmghemite Fe203. Initially produced by
grinding large particles ir! suitable organic solvents and
sieving 131, ionic ferrofluids are now prepared chemically,
mostly by Ibllowing the Massart method 141 which does
not include an obligatory requirement el" organic solvents
or surfactants as in other methods, in the absence of surfactants, the stability of a fcrrol'luid depends on the small
size of the particles and on an equilibrium between their
charge repulsion and their Brownian motion. For the obtaining e r a stable ferrofluid (FF) in physiological media,
ie at neutral pH and appropriate ionic strength, it is necessary to condition the particle surface. To this end the particles are most freqttently coated with dextran 15-91,
II()-121ol ° symhclic Imlymcr.~ ~uch a~ niclllacryl~Ilc.s~md orgmlo.,,ihm¢.,, I 13~ 15 I. The eff¢~;It~rfittest frequently an ~mtihody) is ~lttached It) the pm°lic!e through a
covalent bond to the coating polymer, ~md the st~bility of
this coating determines the stability of the effectoropm~tiele
complex. Indeed, as pointed out by Groman and Josephson
1161. a partial desorption of these coatings has been obo
served in rive, particularly where the coating was not very
well tolerated, and the result was a shedding of the coat°
ing-bound effector followed by an accelerated elimination
of the particles. Also, the copolymerization with the coat°
ing results in larger sized particles favoring their upt~ke by
hepatic Kupfer cells. To overcome these problems, tt difo
ferent type of particle was developed in our i~lb. These
consist of bare maghemite particles in complex with dimer°
caplosuccinic acid (DMSAI which stabilizes the fcrrofluid
at physiological pH and ionic conditions and permits binding of effectors directly to the particle core through the
DMSA, using hetero-bifunctional reagents, such as N-suco
cinin|idyl 3-(2-pyridyldithio) propionate (SPDP) as dee
scribed I 17. i 81.
albunlin
380
Two biomedical applications were studied in this work:
the characterization oferythrocyte membrane modifications
in several physiological and pathological situations and
magnett~ytolysis which signifies a destruction of specific
aetic field.
"tides with
ing a relatively stable union, is a prerequisite for these applications,
and was developed using magnetic cell sorting h~ vitro.
80
....
!"
~' ' ' - I . . . . I . . . . . I . . . . I. . .
i
1
!
I
~
l l l . l l l le1,,lll s I I I I l i ~ l
,lIllIllIllIllIlllllIlllIllllIllllIllllllllll~llIIIIllllllllIlllIllllllIlllIll
!
i
IIIIIlt
|Illlllllllllll~
Iltlltltotl
IIIilllllIIOIIIIIII
I t l liOlllllllIllllllIllllIlllIllOllll
II
lllIllIIIlll|l|!
IIllllIlllllllll
10
ill~|llllllllllll Illll~lllII|IIIIIIIIIIII IIIIIIIIIIIIIIII.IIIIIIIIIIIIIIIIII
~
' ............... i .................. I................ ...............
'
5i
0
~
10
15
i
i
III~|IllIIIIIIIIIIII~II IIIIIIIlIttill
;9
25
Diameter in nm
Synthesis and characterization of ferrofluid
The magnetic nanoparticles were synthesized as described previously 14, 17 I. Briefly, this involves coprecipitating a mixture
of ferrous and ferric chlorides in an anamoniacal solution, oxidation of the resulting magnetite with ferric nitrate at 90°C to
yield maghemite (Fe203). A cationic ferrofluid with an isoelectric point at pH 7.5 was obtained by dispersing the pm'ticles
in nitric acid; their complexation with meso 2,3-dimercaptosuccinic acid (DMSA) in an acidic medium followed by peptization of the flocculate in an alkaline medium and
neutralization yielded a l~rrofluid which is stable at pH values
between 3 and 9 and in physiological saline (0.15 M NaCI at
pH 7.4). Binding of the effector vhs heterobifunctional tea=
gents is tollowed by masking or neutralization of excess l~actire groups and by surlhce inodifications (lor use in i,il,o),
which suppress non-specific interactions and diminish considerably the non-specific trapping of the particles in hepatic
Kupfer cells. Particles, currently synthesized in our laboratory,
have a diameter Dm = 7.5 + 3.15 nm as measured by electron
microscopy (fig l a). Magnetic birefringence, which measures
a hydrodynamic diameter, yielded a value Dn = 72 nm (fig ! b)
in good agreement with a Dll of 74 nm observed by nanosizing
(not shown). In these latter methods, the size polydispersity of
the preparation (t~ = 0.4) is mote heavily weighted towards the
large particles. This and the solvation layer are responsible Ibr
the large difference between Dm and DH.
Some physical properties of magnetic fluid
30
S!alic' l'[l'rcfs'
I:ronl a I~ficros¢opic poin! of view, maguclic par!icl¢,~ nf a FF
alV lUagll~licallyn11modonlai11 with a diameter hetwccll d ~ 3
n m and d ~ 12 rim, These panicles l~a,ru lllilBnl~li¢ l||OlHl#!it
@
0,5
1
1,5
2
Time (ms)
Ftlt I, Slt~ distributitm o1" DMSA-coalcd maghemitc particles as
detennil~ed by elcctl"~m micruscopy (tl} aud by magnclobirefriugcnce {h},
m~ of the ordor of 1(~I. Bohr l UUglletOllS for d ~ I0 nm.
Depending on the ratio o1' the magnetic anisotropic energy E~ to the thermal energy k13T, the particles are either
i'crmmagnetic or superparamagnetic, This anisotropic energy represents the height of the barrier which opposes a
flip-flop t~fthe magnelic moment,
if E~ > kaT, the particles are ferromagnetic, and their
magnetic moments lbllow the easy magnetic axis. The particles can rotate in a field until thc i~ magnetic tnoment
is aligned with the magnetic field H,
If E~ < klff, the particles are superparamagnetic and their
magnetic moments can rotate freely inside the grains.
E,~can be proportional either to the volume of the particle
E~, = K,V or to its surlhce Ea= K,S where V, S ate respectively the volume and the surface of the particle, K,., K, the
anisotmpic constants for the volume or surface.
The magnetization curve is the first data which characterizes a magnetic fluid. The magnetization Ibllows a classical Langevin law if the volume fraction ¢ ot" tile particles is
not large: ¢ < 8%. M = ¢ M, L (~) where ~ = Bo in, H / kaT
is thc__~ange~l parameter tLt~i = cofl~ ~ - t it~!l. AI t,~x
field M = Xo IT wilh Xo = ~ M d l l , / 3 kilT.
Tile inieraclion belween a magnetk" dipole n~, and a homogelleOtlS magnetic field given a lOltlUe 4c'ling on Ihe parlicle
(for I'ciTonlagnclic |~t~.ii<lit'ie)or Oli lhe nl4~2neiil." nlonlelll (for
superparanlagnelic pariicle); Ibis lo~ttuo varies whh !.~0 m~ H
and has, lor example, a vahie of 10 kn~T for H - 104 Aim.
A force is exerled on a magnetic particle h~ a gradienl of
nia~nel:ic field Ibllowln,, an Ol'ienl¢ilion of its nla~rlellc moIllellt aIOI! o ~,
Fp - t.i0 (i n o?)oH with lu = uru,,L (~)
This force is very weak, on Ihc order of 3' l0 is N, in a
realistic experiment: It. =4~t'10~: m , = M,V (M, = 3"1~1~
A/nIL But ;I i]lrce ~,|Clillg 1111~<1nlagnelic particle ;il |he inter°
face IlelWeell a niagnelic Ill.lid and a noli-niagnelic one (a
llleilibrane for example) can allain abotll IO is N. "Pile reason is thai lhe gradient of field is nol lh¢ exlernal ont~, btil
a local one which is equal !o M I H ) t b tvhe|'e M i t t ) i~ ihc
nlagne~i;'.alion of the illagnclic fluid and b a t:haraclcrislic
length representing the nlean distance belween lwo p:u'iicles
(b ,.- d (ll-I/.l; (~) iS tile volulne fraetioll Of lhe FF).
CelLs .s'orlilig with magneli~" parlil'h,s
Induction of a nlagnetoi)hor¢lic effect ou the particles in ;ill
Oxlcrllal gradient of illagnelic field is nol praclical with a
sial)It !:1( il can, however, be shown thai this effccl is indeed
po~sib!c with Ilit!logic'al cells covered with niaglielic parIiclc~, In the case whore N parlicle~ arc bound on Ihe cell
Ih¢ inagnelic force is given I~y:
i:,, : N lill Ilk I. I~) V I!
h! l!lc CXl)crii!!cilt, the co!In arc iiti¢cicd in a tube el
Icnglh 1 and diameter a with a wdum¢ flow rate Q. The tube
is immersed in il constant gradient of magnetic field VH,
perpendicular Io file flow in the tube. The nlcan Iransit lime
is given by it, = t / v where {7 is the inean velocity of the
llow (Q = 7 it a : / 4 1 .
t. = I / ~; = t it p a -~14 Q
We have to conlpare this iransil time ~tt to the time of
trapping 17p which represents lhe mean tinle reqoired I'or a
cell io cover half a diameter wiihoul l'low.
'~p = ii / 2 Vp, where Vp repl'eSelllS the nlagnetophorelic
velocity. Vp is determined by the balance between the magnetic force F,, and the viscous one F, = 3 ix 11 D vp where !1
and D are respectively Ihe viscosity of the carrier fltiid and
the diameler of tile cell.
t i, = (312) (it 11 a D / H e N V H
For trapping the cells on lhe tube. we ueed to have l:p < ttr
which corresponds Io:
Q < 14~ N V tt l a m, L ( ~ ) t 6 q D
In practice, a pood Irappia~ of lhc cell,, is oblaiaod for
q = I0 ~ Pa..,,, a = ] ram, D = ll) u m . I = 5 ~ } c m . # , , = 4 ~
I0-7, N ~ I06, m ~ = M ~ V = 3 I0 -Is Alto 2 V H ~ 1 0 7 . A i m -~
with Q = 15 mmVs (~7 = 2 cmis.
Dynamic el.]'eels q[./~wro/hdd
Two eli:eels conlrol ihe magnetic dynamics of the parallel
susceptibility Xz/((0). depending on the magnetic anisotropic
energy of ~he particle. If the pariicle is ferromagnetic
(E;, > kltT), file Brownian lime 1;B is dominant: lh~ = 11 V /
ki(P, ii' Ihc parlicle is superparanlagneilc, die NCel lime 1;x
is donlinanl: 1;N = 1;o exp KV/KBT (lypically, 1;ct~ l() `j st.
A sinlplc Debye lay,,, can take l!11o accotilll l:hesc relaxation times for lhe magnetic susceptibility X((o):
)OA(o) = Xd (1+i¢o1;) ~ilh
1; = ei~. 1;,,
The transverse susceplibility X r~__(
~ ) has a resonance
corresponding to Ihe t~rron/agnelic resonance IFMR)of the
luacrospin which is the equivalent of the spin resonance of
111o protons in nuclear magnetic resonance.
X! = lLlo '7 M, (os/(( (oZ o-(o2) - 2icz0h co)
where y is the ~yroinagnelic factor of the electron. (~ is a
faclor faking into accounl Ihe dissipalion of Ihe magnetic
momoill inside Ihe crysial. (el is Ihc L;irm~r frcqucilcy:
(,it = 7 II,
B, can be induced by the ;inis~triq~ic field v¢iih ;ill illl¢rll;ii
field It;i -- i{.L/ IlK Ill i,, c;illcd lhc illlrinnic I:MR)ttr by ;it1
exicrniil field (ihc cxli°ii!isic • PMRI. And forn!al!y the iolat
nlagnclic stiseerttibility is given by:
Z((o) = Z~J((oh 2Z~(co) t 3
Figure 2 presents the real tlild iinaginary paris of X(Oii,
showing the N6el relaxation and the intrinsic FMR.
f e l l t a r g e t i n g w i t h efl~etok- b o u n d f e r n ) f l u i d
Cell targeting by fcr|'ofittid (FF) i , vivo requires reagent~
that are, on one hand, suMciently free of nonospccific inter°
actions with non-targeted tissues, nt)iably the hepatic KLipo
for cells and, on the other hand, can cros,~ ihc endothelial
walls, recognize the desired target and fix lhcnis¢lves Io it.
Particles without el'feeler (bill otherwise lreaied to ¢ounlero
act non-specific interactions) were administered to mice at
doses of up to 100 Hmol Fe/kg and, in preliminary experio
menls, were observed 1o circulate for al least 4 h wi!holil
excessive hepatic uptake (unpublished resulis). They seem
382
to pass through endothelial walls and possibly through the
blood-brain barrier as well. Intravenous administration of
up to 400 ~mol Fe/kg to mice did not reveal any overall
toxicity over extended periods.
.
.
.
.
~10Wsa convenient separation of ~,binding cells from nonbinding cells inn magnetic field gradient, and the collection
of the two cell populations[ 17]. As shown previously, binding
of an enzyme, h o ~ i s h
~roxidase, to the p'~icles, either
directly using SPDP or indirectly through an avidin-biotin
bridge, did not alter its enzymatic activity 118]. Likewise,
binding appropriate !¢ctins to FF yielded reagents that recognized correctly and very specifically the corresponding blood
groups (results not shown). One of these lectin-FFs, Ulex europeaus I-IF, was also used for the purification of endothelial
cells [ i 8] in a reaction that could be competed out completely
by ob L-fuco~. The purified cells could be cultured after removal of the p~icles by EDTA treatment. This demonstrated the
efficacy of efl~ctor bound ferrofluid in targeting and sorting
specific cells and paved the way to the use of annexin V-ferrofluid in the detection and characterization of modifications
in erythrocyte membranes.
Annexin V bound fi, rtY~tluid in the characterization
qf cell membranes
production also results in PS exposure, but this has not been
so far demonstrated. Physiologically, PS exposure on the
cell surface is associated with blood coagulation, erythrocyte aging [25] and apoptosis [26l (it is possible that the
disruption of the inner mitochondrial transmembrane potential [27l and the randomization of PS distribution in the
mitochondrial membranes are also associated with a deregulation of ATP metabolism). In rive, PS, on the outer
leaflet, is recognized by specialized macrophage receptors
[28, 291, thus leading to the elimination of the erythrocytes
or apoptotic cells by the reticuloendothelial system. PS exposure on aged or sickled erythrocytes was revealed, in
early work. through its activation of prothrombinase activity or by accessibility to hydrolysis by phospholip~ses II 91,
and. in later work, by the use of t2~l- or FlTC-labeled annexin V 130, 311.
06
0,5
04
.
.
.
.
.
.
.
.
.
...................................
X '° O a
.
.
.
.
.
.
O! .......
Recombinant human annexin V was bound to FF particles
and the ensuing AnxFF complex was used to characterize
phospholipid distribution in cellular membranes, Annexin
V belongs to a family of homologous proteins of 27-~55 kDa
1
0
|f,031
whidt are ubiquitous intracellular proteins that participate
in nlany devdopm~nial pl'O¢osses illohiding pl'Olociioil of
pla~ntue l'ronl thrombolytic activity, initiation and in!or.
ruption of lactation, ere (for review see t~tynal and Pollard
[191L The common feature of the annexins is their ability
to bind to membranous acidic phospholipids (PL) in the
presence of C#*, Annexin V binds preferentially to phosphatMylsefine (PS)[191 and, in view, to cardiolipin as well
[201, It is possible that their binding to PS in the cell cytoplasm is part of their activity in signal transduction (eg
tall), as it is frequently found to ~ concentrated along the
cytoplasmic side of the plasma membrane 1221. Normally,
phospholipids are organized asymmetrically in cell mere.
branes so that PS is found on the cytoplasm-facing side, and
phosphatidylcholine and sphingomyelin on the outer leaflet
of the plasma membrane, This asymmetric distribution is an
active dyt amtc process, accomplished by the continuous
action of an enzymatic system 1231 and a considerable in°
vestment of metabolic energy which counteract the natural
tendency towards an equilibrated d .istri
of membrane
. . . b utica
"
constituents in all its compartments, Inhibition of this
enzyme(s) by incubating the cells with N-ethylmaleimide
1241 results in a gradual exposure of PS on their surface, it
is reasonable to assume that interruption of cellular ATP
.
1 [~ ttl,}
|ftOfi
| g *L)fi
I~*0]
1 (:~* L'IL~
t~+O~
IE+IO
IE*09
IE+IO
%
Y"
05
0
,05
,15
!E*03
IE:.Oa
tE*OS
IE®06
IE,O?
IE~08
Fig 2, Real X" and imaginary X;"paris of the initial ,~usceptibility
very'usthe frequency t"= o~/2~:,)( and X" represent respcctivdy the
real and imaginary pans of the initial susceptibility vep:vus frequency (f = co/2~),X°" and Z" show successively the rdaxation and
the ferromagnetic resonance (l-MR).
383
Our interest in erythrocyte membrane modifications
stemmed from our studies of cerebral malaria in mice which
revealed modifications in the characteristics of red Nood
cells (RBC) already during tile hepatic stage of plasmodiam
infection, well before any RBC, were parasitized [321. associated with an accumulation of a subpopulation of RBC
intermediate between reticulocytes and erythrocytes, and
prone to plasmodial infestation [33]. Recombinant human
annexin V was thus bound to DMSA-complexed FF, and the
ensuing AnxFF reagent was used to probe for PS exposure
on RBC i!7, 34]. In this assay, PS exposure ts revealed by
magnetic cell sorting and estimated from the observed fraction of cells, which are retained in a magnetic field gradient
following their incubation with AnxFE As seen in figure 3,
freshly collected mouse blood does not contain any detectable AnxFF-binding RBC whereas 10% of the cells bound
AnxFF already after storage for 24 h. Similarly, AnxFF
binding to RBC increased steadily during storage of human
blood under blood bank conditions (fig 4).
AnxFF binding to RBC is also enhanced in various pathological situations 11341. As can be seen in table 1, blood
from patients with sickle cell anemia or those displaying a
high blood sedimentation rate contained up to 50% AnxFF
binding cells compared to up to 15% in normal controls.
For stored blood and sickled erythrocytes, the values in
figure 4 and table I, using AnxFF, for both the control and
the experimental cases, are 10-20 times higher compared
with values reported by Kuypers et al [351 and by Wood et
a11361, both using Anx-F!TC, while confirming the general
sense of their results. Likewise, Taverne et al 1371 did not
observe any enhanced binding of FITC-Anx V to RBC at
any time ibllowing the infectio,i of mice with plasmodial
parasites. On the othe," hand, we had observed 1331 modifi.
cations of tile size, cell electrophoretic mobility and resist°
ance to glycerol-induced lysis of eryth,'ocytes from mice
during the early hepatic stage of their hdection with plasmodia, at a time when Ihe RBC themselves were not yet
parasitized, and described an RBC subpopulattt
•
, ' 3n, intermediate between reticulocytes and erythrocytes, which is
the actual target for plasmodial invasion. We thus inquired
[381 whether the appearance of this sub-population was not
preceded by an enhanced PS exposure on RBC, the removal
of which could trigger a recruitment of new cells in the bone
marrow. Indeed, as seen in figure 5, a transient enhancement
of AnxFF binding by RBC was observed 24 h after the
infection of Balb/C or C57/bl mice by P Berghei Anka, and
this was followed by a peak of reticulocytes by day 3 and
of the RBC sub-population by day 4 ! 181, just in time to be
invaded by parasites emerging from the liver. This scenario
is similar to the one operating in the evolution oi' normal
RBC 125, 281 whereby aging erythrocytes, exposing PS, are
recognized by macrophages and eliminated in the reticuIoendothelial system. They are replaced by young cells
emerging from the hematoporetic system as reticulocytes,
and maturing into erythrocytes, thus maintaining a low level
of PS exposing cells. The higher sensitivity of AnxFF, corn-
100-
%RBC
~d 24 H
Baib
80604020-
Control
Balbtc = 0%
Non-retained
Retained
Fig 3. Annexinqerrofluid binding to mouse erylhmcytes as a function of their storage in vilro, "Pailblood was coilecled fl'om Balb/C
mice into citrate buffer and RBC were separaled by centrifugafion
and removal of the buffy coal. AnxFF binding |o RBC was tested,
immediately or following an overnight storage at 4"C, by a 30omm
incubation at 37'~C of 30 IJL of a 3% RBC suspension with 30 ~L
AnxFF in 0.35 mL of Tris buffered saline containing 2.5 mM CaCI2
and O.I% detipidated BSA. The reaction mixture was diluted with
buffer and passed through an electromagnetic field gradient to separate the AnxFFobound cells from the non-bound cell as described
previously [17. 341. Omission of Ca2+ ions in any of the steps
abolished AnxFF binding. Reprinted from [34j with permission.
pared to FITC-Anx, leads to a different description of the pm
tlmgenic process. This is also trite in the case of thymocyte
apoptosis where PS exposure is detected by FITC~An× later
F
60-
% Bound
............. y ~ 2.3728 + 19 987 log(x)
R ,, 0.99436
45-
,J
15~°.e .°'
1
10
Storage (days, logarithmic scale)
100
Fig 4. Effect o1"tile duration of human RBC storage in vitro on the
extent of Anx-FF binding, Normal blood samples, collected by the
'Centre de la Transfusion Sanguine', were tested fl~llowingstorage
in viov for varying limes under the blood bank conditions as described in the legend to figure 3. Reprinted I't'onl 134] with per*
mission.
384
Table I. Percentage of AnxFF binding to flesh RBC fronl human subjects with various pathologies compared to healthy persons. Anonymous
b l ~ sample, of persons requiring blotxl analysis were obtained from a local laboratory and used within 48 h of collection. Other conditions
were as described in legend to figure 3. SR. sedimentation rate; other pathology, normal SR blood flora patients with either a deforming
rheumatic disease era cancer necessitating chemotherapy. Reprinted from 1341 with permission.
Control
Normal SR
19,60
0.(30
14.80
12.(~
11.90
18.00
0.0O
12.60
21.73
16.75
! 9.56
27.59
32.1X)
Sickle cell anemia
50.00
49.00
(~.00
35.00
49.00
53.20
High SR
36.94
36.37
51.26
54.49
49.85
46.80
65.70
Other l,tlthoh,,~,'y
49.76
63.00
63,00
71.00
14,00
5.90
! 2110
8,70
Mean ~ SD
.
.
.
.
.
.
.
.
10.7 :t: 5.9
.
.
.
23,5 ± 6, I
5(1.(} ± 9.3
51,5 ± 12.2
58.5 ± 7.6
.
than by a pmthmmbinase assay 1391 (cfM Sorice, lx~rsonal
communication). The apparent lower sensitivity of FITC-Anx
is also compatible with the fact that results obtained with this
reagent correlated with those obtained by a prothrombinase
assay only when ionophore-treated RBC were utilized 1351.
This interpretation was confirmed in a comparison of 1he different m e t h ~ l s of detecting PS exposure 1401. AnxFF binding
to RBC, stored Ibr various lengths ol' time. correlated well with
the estimation o1" PS exposure by either anti PS-IgG or a
prothrombinase assay, whereas significant ~-'-Sl-Anx or
FITC-Anx binding to the same samples was detected only after
80~ I IX) days of storage when extensive hemolysis was already
evident. The same Kd of I-~sI-Anx binding to RBC was
measured throtlghout the storage of blood (Geldwerth et al, in
preparation), indicating that the same low aMnity sites arc
detected at all time points with this reagent, alld these sites
be¢ollle preponderant only after prolonged storage.
While being caused by many dil't~rent, and thu~ non-specil'ic causes, measuring PS cxposulv o|1 RBC by AnxFl:: bind°
tug i~lay ncvcllhclcss olTcr an assL,'alWCas to the viability in
rive of the Iransl'used RBC. Also, viral (itlcluding tt!V and
hepatitis C} alld bacterial illl'~clions nlanifcsl thelu~elves very
rapidly by strong inflamlnatory reactions. Determining the
fi'action of AnxFF-binding RBC in blot~ donations can thus
be uselid during the time lapse (currently a minimum of 15
days for HIV) between inl~clion and seroconvcrsion.
Eryth..'yte membrane mod(lh'alhms in Alzheimer k disease
10 ......
0
24
45
70
Time after infection (hours)
FIR
$, AnllC~illof~rr~ffluidbinding to mouse erytlm~cytcs immcdi°
ately at~er hlt~ction with malaria l~U,asit~s, Balbtc mice wcrc inI't.'cted with Ph~smmlhu, voelii tclosed circles) or injected with
Ballqc mouse erylhmcyles infested with P Berghei ANKA ~open
ci~les), Controls (o~n ~uares~ were el!her exl~,~sedto non.inl'cctcd
,losquit~s or injected ip with uniu~k,;~:d~:(y~h~.~-~,~:s,Tail bh~d was
colh,'ck,d and its AnxFF binding was measulxxl as descfil'~'d in the
legend to figure 3, Reprinted with permission fix,, 1381,
We had already developed previously a baUcry of tests to
characterize cell membrane modifications in malarial infections 133 I, and considered the possibility that this battery of
tests could provide a diagnostic tool Ibra specific disease
~uch as Alzheimer's disease,
Alzheimer's disease (AD} is a neumdegenerative disease
with an as yc! unknown etiology which usually appears relatively late in Ill}, AD is routinely diagnosed by a ncuro-psychological evaluation of patients according to the criteria
proposed in 'DSM IV' for dementia 14 i I, by Mac Kahnn et
a11421 and by Panisset et a11431 including a 'Mini Mental
Stale Score' (MMS} 1441, Definitive diagnosis must, however, await histological analysis of cerebral tissue (usually
385
postmortem). This reveals in AD, but no| in other dementias, an extensive degeneration of brain tissue with neural
plaques composed of insoluble ~-amyloid (ilself resuhing
of an abnormal processing of the lransmembrane amyloid
precursor pro|ein lAPP) and neurofibrillary tangles containing phosphorylated tau peptide (for review see I45]). Incubation of cells in v#n~ with ~-amyloid results in cell death
[461, but the mechanism initiating the abnormal APP processing is not known, Several genetic variants of the APP
and presenilin are associated with an early onset AD, but
these concern only a small fraction of patients while age is
the major risk factor for this disease. Polymorphic variation
at the apoE locus is associated as a risk or protective factor
for late onset AD [47,481. Other genetic risk factors for AD
include presenilin intronic polymorphic variants 149, 501,
and serotonin transporter polymorphisnl [51 I. Gene|ic markers hold considerable promise to improve diagnosis [52,
53], However, considering apoE phenotype as a screen or
diagnostic marker tbr AD would miss many Irtle cases and
could misclassil~, many normais as AD 1541. Therapeutic
interventions are limited to symptomatic treatment and, recently, to a transient augmentation of cerebral acetylcholine
or a direct activation o1' nicotinic receptors !551. Objective
surrogate markers would be very ht22"d in diagnosis (particularly in early stages) and follow up of patients, and
could thus contribute to a better understanding of the underlying pathogenic processes. It might eventually enable a
rationalization of therapeutic interventions.
Numerous works have described extra neuronal anomalies in AD including abnormal aging of erythrocyte
membranes 156-591, lipid peroxidation I(~01 and con fl icling
reports exist on the internal fluidity of cell membranes 161-
651. O|" parLicuiar imeresl was the ~eporl of Walter m~d
Widen of an ahered electrophorefic mobility of RBC from
AD pafiems in certain polymer solutions I66, 671.
Modifications in RBC membranes from probable AD patients were studied 168] using cell electrophoretic mobility
measurements in polymer (CEMp), AnxFF binding, celt filterability (mean transit time, Mtt), which estimates cell
membrane fluidity [69, 701, and resistance of the RBC to
lysis in a glycerol medium (glycerol resistant cells, GRC).
Both AD and non-AD patients were hospitalized in a geriatric hospital department in a northern suburb of Paris. All
24 AD patients met the criteria of dementia [41 ]. Diagnosis
of a probable AD was established according to the guidelines proposed by Mc Khann et al 1421. The patients had
an MMS score below 21/30, they all had amnesia, visuospathd disorientation and aphasia 143], and their educational
level corresponded to that of a primary school. All ! 8 ageand sex-matched control patients were hospitalized lbr o f
thopedic problems but were free of any neurological symptoms and had an MMS score above 22/30. A duly signed
inlbrmed consent was obtained from the patients and responsible parties in accordance with the regulations of
French hospitals. A group of 18 healthy, 30-52-year-old
volunteers served a~ a healthy young control.
Many of the patients, hospitalized in a geriatric laci!ity,
received various medications, mostly 'comfort' drugs.
Medication included antidepressive, antisecretory and anti°
Iwpertensive drugs. The effect of these drugs was tested in
vitro for the dill)rent l)atures of cell membranes studied
(table il). it was assumed, for simplicity, that the prescribed
dose was distributed homogeneously in vivo in the whole
body. Only the effect on CEM 1711 and AnxFF binding !341
Tahle !1. El'lecl of various prescril~ed drugs, when added in vm'¢~o on the elcclrophoreli¢ mobility (('I:.M)and ;mnexin-|crrolh.d (Anxl:[:)
binding of norlnlll RBC. Control RBC were incubated for 2 i~ at room temperature with diti'c!¢nl ¢oac¢~tr~tion.~ u[ drug. bcl~v¢~:n11,i ;lilt[
l0 times of the estimated 'correcl' concenlralion, washed, and the vat'iotls l~altlrt~,s were measured in comparison with t:l)lreated RBC~
Drugs
Generic name
Nature
None
Fluoxetine
Theophiline
Anti depressive
Anti asmatic
Thioridazine
Neuroleptic
Enoxaparine
Tiapride
Nitroglycerine
Oazepam
Amiodamne
Omeprazole
Hydmxyzine
Enalapryl
FeSO4 B6
Heptaminol
Paracetamol
Paracetamol - codeine
Anti clotting
Neuroleptic
Vasodilator
Tramluilizer
Vasodilator
Enzyme inhibitor
anti depressive
ACE inhibitor
Anti anemic
Anti hypertensive
Analgesic
Analgesic
¢'EM
/tnxFl: himting
¢~tms¢' t V t c m I
Celt ('~ t
!.031
2.t~5
7.10
46.(10
I00.(10
8,fit)
7.t~5
ND
2,70
1.064
i. !()(1
1.057
1.085
1.083
Lysis
I. 108
1.099
I.O37
1.071
1.04 I
1.002
1.007
1.025
1.028
3.8O
6.1(I
1.73
0.84
ND
7.111
ND
3~
is shown in table 11. Patients or controls who required the
drugs producing an effect on the studied parameters were
excluded from analysis.
The mean values of the RBC membrane features in the
and
rices
rand
in the extent of AnxFF binding, cell electrophoretic mobility
in polymer(CEMp), and the fraction of glycerol resistant cells
(GRC), while the difference in filterability (Mtt) was only of
limited significance. Statistical significance is presented only
for the differences between AD patients and their hospitalized
age,. and sex-matched controls (ND). The changes in AD erythrocyte membrane features were seen repeatedly over time in
~veral patients who were tested over an 18-month period, thus
distinguishing this picture from epls
'~,~dtc
' " modifications which
are associated with acute inflammatory events (as discussed
earlier). The magnitude of changes in RBC features was, in
this limited cohort, not different in patient,
'- s of relatively e,'u'ly
(MMS of !5-20) and late stages (MMS < !0) indicating that
the changes may be produced early and persist throughout
disea~ progression. Judging from this ~mple of probable AD
patients, the~ results indicate that the modifications in RBC
membranes are indeed associated with Alzheimer's disease.
The changes in CEM, filterability and glycerol resistance
might be inherent to the alterations in membrane structure in
this disease, However, in other physiological and pathological
situations these modified features at,, characteristic of young,
newly recruited RBC 133, 68, 71 !, As discussed earlier in this
pal~l; PS, exposed on aging or damaged cells, is recognized
by specialized receptors on macrophages 128,201 and the cells
are eliminated in the reticuloendothelial system 1251, Results
presented in I'i~urcs 4 and 5 are compatible with tiffs picture
We, therefore, separated RBC fractions enriched for young,
glycerol resistant ~ells by centrifugation in suitable density
gradients, Unex~ctedly, howevel; the separated young cells
exhibited the same extent of AnxFF binding as the whole
population. This is compatible with an assumption that PS
exposure in AD erythrocytes is brought about by oxidative dinsage that is expected to act with the same efficiency on young and
old cells, especially in view of the apparently high preponderance of young ~ C in AD pauents.
The overlap in test values between AD patients and their
age- and sex-matched controls, seen in table III, would not
permit the use of any one indiwdual feature as a diagnostic
tool. However, a multiparametric ~ogistic analysis 1741 suggested a rather specific predictive value for several threefeature combinations in this limited cohort [6811. The
probability of the patients belonging to the AD group may
be estimated by the formula:
Prob IA D ! = I/( 1,4-e~,~,)
where x can be calculated by one el lhe following combinations (with 75, 80 and 95% correct classification respectively):
Xt = 30.4 + 0.2 x GRC + 0.06 x AnxFF - 35.6 x CEMp;
X2 = 45.5 + O, 12 x AnxFF - 41.8 x CEMp - 4.7 x MTI;
X~ = -97. I + 0.21 x AnxFF + 50.65 x M~'~ + 0.5 x GRC.
The logistic regression using subsets of features permitred quite a good classification assuming t!lat the histologic examination will confirm the AD diagnosis.
Characterization of RBC from a large, multiccntric sample
of patients including cases of other dementias and ncuro~
logical problems will be required in order to de~ermine the
diagnoslic value of this test battery and estimate it~ positive
and negative predictive values.
The observed modificatiou~ oI' erylhrocyte membrane
prol~rties present a seemingly contradictory picture; on one
Table III, Biochemical and biophysical characterization of red bk~d cells from Alzheimer's disease patients. 5 mL blood s',unpies were
~:ollected in citrate vacutaincrs t'rom AD patients, age° and sex-matched non-demented (ND) patients, and from young controls, RBC were
separated by centrifugation and aspiration of the bully coat, and used in determining An×FF binding [3:q, CEM [71 !, Mtt [701 and GRC
172l, L~vels of significance are shown tbr the t:omparisons of values fi'om AD and ND patients,
A,~e
I'l¢clroldtort,tit' I~lobililit, s
f t~oli ,vet' t V t cot)
h,
.valh~e
in polym(,rs
...........................................................................................................................................................................................................................
tyem's ~t
Atolt'A'iPI- V
bimfing f t:~,,J
Glyt'¢ro!
resistant cells
(%)
...............................................................
Al~heimer's patients (AD)
84
45.5
1.45
30.8
± 7
± 18,0
± 0.018
± 0.t)22
± O, 12
± I I, I
Age-matched non-demented
patients (ND)
84
:t 7
27, I
± 14,7
i ,(M3
± 0.020
1,046
± 0.022
i .55
± O. I I
I9,6
± 6.4
35
2,7
±6
± 1,9
1,0f~5
:E0,014
_+0,021
±0.11
0,48
0,02
0, I
Healthy young contrails
18
P = AD vs ND
0,36
0,(X)3
! .028
time ¢MFr~
fins)
n = 24
~I =
1,039
Mt'ZO~transit
1.053
........................................................... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.54
10.2
± 3.1
0.026
.~87
hand, that of an extensive damage 1o RBC membranes is
seen in the enhanced AnxFF binding, and, on the olher hand,
a high content of young, l~cently recruited RBC can be
gleaned from the higher resistance to glycerol lysis, higher
fluidity mid lower electrophomtic mobility 133, 70, 731. We
assume that these changes reflect similm" changes in neuronal cells or result from them. Disruption of membrane
asymmetD, and the consequent exposure of PS on the surface of any ceil, includh, g neural cells, could result from
oxidative damage [ 19| or, at least initially, from an interruption of energy metabolism. A diminution of mitochondrial
energy metabolism has been associated with aging 1751. It
has also been observed in cerebral tissue l~om AD patients
1761 along with a decrease in the expression of the milochondrially encoded cylochrome oxidase genes 1771. Mammalian cells contain a large excess capacity of energy
metabolism I78I and can function normally at 0.4-0.5 of the
normal ATP concentration [79I provided that the metabolic
rate is suMcient to prevent an accumulation of ADP and/or
AMP 178-801. This excess capacity results from a large
number of mitochondria per cell, each with an independent
mitochondrial genome (up to 50/cell). Depletion of cellular
ATP through mutational inactivation of mitochondria thus
requires many independent mutational events and such an
eventuality should have a rather low probability. This could
explain both the relatively late onset of AD and its modulation by various genetic factors. A disruption of the membrane asymmetry of neural cells would lead to their
interaction with the specil'ic receptor(s) on macrophages
(nticmglia in the brain). Unlike activated systemic macrophages, which atv evacuated in the reticuh)endothelial ceils,
brain macmphages remain in place and produce NO or cytokines such as TNF or !!2 which, in turn, may be a source ol" a
continuous insult to neighboring cells including the damage to
RBC (which thus become a suitable surrogalc nlarkerl alld ;lls~,
C
L
To
/
Fig 6. A protot) pc of a magnemoc>tolysisapparatus. C and L are the
capacitance and selI" hlductance of the coil for the l'e~onance circuit.
Ti and To are the temperatures reside and outside the test tube.
When a particle is rigidly bound to a membrane, only the
Ndei response is possible with the characteristic time
!: = I:N = 1:oexp (E,,/klaT). The maximum of energy dissipation occurs for ml:N ~ I. For maghemite particle td ~ 10 nm),
ml:N ~ 1 is in the MHz range.
In order to test this phenomenon the prototype described in
figure 6 was consmtcted: a resonant series circuit with a capa~
citance C and a sell" inductance L. The electronic power sup°
ply permits us to obtain a magnetic field of 0.(11 T inside the
solenoid.
For a given magnetic field (B
0.(11 T) and a ~ivcn
frctitlcncy (f :::: I MIIz)l'igui'c 7 picscnls the inct'¢a~cof the
as relxmed, to T lymphocytes 1591.
50
Magnetocytolysis of ferrofluid-bearing cells
Physical basis
Magnetocytolysis is the destruction o f ferrofluid bearing
cells following incubation in an alternating magnetic field.
A wide spectrum of hyperthermia methods exists in the
radio frequency range (10-100 MHz), microwave range
(> 300 MHz), infi'a-red range using the imaginary part of
the dielectric constant or ultrasonic waves using the acoustic absorption in the tissues. Howeve,', the use of magnetic
nanoparticles in a radio frequency magnetic field actually
seems the best way (Ibr complementary information see the
excellent review paper of Jordan et al 1811).
This phenomenon of hyperthermia in FF is based on the
response of the imaginary part (fig 2) of the magnetic susceptibility of the solution X" = Z0 t0x / (l+t021:z).
jm
40
I,,C1=
~
30
/
W
a. 2B
Ig
'1
IO
VOLUME FRACTION
0
+
O
8.805
F
O.Ol
.F
0.015
i
i
O.B2
Fig 7. Solution heating in an alternatingmagnetic fMd a.sa ftmclion ot' the volume fraction of t~m)fluid.
388
temperature AT as a function of ¢ the volume fraction of the
FE It is possible to describe AT by:
a2 Ao
@
AT8~
I +¢(~:p-K.,.)
whe~ A ~ is the rate of heat generation; Ks and % are respectively the thermal conductivity of the solvent and of the
panicle.
A,@ = toLI2x" (to) / 2 VF
where I, L V~ are respectively the current, the self inductance and the volume of the sample of the magnetic fluid.
it is clear (fig 7) that for a high volume traction 0 > !0~,
a macroscopic heating is induced. This would not be interesting since it is non-specific. Interestingly, however, as
seen in the legend to table IV, magnetocytolysis was produced with FF concentrations smaller than I0 ~,~(5 IJM Fe),
with no macroscopic heating, and only when the particles
were linked to the membrane,
enabled the particles to bind to membrane proteins through
mixed S-S bridges. As seen in table IV, THPI monocytes
and macrophages, obtained by incubation with retinoic acid
or harvested from mice, were destroyed in an alternating
magnetic field only when preincubated with DTT-treated
FF. The FF in these experiments was routinely filtered
through a O. 1 ~ filter to remove aggregates or larger particles. Incubation of macrophages, but not undifferentiated
THPI cells, with unfiltered FF resulted in their destruction
by magnetocytolysis (table IV) presumably owing to their
ingestion by the macrophages, Cell death, seen either by
vital counting using Trypan blue or by the WSTI kit, was
evident only 3-6 h after the lO-min incubation in the magnetic field.
In later experilnents, still in progress, 4-hydroxytamoxil'en or estradiol were fixed onto FF and used to produce a
specific magnetocytolysis of MCF7 human cancer ceils
whereas THPI cells were unaffected under these conditions
(results not shown).
Magnetocytolysis of THP I human monocytic cells and
mac.,i, hages
Conclusion
In the initial experiments, described in table IV, a non-specific binding of FF to ceils was achieved by exposing -SH
on DMSA--coated FF by DTT treatment, Presumably this
The dilTerent lines of experimentation, described in this
paper, were undertaken with the perspective of using specifically targeted ferrofluids both in the elaboration in vitro
of cellular mechanisms such as apoptosis, membrane constitution, elc and in i,il,o lbl"diagnostic (eg MRI) and tllerapeutic (maglletocytolysis) applications which depend on the
magnetic properties of tile particles. The targeting potential
of this l'errtdhdd has beeu established in the magnetic cell
~ol'ting apl~!ica!i(ms and i~ !row heing incorporated iu the
nlagtletocytolysis experiments. II is hoped lhal Ihis approach will buth diminish non ,~pecil'ic e!'t~cts on 11eighbur°
ing c~lls and also facilitate an c l a b o r a l i o u o{' the
mechanisms involved in maglletocytolysis.
Table IV, Magnelocylolysis of humml THPI monocytes aud
macrophages, FF was reduced with dilhiolreiloi (DTT) mtd exccs~
DTT was removed by t'locculalion of !he parlicles at pH 2, The
resultin8 FFDT particles underwent pepti~ation, after retention uu
tl pel'mal+el,mtl~.eh by muspel+dingIhem iu 1), 15 M NaCIo(), I M
Tris--HCI, pH 9 and adjustmel, to pl=l7.i,l. Particles were sterilized
by a {),1 ~tlU filtration except where otherwise stated. Mouse
nla~rophllges ~,ere induced by an intral~ritoneal injection of
0,~ mL thio~ly¢ol~te bl~th, harvested aseptically 48 h later and
sus~nded in RPM11640 culture mediumsupplemented with !0%
fetal calf serum, TPHI human monocytes were cultivated in the
same cultu~ mediumat 37°C in a 5% (702 atlnosphere, Retinoic
acid t~almel'lt consisted of 2~3 day,'s of incubation of O,5~l × IOt,
cellstmL with 0,2=2 × !0~ M retinoic acid, For magnett~ytulysis,
3~8 × 10~ ~ells Wel~ incubated for I h with or without 5 pM of
the various ferrofluid ptepal'ations and subsequently introduced
into the apparatus described in figure 5, and treated tbr I0 rain by
an alternating magnetic field of I{~ Oe at a frequency el" - I Mhz.
Cell survival was estimated 2, 4 and 24 h later by a differential
cell count using the Trypan blue exclusion te~t and by a dye
oxidation test using a WST° I kit (B~hringer), Result present the
percentage of surviving cells f~dlowing magnetocytolysis
Cul.pared to cells not treated in the luagnetic field.
?)v.tme.t
........................
None
FF
FFDT
Non-filter~,d FF
THPI cells
99,3 ~ I,t)
91.8 ± I 1,5
7~,3 ~ 8.4
~).4
IYIPl celt,v+
ret#loic acid
97,8 :k 2.8
8,9, I ~ 9,6
54.0 ~ 8,0
46.5
Mouse
maCrOldmges
I00
nd
48.5
42,4
Acknowledgment
Financial support was prm, ided by Assodatiun pour la Recherche
contre le Cancer ( R().
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