Journal of Colloid and Interface Science 212, 474 – 482 (1999)
Article ID jcis.1998.6053, available online at http://www.idealibrary.com on
Synthesis of Iron Oxide Nanoparticles Used as MRI Contrast Agents:
A Parametric Study
Lucia Babes, Benoı̂t Denizot, 1 Gisèle Tanguy, Jean Jacques Le Jeune, and Pierre Jallet
Vectorisation Particulaire UPRES EA 2169, 1 rue Haute de Reculée, 49045 Angers, France
Received September 21, 1998; accepted December 17, 1998
Colloidal iron oxides play an important role as magnetic reso-
nance imaging (MRI) contrast agents. The superparamagnetic
particles actually used are constituted by solid cores (diameter of
5–15 nm), generally coated by a thick polysaccharidic layer (hy-
drodynamic radii of 30 –100 nm), and formulated by direct copre-
cipitation of iron salts in the presence of polymeric material. To
better control the synthesis, we attempted to formulate new stable
uncoated superparamagnetic nanoparticles. Colloids were gener-
ated by coprecipitation of an aqueous solution of iron salts and
tetramethylammonium hydroxide (TMAOH) solution. The influ-
ence of parameters such as media composition, iron media, injec-
tion fluxes, Fe and TMAOH concentrations, temperature, and
oxygen on size, magnetic and magnetic resonance relaxometric
properties, and colloidal stability of particles were evaluated. We
have determined the relative importance of these parameters as
well as the optimal conditions for obtaining uncoated stable par-
ticles with an average size of 5 nm and interesting relaxivities. The
interpretation of the observed limits takes into account diffusibil-
ities of reactants and product, feeding rates of reactants, and
surface properties of nanoparticles. A model of synthesis, related
to spontaneous emulsification of suspensions, is proposed. © 1999
Academic Press
Key Words: contrast agents; colloidal stability; iron oxide; nano-
particles; relaxivity; size; susceptibility; synthesis.
INTRODUCTION
Colloidal iron oxides play an important role as magnetic
resonance imaging (MRI) contrast agents, to better differenti-
ate healthy and pathological tissues. A wide variety of iron
oxide nanoparticles have been synthesized. They differ in size
and type of coating material used (dextran, starch, albumin,
silicones, and poly(ethylene glycol)), the hydrodynamic parti-
cle size varying between 10 and 3500 nm (1). Intravenously
injected products are constituted of a small nucleus of iron
oxide (diameter of 5–15 nm), coated by a thick layer of
adsorbed polymer (total diameter 30 –150 nm). These nanopar-
ticles present a high vascular remanence and redistribute in the
mononuclear phagocyte system (MPS) (liver, spleen, bone
1
To whom correspondence should be addressed. E-mail: Benoit.
Denizot@univ-angers.fr.
0021-9797/99 $30.00
Copyright © 1999 by Academic Press
All rights of reproduction in any form reserved.
marrow, and lymph nodes) (1). Studies on the different contrast
agents have shown that the biodistribution, as well as the
vascular remanence, depends on the size, charge, and thickness
of the coating of the particles (2, 3).
Two methodologies are currently employed to obtain iron
oxide contrast agents. The first consists of the synthesis of
isolated cores by successive or simultaneous precipitation of
Fe(III) and Fe(II) ions in ammonia (NH 3) or soda (NaOH)
alkaline media (4 – 6). This method results in the synthesis of a
precipitate which needs ulterior processing (centrifugation,
washing, and magnetic separation) before redispersion in water
and eventually an ulterior coating. Magnetite particles may
also be synthesized at high temperature (7) or by hydrolysis
and oxidation of Fe(OH) 2 suspensions at elevated temperatures
(8). The majority of wet processes, such as coprecipitation, air
oxidation, and hydrothermal processes, give rise to products
with a good crystallinity and magnetization, but with bigger
particles (order of micrometers).
The second method consists of a one-step synthesis of par-
ticles covered in situ by different polymers (notably dextran)
(9 –11). Some typical contrast agents, e.g., USPIO (ultrasmall
superparamagnetic iron oxide) (12), are obtained from a sep-
aration of AMI 25 nanoparticles (Advanced Magnetics) on a
column.
In many syntheses of polymer-covered particles, crystal
growth is probably inhibited by the presence of the adsorbing
molecules (as, for example, the polymer in the alkaline media).
The adsorbed polymer layer stabilizes particles by preventing
their aggregation but, due to its brush layer constitution (16), is
responsible for the important hydrodynamic radius. Moreover,
the majority of syntheses give rise to size heterogeneity, with
atypical populations, hardly characterizable, being considered
as potentially toxic for human injection (10). To optimize the
synthesis of these particles with a view toward optimization of
the thickness of the polymeric coating, the composition, and
the relaxometric properties of the cores, we adopted a two-step
process. We then concentrated on the synthesis of bare iron
oxide cores to find a reliable and accessible way to synthesize
particles of reproducible size and relaxometric properties.
This paper describes the synthesis of iron oxide nanopar-
ticles by coprecipitation of acidic solutions of Fe(II) and Fe(III)
474
 SYNTHESIS OF IRON OXIDE NANOCORES
in tetramethylammonium hydroxide (TMAOH). We investi-
gate the influence of different factors on the synthesis, such as
media composition, injection fluxes, concentrations, and the
influence of temperature and oxygen. The obtained colloidal
solutions were characterized by iron analysis, electron micros-
copy, magnetic resonance (MR) relaxivity, and preparation
yield (ratio between Fe concentration of ready-to-use particles
after the different processings and total initial Fe concentra-
tion).
MATERIALS AND METHODS
Colloidal Formulation
Colloids were generated by coprecipitation of an aqueous
solution of Fe 21/Fe 31 iron salts in a TMAOH solution. The iron
mixture (FeCl 3 z 6H 2O and FeCl 2 z 4H 2O, minimum 99% pure,
Sigma Chemical, St. Louis, MO) was injected through a cath-
eter (f int 5 3.8 mm) in the circulating flux (f int 5 8 mm) of the
1 M TMAOH solution (Sigma Chemical). The flux rates of Fe
and TMAOH solutions were controlled by a Pharmacia LKB
pump P-1 (Pharmacia, Sweden) and a Millipore Pump (Mil-
waukee, U.S.A.), respectively. After deposition of aggregates
on magnets (at least 60 min), the basic colloidal suspension
obtained (pH ' 13) was electromagnetically filtered. The
influences of parameters such as media composition (compar-
ison between a TMAOH solution and a mixture of TMAOH
and ammonia (NH 3)), FeCl 2/FeCl 3 ratio (0/10 # Fe 21/Fe 31 #
8/10 M/M), injection fluxes (1 # Fe flow # 5 ml/min and 50 #
TMAOH flux # 800 ml/min), concentrations (12.93 # [Fe] #
116.4 mM and 0.25 # [TMAOH] # 2 M), temperature (20 #
T # 80°C), and oxygen (solutions partly degassed for at least
30 min by plunging a cannula directly into the solution, Ar or
N 2 flow 5 3 liters/min) on size, relaxometric properties, and
stability of particles were evaluated.
Electromagnetic Filtration
Preparations were filtered into a 10-ml plastic syringe body
filled with steel wool (kindly provided by Prof. Dr. De Cuyper)
and placed in an electromagnet Stelltrafo power supply/
PHYWE 0.7 T (Phywe, Frankfurt am Main, Germany) at a
potential of 25 V (about 0.7 T). The filtrate was considered as
the final formulation product and evaluated.
Iron Analysis
Total Fe concentration in suspension was determined after a
2-h digestion of particles in 30% (v/v) HCl (Prolabo) by
spectrophotometric measurements at 340 nm using a Uvikon
940 spectrophotometer (Kontron Instruments S.A., St.-Quentin
Yvelines, France).
475
Preparation Yield
This percentage reflects the ratio between Fe concentration
of particles after electromagnetic filtration and the initial the-
oretical concentration.
Transmission Electron Microscopy
Negatives were obtained with a JEOL 100CX microscope
(JEOL, Tokyo, Japan). Copper grids were covered by a collo-
dion film (2% in amyl acetate). The grids were placed on dilute
drops (1/100 in distilled water) of the different preparations.
After 2 min, the grids were retrieved and dried at room tem-
perature. Photos (magnification 240,000) were scanned using a
600 dpi scanner.
The scanned images were saved as gray scale TIFF files and
processed with NIH-IMAGE (freely available at http://rsb.info.
nih.gov/nih-image/). About 200 particles were measured and
tabulated for size distribution analyses. Particles of size greater
than 200 nm and of regularity (measured perimeter to calcu-
lated ideal rectangular perimeter ratio) greater than 1 were
considered as aggregates and discarded. Particle sizes were
reported as the major axis (i.e., the equivalent diameter of the
particle considered as a sphere). The mean size was obtained
with a lognormal renormalization datasheet, using a laboratory
personal program.
MR Relaxivity
The colloids were diluted in monodistilled water. Water T 1
and T 2 relaxation times were measured at 39°C and 10 MHz
(triplicate runs) with a Minispec Bruker spectrometer (Bruker,
Wissembourg, France) by standard inversion–recovery (T 1 )
and Carr–Purcell–Meiboom–Gill (T 2 ) pulse sequences. Plots
of relaxation rate (T 121 and T 221 ) versus Fe concentration were
fitted as linear and the slopes evaluated as the relaxivities r 1
and r 2 , respectively.
Colloidal Stability
The undiluted samples were kept in closed bottles in the dark
at 4°C for at least 4 weeks. Colloidal stability against time was
studied, comparing water relaxation rates T 121 and T 221 of
different samples at 39°C and 10 MHz (triplicate runs), using
a Minispec Bruker spectrometer.
Two types of studies were carried out. For the first study,
called infield, the sample was diluted in 1 M TMAOH and kept
in the spectrometer magnet for at least 24 h; relaxation times
were measured every hour. For the second study, called out-
field, the particles were diluted in distilled water and relaxation
times were measured once a week for at least 4 weeks. Be-
tween the weekly measurements, the undiluted samples were
kept at 4°C.
Magnetic Susceptibility
Susceptibilities of liquid samples were measured at room
temperature on a Bartington MS2 susceptibilimeter (Barting-
476 BABES ET AL.

The following results were obtained with solutions aged 2

days, magnetically stabilized and electromagnetically filtered.

Influence of Media Composition

FIG. 1. Influence of hydroxide media composition on size and crystal
morphology. TMAOH particles (original magnification: 30,000). These parti-
cles show two distinct populations: the small particles are spheroids, and the
larger ones are well-faceted prismatic crystallites.
Hydroxide media. To study the influence of media com-
position on the formation of the particles, pure 1 M TMAOH
and a mixture of NH 3 and TMAOH media were used.
The images of the two preparations showed differences in
size and crystal morphology. TMAOH particles (Fig. 1)
showed two distinct populations: many small particles ('4
nm) are spheroids, whereas large particles are well-faceted
prismatic crystallites ('15 nm). TMAOH/NH 3 particles (Fig.
2) showed a single population, with a larger distribution and
shapes of smooth angulated spheroids. After magnetic filtra-
tion, the remaining populations of the TMAOH suspensions
were slightly larger than the TMAOH/NH 3 ones; both size
profiles followed a lognormal distribution (Table 1). In terms
of preparation yields, the TMAOH preparation was of 84.4 6
1.6% versus 46 6 2% for TMAOH/NH 3 ( p 5 0.0001).
The relaxivities obtained are summarized in Table 1. For
almost identical r 2 /r 1 ratios, TMAOH nanoparticles are more
effective than TMAOH/NH 3 nanoparticles.
For the study of the infield colloidal stability, the regression
coefficients were statistically nonsignificant and no evolution
of relaxation rates was observed for particles kept in the

ton, Oxford, England). Measurements were carried out on

freshly synthesized preparations once a week for at least 4
weeks.

Experimental Point
At least three preparations were analyzed for each studied
parameter and the results were averaged. A statistical analysis
was performed for each parameter, using a Mann–Whitney
u-test. This test, also called the rank sum test, is a nonpara-
metric test that compares two unpaired groups without any
assumption about their normal distribution functions. The key
result is a p value; if p is small, the populations have different
medians. Differences were considered nonsignificant for p .
0.05.

RESULTS

The influence of parameters such as media composition,

concentrations, injection fluxes, temperature, and oxygen on
size, relaxometric properties, and stability of the particles was
studied. Among these parameters, TMAOH concentration
(0.25–2 M) and synthesis temperature (20 –50°C) showed little
or no influence in our experimental setup (data not shown).
Only the difference of the sizes of the particles prepared at 20 FIG. 2. Influence of hydroxide media composition on size and crystal
and 65°C was statistically significant ( p ' 0.006), with lower morphology. TMAOH/NH 3 particles (original magnification: 30,000). These
relaxivities and sizes with increasing temperature. particles show a single population and shapes of smooth angulated spheroids.
 SYNTHESIS OF IRON OXIDE NANOCORES 477

TABLE 1
Influence of Two Different Media Compositions on the Mean Size, r 1, r 2, and r 2/r 1 Ratio of Particles

r 1 6 SD r 2 6 SD
Hydroxide media Composition Mean size (nm) (s 21 mM 21) (s 21 mM 21) r 2 /r 1 6 SD

TMAOH 1 M TMAOH 4.27 6 0.60 56.4 6 9.4 122 6 17.6 2.1 6 0.05
TMAOH/NH 3 1 M TMAOH/ 3.86 6 0.68 31.6 6 5.4 69.3 6 8.8 2.2 6 0.09
2 M NH 3

Note: TMAOH particles show a slightly higher mean size as well as more important relaxometric properties.

magnet for at least 24 h. No magnetically induced aggregation
seemed to occur during these 24 h.
For the study of the outfield stability, the weekly measure-
ments did not show significant differences of relaxation rates;
solutions were stable for at least 1 month.
Because of the more interesting practical properties of
“pure” TMAOH-based suspensions (higher relaxivity and
lower size), only this type of colloid was further developed.
These particles will be called standard particles in the follow-
ing.
Fe 21/Fe 31 ratio. The Fe 21/Fe 31 ratio varied between 0/10
and 8/10 M/M. No adequate particle formation was observed
with only Fe 21 or Fe 31 ions. Below a Fe 21/Fe 31 ratio of 0.3,
colloids precipitated very quickly after synthesis. For Fe 21/
Fe 31 $ 0.4, macroscopically stable colloids were obtained (.1
day). For ratios over 0.7, the suspension size did not allow
electromagnetic filtration.
Images of particles synthesized with different Fe 21/Fe 31
ratios showed the same morphological characteristics as the
standard TMAOH particles. The mean size increased with the
Fe 21/Fe 31 ratio (Table 2) as well as the proportion of large
particles (over 7 nm), whereas the preparation yield decreased
from 66.3 6 5.5% for Fe 21/Fe 31 5 0.4 to 50.2 6 5.3% for
Fe 21/Fe 31 5 0.6, becoming very low for a ratio of 0.7. No
significant differences were revealed by statistical analysis
between particles synthesized with Fe 21/Fe 31 ratios of 0.4 and
0.5 or for ratios of 0.6 and 0.7.
Relaxivities are shown in Table 2. Only particles synthe-
sized with ratios between 0.4 and 0.6 are effective enough to be
used as contrast agents. For particles synthesized with an
Fe 21/Fe 31 ratio of 0.7, the electromagnetic filtration gave rise
to a suspension with an iron concentration too low to allow the
NMR measurements.
Susceptibilities are presented in Fig. 3. The values increased
with increasing Fe 21/Fe 31 ratios, with significant differences
between preparations synthesized with Fe 21/Fe 31 ratios of 0.4
and 0.48 M/M, respectively.
Weekly measurements showed that the solution aging
entailed an increase of susceptibilities during the first 8 days
after the synthesis; no variations were observed after this
period.
Influence of Flux Rates
Iron flow. Particles were synthesized with Fe solution
flows from 1 to 5 ml/min, while the TMAOH solution flux was
constant at 500 ml/min. All the preparations were colloidally
stable after synthesis.
Images showed the same morphological profile as standard
particles. No significant variations in mean sizes (Table 3)
were observed for different flow rates, except for the prepara-
tion at 1 ml/min, which presents a higher mean (6.4 nm versus
a mean of 4.3 nm, p 5 0.0001) and a lower preparation
percentage (71.0 6 8.0% versus a mean of 85.6 6 0.8% for
standard preparation, p 5 0.04).
No variation of r 1 relaxivity with the increase of the Fe flow
was observed, whereas r 2 and hence the r 2 /r 1 ratios decreased
(Table 3).

TABLE 2
Influence of Fe 21/Fe 31 Ratio on the Mean Size, r 1, r 2, and r 2/r 1 Ratio of Particles

Fe 21/Fe 31 ratio (M/M) Mean size (nm) r 1 6 SD (s 21 mM 21) r 2 6 SD (s 21 mM 21) r 2 /r 1 6 SD

0.4 3.44 6 0.78 26.30 6 3.90 45.50 6 8.00 1.72 6 0.09

0.48 4.27 6 0.60 62.11 6 4.60 115.37 6 9.40 1.85 6 0.04
(TMAOH)
0.5 3.21 6 0.81 68.70 6 5.10 126.80 6 13.80 1.84 6 0.17
0.6 4.99 6 0.48 99.75 234.60 2.37
0.7 4.30 6 0.63 * * *

Note: Relaxivities and r 2 /r 1 ratios increase with the Fe 21/Fe 31 ratio.

* Not available.
478 BABES ET AL.

FIG. 3. Influence of Fe 21/Fe 31 ratio on magnetic susceptibilities of particles. Study of the magnetic behavior of preparations against time. Susceptibilities
increase with Fe 21/Fe 31 molar ratio. Independently of the Fe 21/Fe 31 ratio, an important increase of susceptibilities was noticed between 2 and 8 days, followed
after this period by a stability tray.

TMAOH flux. Colloids were synthesized with TMAOH
fluxes from 50 to 800 ml/min, while the Fe flow was held
constant (2 ml/min). All the preparations were colloidally
stable after synthesis.
Image analysis of all preparations showed the usual lognor-
mal morphological profiles. Mean sizes are presented in Fig. 4.
Although statistically significant, the lower median size of
TMAOH particles is not clearly understood. Except for the
particles synthesized at 250 ml/min, the mean size is higher
than the mean size of TMAOH particles. No important varia-
tions in the preparation yields were noticed, except for the flux
of 800 ml/min, which is slightly lower than the rest.
Relaxivities for different preparations are presented in Fig.
5. Both r 1 and r 2 relaxivities increased with TMAOH fluxes
until 400 ml/min. An optimum “plateau” was observed for both
r 1 and r 2 between 400 and 600 ml/min, followed by a decrease
of r 1 and r 2 values over 600 ml/min, the slightly lower values
at 500 ml/min being viewed as a consequence of the smaller
size. As r 2 /r 1 ratios are more efficient between 500 and 600
ml/min, these two fluxes were considered of interest.
Influence of Fe Concentration
The total Fe concentration varied between 12.93 and 116.4
mM. For [Fe] # 19.15 mM, colloids precipitated rapidly after
synthesis. All the other preparations were stable but after 3
weeks, a decrease in stability with increasing iron concentra-
tion was observed.
Image analysis of all preparations showed the usual mor-
phological profile, with an increase of the large population with
the concentration. For [Fe] $ 51.6 mM, the mean size was
slightly higher than for TMAOH particles (Table 4). Prepara-
tion yields were equivalent for all preparations, except for [Fe]
5 19.15 mM, which was lower than the general mean (67.2 6
4.6% versus ' 85%). For [Fe] $ 58.2 mM, the percentage
decreased slightly with the concentration increase.

TABLE 3
Influence of Fe Flow on Mean Size, r 1, r 2, and r 2/r 1 Ratio of Particles

Fe flow (ml/min) Mean size (nm) r 1 6 SD (s 21 mM 21) r 2 6 SD (s 21 mM 21) r 2 /r 1 6 SD

1 6.38 6 0.41 44.30 6 5.60 93.18 6 15.80 2.10 6 0.18

2 4.27 6 0.60 62.11 6 4.60 115.37 6 9.40 1.85 6 0.04
(TMAOH)
3 5.45 6 0.48 50.20 6 14.90 94.88 6 30.05 1.89 6 0.07
5 5.45 6 0.49 47.35 6 5.80 79.62 6 7.10 1.69 6 0.06

Note: The preparation synthesized with an Fe flow of 1 ml/min presents a significantly higher mean size. r 2 relaxivities and r 2 /r 1 ratios decrease with increasing
Fe flows.
 SYNTHESIS OF IRON OXIDE NANOCORES
FIG. 4. Influence of TMAOH flux on the mean size of particles. Bars are
standard deviations (n 5 3) (****, p , 0.0001; ***, p , 0.001; *, p ,
0.05; p estimated in reference of 500 ml/min flux). Except for the preparation
synthesized with a TMAOH flux of 250 ml/min, the other mean sizes are
higher than the TMAOH mean size.
Relaxivities for different preparations are presented in Table
4. Both r 1 and r 2 increased with Fe concentration, with the
increase more important for r 2 . For nearly constant r 2 /r 1 ratio
averages, variations of standard deviations were very large.
The optimum seems to be between 38.8 and 77.6 mM.
The susceptibilities are presented in Fig. 6. The values
increased with increasing Fe concentration. The differences for
preparations synthesized with [Fe] of 25.86 and 38.80 mM,
respectively, were significant.
The weekly susceptibility measurements showed an impor-
tant difference between the values measured after 2 and 8 days
of aging, respectively, with '50% of increase of the initial
values. No variations were observed after this period.
Influence of Oxygen
Syntheses were realized with solutions partly degassed in an
argon or nitrogen stream.
The same morphological profile was observed for all prep-
arations, but an increase in the proportion of angulous particles
was noticed for degassed preparations. Moreover, the black
precipitate observed after the magnetic deposition was much
more important in this case. Consequently, the preparation
yield decreased from 84.4% for standard particles to 75.5% for
degassed preparations. At the same time, an increase of the
mean size from 4.3 nm for standard particles to 5.5 nm for
degassed preparations was noticed.
The relaxometric properties are presented in Table 5. An
increase of nearly 50% for relaxivities r 1 and r 2 for equivalent
r 2 /r 1 ratios was noticed. If these particles are more efficient
than the standard preparations, they are less stable, too. T 1 and
T 2 measurements become impossible after 1 week (particles
were conserved under air).
479
DISCUSSION
The influence of factors intervening in the synthesis of new
superparamagnetic iron oxide–tetramethylammonium hydrox-
ide nanoparticles, such as the media composition (comparison
of alkaline media: TMAOH alone and a TMAOH/NH 3 mix-
ture; variation of the Fe 21/Fe 31 molar ratio), solution concen-
tration, temperature, and influence of oxygen, has been studied.
The synthesis in TMAOH alone entails the formation of
particles with important relaxivities for r 2 /r 1 ratios almost
identical to those of TMAOH/NH 3 particles, with a qualitative
difference in external shape and, probably, crystallinity. Thus,
the presence of particles of square form is a hint for the absence
of inhibition factors for the crystalline growth of the particles.
These factors could partially explain the shape of particles
obtained in the TMAOH/NH 3 media: the presence of NH 3 in
the synthesis media would tend to stabilize particles toward air
oxidation. In our case, the use of TMAOH alone as alkaline
media allowed the direct synthesis, at one time, of ready-to-use
stable colloidal solutions, with better preparation ratios than
those obtained using the mixture of the two alkalines. More-
over, the square shape of the TMAOH particles has been
described in the literature. It is often observed for particles
synthesized in media presenting an OH 2 excess (17) and would
be due to a very low TMA adsorption on the surface.
Among the other studied factors, the concentration of the
base does not seem to influence greatly the nanoparticle syn-
thesis. The particle sizes decrease strongly with increasing pH
and ionic force of the precipitation media (6, 14). In our case,
the synthesis takes place in an area where the ionic force is
FIG. 5. Influence of TMAOH flux on relaxometric properties of particles.
For low fluxes (,200 ml/min), relaxivities and r 2 /r 1 ratios tend to decrease.
For fluxes higher than 200 ml/min, r 1 , r 2 , and r 2 /r 1 ratios increase until 400
ml/min and then become stable for flux values above 400 ml/min.
480 BABES ET AL.

TABLE 4
Influence of [Fe] on Mean Size, r 1, r 2, and r 2/r 1 Ratio of Particles

[Fe] (mM) Mean size (nm) r 1 6 SD (s 21 mM 21) r 2 6 SD (s 21 mM 21) r 2 /r 1 6 SD

19.15 * 26.07 6 8.40 52.51 6 18.90 1.99 6 0.17

25.86 * 39.16 6 4.60 72.46 6 7.80 1.85 6 0.04
38.80 4.27 6 0.60 62.11 6 4.60 115.37 6 9.40 1.85 6 0.04
(TMAOH)
51.60 5.77 6 0.41 72.44 6 3.80 137.60 6 3.40 1.90 6 0.12
58.20 5.33 6 0.46 65.09 6 3.40 119.21 6 9.10 1.89 6 0.07
77.60 5.48 6 0.49 84.16 6 10.30 166.94 6 20.90 1.99 6 0.18
116.40 5.52 6 0.48 80.36 6 4.70 149.12 6 17.70 1.85 6 0.11

Note: The mean sizes are equivalent and slightly higher than that of the preparation synthesized with [Fe] 5 38.8 mM. Relaxivities (and more particularly
r 2 ) increase with increasing [Fe] for almost identical r 2 /r 1 ratios.
* Not available.

between 1 and 3 M and the pH is higher than 13, inducing a
quasi-stability of sizes, explained by the highly charge-induced
superficial energy fall.
Temperature influences the synthesis little, with a small
diminution in sizes, as well as r 1 and r 2 relaxivities and r 2 /r 1
ratios, as reported in the literature (13, 18).
Hence, at constant temperature, the nanoparticle size de-
pends on four parameters: pH, ionic force, nature of the ions,
and aging time of suspensions (14). Due to the very high pH
($12), no molecular exchange analogous to Oswald ripening
occurs during the storage (14).
In our setup, the most important factor is the Fe 21/Fe 31
molar ratio: the core formation occurs only in the presence of
the Fe 21/Fe 31 redox pair in a molar ratio close to 0.5. The
presence of only one ionic kind entails the formation of unsta-
ble colloidal solutions. Using only Fe 31 leads to preparations
evoking paramagnetic iron (r 2 /r 1 ' 1). The decrease of the
Fe 21/Fe 31 ratio induces the appearance of an initial deposit,
and for values of this ratio above 0.6, the average particle size
increases, too. These results are corroborated by literature data
(4, 6, 15).
The second most important factor influencing the synthesis
is the iron concentration, with an optimum between 39 and 78
mM. We have noticed a decrease of stability with increasing
concentration but also a plateau of the magnetic properties for
[Fe] above 38.8 mM. Thus, it seems interesting to work with
[Fe] in the plateau region, which allows the choice of a contrast
agent concentration adapted to MRI needs (increasing relax-
ivities with concentration).
Injection flux rates do not seem to have a preponderant

FIG. 6. Influence of [Fe] on magnetic susceptibilities of particles. Study of the magnetic behavior of preparations against time. For [Fe] , 38.8 mM,
susceptibilities increase with Fe concentration. A tray of stability is noticed for particles synthesized with [Fe] . 38.8 mM. Independently of the initial
concentration, an important increase of susceptibilities was noticed between 2 and 8 days, followed after this period by a stability tray.
 SYNTHESIS OF IRON OXIDE NANOCORES
TABLE 5
Example of Oxygen Influence on Relaxometric Properties of
Particles Synthesized in Different Media
Preparation r 1 (s 21 mM 21) r 2 (s 21 mM 21)
TMAOH/Ar 90.56 187.83
TMAOH/N 2 89.75 166.98
TMAOH/air 64.51 126.94
Note: Although r 2 /r 1 ratios are equivalent, relaxivities are more important
for preparations synthesized with partially degased solutions.
influence on the nanoparticle synthesis. It is nevertheless pref-
erable to work in an “ideally shaken” media, with values of Fe
and TMAOH fluxes belonging to the observed trays of opti-
mum, and to avoid extreme values of the ranges studied (name-
ly, 1 and 5 ml/min of iron flow and fluxes of TMAOH less than
or equal to 250 ml/min and greater than 600 ml/min). Con-
cerning the Fe flow, the best results were obtained with a value
of 2 ml/min. For the TMAOH flux, the optimum was observed
for values between 400 and 600 ml/min, with the best r 2 /r 1
ratios between 500 and 600 ml/min.
Synthesis under Ar gas bubbling limits the oxidation by air
oxygen. Proceeding in this way increases the relaxivities and
r 2 /r 1 ratios but also the colloidal and relaxometric instabilities.
This phenomenon is independent of the conservation mode
(under air or argon). The size profile is slightly modified, with
an increase of the average size, but an important iron deposit
appears. The complex phenomena occurring in the Fe 21/Fe 31
electronic exchange (6) were further investigated.
The exact nature of the limiting phenomena during particle
formation is not perfectly established in our setup. The drop of
the solubility product in alkaline media leads to the formation
of numerous nuclei during the reciprocal diffusion of the basic
and ferrous and ferric ions. Diffusion and adsorption of ferro–
ferric ions after this nucleation step are certainly limited by
weak solubility; similarly, the Oswald ripening is probably
minor in our system, because of the high pH values.
Taking these preliminary remarks into account, it seems
possible that particle formation, and therefore the size of the
particles, is governed by a phenomenon of fragmentation of the
acidic flow in the basic surrounding media, comparable to a
process of microemulsification or the formation of polymeric
organic particles (19). Following this theory, to get a 5-nm
particle, a cubic aqueous volume of approximately 86 nm 3 is
necessary when an iron concentration of 38.8 mM is assumed.
An isolated droplet of this size is probable if one assumes a
solvent pair of low interfacial tension. Taking into account the
high molarity of the basic media, the time of interdiffusion of
the solutions would be about 5 3 10 26 s. For the given fluxes
of the alkaline and iron solutions and the diameters of the
catheters, this corresponds to a displacement of the droplet of
approximately 50 mm during this time. This is a possible
explanation for the weak influence of the modification of the
481
flux rates on the particle size during synthesis. This means
(taking into account that the mixing is not perfected instanta-
neously) that each “microdroplet” of one of the solutions
serves as an autonomous reactor for the particle synthesis, as
r 2 /r 1
do microemulsion objects. In these cases, phenomena observed
2.07 under extreme conditions (very high or very low flow values
1.86 with an increase or a decrease in particle size, respectively)
1.96 could be interpreted, on one hand, as a diffusional limitation to
the mouthpiece of the catheter (lower flows inducing total
obstruction) and, on the other hand, as an effect of turbulences
intervening at this mouthpiece that would “artificially” increase
the segmentation of the liquid column for very high flow rates.
The aging studies showed an increase of the susceptibility
values during the first 8 days of aging, without any modifica-
tions after this period. It is well known that magnetic properties
depend on the grain size, the degree of anisotropy, the micro-
structure, and the presence of oxidation elements (13). Since
we did not notice any variation of mean sizes and relaxivities
during 1 month, we consider that the susceptibility variations
are related to a structural modification of the crystal during the
first 8 days after the synthesis. In our case, the iron oxide
nucleus seems to be composed of a mixture of magnetite and
maghemite, with small differences of magnetic properties.
After the synthesis, these two components could be in the form
of small domains responsible for the observed relaxometric
properties, but these domains are subjected to rearrangements.
The crystal seems to reach its final stabilized structure 8 days
after synthesis, and this structure is responsible for the ob-
served magnetic properties.
CONCLUSION
This work describes a formulation of uncoated superpara-
magnetic iron oxide nanoparticles. This synthesis is reproduc-
ible over a wide range and is independent of factors such as
temperature and hydroxide concentration. Furthermore, the
control of the initial iron concentration allows the synthesis of
nanoparticles of equivalent sizes but different relaxivities. In-
dependently of the synthesis conditions, uncoated nanopar-
ticles always have a size under 10 nm and the solutions are
colloidally stable with time. Thus, the advantage of this method
is that it allows the decoupling of the synthesis of the core and
its coating. Consequently, it allows the study of various bio-
distributions with coatings which have to be applied under
well-defined conditions (concentration, pH, surface/polymer
ratio, etc.).
To study their dynamic biodistributions, pharmacokinetics,
and complement activation, these particles have to be covered
by different polymers for animal experimentation.
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