Colloids and Surfaces B: Biointerfaces 51 (2006) 101–106

Synthesis and characterization of iron oxide/polymer composite

nanoparticles with pendent functional groups夽
Shupeng Liu a,c,1 , Xiaohui Wei b,1 , Maoquan Chu b , Jinliang Peng a , Yuhong Xu b,∗
a Department of Biomedical Engineering, College of Life Science and Biotechnology, Shanghai Jiao Tong University,
1954 Hua-Shan Road, Shanghai 200030, PR China
b School of Pharmacy, Shanghai Jiao Tong University, 1954 Hua-Shan Road, Shanghai 200030, PR China
c Institute of Biomedical Engineering, Shanghai University, 149 Yan-Chang Road, Shanghai 200072, PR China

Received 3 February 2006; received in revised form 27 April 2006; accepted 30 May 2006
Available online 6 June 2006

Abstract
We describe in this paper an approach to synthesize superparamagnetic iron oxide nanoparticles in the presence of polymerized lactic acid. The
resulted particles consisted of clusters of iron oxide monocrystals, embedded inside the polymer chains. The composite particles synthesized in situ
were highly dispersible in aqueous solution with good stability. X-ray diffraction and magnetometer data all confirmed the crystalline structure and
super-paramagnetic property of the particles. They exhibited narrow size distribution with hydrodynamic diameters close to 80 nm. In addition, the
particles were shown to have abundant surface carboxyl groups, which can be used to conjugate various biomolecules. Such a preparation would
be especially useful for developing target specific MRI contrast agents or drug delivery vehicles.
© 2006 Published by Elsevier B.V.

Keywords: Iron oxide; Superparamagnetic; Nanoparticles; Contrast agent

1. Introduction are also very useful in bioseparation applications such as cell

Paramagnetic particles in nanosizes possess many advanta-
geous properties. Particles such as crosslinked iron oxide (CLIO)
[1–3], ultrasmall superparamagnetic iron oxide (USPIO) [4–8],
and monocrystalline iron oxide nanoparticles (MIONs) [9–11]
had all been developed as imaging agents in magnetic resonance
imaging (MRI). After administration in vivo, these particles
would distribute through out the body following specific patterns
which can be detected using MRI systems. Some of the particles
are likely to be taken up by macrophages and immune cells, and
can be used to image lymph nodes and inflammatory tissues.
In other studies, specific ligands were attached on the particle
surface, in order to image the localization pattern of the target
molecules [2]. Further more, paramagnetic micro/nanoparticles
夽 This work was supported by the National Natural Science Foundation of
China, No. 30470513/C03031801 and by Shanghai Nanotechnology Promotion
Center, No. 0352nm103.
∗ Corresponding author. Tel.: +86 21 34202739; fax: +86 21 34202740.
E-mail address: yhxu@sjtu.edu.cn (Y. Xu).
1 These authors contributed equally to this study.
sorting, and protein or DNA purification, etc. [12,13], which
also requires the attachment of specific bioactive molecules on
the surfaces.
There have been various methods developed for the prepa-
ration of paramagnetic nanoparticles [14]. The most commonly
used protocol involves co-precipitation of ferrous and ferric ions
in basic solutions. Polymers such as dextran, polyvinyl alcohol
(PVA), and DEAE-starch were added to coat the particles for
better stability, before or after the formation of iron oxide par-
ticles [15,16]. However, the loose association of polymers may
fall off after injected into body. An improved approach is to initi-
ate the iron oxide co-precipitation process in polymer solutions
such as carboxylated dendrimer [17]. In these cases the poly-
mers were thought to provide templates for mineral nucleation
and to regulate particle growth.
For molecular recognition, the particles surface would need
to be modified by covalently attaching ligands. In order to
introduce surface reactive groups, carboxymethylated dextran
were commonly used to coat the particles surface. However,
the coating as well as the conjugated ligands may still be
loose.

0927-7765/$ – see front matter © 2006 Published by Elsevier B.V.

doi:10.1016/j.colsurfb.2006.05.023
102 S. Liu et al. / Colloids and Surfaces B: Biointerfaces 51 (2006) 101–106
With all these considerations in mind, we developed a new
and simple method of preparing iron oxide/polymer compos-
ite nanoparticles with narrow size distribution, strong polymer
association and abundant surface reactive groups. These parti-
cles may be of potential use in many biological applications that
requires target specificity and surperparamagnetism.
2. Materials and methods
2.1. Materials
The polymerized lactic acid was synthesized and kindly
provided by Ms. Suping Qian from Shanghai Biopolymer
Co. They were photochemically synthesized by dissolving
l-lactic acid in ethyl acetate, mixing with trace amount of
benzophenone and illuminating with a 500 W high-voltage
mercury lamp. The resulted polymers were extracted and
purified in ethyl acetate. Other reagents including NaOH,
K2 CO3 , FeCl2 ·4H2 O and FeCl3 ·6H2 O were bought from
Shanghai Chemistry Reagent Company (Shanghai, China).
4-Bromomethyl-7-methoxycoumarin was bought from Acros
Organics (New Jersey, USA). Centriplus® ultrafiltion tube
10000 MWCO was from Millipore (BedFord, MA, USA). The
water used in the experiments was prepared by Milli-Q water
purification system (Millipore, BedFord, MA, USA).
2.2. Preparation of the iron oxide/polymer composite
nanoparticles
A 730 mg of FeCl3 ·6H2 O and 300 mg of FeCl2 ·4H2 O were
dissolved in 20 ml of deaired water with fixed Fe2+ to Fe3+ molar
ratio of 1:1.8. The polymerized lactic acids were then added
to reach specific polymer concentrations. To initiate iron oxide
formation, NaOH solution (5 M) or 28% NH3 ·H2 O was used to
basify the solution by drop-wise addition until the pH of the sys-
tem reached between 9.0 and 11.0 and remained stable for a half
hour. The reaction was carried out at 2–4 ◦ C with vigorous mix-
ing under nitrogen. Afterwards the solution was heated to 80 ◦ C
for 60 min for crystalline maturation. Magnetic nanoparticles
were purified and harvested by ultrafiltration with Centriplus®
tube after ultracentrifugation at 10,000 rpm for 20 min. They
were then lyophilized.
2.3. Transmission electron microscope (TEM) study
Drops of the diluted solutions of the preparations were
deposited on carbon films supported by copper grids and air-
dried in ventilation cabinet. TEM morphology study was per-
formed using a Philips CM120 TEM.
2.4. Particle size analysis
The particle size distribution of the magnetic nanoparticles
was determined by photon correlation spectroscopy (Zetasizer
3000HSA , Malvern, UK). The average hydrodynamic diameter
and polydispersity index were evaluated.
2.5. XRD study
The lyophilized magnetic nanoparticles powder was charac-
terized by XRD on a Bruker-Axs D8 Advance X-ray diffrac-
tometer (BRUKER, Germany) employing Cu-K␣ radiation at
40 kv and 40 mA.
2.6. Magnetometry measurement
Magnetic characterization of the nanoparticles was carried
out in a JDM-13 vibrating sample magnetometer (Jilin Univer-
sity, China) at room temperature with maximum applied field of
1.8 T.
2.7. FT-IR characterization
The lyophilized magnetic nanoparticles were grounded with
KBr for FT-IR measurement by EQUNOX 55 Fourier Trans-
formed Infrared Spectrometer (Bruker, Germany). Samples of
blank iron oxide particles prepared parallel under the same con-
dition but without the polymer were utilized as the control.
2.8. Characterization of the surface reactive carboxyl
groups
To demonstrate and characterize surface carboxyl groups
on the magnetic particle, we used 4-bromomethyl-7-
methoxycoumarin (BrMmC) as the probe [18]. The haloid group
of BrMmC can react with carboxyl group using 18-crown-6
ether and K2 CO3 as catalysts to form acylamide, which can be
easily detected by fluorescence spectroscopy. The lyophilized
nanoparticles were dissolved in anhydrous acetone in the
concentration of 10 mg/ml. A 100 mg potassium carbonate was
added into 400 ␮l of the solution for basification. Then 400 ␮l
of 2.5 mg/mL BrMmC (Acros Organics, New Jersey, USA) and
200 ␮l of 10 mg/mL 18-crown-6 were added in. The mixtures
were kept with gentle shaking at room temperature (25 ◦ C)
for 1 h, and then separated by applying magnetic fields. The
extracted magnetic nanoparticles were washed five times using
water. Fluorescence analysis was performed on a fluorescence
spectrophotometer (F-2500, Hitachi, Japan).
3. Results
3.1. Preparation of the iron oxide/polymer nanoparticles
The preparation protocol of the iron oxide particles was opti-
mized by varying the polymer concentration, alkalization rate
and the end point pH. The reaction solution usually started with
a yellow/green color and gradually turned to orange and then to
dark green with the addition of alkaline ions. After heating the
solution to 80 ◦ C for crystalline maturation, its color then usually
turned to dark red or black. The resulted nanoparticles were puri-
fied by ultrafiltration with Centriplus® tube (10000 MWCO).
The size distribution and stability of the resulted particles were
evaluated. The most stable and well-dispersed composite par-
ticles were obtained under the following conditions: 80 mg/ml
 S. Liu et al. / Colloids and Surfaces B: Biointerfaces 51 (2006) 101–106 103

Fig. 1. TEM images of the resulted composite nanoparticles (A) and Fe3 O4 without polymer (B).

polymer concentration; 35 mg/ml Fe3+ concentration; and end

point pH at 10.

3.2. Morphology of the composite particles

The resulted composite particles were examined under TEM

and were seen as clusters of a few small iron oxide dots (Fig. 1A).
Each individual dot was about 10 nm in diameter. But they clus-
tered together to form dispersed particles at about 30–50 nm.
As a control, iron oxide synthesized in the absence of the poly-
mer was shown in Fig. 1B. The particles aggregated and usually
precipitated during preparation.
Dynamic light scattering measurements of the particles
reported the hydrodynamic diameters of the composite particles
were about 60–90 nm (Fig. 2). The numbers were a bit bigger
than what we observed under TEM, suggesting the presence of
associated bulky polymer chains although they were not visible
under TEM.

3.3. Crystalline structure and magnetic properties of the

nanoparticles

The X-ray diffraction pattern of the dried magnetic nanopar-

ticles was shown in Fig. 3. Standard Fe3 O4 crystals in inverse
cubic spinal structure have six diffraction peaks: {2 2 0}, {3 1 1},
Fig. 2. The PCS of magnetic nanoparticles (data based on light intensity anal-
{4 0 0}, {4 2 2}, {5 1 1} and {4 4 0}, as shown by red spikes in ysis).
Fig. 3. The positions and relative intensities of XRD peaks of
the magnetic nanoparticle were consistent with the standard pat-
tern, except for the broadening of the peaks. The broadening of
the peaks was probably due to the matrix constrain of the nano-
sized particles. The calculated mean size of the monocrystals
was about 12 nm.
The magnetization curve of the nanoparticles confirmed that
the composite nanoparticles indeed have superparamagnetic
properties (Fig. 4). The saturation magnetization was about
50 emu/g Fe, and the coercive force (jHc) was 0 Oersteds (Oe).
These data were comparable to what had been reported in the
literature for iron oxide nanoparticles synthesized using dextran
or PVA. Fig. 3. XRD pattern of resulted composite nanoparticle.
104 S. Liu et al. / Colloids and Surfaces B: Biointerfaces 51 (2006) 101–106
Fig. 4. Magnetization curve of magnetic composite nanoparticles.
3.4. FT-IR study of the composite particles
The magnetic nanoparticles were purified by centrifugation
at 10,000 rpm for 20 min to remove the excessive polymer, and
washed with water for two more times. The purified nanopar-
ticles suspension was lyophilized and examined using FT-IR
spectroscopy. As shown in Fig. 5, there was an intense and broad
band in the region of 3400 cm−1 , which was assigned as the O–H
stretching vibration. The peak at 1622 cm−1 corresponded to
C O stretching vibration. These two peaks indicated that the
composite nanoparticles contained carboxyl and/or hydroxyl
groups, presumably on the polymer chain. The weak peak at
1378 cm−1 may be due to ␦CH.
3.5. Characterization of the surface reactive carboxyl
groups
The free carboxyl groups hanging from the polymer chains
in the composite particles can be used to conjugate bioactive
molecules. To characterize the reactivity of these functional
Fig. 5. FT-IR spectra of the magnetic composite nanoparticles.
Fig. 6. Room temperature fluorescence spectra of the nanoparticles conjugated
with BrMmC. (a) The fluorescence spectra of BrMmC conjugated nanoparticle;
(b) the fluorescence spectra of the BrMmC mixture with iron oxide particle pre-
pared without polymer; (c) the fluorescence spectra of BrMmC mixture with iron
oxide/polymer composite particle, but without the catalysts. Excitation wave-
length: 320 nm.
groups, BrMmC was used to specifically react with them and
the resulted fluorescence was recorded. Fig. 6 showed the fluo-
rescence spectrum of BrMmC before and after reaction with the
nanoparticles. The fluorescence peak at 400 nm confirmed the
formation of the covalent bond. On the other hand, the spectra of
BrMmC alone and the BrMmC–nanoparticles mixture without
the catalysts showed no peak at 400 nm.
4. Discussion
Superparamagnetic nanoparticles have long been of sig-
nificance for biological application and medical imaging.
SPIO and USPIO have been approved for clinical use as MRI
contrast agents based on their higher magnetic susceptibility
than that of conventionally used gadolinium-based agents.
Accordingly, many different preparation methods have been
developed. One of the most common protocols is the ferric
and ferrous ions co-precipitation method. In order to achieve
good dispersity and to maintain size stability of the resulted
magnetic particles, hydrophilic polymers such as dextran and
PVA are usually added to act as coating agents or stabilizing
matrixes. The physical association of the polymers with iron
oxide particles is thought to be due to hydrogen bonds between
the polymer hydroxyl groups and the particle surface oxide
and hydroxyl groups [19]. Similarly, proteins and polymers
containing free carboxyl groups were used as templates for in
situ synthesis of nanoparticles [20]. It was suggested that free
carboxyl groups on these structures can act as mineral nucle-
ation site, and therefore provided templates for the growth of
nanoparticles.
Based on these studies, we found a new poly-lactic acid
matrix that can be used for efficient synthesis of iron oxide
nanoparticles. The poly-lactic acid chains were biodegradable
and considered to have high biocompatibility. The unique photo-
 S. Liu et al. / Colloids and Surfaces B: Biointerfaces 51 (2006) 101–106
chemical polymerization protocol resulted in a polymer structure
which is highly branched and significant different from the con-
ventional linear Poly-lactic acid structure. Both pH titration
studies and FT-IR spectroscopy study of the polymer confirmed
the presence of abundant pendent carboxyl group in the structure
[21]. We hypothesize that such a matrix would not only serve as
templates for the formation of iron oxide crystals but also pro-
vide a scaffold for maintaining the particle stability in solution.
During the co-precipitation process, some of the pedant carboxyl
groups may form strong chelating bonds with ferric or ferrous
ions and provide the nucleation point for iron oxide formation.
At the same time, the highly branched polymer chains may limit
the rapid growth of the crystals and blocked the aggregation
of different crystals. Indeed, based on the TEM examination,
the particles were consisted of clusters of tiny distinctive dots
with sizes of a few nanometers. We think these little dots were
monocrystalline iron oxide particles, embedded inside polymer
matrixes. The polymer chains were not visible under TEM, but
they can be estimated by the general morphology and arrange-
ment of iron oxide clusters. Therefore, although some of the
monocrystalline spheres were closely packed together, they were
still distinctively separated with clear boundaries.
In addition, the polymer chains would also help to stabi-
lize the whole particle and provide great aqueous solubility.
Compared to the loose dextran coating after iron oxide co-
precipitation, the poly-lactic acid matrix with free carboxyl
groups became complexed with iron oxide nanocrystals dur-
ing the process of synthesis. Therefore there was much tighter
association, and the resulted nanoparticles were more stable.
Furthermore, we demonstrated that there were still reac-
tive carboxyl groups that can be used to conjugate bioactive
molecules on the particle surfaces. Such surface modifications
could make specific in vivo detection or imaging of biomolecules
or molecular functions possible. There have been quite a few
interesting applications developed in literature. Schellenberger
et al. had reported the attachment of Annexin V to CLIO surfaces
for MRI imaging of apoptotic cells [2]. Funovics et al. conju-
gated antibodies against Her2/neu for the imaging of Her2/neu
positive tumor cells [9]. Therefore, we believe the abundant
hydroxyl groups available on our magnetic composite particles
would be very useful in further studies to develop target specific
contrast agents or drug delivery vehicles.
The preparation method of the iron oxide/polymer compos-
ite nanoparticle is quite simple and economical. We found there
were two important factors affecting the resulted size and sta-
bility of the nanoparticles: polymer concentration and reaction
pH. The stabilization effect of the polymer is clearly concentra-
tion dependent. For 37 mg/ml Fe3+ ion concentration, at least
50 mg/ml polymer concentration was required to maintain the
stability of the iron oxide nanocrystals. Without the polymer or
with a polymer concentration less than 50 mg/ml, the particles
were seen aggregated and precipitated. The higher the polymer
concentration, the smaller the particles, until the polymer con-
centration approached 400 mg/ml when no more particles could
form. In addition, the reaction pH was also important, since it
may affect the kinetics of nucleation and crystal growth. When
the pH was lower than 8, there was no solid particles formed.
105
When the pH approached 12, evident precipitation would result.
In order to obtain nanosized stable magnetic composite particle,
we find the reaction pH of 10 to be the optimal.
In summary, the novel polymer matrix structure provided a
nice template for the synthesis and stabilization of iron oxide
nanoparticles. The resulted paramagnetic composite particles
were well dispersed in water, with good stability over time, and
have the potential to be conjugated with bioactive molecules for
interesting applications.
Acknowledgement
Thanks to Professor Ren Jicun in Shanghai Jiao Tong Uni-
versity for helpful discussion.
References
[1] P. Wunderbaldinger, L. Josephson, R. Weissleder, Crosslinked iron
oxides (CLIO): a new platform for the development of targeted MR
contrast agents, Acad. Radiol. 9 (Suppl. 2) (2002) S304–S306.
[2] E.A. Schellenberger, A.J. Bogdanov, D. Hogemann, J. Tait, R.
Weissleder, L. Josephson, Annexin V–CLIO: a nanoparticle for detecting
apoptosis by MRI, Mol. Imaging 1 (2) (2002) 102–107.
[3] M.F. Kircher, J.R. Allport, E.E. Graves, V. Love, L. Josephson, A.H.
Lichtman, R. Weissleder, In vivo high resolution three-dimensional
imaging of antigen-specific cytotoxic T-lymphocyte trafficking to tumors,
Cancer Res. 63 (20) (2003) 6838–6846.
[4] M.E. Kooi, V.C. Cappendijk, K.B. Cleutjens, A.G. Kessels, P.J. Kit-
slaar, M. Borgers, P.M. Frederik, M.J. Daemen, J.M. van Engelshoven,
Accumulation of ultrasmall superparamagnetic particles of iron oxide
in human atherosclerotic plaques can be detected by in vivo
magnetic resonance imaging, Circulation 107 (19) (2003) 2453–
2458.
[5] T.M. Keller, S.C. Michel, J. Frohlich, D. Fink, R. Caduff, B. Marincek,
R.A. Kubik-Huch, USPIO-enhanced MRI for preoperative staging of
gynecological pelvic tumors: preliminary results, Eur. Radiol. 14 (6)
(2004) 937–944.
[6] M.G. Harisinghani, W.T. Dixon, M.A. Saksena, E. Brachtel, D.J. Blezek,
P.J. Dhawale, M. Torabi, P.F. Hahn, MR lymphangiography: imaging
strategies to optimize the imaging of lymph nodes with ferumoxtran-10,
Radiographics 24 (3) (2004) 867–878.
[7] C. Corot, K.G. Petry, R. Trivedi, A. Saleh, C. Jonkmanns, J.F. Le Bas,
E. Blezer, M. Rausch, B. Brochet, P. Foster-Gareau, D. Baleriaux, S.
Gaillard, V. Dousset, Macrophage imaging in central nervous system
and in carotid atherosclerotic plaque using ultrasmall superparamagnetic
iron oxide in magnetic resonance imaging, Invest. Radiol. 39 (10) (2004)
619–625.
[8] M. Schroeter, A. Saleh, D. Wiedermann, M. Hoehn, S. Jander, Histo-
chemical detection of ultrasmall superparamagnetic iron oxide (USPIO)
contrast medium uptake in experimental brain ischemia, Magn. Reson.
Med. 52 (2) (2004) 403–406.
[9] M.A. Funovics, B. Kapeller, C. Hoeller, H.S. Su, R. Kunstfeld, S. Puig,
K. Macfelda, MR imaging of the her2/neu and 9.2.27 tumor antigens
using immunospecific contrast agents, Magn. Reson. Imaging 22 (6)
(2004) 843–850.
[10] M.H. Krause, K.K. Kwong, E.S. Gragoudas, L.H. Young, MRI of
blood volume with superparamagnetic iron in choroidal melanoma
treated with thermotherapy, Magn. Reson. Imaging 22 (6) (2004) 779–
787.
[11] E.X. Wu, H. Tang, K.K. Wong, J. Wang, Mapping cyclic change of
regional myocardial blood volume using steady-state susceptibility effect
of iron oxide nanoparticles, J. Magn. Reson. Imaging 19 (1) (2004)
50–58.
[12] T. Lea, F. Vartdal, K. Nustad, S. Funderud, A. Berge, T. Ellingsen, R.
Schmid, P. Stenstad, J. Ugelstad, Monosized, magnetic polymer parti-
106 S. Liu et al. / Colloids and Surfaces B: Biointerfaces 51 (2006) 101–106
cles: their use in separation of cells and subcellular components, and in
the study of lymphocyte function in vitro, J. Mol. Recogn. 1 (1) (1988)
9–18.
[13] B.I. Haukanes, C. Kvam, Application of magnetic beads in bioassays,
Biotechnology (NY) 11 (1) (1993) 60–63.
[14] S. Mornet, S. Vasseur, F. Grasset, E. Duguet, Magnetic nanoparticle
design for medical diagnosis and therapy, J. Mater. Chem. 14 (2004)
2161–2175.
[15] J. Lee, T. Isobe, M. Senna, Preparation of ultrafine Fe3 O4 particles by
precipitation in the presence of PVA at high pH, J. Coll. Interf. Sci. 177
(1996) 490–494.
[16] C. Bergemann, D. Muller-Schulte, J. Oster, L.a. Brassard, A.S. Lubbe,
Magnetic ion-exchange nano- and microparticles for medical biochem-
ical and molecular biological applications, J. Magn. Magn. Mater. 194
(1999) 45–52.
[17] E. Strable, J.W.M. Bulte, B. Moskowitz, K. Vivekanandan, M. Allen, T.
Douglas, Synthesis and characterization of soluble iron oxide-dendrimer
composites, Chem. Mater. 13 (2001) 2201–2209.
[18] J.C. Ren, A. Ulvik, H. Refsum, P.M. Magne Ueland, Uracil in human
DNA from subjects with normal and impaired folate status as determined
by high-performance liquid chromatography–tandem mass spectrometry,
Anal. Chem. 74 (1) (2002) 295–299.
[19] W.J. Chu, Surface properties of superparamagnetic iron oxide MR con-
trast agents: ferumoxides, ferumoxtran, ferumoxsil, Magn. Reson. Imag-
ing 13 (5) (1995) 675–691.
[20] M.K. Ryan, L. Chester, C.C. Daniel, O.S. Morley, R.N. Rajesh, Engi-
neered protein cages for nanomaterial synthesis, J. Am. Chem. Soc. 126
(41) (2004) 13282–13286.
[21] S.P. Qian, Y.H. Xu, A polymerized lactic acid and the preparation
method, Chinese Patent Application (2003) CN03115760.2.