ticles coated with transferrin are not taken up by cells in this particular case, and demonstrate an increase of
cell number.
8
SPION conjugated with antibodies, en- zymes, and proteins have also been tested to specific
targeted cells as DDS.
9
However, there are several significant demands which should be considered for in
vivo applications. The particle size of uncoated SPION has to be less than 20 nm and may not exceed 50 nm
after coating due to the limitation of extra-cellular space and biological capillary dimensions. The large surface
area and specific magnetic properties of SPION may cause formation of clusters before they reach the target
cell. Therefore, the colloidal stability of SPION is one of the important parameters to consider for biomedical
applications. Several materials have been introduced as coating agents to improve colloidal stability of the
ferrofluid, namely BSA, starch, polyethylene glycol PEG, and polyvinyl alcohol PVA.
2,3,10
However, the stabilization mechanism after modifying the surface of
SPION has not yet been intensively studied by consid- ering the required factors for in vivo and in vitro
applications. Currently, several types of SPION-like magnetic particles are commercially available, but their
properties do not always satisfy the requirements for biomedical applications, such as monodispersed particle,
intrinsic magnetization, and surface functionality. BSA was selected from among several types of protein
molecules for immobilization on the surface of SPION. The special functional groups are required on the
surface of the SPION to covalently attach the BSA molecules. Because the SPION do not have such func-
tional groups, they must be introduced. The phagocytic uptake of SPION has been investigated by using surface-
modified SPION with butylamine and ethanolamine.
11
Recently, SPION functionalized with different numbers of amine groups have been tested for DNA extraction
processes.
12
Aspartic and glutamic acids, nonessential amine acids, are two of the most promising candidates to
introduce the functional amine groups on the surface or SPION for in vivo applications. Aspartic acid is
reported as a helpful chemical to control mental capaci- ties and is essential in the urea cycle for proper
elimination of waste products from dietary protein, whereas glutamic acid is significant in the metabolism
of sugars and fats, and aids in transportation of potas- sium across the blood-brain barrier BBB. Recently,
the adsorption of aspartic and glutamic acids on maghemite nanoparticles has been demonstrated.
13
These particles also show a high colloidal stability in physiological media.
In this study, an attempt to simplify and optimize the process of SPION surface modification with amine
groups is mainly focused for second functionalization with biocompatible substances. The synthesis of SPION,
introduction of amine groups via silanization with APTMS, and chemisorption of LAA on the surface of
SPION are briefly summarized. The binding capacity is estimated on the basis of magnetic measurements as
well as thermal and chemical analyses. Furthermore, BSA-APTMS coated SPION were incubated with hu-
man dermal fibroblasts in vitro to investigate the cell response. Light microscopy was used to visualize cell
morphology. In addition, fluorescent observations of the cell cytoskeleton and intracellular clathrin localization
have also been evaluated.
2. Experimental Section
Ferric chloride hexahydrate FeCl
3
‚6H
2
O 99, ferrous chloride tetrahydrate FeCl
2
‚4H
2
O 99, sodium hydroxide 0.5 M in water, hydrochloric acid diluted in water from HCl
37, sulfuric acid 96, nitric acid 65, hydrogen per- oxide 30, 3-aminepropyltrimethoxysilane APTMS, 1-ethyl-
3-3-dimethylaminepropyl carbodiimide hydrochloride EDC, N
-hydroxysuccinimide NHS, bovine serum albumin BSA, sodium azide and BCA kit for protein analysis, Natriumdihy-
drogenphospate-1-hydrate and Natriumdihydrogenphospate anhydrous, and
L
-aspartic acid LAA were purchased from Aldrich. All chemicals were used as received except methanol
which was dried before use in the silanization process. Milli-Q purified water was used for all experiments.
Synthesis of Superparamagnetic Iron Oxide Nano- particles SPION. An aqueous suspension of SPION with
an average particle size of 8-10 nm was prepared using controlled coprecipitation as reported before.
3
Briefly, 25 mL of 1 M FeCl
3
‚6H
2
O, 0.5 M FeCl
2
‚4H
2
O, and 0.4 M HCl was prepared as a source of iron by dissolving the respective
chemicals in Milli-Q water under vigorous stirring. The coprecipitation of SPION was carried out in a reactor with
high-speed mechanical stirring 2000 rpm by adding the iron solution to 250 mL of 0.5 M NaOH, which was preheated to
80 °C before the coprecipitation reaction. N
2
was used during the reaction to prevent critical oxidation.
3
Black powder was collected by sedimentation with a help of an external magnetic
field and washed several times with Milli-Q water until stable ferrofluid was obtained. Finally, the particles were redispersed
in an aqueous solution by changing the pH to 11 with TMAOH 5 wt .
Surface Modification of SPION with APTMS. The
SPION suspension was prepared as described above. After- ward, 100 mg of SPION was washed several times with
analytical grade methanol 99.5 with the help of a strong magnet to remove the SPION from the residual water. The
amount of APTMS required to coat 100 mg of SPION with a diameter of 8-10 nm was calculated, taking into account the
surface area of single particles. A 3 mM APTMS solution was prepared in 100 mL of a toluenemethanol 1:1 vv mixture.
APTMS in the toluenemethanol solution 200 µL was added to the SPION suspension. The ferrofluid suspension was
transferred into a three-necked flask with water-cooled con- denser, temperature controller, and N
2
gas flow. The silaniza- tion was performed at 110 °C for 24 h under vigorous stirring.
APTMS acts as a coupling agent, where silanization takes place on the particle surfaces bearing hydroxyl groups in the
organic solvent. As a result, a three-dimensional polysiloxane network was formed. The silanization taking place during the
reflux of APTMS results in the formation of an APTMS coating with a thickness of two or three molecular layers tightly cross-
linked with a large surface density of amines. The powder was collected by applying an external magnetic field after the
silanization process, washed with methanol several times, and finally redispersed in an aqueous medium.
8 Berry, C.; Curtis, A. J. Phys. D: Appl. Phys. 2002, 35, R1.
9 a Pulfer, S.; Gallo, J. In Scientific and Clinical Applications of Magnetic Carriers
; Ha¨feli, U., Schutt, W., Teller, J., Zborowski, M., Eds.; Plenum Press: New York and London, 1997; p 445. b Li, J.;
He, X.; Wu, Z.; Wang, K.; Shen, G.; Yu, R. Anal. Chim. Acta 2003, 481
, 191. 10 a Chatterjee, J.; Haik, Y.; Chen, C. J. Magn. Magn. Mater.
2002, 246, 382. b Kim, D. K.; Mikhaylova, M.; Wang, F. H.; Kehr, J.;
Bjelke, B.; Zhang, Y.; Tsakalakos, T.; Muhammed, M. Chem. Mater. 2003, 15, 4343.
11 Roser, M.; Fischer, D.; Kissel, T. Eur. J. Pharm. Biopharm.
1998, 46, 257.
12 a Yoza, B.; Matsumoto, M.; Matsunaga, T. J. Biotechnology
2002, 94, 217. b Ma, M.; Zhang, Y.; Yu, W.; Shen, H.; Zhang, H.; Gu, N. Colloids Surf. A 2003, 212, 219.
13 Sousa, M.; Rubim, J.; Sobrinho, P.; Tourinho, F. J. Magn. Magn. Mater
. 2001, 225, 67.
Chemisorption of LAA on the Surface of SPION. A 500-
mg portion of uncoated SPION was washed with deionized water and mixed with 200 mL of 0.05 M LAA solution prepared
in nitric acid pH ≈ 2. The mixture was sonicated for 10 min and vigorously stirred for 5 h at room temperature RT. The
particles were recovered by applying an external magnetic field and washed several times with Milli-Q water.
Immobilization of Bovine Serum Albumin BSA on SPION: Double-Step Immobilization Process via 1-Ethyl-
3-3-Dimethylaminepropyl Carbodiimide Hydrochlo- ride EDC Activation. The first step of the immobilization
process represents the possibility of direct immobilization of BSA on the surface of SPION to produce either partially or
fully covered SPION. As a result, SPION were coated with a thin layer of BSA. The second step includes the coupling of
SPION with a second layer of BSA through the activation of a cross-linking agent. The stock solution of 10 mgmL BSA
was prepared in a phosphate buffer solution PBS, pH 7.3 and stored at 4 °C. The mixture of iron salts solutions and BSA in
the ratio of 1:1 was added dropwise into alkaline solution under continuous vigorous stirring. The product was washed
several times with Milli-Q water and then placed in a dialysis membrane against 5 L of Milli-Q water. The dialysis was
carried out for 48-72 h and finally BSA-coated SPION were redispersed in a weak alkaline solution in the presence of
NaN
3
. In the second step, 13 mg of SPION partially coated with BSA was redispersed by sonication in 75 mL of PBS.
Appropriate amounts of EDC, NHS, and BSA were dissolved in 25 mL of PBS. The mixture of preliminary BSA-coated
SPION, BSA, EDC, and NHS was finally shaken for 24 h. The suspension was then dialyzed against 5 L of Milli-Q water at
RT. Subsequently, the particles were collected by applying an external magnetic field and conserved in buffer solution at 4
°C.
Immobilization of BSA on APTMS-Modified SPION.
BSA 5 mg, EDC 5 mg, and NHS 6 mg were dissolved in a 3 mM phosphate buffer solution. This mixture was added to
10 mg of SPION modified with APTMS as described above. The mixture was then sonicated for 10 min at 4 °C and shaken
for 24 h at RT. The particles were collected under a strong external magnetic field and washed several times with PBS.
The SPION were recovered by applying a magnetic field and redispersed in aqueous media.
Cell Culture. Infinity Telomerase Immortalized primary
human fibroblasts hTERT-BJ1, Clonetech Laboratories, Inc., Palo Alto, CA were seeded onto glass coverslips D 13 mm
at a density of 1 × 10
4
cells per disk in 1 mL of complete medium. The medium used was 71 Dulbeccos Modified
Eagles Medium DMEM Sigma, UK, 17.5 Medium 199 Sigma, UK, 9 fetal calf serum FCS Life Technologies,
UK, 1.6 200 mM
L
-glutamine Life Technologies, UK, and 0.9 100 mM sodium pyruvate Life Technologies, UK. The
cells were incubated at 37 °C with a 5 CO
2
atmosphere for 24 h. At this time the cells were incubated in complete medium
supplemented with 0.05 mg mL
-1
BSA-coated SPION for a further 6-24 h. All control cells were cultured in the absence
of any particles.
X-ray Diffraction XRD. The crystal structure of the
precipitated and coated powders was obtained by analyzing the XRD pattern of each sample recorded with a Philips PW
1830 diffractometer, using a monochromatized X-ray beam with nickel-filtered Cu KR radiation. The lattice constants
were estimated using CELREF, a crystal cell parameters refinement program for powder X-rays, using at least squares
refinement method.
Transmission Electron Microscopy TEM. The size and
morphology of the particles were measured by a JEOL-2000EX TEM. The specimen was prepared by dispersing the SPION
in Milli-Q water by using sonication for 3 min. After the dispersion, 1 mL of SPION suspension was centrifuged for 5
min at 14 000 rpm. A drop of well-dispersed supernatant was placed on a carbon-coated 200-mesh copper grid, followed by
drying the sample under ambient conditions.
ζ-Potential Measurements. Surface charge measure-
ments were performed with a Matec Applied Sciences ESA- 9800 system. The dynamic mobility, also called the acoustic
pressure amplitude ESA, was generated by colloidal particles in alternating high-frequency electric fields. A 250-mL portion
of 1.0 M NaOH solution was placed in a Teflon sample container. Iron stock solution 1mL was pumped by a MI-
CROLAB 500 Digital buret and stabilized for 10 s before measurements were taken. The electro-acoustic signal, electri-
cal conductivity, and temperature were recorded as a function of pH while using the titration cell. The stirring rate was kept
at a maximum value.
Fourier Transform Infrared FT-IR Spectroscopy.
FT-IR spectra were recordered by using a Nicolet Avatar 360 FT-IR spectrometer with the Smart DuraSamplIR Diamond
ATR Accessory. Spectra were recordered with a resolution of 1 cm
-1
.
Thermal Study. Thermogravimetric analysis TGA was
performed for powder samples ∼10 mg with a heating rate of 10 °Cmin using a Perkin-Elmer TGA 7 thermogravimetric
analyzer in synthetic air atmosphere up to 700 °C.
Magnetic Measurements. Magnetic properties were mea-
sured with a Princeton Applied Research vibrating sample magnetometer VSM at RT, with magnetic fields up to 15 Oe.
Chemical Analysis. The total concentration of iron was
measured by using a SpectrAA-200. The amount of BSA immobilized on the surface of SPION was calculated by
measuring the BSA concentration in solution before and after the reaction. The concentration of BSA was analyzed by using
a BCA kit designed for protein analysis.
Light Microscopy Staining for Cell Morphology. The
fibroblasts were cultured in the presence of SPION for 6 and 24 h. At each time point, the cells were washed in PBS and
then fixed in 4 formaldehydePBS for 15 min at 37 °C. The cells were subsequently stained for 2 min in 0.5 Coomassie
blue in a methanolacetic acid aqueous solution, and washed with double distilled water to remove excess dye. Samples
could then be observed by light microscopy, and digital images were captured using a Hamamatsu Argus 20 for image
processing. Control cells were cultured in complete medium only.
Clathrin Immunofluorescence and Cytoskeletal Ob- servation. After both 6 and 24 h culture with the SPION,
cells were fixed in 4 formaldehydePBS with 1 sucrose at 37 °C for 15 min. The samples were then washed with PBS,
and permeabilizing buffer was added at 4 °C for 5 min, followed by incubation at 37 °C for 5 min in 1 BSAPBS. This
was followed by the addition of anti-tubulin or anti-clathrin primary antibody 1:100 in 1 BSAPBS, monoclonal anti-
human raised in mouse IgG1, Sigma, Poole, UK for 1 h 37
Figure 1. Transmission electron micrograph left and size
distribution right of uncoated SPION.
Table 1. Particle Size Determined by TEM and Magnetization Analysis
sample D
TEM
Å D
MAG
Å uncoated SPION
80 109
LAA-coated SPION 92
APTMS-coated SPION 100
BSA-coated SPION 110
BSA-APTMS-coated SPION 120
108
°C. Simultaneously, rhodamine-conjugated phalloidin was added for the duration of this incubation 1:100 in 1 BSA
PBS, Molecular Probes, Eugene, OR. The samples were then washed in 0.5 Tween 20PBS, and a secondary, biotin-
conjugated antibody 1:50 in 1 BSAPBS, monoclonal horse anti-mouse IgG, Vector Laboratories, UK was added for 1 h
37 °C, followed by more washing. A FITC-conjugated strepta- vidin third layer was added 1:50 in 1 BSAPBS, Vector
Laboratories, UK at 4 °C for 30 min, and given a final wash. Samples were then observed by fluorescence microscopy Vick-
ers M17.
3. Results and Discussion
