ARTICLE IN PRESS

Biomaterials 26 (2005) 20612072

www.elsevier.com/locate/biomaterials

Epithelial internalization of superparamagnetic nanoparticles and

response to external magnetic eld
Kenneth Dormera,, Charles Seeneyb, Kevin Lewellingc, Guoda Liand, Donald Gibsonc,
Matthew Johnsond
a

Department of Physiology, Oklahoma University Health Sciences Center, 940 S.L. Young Blvd., Room 634, Oklahoma City, OK 73190, USA
b
Chemistry Department, NanoBioMagnetics Inc., 124 N. Bryant Ave., Suite C3, Edmond, OK 73034, USA
c
Physics Department, Oklahoma Christian University, 2501 E. Memorial Rd, Edmond, OK 73013, USA
d
Department of Physics and Astronomy, University of Oklahoma, 131 Nielsen Hall, Norman, OK 73019, USA
Received 5 April 2004; accepted 25 June 2004
Available online 10 August 2004

Abstract
Superparamagnetic magnetite nanoparticles (MNP) coated with silica were synthesized and chronically implanted into the middle
ear epithelial tissues of a guinea pig model (n 16) for the generation of force by an external magnetic eld. In vivo limitations of
biocompatibility include particle morphology, size distribution, composition and mode of internalization. Synthesis of MNP was
performed using a modied precipitation technique and they were characterized by transmission electron microscopy, X-ray
diffractometry and energy dispersive spectroscopy, which veried size distribution, composition and silica encapsulation. The
mechanism for internalizing 1672.3 nm diameter MNP was likely endocytosis, enhanced by magnetically force. Using sterile
technique, middle ear epithelia of tympanic membrane or ossicles was exposed and a suspension of particles with uoroscein
isothiocyanate (FITC) label applied to the surface. A rare earth, NdFeBo magnet (0.35 T) placed under the animal, was used to pull
the MNP into the tissue. After 8 days, following euthanasia, tissues were harvested and confocal scanning laser interferometry was
used to verify intracellular MNP. Displacements of the osscicular chain in response to an external sinusoidal electromagnetic eld
were also measured using laser Doppler interferometry. We showed for the rst time a physiologically relevant, biomechanical
function, produced by MNP responding to a magnetic eld.
r 2004 Elsevier Ltd. All rights reserved.
Keywords: Nanoparticles; Middle ear; Biomechanics; Hearing; Endocytosis

1. Introduction
Superparamagnetic nanoparticles (MNP), such as
magnetite, have been widely used for biomedical
applications [1]. Utilization of MNP to produce forces
in living cells, likewise, is not entirely a new concept [2].
Magnetic twisting cytometry was previously developed
to generate torque on cells in culture to assess the role of
mechanical stress during development, notably in
developing pulmonary epithelium [35]. Biomedical
Corresponding author. Tel: +1-405-271-2226-1221; fax: +1-405271-3181.
E-mail address: ken-dormer@ouhsc.edu (D. Kenneth).

0142-9612/$ - see front matter r 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biomaterials.2004.06.040

applications of MNP include magnetic resonance

imaging contrast enhancement, hyperthermia, intravascular targeted delivery of therapeutics, biosensors and
others [1,69]. Targeted delivery of therapeutics requires
adequate particle susceptibility to and directional
control by an external magnetic eld. Chronically
implanted MNP for assisted biomechanical organ or
tissue functions is feasible only if particle susceptibility is
commensurate with directionality and strength of
external magnetic elds [2]. Physiological mechanisms
for cellular internalization of particles include uid
phase or receptor-mediated endocytosis, phagocytosis
and non-endocytic pathways. Enhancement of MNP
internalization by magnetic forces and subsequent long-

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Kenneth et al. / Biomaterials 26 (2005) 20612072

term viability are not understood [1]. Additionally, the

limits of intracellular MNP mass sufcient to exert
forces without harming cells are unknown. Little is also
known of intracellular trafcking following magnetically enhanced endocytosis and potential iron cytotoxicity from particle degradation [10]. Additionally, the
effects of cytosolic MNP on cellular apoptotic processes
are untested.
In recent years electromagnetic implantable hearing
devices for patients with sensorineural hearing loss have
been developed. Permanent magnets, such as neodymiumironboron, are surgically implanted into the
middle ear, wherein the implants interact with external
electromagnetic elds mechanically directly driving the
ossicular chain [11]. Resulting vibrations representing
acoustic input are conveyed into the inner ear (cochlea),
creating the percept of sound [12]. Such direct drive
hearing device technology has generated high delity,
amplitude and frequency sounds. Since implanted hard
magnets can generate sufcient force to produce sound,
we hypothesized that comparable vibratory forces could
be produced by MNP, with sufcient mass implanted in
the ossicular epithelium or tympanic membrane. Perhaps, hearing amplication could serve as a model of
tissue biomechanics from implanted MNP responding
to an external magnetic eld.
Of prospective mechanisms for internalization, we
hypothesized that endocytic processes will be enhanced
by forces pulling silica encapsulated MNP into the
tympanic membrane and middle ear epithelium. Widder
and colleagues have shown in magnetic drug targeting
studies that external magnetic elds can concentrate
MNP in target tissue, enhancing cell entry by endocytosis [13]. By contrast, intravascular MNP targeting of
endothelial receptors must contend with opsonization
by proteins and phagocytosis by the reticuloendothelial
system. Hence, coatings such as polyethylene glycol
(PEG) have been used to reduce macrophage recognition and uptake [1]. Silica encapsulation of MNP with
negative surface charge (zeta potential) is useful for
corrosion protection, colloidal dispersion and as substrate for particle functionalisation [14,15]. Silica does
not substantially interfere with magnetic susceptibility
[16,17]. We directed forces on silica encapsulated MNP
using an external magnetic eld to more effectively
internalize these particles, irrespective of endocytic and
non-specic mechanisms [1,18].
A requirement for MNP generating forces in cells and
tissues is long-term viability. Unlike MNP for drug
delivery, chronic implantation requires cell compatibility,
hermetic encapsulation, and no exocytosis. Potential iron
leaching and toxicity, an aspect of long-term MNP
viability, has limited data. Normal intracellular iron
homeostasis involves internalization of the complex of
ironbound transferrin via the transferrin receptor but
excessive intracellular iron ions are potentially toxic

through the formation of oxygen radicals and peroxidative

damage [10,19]. Intracellular iron balance could be upset if
MNP were to leach iron. Nevertheless, studies on magnetic
resonance imaging showed (I.V.) ferrite nanoparticles as
compatible with hepatic reticuloendothelial cells and
causing no hepatocellular injury [20].
We report here on a model for generation of forces in
living tissues, implantation of superparamagnetic nanoparticles in the middle ear epithelium. Magnetite
nanoparticles (Fe3O4) were synthesized by a modied
Massart technique [21] and silica encapsulated for
hermeticity and functionalisation. Particles were characterized using X-ray diffraction (XRD) and transmission electron microscopy (TEM) equipped with energy
dispersive X-ray spectroscopy (EDS). Middle ear
epithelial cell internalization was enhanced by an
external magnetic eld and conrmed by observing
conjugated uoroscein isothiocyanate (FITC) intracellular uorescence under confocal microscopy. Neither
the mechanisms of MNP endocytosis nor their intracellular trafcking were the focus of this study.
The ability to generate sinusoidal, vibratory forces by
the interaction of MNP with an external magnetic eld
was also tested. The tip of an electromagnetic coil was
placed 12 mm from either the implanted tympanic
membrane or incus epithelium. One and two kHz
sinusoidal vibration of the ossicular chain was produced. Thus, we concluded that intracellular MNP,
implanted up to 15 days, remained viable in situ and
could be used to produce ossicular vibrations consistent
with the perception of sound in humans.

2. Materials and methods

2.1. Synthesis and characterization of magnetite
nanoparticles (MNP)
2.1.1. Preparation of magnetic nanoparticles
Nanoparticle synthesis employed criteria for size,
superparamagnetism, mass, hermetic encapsulation,
substrate for linkers and viability in tissues. Minimal
size optimizes ease of entry across cell membranes
[21,22]. To enhance endocytosis by magnetic forces,
MNP less than 30 nm diameter were sought. Magnetite
less than 3050 nm also exhibits superparamagnetism
due to single domain crystalline structure. Superparamagnetism exhibits no remanence, dispromoting agglomeration that occurs with magnetized particles The
spherical magnetite particles (Fe3O4) with relatively high
magnetic susceptibility included an 5 nm shell of silica
(SiO2). Encapsulation of the magnetite by SiO2 prohibited corrosion and provided an anionic surface charge
that promoted endocytosis [15] as well as a substrate for
attachment of amines that can serve as linkers to other
molecules.

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To synthesize MNP we used a modied procedure of

Massart [21,23] in an oxygen-free medium to prevent
formation of maghemite. A 2 M iron (II) sulfate
heptahydrate solution was prepared in 2 M HCl and
combined with 1 M iron (III) chloride hexahydrate
aqueous solution in a molar ratio of 2:1 Fe3+:Fe2+.
The solution was mixed and added to 0.7 M ammonium
hydroxide with rapid stirring. The resulting precipitate
was stirred for 30 min, restrained using a permanent
magnet (300 gauss) and the supernatant was discarded.
After several washes, the precipitate was re-suspended in
0.7 M ammonium hydroxide and peptized through the
addition of 1 M tetramethylammonium hydroxide aliquots. The total volume was then taken to 250 ml.
2.1.2. Surface modification of magnetite nanoparticles
with silica
The particles were next coated with SiO2 [24]. A
suspension of magnetite nanoparticles was vigorously
stirred and a 4 ml aliquot taken to a volume of 100 ml
with distilled water. A solution of 0.54% sodium silicate
was prepared at pH 10.5, and 4 ml was added to the
previously prepared MNP suspension. The suspension
was pH adjusted to 10.0 then stirred for 2 h and allowed
to stand for 4 days. Excess silica was removed by several
washes, a magnet retaining the MNP. To funtionalise
the silica coating with amine groups, particles were next
treated with 3-aminopropyltrimethoxysilane. A 1 ml
aliquot of the suspension was brought to a volume of
5 ml with distilled water and sufcient 3-aminopropyltrimethoxysilane added for a nal concentration of 5%
[25]. The reaction system was stirred at room temperature for an hour. After the incubation period, the
particles were again washed with distilled water.
The Kaiser assay [26] was used to conrm the
presence of free amines on the silica-encapsulated,
amine-functionalised MNP. A sample of particles was
reacted with 0.28 M ninhydrin, 76% phenol in ethanol
and 0.0002 M KCN. After a short incubation in boiling
water, a blue color indicates the presence of free amines
(yellow color indicates the absence of amines).
2.1.3. Functionalisation of silica coated magnetic
nanoparticles
Using the amine linker, FITC was conjugated to the
MNP for subsequent location in cells using confocal
uorescence microscopy. Particles were functionalised
following standard conjugation protocol (Molecular
Probes, 2003, Eugene, OR). After the conjugation,
several rinses with sterile 0.9% saline removed excess
non-conjugated, FITC and promoted sterility. The
particles were stored in saline at 20 1C until surgery.
2.1.4. Nanoparticle characterization
2.1.4.1. XRD analysis. The MNP crystal structure,
morphology, size distribution, elemental and chemical

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composition were investigated using XRD, TEM, and

EDS. Crystal structure (phase) and average nanoparticle
size found in a macroscopic sample can be determined
using XRD techniques. XRD analyses were performed
using a diffractometer (Scintag XTRA, Applied Research laboratories, Ecublens, Switzerland).
2.1.4.2. TEM and EDS analysis. For this work,
crystallinity, phase and crystal size are important properties because they affect particle magnetic susceptibility.
TEM analysis allows individual particles to be directly
imaged, whereupon images are used to determine the
specic morphology, chemical composition, as well as
particle size and size distribution. High-resolution TEM
(HRTEM) and electron diffraction can show whether an
individual MNP is a single crystal or polycrystalline.
Under the appropriate imaging conditions, silica-coated
iron-oxide nanoparticles can be differentiated from
uncoated particles. The elemental constituents can also
be unambiguously determined using EDS. The TEM
experiments, including selected area electron diffraction
(SAED), HRTEM and EDS, were all performed using a
JEOL 2000FX electron microscope (JEOL, Japan)
operated at 200 kV equipped with an EDS system
(KEVEX, Thermo Kevex X-ray, Scotts Valley, CA).
Initial assessment of the nature and extent of silica
surface treatments was tested. Particle samples that
ranged in silica content, based on reaction feed ratio
and time conditions, were dispersed in 0.9% saline and
the settling rates were observed as a function of time.
Particles with higher ratios in the reaction feed have the
longest settling rates. Hermeticity of the silica coating and
nascent resistance to corrosion of uncoated magnetite
nanoparticles was tested by soaking both uncoated and
silica-coated nanoparticles in 10% NaCl solution for 2
days at room temperature and then 2 days at 40 1C (total
of 4 days). A standard Sodium Thiocyanate (NaSCN)
assay was used periodically over 96 h to reveal the
presence of Fe ions. Up to 6 months later tests for
corrosion were also performed on the MNP stock
solution, an unprecipitated ferrouid in tetramethylammonium hydroxide. Additionally, precipitated MNP that
were placed in aqueous solution, double distilled water at
pH=13 were tested after 5 months of soaking using the
NaSCN test for the Prussian Blue Reaction.
Magnetic susceptibility (emu/g) of both unmodied
and silica modied MNP were measured using Vibrating
Sample Magnetometry (VSM, ADE/DMS Model 880,
Arkival Technologies, Nashua, NH) which provides a
quantitative measure and is necessary for assessing the
ability of MNP to respond to an external magnetic
eld [27].
The zeta potential for the SiO2 encapsulated MNP,
without the amine linkers used to conjugate FITC, was
measured by dispersing the particles in distilled water at
pH=7.5 adjusted using sodium hydroxide. The MNP

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Kenneth et al. / Biomaterials 26 (2005) 20612072

suspension was sonicated in a water bath for 15 min then

diluted into concentrations of 42, 40 and 38 mg/ml,
sonicated again and their electrophoretic mobility measured to determine zeta potentials (Zeta-Meter Inc.,
Stanton, VA).
2.2. Cellular uptake of magnetite nanoparticles
2.2.1. Surgical approach
Guinea pigs (n 16) of either sex, weighing 375450 g
were anesthetized with Ketamine and Xylazine (35 and
15 mg/kg IP, respectively). Skin surrounding the left or
right pinna was shaved and sterilized and using sterile
technique, a retro-auricular incision exposed the temporal bone over the middle ear cavity. The middle ear
space was drilled opened, exposing the incus as the
target for implantation (MicroCraftTM surgical drill,
Xomed Inc., Jacksonville, FL).
A sterile solution of MNP in physiological saline at
pH=7.4 was sonicated (Sonicor, Copaigue, NY) for
34 min before implantation. Next, 25 ml of the MNP
solution, adherent to the tip of a gentle curved pick, was
touched to the tissue surface. This application was
repeated to place a total volume of 5075 ml on either the
incus or tympanic membrane. Nine animals were
implanted in the incus epithelium (n 3 controls,
contralateral ear). Two animals were implanted on the
tympanic membrane (n 1 control, contralateral ear).
The operative skin site was closed using 3-0 absorbable
suture and the animals were monitored during simultaneous external magnetic eld exposure and recovery
from anesthesia.
2.2.2. Epithelial internalization of nanoparticles
During recovery from anesthesia the animals head
was laid on the pole face of a 4 in cube permanent
magnet (NdFeBo, 50 MGO) with the implanted ear
facing upward. Thus, the MNP solution held by surface
tension was magnetically pulled downward, into the
epithelia. The MNP solution on epithelia at distances of
1 in from the pole face of the magnet experienced a eld
of 0.35 T. Each animal was exposed to this magnetic
eld for 2030 min then was subsequently returned to its
cage for 115 days of monitored recovery.
2.3. Histology and fluorescence microscopy
Recovering 815 days, animals were re-anesthetized
and euthanasia caused by an injection of sodium
pentobarbital (6 grains, IM). The experimental incus
was removed and placed in 80% ethanol, 10% normal
saline for a minimum of 24 h. Next, the incii were
decalcied (Decalcication SolutionTM, Richard Allen
Scientic, Kalamazoo, MI) for 3 days then processed for
parafn sectioning (Tissue-Tek VIPTM, Sakura Finetek,
Torrance, CA). Aldehydes were excluded to prevent

autouorescence of tissues. Transverse 5 mm microtome

sections of the incii or tympanic membrane in parafn
blocks were mounted and either stained with hematoxylin and eosin for histopathology or unstained for
confocal laser microscopy.
Transverse epithelial sections of a decalcied incus
from experimental ears having been implanted for 5
days were given to a Veterinary Pathologist who
compared the (unlabeled) experimental and control
tissues using light microscopy (40100  ). Unstained
transverse sections also were examined using an Argon
laser scanning Spectral Confocal and Multiphoton
Microscope (Leica Model TCS SP2, Leica Microsystems, Mannheim, Germany). Stacks of 1020 scans were
made through the epithelium lining the incii, or the
tympanic membrane epithelium while looking for
intracellular uorescence, indicating the presence of
MNP.
2.4. Laser Doppler interferometry measurement of
middle ear displacements
Conrmation of epithelial implantation with MNP
was made using laser Doppler interferometry (LDI),
providing contactless velocity and displacement measures with a frequency range of 0150 kHz [28].
Following euthanasia, the animal was placed in a lateral
recumbent position, implanted ear up. The pinna was
removed for visualization of the bony ear canal and
tympanic membrane through an operating microscope.
The surgical site was reopened and a 1  1 mm2
reective tape (3-M, Minneapolis, MN) placed either
near the umbo on the tympanic membrane or on the
lateral incus. Middle ear displacements were measured
using single point, helium neon laser LDI (Model OFV
501 and Model 3000 Controller, Polytec PI, Tustin,
CA). When an electromagnetic coil (8 mm length, 2 mm
width, 6.5 mH) was placed 12 mm from the epithelial
surface and activated with 500 or 1000 Hz sine waves at
58 V, peak to peak (Model 80 Function Generator,
Wavetek, San Diego, CA and Model 2706 Precision
Amplier, Bruel & Kjaer, Denmark), the resulting
magnetic eld vibrated the MNP. The middle ear was
intact in these studies; therefore, LDI displacement
measurements reected movements of the ossicular
chain.

3. Results
3.1. MNP characterization
In order to conrm the iron oxide phase and size of
the core in the MNP, uncoated, bare magnetite particles
were characterized before applying a SiO2 shell. Fig. 1
shows an XRD spectrum of the uncoated MNP. The

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spectrum displays peak positions (labeled) and relative

intensities consistent with the crystal structure of
magnetite, Fe3O4. The broad background peak is from
the glass substrate used during XRD scanning. XRD
analyses conrmed the absence of other common phases
of iron oxide species. XRD analysis determined the
diameter of these uncoated MNP, using Scherrers
formula [29], with an average of 10 nm. Fig. 2 shows
TEM results for uncoated MNP with a typical diameter
of 10 nm, in agreement with the XRD results. Selected
area electron diffraction (SAED) patterns in Fig. 2b
conrm the phase of the uncoated MNP to be crystalline
magnetite, again in agreement with the XRD analysis.

1.1

(311)
1

(220)

Intensity (a.u.)

0.9

0.8

(400)
0.7

(511)
(440)

0.6

0.5

0.4
5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

2 (o)
Fig. 1. XRD pattern of uncoated superparamagnetic, ferrite nanoparticles; the indexing corresponds to Fe3O4 magnetite.

2065

Finally, Fig. 2c is an HRTEM image of an individual

particle as marked in Fig. 2a. This image clearly shows
lattice planes with spacing of 4.8 A, corresponding to
Fe3O4 magnetite (111 lattice spacing).
Silica-coated magnetite particles were also investigated using TEM and EDS. Fig. 3a is a TEM
micrograph of an ultra-thin (o15 nm) layer of silicacoated MNP. For unambiguous statistical analysis of
MNP it is crucial to prepare such ultra-thin layers, so
that individual rather than aggregates of nanoparticles
are imaged. For these silica-coated particles we reduced
agglomeration by using a polyelectrolyte, polydiallyldimethylamonium chloride (PDDA), to positively charge
the ultra-thin carbon coated, copper TEM grid surface
before applying a dilute, basic, aqueous suspension of
negatively charged MNP. Statistical analysis of coated
NPs indicated an average particle diameter of 16 nm
with a standard deviation of 2.3 nm (n 80). Fig. 3c is
an enlarged view of a sampled individual particle, which
shows a magnetite core (dark) covered by a layer of
silica (light). Figs. 4a and b show the X-ray spectra
taken from shell and core regions of a silica-coated
particle as determined from a HRTEM image. The
spectrum from the silica shell shows very low iron
content, whereas that from the core shows much higher
iron content. The small amount of iron seen in the SiO2
coating is not likely from iron present in the SiO2 shell,
but rather from nearby iron oxide excited due to
electron beam-broadening inherent in the EDS technique. Based on the EDS analysis, it was conrmed that
the outer shell is SiO2 and the core is iron oxide as
expected.
Zeta potentials (n 6) of silica-coated MNP ranged
from 15 to 20 mV, though 30 mV is ideal according

Fig. 2. (a) Transmission electron micrograph of uncoated magnetite nanoparticles. (b) SAED pattern from large area of NPs. (c) Magnication
showing high-resolution electron micrograph of an individual nanoparticles as marked in (a).

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Fig. 3. (a) Transmission electron micrograph of silica-coated superparamagnetic, magnetite nanoparticles; size distribution of coated nanoparticles is
shown in (b) and detailed core-shell structure of an individual MNP is shown in (c).

EDS on Silica Shell of Particle

200
150

Fe K

Si K

50

Cu K

Cu K

100
OK
Cu L

Counts (a.u.)

250

0
0

10

Energy (keV)

(a)

EDS on Dark Core of Particle

250

50

Cu
K

100

Cu K
Fe K

150

CK
OK
Fe L
Si K

Counts (a.u.)

Fe K
200

0
0
(b)

10

Energy (keV)

Fig. 4. Energy dispersive X-ray spectroscopy spectra from a silica shell

(a) and the iron oxide core of silica-coated nanoparticles (b).

to Widder and colleagues [13]. Nevertheless, the

particles remained dispersed in aqueous solution for
1 h and did not show visible precipitation until 24 h
later, indicating electrostatic repulsive charge moderately cancelled van der Walls attraction forces. Magnetic
susceptibility of the MNP used in this study had values
of 31 and 23 emu/g for unencapsulated and silicaencapsulated MNP, respectively.

Saline corrosion testing of the MNP by the NaSCN

Prussian Blue Reaction showed that 48 h exposure to
corrosive saline caused no leaching of iron or particle
degradation. Fig. 5a shows control NaSCN test result
following 4 days exposure of commercially obtained
ferrite nanopowder in 10% saline (Tal Materials, Ann
Arbor, MI). After 4 days the presence of Fe ions in the
media was revealed by the NaSCN reaction. Fig. 5b
shows the negative Prussian Blue Reaction, tested
concurrently on unencapsulated MNP of the type used
in this study with the addition of silica encapsulation.
No Fe ion leaching occurred, indicating the magnetite
crystalline structure was nonreactive and suggesting that
silica coating would provide additional protection
against corrosion during short-term implantation. However, both the silica-coated MNP in the ferrouid state
and those in pH adjusted double distilled water did
show evidence of long-term corrosion, following 6 and 5
months of soaking, respectively. Thus, long-term
hermeticity of these MNP must be demonstrated for
long-term viability in vivo.
3.2. Uptake of MNP by epithelial cells
Using confocal laser and epiuorescence microscopy
we showed that FITC-labeled MNP were present in
epithelial cells of the incus and tympanic membrane
after 20 min of magnetic eld exposure. Quantication
of label or number of particles was not performed but
increased uorescence on the upper surface of the incii
indicated a moderate mass of MNP had been internalized (Fig. 6a) as well as in the tympanic membrane
(Fig. 6b). Epithelia from control ears, not exposed to the
external magnetic gradients, showed reduced intracellular uorescence, suggesting that endocytosis is also

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Fig. 5. The Prussian Blue Reaction was initiated to test hermeticity of the coated magnetite. In (a) bare ferrite nanoparticles elicited the reaction
while in (b) the test was negative on the silica-coated magnetite particles, demonstrating no free iron and corrosion protection.

internalizing MNP. Fluorescence labeling 15 days

following implantation suggested that cells had not
totally expelled the MNP by exocytosis.
Silica encapsulated magnetite in the cytosol was
apparently non-toxic as no indications of cell pathology
was noted in implanted incii epithelia relative to control
tissues. No giant cells or other indicators of inammatory processes were noted no visible indications of
apoptosis or necrosis were seen following 215 days of
implantation. A freshly explanted incus from a guinea
pig following 8 days of implantation with MNP is
shown in Fig. 7. The dark coloration is from implanted
MNP on the lateral (surgically upward) surface of the
apparently healthy incus.

3.3. Vibration of the ossicular chain

Implanted epithelia of incus and tympanic membrane
responded to an external magnetic eld and could be
measurably vibrated at 2000 Hz. When exposed to the
electromagnetic sine wave (1000 Hz), frequency doubling occurred in the ossicular vibration due to the
superparamagnetic property of MNP. Particles attracted to both north and south polarities produced a
sine wave vibration of 2000 Hz. The recorded frequency
was a pure doubling of the sinusoidal input, with no
discernable distortion. Table 1 shows the coil activation
parameters and resulting laser interferometer displacements as measured from the tympanic membrane or
incus in three guinea pigs implanted with MNP.

4. Discussion
4.1. Characterization of MNP for generation of forces in
tissues
XRD and TEM analyses indicated that uncoated
MNP had a uniform size distribution and magnetite
phase. The average size of unencapsulated MNP was
about 10 nm and HRTEM images indicated that they
were single crystal domain. For the silica-coated MNP,
TEM images and EDS spectra showed most MNP were
coated by amorphous silica with an average size of
1672.3 nm. This size is below the 2030 nm criteria for
achieving optimal membrane binding [30,31].
Agglomeration impairs the implantation of nanoparticles using external magnetic forces, as aggregates of
particles are less likely to cross cell membranes.
Magnetite nanoparticles naturally agglomerate in aqueous solutions at physiological pH levels. Greater net
surface charge lessens agglomeration. The isoelectric
point for bare magnetite nanoparticles, as measured by
the Zeta-Meter, is 6.8 within the pH range of 610.
Previous studies have shown that the isoelectric point is
shifted by silica encapsulation, increasing the zeta
potential and decreasing the propensity to agglomerate
[14,32]. Absolute zeta potentials 430 mV generally
assure mutual repulsion of MNP and no agglomeration
[13,33]. Our MNP placed on the middle ear epithelium
had sufcient surface charge to create a nominally
homogenous dispersion at pH44, which contributed to
the effective internalization in the guinea pig model.

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Fig. 6. (a) A 40  confocal laser micrograph is shown from the single cell epithelial layer of the incus epithelium with uorescent magnetite
nanoparticles; the ossicle was decalcied and the soft tissue then processed and sectioned to reveal uorescenct intracellular nanoparticles. The light
areas of the tissue depict intracellular aggregates of nanoparticles. (b) A 100  confocal scanning laser micrograph of a section from guinea pig
tympanic membrane having been implanted for 8 days with FITC-labeled MNP. The light areas depict intracellular locations of aggregates of
nanoparticles.

Dispersion was important, hence, for implantation all

particles were also sonicated in isotonic saline at
pH=7.4.
Key criteria for chronic implantation of MNP and
production of force involved the ferrite formula, its mass
and magnetic susceptibility. Minimizing size is important for ease of cellular internalization, but reduces
susceptibility [34]. Theoretically, magnetite crystals can
have susceptibilities as high as 90 emu/g, depending on
such factors as synthesis, isolation conditions and

particle size. In biological applications, other investigators have used polymer-coated magnetic nanoparticles
in the range of 30 emu/g, which is comparable to our
model [27]. Our somewhat lower susceptibilities were
primarily are due to the silica encapsulation.
Long-term hermeticity provided by SiO2 encapsulation remains unclear. Related in vitro testing showed
bare magnetite nanoparticles did not corrode while
suspended in 10% saline over 4 days. Such corrosion
resistance was likely due to the tight crystalline structure

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Fig. 7. Incus of guinea pig explanted following 8 days of implantation. Darker lateral surface indicated by the arrow shows where magnetite
nanoparticles were placed intraoperatively and then subsequently internalized by an external magnetic eld. Implanted epithelia did not display any
inammatory responses when examined by a histopathologist.

Table 1
Laser Doppler interferometry data on displacements of middle ear epithelia containing superparamagnetic nanoparticles
Animal Epithelium
GP17
GP18
GP24

Incus

Excitation freq. (Hz) Frequency out (Hz) Voltage to coil (V) Displacement PP  104 m Displacement (A)

500
1000
Incus
500
1000
Tympanic membrane 1000

1000
2000
1000
2000
2000

5.5
5.5
8.0

0.106
0.028
0.106
0.067
0.165

0.12
0.03
0.12
0.07
0.17

In three animals the ability to vibrate implanted middle ear tissues by an external magnetic eld was veried using single point interferometry. Note
that the tympanic membrane was vibrated more easily than the incii, consistent with its greater compliance as a membrane unattached to a substrate.
Vibrations were obtained up to 3200 Hz.

of magnetite. Nevertheless, after 56 months in saline

both naked and silica-encapsulated particles showed
some corrosion (detection of free iron). Our biomechanical assay, ossicular chain vibrations in vitro by an
external magnetic eld, was effective after 8 days of
implantation. One explanation for our successful results
is that the majority of the MNP were hermetically sealed
and other particles, incompletely encapsulated, were not
implanted long enough for corrosion to occur. Certainly
for chronic implantation, 100% hermeticity of MNP
would be required.
4.2. Internalization of MNP by magnetic forces
Physiological intracellular trafcking of substances
begins with internalization and culminates with exocytosis. If MNP were internalized solely by endocytosis,
then lysosomal pH of 55.5 could potentially promote
corrosion of SiO2 encapsulation [14,17]. Endocytic

processes normally are concentration-, time- and energy-dependent with internalization occurring after
about 1 min of incubation in cell culture. Other studies
have shown that nanoparticle exocytosis begins once an
extracellular concentration gradient is removed, with
about 65% of an internalized fraction of particles
(9773 nm diameter) undergoing exocytosis in 30 min
[35]. In our studies, silica encapsulation (negative zeta
potential) should repulse negative membrane surface
charges. Our MNP aminated (positive surface charge)
for FITC conjugation should be attractive to membrane
surfaces, just as fusegenic cationic polymers promote
internalization [22]. A new class of albumin coated,
anionic, maghemite nanoparticles, however, has shown
high afnity for cell membranes with endocytic
efciency three times greater than dextran coated
nanoparticles [15]. Other studies also have shown
negative nanoparticles non-specically taken up more
readily than neutral or positive charged particles [36].

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Kenneth et al. / Biomaterials 26 (2005) 20612072

Irrespective of natural endocytosis and these conicting

studies on non-specic, surface charge related interactions, internalization was greatly enhanced by magnetic
forces in these in vivo studies.
Since nanoparticle retention increases with concentration in cell culture [35], it may be that our middle ear
epithelia were continually endocytic due to excess MNP
adsorbed to the surfaces. Since MNP remained intracellular up to 15 days following implantation, magnetically enhanced endocytic mechanisms may have
contributed to their substantial internalization. What
remains to be proven is long-term viability of these
MNP, with sinusoidal generation of forces, and within
normal apoptotic lifetimes of these tissues.

elements [40]. Tensegrity cell models predict complex

cell behaviors, including how cells change shape when
they adhere to rigid extracellular matrices, as in the
epithelium covering middle ear ossicles. Such models
also predict that cells and nuclei will immediately
respond to mechanical stresses transmitted over cell
surface receptors that physically connect the cytoskeleton with the extracellular matrix [41]. Thus, transegrity
models would predict that chronic stresses imposed on
the middle ear epithelium will result in cell stiffness
changes to accompany the continual internal stress on
the cytoskeleton during ossicular displacments.

4.3. Generation of forces in tissues

Short-term viability of intracellular MNP was demonstrated by the presence of particles 15 days after
implantation by the absence of histopathology and by
effective mechanical transduction of the middle ear.
Magnetite microparticles have been shown to be less
sensitive to oxidation than magnetic transition metal
particles [27]. Nevertheless, long-term cell viability from
potential iron toxicity remains a concern, if silica
encapsulation should fail. Normal homeostasis of iron
is primarily regulated at the level of messenger RNA
translation, where regulatory proteins control uptake,
storage and utilization [19]. Regarding longer in vitro
stability, we noted oxidation of MNP only after 5
months of soaking in hypertonic saline.
The only data on in vivo iron particles showed nontoxicity when ferrite nanoparticles were administered
(I.V.) to patients for enhanced magnetic resonance
imaging. Iron determinations in the liver, spleen, lung
and kidney after a massive 250 mg iron/kg body weight
dose showed that particles were taken up by hepatic
reticuloendothelial cells, with no evidence of mitochondrial or microsomal lipid peroxidation or organelle
dysfunction, sensitive indicators of iron-induced hepatocellular injury [20]. Incus MNP released in humans by
exocytosis or apoptosis would pass down the Eustachian
tube to the throat, be swallowed and eliminated from
the body.

For the rst time we have shown that implanted

superparamagnetic nanoparticles have produced sinusoidal forces in vivo. Displacements of the guinea pig
ossicular chain at an auditory frequency were relatively
greater than those recorded from human ears. In
previous studies using fresh-frozen temporal bones
(n 17), 90 dB SPL calibrated sinusoidal 1000 Hz
sounds were presented 2 mm from the surface of
tympanic membranes and LDI measured average
displacements of 0.034 mm (peak to peak) for 1000 Hz
input [28,37]. Incus and tympanic membrane displacements in this study using the apparatus were 0.106 and
0.165 mm, respectively (Table 1). Though indicating
substantial amplication relative to the human, the
guinea pig middle ear has much less mass and the coil
voltages used were greater than those typically used in
human implantable hearing devices [11]. Nevertheless,
hearing amplication was not the goal of this study, but
rather feasibility of particle generation of force. Our
results are consistent with biomechanics of the middle
ear in that greater displacements were recorded from the
tympanic membrane (Table 1), which is consistent with
the ossicular lever ratio.
Other applied forces using magnetic particle methodology has been magnetic twisting cytometry, probing
micromechanical properties of epithelia in cell lines [38].
Magnetic microbeads (5 mm) bound to cell surface
ligands applied sinusoidal (0.0316 Hz) torque in response to an external magnetic eld [39]. Cell deformability is important to understanding cell motility,
apoptosis or DNA synthesis, and in our study deformability of the middle ear epithelium relates to the longterm ability to generate acoustically relevant displacements.
Long-term viability of MNP in middle ear epithelial
cells also depends upon the response of these cells to
newly imposed forces. Cells may use tensegrity architecture to structure and stabilize themselves through
continuous tension distributed across the structural

4.4. Non-toxicity of MNP

5. Conclusion
In summary, we have shown that silica encapsulated,
magnetite nanoparticles can be used in conjunction with
an external magnetic eld to produce biomechanical forces
in the middle ear. MNP, customized for ease of
internalization, and magnetic susceptibility can be implanted in epithelia without producing toxic effects and
remain viable for at least 15 days. Displacements in tact
ossicular chain at auditory frequencies have been generated in the guinea pig using a miniature electromagnetic
coil. Amplitudes of these displacements are comparable to

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Kenneth et al. / Biomaterials 26 (2005) 20612072

those produced by 90 dB SPL at the tympanic membrane

in fresh frozen human temporal bones.

Acknowledgements
Our appreciation extends to Arif Mamedov, Ph.D. for
assistance in synthesis, Wanda Day, HTL II for her
histological processing of tissues, Fadee Mondalek,
B.S., for technical support, Don Nakmali, BSEE,
Hough Ear Institute for assistance with interferometry
and Stanley Kosanke, DVM, Ph.D., for evaluating the
histopathology. This work was performed under NIH
SBIR Grant 1 R43 DC05528-01 to NanoBioMagnetics
Inc.
Conict of Interest statement:
Charles E. Seeney and Kenneth J. Dormer are
Ofcers of NanoBioMagnetics Inc., recipient of the
above NIH-SBIR grant.

References
[1] Zhang Y, Kohle N, Zhang M. Surface modication of superparamagnetic magnetite nanoparticles and their intracellular
uptake. Biomaterials 2002;23:155361.
[2] Valberg PA, Butler JP. Magnetic particle motions within living
cells. Physical theory and techniques. Biophys J 1987;52:53750.
[3] Feringa B. In control of motion: from molecular switches to
molecular motors. Acc Chem Res 2001;24(6):50413.
[4] Laurent VM, Henon S, Planus E, Fodil R, Balland M, Isabey D,
Gallet F. Assessment of mechanical properties of adherent living
cells by bead micromanipulation: comparison of magnetic
twisting cytometry vs optical tweezers. J. Biomech Eng
2002;124(4):40821.
[5] Ingber DE. Cellular basis of mechanotransduction. Biol Bull
1998;194:3237.
[6] Freitas Jr. R. Nanomedicine. Austin: Landes Bioscience; 1999.
[7] West J, Halas NJ. Applications of nanotechnology to biotechnology. Curr Opin Biotechnol 2000;11:2157.
[8] Igartua M, Saulnier P, Heurtault B, Pech B, Proust JE, Pedraz JL,
Benoit JP. Development and characterization of solid lipid
nanoparticles loaded with magnetite. Int J Pharm
2002;233:14957.
[9] Browning VM. Potential department of defense (DOD) applications of magnetic carriers. Eur Cells Mater 2002;3(Suppl. 2):56.
[10] Lacava ZGM, Asevedo RB, Martins LM, Freitas MLL, et al.
Biological effects of magnetic uids: toxicity studies. J Magn
Magn Mater 1999;201(13):4314.
[11] Dormer KJ. Implantable electronic otologic devices for hearing
restoration. In: Finn WE, Lopresti PG, editors. Handbook of
neuroprosthetic research methods. Boca Raton, FL: CRC Press;
2003.
[12] Austin DF. Acoustic mechanisms in middle ear sound transfer.
Otolaryngol Clin N Am 1994;27(4):64154.
[13] Widder KJ, et al. Selective targeting of mangetic albumin
microspheres to the Yoshida sarcome: ultrstructural evaluation
of microsphere deposition. Eur J Cancer Clin Oncol
1983;19:1417.
[14] Mornet S, Grasset F, Portier J, Dugnet E. Maghemite silica
nanoparticles for biological applications. Eur Cells Mater
2002;3(Suppl 2):1103.

2071

[15] Wilhelm C, Billotey C, Roger J, Pons J, Bacri J, Gazeau F.

Intracellular uptake of anionic superparamagnetic nanoparticles
as a function of their surface coating. Biomaterials 2003;24(6):
100111.
[16] Qhobosheane M, Santra S, Shang P, Tan W. Biochemically
functionalized silica nanoparticles. Analyst 2001;126(8):12748.
[17] Aliev FG, Correa-Duarte MA, Mamedov A, Ostrander JW,
Giersig M, Liz-Marzan LM, Kotov NA. Layer-by-layer assembly
of core-shell magnetite nanoparticles. Effect of silica coating on
interparticle interactions and magnetic properties. Adv Mater
1999;11(12):100610.
[18] Panyam J, Zhou WZ, Prabha S, Sahoo SJ, Labhasetwar V. Rapid
endo-lysosomal escape of poly(DL-lactide-co-glycolide) nanoparticles: implications for drug and gene delivery. FASEB J
2002;16:121726.
[19] Crichton RR, Ward RJ. An overview of iron metabolism:
molecular and cellular criteria for the selection of iron chelators.
Curr Med Chem 2003;10:9971004.
[20] Bacon BR, Stark D, Park CH, Saini S, Groman EV, Hahn PF,
Compton CC, Ferrucci Jr. JT. Ferrite particles: a new magnetic
resonance imaging contrast agent. Lack of acute or chronic
hepatotoxicity after intravenous administration. J Lab Clin Invest
1987;110(2):16471.
[21] Massart R. Preparation of aqueous magnetic liquids in alkaline
and acidic media. Trans Magn 1981;Mag-17(2):12479.
[22] Akinc A, Lynn DM, Anderson DG, Langer R. Parallel synthesis
and biophysical characterization of a biodegradable polymer
library for gene delivery. J Am Chem Soc 2003;125:531623.
[23] Philipse AP, vanBruggen MPB, Pathmamanoharan C.
Magnetic silica dispersions: preparation and stability of surface
modied silica particles with a magnetic core. Langmuir
1994;10(1):929.
[24] Correa-Duarte MA, Giersig M, Kotov NA, Liz-Marzan LM.
Self-assembled monolayers of magnetite nanoparticles with and
without SiO2 coating by microwave irradiation. Langmuir
1998;14:64305.
[25] Wong C, Burgess JP. Modifying the surface chemistry of silica
nano-shells for immunoassays. J Young Invest 2003;6(1):114.
[26] Kaiser ET, Colescott RL, Bossinger CD, Cook PI. Color test for
detection of free amino groups in the solid-phase synthesis of
peptides. Anal Biochem 1970;34(2):5958.
[27] Harris LA, Goff JD, Carmichael AY, Rife JS, Harburn JJ,
St. Pierre TG, Saunders M. Magnetite nanoparticle
dispersions stabilized with triblock polymers. Chem Mater
2003;15:136777.
[28] Gan RZ, Dyer RK, Wood MW, Dormer KJ. Mass loading on
ossicles and middle ear function. Ann Otol Rhinol Laryngol
2001;110(5):47885.
[29] Cullity BD. Elements of X-ray diffraction. 2nd ed. Reading, MA:
Addison-Wesley Publishing Inc.; 1978. p. P102.
[30] Harrison RJ, Dunin-Borkowski RE, Putnin A. Direct imaging of
nanoscale magnetic interactions in minerals. Proc Natl Acad Sci
2002;99(26):1655661.
[31] Bergemann C, Muller-Schulte D, Oster J, Brassard LA, Lubbe
AS. Magnetite ion-enchange nano- and micro-particles for
medical, biochemical and molecular biological applications.
J Magn Magn Mater 1999;194:4552.
[32] Sameti M, Bohr G, Ravi-Kumar MN, Kneuer C, Bakowsky M,
Nacken H, Schmidt H, Lehr CM. Stabilisation by freeze-drying of
cationically modied silica nanoparticles for gene delivery. Int J
Pharm 2003;266(12):5160.
[33] Harris LA. Polymer stabilized magnetite nanoparticles. Blacksberg: Virginia Polytechnic Institute; 2002.
[34] Popplewell JaS L. The dependence of physical and magnetic
properties of magnetic uids on particle size. J Magn Magn Mater
1995;149:728.

ARTICLE IN PRESS
2072

Kenneth et al. / Biomaterials 26 (2005) 20612072

[35] Panyam JaL V. Dynamics of endocytosis and exocytosis of

Poly(D,L-Lactide-co-Glycolide) nanoparticles in vascular smooth
muscle cells. Pharm Res 2003;20(2):21220.
[36] Billotey C, Wilhelm C, Devaud M, Bacri JC, Bittoun J, Gazeau F.
Cell internalization of anionic magnemite nanoparticles: quantitative effect on magnetic resonance imaging. Magn Resonance
Med 2003;49:64654.
[37] Gan RZ, Sun Q, Dyer RK, Chang KH, Dormer KJ. Three
dimensional modeling of middle ear biomechanics and its
applications. Otol Neurotol 2002;23:27180.
[38] Puig-De-Morales M, Grabulosa M, Alcaraz J, Mullol J, Maksym
GN, Fredberg JJ, Navajas D. Measurement of cell microrheology

by magnetic twisting cytometry with frequency domain demodulation. J Appl Physiol 2001;91:11529.
[39] Crick FHC, Hughes AFW. The physical properties of the
cytoplasm. A study by means of the magnetic particle method.
Part 1. Exp Cell Res 1950;1:3780.
[40] Ingber DE. Tensegrity: the architectural basis of cellular
mechanotransduction. Ann Rev Physiol 1997;59:57599.
[41] Wang N, Naruse K, Stamenovic D, Fredberg JJ, Mijailovich SM,
Tolic-Norrelykke IM, Polte T, Mannix R, Ingber DE. Mechanical
behavior in living cells consistent with the tensegrity model. Proc
Natl Acad Sci 2001;98:776570.