A Nanodot Array Modulates Cell Adhesion and Induces an Apoptosis-Like Abnormality in NIH-3T3 Cells.

Abstract

Micro-structures that mimic the extracellular substratum promote cell growth and differentiation, while the cellular reaction to a nanostructure is poorly defined. To evaluate the cellular response to a nanoscaled surface, NIH 3T3 cells were grown on nanodot arrays with dot diameters ranging from 10 to 200 nm. The nanodot arrays were fabricated by AAO processing on TaN-coated wafers. A thin layer of platinum, 5 nm in thickness, was sputtered onto the structure to improve biocompatibility. The cells grew normally on the 10-nm array and on flat surfaces. However, 50-nm, 100-nm, and 200-nm nanodot arrays induced apoptosis-like events. Abnormality was triggered after as few as 24 h of incubation on a 200-nm dot array. For cells grown on the 50-nm array, the abnormality started after 72 h of incubation. The number of filopodia extended from the cell bodies was lower for the abnormal cells. Immunostaining using antibodies against vinculin and actin filament was performed. Both the number of focal adhesions and the amount of cytoskeleton were decreased in cells grown on the 100-nm and 200-nm arrays. Pre-coatings of fibronectin (FN) or type I collagen promoted cellular anchorage and prevented the nanotopography-induced programed cell death. In summary, nanotopography, in the form of nanodot arrays, induced an apoptosis-like abnormality for cultured NIH 3T3 cells. The occurrence of the abnormality was mediated by the formation of focal adhesions.

Full text

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NANO EXPRESS
A Nanodot Array Modulates Cell Adhesion and Induces
an Apoptosis-Like Abnormality in NIH-3T3 Cells
Hsu-An Pan Æ Æ Yao-Ching Hung Æ Æ Chia-Wei Su Æ Æ
Shih-Ming Tai Æ Æ Chiun-Hsun Chen Æ Æ
Fu-Hsiang Ko Æ Æ G. Steve Huang
Received: 31 March 2009/Accepted: 24 April 2009/Published online: 19 May 2009
? to the authors 2009
Abstract
substratum promote cell growth and differentiation, while
the cellular reaction to a nanostructure is poorly defined.
To evaluate the cellular response to a nanoscaled surface,
NIH 3T3 cells were grown on nanodot arrays with dot
diameters ranging from 10 to 200 nm. The nanodot arrays
were fabricated by AAO processing on TaN-coated wafers.
A thin layer of platinum, 5 nm in thickness, was sputtered
onto the structure to improve biocompatibility. The cells
grew normally on the 10-nm array and on flat surfaces.
However, 50-nm, 100-nm, and 200-nm nanodot arrays
induced apoptosis-like events. Abnormality was triggered
after as few as 24 h of incubation on a 200-nm dot array.
For cells grown on the 50-nm array, the abnormality started
after 72 h of incubation. The number of filopodia extended
from the cell bodies was lower for the abnormal cells.
Immunostaining using antibodies against vinculin and actin
filament was performed. Both the number of focal adhe-
sions and the amount of cytoskeleton were decreased in
cells grown on the 100-nm and 200-nm arrays. Pre-
coatings of fibronectin (FN) or type I collagen promoted
cellular anchorage and prevented the nanotopography-
Micro-structures that mimic the extracellular
induced programed cell death. In summary, nanotopogra-
phy, in the form of nanodot arrays, induced an apoptosis-
like abnormality for cultured NIH 3T3 cells. The occur-
rence of the abnormality was mediated by the formation of
focal adhesions.
Keywords
Fibronectin ? Fibroblasts
Cell adhesion ? Nanotopography ? Apoptosis ?
Introduction
Surface topology encodes information that directs cell
behavior [1–5]. Cells detect and respond to the specific
ligands and the spatial organization of the scaffoldings
known as the extracellular matrix (ECM). The ECM con-
sists of collagen and elastin fibers of 10–300 nm diameters
intertwined into a landscape of peaks, valleys, and pores
[6]. Since ECM contains structures from micro-scale down
to nanoscale, it is hypothesized that cells respond to both
micro-structure and nanostructure.
Micro-scaled landscapes have been fabricated to direct
growth of cultured cells. When cultured on ridges and
grooves of nanoscale dimensions, cells migrated more
extensively to the ridges than into the grooves. The cells’
shapes were aligned and extended in the direction of the
grooves [3, 7]. It has been shown that a three-dimensional
micro-structure that mimics ECM provides an environment
for the in vivo growth of cells. Osteoblasts grown on a
fibrous matrix composed of multiwalled carbon nanofibers
(100 nm in diameter) exhibited increased proliferation
compared to those grown on flat glass surfaces [8, 9].
Breast epithelial cells proliferate and form multicellular
spheroids on interwoven polyamide fibers fabricated by
electrospinning polymer solution onto glass slides [10].
H.-A. Pan ? C.-W. Su ? S.-M. Tai ? F.-H. Ko ?
G. Steve Huang (&)
Institute of Nanotechnology, National Chiao Tung University,
1001 University Road, Hsinchu 300, Taiwan, ROC
e-mail: gstevehuang@mail.nctu.edu.tw
Y.-C. Hung
Section of Gynecologic Oncology, Department of Obstetrics and
Gynecology, China Medical University and Hospital,
91 Hsueh Shih Rd, Taichung 404, Taiwan, ROC
C.-H. Chen
Department of Mechanical Engineering,
National Chiao Tung University, Hsinchu, Taiwan, ROC
123
Nanoscale Res Lett (2009) 4:903–912
DOI 10.1007/s11671-009-9333-7

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Nanofibers with 100 nm diameters have been fabricated to
mimic the three-dimensional fibrous structure of the
extracellular matrix [5, 9]. 3-D nanofibrillar surfaces
covalently modified with tenascin-C-derived peptides
enhance neuronal growth in vitro [11]. The three-
dimensionality and nanofibrillar architecture of the ECM
may represent another essential element in signal trans-
duction pathways and cellular physiology. Nanotopography
can activate the small GTPase Rac [12]. This activation of
Rac was accompanied by changes in cell morphology and
proliferation, Rac localization, fibronectin deposition, and
the organization of actin filament-based networks [10].
Although cellular response to micro-topography has been
extensively investigated, the nanotopography that cells
respond to and the molecular apparatus that senses and
transmit the spatial signal from the membrane to the
nucleus are not clearly defined at the present time.
Nanotopography-induced cellular response has been
explored using nanoislands. Nanoislands were fabricated
through varying the polymer blend and allowing sponta-
neous demixing [13]. Strong influence on the formation of
focal adhesions, reorganization of cytoskeleton, and change
in the mobility were observed [12]. The cells manage an
initial fast organization of the cytoskeleton in reaction to the
islands [14]. It has been observed that 13-nm-high islands
induce cell spreading and proliferation, while 160-nm
islands retard the attachment of filopodia. A gene expres-
sion study using a microarray indicates the down regulation
of genes associated with the cytoskeleton for cells grown on
95-nm deep nanoislands. The cells responded to the islands
with broad gene up-regulation, notably those involved in
cell signaling, proliferation, the cytoskeleton, and the pro-
duction of extracellular matrix protein [15]. Nonetheless,
the topography consists of nanoscale islands with control-
lable heights of tens to hundreds of nanometers, however,
with large variation in diameter [16].
The current study is based on the hypothesis that
signal transduction pathways must exist that transmit a
Fig. 1 Fabrication of tantalum-based nanodot arrays using AAO
processing. a Schematic representation of fabrication procedure.
b SEM images of the fabricated nanodot arrays. c AFM images of the
fabricated nanodot arrays. Images are arranged from left to right:
unprocessed silicon (Si), 10-nm nanodot array (10 nm), 50-nm
nanodot array (50 nm), 100-nm nanodot array (100 nm), and
200-nm nanodot array (200 nm)
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nanotopography-induced special signal, directs cellular
behavior from the extracellular domain to the nuclear area
where genetic control occurs [12, 17]. Arrays of nanodots
with defined diameters and depths can be fabricated using
aluminum nanopores as a template during the oxidation of
tantalum thin films [16]. The pore size of the aluminum
Fig. 2 SEM images of cells seeded on the nanodot arrays. NIH-3T3
cells were seeded on a flat silicon surface, 10-nm nanodot array
(10 nm), 50-nm nanodot array (50 nm), 100-nm nanodot array
(100 nm), and 200-nm nanodot array (200 nm). The cells were
harvested at 24 h (Day 1), 48 h (Day 2), 72 h (Day 3), and 96 h
(Day 4) after seeding. SEM images were taken. Representative
images are shown: a top view, b side view
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oxide is controllable and uniformly distributed, whereas the
depth of the dots depends on the voltage applied; thus, this
can serve as a convenient mold for fabricating tantalum
into a nanodot array of specific diameter and depth. The
structure containing nanodots of uniform size can serve as
a comparable nanolandscape to those probing cellular
response at the molecular level.
Materials and Methods
Chemicals
Glutaraldehyde and osmium tetroxide were purchased from
Electron Microscopy Sciences (USA). Cytochalasin D was
purchased from Calbiochem (USA). Anti-vinculin mouse
antibody was purchased from Abcam (USA). Alexa Fluor
594 phalloidin, Alexa Fluor 488 goat anti-mouse IgG, and
EnzChek Caspase-3 Assay Kit #2 were purchased from
Invitrogen (USA). Fibronectin (FN), type I collagen,
L-glutamine, and trypsin were purchased from Sigma
(USA). Other chemicals of analytical grade or higher were
purchased from Sigma or Merck.
Fabrication of Nanodot Arrays
Nanodot arrays were fabricated as described previously
[16]. A TaN thin film of 150 nm thickness was sputtered
onto a 6-inch silicon wafer, followed by the deposition of
3 lm-thick aluminum onto the top of the TaN layer.
Anodization was carried out in 1.8 M sulfuric acid at
5 Volts for the 10 nm nanodot array, or in 0.3 M oxalic
acid at 25, 60, and 100 V for the 50, 100, and 200 nm
nanodot arrays, respectively. Porous anodic alumina was
formed during the anodic oxidation. The underlying TaN
layer was oxidized into tantalum oxide nanodots using the
alumina nanopores as template. The porous alumina was
removed by immersion in 5% (w/v) H3PO4overnight. A
thin layer of platinum (*5 nm) was sputtered onto the
structure to improve biocompatibility. The dimension and
homogeneity of the nanodot arrays were measured and
calculated from images taken by JEOL JSM-6500 TFE-
SEM and by atomic force microscopy (AFM).
Coating of BSA, FN, and type I collagen were per-
formed by covering the nanodot arrays with 0.1 mg/mL
protein solution at 4 ?C for 8 h followed by rinsing with
PBS three times before use.
Cell Culture
To eliminate possible contamination of nanomicro parti-
cles, the cell culturing was performed in a class-10 clean
room. NIH-3T3 cells were cultured in Dulbecco’s Modified
Eagle’s Medium complimented with 10% FBS and 5%
CO2and incubated at 37 ?C.
Treatment of Cells for Scanning Electron Microscopy
The harvested cells were fixed with 1% glutaraldehyde in
PBS at 4 ?C for 20 min, followed by post-fixation in 1%
osmium tetroxide for 30 min. Dehydration was performed
through a series of ethanol concentrations (5-min incuba-
tion each in 50, 60, 70, 80, 90, 95, and 100% ethanol) and
air drying. The specimens were sputter-coated with plati-
num and examined by JEOL JSM-6500 TFE-SEM at an
accelerating voltage of 10 keV.
Fig. 3 Apoptosis occurred in cells cultured on nanodot arrays. a The
percentage of cells with abnormal morphology calculated from
SEM images. Bars depict percent apoptotic cells grown on the flat
silicon surface (gray), 10-nm nanodot array (vertical line), 50-nm
nanodot array (empty), 100-nm nanodot array (horizontal line), and
200-nm nanodot array (filled). b Caspase-3 activity for cells cultured
96 h on the nanodot arrays. Values were averaged from six sets of
independent experiments and were expressed as mean value ± stan-
dard deviation
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Caspase-3 Activity Assay
The EnzChek Caspase-3 Assay Kit #2 (Invitrogen, USA)
was applied to evaluate the caspase-3 activity, using the
procedures provided by the manufacturer. Cells were har-
vested and counted, followed by incubation with the lysis
buffer for 30 min. The cell lysate was centrifuged, and the
supernatant was transferred to microplate wells containing
Z-DEVD-R110-substrate-working solution followed by a
30 min incubation at room temperature. The fluorescence
was measured using an ELISA microplate reader (Perkin
Elmer, USA) with an excitation wavelength at 496 nm and
an emission wavelength at 520 nm. The caspase-3 activity
was normalized to cell counts.
Immunostaining of Actin Filament and Vinculin
Cells were harvested and fixed with 4% paraformaldehyde
in PBS for 15 min, followed by three-three PBS washes.
The membrane was permeated by incubating in 0.1% Triton
X-100 for 10 min, followed by a three PBS washes,
blocking with 1% BSA in PBS for 1 h, and three PBS
washes. The sample was incubated with anti-vinculin
antibody (properly diluted in 0.5% BSA) and phalloidin for
1 h, followed by incubating with Alexa Fluor 488 goat anti-
mouse antibody for 1 h and followed by three PBS washes.
Results and Discussion
Fabrication of Nanodot Arrays
Nanodot arrays made using anodic aluminum oxide (AAO)
template are highly packed and uniformly distributed in
size and shape. This processing defines a series of nanot-
opologies and can serve as an excellent model system
for studying how physical topography affects cellular
behavior.
Nanodot arrays were fabricated, as described previ-
ously, by AAO processing on tantalum-coated wafers
[16]. Tantalum oxide nanodot arrays with dot diameters
of 10, 50, 100, and 200 nm were constructed on the
silicon wafers. To provide a biocompatible and unique
interacting surface, *5-nm-thick platinum was sputter-
coated onto the top of the nanodots. Scanning electron
microscopy (SEM) and AFM images showed diameters of
15 ± 2.8, 58.1 ± 5.6, 95.4 ± 9.2, and 211.5 ± 30.6 nm
for the 10, 50, 100, and 200 nm dot arrays, respectively
(Fig. 1). The average heights were 11.3 ± 2.5, 51.3 ±
5.5, 101.1 ± 10.3, and 154.2 ± 27.8 nm, respectively.
Dot-to-dot distanceswere
108.1 ± 2.3, and 194.2 ± 15.1 nm, respectively. The
dimensions of the nanodots were well controlled and
highly defined.
22.8 ± 4.6, 61.3 ± 6.4,
Fig. 4 SEM images of the used
nanodot arrays. Nanodot arrays
of 100-nm and 200-nm were
cleaned and washed thoroughly
after culturing cells. SEM
images were taken on the
cleaned nanodot arrays of (a, b)
100-nm and (c, d) 200-nm
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Cellular Response to Nanodot Arrays
NIH-3T3 cells were cultured on fabricated nanodot arrays
and on flat wafers at a density of 1,000–5,000 cells per
square centimeter. Cells were harvested at 24 h (day 1),
48 h (day 2), 72 h (day 3), and 96 h (day 4) after seeding.
SEM was performed to examine the morphology of the cells
(Fig. 2). The side view of the SEM images provided alter-
native angles for evaluating the morphological change of
cultured cells. Cells grown on the control surface and the
10-nm nanodot array remained flat and extended throughout
the course of incubation. Cells grown on the 50-nm nanodot
array began to show an abnormal appearance on day 4. The
abnormal cells underwent a transformation of the main cell
Fig. 5 SEM images of NIH-3T3 cells cultured on nanodot arrays to show the filopodia extended from cells. Typical the cells were shown to
elicit the detail of cellular structure
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body into sub-cellular spheres of *5 lm in diameter. On
day 4, spherical sub-cellular cell bodies were visible. For
cells grown on the 100-nm nanodot array, a comparable
morphology occurred as early as day 3, while for the
200-nm nanodot array, the morphological aberration started
from day 2. The proportion of cells undergoing this mor-
phological change was higher, and the event was triggered
earlier, on the 100- and 200-nm nanodot arrays (Fig. 3a).
The morphology of the abnormal cells resembled cells
proceeding in programed cell death. Caspase activity is the
hallmark for apoptosis. Thus, the occurrence of apoptosis-
like events was verified by a caspase-3 activity assay per-
formed on cells seeded on nanodot arrays following the
time course (Fig. 3b). The onset, time-dependent accu-
mulation, and size-dependent profile of caspase-3 activity
matched the proportion of cells undergoing morphological
transformation on the nanodot arrays. The nanotopography
triggered apoptosis-like events for cultured cells in a size-
dependent and time-dependent manner.
Cells grown on the nanoscaled structure could engulf any
loose nanoparticles. The endocytosis of the remnants might
induce the observed abnormalities. To exclude the possi-
bility that the abnormality was due not to the growth on the
nanostructure but to the endocytosis of nanodots, the used
arrays were thoroughly cleaned and examined under an
electron microscope. The SEM images indicated that the
nanodot structures of the used 100-nm and 200-nm nanodot
arrays were intact even after a prolonged culturing of cells
(Fig. 4).
Cell Adhesion and Reorganization of the Cytoskeleton
were Required for the Nanotopography-Induced
Cellular Abnormality
The formation of focal adhesions, reflected by the attach-
ment of filopodia to the substratum, indicates normal
growth for cultured cells [15]. The number of filopodia
extended from the cells decreased for cells grown on
nanodot arrays larger than 50 nm (Fig. 5). For cells seeded
on the 200-nm nanodot array, very few filopodia were
found. Cells grown on larger-sized nanodot arrays lost the
ability to establish filopodia attachment. Further examina-
tion indicated that cellular attachment was defective for
cells grown on the 100 nm nanodot array (Fig. 6).
Topology and surface chemistry might share a common
pathway for directing cell behavior. Focal adhesions are
mediated by cell adhesion through receptor–ligand binding
[18, 19]. The inability of cells to establish filopodia
attachment on a nanolandscape might be prevented by a
surface modification with ligands. We coated the 100-nm
nanodot array with BSA, FN, or type I collagen. Pre-
treatment with BSA did not prevent the nanotopography-
induced apoptosis-like abnormality, while FN and collagen
I coating completely averted cellular abnormality (Fig. 7).
FN and collagen are native substrates of integrins, the key
transmembrane proteins of focal adhesions. The prevention
of programed cell death by FN- or type I collagen-enforced
cell anchorage indicates that the topography-induced
apoptosis-like abnormality could be overridden by recep-
tor-mediated cell adhesion.
To evaluate the role of adhesion molecules in the
nanotopography-induced apoptosis-like events, immuno-
staining specific to actin filaments and vinculin was per-
formed on cells grown on the nanodot arrays (Fig. 8).
Well-organized actin filaments were visible for cells grown
on the flat wafer and on the 10-nm nanodot array. This tight
arrangement was gradually lost in cells grown on the
50-nm array and completely disappeared on the 100-nm
and 200-nm arrays. Vinculin staining indicated formation
of focal adhesions. Vinculin was detected and well-dis-
tributed for cells grown on the flat surface and on the
Fig. 6 SEM side-view images showing the poor cell attachment
of NIH-3T3 cells grown on the 100 nm nanodot array
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10-nm array. The amount of vinculin staining decreased for
the 50-nm array and almost disappeared for the 100-nm
and 200-nm arrays. This immunostaining indicated that the
nanotopography retarded or inhibited the assembling of
focal adhesions.
Micro-topography has been shown to be advantageous
to cell growth. Three-dimensional fibrous structures pro-
vide an in vivo-like environment that enhances the growth
of cells. Micro-scaled grooves and valleys direct the
growth of cells. In the current study, the nanotopography of
the nanodot arrays generated an apoptotic signal leading to
the suicide of cells. Since a micro-topography of 100-nm
nanofibers promotes cell proliferation and adhesion, the
apoptosis induced by the nanotopography is unexpected.
For cells cultured on 13-nm deep nanoislands, increased
cell adhesion, proliferation, cytoskeleton, and extracellular
matrix remodeling were observed [20, 21]. A reduced cell
adhesion and cytoskeletal organization was shown for cells
cultured on 95-nm deep nanoislands. Although the exact
shape and topology is different for the nanodot arrays
applied in the current study, the results from both studies
shared a common theme that a dot-like nanotopology with
dimensions at about 100 nm reduced cytoskeletal organi-
zation. Although cellular abnormality was not stated, the
result from the nanoisland study is consistent with the
current study.
Fig. 7 Effects of BSA-, FN-, and type I collagen-coating on the
nanotopography-induced apoptosis-like abnormality. Cells were
seeded on 100-nm nanodot arrays and flat wafers pretreated with
BSA-, FN-, and type I collagen. Cells were harvested on day 4. SEM
was performed to visualize the morphology of the cells (a).
Apoptosis-like events occurred were quantified by the caspase-3
activity assay (b)
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Our evidence supports the hypothesis that the formation
of focal adhesions and the reorganization of the cytoskel-
eton are part of the apoptotic pathway triggered by
nanotopography. The number of focal adhesions was
decreased for cells cultured on the 50-nm arrays and was
completely absent for cells on the 100-nm and 200-nm
arrays. The organization of actin filaments was observed in
cells cultured on flat surfaces and on the 10-nm nanodot
arrays, but was absent in cells cultured on the 100-nm and
200-nm nanodot arrays. Pretreatment with FN and collagen
forced cell adhesion and the formation of focal adhesions,
which prevented the apoptosis-like abnormality of cells
culturing on the 100-nm arrays. Since focal adhesions and
the cytoskeleton play important roles in the nanotopology-
induced apoptosis-like abnormality, it is likely that inte-
grins are the receptors mediating the suicidal signal.
Nanotopography-induced apoptosis shares some common
featureswithanoikis,theapoptosisinducedbythelossofcell
adhesion [22]. Both events were initiated at the bio-nano
interface. The loss of focal adhesions and organization of
actin filaments were key features of both phenomena. How-
ever, anoikis istriggered byforcing epithelialcells togrowin
suspension, and signaling is detectable in minutes to hours
[23]. Nanotopography-induced apoptosis-like events became
evident only after days of incubation. We noticed that cells
seemed to lose adhesion when grown on the 100-nm and
200-nm arrays (Fig. 6). Serious deformation of cells was
observed. The loss of attachment might be due to an imbal-
anced shear force caused by the uneven evaporation of sol-
vent during the dehydration process. However, cells cultured
on other nanodot arrays maintained decent adhesion with the
surface, indicating that the cells grown on the 100-nm and
Fig. 8 Immunostaining to show
organization of actin filament
and distribution of vinculin in
cells cultured on 10-nm, 50-nm,
100-nm, and 200-nm nanodot
arrays and on flat surfaces. Cells
were seeded on the arrays for
96 h before harvest. The sample
was incubated with anti-
vinculin antibody (properly
diluted in 0.5% BSA) and
phalloidin, followed by
incubating with Alexa Fluor 488
goat anti-mouse antibody
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200-nm arrays exhibited relatively weak binding affinity to
the surface. To exclude the possibility of anoikis, measuring
the number of non-adherent cells at various time intervals
will be performed in future experiments.
Nano- to micro-structure enhance surface hydrophobic-
ity [24–26]. Since hydrophobicity plays an important role in
protein folding and in the protein–protein interaction,
hydrophobicity might be an important factor in the nanot-
opology-induced apoptosis-like events. Contact angle
measurements indicated that hydrophobicity increased as
the dot-size increased (Fig. 9a). The coating of BSA elim-
inated the difference of the hydrophobicity between arrays
(Fig. 9b); this coating of BSA did not prevent the nanoto-
pology-induced apoptosis-like abnormality (Fig. 7). Thus,
factors other than surface hydrophobicity must be involved
to trigger apoptosis-like events at the bio-nano interface.
Acknowledgment
Science Council Grant NSC94-2320-B-009-003 and Bureau of
This study was supported in parts by National
Animal and Plant Health Inspection and Quarantine Council of
Agriculture Grant 95AS-13.3.1-BQ-B1 and 95AS-13.3.1-BQ-B6.
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10.1021/la034961d
Fig. 9 Contact angle measurements for the nanodot arrays. Contact
angles were measured for untreated nanodot arrays (a) and BSA-
treated nanodot arrays (b)
912Nanoscale Res Lett (2009) 4:903–912
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