High content analysis of the biocompatibility of nickel nanowires

Article abstract of DOI:10.1016/j.jmmm.2009.02.035

Nickel nanowires, 20 μm long and 200 nm in diameter, were fabricated by electrodeposition into alumina templates, and characterised by superconducting quantum interference device (SQUID) magnetometer, X-ray diffraction and scanning electron microscopy. Bi

Full text of DOI:10.1016/j.jmmm.2009.02.035

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Journal of Magnetism and Magnetic Materials 321 (2009) 1341–1345
Contents lists available at ScienceDirect
Journal of Magnetism and Magnetic Materials
journal homepage: www.elsevier.com/locate/jmmm
High content analysis of the biocompatibility of nickel nanowires
a,
Ã
a
c
b
b
Fiona Byrne , Adriele Prina-Mello , Aine Whelan , Bashir M. Mohamed , Anthony Davies ,
c
a
a,b
Yurii K. Gun’ko , J.M.D. Coey , Yuri Volkov
a
CRANN and School of Physics, Trinity College, Dublin 2, Ireland
b
Department of Clinical Medicine, Trinity College Health Sciences Centre, Dublin 8, Ireland
CRANN and School of Chemistry, Trinity College, Dublin 2, Ireland
c
a r t i c l e i n f o
a b s t r a c t
Available online 20 February 2009
Nickel nanowires, 20 mm long and 200 nm in diameter, were fabricated by electrodeposition into
alumina templates, and characterised by superconducting quantum interference device (SQUID)
Keywords:
magnetometer, X-ray diffraction and scanning electron microscopy. Biocompatibility studies of nickel
Magnetic nanowire
Nickel
nanowires with differentiated THP-1 cell line-derived macrophages were carried out. From
a
Electrodeposition
Cytotoxicity
multiparametric assay, using high content analysis (HCA), the critical time points and concentrations
of nickel nanowires on THP-1 cellular response were identified. The nanowires displayed little or
no toxic effects on THP-1 cells over short incubation times (10 h), and at low concentrations
Biocompatibility
High content analysis
(o100 nanowires per cell). Our findings indicate the potential suitability of these wires for biological
and clinical applications.
&
2009 Elsevier B.V. All rights reserved.
The interaction between nanomaterials and biological speci-
mens has been attracting much interest recently due to their
potential use in biomedical and industrial applications, but
information regarding their toxicity is limited. The ability to use
nanowires and nanoparticles as carriers for various antibodies and
fluorescent labels, thanks to advances in surface coordination
chemistry, has enabled their successful use in drug delivery and
biosensing [1,2]. The magnetic properties of these nanomaterials
have also allowed their application in cell separation and
manipulation. Meyer et al. first demonstrated the potential use
of magnetic nickel nanowires for these applications [3]. They
observed that fluorescent and non-fluorescent wires orientated in
a head-to-tail configuration when a small magnetic field was
applied, whereas magnetic nanoparticles ordered in close packed
arrays. The separation efficiency of ferromagnetic nickel nano-
wires compared to commercially available superparamagnetic
beads has also been investigated [4]. It was shown that nanowires
outperformed magnetic beads by a factor of two. This was
attributed to the larger magnetic moment and larger surface area
of the wires. The large remnant magnetisation exhibited by
nanowires enables their use in low-field environments, thus
reducing strain effects experienced by cells and maximising their
possible applications [5].
Previous studies have been carried out to examine the cytotoxicity
of nickel nanowires in vitro. After a 24 h incubation period at a low
number of nanowires to cells (ratio 1:3), Reich et al. reported a
non-toxic effect on mouse fibroblast cells after internalisation of
the wires [6–8]. This may have been due to the low number of
nanowires binding to individual cells. Prina-Mello et al. demon-
strated the internalisation of nickel nanowires by rat marrow
stromal cells (MSC), MC3T3-E1 osteoblast cells and UMR-106
osteosarcoma cells [9]. It was reported that the cell survival rate
was greater than 95% up to 5 days after internalisation. This
indicated that nickel nanowires could be used for various
biological applications with no disruption to the cellular growth
cycle.
Fabricating metal wires by electrodeposition into templates
with nanometre diameters was first demonstrated in the late
1960s [10]. It provides a method of producing large quantities
of nanowires with desired dimensions. One of the advantages of
using electrodeposition is that the wires are always continuous.
Continuity is guaranteed because if any breakage in the nanowire
occurs, then conductivity will be lost and growth of that particular
nanowire will stop [11]. The fabrication of multisegmented
nanowires composed of multiple materials along the wire with
various segment lengths enables the specific functionalisation of
different receptors of interest onto a single nanowire [12]. They
have an advantage over magnetic beads, in that the precise
location of molecular markers can be positioned on particular
segments of the nanowire.
On the other hand, with advances in fabrication, functionalisa-
tion and applications of nanowires and nanoparticles there is a
pressing need to understand their possible cytotoxic effects.
High content analysis (HCA) has proven to be a powerful tool
for the evaluation of nanoparticle interactions with in vitro
cellular systems of different origin. It is a fully automated system
Ã
Corresponding author. Tel.: +35318962171; fax: +35316711759.
E-mail address: fibyrne@tcd.ie (F. Byrne).
0304-8853/$ - see front matter & 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.jmmm.2009.02.035

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that allows for a quantitative and qualitative study of cellular
properties in a fast and high-throughput manner. HCA approach
enables cellular signaling and morphology of cells to be
automatically analysed using fluorescent-based reagents.
Dimensions of the nanowires were examined using a scanning
electron microscopy (SEM; Carl Zeiss EVO-50) operating at 10 kV.
From Fig. 1 it can be seen that nickel nanowires had a diameter of
200720 nm with an average length of 2072 mm and were
Recently, HCA has been employed to study the cellular effect of
silica-coated quantum dots (QDs) [13]. The use of this tool enabled
relatively uniform in length.
Powder X-ray diffraction (XRD) was used to determine the
crystallographic structure of the sample. A Philips X’Pert Dif-
fractometer system was used with a copper X-ray tube operating
a
comprehensive analysis of the multiple cellular features
indicating possible cytotoxicity of QDs. Recent data demonstrated
the need to understand not only the possible toxic effects of QDs
but also the mechanisms of their interaction with cells [14,15].
With the aid of HCA detailed studies of different QDs with various
cell lineages provided a quantitative analysis of their toxic effects
and localisation within cellular compartment [14,16].
at 40 kV and 40 mA. The wavelength of the strongest Cu radiation
˚
(Ka) was 1.5406 A with a corresponding energy of 8.047 keV.
A few milligrams of dried nanowire powder were placed on a
PW3064 sample spinner and data were collected in a step scan
mode from 301 to 1001 at a step size of 0.008 and a rate of
120 s per step using an X’Celerator detector. The scan data
were then analysed by comparing the peak positions and relative
intensities with standard data on nickel provided by the
International Centre for Diffraction Data (ICDD). Fig. 2 shows
the XRD pattern of the nickel nanowires. The sample had a face-
In this paper, we report the synthesis and characterisation of
magnetic nickel nanowires fabricated by electrodeposition. We
also introduce the concept of HCA as a new technique to examine
the possible cytotoxic effects of nickel nanowires in vitro. We have
chosen the model of human phagocytic cells differentiated from
the THP-1 cell line since macrophages represent the first line of
immunodefence against artificially engineered nanomaterials in
the human body. HCA is a reliable and unbiased system for
examining the cytotoxicity of nanoparticles in vitro in a fast and
standardised method. However, to date no study has been
implemented using a high-throughput method to examine the
multiple parameters indicating the possible cytotoxicity of
nanowires in vitro. A unified screening approach is essential for
the future role of nanomaterials in biomedical applications due to
their varying size, shape, chemical composition and surface
functionalisation.
0
centered cubic (FCC) structure with a lattice parameter a of
0.353 nm. There was some evidence of (111) texture.
Magnetisation measurements on the nanowire sample were
carried out at room temperature using a Quantum Design MPMS
1.
Nanowire fabrication
The experimental set-up for electrodeposition involved an
electrolyte bath containing 2 M NiSO
4
ꢀ 6H
2 3 3
O with 0.6 M H BO
and a three-electrode cell with a platinum counter electrode and
an Ag/AgCl (saturated KCl) reference electrode. Anopore inorganic
alumina membranes (Anodisc 25; Whatman, UK), 20 mm in
diameter and 60 mm thick, with 200 nm parallel pores served as
the working electrode. Firstly, a gold layer, a few hundred
nanometres in thickness, was sputtered onto the back of the
membrane to provide a conducting substrate. Nickel was then
deposited into the pores of the membrane at a potential of ꢁ0.9 to
Fig. 1. SEM image of nickel nanowires with a diameter of 200720 nm and an
average length of 2072 mm. Scale bar 100 mm.
ꢁ
1.0 V relative to the reference electrode. Electrons from the
conducting layer reduced the nickel ions to metallic form
resulting in the growth of nickel nanowires within the pores.
The length of these wires could easily be controlled by altering the
deposition time.
The nickel nanowires were removed from the template by
dissolving the alumina membrane in 1 M NaOH. The solution was
heated to 40 1C for 5 min and then sonicated in an ultrasonic bath
(Grant XB3; UK) for 1 h. The NaOH solution was removed and
replaced several times with fresh 1 M NaOH to ensure the
membrane was completely dissolved and the nickel nanowires
were liberated. Finally, the nickel nanowire stock sample was
washed multiple times and resuspended in deionised water by
centrifugation in order to remove the last traces of NaOH. The pH
was checked to confirm NaOH had been completely removed
before proceeding.
2.
Sample characterisation
The nanowire samples were characterised using a variety of
techniques in order to determine their overall dimensions,
magnetic properties, crystallographic structure and concentration.
Fig. 2. X-ray diffraction pattern of nickel nanowire powder.

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1343
XL superconducting quantum interference device (SQUID)
magnetometer with applied magnetic fields up to 5 T. From
of nickel nanowire sample onto an Si/SiO
2
wafer and visualising it
under a microscope (Nikon Eclipse LV series; UK) containing a DIC
prism positioned between two crossed polarisers. Fig. 4 shows the
comparative analysis between counting the dispersed nickel
nanowires on the wafer using a tally counter versus using a
defined algorithm on the Image-Pro software (Media Cybernetics,
UK). Data from the Image-Pro software, within a predefined object
range, demonstrated the accuracy of nanowire measurement
using a tally counter. Both methods enabled the total number of
2
ꢁ1
Fig. 3, the saturation magnetisation was found to be 40 Am kg
,
which is significantly less than the expected value for bulk nickel
2
ꢁ1
(
55.4 Am kg ). A likely source of this discrepancy is due to the
oxidation of the surface layer of nickel when exposed to NaOH
during removal of the nanowires from the alumina template.
A surface coating of oxide, approximately 15 nm thick, can explain
this reduced magnetisation. The nanowires also exhibited a
ꢁ
1
coercive field of 48 mT and
a
remnant magnetisation of
nanowires per millilitre (NW mL ) to be determined.
2
ꢁ1
0
2
.26 Am kg . The average magnetic moment per wire was
ꢁ13
2
.2 ꢂ10
Am .
3
.
Cell culture and high content analysis
A novel technique was developed using Nomarski imaging, also
known as differential interference contrast (DIC) imaging, to
determine the concentration of nanowires defined as number of
To examine the effect of nickel nanowires on cellular response,
ꢁ1
THP-1 suspension cells were used. They are a human monocytic
leukemia cell line with a diameter of 40 m. These cells were
cultured in RPMI 1640 media supplemented with 10% fetal bovine
nanowires per millilitre (NW mL ) rather then conventional
ꢁ
1
m
method of nanowire mass per millilitre (ng mL ). This was
performed in order to quantify the number of nanowires affecting
cellular response in cytotoxicity experiments. Nomarski imaging
is a type of phase contrast microscopy where an image is
produced from refractive index inhomogenities in the sample
rather than absorption inhomogenities. Light is passed through
the sample, which is made up of elements whose refractive
indices differ. As a result, the phase of the wavefront is altered.
A contrast image is produced by converting these phase changes
into amplitude changes. The procedure involved pipetting a drop
ꢁ
1
ꢁ1
serum (FBS), 2 mM L
L-glutamine and 100 mg mL penicillin–-
streptomycin (Sigma-Aldrich), to inhibit bacterial contamination.
Cells were cultured in T75 tissue culture flasks and incubated at
3
2
7 1C and 5% CO until highly confluent. In order to seed THP-1
cells in 96-well, flat-bottom plates (Nunc, USA), they were first
stimulated with phorbol 12-myristate 13-acetate (PMA) (Sigma-
Aldrich). This chemical induces cells to differentiate into adherent
macrophages and stop their natural proliferation. THP-1 cells and
ꢁ1
2
5 ng PMA mL in RPMI media were dispensed at 200
into desired wells of the 96-well plate using a Matrix WellMate
Thermo Fisher Scientific, USA). This improved the accuracy of cell
mL/well
(
plating and ensured cell viability due to quick dispensing speed.
Two hundred microlitre RPMI 1640 media were also dispensed
into the outer wells of the plate to prevent edge effects occurring.
Plates were incubated for 72 h in the above conditions to allow
THP-1 cells to adhere to the bottom of the wells.
Once THP-1 cells had successfully adhered to the wells, nickel
nanowire samples could be plated at various concentrations
ranging from 10 nanowires to every cell (10:1) to 500 nanowires
to every cell (500:1). To accurately plate the various ratios of
nanowires to cells, an initial reference cell count was carried on
specific wells on all 96-well plates. This was achieved by
ꢁ
1
incubating 1
m
g mL
Hoechst 33342 fluorescent dye (Sigma-
that stained and labelled
Aldrich) for 30 min at 37 1C and 5% CO
2
DNA and in turn made it possible to visualise the nuclei of the
viable cells. Plates were then read on HCS KineticScan Reader
(
KSR) (Thermo Fisher Scientific, USA) where the cells were
Fig. 3. SQUID magnetisation curve measured at room temperature for nickel
exposed to a UV lamp for approximately 30 ms in a controlled
environment to maintain cell viability. The cell count was
nanowires in a diamagnetic sample holder.
Fig. 4. Nomarski images of nickel nanowires taken to determine sample concentration using (a) a tally counter and (b) a defined algorithm on Image-Pro software. Scale bar
m.
50 m

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evaluated from the output data and thus the nanowire to cell
concentration was calculated ahead of the plating step procedure.
The nickel nanowire solution was sonicated for approximately
simultaneously using a number of different fluorescent dyes.
These properties include (1) cell viability, (2) nuclear morphology
and size, (3) cell membrane permeability and (4) lysosomal
mass–pH. After the desired exposure time of THP-1 cells to nickel
nanowires, each well was stained appropriately with the HitKit, as
indicated in our previous work with slight modifications [14–16].
Plates were then read on the HCS KSR using three detection
channels with different excitation filters. These included a DAPI
filter, which detected a blue fluorescence indicating nuclear
intensity at a wavelength of 461 nm; FITC filter, which detected
a green fluorescence indicating cell permeability at a wavelength
of 509 nm and a TRITC filter, which detected lysosomal mass and
pH changes with a red fluorescence at a wavelength of 599 nm.
Once the scan was completed, data were retrieved and analysed to
obtain information about the cellular properties.
1
h to obtain a uniform dispersion. The nanowire sample was
pipetted multiple times with THP-1 cells in each of the wells to
ensure a homogeneous mixture. Each of the nanowire to cell
ratios was repeated in triplicate to improve the accuracy of the
experiment. Negative controls of THP-1 cells plated with no nickel
nanowires and a positive control of THP-1 cells plated with 1 M
NiSO
of NiSO
4
solution were also included in triplicate. This concentration
was chosen to sufficiently induce cell death. To
4
thoroughly examine the effect of nickel nanowires on THP-1 cells
a time course study was also implemented. Nickel nanowires
were incubated with cells for a few hours up to a day in standard
conditions to evaluate the time dependence of wires on cell
viability.
An appropriate assay for high content analysis was selected to
examine the main factors indicating toxicity. The multiparameter
cytotoxicity 1 HitKit (Cellomics, PA, USA) is an in vitro assay that
allows changes in many cellular properties to be examined
A typical HCA output image can be seen in Fig. 5. Cell viability
and nuclear size/morphology are indicated by a blue fluorescence,
cell membrane permeability by
a green fluorescence and
lysosomal mass and pH with a red fluorescence. A specific
algorithm was written in order to extract the quantitative data,
shown in Fig. 6, from these images.
The results of the cytotoxicity studies are summarised in Fig. 6.
Data are represented as mean values normalised relative to the
untreated control cells7standard deviation for three replicates
(n ¼ 3). THP-1 cell viability can be seen in Fig. 6a while cell
membrane permeability is indicated in Fig. 6b. Nuclear morphol-
ogy/size and changes in lysosomal mass–pH were also investi-
gated (data not shown). Nickel nanowires appear to have no
considerable effect on THP-1 cellular response after incubation
times of 3 and 6 h regardless of the nanowire concentration. Cell
viability decreased by less than 30% for 500 nanowires plated to
every cell. Membrane permeability showed a slight increase
in intensity after these time points. After incubation periods of
1
5 and 24 h, THP-1 cells remained viable with a marginal decrease
Healthy cell
in cell count for 100 nanowires per cell (Fig. 6a). However, the
lethal dose concentration occurred at 500 nanowires per cell
when there was a 50% loss in cell viability for both 15 and 24 h
time points. This evidence was supported by a dramatic increase
in membrane permeability intensity indicating the cell membrane
had been compromised (Fig. 6b). The intensity increased due to a
large volume of fluorescent dye entering the cell’s nucleus, often
associated with an ongoing apoptotic response. The natural
phagocytic behaviour of THP-1 cells induced by prolonged
exposure to the nanowires and their affinity to bind to oxidised
Permeabilised cell
Fig. 5. HCA images of nickel nanowires plated with THP-1 cells for 24 h. Images
show THP-1 cells plated with (a) no nickel nanowires i.e. negative control, (b) 100
nanowires per cell and (c) 500 nanowires per cell. An increase in cell membrane
permeability, green fluorescence can be seen with increasing concentration of
nanowires. This is accompanied by a decrease in both blue and red fluorescence,
indicating cell viability and the number of lysosomes present, respectively. These
changes in fluorescence intensity are indicative of cell death.
Fig. 6. Investigation of possible cytotoxicity effects of nickel nanowires on (a) cell viability and (b) cell membrane permeability. The dose and time dependence of nickel
nanowires on THP-1 cellular response is shown.

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1345
hydrophilic surfaces, via integrins, resulted in reduced cell counts
and membrane permeabilisation. Whereas, an increase in cell
count for the 1 M NiSO positive control is due to the possible
fragmentation of the cells.
Acknowledgements
4
We would like to acknowledge financial support from CRANN,
Science Foundation Ireland (SFI) under project PR31, The EU
BIOMAGSENS and NanoInteract Projects, HRB and HEA PRTLI
Cycle 3.
References
4.
Conclusions
The results of this high content analysis study established that
[
[
1] K.K. Jain, Drug Discovery Today 10 (2005) 1435.
2] N.L. Rosi, C.A. Mirkin, Chem. Rev. 105 (2005) 1547.
there was a time and concentration dependence of nickel
nanowires on THP-1 cellular response. THP-1 cells remained
viable for 24 h with no significant decrease in cell count or affect
on cellular membrane for a concentration of 10 nanowires plated
to every cell. It was also shown that THP-1 cells remain viable for
periods in the order of 10 h even after ingesting surface-oxidised
nickel wires at concentrations up to 100 nanowires per cell in the
culture media. These results point the way to the application of
nickel nanowires for in vitro magnetic manipulation and labelling
of macrophage cells for future biological and clinical diagnostic
applications, including cell manipulation and separation.
[3] M. Tanase, L.A. Bauer, A. Hultgren, et al., Nano Lett. 1 (2001) 155.
[4] A. Hultgren, M. Tanase, C.S. Chen, et al., IEEE Trans. Mag. 40 (2004) 2988.
[
[
[
5] D.H. Reich, M. Tanase, A. Hultgren, et al., J. Appl. Phys. 93 (2003) 7275.
6] A. Hultgren, M. Tanase, C.S. Chen, et al., J. Appl. Phys. 93 (2003) 7554.
7] A. Hultgren, M. Tanase, E.J. Felton, et al., Biotechnol. Prog. 21 (2005) 509.
[8] M. Tanase, E.J. Felton, D.S. Gray, et al., Lab Chip 5 (2005) 598.
9] A. Prina-Mello, Z. Diao, J.M.D. Coey, J. Nanobiotechnol. 4 (2006).
[
[
[
10] G.E. Possin, Rev. Sci. Instrum. 41 (1970) 772.
11] K. Nielsch, F. M u¨ ller, A.P. Li, et al., Adv. Mater. 12 (2000) 582.
[12] L.A. Bauer, D.H. Reich, G.J. Meyer, ACS Langmuir 19 (2003) 7043.
13] T. Zhang, J.L. Stilwell, D. Gerion, et al., Nano Lett. 6 (2006) 800.
14] S.J. Byrne, Y. Williams, A. Davies, et al., Small 3 (2007) 1152.
15] I. Nabiev, S. Mitchell, A. Davies, et al., Nano Lett. 7 (2007) 3452.
[
[
[
[16] E. Jan, S.J. Byrne, M. Cuddihy, et al., ACS Nano. 2 (2008) 928.