Applied Surface Science 255 (2008) 357–360

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Applied Surface Science

journal homepage: www.elsevier.com/locate/apsusc

Preparation of nano-hydroxyapatite particles with different morphology and their

response to highly malignant melanoma cells in vitro
Bo Li a, Bo Guo a,b, Hongsong Fan a,*, Xingdong Zhang a
a
National Engineering Research Center for Biomaterial, Sichuan University, Chengdu 610064, China
b
West China Eye Center of Huaxi Hospital, Sichuan University, Chengdu 610064, China

A R T I C L E I N F O A B S T R A C T

Article history: To investigate the effects of nano-hydroxyapatite (HA) particles with different morphology on highly
Available online 26 June 2008 malignant melanoma cells, three kinds of HA particles with different morphology were synthesized and
co-cultured with highly malignant melanoma cells using phosphate-buffered saline (PBS) as control. A
PACS: precipitation method with or without citric acid addition as surfactant was used to produce rod-like
81.07.Wx hydroxyapatite (HA) particles with nano- and micron size, respectively, and a novel oil-in-water
81.10.Dn emulsion method was employed to prepare ellipse-like nano-HA particles. Particle morphology and size
81.16.Be distribution of the as prepared HA powders were characterized by transmission electron microscope
(TEM) and dynamic light scattering technique. The nano- and micron HA particles with different
Keywords:
Hydroxyapatite morphology were co-cultured with highly malignant melanoma cells. Immunofluorescence analysis and
Morphology MTT assay were employed to evaluate morphological change of nucleolus and proliferation of tumour
Nano cells, respectively. To compare the effects of HA particles on cell response, the PBS without HA particles
Malignant melanoma cells was used as control. The experiment results indicated that particle nanoscale effect rather than particle
morphology of HA was more effective for the inhibition on highly malignant melanoma cells
proliferation.
ß 2008 Elsevier B.V. All rights reserved.

1. Introduction influence of the degraded particles of HA with different morphol-

Hydroxyapatite (Ca10(PO4)6(OH)2, HA), due to its excellent
biocompatibility and bioactivity, is widely used in the clinic of
orthopedic, dental, and maxillofacial applications [1–3]. However,
previous studies demonstrated that HA ceramics could produce
debris or particles which would deposit between the prosthetic
interface and surrounding tissue [4,5]. Accumulated particles would
act as a stimulus to irritate cells such as monocytes or macrophages
to release inflammatory mediators, cytokines and matrix metallo-
proteinases for a long time [6,7], which would induce cytotoxicity,
pathologic bone resorption and so on [8]. Different characteristics of
HA particles, such as morphology, size and crystallinity, would cause
apparently different consequences [9].
On the other hand, it was reported that nano- or micron HA
particles had suppressive effect on the proliferation of tumor cells
[10,11]. Malignant tumor cells, especially those with high
malignancy, were characterized by their capability of rapid
proliferation, local invasion and distance migration. Since it was
almost impossible to excise malignant tumor completely, the
ogy, size and ions substitution on tumor cells, especially highly
malignant ones, was significant in clinic [9]. But to our best
knowledge, few studies focused on the biological properties of
nano-particles with different morphology, especially the response
to highly malignant melanoma cells in vitro. In this study, a
precipitation method with or without citric acid addition as
surfactant was used to produce rod-like HA particles with nano-
and micron size, respectively, and a novel oil-in-water emulsion
method was employed to prepare ellipse-like nano-HA particles.
The nano- and micron HA particles with different morphology
were co-cultured with highly malignant melanoma cells. To
compare the effects of HA particles on cell response, the PBS
without HA particles was used as control. Immunofluorescence
analysis and MTT assay were employed to evaluate morphological
change of nucleus and proliferation of tumour cells, respectively.
2. Materials and methods
2.1. Particles preparation and characterization

The method of preparing rod-like HA particles under 100 nm

* Corresponding author. Tel.: +86 28 85410703; fax: +86 28 85410246. was similar to the previous report [12], followed by freeze-drying
E-mail address: leewave@126.com (H. Fan). method to get more uniform morphology. On the other hand, the

0169-4332/$ – see front matter ß 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.apsusc.2008.06.114
358 B. Li et al. / Applied Surface Science 255 (2008) 357–360
previous experiment without citric addition was performed to
produce micron size HA particles. A novel emulsion method was
used to produce ellipse-like HA particles (which will be discussed
in detail somewhere else, and we have submitted it to Materials
Letters). To get the precise information of particle size and
morphology, the dried powders were investigated by transmission
electron microscope (TEM, JEM-100cx, JEOL, Japan). The powders
were dispersed in deionized water and then a few droplets were
put on copper grids coated with carbon film and observed under
TEM. The particle size distribution was further characterized by
dynamic light scattering technique (Malvern Co., UK). Generally,
the as prepared HA powders were ultrasonically dispersed in
deionized water to form a dilute sol, and then the sol was dropped
into the sample cell to determine the particle size distribution.
Each specimen was tested three times and the average value would
be used. Prior to cell experiment, samples were dispersed in PBS to
form sol.
2.2. Cell culture
Malignant melanoma cells (A875, received it as a gift from Dr. Z.
Hao, Department of Pathology, Sichuan University, China) were
cultured in RPMI1640 medium (Gibco, USA) supplemented with
100,000 units/l penicillin G, 100 mg/l streptomycin, and 10% fetal
calf serum at 37 8C in a humidified atmosphere of 5% CO2.
2.3. Immunofluorescence analysis
Malignant melanoma cells were placed into 6-well plates
(2  105/well) and incubated with the HA particles. Nuclear
morphology was evaluated by immunofluorescence analysis after
48 h in culture. The samples were fixed in 4% paraformaldehyde at
4 8C and washed with PBS for 5 min and 3 times. Then the
incubated cells were counterstained with DAP (40 ,6-diamidino-2-
phenylindole, dihydrochloride, molecular probes, USA) for 5 min at
37 8C, and controls were obtained by omitting the nuclear
counterstain. After washing with PBS for 5 min and 3 times,
samples were evaluated by fluorescence microscope (TE2000-U,
Japan).
2.4. Cell proliferation
Malignant melanoma cells proliferation rate was evaluated by
MTT assay. Cells, seeded in 96-well plate, were cultured with three
kinds of HA particles described above. PBS was used as control and
four parallel samples were used. After day 1, 2, 3, and 4, 20 ml MTT
Fig. 1. TEM micrographs of rod-like (a) and ellipse-like (b) HA particles.
solution (5 mg/ml) was added to each well, and plates were placed
in incubator at 37 8C for 4 h. Then, 150 ml dimethyl sulfoxide
(DMSO, Sigma) was added after supernatant medium was
removed. The absorbency value (O.D. value) was recorded at a
wavelength of 570 nm.
2.5. Statistical analysis
Significant differences between the groups were identified by
an analysis of t-test with SPSS10.0 and a level of P < 0.05 was
considered significant.
3. Results
3.1. HA particles analysis
The different morphology of nano-HA particles is shown in
Fig. 1. Fig. 1(a) exhibits the morphology of the as prepared HA
powders with citric acid addition as surfactant. The TEM
micrograph confirms that the HA particles are rod-like with the
width and length of 10–20 nm and 50–70 nm, respectively.
Fig. 1(b) shows that ellipse-like HA particles with the width of
20–40 nm and the length of 50–60 nm were produced with
emulsion route. The particle size distribution of the three samples
was further analyzed by dynamic light scattering technique. From
Fig. 2, it is examined that the particle size of rod-like and ellipse-
like HA particles is uniform. The particle size distribution of rod-
like and ellipse-like HA is about 20–120 nm, and is concentrated at
65 nm approximately. In terms of the micron HA particles
produced by co-precipitation method without citric acid, particle
size analysis shows that size distribution is from 500 to 1100 nm,
and is concentrated at about 1000 nm. It could be seen that the
results of particles size distribution were in accord with TEM
micrographs mostly since the particle size distribution measured
with this method is the size of agglomerate.
3.2. Immunofluorescence analysis
The effects of HA particles on malignant melanoma cell nucleus
were investigated by immunofluorescence analysis. In blank
control and micron sized groups, the cell nucleus are smooth
and the chromatin is uniform, as can be seen from Fig. 3(c and d).
When nano-sized HA particles were used, the morphology of
malignant melanoma nucleus changed greatly compared with the
blank control. The membrane of nucleus contracted and some cell
nucleus broke into debris, as could be seen from Fig. 3(a and b).
 B. Li et al. / Applied Surface Science 255 (2008) 357–360
Fig. 2. Particle size distribution of rod-like nano-powder (a), ellipse-like nano-
powder (b) and micron HA powder (c) by dynamic light scattering technique.
Tumor cells showed the trend of being genetic altered by small
sized HA particles, especially nano-sized ones. However, there was
little difference between the samples incubated with rod-like HA
particle and ellipse-like ones by immunofluorescence analysis, and
another analysis technique should be further applied to investigate
the effect subsequently.
3.3. Cell proliferation and statistical analysis
The results of MTT assay is shown in Fig. 4. When the three
kinds of particles were co-cultured with malignant melanoma cells
for 1 day, there were little differences among the O.D. value.
Whereas, from day 2 to day 4, the O.D. value of rod-like and ellipse-
like nano-groups was lower than the control. Statistical signifi-
cance was found between the nano-groups and the control
(P < 0.05), and no statistical significance was found between the
micron sample and the control except those were co-cultured with
tumour cells at day 3 (P > 0.05), which was consisted with our
previous studies [13,14]. On the other hand, there was no
statistical significance between rod-like sample and ellipse-like
one (P < 0.05). This meant the proliferation of tumor cells was
suppressed apparently by nano-HA particles.
4. Discussion
HA ceramics were successfully used in bone repair and
substitution application, however, the production of degradation
such as particles and debris was often regard as negative effect on
human body [15]. It was suggested that those particles would
accumulate at prosthetic interface and influence the cell behaviors
Fig. 3. Immunofluorescence images of malignant melanoma cells co-cultured with rod-like (a), ellipse-like (b) and micron (c) HA particles at day 2, PBS as control (d).
359
Fig. 4. Proliferation of malignant melanoma cells cultured with different HA
particles with MTT assay (compared with PBS without HA particles, OP > 0.05 and
*P < 0.05).
such as morphology, proliferation and cytokines production [6,16].
Bloebaum demonstrated that the cell multiplication could be
influenced by HA particles, and small sized ones had more obvious
effect [4,5]. The degraded particles of implanted hydroxyapatite
ceramics were approximately 10–60 mm, particles less than 5 mm
could induce more obvious cytotoxicity [5].
In our experiment, immunofluorescence analysis combined
with MTT assay indicated that particles about 1 mm had no
influence on malignant melanoma cells, whereas, nano-HA
particles with either rod-like or ellipse-like morphology had great
effect on tumor cells. It suggested that nanoscale effect, including
high solubility, surface energy, ion exchange capability and
polarization, changed tumor cell behaviors. It is not well know
that the molecular mechanism of cell apoptosis caused by nano-
particles although some researchers reported that up-regulated
expression of MMP2 has correlation with invasion and metastasis
of tumor cell [17]. Further studies should be carried out. Current
study indicated that nano-HA particles inhibited the proliferation
of malignant melanoma cells. Compared with micron HA sample,
particles nanoscale effect rather than particle morphology showed
inhibition on highly malignant melanoma cells proliferation.
5. Conclusions
Rod-like and ellipse-like nano-HA particles were prepared
successfully. Particle nanoscale effect rather than particle mor-
phology showed inhibition on highly malignant melanoma cells
360 B. Li et al. / Applied Surface Science 255 (2008) 357–360

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