Preparation of nano-hydroxyapatite particles with different morphology and theirresponse 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, Chinab West China Eye Center of Huaxi Hospital, Sichuan University, Chengdu 610064, China

Applied Surface Science 255 (2008) 357–360

A R T I C L E I N F O

Article history:

Available online 26 June 2008

PACS:

81.07.Wx

81.10.Dn

81.16.Be

Keywords:

Hydroxyapatite

Morphology

Nano

Malignant melanoma cells

A B S T R A C T

To investigate the effects of nano-hydroxyapatite (HA) particles with different morphology on highly

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

precipitation method with or without citric acid addition as surfactant was used to produce rod-like

hydroxyapatite (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. Particle morphology and size

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

morphology were co-cultured with highly malignant melanoma cells. Immunofluorescence analysis and

MTT assay were employed to evaluate morphological change of nucleolus and proliferation of tumour

cells, respectively. To compare the effects of HA particles on cell response, the PBS without HA particles

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.

� 2008 Elsevier B.V. All rights reserved.

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1. Introduction

Hydroxyapatite (Ca10(PO4)6(OH)2, HA), due to its excellentbiocompatibility and bioactivity, is widely used in the clinic oforthopedic, dental, and maxillofacial applications [1–3]. However,previous studies demonstrated that HA ceramics could producedebris or particles which would deposit between the prostheticinterface and surrounding tissue [4,5]. Accumulated particles wouldact as a stimulus to irritate cells such as monocytes or macrophagesto 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 ofHA particles, such as morphology, size and crystallinity, would causeapparently different consequences [9].

On the other hand, it was reported that nano- or micron HAparticles had suppressive effect on the proliferation of tumor cells[10,11]. Malignant tumor cells, especially those with highmalignancy, were characterized by their capability of rapidproliferation, local invasion and distance migration. Since it wasalmost impossible to excise malignant tumor completely, the

* Corresponding author. Tel.: +86 28 85410703; fax: +86 28 85410246.

E-mail address: leewave@126.com (H. Fan).

0169-4332/$ – see front matter � 2008 Elsevier B.V. All rights reserved.

doi:10.1016/j.apsusc.2008.06.114

influence of the degraded particles of HA with different morphol-ogy, size and ions substitution on tumor cells, especially highlymalignant ones, was significant in clinic [9]. But to our bestknowledge, few studies focused on the biological properties ofnano-particles with different morphology, especially the responseto highly malignant melanoma cells in vitro. In this study, aprecipitation method with or without citric acid addition assurfactant was used to produce rod-like HA particles with nano-and micron size, respectively, and a novel oil-in-water emulsionmethod was employed to prepare ellipse-like nano-HA particles.The nano- and micron HA particles with different morphologywere co-cultured with highly malignant melanoma cells. Tocompare the effects of HA particles on cell response, the PBSwithout HA particles was used as control. Immunofluorescenceanalysis and MTT assay were employed to evaluate morphologicalchange 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 nmwas similar to the previous report [12], followed by freeze-dryingmethod to get more uniform morphology. On the other hand, the

B. Li et al. / Applied Surface Science 255 (2008) 357–360358

previous experiment without citric addition was performed toproduce micron size HA particles. A novel emulsion method wasused to produce ellipse-like HA particles (which will be discussedin detail somewhere else, and we have submitted it to MaterialsLetters). To get the precise information of particle size andmorphology, the dried powders were investigated by transmissionelectron microscope (TEM, JEM-100cx, JEOL, Japan). The powderswere dispersed in deionized water and then a few droplets wereput on copper grids coated with carbon film and observed underTEM. The particle size distribution was further characterized bydynamic light scattering technique (Malvern Co., UK). Generally,the as prepared HA powders were ultrasonically dispersed indeionized water to form a dilute sol, and then the sol was droppedinto the sample cell to determine the particle size distribution.Each specimen was tested three times and the average value wouldbe used. Prior to cell experiment, samples were dispersed in PBS toform sol.

2.2. Cell culture

Malignant melanoma cells (A875, received it as a gift from Dr. Z.Hao, Department of Pathology, Sichuan University, China) werecultured in RPMI1640 medium (Gibco, USA) supplemented with100,000 units/l penicillin G, 100 mg/l streptomycin, and 10% fetalcalf 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. Nuclearmorphology was evaluated by immunofluorescence analysis after48 h in culture. The samples were fixed in 4% paraformaldehyde at4 8C and washed with PBS for 5 min and 3 times. Then theincubated cells were counterstained with DAP (40,6-diamidino-2-phenylindole, dihydrochloride, molecular probes, USA) for 5 min at37 8C, and controls were obtained by omitting the nuclearcounterstain. 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 byMTT assay. Cells, seeded in 96-well plate, were cultured with threekinds of HA particles described above. PBS was used as control andfour parallel samples were used. After day 1, 2, 3, and 4, 20 ml MTT

Fig. 1. TEM micrographs of rod-like (a

solution (5 mg/ml) was added to each well, and plates were placedin incubator at 37 8C for 4 h. Then, 150 ml dimethyl sulfoxide(DMSO, Sigma) was added after supernatant medium wasremoved. The absorbency value (O.D. value) was recorded at awavelength of 570 nm.

2.5. Statistical analysis

Significant differences between the groups were identified byan analysis of t-test with SPSS10.0 and a level of P < 0.05 wasconsidered significant.

3. Results

3.1. HA particles analysis

The different morphology of nano-HA particles is shown inFig. 1. Fig. 1(a) exhibits the morphology of the as prepared HApowders with citric acid addition as surfactant. The TEMmicrograph confirms that the HA particles are rod-like with thewidth and length of 10–20 nm and 50–70 nm, respectively.Fig. 1(b) shows that ellipse-like HA particles with the width of20–40 nm and the length of 50–60 nm were produced withemulsion route. The particle size distribution of the three sampleswas further analyzed by dynamic light scattering technique. FromFig. 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 at65 nm approximately. In terms of the micron HA particlesproduced by co-precipitation method without citric acid, particlesize analysis shows that size distribution is from 500 to 1100 nm,and is concentrated at about 1000 nm. It could be seen that theresults of particles size distribution were in accord with TEMmicrographs mostly since the particle size distribution measuredwith this method is the size of agglomerate.

3.2. Immunofluorescence analysis

The effects of HA particles on malignant melanoma cell nucleuswere investigated by immunofluorescence analysis. In blankcontrol and micron sized groups, the cell nucleus are smoothand the chromatin is uniform, as can be seen from Fig. 3(c and d).When nano-sized HA particles were used, the morphology ofmalignant melanoma nucleus changed greatly compared with theblank control. The membrane of nucleus contracted and some cellnucleus broke into debris, as could be seen from Fig. 3(a and b).

) and ellipse-like (b) HA particles.

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.

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).

B. Li et al. / Applied Surface Science 255 (2008) 357–360 359

Tumor cells showed the trend of being genetic altered by smallsized HA particles, especially nano-sized ones. However, there waslittle difference between the samples incubated with rod-like HAparticle and ellipse-like ones by immunofluorescence analysis, andanother analysis technique should be further applied to investigatethe effect subsequently.

3.3. Cell proliferation and statistical analysis

The results of MTT assay is shown in Fig. 4. When the threekinds of particles were co-cultured with malignant melanoma cellsfor 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 themicron sample and the control except those were co-cultured withtumour cells at day 3 (P > 0.05), which was consisted with ourprevious studies [13,14]. On the other hand, there was nostatistical significance between rod-like sample and ellipse-likeone (P < 0.05). This meant the proliferation of tumor cells wassuppressed apparently by nano-HA particles.

4. Discussion

HA ceramics were successfully used in bone repair andsubstitution application, however, the production of degradationsuch as particles and debris was often regard as negative effect onhuman body [15]. It was suggested that those particles wouldaccumulate at prosthetic interface and influence the cell behaviors

Fig. 3. Immunofluorescence images of malignant melanoma cells co-cultured with rod

such as morphology, proliferation and cytokines production [6,16].Bloebaum demonstrated that the cell multiplication could beinfluenced by HA particles, and small sized ones had more obviouseffect [4,5]. The degraded particles of implanted hydroxyapatiteceramics were approximately 10–60 mm, particles less than 5 mmcould induce more obvious cytotoxicity [5].

In our experiment, immunofluorescence analysis combinedwith MTT assay indicated that particles about 1 mm had noinfluence on malignant melanoma cells, whereas, nano-HAparticles with either rod-like or ellipse-like morphology had greateffect on tumor cells. It suggested that nanoscale effect, includinghigh solubility, surface energy, ion exchange capability andpolarization, changed tumor cell behaviors. It is not well knowthat the molecular mechanism of cell apoptosis caused by nano-particles although some researchers reported that up-regulatedexpression of MMP2 has correlation with invasion and metastasisof tumor cell [17]. Further studies should be carried out. Currentstudy indicated that nano-HA particles inhibited the proliferationof malignant melanoma cells. Compared with micron HA sample,particles nanoscale effect rather than particle morphology showedinhibition on highly malignant melanoma cells proliferation.

5. Conclusions

Rod-like and ellipse-like nano-HA particles were preparedsuccessfully. Particle nanoscale effect rather than particle mor-phology showed inhibition on highly malignant melanoma cells

-like (a), ellipse-like (b) and micron (c) HA particles at day 2, PBS as control (d).

B. Li et al. / Applied Surface Science 255 (2008) 357–360360

proliferation. Nano-HA particle was potential to treat cancer, andthe effect mechanism would be discussed somewhere else.

Acknowledgement

This work was financially supported by the key basic researchproject of China, contract no. G2005CB623900.

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