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Evaluation study of doping hydroxyapatite Nano composite with gold

nanoparticles

Article · March 2015

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 ISSN 2321-807X

EVALUATION STUDY OF DOPING HYDROXYAPATITE NANO-

COMPOSITE WITH GOLD NANOPARTICLES
E. Ismail1, M.B. Mohamed2, A. Mostafa3, H. Oudadesse4, G. Kamal1
1
Physics dep., Al -Azhar University,Girls branch,Cairo, Egypt
[email protected] & [email protected]
2
National Institute of Laser enhanced Sciences (NILES), Cairo University, Egypt
3
Biomaterials Department, National Research Centre, Giza, Egypt
4
University of Rennes 1, Rennes, France.
ABSTRACT
Hydroxyapatite (HA) possesses an exceptional biocompatibility and bioactivity properties with respect to bone tissue,
probably due to its similarity with the hard tissues of the body. Recently a great interest has been directed to incorporation
of bioactive HA with other materials (polymer; metals) for biomedical applications, such as synthetic bone graft. In this
study, hydroxyapatite/PVA nano-composite has been synthesized and doped with gold nano particles (NPs). Assesment
of the prepared gold NPs were established using UV-VIS spectroscopy. Furthermore, gold NP shape and size were
revealed using Transmission Electron Microscopy (TEM). Effect of doping with gold NPs on the Physicochemical
properties of the prepared HA was estimated using X-ray diffraction (XRD), Transmission Electron Microscopy (TEM),
Fourier Transform Infra-Red spectroscopy (FTIR). The thermal behaviour for the prepared composite was investigated
using Thermo Gravimetric Analysis (TGA) and Differential Thermal Analysis (DTA). The prepared gold NPs exhibited
nanosphere particles with an avarege diameter of 3-13 nm as confirmed by UV-VIS spectroscopy and TEM results. The
morphology of HA/PVA nanocomposite doped with gold NPs was found to be highly affected by gold NPs, exhibiting nano-
rod HA similar to the biological apatite as confirmed by TEM results. . The X-ray result demonstrated no change in the
phase formation even after doping process. However, the FT-IR indicates some changes in the functional groups,
especially for the OH groups. These combined results suggested that the HA/PVA nanocomposite doped with gold NPs
may be suitable for advanced tissue engineering applications.

Keywords
Hydroxyapatite, Polyvinyl alcohol, Gold NPs, composite, doping of hydroxyapatite.

Academic Discipline And Sub-Disciplines

Biophysics, Nanobiotechnology.

SUBJECT CLASSIFICATION
Materials Subject Classification

TYPE (METHOD/APPROACH)
In situ polymerization synthesis technique, Expermintal study of Hydroxyapatite composites.

Council for Innovative Research

Peer Review Research Publishing System
Journal: Journal of Advances in Chemistry
Vol.11, No. 5
[email protected]
www.cirjac.com
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1. INTRODUCTION
Hydroxyapatite (HA) has been considered as an ideal biomaterial particularly for applications in bone tissue engineering
[1]. This is due to its superior biocompatibility, bioactivity and chemical reactivity. However, the poor mechanical properties
of the synthesized HA, such as high elastic modulus and low fracture toughness, restrict its applications. Extensive efforts
have been made to produce synthetic nano-HA materials through different methods [2-4]. However, the biomimetic
approach to produce HA powder has the challenge to provide the desired characteristics such as high specific surface
area, fine grain size and mono-disperse size distribution with very limited agglomeration [5]. Thus, many researches
focused on the synthesis of HA/polymer composite to retain the useful bioactive properties as well as enhancement of the
mechanical properties [6-10]. These composites have attracted much attention as materials suitable for repairing and
substituting for hard tissues. Their clinical applications are gradually expanding because of their excellent safety,
osteoconductive properties and similarity to the mineral components found in hard tissues [11-14]. Poly (vinyl alcohol)
presents many excellent properties such as chemical stability, the availability, good biocompatibility. It has been widely
studied as a potential artificial replacement material [15-17].
Recent in vitro studies showed that gold nanoparticles (Au NPs) have no toxic effect on human cells [18]. Therefore,
it has received tremendous interest for modern biomedical sciences. Many scientisits had reported different preparatin
methos for gold NPs synthesis in a different range of size and shape distributions via different techniques such as
citrate reduction of HAuCl4 in water [19, 20], seed-mediated growth method [21], metal vapour synthesis, electrochemical
method through gold ionization and reduction [22]. Citrate reduction method has been selected for this work.
In this work, we aimed to study the effect of doping hydroxyapatite/PVA composite with gold NPs. Characterization of the
synthesized nano-composite had been done using different techniques. The physic chemical propreties were evaluated
using Perkin Elmer Lambda 40 double beam UV-visible Absorption spectrophotometer, Philips PW3710 diffractometer
with Cu Kα radiation, TEM equipment by Philips CM-20 operated at 200 KV and FTIR (BRUKER EQUIPED 55). While the
thermal characterization technique that has been used was the differential thermal analyser Labsys 1600.

2. MATERIALS AND METHODS

2.1 Chemicals:
Tetrachlorauric acid H(AuCl4).H2O . F.W 357.79 [Electron Microscopy Sciences, USA]; tri-Sodium Citrate 2- hydrate PA-
ACS (C6H5Na3O7.2H2O) M.W.294.10 [Panreac Quimica, Barcelona, Spaña]; Polyvinyl alcohol (PVA) M.W. approx. 
77000 [Loba Chemicals, India]; Calcium chloride dehydrate (CaCl 2.2H2o) (extra pure AR) M.W.147.02 [Sisco Research
Laboratories, India]; Di-Ammonium hydrogien Phosphate (Grade:LR) M.W.132.06 [Arabian Medical & Scientific
Lab.Sup.co., U.A.E.]; Sodium Hydroxide [Modern Lab., Egypt]
2.2 Synthesis of Gold NPs
Gold NPs were prepared by citrate reduction method of HAuCl 4.H2o. One gram of PVA was dissolved in 30 ml H2O. After
that, a solution of 0.05 M Citrate was added to the previous PVA solution. The mixture was left for continuous stirring. An
-3
aqueous solution of (5x10 M) HAuCl4.H2O was added to the previous mixture. The observation indicated changing in the
solution colour from pale yellow to deep red. The solution was left for stirring additional 10 min .
2.3 Synthesis of HAA nano-composite
Hydroxyapatite/PVA composite have been prepared according to our method that has been published before [23-25]. The
“In situ” preparation of nano-hydroxyapatite n-HA was carried out in the presence of polyvinyl alcohol (PVA). Calcium
o
chloride di-hydrate solution (1.388 M) slowly added to the PVA solution under continuous stirring for 10 min at 60 C. The
pH was adjusted to 11 by sodium hydroxide. After that, a solution of di-ammonium hydrogen phosphate (0.833 M) was
o
added gradually to the previous mixture. pH was adjasted again to 11. The mixture left for 3 hours stirring at 90 C. The
sample was lifted to age at room temperature for 24 hrs. Then, centrifuged at 4500 rpm , washed with dist. Water and
o
dried overnight in an oven at 60 C.
2.4 Synthesis of gold NPs doped HAA nano-composite
-3
A stock of (5x10 M) gold NPs have been doped to one gram of hydroxyapatite/PVA composite (HAA). The mixture was
o o
kepted into the ultrasonic path for 30 min at 60 C., thereafter, dried at 60 C for 24 hours. The new composite denoted as
HAU.

3. Results and discussion

3.1 UV-Visible spectroscopy
The very small size of nanoparticles (< 100nm) imparts a large surface to volume ratio, and thus physical and chemical
properties are very different from those of the same material in the bulk form. These properties include enhanced
photoemission, high electrical and heat conductivity, and improved surface catalytic activity [26, 27]. Gold NPs have
unique optical properties in the visible region [28]. The origin of the unique optical properties of Au NPs is a phenomenon
known as Surface Plasmon Resonance (SPR). The Au NP has to be much smaller than the wavelength of the incidence
wavelength, as referred to as the quasi-static approximation. These oscillations are known as SPR; they occur within
visible frequencies and result in strong optical absorbance and scattering properties of Au NPs [29]. UV–visible absorption
spectroscopy is the most widely used method for characterizing the optical properties and electronic structure of

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 ISSN 2321-807X
nanoparticles, as the absorption bands are related to the diameter and aspect ratio of metal nanoparticles. Figure (1)
represents the UV absorption spectra of the prepared Au NPs stabilized by citrate reduction method. The spectra revealed
a typical surface plasmon resonance band at 520 nm which is characteristic of spherical Au NPs [30]. This result will be
confirmed by TEM resultus.

0.35

0.30

520 nm
Absorbance (a.u.)

0.25

0.20

0.15

0.10

450 500 550 600 650 700

Wavelenght (nm)

Fig. 1: UV-vis spectra for the prepared gold NPs.

3.2 X-ray diffraction analysis

X-ray diffraction (XRD) patterns of HAA and HAU samples are shown in Figure (2). The results revealed the presence of
all major HA peaks, matching the ICDD standard for HA (JCPDS 09-432). After doping with gold NPs, the pattern is found
to be nearly similar and correspond to the hexagonal HA crystal (JCPDS 9-432). The XRD results show increasing in the
crystallinity after doping with Au NPs. However, there is no appearance of peaks corresponding to Au NPs [31]. The
disappearance of Au peaks may be due to the completely encapsulated of gold NPs within HA/PVA nanocomposite . No
other calcium phosphate phases were detected indicating the formation of pure hydroxyapatite phase.

5000
211 HAU
4500
HAA
4000

3500

3000
112

2500 222
002 300

2000 202 213

312
310
321 004 511
1500

1000

500

-500
10 20 30 40 50 60 70 80 90 100 110

2
Fig. 2 : X-ray diffraction (XRD) patterns of HAA and HAU samples

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3.3 Transmission Electron Microscopy results
TEM image in figure (3 - a), shows highly dispersed sphere gold NPs with a diameter ranging from 3-13 nm. Also, HAA
sample seemed to be nearly elongated sphere in shape with a diameter ~ 40 nm as indicated in figure (3 - b). Interesting
result has been revealed in figure (3 - c) after doping with gold NPs. It revealed rod shaped hydroxyapatite nano-
composite nearly similar to the biological apatite. These results may be explained due to polymer presence, which could
have altered the hexagonal crystal structure of HA which in turn affected on shaping the hydroxyapatite nanocomposite
sample. It could be deduced that, the particles tend to preferentially grow along the <002> plan direction as described by
Zhang and Lu. [32]. Actually, the presence of PVA only is not enough to acheive rod shaped hydroxyapatite. However, the
doping process with highly dispersed sphere gold NPs facilitated the formation of rod like shape. Also, the TEM results
shows that the gold NPs has been deposited on the surface of HAA as seen in figure (3 - d).

(a) (b)

(c) (d)

Fig. 3: TEM images of (a)- gold NPs, (b)- HAA composite, (c) and (d) HAU composite samples
3. 4 Fourier Transform Infrared Spectroscopy results
The main constituting elemental groups of prepared powders determined by FTIR spectroscopy. FTIR spectra of samples
HAA and HAU revealed in Figure (4 - a, b), respectively. It is clear that, both samples exehibited the same characteristic
-2
bands for the nano-crystalline HA as reported before [33]. They showed bands corsponding to carbonate (CO3 ) v3
-1 -1 -1
stretching mode (around 1403 and 1452 cm ) and of phosphate at (1039, 1086 cm ) [34]. The band at 864 cm
-2
represented the CO3 v2 in plane bend. The presence of carbonate inclusions attributed to the air dried atmosphere so
-1 3-
that a little carbon dioxide from air was integrated [33]. The band at 566 cm is due to apatite phosphate PO4 [35]. The
−1
broad band at 3423 cm assigned to hydroxyls (νOH) stretching mode due to the strong hydrogen bond of intermolecular
−1
and intermolecular type of PVA [36, 37]. The band at 2930 cm was attributed to alkyl stretching mode (νCH), while it
-1 −1
shifted to 2940 cm in HAU due to the effect of doping with gold NPs. The band at 607 cm is attributed to the OH
structural band of hydroxyapatite [38]. Due to the doping process of Au NPs; the bands revealed a little shift and increase
-1 -1
in the intensity of the OH bands around 3451 and 1653 cm . Also a new band has been observed at 1294 cm which may
be attributed to the vibrational mode of δ (O C–O) of the tri-sodium citrate reduced gold NPs [39].

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1.0
-2

HAU
CO
0.8 3

-2
0.6 CO
3
(b)
o=c-o
OH
0.4 PO4
3-

0.2 3- OH
PO4
OH
absorbance

0.0
0 500 1000 1500 2000 2500 3000 3500 4000 4500

1.0

0.8 HAA
-2 OH
CO -2
0.6 3 CO (a)
3

0.4 OH
OH
3-
PO4
0.2
3-
PO4
0.0
0 500 1000 1500 2000 2500 3000 3500 4000 4500
-1
Wavelenght cm

Fig. 4 : FTIR spectra of composite samples (a)- HAA, and (b)- HAU.
3.5 Thermal behavior of the prepared gold NPs doped HA/PVA nanocomposite
Figure (5) represents the results of TGA and DTA for HAA and HAU nanocomposites. The thermo gravimetric analysis of
o o o
the prepared nano composites powder was carried out between 35 C and 1200 C in air with a heating rate of 20 C /min.
The results revealed a clear variation in the total weight loss from 16 % to 55% for HAA and HAU composites, respectively
o o
after heating up to 1200 C. For HAA sample that the first stage of the weight loss up to 100 C corresponding to the
o
removal of adsorbed water humidity. A second weight loss 200- 300 C is due to removal of lattice water. While 400 – 650
o
C owing to the decomposition of main chain of PVA [40]
For HAU sample the results indicate strong removing of adsorbed and lattice water at the same temp. This result
explained the increasing in the total weight loss for this sample up to 55%. This result is in agreement with the FTIR result,
indicating the strong effect of doping with gold NPs on the effective OH function groups of hydroxyapatite sample.

3
HAA 0

2 -2

-4
heat flow (µV)

1 -6
Dm(%)

-8

0
-10

-12
-1
-14

-16
-2
100 200 300 400 500 600 700 800 900 1000 1100 1200

T(°C)

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2 10

1 HAU
0

0
-10

-1

Heat flew (V)

-20
-2

Dm %
-30
-3
-40
-4

-50
-5

-6 -60

0 100 200 300 400 500 600 700 800 900 1000 1100 1200
o
TC

Fig. 5 :TGA and DTA for the prepared composites HAA and HAU.
In the DTA curves several steps are observed. The first step, showing a small decrease in weight, is associated with the
o o
adsorbed water-removing when heated above 90 C [41]. DTA appeared from 200 to 500 C corresponding to the loss of
o
lattice water partly and/or hydroxyl ions (loss until 400 C), according to other authors [42-44]. For HAU composite,the
temperature shifts to a higher temperature, Indicating the positive effect of doping with gold NPs whithout altring the
o
stability of the composite. Also, a strong appearance of an endothermic peak at 290 C could be attributed to the
evaporation of lattic water. The effect of doping on the OH group in HAU composite has been confirmed in the FT-IR
o –2
result. The third step (700-900 C) was attributed to the degradation of PVA matrix releasing CO2 gas or due to CO3
decomposition [41, 45].

4. Conclusion
In this work, HA/PVA nanocomposite was sucssfuly doped with gold NPs. The TEM results revealed the positive effect on
doping with gold NPs facilating the formation of rod shaped nano-HA which is similar to the biological apatite. In addition to
that, doping with gold NPs demonstrated no effect on the formed phase of HA as confirmed by XRD measurments. From
The FTIR result. its clear the enhancement of the hydroxyl groups due to doping with gold NPs. As the main role of
hydroxyl groups in HA structure is balancing the stoichiometric amount of Ca/P for stabilizing the structure to prevent
phase transformations. So that, doping with Au NPs enhance stabilizing the formed phase and degree of crystallineity.
These results also confirmed by thermal analysis.

Acknowledgment
This research program was generously supported by grants from the Egyptian National Research centre, Academy of
Scientific Research and Technology, Egypt and Rennes, France. The author is also affiliated to the Physics Dept., Faculty
of science "girls branch", Al Azher University, Egypt. She is thankful to Mr. Yann Legal, Rennes 1 University Rennes,
France for his assistance in the charachterization process.

Future work
In-vitro study of the bioactivity of this composite. Studying the effect of doping HA with different conc. of gold NPs. and
evaluate the effect on the physical properties of the composite.

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Author biography
Mrs. Enas Ismail
- Assistant lecturer in the Faculty of Science "Girls branch", Al Azher
University, Egypt.

- PhD Student at Collegue of Science, Engineering and Technology,

university of South Africa, South Africa.
Education:

- Master degree (M. Sc.) in Biophysics, specialist in material science,

physics dep., Al-Azhar University [2011]

- B.Sc. of science in Biophysics, Excellent with honor degree, physics

dep., Al-Azhar University [2006]
I am interesting in; nano-materials, bio-materials, nano-biotechnology, green
chemistry, medical applictions.

Corresponding auther e mail: [email protected] & [email protected]

http://www.linkedin.com/profile/view?id=247907437&trk=nav_responsive_tab_p
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