18

CHAPTER 2

SYNTHESIS AND CHARACTERIZATION OF

HYDROXYAPATITE AND TITANIUM DIOXIDE

NANOPARTICLES

2.1 INTRODUCTION

The applications of nanotechnology in medicine have increased

enormously. Nanomaterials have been used as carriers of active

pharmaceutical drugs in delivery, targeting applications and for medical

imaging. The advantages of nanoparticle drug delivery systems are manifold:

high stability, high drug carrying capacity, feasibility of incorporation of both

hydrophilic and hydrophobic substances, and compatibility with different

administration routes (Intraperitonial, intravenous, oral, inhalation, etc).

Nanoparticles can also be designed to allow controlled (sustained) drug

release from the matrix. All of these properties could result in improvements

in drug bioavailability values and dosing frequency, and could resolve the

common problem of non-compliance to prescribed therapy. The use of

nanoparticles as drug carriers may also reduce the toxicity of the incorporated

drug. The nanomaterials used in medicine can be categorized into carbon-

based materials such as fullerenes, carbon nanotubes, inorganic nanoparticles,

and including those based on metal oxides (iron oxide, cerium oxide, titanium

dioxide, silicon dioxide, Zinc oxide etc), metals (gold and silver), and

semiconductor nanoparticles (typically cadmium sulfide and cadmium

selenide) (Fadeel and Bennett 2010).

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Hydroxyapatite (HAp), Ca10(PO4)6(OH)2, is one of the calcium

phosphate (Ca-P) based bioceramic materials which make up the majority of

the inorganic components of human bones and teeth (Bogdanoviciene et al

2006). Bioceramics are a class of advanced ceramics which are used in

medical applications (Paul and Sharma 2005). They are biocompatible, inert,

bioactive, and degradable in a physiological environment. These

charecteristics make it an ideal biomaterial. The other calcium phosphate

components are monocalcium phosphate (Ca(H2PO4)2, dicalcium phosphate

CaHPO4, α-Tricalcium phosphate α-Ca3(PO4)2, -Tricalcium phosphate -

Ca3(PO4)2, Tetracalcium phosphate Ca4(PO4)2, Octocalcium phosphate

Ca8H2(PO4)6·5H2O, and Oxyapatite Ca10(PO4)6O. HAp is highly crystalline

and the most stable Ca-P in an aqueous solution. It is also the most

biocompatible Ca-P (Kanazawa 1989). The biocompatibility, biodegradability

and the close similarity with the inorganic component of vertebrate bone

makes HAp a suitable material for biological application.

Biomaterials used in biological applications such as drug delivery

systems should be in submicron range. There needs to be a possibility for

surface modification. It should have high drug loading capacity. There should

be no toxic side effects. Nano sized HAp, when used as a carrier for the

delivery of drug and other therapeutic agents, enhances bioavailability,

predictable therapeutic response, greater efficacy and safety, and controlled

and prolonged release. The usage of HAp for a drug delivery is promising

(Ong et al 2008, Han et al 2009). HAp nanoparticles are used as a drug

delivery system for various classes of drugs. It is used for the delivery of

protein drugs (Ijnteme et al 1994), and it is used for the delivery of enzymes

(Reibeiro et al 2004). In order to attract antigen presenting cells to the

vaccination site it is used as a medium (Ciocca et al 2007). Europium doped

HAp has been used for the delivery of Ibuprofen and various classes of

antibiotics (Yang et al 2008, Ste´phane et al 2009). Recombinant protein like

20

human bone morphogenic protein 2(rh-BMP2) was also delivered in the bone

using HAp (Sotomea et al 2004). HAp is used extensively for the delivery of

chemotherapy agents. The delivery of 5-flurouracil (Santos et al 2009), and

Adriamycin (Yamamura et al 1994) have been reported. HAp is also used as

an inorganic drug (Xian et al 2009). It has high affinity towards proteins,

DNA, chemotherapy drugs and antigens (Cheng and Kuhn 2007). The usage

of HAp nanoparticles for targeted and controlled release of therapeutic agents

(targeted-delivery) in biomedical applications is promising.

Titanium dioxide (TiO2) is used extensively as an industrial

nanomaterial. TiO2 is employed on a wide scale in chemical industry. It also

finds application in the production of ceramics, as a pigment for paint and

varnish, plastics, paper, rubber, ceramics, and in the pharmaceutical, food,

and cosmetics industries (Grzmil et al 2004). TiO2 can be obtained in three

crystalline phases: anatase, rutile, and brookite. The most stable phase is rutile

and is usually obtained after annealing at temperature above 723 K (Abazovic

et al 2009). In nanoparticles, the surface energy contributes more to the total

energy than in bulk materials. The surface energy of anatase is lower than for

rutile and brookite. Anatase TiO2 possesses a wider absorption gap and a

smaller electron effective mass, which results in a higher mobility of charged

carriers and a higher efficiency in the generation of Reactive oxygen species

(ROS) (Fadeel and Bennett 2010).

TiO2 nanoparticles are one of the most promising nanomaterials

capable of a wide variety of applications in medicine and life science. It is

used in photodynamic therapies for cancers. Nano-TiO2 is chemically stable,

relatively nontoxic and environment friendly. TiO2 particles show weak or no

toxicity in vitro and in vivo. TiO2 photocatalyst kills bacterial cells in water

due to the generation of the ROS. Photoexcited TiO2 nanoparticles have

effectively induced cytotoxicity against HeLa cancer cells and human colon

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carcinoma cells. It is also used in the photo dynamic therapy for cancer (Song

et al 2006).TiO2 has been considered biologically inactive in experiments on

animals and humans (Liu et al 2010). Nanoporous TiO2 films have been used

to incorporate dexamethasone (Ayon et al 2006).

The synthesis of HAp and TiO2 nanoparticles by a wet chemical

method is discussed in this chapter. The HAp and TiO2 nanoparticles were

annealed at different temperatures and their properties have been evaluated

using X-ray diffraction (XRD), Raman spectroscopy, transmission electron

microscopy (TEM) and Dynamic Light scattering (DLS) studies.

2.2 EXPERIMENT

2.2.1 Synthesis of Hydroxyapatite Nanoparticles

Hydroxyapatite nanoparticles were synthesized by a wet chemical

precipitation reaction:

10Ca (OH)2 + 6 H3PO4 →Ca10(PO4)6 (OH)2 +18H2O (2.1)

The aqueous suspension of calcium hydroxide (Ca(OH)2) and

orthophosphoric acid (H3PO4, 85 %), both of analytical grade, were used as

reagents for the preparation. One litre of an aqueous suspension of H3PO4 (0.6

M) was slowly added drop by drop to one litre of an aqueous suspension of

Ca(OH)2 (1 M) while stirring for 2 h at room temperature (Loo et al 2008).

Concentrated NaOH was added until a final pH of 11 was obtained. The white

suspension obtained was washed using deionized water and dried in an oven

at 353 K for 24 h (Mateus et al 2007). The dried powder was annealed at 773,

873, 973 and 1073 K for 3 hours.

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2.2.2 Synthesis of Titanium Dioxide Nanoparticles

7.4 ml of titanium tetra isopropoxide was added, drop by drop, to

30 ml of 1 M HNO3 aqueous solution, and then agitated for 2 h to give a

transparent sol, in which 2.0 g TiO2 are contained. The pH of the colloidal

solution was adjusted to pH 3, with the addition of 1 M NaOH solution after

dilution of the colloid with 100 ml water, resulting in a turbid TiO2 colloid.

The suspension was agitated at room temperature, centrifuged and then

washed with distilled water. The isolated TiO2 was dried for 1 h at 373 K in

air. The resulting powder was then annealed at 573, 623, 673 and 723 K for

3 h (Robert and Weber 1999, Manivannan et al 2008).

2.3 CHARACTERISATION

2.3.1 X-Ray Diffraction (XRD) Analysis of Hydroxyapatite

Nanoparticles

X- ray diffraction (XRD) is widely used to study the structural

properties of materials. Copper Kα1 line was used, and this was filtered using

a bent quartz monochromator. The focussing monochromator geometry

results in narrow diffracted peaks and low background with highly

monochromatized X-ray beam for diffraction studies. The sample is mounted

tangent to the Siemann-Bohlin focussing circle with the scintillation counter

tube moving along the circumference of it. It is possible to record the

diffracted beam from 2º to 90º with camera mounted in different positions.

The diffractometer is connected to a computer for the collection of data and

analysis. When the grain size of a polycrystalline material is very small, say

in the nanometer range, the crystals cause broadening of the diffracted beam

due to diffraction at an angle near to, but not equal to the exact Bragg’s angle.

The crystallite size (D) and the full-width at half-maximum (FWHM) of the

diffracted line (in radian) are related by,

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D=0.λλ/ cosθ (2.2)

where λ is the wavelength of X-rays used and θ is Bragg’s angle of the peak

in degree. The above formula is known as Scherrer formula.

Figure 2.1 shows the XRD pattern of HAp nanoparticles annealed

at different temperatures. It shows the formation of single phase HAp, and the

diffraction peak matches with the JCPDS values (09-0432). The major peaks

indicate the crystalline form of HAp. At 723 K the HAp structure started to

crystallize with peaks corresponding to the (200), (111), (002), (211), (112),

(300), (202), (310), (222) and (213) reflections. After heating at 1073 K the

samples became more crystalline. Diffraction peaks of the annealed HAp

particles were narrow and well-separated as compared to the broad X-ray

diffraction peaks of as-synthesized HAp nanoparticles. It confirms that the

sample is of improved crystallanity and increased crystallite size.

Figure 2.1 XRD of HAp nanoparticles a) as synthesised b) annealed at

773 K c) annealed at 873 K d) annealed at 973 K e) annealed

at 1073 K

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2.3.2 Transmission Electron Microscope Analysis of Hydroxyapatite

Nanoparticles

The most significant characteristics of TEM is that it allows

observation of both an image and an electron diffraction pattern from the

same area. Electrons from the electron gun, either thermionic or field

emission, are accelerated to a very high voltage that is allowed to pass

through a specimen and focusing lenses. The lens-systems consist of electrical

coils to generate an electromagnetic field. The ray of electrons is first

focussed by a condenser and then it passes through the specimen, where it is

partially deflected. The degree of deflection depends on the electron density

specimen and lattice parameter of the specimen. If the intermediate lens is

adjusted so that its object plane is the image plane of the projector lens, then

an image is projected on the screen. The major advantage of TEM is that it

can resolve to the order of few Å. When a crystal of lattice spacing d is

irradiated with electrons of wavelength λ, diffracted waves will be produced

at specific angles βθ, satisfying the Bragg conditionμ

2dsinθ = n λ (2.3)

The diffracted waves form a spot diffracted pattern for the single

crystal specimen, but in the case of a polycrystalline specimen, the diffraction

pattern will look like a superposition of single crystal spot patterns: a series of

concentric rings resulting from many spots very close together at various

rotations around the centre beam spot. From the diffraction rings, one can

determine the crystal structure and lattice parameter using the following

relation:

Rd= λL (2.4)

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where R is the radius of the ring, d is the lattice spacing, and L is the camera

length. The radius of each ring is characteristic of the spacing of the

diffracting planes in the specimen and the magnification settings of the

microscopic lenses. In this research work, morphology and crystal structure

were carried out using JEOL 2000Fx-II operated at 200kV, High Resolution,

analytical TEM with a tungsten-source and a point-point resolution of 2 Å. In

order to perform this measurement, powder samples were dispersed in

methanol for 15 minutes using an ultrasonic agitator and allowed to settle for

5 minutes. A couple of drops of the suspension from the top were placed on a

carbon coated copper-supported grid and were allowed to dry. This was used

in TEM to observe the morphology and crystal structure. Dynamic light

Scattering (DLS) studies were done using Malvern Zetasizer Nano-S to find

out the particle size distribution.

Figure 2.2 shows the TEM images and DLS analysis of HAp

nanoparticles annealed at different temperatures. All the HAp samples show

spherical morphology. An increase in the annealing temperature gives the

system adequate kinetics to permit further growth of the HAp grains. The

SAED analyses confirmed the formation of the hexagonal structure of HAp

and are in agreement with the XRD results. Whisker morphology remained in

the as prepared samples annealed at 773 and 873 K. At 973 and 1073 K the

tip of the crystals became more rounded off. For the as-prepared HAp, the

size of the particle is around 10 nm (60%). It is interesting to note that with

increase in the annealing temperature, the size of the particles increase. The

size is 14 nm (70%) at 773 K, while it is 24 nm (75%) at 873 K, 31 nm (70%)

at 973 K and is 60 nm ( 72%) at 1073 K. All the samples have a narrow size

distribution.

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Figure 2.2 TEM image and particle size distribution of HAp

nanoparticles a) as synthesised b) annealed at 773 K

c) annealed at 873 K d) annealed at 973 K e) annealed at

1073 K

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2.3.3 Micro Raman Spectroscopy Analysis of Hydroxyapatite

Nanoparticles

Raman analysis is one of the powerful non-destructive technique

which can provide key information dealing with the chemical composition

and the structure of the investigated materials. Localized or extended

(mapping) investigations can be done under a microscope using micro-Raman

spectroscopy.

When photons interact with matter, such as when light is focussed

onto a sample in a microscope, it can either be reflected, absorbed, or

scattered. Raman scattered light is due to the interaction of light with the

vibrational modes of molecules. The spectrum obtained allows for

identification of the molecules and their functional groups. (Raman

spectroscopy is about the evaluation of wavelength and intensity of

inelastically scattered light from atoms and molecules). Raman scattered light

occurs at wavelengths that are shifted from the incident light by the energies

of molecular vibrations. It is a standard optical characterization technique for

studying the structural aspects of solids. Raman spectroscopic studies were

carried out using a Horiba Jobin Yvon-HR 800 UV micro-Raman setup. The

325 nm line of He-Cd laser was used as the excitation source with a

2400 grooves mm-1 grating in the backscattering geometry. A 500 m

confocal pinhole was used to obtain high resolution Raman spectra.

The micro Raman spectrum of HAp is presented in Figure 2.3.

HAp has υ1(PO4) bands, υ2(PO4) bands, υ3(PO4) bands, and υ4(PO4) bands.

The Raman spectrum of the υ1(PO4) band at 963 cm-1 is characteristic of

HAp. This mode is associated with the totally symmetric υ1(PO4) A1

stretching mode of the ‘free’ tetrahedral phosphate ion. The non-degenerate

wave number band, the symmetric stretching mode, υ1, is the most intense

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and polarized in the Raman spectrum. The phosphate modes 1048 cm-1

(υγ(PO4), and1029 cm-1(υγ(PO4)), 963 cm-1 (υ1(PO4), 608 cm-1 (υ4(PO4), 592

cm-1 (υ4(PO4)), 580 cm-1(υ4(PO4)), 443 cm-1 and 432 cm-1 (υβ(PO4) are

observed. At 1076 cm-1 one has the (υ1(CO3) mode. It is seen from the figure

that all the calcined powders have high intensity Raman peak at 963 cm-1

indicating the strong characteristic of HAp.

Figure 2.3 Raman spectrum of HAp nanoparticles a) as synthesised

b) annealed at 773 K c) annealed at 873 K d) annealed at

973 K e) annealed at 1073 K

2.3.4 X-Ray Diffraction (XRD) Analysis of Titanium Dioxide

Nanoparticles

Figure 2.4 shows the X-ray diffraction patterns for TiO2 as a

function of annealing, where the diffraction peaks correspond to the anatase

phase. From 573 to 723 K only peaks related to anatase structure were

29

evident. The existence of strong crystalline peaks at βθ values of β5.β0°,

37.80°, 38.57°, 48.04°, 53.89°, 55.06° and 62.68° corresponding to the crystal

planes of (101), (004), (112), (200), (105), (211) and (204) indicates the

formation of anatase TiO2 (JCPDS card No. 21-1272). The analysis confirms

that they are in tetragonal crystal structure without any impurity phases within

the detection limits of XRD instrument and within the scanned region 20° to

70°. As the annealing temperature increases above 573 K, the intensity of

(101) peak increases and the line width of the same decreases. This indicates

that after annealing the crystallinity of the sample becomes better and the

crystallite size increases.

Figure 2.4 XRD of TiO2 nanoparticles a) as synthesised b) annealed at

573 K c) annealed at 623 K d) annealed at 673 K

e) annealed at 723 K

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2.3.5 Transmission Electron Microscopy (TEM) Analysis of

Titanium Dioxide Nanoparticles

The TEM images and DLS analysis of TiO2 nanoparticles annealed

for 3 h at different temperature are given in Figure 2.5. TEM images confirm

the formation of spherical particles. As the annealing temperature increases,

the average particle size increases. The increase in the average particle size is

not significant up to about 723 K. This agrees well with the results of Siegel

et al (1988). Grains with much agglomeration are clearly observable from the

images of low temperature heat treated samples. The particle size of TiO2

varies from 6 to 12 nm. The SAED analyses confirmed the formation of

tetragonal structure of TiO2, and are in agreement with the XRD results. For

the as-prepared TiO2, the size of particle is around 6 nm (65%). It is observed

that with the rise in temperature, the size of the particles increases. The size is

around 8nm (70%) at 573 K, while it is around 9 nm (75%) at 623 K, around

10 nm (78%) at 673 K and around 12 nm (80%) at 723 K. All the samples

have a narrow size distribution.

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Figure 2.5 TEM image and particle size distribution of TiO2

nanoparticles a) as synthesised b) annealed at 573 K

c) annealed at 623 K d) annealed at 673 K e) annealed at

723 K

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2.3.6 Raman Spectroscopy Analysis of Titanium Dioxide

Nanoparticles

Micro Raman spectra of TiO2 samples annealed at different

temperatures are given in Figure 2.6. In the spectra of all TiO2 samples, the

dominant Raman modes can be assigned to the Raman active modes of the

anatase crystal 144 cm-1 (Eg(1)), 197 cm-1 (Eg(2)), 399 cm-1 (B1g(1)), 519 cm-1

(combination of A1g and B1g(2) that cannot be resolved at room temperature)

and 639 cm-1 (Eg(3)) (Ma et al 1998). The dependence of the shape of the

Raman peaks from the crystal dimension may have various explanations. A

pressure effect on the grains, induced by the surrounding grains or by the

surface tension, has been invoked often in similar cases. This mechanism

usually gives large frequency shifts. Moreover, Raman measurements as a

function of the external pressure show behaviour, for some of the peaks,

which is the opposite of that observed here. Therefore we believe that the

changes in the Raman line widths have a different origin.

A possible cause of the Raman line broadening could be non-

stoichiometry of the sample i.e., an oxygen deficiency or the disorder induced

by minor phases, which in titania samples obtained with other preparation

methods play an important role. In our case, the thermal treatments are

performed in air and the non stoichiometry is expected to be negligible. In

addition, Raman and XRD measurements suggest that only the anatase phase

is present (Zhang et al 2000).

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Figure 2.6 Raman spectrum of TiO2 nanoparticles a) as synthesised

b) annealed at 573 K c) annealed at 623 K d) annealed at

673 K e) annealed at 723 K

Therefore, an attempt has been made to explain the observed line

broadening with the breakdown of the phonon momentum selection rule q=0,

specific to the Raman scattering in ordered systems. In the case of crystals of

very small size, this rule is no longer valid, as the phonons are confined in

space and all the phonons over the Brillouin zone will contribute to the first

order Raman spectra. The weight of the off-center phonons increases as the

crystal size decreases, and the phonon dispersion causes an asymmetrical

broadening and the shift of the Raman peaks. A simple confinement model

may then be used to calculate the shape of the 144 cm-1 mode of the anatase at

various nano crystal sizes. For spherical nano crystals and first-order

scattering, the Raman intensity I (ω) can be written as

34

( ) 1 cos( )oq q a

bz

qd

q

Lq3

2

02

22

2)(

)2

exp()I(

(2.5)

where q is wave vector expressed in units of π divided by the value of the unit

cell parameter, L is the diameter of the particle, Γ0 is the intrinsic line width

of Raman mode at given temperature, d is the crystallite size, and ω (q) is q-

dependent phonon frequency.

By taking a spherical Brillouin zone and an isotropic dispersion

curve, the dispersion relationship for the Eg Raman mode in the anatase phase

could be expressed as (Golubovic et al 2009).

(2.6)

where ω0 is the zone-centre frequency of the Eg(1) mode at room temperature

∆= 20 cm−1, a= 0.3768 nm, and ω0= 144 cm−1.

Figure 2.7 shows the Raman intensity calculated using the phonon

confinement model for different crystal sizes (diameter 6, 6.5, 7, 8.25 and

11nm). As the crystallite size decreases, the frequency and line width of the

Eg Raman peak show a blue shift and an increase, respectively. At the

crystallite size of 6 nm, the Raman peak exhibits a significant asymmetric

broadening. Obviously, the calculated results based on the phonon

confinement model are in qualitative agreement with our experimental data.

The main Eg Raman modes of the TiO2 nano crystals annealed at various

temperatures are displayed in Figure 2.8. The broadening of the peak

becomes important only for the smallest crystals, when the confinement

involves the phonons far from the zone center. When the size is less than 3–4

nm the model cannot be used, as the details of the dispersion curve and of the

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shape of the Brillouin zone become critical. The calculated broadening and

blue shift of the Raman peak fairly describe those observed in nano crystals

with different sizes. This indicates that the phonon confinement is the

dominant mechanism responsible for the blue shift of the Raman peak in

small-sized nano crystals, and the non-stoichiometry has only a little

influence on the blue shift.

Figure 2.7 Raman intensity calculated using phonon confinement

model for different crystal sizes

Figure 2.8 Raman modes Eg of the TiO2 nanoparticles annealed at

various temperatures

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2.4 CONCLUSIONS

Hydroxyapatite and titanium dioxide nanoparticles were

synthesised. A single phase HAp and pure anatase TiO2 were obtained. XRD

analysis confirms the formation of pure HAp and anatase TiO2 nanoparticles.

As the annealing temperature increases, the particle size increases which is

very much confirmed by the decrease in the XRD peak width and the increase

in the intensity. TEM analysis confirms that the particle size increases with

the increase in the annealing temperature. Raman spectroscopy analysis

confirms the formation of pure HAp nanoparticles. In TiO2 nanoparticles,

Raman spectroscopy confirms that the phone confinement is the dominant

mechanism responsible for the blue shift of the Raman peak. The HAp and

TiO2 nanoparticles were further investigated for their in vitro anticancer

activity and their ability to be a drug carrier in targeted drug delivery systems.