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Room temperature synthesis of highly hemocompatible hydroxyapatite, studyof their physical properties and spectroscopic correlation of particle size†

Nagaprasad Puvvada, Pravas Kumar Panigrahi and Amita Pathak*

Received 18th August 2010, Accepted 26th August 2010

DOI: 10.1039/c0nr00611d

Needle shaped nanoparticles of hydroxyapatite (HA) have been synthesized at room temperature using

orthophosphoric acid as the source of (PO4)3� ions, while calcium chloride, the calcium source, is

suitably complexed with citric acid/tartaric acid/acetic acid. The presence of ligands inhibits the growth

along [001] and [100] directions of the crystal and thus, helps in formation of needle shaped

nanoparticles. The chemical compositions of the samples have been established through AAS and

FTIR spectroscopy, while the crystallinity has been assessed through XRD and by the spectral changes

in the y1 and y3 frequencies of the phosphate group in the respective FTIR spectra. The particle sizes of

the samples have been determined from line broadening studies and correlations have been established

between the curve fitted percentage area of FTIR and full width half height (FWHH) of the XRD

peaks. TEM studies revealed the particle to be needle-shaped with a length and diameter in the range of

20–65 nm and 4–11 nm respectively. Changes in the surface charge of the water dispersed HA samples

have been determined at different pH and the isoelectric point for the samples have been found in the

range of 3.1–3.4. Finally, the morphology, surface area and hemocompatibility characteristics of the

HA samples, prepared by using different complexing agents, have been compared.

Introduction

Hydroxyapatite (HA), a mineral with hexagonal symmetry, is the

most stable form of calcium phosphate at room temperature and

in the pH range of 4–12.1 There have been extensive efforts to

synthetically produce HA in the nanocrystalline form since, it is

the prototype of biological apatites that occur as the main inor-

ganic component in bone, teeth and other calcified tissues.2 The

properties of synthetically prepared HA have been reported to be

influenced by the size and morphological characteristics of their

particles.3 For example, smaller crystal size and more imperfect

crystals, being subjected to dissolution,4,5 may affect the extent of

bone loss in osteoporosis and other metabolic diseases.6 HA in the

nanocrystalline form, enhances densification and improves the

fracture toughness, and thus finds application as a bioactive and

osteoconductive bone substitute material in clinical surgery. It is

essential that the biomaterials used for drug delivery and

biomedical application needed to be hemocompatible. This can be

analyzed by hemolytic assay using rat red blood cells.7,8 Being a

biocompatible material, they are promising drug delivery systems

for the delivery of antitumor agents and antibodies in the treat-

ment of cancer.9–11 HA has been used for the removal of numerous

heavy metal ions (especially lead and cadmium ions) from waste

water through ion-exchange process.12

The method of preparation, along with the type of reagents

used and control of the experimental parameters are known to

affect the size and morphology of the particles, and consequently

influence the properties of the final product. Therefore, HA

Department of Chemistry, Indian Institute of Technology Kharagpur,Kharagpur, 721302, West Bengal, India. E-mail: ami@chem.iitkgp.ernet.in

† Electronic supplementary information (ESI) available: Table S1 andFig. S1–S5. See DOI: 10.1039/c0nr00611d

This journal is ª The Royal Society of Chemistry 2010

sample with desired properties could be tailored through

appropriate choice of the preparation route. Various methods

that have been reported for the preparation of nanocrystalline

HA include; chemical precipitation,13 in some cases followed by

mechano-chemical,14,15 spray drying,16 electro-deposition,17

hydrothermal method,18 sol–gel,19 micro emulsion,20 wet chem-

ical methods incorporating a freeze drying step21 and microwave

irradiation.22 The wet chemical process based on precipitation

route, is however the most convenient and commonly used

process for the synthesis of nanocrystalline HA material. The

process is simple, easy to use, and generates nanocrystalline HA

at low temperatures. The process is also suitable for the precip-

itation of the appreciable quantities of apatites necessary for

developing ceramic/ceramic, ceramic/metal and ceramic/polymer

nanocomposites,23,24 which are widely used in medicine stoma-

talogy for repair of bone tissue.

In most of the conventional precipitation methods, the factors

governing the precipitation, such as the pH, the temperature and

the Ca/P mole ratio etc., are however not always precisely

controlled, which results in the formation of HA nanocrystals

with varying morphologies and sizes. Rodriguez-Lorenzo and

Vallet-Regi25 et al. have used the conventional precipitation

method to produce nanosized HA powders with variable higher

dimensions by using (NH4)2HPO4 as a phosphate source. Van

Der Houwen et al. have synthesized calcium phosphate with

citric acid and acetic acid as a complexing agent under titration

method by using sodium hydrogen phosphate as a source of

phosphate ion at pH 7.12 Based on the understanding that the

grain-growth during the precipitation process could be con-

trolled by capping the nuclei with organic complexing agents,

Pramanik et al. synthesized nanoparticles of hydroxyapatite

through chemical precipitation method by using various com-

plexing agents like triethanolamine (TEA), ethylenediamine

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tetraacetic acid (EDTA), diethanolamine (DEA) and ethylene

glycol (EG). They found that the smallest particles with 5–8 nm in

diameter and 30–56 nm in length were formed, when TEA was

used as capping agent, whereas the biggest particles with 12–16 nm

in diameters and 80–120 nm in lengths were formed by using EG as

capping agent.13 The specific surface areas for these particles

ranged between 97 and 64 m2/gm respectively. The process is con-

tributed by grain growth during the precipitation, which gives rise

to particles with high aspect ratio. This problem can be overcome

by arresting the growth of the nuclei using an organic complexing

agent.26 Chemisorption of the organic ligands to the surface of the

nucleating particles may inhibit the growth of the precipitating

crystal in all the three dimensions but may, on the other hand,

facilitate the preferential growth in one direction, consequently

leading to formation of HA particles with higher aspect ratio (>4).

Besides, the functional groups from the complexing agent may

functionalize the surface of the nanocrystals, and make them more

acceptable to the host tissues in biological applications. Hence, our

aim is to prepare agglomerate free, less particle size of HA samples

with high surface area.

The apatite crystal size has been determined by X-ray diffrac-

tion but directly cannot be applied to microscopic samples with

rapid spatial variation in mineral structure such as biological

tissue specimens. However, structural information of the biologi-

cally important apatite analogues, which are microscopic samples,

can be readily obtained from IR spectroscopy. Here, we depict an

IR-spectra based method to assess the crystallinity of HA mineral,

based on the changes in the phosphate spectral region such as 900–

1200 cm�1. This method can also be applied to the spectra of

biologically applicable hydroxyl, carbonate, and fluoride substit-

uted apatites.27 Fourier self-deconvolution, curve fitting and

derivative spectroscopy technique was recently utilized to analyze

the spectra of both synthetic as well as biological calcium phos-

phates.28,29 By using curve fitting, the percentage area of one of the

subcomponents in the y1 and y3 phosphate (900–1200 cm�1) region

is correlated with the changes in the particle size along the c-axis

dimension which is measured through XRD line broadening

analysis30 of the synthesized HA material.

In this paper, effort was made to synthesize nanosized HA

particles through precipitation method starting from phosphoric

acid and calcium chloride in presence of ammonia solution and

different organic modifiers such as, acetic acid (AC), tartaric acid

(TAT) and citric acid (CIT). Synthesized HA was characterized by

FT-IR, XRD, SEM, HRTEM, BET, AAS, Zeta potential and

Hemolytic assay. Further correlations between infrared subcom-

ponents in the y1 and y3 phosphate region and changes in X- ray

parameters were carried out. Finally, control growth mechanism

was discussed for the prepared HA in presence of ligands.

Experimental section

Materials and methods

All the chemicals were used of analytical grade and available

from commercial sources. CaCl2 (Merck Ltd, Mumbai, $ 98%),

Glacial acetic acid (Merck Ltd, Mumbai, $99.5%) Tartaric acid

(Merck Ltd, Mumbai, 99.7%), Citric acid (Merck Ltd, Mumbai,

98%) phosphoric acid (Merck (India) Ltd, Bombay, 85%) and

ammonia (Merck Ltd, Mumbai, 25%) were used.

2632 | Nanoscale, 2010, 2, 2631–2638

Synthesis of hydroxyapatite nanoparticles

HA sample was synthesized by using calcium chloride and phos-

phoric acid as the source of calcium and phosphate ions respec-

tively. Aqueous solution of calcium chloride (0.1M) and

phosphoric acid (0.5 M) were mixed together as per required

stoichiometry maintaining that Ca/P mole ratio is at 1.67. The pH

of the resultant solution was maintained at 10.5 by adding con-

centrated ammonia to obtain white precipitate. It was then dried

at 80 �C in a vacuum oven and grounded to fine powders and then

characterized by different techniques. The resulted sample was

named as HAP. The procedure for the preparation of HAP was

further repeated using aqueous solution of chelated complexes in

the starting solution by using three different ligands such as, acetic

acid (AC), tartaric acid (TAT) and citric acid (CIT) and the

resulted samples were respectively named as HAPAC, HAPTAT

and HAPCIT. In the above synthesis procedures, the required

stoichiometric ratios of calcium to ligand were maintained. A

cloudy solution was obtained after about 5 min, when ammonia

solution was added into the resultant solution, maintaining a pH

around 10.5 (The organic modifiers were added to calcium ions

solution before adding the phosphoric acid and ammonia solution

in order to control the growth process).

The possible overall chemical reaction involved in the process

can be represented as follows:31

10Ca2+ + 6PO3�4 + 2 OH�/Ca10(PO4)6(OH)2 (i)

Eqn (i) may include the following set of intermediate reaction

steps, shown by the eqn (ii)–(v).31–33

CaCl2 + 2H2O/CaCl2$2H2O (ii)

CaCl2$2H2O + X R–COOH/Ca(RCOO)X + 2Cl� + (X + 2)H+

+ 2OH� (iii)

10 Ca(R–COO)X + 6H3PO4 + 2OH�/6CaHPO4$2H2O +

4Ca2+(R–COO�)Y + (X � Y)R–COOH (iv)

6CaHPO4$2H2O + 4Ca2+ + 8OH�/Ca10(PO4)6(OH)2 +

6H2O (v)

It is known that anhydrous calcium chloride reacts with water

molecule to form hydrous calcium chloride (eqn (ii)). This

hydrous calcium chloride then reacts with carboxylic acid

groups, which is present in the organic modifiers used in the

different reaction mixtures, to form calcium carboxylate complex

according to eqn (iii). The reaction of Ca(R–COO)x with H3PO4

produces an water soluble compound, i.e., CaHPO4$2H2O,

which is also reported as an intermediate in the modified Gee and

Deitz method.32 The intermediate formed, in eqn (iv), finally

generates HA in the presence of ammonium hydroxide (eqn (v)).

Characterization of nano sized hydroxyapatite

The functional group analysis in the as-dried powder of HA were

carried out using Fourier transformed infrared (FTIR) spec-

troscopy (Perkin-Elmer Spectrum RXI instrument, within the

scan range 4000–450 cm�1). Phase analysis of the powders were

This journal is ª The Royal Society of Chemistry 2010

Table 1 Assignments of the functional groups to the observed infraredfrequencies (cm�1) for different synthesized HA samples

Assignment(hydroxyapatite)

LiteratureIR bands HAP HAPAC HAPTAT HAPCIT

O–H stretching 3571 3566 3565 3565 356563433,34 635 634 635 637

PO–H stretching 236535 2366 2364 2368 2367C]O (carbonate ion)

stretching139835 1400 1400 1406 1400

PO stretching (y3) 109636 1094 1091 1091 1091PO stretching (y3) 103237 1037 1036 1034 1034PO stretching (y1) 96234–37 962 962 963 963PO stretching (y2) 47237 471 469 477 471PO bending (y4

0 0 0) 56334,36 564 564 563 564PO bending (y4

0) 60334,37 603 603 603 603

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carried out by X-ray diffraction (XRD) using Cu-Ka radiation

over 2q range of 20�–60� at a scan rate of 1.1� min�1 and with a

sampling interval of 0.02 at 30 mA and 40 kV by using Philips

PW 1710 diffractometer. The powders aggregations in the

samples were analyzed by digital imaging and scanning elec-

troscope using the model JEOL JSM-5800 microscope at an

accelerating voltage of 5 kV. The synthesized HA nano-

particles morphology and the particle size were measured by

high-resolution transmission electron microscope (HRTEM) of

JEOL JEM-2100 model with an acceleration voltage 200 kV.

Particle size distribution was performed by a Laser 90 Plus

particle size analyzer. Specific surface area measurements have

been done by using Quantachrome Instruments (Autosorb-1,

Model. No. ASI-C-9) BET surface area analyzer. Surface charge

of the various samples has been investigated through zeta

potential measurement using Zetasizer-4, Malvern instruments,

U K. Atomic absorption Spectroscopy (AAS) has been per-

formed to determine the concentration of calcium ion in HA

samples by using Perkin Elmer, AAnalyst 700. Hemolytic activity

was determined by measuring the absorption at 570 nm (Biorad

Microplate reader 5804R).

Fig. 2 XRD patterns of the synthesized hydroxyapatite samples; (a)

HAP (b) HAPAC (c) HAPTAT (d) HAPCIT.

Results and discussion

In order to analyze the functional groups present in the various

synthesized HA samples, FT-IR spectroscopy was carried out

after overnight drying in vacuum oven at 80 �C. KBr pellets of

the samples were prepared using 0.5 mg sample/100 mg KBr

mixture by crushing and making translucent pellets in mechan-

ical die press. Fig. 1. shows the FTIR spectra of different

samples, recorded with in the scan range of 4000–450 cm�1. The

observed bands were assigned to the corresponding possible

functional groups and were listed in Table 1.

The phase analysis of hydroxyapatite nanoparticle was

analyzed by XRD (Fig. 2.). All the XRD peaks were indexed to

the lattice planes of hexagonal crystal structure with P63/m

symmetry of synthesized HA nanoparticles with various ligands.

The diffraction data are consistent with JCPDS file no. 09-432.

The system has hexagonal symmetry and unit cell representation

as depicted in schematic diagram in Fig S1.† The XRD peaks are

Fig. 1 FTIR spectra of hydroxyapatiteprepared (a) in the absence of ligand,

with different ligands; (b) acetic acid (c) tartaric acid and (d) citric acid.

This journal is ª The Royal Society of Chemistry 2010

broad in nature, which may be attributed to the lattice strain and

low crystalline size of the formed apatite.38 The degree of crys-

tallinity (Xc), corresponding to b002 is FWHH (�) of reflection

(002) was evaluated by using the relation Xc ¼ k

b002

� �3

; where k

is constant and found to be 0.24 for a very large number of

different HA powders.39 Crystallinity degree is decreased as

shown in Table 2. The synthesized HA with various ligands

nanoparticles have broader XRD peaks indicates nano-

crystalline nature. The crystallite size has been calculated by

using Scherrer’s equation, i.e. D ¼ 0:9l

bcos q; where D is the crys-

tallite size, l is the wavelength of the target material (1.5406 �A),

b is full width at half height (FWHH) in radians, and q is

Table 2 Summary of particle size and cell parameters calculated fromXRD patterns of the synthesized hydroxyapatite samples

SampleNo.

Ligandsused

Particlesize/nm

Crystallinity(Xc)

Latticeparameters

a c

1 None 17–24 0.21 9.4156 6.87042 AC 15–21 0.18 9.4172 6.85273 TAT 13–17 0.15 9.4225 6.84364 CIT 11–15 0.13 9.4175 6.8592

Nanoscale, 2010, 2, 2631–2638 | 2633

Fig. 4 The second order derivative plots obtained from FT-IR spectra

shown in Fig. 3.

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diffraction angle. The order of the crystalline size obtained

using various ligands were found to be in the order: HAP >

HAPAC >HAPTAT > HAPCIT. Hexagonal cell parameters

(a and c) has been calculated by using the following relationship

d ¼ 1ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi4

3:h2 þ hk þ k2

a2þ l2

c2

r . The cell parameters are shown in

Table 2.

Earlier X-ray diffraction technique was widely used as the main

method for determination of apatite crystal size, while this method

though directly cannot be applied for microscopic biological

samples with spatial variation of crystal structure.30 The broad

nature of this contour in biologic HA results from factors such as,

vacancies in the HA lattice and lowering symmetry due to

substitute ions either in the HA lattice or on the surface of the

particles.28,29 Spectral changes of the phosphate contour in poorly

crystalline biologic apatite’s are characterized by determining the

variations in the broad FT-IR contours of the poorly crystalline

HA materials (Fig. 3.). Data reduction techniques such as Fourier

deconvolution, curve fitting, and derivative spectroscopy were

used for the determination of spectral structure correlation.

Second derivative spectroscopy27 was utilized in the assignment of

the factor group elements to peak positions in the y1, y3, and y4

regions of HA. In view of the above drawback of XRD, IR

method was developed40 to determine the percentage of cris-

tallinity in apatite minerals at microscopic as well as macroscopic

levels based on the changes in phosphate y4 mode. In this method,

the broad y4 absorbance bands in 500–700 cm�1 region arise

primarily from antisymmetric P–O bonding modes of the phos-

phate group. This mode, which is triply degenerate (F2 and Td

symmetry) is resolved into at least two well defined peaks in HA

materials.30 However, analysis of this spectral region does not give

any determinative spectral-structural correlations.

IR method was used for the study of crystallinity of HA

minerals, which is based on the changes in the phosphate y1 and

y3 absorbance in 900–1200 cm�1. These spectral features arise

primarily from the symmetric (y1) and antisymmetric (y3) P–O

stretching modes of the synthesized HA phosphate groups.28

Therefore, we expect that the analysis of this spectral region

would lead to multiple bands arising from both the phosphate y1

and y3 modes.27 Changes in this spectral region for a series of

synthetic hydroxyapatites with crystalline sizes 6–20 nm were

Fig. 3 FT-IR spectra of synthesized hydroxyapatite nanoparticles; (a)

HAP (b) HAPAC (c) HAPTAT (d) HAPCIT.

2634 | Nanoscale, 2010, 2, 2631–2638

analyzed with curve fitting and second derivative spectroscopy.

Analysis was done with peak fitting software and the peak-fitting

algorithm creates Lorentzian-Gaussian bands that are added to

produce a computed spectrum, which was compared with the

experimental spectrum. To enhance the visibility of the changes in

the FT-IR absorbance spectra accompanying maturation of the

crystals, second-derivative spectroscopy was used to define the

underlying bands in a better way. A mixed Gaussian-Lorentzian

band shape was used to fit the region. Fig. 4. displays spectra

representative of poorly crystalline HA, with the experimental

and calculated contours overlaid along the individual sub-bands.

It was reported that six or more components are necessary

and sufficient for a suitable fit in this spectral region.27 To

correlate the subtle changes in the FT-IR absorbance spectra

Fig. 5 Curve fit of the FTIR spectra of the synthesized hydroxyapatite

samples; (a) HAP, (b) HAPAC, (c) HAPTAT, and (d) HAPCIT and (e),

(f), (g) and (h) represents the peak fit of X-ray diffraction patterns of the

corresponding hydroxyapatite samples.

This journal is ª The Royal Society of Chemistry 2010

Fig. 6 Relationship plot between crystalline size (from XRD patterns)

and percentage area (from FT-IR spectra) of different hydroxyapatite

samples; (a) without ligand, (b) acetic acid, (c) tartaric acid, (d) citric acid.

Fig. 7 TEM micrographs of the synthesized hydroxyapatite samples; (a)

HAP (b) HAPAC (c) HAPTAT (d) HAPCIT and the insets represent the

corresponding SAED patterns.

Table 3 Comparison of particle sizes of synthesized samples fromvarious measurements

SerialNo. Sample

XRD/nm

PSD/nm

TEM

BET/nmLength/nm

Diameter/nm

1 HAP 17–20 135–250 49–65 7–11 172 HAPAC 15–18 110–225 26–45 6–9 153 HAPTAT 13–16 100–145 23–31 4–7 144 HAPCIT 12–15 75–140 20–29 3–7 13

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with those of the XRD patterns, a curve fitting algorithm was

used, the underlying components of both the FT-IR and X-ray

data of synthesized HA was calculated. Fig. 5. depicts typical

curve fit data of the FT-IR spectra and XRD patterns of all the

poorly crystalline HA samples. For each of the HA synthesized

samples, the crystal sizes calculated from the FWHH of the

XRD peaks, after curve fitting were compared with the

percentage areas of the FT-IR sub-bands. The percentage areas

of the FT-IR sub-bands were plotted against the calculated

crystal sizes using the FWHH of the corresponding X-ray

diffraction peaks to obtain spectra-structure correlations as

shown in Fig. 6.

FT-IR spectra of the synthesized nano hydroxyapatites, whose

principal mineral components in each instance are poorly crys-

talline, agree extremely well with the applicability of the index of

the crystallinity in biological materials.41 Nonlinear regression

analysis led to the following relationship between fractional area

of the FT-IR and crystalline size (Fig. 6.), calculated from the

XRD, in the following relationship:

Y ¼ a Xb + c (r ¼ 0.87)

Y ¼ Crystalline size (�A), X ¼ Percent area, a ¼ �28.03334, b ¼0.3497, and c ¼ 178.4

SEM micrographs (Fig S2†) reveal that the synthesized

hydroxyapatite powders tend to aggregate in all the systems

owing to the high surface energy of the nanosized particles. So

the morphologies of the particles are unclear in the SEM

micrographs. In the presence of the ligand, the aggregation

tendency was found to decrease with increase in ligand coordi-

nation.

TEM studies were carried out for nanocrystalline HA samples

and representative micrographs of HAP, HAPAC, HAPTAT

and HAPCIT are shown in Fig. 7. The micrographs revealed the

needle shaped morphology of the hydroxyapatite particles with

highest degree of aggregation in these samples (which were

prepared without using any ligand) while the aggregation was

minimum for HAPCIT sample. The length and diameter of the

particles of the different HA samples were determined from their

respective micrographs, where the smallest visible aggregate was

This journal is ª The Royal Society of Chemistry 2010

considered as a single particle. A summary of the particle

dimension obtained from the TEM studies of the different

hydroxyapatite samples were tabulated in Table 3. The corre-

sponding selected area electron diffraction (SAED) patterns,

shown in the insets of Fig. 7., indicate clearly visible rings, whose

interplanar spacing are in good agreement with the characteristic

spacing of apatite-like structure. On the basis of the aforemen-

tioned results, it is feasible to use the employed method for the

preparation of needle-like HA particles with a high aspect ratio.

The control of shape and morphology of these prepared

samples is an actual challenge and further research is needed to

validate the exact mechanism that determines nanocrystalline

morphology.42 Pramanik et al. hypothesized that the morphology

and shape of HA samples related to various complexing agents in

which they were synthesized.13 From the morphological study of

the particles, it was observed that HAPCIT sample depicted the

highest aspect ratio among all the prepared samples while the

HAPAC sample had the minimum value. The distribution in

aspect ratio and average aspect ratio in each of the prepared HA

samples are depicted in Fig. 8 (in this figure each particle is equal

to average of ten particles aspect ratio). It was also observed that

aspect ratio of the HAP sample was higher than the HAPAC

sample, but in case of other ligands the aspect ratio was increased.

It may be due to strong surface bound ligands but the particle size

was decreased among all ligands. It is worthy to note that the

diameters and lengths of the synthesized HA samples range from

3–11 nm and 20–65 nm, respectively. The difference in aspect

Nanoscale, 2010, 2, 2631–2638 | 2635

Fig. 8 Histograms of average aspect ratios of different hydroxyapatite

samples; (a), (b), (c) and (d) represents HAP, HAPAC, HAPTAT and

HAPCIT respectively.

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ratios of these HA samples can satisfy the demands for potential

applications in artificial bone and teeth.

The specific surface area of prepared HA samples were deter-

mined by nitrogen absorption analysis (BET) using a quanta

chrome surface area analyzer. Samples were degassed at 150 �C

for 4.5 h under vacuum prior to analysis. The BET surface area of

the HAP, HAPAC, HAPTAT and HAPCIT powders were found

to be 107.21, 121.26, 134.67 and 141.20 m2 g�1 respectively

(Fig. 9). The corresponding average grain sizes of these HA were

17, 15, 14 and 13 nm as estimated by using the empirical equation

d ¼ 6/(rS) (where d is the average grain size in micron, r is the

density in g/cc and S is the specific surface area in m2 g�1).43 These

values of grain sizes are in reasonable agreement with the results

of the TEM observation and the average crystalline size esti-

mated from the respective XRD peaks. The ligating ability of

various complexing agents used in aqueous solution to form

complex with the metal chloride may be responsible for the

observed difference in the surface area of the synthesized samples.

From the observations, it can be noted that as the density of

carboxyl groups increases the yield of particle size decreases and

surface area increases. However, we have also carried out a series

of reactions to optimize the concentration of different ligands.

However, the result of the optimization study has not been

included in the present manuscript. After analyzing the morpho-

logical and surface area measurement results, we found that

particles with minimum size and maximum surface area can be

obtained by using the ligands at the concentrations mentioned in

the paper.

Fig. 9 BET surface area of the prepared HA samples (1) HAP (2)

HAPAC (3) HAPTAT (4) HAPCIT.

2636 | Nanoscale, 2010, 2, 2631–2638

The dried particles were dispersed in water to evaluate the zeta

potential and particle size distribution (PSD). The particles

showed negative potential at pH 10, the potential which

increased with decreasing pH as shown in Fig. 10. The isoelectric

point of the prepared HA samples are found to be in the range of

3.1–3.4. Zeta potential is very sensitive to the particle surface

conditions, ions adsorbed on the particle surfaces, and the kind

and concentration of ions in the solution. The variations in zeta

potential are likely caused by difference in the surface conditions

of the HA particles and interaction between particles and ions in

the solution. The PSD analysis of the nanoparticles prepared

with various ligands such as AC, TAT, and CIT shows

substantial distribution of colloidal systems in PSD range of 110–

225, 100–145 and 75–140 nm respectively (Fig S3†), while the

HAP sample prepared without ligand shows PSD between 135–

250 nm (Fig S3†). Further, the PSD analysis reveals the res-

pective values of the mean particle sizes of HAP, HAPAC,

HAPTAT and HAPCIT samples as 198, 155, 117 and 114 nm.

All these values are found to be larger than those obtained from

TEM observation. The aggregation of HA samples may be due

to the relative bigger particle size observed under nanosizer.

However, these aggregations were less pronounced in case of the

nanoparticles prepared in the presence of ligands compared to

that synthesized in the absence of any ligand. The details

comparisons of particle sizes of synthesized samples from various

measurements were incorporated in Table 3.

Calcium ions content in the HA samples were determined

using calcium ion calibration curve obtained from AAS. For the

determination of calcium content by Flame Atomic Absorption

Spectroscopy (FAAS) mode, the samples were dissolved in dilute

HCl. The calibration curve was prepared over the concentration

range of 0.1–30.0 mg ml�1 of calcium ions and the absorbance

were measured at 422.7 nm beam produced by the hollow

cathode lamp.

The mass fraction of calcium ions concentration in HAP

samples were determined as 38.92% (HAP), 39.06% (HAPAC),

39.19% (HAPTAT) and 39.22% (HAPCIT). It should also note

that the percent of mass fraction for all the prepared samples

was increased in presence of complexing agents. It is due to

strongly bound carboxylic groups to form complexes with the

calcium ions.

The samples (HAP, HAPAC, HAPTAT, and HAPCIT) were

dispersed in HEPES buffer to 10 mg ml�1 concentration. Hemo-

compatibility of the samples were analyzed using a protocol7

Fig. 10 Zeta potential of hydroxyapatite samples; (a) HAP (b) HAPAC

(c) HAPTAT (d) HAPCIT.

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Fig. 12 HRTEM image of HA prepared with citric acid ligand (a) (Inset:

FFT image of corresponding HA), Schematic representations of esti-

mated morphology of HA with ligands (SHAPE software) (b).

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with some alteration. In brief, blood was obtained from 6-week-

old BALB/c male mice and red blood cells (RBC) were collected

by centrifugation (1500 g, 5 min, 4 �C) of the blood. The collected

RBC pellet was diluted in 20 mM HEPES buffered saline (pH

7.4) to give a 5% (v/v) solution. The RBC suspension was added

to HEPES-buffered saline containing 1% Triton X-100 and

samples (HAP, HAPAC, HAPTAT, and HAPCIT) and incu-

bated for 30 and 60 min at 37 �C. After completion of the

incubation, all the control and samples were centrifuged

(Heraeus table top centrifuge 5805R) at 12,000 rpm at 4 �C and

the supernatants were transferred to a 96-well plate. Hemolytic

activity was determined by measuring the absorption at 570 nm

(Biorad Microplate reader 5804R). Control samples of 0% lysis

(-ve control) (in HEPES buffer) and 100% lysis (+ve control) (in

1% Triton X-100) were employed in the experiment.44 All the

assays were performed in triplicate. Hemolytic effect of each

treatment was expressed as percent cell lysis relative to the

untreated control cells (% control) defined as: [(Abs570 samples)/

(Abs570 control cells)] � 100, where absorbance is abbreviated

as Abs.

Previous report indicates that the percentage of hemolysis less

than 5% and 5–10% are considered as highly hemocompatible

and hemocompatible material respectively.45 It is found that all

the synthesized nanoparticles showed significantly lower hemo-

lytic activity (less than 5%) with respect to control samples

(Fig. 11). But no significant difference was found in hemolytic

activity among these samples. The high hemocompatibility of the

synthesized hydroxyapatite nanoparticles can make them to be

used for potential application in biomedical engineering. This

high hemocompatibility of the nanoparticles may be attributed

to the presence of surface hydrophilic groups (carboxy, hydroxyl

functional groups).8

HRTEM analysis reveals particulars related to the growth

mechanism that leads to formation of HA by controlling the

growth in presence of various ligands. The dhkl values found from

the FFT analysis agree with the reflection of HA. The most

important surface, i.e., (001) is estimated through study of

HRTEM. On the basis of measured reflections, it is envisaged

that the control of the growth occurs along the (001) and (100)

Fig. 11 Hemolytic assay of samples; (c) HAP, (d) HAPAC, (e)

HAPTAT, and (f) HAPCIT at 10 mg ml�1 concentration. (a) and (b)

respectively represent +ve control 100% lysis (in 1% Triton X-100) and

�ve control samples of 0% lysis (in HEPES buffer) employed in the

experiment. The bars indicate the means � SD (n ¼ 3). Significant

difference is shown as ***p < 0.001 versus +ve control.

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surface (Fig. 12) which is in agreement with recent study by

Kwon et al.46 These face act as the binding site for many ionic

species, including small molecules, polymers, and anionically

modified cell surfaces. In this study, we have used literature

which were reported on molecular modeling technique to study

the binding of organic ligands on HA crystal surface, which

eventually affects the crystal growth.47,48 The representative

atomistic models of HA surfaces help in investigating optimized

binding geometries and calculating binding energies by using

parameters from the universal force field.48 The literature

reported results confirm the preferential adsorption of ligands on

the (001) and (100) surface.47–49 In order to have better under-

standing of the growth control of the particles, we have also

prepared samples at a time interval of 6 h. The morphology (refer

to the TEM images shown in Fig. S4†) and particle size (Table

S1†) of the prepared samples is found to be comparable with

those of the samples obtained after 5 min of reaction time as

shown in Table 3. The data are supported by the zeta potential

measurement, which shows nearly same zeta potential of the

samples obtained after 5 min of reaction time (Fig. S5†). Thus, it

can be said that the control of morphology may be due to the

negatively charged carboxyl and hydroxyl groups present in the

ligands, which are bound to calcium ions to form HA. Electro-

static interactions are therefore believed to occur between the

cationic sites in the HA mineral and the anionic domains in the

complexing agents. With the increasing densities of carboxyl

groups, the abundant supply of coordination sites for complex-

ation with calcium ions leads to a very large number of nuclei for

the growth of HA particles, resulting in smaller crystal size.46

This may be the plausible elucidation for controlling the growth

of the material. The proposed mechanism is also supported by

the increase in the value of lattice parameter ‘a’ and decrease in

the value of ‘c’ with reducing crystallite sizes.50 The high surface

area and control of particle size have many more applications.

For example, apoptotic study on cancer cells depends on the

surface area and size particles.51

Conclusion

In summary, we have successfully synthesized HA nanoparticles

with high surface area by co-precipitation method using com-

plexing agents. All the synthesized samples are characterized by

various characterization techniques, such as XRD, TEM, PSD

and SEM analysis. These analyses suggest that the synthesized

hydroxyapatite powders under various complexing agents show

smaller crystallites, particle sizes, less agglomeration compared

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to HA, synthesized in the absence of any ligand. Though,

HRTEM images of the hydroxyapatite nanoparticles indicate the

formation of acicular needle-like morphologies under all reaction

conditions, the HA with complexing agents has shown a reduc-

tion in particle sizes. This observation has been attributed to the

effective restriction of the nuclei growth (control the growth

along the [001] and [100] axis) of the synthesized hydroxyapatite

samples by ligands during precipitation process. No significant

change in the particle size of HA is observed even after increasing

reactive time interval. The correlation between infrared and

X-ray parameters of these nanomaterials is found to be well

matched. The synthesized HA nanoparticles shows highly

hemocompatibility in bulk aqueous media, which depends on the

shape of the particles (i.e., needle-like shape). The present study

has provided not only novel building blocks for the construction

of artificial bones with novel mechanical properties but also

a new strategy for the controlled growth of inorganic nano-

particles. This work can further be extended for investigating the

effect of high surface area of the HA nanoparticles on the anti-

tumor activity, apoptosis and apoptotic signaling activation

protein levels.

Acknowledgements

Authors would like to acknowledge M.H.R.D., Govt. of India,

for the financial support. The authors are grateful to Prof. M.

Mahitosh for supporting the Hemolytic assay measurement,

School of Medical Science and Technology, IIT-Kharagpur,

India.

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