Materials Chemistry and Physics 183 (2016) 220e229

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Materials Chemistry and Physics

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

Controllable synthesis of spherical hydroxyapatite nanoparticles using

inverse microemulsion method
Xiaoyu Ma a, b, Yaying Chen b, Jiangchao Qian c, Yuan Yuan a, b, c, *, Changsheng Liu a, b, c, **
a
Key Laboratory for Ultrafine Materials of Ministry of Education, East China University of Science and Technology, Shanghai 200237, PR China
b
Engineering Research Center for Biomedical Materials of Ministry of Education, East China University of Science and Technology, Shanghai 200237, PR China
c
The State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, PR China

h i g h l i g h t s

Spherical nano-sized hydroxyapatite particles were successfully synthesized through inverse microemulsion method.

A systematical study was performed to illustrate correlation between microemulsion stability and particle morphology.

A possible mechanism for the growth of HAp was proposed.

a r t i c l e i n f o a b s t r a c t

Article history: Spherical nanosized hydroxyapatite (HA) was successfully synthesized via a water-in-oil microemulsion
Received 6 March 2016 route at room temperature in short time. The effect of water-to-oil and water-to-surfactant ratios on the
Received in revised form stability of microemulsion system was investigated to establish a stable reaction system, with span-80,
1 July 2016
cyclohexane and Ca(NO3)2$4H2O((NH4)2HPO4) solution as surfactant, oil phase, and water phase,
Accepted 13 August 2016
Available online 18 August 2016
respectively. Size and morphology change of obtained HA under optimal microemulsion system were
further studied under various reaction time and temperature, and a possible growth mechanism was
proposed. A moderate reaction temperature of 25  C and reaction time of 5 h facilitated the formation of
Keywords:
Microemulsion
spherical HA particles in the dimension of 70 nm with good uniformity and regularity, which served as a
Hydroxyapatite great candidate in biomedical applications.
Spherical nano particle © 2016 Elsevier B.V. All rights reserved.

1. Introduction
Hydroxyapatite(Ca10(PO4)6(OH)2, HAp), as a main inorganic
composition in bone tissue, has been widely used in various
biomedical fields such as bone tissue engineering [1e3], drug de-
livery [4e7] and cell imaging [8,9], due to its excellent biocom-
patibility, bioactivity and biodegradability. Among different
hydroxyapatite structures, nano-sized ones have stimulated great
interests for its outperforming characteristics compared to corre-
sponding counterparts with larger dimension. Studies revealed that
a series of unique properties of hydroxyapatite are closely related to
the size [10,11], shape [12], surface morphology and surface area
[13]. For example, Tang [12] reported that nano-sized hydroxyap-
atite could exhibit unique anti-tumor ability. In other studies, Zhou
[1] proposed that HAp with microstructure might lead to magnif-
icent improvements in mechanical properties of resulting scaffolds.
It is thus of significant importance to fabricate hydroxyapatite
nanoparticles, particularly with regular shape and uniform size to
achieve desired properties.
At industrial scale, hydroxyapatite have been synthesized by
spray drying method [14], NETmix technique [15] and traditional
wet chemistry method involving precipitation or hydrolysis [16].
For example, a series of spherical HAp manufactured by NETmix
technique [17e19] show great uniformity and regularity within the

Abbreviations: HA(p), hydroxyapatite(particle); W/0, water to oil ratio; O/SC, oil to surfactant ratio; DLS, dynamic light scattering; XRD, x-ray diffraction; LHME, hy-
droxyapatite by micro-emulsion at low temperature; HME, hydroxyapatite by micro-emulsion at optimal temperature; HHME, hydroxyapatite by micro-emulsion at high
temperature.
* Corresponding author. Engineering Research Center for Biomedical Materials of Ministry of Education, East China University of Science and Technology, Shanghai 200237,
PR China.
** Corresponding author. Engineering Research Center for Biomedical Materials of Ministry of Education, East China University of Science and Technology, Shanghai 200237,
PR China.
E-mail addresses: yyuan@ecust.edu.cn (Y. Yuan), liucs@ecust.edu.cn (C. Liu).

http://dx.doi.org/10.1016/j.matchemphys.2016.08.021
0254-0584/© 2016 Elsevier B.V. All rights reserved.
 X. Ma et al. / Materials Chemistry and Physics 183 (2016) 220e229
micro-scale range. For other commercialized nano HAp under
150 nm, they fail to maintain regular spherical shape due to the
heterogeneity when scaling up. As a result, works still have to be
done to achieve nano-sized HAp with uniform spherical shape.
Meanwhile, diverse synthetic methods [20] have been devel-
oped in the lab to prepare nano-HAps with precise control over
microstructure, including chemical precipitation [21,22], sol-gel
method [23,24], hydrothermal process [25,26] and so on. Howev-
er, due to the intrinsic crystal growth preference along c-axis, hy-
droxyapatite nano-particles often tend to generate elongated rod-
like or sheet-like morphology with polydispersive distribution.
Noteworthily, there were few studies focused on the fabrication of
spherical HAp particle in nano-scaled dimension, which might be a
promising candidate in biomedical applications for its high surface
area and easiness to penetrate cytomembrane. Up to now, most
chemical methods still cannot fulfill the precise control over
spherical morphology or the uniform size distribution, mainly due
to complicated process used in the synthesis, uncontrolled long-
term aging before sintering and severe aggregation and agglom-
eration in the particle formation. Consequently, it is in urgent need
to find a specific way to meet the above demands.
Among the existing synthetic routes, microemulsion [27,28]
could serve as the method of choice in controllable synthesis of
spherical hydroxyapatite nanoparticles. Microemulsions are ther-
modynamically stable and optically transparent dispersions of two
immiscible liquids (water and oil usually), stabilized and coexisted
in the presence of surfactants. By varying primary parameters such
as water to oil ratio and surfactant concentration, the method could
offer precise control over size and shape. In the preparation of
nano-hydroxyapatite, microemulsion method provides mono-
dispersive yet stable droplets, working as small reactor to confine
the HAp nuclei growth and hinder inter-grain agglomeration, so as
to achieve spherical shape in nano dimensions.
In this paper, an inverse microemulsion system was established
to prepare spherical hydroxyapatite nanoparticles with good uni-
formity and regularity. The effect of water to oil ratio and the sur-
factant concentration on the stability of microemulsion system was
discussed. A clear and transparent microemulsion with good sta-
bility and no phase separation for over 24 h was prepared under
water-oil ratio of 1/100 and O/SC of 7 with ultrasonic emulsifica-
tion. Furthermore, the influence of reaction time and temperature
were also investigated in the study. It is observed that a relatively
short reaction time and moderate reaction temperature (5 h and
25  C) were beneficial to the stability of microemulsion system,
thus facilitating the shape and size control of resulting particles
(spherical HA with diameter of 70 nm).
2. Materials and experimental methods
2.1. Materials
In the synthesis of hydroxyapatite nanoparticles, Ca(N-
O3)2$4H2O (purity: 99.0%), (NH4)2HPO4 (purity: 99.0%), ethanol
(purity: 99.7%), Span-80 were all bought from Sinopharm
Chemical Reagent Co., Ltd., (Shanghai, China). n-amyl alcohol
(purity:99.0%), cyclohexane (purity: 99.5%) and ammonium
hydroxide (purity: 25.0%e28.0%) were purchased from Shanghai
Lingfeng Chemical Reagent Co., Ltd., (Shanghai, China). All the
materials were used directly without further purification.
2.2. Preparation of HAp nanoparticles
HAp nanoparticles are synthesized via microemulsion process
according to a previous report [27], with a slight modification. For
the preparation of microemulsions, briefly, 58.5 g n-amyl alcohol,
221
117 g Span-80 were added to 500 g cyclohexane solution in
vigorous stirring to form the oil phase at room temperature. A total
volume of 0.6 mL of 0.5 M Ca(NO3)2 solution and 0.3 M (NH4)2HPO4
solution was added dropwise into 60 mL of the above oil e sur-
factant mixture under condition (ultrasound power 40%, ultrasonic
time 2.5 s, interval time 0.5 s, and total ultrasonic time 7 min) to get
Ca microemulsion and P microemulsion respectively. For the syn-
thesis of HAp nanoparticles, the above Ca and P microemulsions
were mixed at the volume ratio of 1:1 at room temperature over-
night for different hours under agitation. After reaction, the pre-
cipitates were collected through filtration. The resulting product
was further washed repeatedly with ethanol for at least three
times, freeze dried for 24 h and grinded. After 5 h of sintering under
600  C to remove remaining organic molecules, pure HAp nano-
particles were obtained. The preparation process of HAp nano-
particles was summarized as in Fig. 1.
2.3. Establishment of microemulsion phase diagram
Firstly, the phase stability of microemulsion system of different
span-80/cyclohexane/Ca(NO3)2aqueous solution ratio was tested. In
detail, span-80 with various mass was dissolved in a certain amount
of cyclohexane. Ca(NO3)2 aqueous solution was slowly injected into
the oil-surfactant mixture under ultrasonic emulsification to
observe the transparency variation of microemulsion system. The
amount of Ca(NO3)2 aqueous solution added was recorded at the
moment when microemulsion system become turbid. Upon the data
obtained, local phase diagram of span-80/cyclohexane/Ca(NO3)2
aqueous solution three-phase system was established.
2.4. Characterization
Zetasizer Nano ZS (Malvern Instruments Ltd, UK) was applied to
determine the size and size distribution of microemulsion droplets
and synthesized hydroxyapatite nano particles in microemulsion
system. The microemulsion was directly used in the test without
any dilution. The microstructure of synthesized hydroxyapatite
nanoparticles was observed through transmission electron micro-
scope (JEM-2100, Japan) and information of surface morphology
was obtained via transmission electron microscope (JEM-1400,
Japan). Freeze-dried HAps were used during the TEM observation.
The accelerating voltage was kept at 200 kV during the process. X-
ray diffraction (Rigaku D/max2550 VB/PC, Japan) was utilized to
analyze the phase composition and inner atomic structure of the
prepared nano particles under 40kV/100 mA and 3500CPS for Cu
target.
3. Results and discussion
3.1. Establishment of microemulsion system
Before synthesis of hydroxyapatite nanoparticles, it is top pri-
ority to establish a microemulsion environment with adequate
stability. According to previous reports [29e31]and considering the
HLB value of common surfactants, span-80, cyclohexane and Ca(P)
aqueous solution were chosen as surfactant, oil phase, and water
phase respectively in the current system. A ternary phase diagram
of the above three phases was presented in Fig. 2(a). It can be seen
that stable microemulsion area only existed on the right of the
curve with a narrow range, which indicated a low water bearing
capacity below 10% in the system. However, after the addition of n-
amyl alcohol as a co-surfactant, the maximum water content in
microemulsion was increased to about 20%, as illustrated in
Fig. 2(b). It was confirmed by the phase diagrams that the addition
of co-surfactant into microemulsion system could greatly enhance
222 X. Ma et al. / Materials Chemistry and Physics 183 (2016) 220e229
Fig. 1. Synthesis process of nano-HAps.
the maximum water content, leading to higher synthesis efficacy
and productivity. In microemulsion systems, the use of single sur-
factant would not satisfy the strict requirement on interfacial film.
Thus, the addition of alcohol could modify the surfactant packing
parameters by absorbing to the interfacial film and stabilizing the
radius of curvature of microemulsion droplets [32]. Here, n-amyl
alcohol functioned in a similar way to keep the droplets intact even
at higher water content [33].
3.2. Effect of n-amyl alcohol/span-80/cyclohexane/Ca(NO3)2
aqueous solution ratio on microemulsion system
As observed from ternary phase diagram, the ratio between
each composition in the microemulsion system played a vital role
in maintaining the stability and properties of microemulsion.
Therefore, in the study, we first study the effect of water/oil ratio
(W/O) and oil/surfactant ratio (O/SC) on size distribution of emul-
sion droplets of Ca microemulsion.
In Fig. 3., the size distribution of Ca microemulsion droplets
under different W/O ratio, 1/250, 1/200, 1/167, 1/125, 1/100, and 1/
80, were labeled as curve (a), (b), (c), (d), (e) and (f) respectively. It
could be seen that at the W/O ratio of 1/125 and 1/100, size
dimension of droplets were in the range of 10 nme100 nm.
Meanwhile, the sharp peak and uniform distribution revealed the
specific characteristics of microemulsion. Compared to 1/125 ratio,
Fig. 2. Ternary phase diagram of the microemulsion system: (a) span-80/cyclohexane/Ca(NO3)2$4H2O solution system; (b) n-amyl alcohol/span-80/cyclohexane/Ca(NO3)2$4H2O
solution system.
Fig. 3. Ca-microemulsion particle size distribution curves with different water-oil ra-
tio: (a) 1/250, (b) 1/200, (c) 1/167, (d) 1/125, (e)1/100, (f) 1/80.
in which two peaks appeared, 1/100 ratio showed single distribu-
tion peak and smaller size dimension between 1 and 10 nm, sug-
gesting more stable and smaller droplets were formed under the
latter condition. At other W/O ratios, namely 1/250, 1/200, 1/167
and 1/80, the size increased to micrometer scale due to closer
interaction between droplets which led to collision and merge of
those droplets. Based on the above results, it could be concluded
that it was of great importance to select an appropriate W/O ratio.
With the increase of water phase, the emulsion droplets first
decrease and then increase to micron order. Herein, 1/100 W/O
ratio was adopted for preparation of hydroxyapatite nanoparticles.
Fig. 4 illustrated the size distribution of microemulsion droplets
under different oil/surfactant (O/SC) ratio while keeping the same
water/oil ratio at 1/100. It can be seen that the minimum size of
droplets between 1 and 10 nm was achieved at O/SC ratio of 8 and 7
(Fig. 4(c) and (d)). However, when the ratio of O/SC increased
further to 9 and 10 or decreased to 6, two peaks appeared, indi-
cating the non-uniformity of size distribution. In particular, when
O/SC was at 6, the size of the droplets was as large as above
1000 nm, which can no longer be considered as microemulsion. It
was worth noting that a higher or lower oil/surfactant proportion
would hamper the formation of a stable microemulsion. An inad-
equate amount of surfactant would result in an unstable micro-
emulsion environment. On the other hand, too much surfactant
would lead to an overgrowth of microdroplets. Since the purpose
for introducing appropriate amount of surfactant is to reach a
balanced state between hydrophilicity and hydrophobicity in the
system, with the over-addition of surfactant, the balance is yet
broken and the interface between water and oil tend to be unstable,
 X. Ma et al. / Materials Chemistry and Physics 183 (2016) 220e229
Fig. 4. Ca-microemulsion particle size distribution curves with different O/SC: (a) 10,
(b) 9, (c) 8, (d) 7, (e) 6.
leading to the breakage of droplets that subsequently collide with
surrounding droplets through an unpredictable approach. The
result was also consistent with previous report for nanosized silica
synthesized through microemulsion [34]. Confirmed by the above
experiments, an O/SC ratio of 7 was set as the optimal condition in
the HAp synthesis.
3.3. Long-term stability of the microemulsion system
After the initial selection of W/O and O/SC proportion, the sta-
bility of Ca-microemulsion system was further investigated by DLS
characterization on size change at various standing time. In
Fig. 5aed represented size distribution at initial state of micro-
emulsion and after 5, 10 and 20 h. As shown in Fig. 5, with the pass
of time, the size of droplets became larger than that of the initial
Fig. 5. Comparison diagram of Ca-microemulsion stability with different water-oil ratio: (a) 1/250, (b) 1/200, (c) 1/167, (d) 1/125, (e) 1/100, (f) 1/80.
223
state. At the W/O ratio of 1/125 and 1/100, the size slightly
increased from 1 nm to 2 nm after 20 h. However, for other W/O
ratios (1/250, 1/200 and 1/167 illustrated in a, b, c), droplet size
significantly expanded from 1 mm to 3 mm in a similar way,
revealing the unstable nature of microemulsion system at a lower
W/O condition. Particularly, for W/O ratio of 1/80 in Fig. 5(f), the
size grew from 1 mm to 5 mm, indicating that the overload of water
imposed adverse effect on the stability of microemulsion. The re-
sults were in good agreement with what we concluded in 3.2,
namely, 1/100 water/oil proportion served as the optimal condition
for current microemulsion system since there's no obvious change
in size compared to others. Apart from the certain W/O ratio,
inadequate water would impede the interaction between water and
oil phase while too much water could not maintain the stability of
the microemulsion environment.
To directly observe the stability of microemulsion, optical pho-
tographs were taken at different time intervals (right after emul-
sification and 20 h later) on various W/O ratios (1/200, 1/100 and 1/
70) (see Fig. 6). After the end of emulsification, it was shown that
the microemulsion was slightly turbid and translucent for 1/200
and 1/70 group while the 1/100 group was totally clear. A micro-
emulsion is an emulsion where the volume of water in oil is small,
thus the emulsion remains transparent. After 20 h, an obvious
phase separation was identified for 1/70 group. Nonetheless,
there's no significant change on 1/200 and 1/100 group, suggesting
excellent stability of microemulsion and the ultrafine droplets
achieved in certain condition, which was well accorded to the re-
sults gained by DLS in Fig. 5.
So far, for Ca microemulsion, an optimal proportion of n-amyl
alcohol/span-80/cyclohexane/Ca(NO3)2 aqueous solution was fixed
(W/O ¼ 1/100, O/SC ¼ 7). Here, Ca microemulsion was selected as a
model system for the stability test of the reaction system. We further
verified the proportion on P microemulsion (n-amyl alcohol/span-
80/cyclohexane/(NH4)2HPO4 aqueous solution). As illustrated in
Fig. 7 and 24 h later after emulsification, the microemulsion was still
clear and transparent, proving that the optimal proportion was also
224 X. Ma et al. / Materials Chemistry and Physics 183 (2016) 220e229
applicable to P microemulsion in maintaining stability.
3.4. Morphological difference of HAp between microemulsion and
emulsion
A morphological comparison was conducted between two
conditions to verify the difference between microemulsion and
emulsion systems. Two synthesis systems based on the obtained
phase diagram were tested, which we hereinafter designate as
system (a) and (b). The optimal ratio was selected at (a) and an
increased W/O ratio was adopted at non-microemulsion region at
(b). The hydroxyapatite particles synthesized at system (a) was
labelled as HME (hydroxyapatite in microemulsion) and those at
system (b) were labelled as HE (hydroxyapatite in emulsion). The
detailed synthesis conditions were summarized in Table 1.
The morphological difference was investigated by TEM. It could
be seen in Fig. 8 that there's huge difference on the morphology
between the two conditions. HME presented near spherical shape
with a uniform size distribution within 100e150 nm. However, HE
was short rod-like with a size distribution of 30e60 nm, diameter
of 10e30 nm and length to width ratio of 2e3. The great variation
in morphology was likely due to the overload of water, which
inhibited the formation of microemulsion. On the other hand, the
provided synthesis environment was close to aqueous precipitation
so that the short rod-like morphology of HAp particle was also
similar to those prepared by homogeneous precipitation in previ-
ous reports [35,36]. In conclusion, microemulsion system could
offer a favorable environment for the formation of nano-sized
droplets, leading to synthesized HAp with uniformity and intact
spherical shape.
Fig. 6. Photos of Ca-microemulsion.
3.5. Study on growth process of hydroxyapatite nanoparticle
In microemulsion, the dispersed phase (here water phase) was
confined in nanometer regimes of droplets, which was capable of
delivering uniform nano-sized spherical HAp when the reaction
was restrained in the domains. It was similar as a micro liquid
precipitation, confined in “water pools”, in which HAp nucleated
and then grew. Based on the previous work, it took 24 h to convert
the reagents to hydroxyapatite under room temperature [37]. As a
result, a reaction time of 24 h, W/O of 1/100, O/SC of 7 was adopted
in microemulsion to prepare hydroxyapatite under room
temperature.
After the addition of Ca and P, as well as the adjustment of pH by
ammonium hydroxide, the reaction immediately initiated accord-
ing to the following equation:
10CaðNO3 Þ2 þ 6ðNH4 Þ2 HPO4 þ 8NH4 OH/Ca10 ðPO4 Þ6 ðOHÞ2
þ 20NH4 NO3 þ 6H2 O
First of all, the relationship between particle size and reaction
time was investigated to understand the nucleation and growth of
hydroxyapatite. As shown in Fig. 9(a) to represented size distribu-
tion with different reaction time, ranging from 0 to 24 h. At the
beginning of the reaction, the size distribution was between 1 and
10 nm, in according with the microemulsion droplets. At this stage,
it was possible that the reaction had not yet completely started due
to the inadequate collision between Ca and P droplets. In the re-
action system, the majority of the particles were still micro-
emulsion droplets instead of solid particles. 30 min later, the curve
in (b) demonstrated a broad distribution in between 100 and
200 nm, which might be attributed to both the diverse growth
speed of calcium phosphate particles and agglomeration. In (c) and
 X. Ma et al. / Materials Chemistry and Physics 183 (2016) 220e229
Fig. 7. Photos of P-microemulsion (a) after emulsification (b) 24 h later.
Table 1
Synthesis parameters of HAPN-ME.
Concentration of Ca and P
Ca(NO3)2$4H2O (NH4)2HPO4 W/O O/SC Temperature
(a) HME 0.5 M 0.3 M 100 7 25  C
(b) HE 0.5 M 0.3 M 3 7 25  C
(d), a similar tendency was observed that particles were growing in
a uniform and stable approach in 10 h with a size dimension of
200 nm. However, 15 h after the initial reaction, size of few particles
had overgrown to micrometer dimension. Two peaks appeared in
(e), indicating the rapid growth of particles and unstability of the
microemulsion system. With further extension of reaction time to
24 h, the particle size greatly expanded to 5 mm which was also the
maximum detection range of DLS, suggesting the severe agglom-
eration and unstability of the reaction system.
The morphologies of HAPNs under different reaction time by
TEM are shown in Fig. 10. Particles were collected at different time
intervals, namely, 0.5 h, 5 h, 15 h and 24 h after the initiation of the
reaction. Due to the remaining surfactants and organic phase
absorbed on the particles, the HAp in solution revealed agglomer-
ation. In microemulsion systems, a large amount of organic solvent
and surfactants employed in the reaction could not be completely
removed, even after repeated washing by water and ethanol. Thus,
under the high-energy electron irradiation employed in TEM at
large magnifications, particles exhibited slight aggregation led by
evaporation and decomposition of the organic solvent and surfac-
tant. As illustrated in Fig. 10 (a)., 0.5 h after the initiation of the
Fig. 8. Microemulsion phase diagram and TEM images of HAP: (a) HME, (b)HE.
225
Fig. 9. Particle size distribution curve of HAPN-ME at different reaction times: (a)
initial, (b)0.5 h, (c)5 h, (d)10 h, (e)15 h, (f)24 h.
reaction, the achieved particles presented regular and uniform
spherical-like shape with smooth surfaces within the dimension of
70e100 nm. 5 hr later, there was no obvious change on the size of
particles. However, small granules of tenth of nanometers appeared
and attached onto the surface of particles. The covering micro-
emulsion droplets broke along with the overgrowth of particles,
resulting in the direct contact of solid granules with oil phase. On
the other hand, the break-down of intact microemulsion droplets
would drive the small granules in HAp precursor to attach to the
surface of the particles, which led to the disruption of original
spherical shape. With the continuous growth, the size of particles
grew into 100e200 nm after 15 and 24 h. It was observed that the
particle presented near spherical shape, a rougher surface and a
broader size distribution compared to those collected after 0.5 and
5 h. As shown in (d), at the end of reaction, the particle became
larger with severe agglomeration. Spherical particles below 100 nm
and irregular aggregates were coexisted in the system.
Fig. 11(a) demonstrated the XRD patterns of the obtained par-
ticles under microemulsion systems at various reaction times after
calcinations. It was clear that all the peaks appeared could be
attributed to hydroxyapatite structure and no other crystalline
phases other than HA were observed. In addition, the narrow
226 X. Ma et al. / Materials Chemistry and Physics 183 (2016) 220e229

Fig. 10. TEM images of HAP at different reaction times: (a) 0.5 h, (b)5 h, (c)15 h, (d)24 h.

diffraction peaks indicated high crystallinity of the obtained OH stretching from hydroxyapatite and absorbed water. It was
products. The crystallinity of the particles achieved at 24 h was worth noting that additional bands at 1450 cm1 were attributed to
slightly higher than those obtained at shorter reaction time. It could carbonates which might have substituted certain phosphate or
be inferred from the XRD patterns that different reaction time only hydroxyl positions in the lattice of HAps.
led to change of morphology, but did not change the phase
composition of the obtained products. On the other hand, as shown
3.6. Effect of reaction temperature on the morphology of HAp
in (b), characteristic bands for phosphate stretching and phosphate
bending appeared in the regions 950e1250 cm1 and
The HAp prepared above was under the optimal condition under
500e600 cm1. Broad peaks around 3300e3500 cm1 came from
room temperature (25  C). However, to investigate the effect of

Fig. 11. (a). XRD patterns for particles obtained from microemulsion system 0.5, 5, 15 and 24 h after initiation of reaction (b). FT-IR spectra for particles obtained from micro-
emulsion system 0.5, 5, 15 and 24 h after initiation of reaction.
 X. Ma et al. / Materials Chemistry and Physics 183 (2016) 220e229 227

Table 2 morphology with a size distribution between 100 and 150 nm with
Synthesis parameters of LHME, HME and HHME. much better dispersibility and uniformity.
Concentration of Ca and P It could be inferred from the above results that an either high or
Ca(NO3)2$4H2O (NH4)2HPO4 W/O O/SC Temperature
low temperature would inhibit the precise control over particle
morphology via microemulsion method. With the change of reac-
(a) LHME 0.5 M 0.3 M 3 7 5 C
tion temperature, the viscosity of both oil phase and surfactants
(b) HME 0.5 M 0.3 M 3 7 25  C
(c) HHME 0.5 M 0.3 M 3 7 60  C would also change, thus altering the surface tension of water-oil
interface and undermining the thermodynamic stability of micro-
emulsion system. For instance in higher temperature of 60  C, the
reaction temperature on the morphology of HAps, two additional
hydrophobicity of surfactant molecules was increased. After the
reaction temperature, 5 and 60 25  C, was adopted. The detailed
addition of Ca2þ, the microemulsion droplets could no longer serve
reaction parameters were concluded in Table 2.
as a stable micro-reactor to confine the HAp formation. Meanwhile,
The morphological difference among the three types of HAp was
due to the increased temperature, the crystal growth of HAp would
illustrated in Fig. 12. It was revealed in (a) that at a relatively low
be faster [38], thus leading to the overgrowth of particles into
reaction temperature, the obtained HAp presented irregular shapes
irregular shape and larger size.
including small granules below 100 nm and plate-like particles
within 500e800 nm. Similarly, no uniformity in size and
morphology was observed at a specific reaction temperature of 3.7. Growth mechanism of spherical HA nanoparticle
60  C. The HHME particles suffered from significant agglomeration
and turned into bulk structure. Only under optimal reaction tem- A possible mechanism of HAp formation could be identified as
perature at 25  C, the resulted HME particles exhibited spherical illustrated in Fig. 13. In the beginning of the reaction, micro-
emulsion system could successfully control the morphology of

Fig. 12. TEM images of HAP: (a)LHME, (b)HME, (c)HHME.

Fig. 13. Possible mechanism growth spherical HA nanoparticles.

228 X. Ma et al. / Materials Chemistry and Physics 183 (2016) 220e229
particles by maintaining the integrity of droplets. The lower su-
persaturation we selected in the system led to moderate provision
of the reactants which resulted in lower nucleation frequency, thus
keeping the droplets from disruption and inhibiting rapid growth
of particles [39]. At this stage, calcium phosphate nucleation
occurred within the micelle cavity and the particle was spherical-
like in the dimension of 100 nm with excellent uniformity. With
the continuity of reaction, the growth of particles caused physical
forces, such as more frequent collisions, and change of viscosity
mainly caused by the variation of reaction temperature, which
might disrupt the integrity of interfacial film and disturb the sta-
bility of the whole system. The adhesion of small granules from
incompletely-reacted precursors onto the surface of particles
resulted in the change of standard spherical morphology into near
spherical ones with poor uniformity and regularity. It was indicated
that a relatively short reaction time, together with a moderate re-
action temperature could define the particle morphology by con-
trolling the stability of microemulsion droplets. On the contrary, a
sustained reaction time and higher or lower reaction temperature
would hamper the stability of whole microemulsion system and
lead to poor shape retention of spherical HAp.
4. Conclusion
We presented a versatile method for the preparation of nano-
sized spherical hydroxyapatite. HAp with good uniformity and
regularity was synthesized within water in oil microemulsion
system under optimized condition. It was demonstrated that the
thermodynamic stability of the microemulsion droplets was of
great importance on the control over HAp size and morphology. By
optimizing the parameters of microemulsion system with W/O of 1/
100 and O/SC of 7, and selecting an appropriate reaction tempera-
ture of 25  C and time of 5 h, well-dispersed spherical hydroxy-
apatite nanoparticles could be achieved. Among different methods,
microemulsion method can best restrict the particle growth in the
droplet to form spherical shape, in stead of the instinctive rod-like
shape [1]. Meanwhile, we have easy access to the reactants,
compared to the expensive and self-designed additives employed
in non-surfactant microemulsions. The obtained particles could
serve as promising candidates in biomedical applications, such as
bone tissue repairing for better cell-material interaction [40] and
drug loading for cancer therapy [41].
Acknowledgements
This work was supported by grants from the National Basic
Research Program of China (973 Program, No. 2012CB933600), the
111 Project (B14018), the National Natural Science Foundation of
China (No. 31271010, 31330028 and 31470924).
References
[1] H. Zhou, J. Lee, Nanoscale hydroxyapatite particles for bone tissue engineer-
ing, Acta Biomater. 7 (2011) 2769e2781.
[2] S.V. Dorozhkin, Bioceramics of calcium orthophosphates, Biomaterials 31
(2010) 1465e1485.
[3] Q. Wu, C. Liu, L. Fan, J. Shi, H. Jia, Q. Qi, L. Sun, F. Chen, Fabrication of hepa-
rinized hierarchically hollow hydroxyapatite microspheres as bone substitute
for controlled growth factors delivery, RSC Adv. 3 (2013) 7486.
[4] M. Yu, K. Zhou, F. Zhang, D. Zhang, Porous HA microspheres as drug delivery:
effects of porosity and pore structure on drug loading and in vitro release,
Ceram. Int. 40 (2014) 12617e12621.
[5] S.R. Rout, B. Behera, T.K. Maiti, S. Mohapatra, Multifunctional magnetic cal-
cium phosphate nanoparticles for targeted platin delivery, Dalton Trans. 41
(2012) 10777e10783.
[6] A. Bouladjine, A. Al-Kattan, P. Dufour, C. Drouet, New advances in nano-
crystalline apatite colloids intended for cellular drug delivery, Langmuir ACS J.
Surf. Colloids 25 (2009) 12256e12265.
[7] T. Long, Y.-P. Guo, Y.-Z. Liu, Z.-A. Zhu, Hierarchically nanostructured
mesoporous carbonated hydroxyapatite microspheres for drug delivery sys-
tems with high drug-loading capacity, RSC Adv. 3 (2013) 24169.
[8] J. Hui, X. Zhang, Z. Zhang, S. Wang, L. Tao, Y. Wei, X. Wang, Fluoridated HAp:
Ln3þ (Ln ¼ Eu or Tb) nanoparticles for cell-imaging, Nanoscale 4 (2012)
6967e6970.
[9] X. Zhang, J. Hui, B. Yang, Y. Yang, D. Fan, M. Liu, L. Tao, Y. Wei, PEGylation of
fluoridated hydroxyapatite (FAp):Ln3þ nanorods for cell imaging, Polym.
Chem. 4 (2013) 4120.
[10] M. Motskin, D.M. Wright, K. Muller, N. Kyle, T.G. Gard, A.E. Porter, J.N. Skepper,
Hydroxyapatite nano and microparticles: correlation of particle properties
with cytotoxicity and biostability, Biomaterials 30 (2009) 3307e3317.
[11] K.H. Muller, M. Motskin, A.J. Philpott, A.F. Routh, C.M. Shanahan, M.J. Duer,
J.N. Skepper, The effect of particle agglomeration on the formation of a
surface-connected compartment induced by hydroxyapatite nanoparticles in
human monocyte-derived macrophages, Biomaterials 35 (2014) 1074e1088.
[12] W. Tang, Y. Yuan, C. Liu, Y. Wu, X. Lu, J. Qian, Differential cytotoxicity and
particle action of hydroxyapatite nanoparticles in human cancer cells, Nano-
medicine 9 (2014) 397e412.
[13] X. Zhao, B.C. Heng, S. Xiong, J. Guo, T.T. Tan, F.Y. Boey, K.W. Ng, J.S. Loo, In vitro
assessment of cellular responses to rod-shaped hydroxyapatite nanoparticles
of varying lengths and surface areas, Nanotoxicology 5 (2011) 182e194.
[14] W. Liu, X.D. Chen, C. Selomulya, On the spray drying of uniform functional
microparticles, Particuology 22 (2015) 1e12.
[15] J.C.B. Lopes, P.E.M.D.S. Da, C. Laranjeira, et al., Network mixer and related
mixing process: U.S. Patent 8,434,933[P], 5e7, 2013-.
[16] M.C. Wang, H.T. Chen, W.J. Shih, et al., Crystalline size, microstructure and
biocompatibility of hydroxyapatite nanopowders by hydrolysis of calcium
hydrogen phosphate dehydrate (DCPD), Ceram. Int. 41 (2) (2015) 2999e3008.
[17] M.J. Lima, A.M.T. Silva, C.G. Silva, et al., An innovative static mixer photo-
reactor: proof of concept, Chem. Eng. J. 287 (2016) 419e424.
[18] P.E. Laranjeira, A.A. Martins, J.C.B. Lopes, et al., NETmix®, a new type of static
mixer: modeling, simulation, macromixing, and micromixing characteriza-
tion, AIChE J. 55 (9) (2009) 2226e2243.
[19] P.J. Gomes, V.M.T.M. Silva, P.A. Quadros, et al., A highly reproducible contin-
uous process for hydroxyapatite nanoparticles synthesis, J. Nanosci. Nano-
technol. 9 (6) (2009) 3387e3395.
[20] M. Sadat-Shojai, M.T. Khorasani, E. Dinpanah-Khoshdargi, A. Jamshidi, Syn-
thesis methods for nanosized hydroxyapatite with diverse structures, Acta
Biomater. 9 (2013) 7591e7621.
[21] N.E. Tari, M.M. Kashani Motlagh, B. Sohrabi, Synthesis of hydroxyapatite
particles in catanionic mixed surfactants template, Mater. Chem. Phys. 131
(2011) 132e135.
[22] Y. Wang, J. Chen, K. Wei, S. Zhang, X. Wang, Surfactant-assisted synthesis of
hydroxyapatite particles, Mater. Lett. 60 (2006) 3227e3231.
[23] W. Feng, L. Mu-sen, L. Yu-peng, Q. Yong-xin, A simple solegel technique for
preparing hydroxyapatite nanopowders, Mater. Lett. 59 (2005) 916e919.
[24] H. Eshtiagh-Hosseini, M.R. Housaindokht, M. Chahkandi, Effects of parameters
of solegel process on the phase evolution of solegel-derived hydroxyapatite,
Mater. Chem. Phys. 106 (2007) 310e316.
[25] Y. Yang, Q. Wu, M. Wang, J. Long, Z. Mao, X. Chen, Hydrothermal synthesis of
hydroxyapatite with different morphologies: influence of supersaturation of
the reaction system, Cryst. Growth Des. 14 (2014) 4864e4871.
[26] X. Jin, J. Zhuang, Z. Zhang, H. Guo, J. Tan, Hydrothermal synthesis of hy-
droxyapatite nanorods in the presence of sodium citrate and its aqueous
colloidal stability evaluation in neutral pH, J. Colloid Interface Sci. 443 (2015)
125e130.
[27] G.C. Koumoulidis, A.P. Katsoulidis, A.K. Ladavos, P.J. Pomonis, C.C. Trapalis,
A.T. Sdoukos, T.C. Vaimakis, Preparation of hydroxyapatite via microemulsion
route, J. Colloid Interface Sci. 259 (2003) 254e260.
[28] C. Lai, S. Tang, Y. Wang, K. Wei, Formation of calcium phosphate nanoparticles
in reverse microemulsions, Mater. Lett. 59 (2005) 210e214.
[29] M. Sanchez-Dominguez, G. Morales-Mendoza, M.J. Rodriguez-Vargas,
C.C. Ibarra-Malo, A.A. Rodriguez-Rodriguez, A.V. Vela-Gonzalez, S.A. Perez-
Garcia, R. Gomez, Synthesis of Zn-doped TiO2 nanoparticles by the novel oil-
in-water (O/W) microemulsion method and their use for the photocatalytic
degradation of phenol, J. Environ. Chem. Eng. 3 (2015) 3037e3047.
[30] G. Guo, Y. Sun, Z. Wang, H. Guo, Preparation of hydroxyapatite nanoparticles
by reverse microemulsion, Ceram. Int. 31 (2005) 869e872.
[31] H. Li, M. Zhu, L. Li, C. Zhou, Processing of nanocrystalline hydroxyapatite
particles via reverse microemulsions, J. Mater. Sci. 43 (2007) 384e389.
[32] Y. Sun, G. Guo, Z. Wang, H. Guo, Synthesis of single-crystal HAP nanorods,
Ceram. Int. 32 (2006) 951e954.
[33] C. Lai, Y.J. Wang, K. Wei, Nucleation kinetics of calcium phosphate nano-
particles in reverse micelle solution, Colloids Surf. A Physicochem. Eng. Asp.
315 (2008) 268e274.
[34] F.J. Arriagada, K. Osseo-Asare, Synthesis of nanosize silica in a nonionic water-
in-oil microemulsion: effects of the water/surfactant molar ratio and
ammonia concentration, J. Colloid Interface Sci. 211 (2) (1999) 210e220.
[35] W.Y. Zhou, M. Wang, W.L. Cheung, B.C. Guo, D.M. Jia, Synthesis of carbonated
hydroxyapatite nanospheres through nanoemulsion, J. Mater. Sci. Mater. Med.
19 (2008) 103e110.
[36] S.K. Saha, A. Banerjee, S. Banerjee, S. Bose, Synthesis of nanocrystalline hy-
droxyapatite using surfactant template systems: role of templates in con-
trolling morphology, Mater. Sci. Eng. C 29 (2009) 2294e2301.
[37] Changsheng Liu, Y. Huang, Wei Shen, Kinetics of hydroxyapatite precipitation
 X. Ma et al. / Materials Chemistry and Physics 183 (2016) 220e229 229

at pH 10 to 11, Biomaterials 22 (2001) 301e306.
[38] K. Sato, Y. Hotta, T. Nagaoka, M. Yasuoka, K. Watari, Agglomeration control of
hydroxyapatite nano-crystals grown in phase-separated microenvironments,
J. Mater. Sci. 41 (2006) 5424e5428.
[39] T. Furuzono, D. Walsh, K. Sato, Effect of reaction temperature on the
morphology and size of hydroxyapatite nanoparticles in an emulsion system,
J. Mater. Sci. Lett. 20 (2001) 111e114.
[40] A. Bernhardt, R. Dittrich, A. Lode, et al., Nanocrystalline spherical hydroxy-
apatite granules for bone repair: in vitro evaluation with osteoblast-like cells
and osteoclasts, J. Mater. Sci. Mater. Med. 24 (7) (2013) 1755e1766.
[41] B. Kundu, D. Ghosh, M.K. Sinha, et al., Doxorubicin-intercalated nano-
hydroxyapatite drug-delivery system for liver cancer: an animal model,
Ceram. Int. 39 (8) (2013) 9557e9566.