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nano-hydroxyapatite with tailored morphologies

Cite this: DOI: 10.1039/c7tb02487h
for biomedical applications†
Xiaoyu Ma,ab Yuanyuan Li,c Chengwei Wang,ab Yi Sun,c Yifan Ma,ab Xiuling Dong,c
Jiangchao Qian,c Yuan Yuan *abc and Changsheng Liu*abc

Received 16th September 2017,
Accepted 30th October 2017
DOI: 10.1039/c7tb02487h
rsc.li/materials-b
Hydroxyapatites (HAps) with nano-sized structures are promising materials in various biomedical areas,
but the synthesis of high quality particles is still challenged by the insufficient precision of size and
morphology, as well as the presence of severe agglomeration. An inadequate knowledge of the early
nucleation, growth and transformation might limit our exploration and application of HAp. Here, we
report a novel oil/water microemulsion–hydrothermal hybrid strategy for the preparation of highly
dispersive HAps with tailored morphologies and controlled size. Through the synergetic effect of the
oleic acid and microemulsion system, a well-dispersed HAp nucleus was first generated at 2 h. By tuning
the ensuing hydrothermal conditions from room temperature to 140 1C, the nucleus would grow from
spherical to needle-like nanoparticles. The size of the particles could be regulated by the alteration of
the hydrothermal temperature. In addition, we experimentally demonstrated the complete evolution of
HAp growth and transformation at a critical temperature of 90 1C by quenching the reaction at various
intervals. The obtained particles were explored as potential cellular delivery carriers and polymer fillers.

1. Introduction largely depend on their size and morphology, it is of great

As a major mineral component in bone tissue,1 hydroxyapatite
(Ca10(PO4)6(OH)2, HAp), with prominent bio-compatibility,
excellent bio-degradability, high bio-activity and non-toxicity,1–3
has already drawn great attention and has been extensively inves-
tigated in biomedical areas, acting as drug delivery vehicles,4,5
scaffolds in bone tissue engineering6 as well as active
coatings7 or scaffolds8 in orthopaedic and dental applications.
Compared to bulk HAp at the micrometer scale, HAps with nano-
sized structures9–11 exhibit better biological performance,12
including high drug-loading capacity, excellent osteogenesis
and osteointegration6,13,14 and unique selective anti-cancer
ability.15,16 Since the biological responses of the HAp nanoparticles
a
Key Laboratory for Ultrafine Materials of Ministry of Education, East China
University of Science and Technology, China. E-mail: yyuan@ecust.edu.cn,
liucs@ecust.edu.cn
b
Engineering Research Center for Biomedical Materials of Ministry of Education,
East China University of Science and Technology, China
c
The State Key Laboratory of Bioreactor Engineering, East China University of
Science and Technology, China
† Electronic supplementary information (ESI) available: Detailed TEM photos,
zeta potential, loading and releasing profile, hemolysis test for obtained particles.
In vitro experiment including cell culture, cytotoxicity study and intracellular
distribution of DOX/HAp samples with different amounts of HAp incorporation.
See DOI: 10.1039/c7tb02487h
significance to prepare particles with a nano-sized structure to
meet the increasing needs in the biomedical field. Unfortu-
nately, uncontrollable morphology and size, together with the
inherently high surface energy-induced severe agglomeration
and aggregation,17 greatly hamper its practical applications.
Traditional synthetic methods,3 such as chemical precipitation18 or
the sol–gel method,19,20 are insufficient to achieve nano-crystals
with excellent size control, fine regularity and uniformity. Thus,
there is still an urgent need to develop an effective route to precisely
engineer HAps, while avoiding subsequent agglomeration.
In recent years, synergistic strategies by coupling various
synthetic methods have been developed to achieve control over
HAp. Among them, microemulsion–hydrothermal synthesis
has attracted great interest, due to the narrow distribution
and prevention of both hard and soft agglomeration provided
by the microemulsion,2,21,22 and the high crystallinity free
of post-sintering offered by the hydrothermal method.22–24
A single crystal HAp with improved dispersibility and narrow
size distribution was prepared by Sun25 and Lin26 and various
ion-doped HAps, such as lanthanide27 or silicon,28 can also be
obtained via this hybrid strategy. However, in this process,
a reverse microemulsion is usually a necessity to load the
reactants since most Ca and P sources can only be dissolved
in water, thus leading to an excessive amount of organic solvent
and low water content, restricting the scale of reaction.

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In addition, the high surface energy of nHAp will easily cause
the generation of clusters via hydrogen bonding and further
development into larger agglomeration through crystalline
bridges in the water phase.29
To overcome the drawbacks of the traditionally reverse
microemulsion–hydrothermal synthesis, an improved artificial
microemulsion strategy based on interface-mediated synthetic
approaches30,31 was applied to synthesize semiconducting
metal chalcogenides,32 rare-earth compounds33 and other
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nanoparticles. Compared to the reverse microemulsion, the
interface in this system firstly provides the place for the
initiation of the reaction, then transfers the nanoparticles into
the oil phase for further confinement during ripening. Mean-
while, the nano-crystals generated at the interface were then
stabilized with oleic acid, transferred and confined in oil cores to
prevent the clustering induced by the capillary effect of water. As
inspired by the interface-mediated synthetic approach, it is
believed that this improved artificial microemulsion/hydrothermal
method could be extended to the synthesis of high quality calcium
phosphate-based nanoparticles.
In this study, we first introduced an interface-mediated
artificial oil/water microemulsion–hydrothermal hybrid strategy
for the preparation of highly dispersive nHAps with tailored
spherical and needle-like morphologies as illustrated in Fig. 1.
The microemulsion system composed of oleic acid, ethanol,
water and cyclohexane allows an excellent chemical homogeneity
under mild conditions and high yield with less organic reagents,
and thus is both ecologically and economically advantageous. In
this system, a well-dispersed HAp nucleus was firstly generated
and protected by the synergetic effect of the oleic acid and
microemulsion system. Hydrothermal treatment further induced
the transformation and growth of the nucleus into mature
HAps. The effects of time and temperature in the hydrothermal
treatment on particle size and morphology, and the possible
mechanism involved were systematically investigated. Finally,
the spherical and needle-like HAps with different crystallinities
were used as drug carriers and enhancers in polymers.
2. Experimental section
2.1 Materials
In the synthesis of HAp, Ca(NO3)2
4H2O (purity: Z99.0%),
(NH4)2HPO4 (purity: Z99.0%), and ethanol (purity: Z99.7%)
Fig. 1 Hybrid strategy of microemulsion with hydrothermal treatment for
the preparation of HAps.
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were all obtained from Sinopharm Chemical Reagent Co., Ltd,
(Shanghai, China). Oleic acid (density: 0.890–0.910 g mL 1 at
20 1C) and cyclohexane (purity: Z99.5%) were purchased from
Shanghai Lingfeng Chemical Reagent Co., Ltd, (Shanghai, China).
Deionized water was used in the particle synthesis process and all
the materials were used directly without further purification.
2.2 Preparation of HAp nanoparticles
HAps were prepared through the oil/water interface in a normal
microemulsion according to a previous study2 with some
modifications. First of all, a favorable microemulsion system
was designed as the reaction system. In detail, 3.2 g of sodium
hydroxide was dissolved into a mixed solution of water and
ethanol (v : v = 160 mL : 240 mL) in an 800 mL beaker under
continuous stirring. Then, 60 mL of oleic acid and 30 mL of
cyclohexane were slowly added into the beaker to form a bright
yellow and transparent microemulsion solution. Meanwhile,
two aqueous solutions of Ca(NO3)2
4H2O and (NH4)2HPO4
(0.5 mmol) with a volume of 25 mL were separately prepared
and gradually poured into the above microemulsion system one
after the other. After the end of the reaction, cyclohexane
was added into the system to disturb the balance of the micro-
emulsion and separate the solution into two phases, namely
the oil phase and the water phase. The oleic acid covered
nanoparticles were thus concentrated and extracted in the upper
oil phase. After precipitation by ethanol, the white precipitate
could be collected and re-dispersed into cyclohexane solution.
The particles were washed several times by alternating the
precipitating and re-dispersing process, then collected and
freeze-dried before further use.
For the subsequent hydrothermal treatment of the nano-
particles, the microemulsion was transferred into a stainless
steel Teflon-lined autoclave and heated for different durations
under various temperatures. The resultant products were washed
and collected according to the protocol mentioned above.
For the investigation of the function of oleic acid and the
pre-treatment of the microemulsion in the synthetic process, a
slight modification was carried out in a non-microemulsion
system without oleic acid, the oleic acid-replaced micro-
emulsion system and the oleic acid-only system without micro-
emulsion. In detail, the oil phase and oleic acid were moved out
for the non-microemulsion system without oleic acid while
the other compositions still remained at the same ratio and
followed the same synthesis process. As for the oleic acid-
replaced microemulsion system, 60 mL of polyethylene glycol
monooleyl ether (Brij-35) was applied to replace the oleic
acid, serving as a non-ionic surfactant to stabilize the micro-
emulsion. As for the oleic acid-only system, oleic acid was
added into the reaction system, but no oil phase was intro-
duced. The rest of the synthesis process and hydrothermal
treatment followed the same steps above.
2.3 Particle characterization
A Zetasizer Nano ZS (Malvern Instruments Ltd, UK) was used
for the detection of size and the size distribution of the
synthesized HAp nanoparticles in the microemulsion system.
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The dried particles were dispersed in cyclohexane before detec-

tion. The microstructure, including the size and morphology
of synthesized nanoparticles was observed through a trans-
mission electron microscope (JEM-2100, Japan) and a second
transmission electron microscope (JEM-1400, Japan). The samples
were prepared by dropping a 10 mL particle suspension onto the
300 mesh copper grid. The accelerating voltage was kept at
200 kV and 120 kV during the process. X-ray diffraction (Rigaku
D/max2550 VB/PC, Japan) was utilized to analyze the phase
Fig. 2 (A) Correlation between particle size and reaction time; (B) optical
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composition and inner atomic structure of the prepared nano- photos of microemulsion prior to and after addition of Ca and P.
particles under 40 kV/100 mA and 3500 CPS for the Cu target.
Scanning electron microscopy (SEM, JSM-6360LV, JEOL, Japan)
was used to determine the section morphology of HAp incor- An optimal composition ratio for the O/W microemulsion was
porated PLA. The Fourier-transform infrared (FTIR) spectra selected according to the empirical phase diagram proposed
were investigated by using a Perkin Elmer System 2000 spectro- by Li and Shan,2,33 and is detailed in Section 2.2, under the
meter (Nicolet Magma-550 series II, Midac, USA) from KBr rationale of ensuring the stability of the oil droplets while
pellets at wavelengths ranging from 4000 to 400 cm 1 at a retaining the minimum usage of organic content.
resolution of 1 cm 1 with an average of 64 scans. Inductively Based on the chosen composition ratio, the correlation
coupled plasma-atomic emission spectroscopy (ICP-AES, IRIS between the HAp growth and reaction time during the nucleus
1000, Thermo Elemental, USA) was also utilized to determine formation stage was found in a time-dependent manner
the degradation and dissolution of calcium phosphate nano- (Fig. 2A). During the first 2 hours, the size was predominantly
particles. Aliquots of the samples were prepared by dissolving maintained below 10 nm without further agglomeration and
the particles in nitric acid before the test. Surface analysis of overgrowth. However, with the extension of reaction time to 12
the obtained calcium phosphate particles was measured by and 24 h, the size enlarged to 70 nm. It is worth mentioning
N2 adsorption/desorption using a Micromeritics ASAP2010 that at the endpoint of 24 h, a cluster of nuclei of about 543 nm,
sorptometer (Micromeritics, USA). Prior to the measurement, accounting for nearly 1% of the total amount, was observed.
all the samples were kept at 100 1C under vacuum for 4 h for The optical photos in Fig. 2B confirmed the disturbance of
degassing. The specific surface area was determined by the the homogeneity with the increase of reaction time. Hence, a
Brunauer–Emmett–Teller (BET) model. relatively short reaction time under 2 h was adopted to ensure a
well-distributed HAp nucleus for the following hydrothermal
2.4 Potential applications treatment.
Furthermore, the morphology and distribution of the initial
Drug loading and in vitro release of DOX. The drug-loaded
nucleus obtained under 2 h was observed under TEM. It can be
particles were prepared. UV-vis spectroscopy was utilized to
seen in Fig. 3A that the particles are of excellent dispersibility
evaluate the adsorption ability of the particles. Meanwhile, the
and regular spherical structure within 10 nm. In addition, as
release profile was determined at both pH 7.4 and 5.4. The
observed in Fig. 2B, the microemulsion incorporated with oleic
details of loading and release, as well as the in vitro experi-
acid remained bright yellow and transparent after the addition of
ments, including cell culture, cytotoxicity study, intracellular
Ca while it gradually turned white and turbid after the addition
distribution of DOX/HAp and hemolysis test, are demonstrated
in the ESI.† The study was carried out in strict compliance with
the NIH guidelines for the care and use of laboratory animals
(NIH Publication No. 85e23 Rev. 1985) and was approved by
the Research Center for Laboratory Animal of the Shanghai
University of Traditional Chinese Medicine.
Mechanical test of HAp incorporated PLA. Different amounts
of HAp were incorporated into PLA, and the PLA/HAp composite
was formed by solvent evaporation. The samples were then
evaluated through SEM observation and the tensile strength test
as detailed in the ESI.†

3. Results and discussion

3.1 Formation of microemulsion and initiation of calcium
phosphate nucleus
In this study, a normal oil/water microemulsion system was
first established for the formation of the stable HAp nucleus.
Fig. 3 Particles obtained under different conditions at 2 h (A) oleic acid
and microemulsion (B) Brij35 and microemulsion (C) oleic acid without
microemulsion (D) without oleic acid and microemulsion.

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of the P precursors, indicating the system slowed down the Ca/P

complexation and offered stability to the generated nucleus.
To explore the roles that the oleic acid and microemulsion
played, a set of experiments were conducted and compared in
Fig. 3B–D. Polyethylene glycol monooleyl ether (Brij-35), a non-
ionic surfactant with a similar HLB value of 17, was substituted
for oleic acid in the same synthesis route. Though a steady
microemulsion system was formed by replacing oleic acid with
Brij-35, the obtained particles succumbed to severe agglomera-
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tion and aggregation. Meanwhile, when oleic acid was applied

in the system alone without microemulsion, the subsequent
particles were of short-rod shape with a broad size distribution
from B5–50 nm. Though the incorporation of oleic acid offered
control of the morphology and dispersibility, it did not contribute
to the improvement in particle regularity, in both shape and size
distribution. These results confirmed the indispensable effects of
both oleic acid and the microemulsion.

3.2 Effect of hydrothermal temperature under a critical

temperature of 90 8C on particle evolution
On the foundation of the obtained well-defined and distributed
nuclei, additional hydrothermal treatment was then conducted
to guide the nHAp growth and to improve crystallinity. It is well-
known that in hydrothermal treatment, temperature and pressure
are elevated above the boiling temperature of solvents to increase
the activity of reactants, induce the formation and precipitation of
nucleus, and facilitate the growth of particles for the generation of
high quality nHAp. Considering the preliminary study where the
mixture boiled at around 90 1C, the hydrothermal temperature
investigated in this study was divided into two regions: below and
above the critical point of 90 1C.
A series of hydrothermal temperatures below and at 90 1C
was first carried out. As seen in Fig. 4, the products were of
spherical shape with good dispersibility and uniformity. Under
the boiling temperature of the solvents, the reaction can no
longer be considered as a genuine hydrothermal process, thus
failing to control the crystal growth. Therefore, the synthesized
particles were HAp with low crystallinity, as suggested by the
spherical shape and substantiated by XRD patterns. In addi-
tion, the Ca/P ratio was about 1.60 for the three samples, which
was close to the stoichiometric ratio of the standard HAp as
identified by ICP-MS. Nonetheless, the temperature is still a
crucial factor in the control of particle size. The size of particles
prepared at room temperature around 25 1C was 8 nm to 10 nm
while the diameter of the HAps could enlarge to B30 nm at
70 1C. The size and size distribution test performed by DLS
accorded well with the direct observation in TEM, indicating
the size of particles could be tuned by the variation of the
reaction temperature.
Specifically, as indicated in Fig. 4C, it was fascinating to find
that at a critical temperature of 90 1C, both spherical and
needle-like morphologies coexisted in the current system. The
size of the spherical particles increased to around 50 nm, and
the length of the needle-like particles was below 50 nm. It was
speculated that at the current reaction temperature, the energy
provided by the hydrothermal process was adequate to induce
Fig. 4 TEM images of hydrothermally treated HAp at different tempera-
tures under 90 1C for 2 h (A, B, C) room temperature around 25 1C, 70 1C,
90 1C; (A3 and B3) XRD patterns and (A4 and B4) DLS for the measurement
of particle size at different temperatures.
the crystal growth of HAp. The variation in size and shape
suggested that the further growth and transformation could
involve a dissolution and recrystallization process.
To deepen our understanding of the transformation and
growth of nHAp, the morphology change was then traced at 4, 8
and 12 h at the critical point of 90 1C in Fig. 5. It was found that
at 4 h, both spherical and needle-like particles coexisted in the
reaction system. Compared to that at 2 h, the particles still
remained intact with a spherical shape, but the diameter of the
particles significantly decreased from 50 nm to around 10 nm.
Meanwhile, the number of needle-like HAp started to increase
and the length was further extended. The shrinkage of the
spherical ones indicated that the particles had first undergone
the dissolution stage. However, after 8 hours of hydrothermal
treatment, the small spherical particles could no longer main-
tain their shape and further elongated into spindle-shaped
ones with a width of 5–10 nm and a length of 15–20 nm, where
a structural rearrangement along the c-axis occurred with a
clear and distinct increase in the length-to-width ratio. When
the hydrothermal time further increased to 12 h, uniform and

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Fig. 5 TEM images for HAp evolution under 90 1C at 4, 8, 12 h A, B, C (1–2):
HAp morphology; A, B, C (3): XRD patterns; A, B, C (4): FT-IR spectra.
well-distributed needle-like HAps of 50–80 nm in length over-
whelmed the field of view during the TEM evaluation, and
almost no spherical or spindle-shaped particles could be
observed. The alteration in morphology, therefore, presented
direct verification of the dissolution and recrystallization
mechanism. To confirm the transformation in the crystalline
structure, the phase composition was also examined by XRD at
different time intervals. HAp with a relatively low crystallinity
was observed at the first few hours (less than four) while
particles with a higher degree of crystallinity could be obtained
with increasing time. In addition, the typical CaP phase com-
position was identified by using FTIR spectra in A, B, C (4). The
absorption peak at 1070 cm 1, as well as at 559 cm 1 and
719 cm 1 could be ascribed to the stretching and bending
vibration of the PO43 . Broad peaks around 3100–3500 cm 1
came from the OH stretching from HAp and absorbed water.
Specifically, a small peak at 1700 cm 1 and a sharp peak at
2850 cm 1 may belong to the stretching vibration of CQO and
C–H in the remaining oleic acid on the products. In summary,
at 90 1C, the boiling temperature of the mixture, the hydro-
thermal reaction began and remained at a relatively low speed,
so as to avail us with the opportunity to explore the transforma-
tion and growth of nHAps. The products went through a series
of evolutions, with an elongation along the preferential axis of
HAp and the perfection of crystallization.
3.3 Effect of hydrothermal temperature above the critical
temperature of 90 8C on particle evolution
The hydrothermal temperature was further increased to inves-
tigate the influence on particle size and morphology (Fig. 6).
The initial nuclei would directly grow into elongated ones and
become highly crystallized and more intact in morphology.
With the energy input from hydrothermal treatment, spherical
particles were able to further elongate and the size was tuned by
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Fig. 6 TEM images and XRD patterns for hydrothermally treated HAp
at different temperatures above 90 1C for 2 h (A: 100 1C; B: 120 1C;
C: 140 1C).
the change of temperature. At 100 1C, most HAps were about
80–100 nm in length. With the temperature reaching 140 1C,
the length of the needle-shaped HAp increased to 200 nm.
However, the as-synthesized particles did not show an obvious
change in both size and morphology at a higher temperature,
indicating that 140 1C was adequate to guarantee the full
growth of HAp. On the other hand, after the treatment at
100–140 1C, the obtained XRD patterns exhibited that well-
crystalized HAps were synthesized.
The hydrothermal time was also tuned for the particles
synthesized at 120 1C, as a demonstration of particle growth
at normal hydrothermal temperature. Shown in Fig. 7, all the
HAps prepared at 120 1C were needle-like shaped, with a length
of around 200 nm. There’s no obvious difference in the particle
length at various reaction times. However, in the TEM observa-
tion, a small portion of subtle granules appeared at 2 h and 6 h,
but almost no granules could be seen at 12 h. It was inferred
that an adequate reaction time could lead the tiny granules to
Fig. 7 TEM images for hydrothermally treated HAp at 120 1C for different
times (A: 2 h; B: 6 h; C: 12 h).
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Fig. 8 Relations among the size, morphology, reaction temperature
and time.
further grow along the c-axis to form needle-like particles with
good regularity and uniformity. In short, compared to the
critical temperature of 90 1C, the extension of the reaction time
to other temperatures could facilitate the complete growth of
HAp, but did not alter the particle size.
With a comprehensive consideration of the above results,
the reaction temperature has a significant influence on the
size, morphology and crystallization of HAp in the synthesis.
In contrast, the properties of the obtained particles were not
sensitive to reaction time, though the extension of time could
facilitate growth and crystallization (Fig. 8).
3.4 Growth mechanism of HAp nanoparticles
Based on the above results obtained, a possible growth mecha-
nism of nHAps could be summarized. In the microemulsion
system, stable droplets were first formed with negatively charged
COO provided by the oleic acid at the outside layer. A stable
complex of calcium oleate23,34,35 via the Coulomb attraction
between COO and Ca2+ was then formed at the exterior of the
droplets, surrounding the oil core. Upon addition of PO43 , the
nucleus was generated at the interface of the oil droplets, due to
the lower Ksp of CaP (B10 25–10 23) than that of calcium oleate
(B10 17.4). The PO43 gradually replaced the oleic acid, thus
slowing down the CaP formation to endow precise control on
the regularity and uniformity of the HAP nucleus. Since the
oleic acid can cover the freshly formed nucleus, rendering the
hydrophobicity, the as-synthesized particles, capped with
the hydrophobic oleic acid chain thus moved from the interface
layer into the oil core,34,36 and stabilized the droplets in the
microemulsion system as a surfactant.37 And the microemulsion
acted as nano-reactors to confine the particle size and distribu-
tion. As protected by the microemulsion droplet, the HAp
nucleus was within the micelle cavity, separated from the outer
water phase and the resultant particles maintained a spherical-
like shape with low crystallinity. On the other hand, the chances
for droplets containing HAp nuclei to collide and merge with
others decreased due to the capping. The oleic acid and micro-
emulsion worked together to guarantee the regularity and
uniformity of the initial nucleus, which is of great importance
in generating the following high quality particles, since the
heterogeneous state at the nucleus formation could lead to
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irregularity and severe agglomeration after the next hydrother-
mal stage, as shown in Fig. S1 (ESI†).
The amorphous nature of the particle may sometimes limit
its application; thus, a hydrothermal process was employed in
the study. During the post-treatment, the boiling temperature of
the solvent at 90 1C, was extraordinarily important and served as
the critical temperature for the transformation of morphology,
and the crystallization of the HAp. Below the critical temperature
of 90 1C, the energy provided by the post-treatment was not
adequate to promote the crystal growth of HAps, but did, to
some degree, have an influence on the size of the resultant
particles. In this study, starting at 90 1C, small needle-like
particles gradually appeared. The width of the needle-like parti-
cles was far less than the diameter of the spherical ACP,
suggesting further growth took place in the dissolution and
recrystallization process.38 The coexistence of both the spherical
and needle-like particles indicated a synergic evolution of both
the outer morphology and the inner crystalline phase. As
reported by Liu,39 the transformation kinetics could be treated
with the Arrhenius equation, where a high activation energy was
acquired for the particle surface reaction controlled process of
transforming ACP to highly crystalized nHAp. The dissolution
and precipitation of the structural transformation was typically a
slow process, but the energy provided by the heat and pressure
involved in the hydrothermal treatment greatly accelerated the
process, identified at 90 1C with a clear morphology evolution. It
was revealed that at higher temperatures, the particles remained
at the same morphology with a needle-like shape. According to
previous reports,40 the intrinsic crystal growth of HAp has its
preference along the c-axis, leading to an elongated rod-like
morphology under most circumstances. With the increase of
hydrothermal temperature, the length of the particles could be
tuned from less than 50 nm to more than 200 nm with high
crystallinity. The needle-like particles elongated by adsorbing
dissolved Ca2+ and PO43 through an oriented attachment route
along the c-axis. HAps belong to the hexagonal system, with
L6PC symmetry where the a- and b-axes are perpendicular to the
c-axis. In the growth process of HAp, the difference of the major
unit cells at the a-axis (Ca2+) and the c-axis (OH ) could lead to
different crystal growth rates. In previous reports, it was found
that the COOH groups would surround the Ca2+ ions,41,42 form a
stable complex due to their strong coordination ability and
absorb on specific crystallographic planes of the resultant
HAp, which subsequently inhibited the normal growth of parti-
cles by shortening the a-axis and lengthening the c-axis38 of the
unit cell. Given the adequate energy input in the hydrothermal
treatment here, the covered oleic acid on the particles, rich
in COOH groups, could facilitate anisotropic growth along the
c-axis and inhibit particle growth in the other two directions.
In addition, as reported by Mann,43,44 the appropriate chain
lengths of oleic acid (B0.5 nm) were a good match to the
distance between the coplanar Ca2+ cations in HAp, thus further
justifying a preferred orientation that gives rise to an oriented
growth along the c-axis into a needle-like shape with high
quality.45 Meanwhile, the modification of oleic acid on the
HAp surface further decreases the zeta potential, to improve
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Fig. 10 (a) MTT for free DOX, DOX-loaded CaP NPs in HeLa cells (b) MTT
for pure CaP NPs in both HeLa and L02 cells; (c) confocal photos of free
DOX and DOX-loaded CaP NPs s after 4 h and 24 h uptake (statistical
significance was set at *P o 0.05 while extreme significance was set
at **P o 0.01).

Fig. 9 Possible mechanism of the growth of HAp nanoparticles.

the dispersibility through charge repulsion among particles,

as in Fig. S2 (ESI†). The whole process, involving nucleation,
transformation and growth is illustrated in Fig. 9.

3.5 Application of HAp nanoparticles

Spherical HAps as drug delivery vehicles. DOX, a model drug,
could be loaded more efficiently onto spherical particles by means
of physical adsorption and electrostatic interactions (Table S1
and Fig. S3a in ESI†). Meanwhile, the release profile indicated a
pH dependent manner (Fig. S3b, ESI†) in a controllable way.
DOX/HAp exhibited a better cytotoxicity in HeLa cells, as in
Fig. 10(a), in a concentration-dependent manner, where the cell
viability drastically decreased to around 10% when the loaded
DOX reached 2 mg mL 1. The cell uptake efficiencies in (c) show
that the DOX/HAp nano-carrier could effectively transport DOX
into HeLa cells in a much faster way. Cell viability tests were
carried out in both cancer cells (HeLa) and normal cells (L02) in
Fig. 10(b), respectively. It was shown that the CaP particles were
non-toxic in normal cells while exhibiting specific toxicity to
tumor cells. The lower pH inside tumor cells might accelerate
the Ca2+ release of HAp, interfere with the normal metabolic
process and lead to the necrosis and apoptosis of tumor
cells. In addition, the safety of the nanoparticles was further
corroborated by the hemolysis test, proving that the particles, Fig. 11 (a) Stress–strain curve of HAp incorporated PLA; (b and c) optical
even when loaded with DOX, had no hemolysis risks in the and SEM images of HAp incorporated PLA (d) elemental mapping images
investigation (shown in Fig. S4, ESI†). of Ca and P for PLA/10HA.
Needle-like HAp nanoparticles as polymer fillers. By incor-
porating of HAp into PLA in Fig. 11(a), the strength of the
extension was slightly lowered, but the tenacity was greatly as in HAp ceramics. (Immediate break of sample after cutting
promoted, with an increase of elongation at break from nearly shown with 50% HAp incorporation in Fig. S5, ESI†).
4% to 160% while the HAp content reached 10%. The stress We believe that the underlying mechanism for the improve-
whitening appeared (Fig. 11b) confirming that 10% HAp incor- ment in mechanical properties might be attributed to the
poration best augmented the ductility of PLA. However, when following two reasons. Firstly, the oleic acid-modified HAp
further increasing the HAp amount, the maximum strain with hydrophobicity, facilitated homogeneous dispersion and
decreased and the sample was apt to show a brittle fracture accommodation into PLA as observed in Fig. 11(c and d).

This journal is © The Royal Society of Chemistry 2017 J. Mater. Chem. B


Paper
The excellent dispersion further guaranteed the particles’ entry
into the PLA phase to mix and entangle with the long chains of
the polymer matrix, leading to the nanofiber-strengthening
effect. In contrast, the traditional precipitated HAp showed a
clear agglomeration and clear phase separation as in Fig. S6
(ESI†). Secondly, the sparsely-dispersed particles can share the
applied load efficiently through the interaction between
HAp and PLA, which thereby enhances the particle-induced
absorbance and dispersion of extra mechanical stress to
Published on 02 November 2017. Downloaded by University of Reading on 17/11/2017 13:01:12.
improve tensile strength. Specifically, the needle-like structure
with a high length-to-width ratio could increase the contact area
between the polymer and HAp to form a firm organic/inorganic
interface.46,47 Additionally, it was reported that calcium
phosphate-based materials could generate a strong interaction
with PLA,48 by forming a weak ionic bond between Ca and the
ester group double bond in PLA. When external load is applied
to the composite, cracks would be propagated through particles,
and extra energy was needed to pull out the needle-like fillers49
to overcome the mechanical interlocking and improved inter-
facial bonding50 over that of the spherical ones during the
material breakage. A large number of HAp needles and traces
of pull-out would consume great energy in the fracture, thus
significantly improving the mechanical properties.
4. Conclusions
In summary, a hybrid route of O/W microemulsion and hydro-
thermal treatment was developed to synthesize nano-sized
HAps with tailored size and morphology. In the oleic acid-
assisted microemulsion system, the HAp nucleus was firstly
formed, presenting a spherical shape with excellent dispersi-
bility and regularity. After the subsequent hydrothermal treat-
ment, the nucleus continued growing to form needle-like
particles with a high length-to-width ratio while maintaining
good dispersibility. Prominently, altering the hydrothermal
temperature could lead to precise control over the particle size
and morphology of the resultant nanoparticles. A dissolution
and recrystallization process was identified during the complete
evolution of HAp growth and transformation at the critical
temperature of 90 1C by quenching the reaction at various
intervals. Due to the high dispersibility, regularity and uniformity,
as well as its flexibility in size and shape control, the obtained
nHAp could satisfy the requirements in the biomedical field
as a drug delivery vehicle or as a reinforcing material for
compound scaffolds.
Abbreviations
(n)HAp (nano) Hydroxyapatite particles
CaP Calcium phosphate
ACP Amorphous calcium phosphate
Conflicts of interest
There are no conflicts to declare.
J. Mater. Chem. B
View Article Online
Journal of Materials Chemistry B
Acknowledgements
This work was supported by grants from the National Natural
Science Foundation of China for Innovative Research Groups
(No. 51621002), the National Natural Science Foundation of China
(No. 31330028 and 31470924), and the 111 Project (B14018).
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