Polymer xxx (2015) 1e8

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Polymer
journal homepage: www.elsevier.com/locate/polymer

Synthesis of hydroxyapatite particles with in situ immobilized

ATRP initiator
_ Wodarczyk a, Rachel Ferebee b, Michael R. Bockstaller b, Joanna Pietrasik a, *
Bazej
a
b

Institute of Polymer and Dye Technology, Lodz University of Technology, Stefanowskiego 12/16, 90-924 Lodz, Poland
Department of Materials Science and Engineering, Carnegie Mellon University, Wean Hall 4307, 5000 Forbes Ave., Pittsburgh, PA 15213, USA

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 22 December 2014
Received in revised form
14 March 2015
Accepted 17 March 2015
Available online xxx

A novel method of in situ incorporation of an ATRP initiator during synthesis of hydroxyapatite particles
is described. Calcium nitrate tetrahydrate, ammonium hydrogenphosphate and 2-bromo-2methylpropionic acid were used as the functionalized hydroxyapatite precursors. Particles with homogenous size distribution within the diameter range of 300500 nm and controlled initiator content
were synthetized. The synthetic procedure of hydroxyapatite was investigated in detail to determine the
inuence of numerous parameters such as pH of reaction mixture, ATRP initiator concentration, dilution
of ammonium hydroxide, on the nal properties of the particles. It appeared the optimal conditions were
those in which reaction was carried out under basic conditions, namely pH 10, attained by slow
addition of the diluted ammonium hydroxide and using no more than 1 M initial concentration of ATRP
initiator. The effectiveness of incorporated ATRP initiator was veried on the basis of surface initiated
polymerization of methyl methacrylate. Particles and hybrids properties were investigated by DLS,
elemental analysis, XRD, TEM, FT-IR and GPC.
2015 Elsevier Ltd. All rights reserved.

Keywords:
ATRP
Polymer brushes
Hydroxyapatite

1. Introduction
Hydroxyapatite (Ca10(PO4)6(OH)2) (HAP) is a naturally occurring
mineral that is also a constituent of human bones and teeth and has
been the object of intensive studies, mainly because of its potential
utility for bio-related applications [1]. While HAP was originally
mostly considered as bone substitute and scaffold for hard tissue
engineering more and more attention has been directed towards
materials in which HAP is responsible for producing a reinforcing
effect, for example in polymer composites [2]. The broad applicability of HAP has resulted in a multiplicity of efforts that have
focused on the controlled synthesis of HAP derivatives, aiming at
varied composition, structure, size and morphology of the materials, each depending on the nal specic application [3,4].
One of the main problems that is usually addressed once HAP is
selected as a part of a composite, or a blend, regardless the HAP
properties itself, is its homogeneous dispersion. It is well known
that a surface modication process is very often required in order to
make inorganic particles compatible with an organic matrix. Silanes are the most typically utilized surface modifying agents [5e7]

* Corresponding author.
E-mail address: joanna.pietrasik@p.lodz.pl (J. Pietrasik).

but an alternative approach is immobilization of a polymer on the

particles surface that, as a consequence, induces miscibility with
the appropriate polymer matrix [8,9].
Polymer immobilization on a particles surface can be chemical
or physical in nature. There are two approaches, which allow for
such procedures, namely grafting to and grafting from [10e12].
In the grafting to approach already synthesized polymer chains,
containing appropriate complementary linking functional groups,
interact with the particles surface. The number of polymer chains
per surface area can be signicantly higher if the grafting from
approach is used. Polymerization from the surface can be carried
using different chain growth mechanisms [13]. Controlled/radical
polymerization methods are commonly used since the molecular
weight of synthesized polymers increases linearly with the reaction
time, providing copolymers with narrow distribution since transfer
and termination reactions are signicantly reduced [14,15]. Therefore using atom transfer radical polymerization ATRP [16e19],
reverse addition fragmentation chain transfer polymerization RAFT
[20,21], or in some cases nitroxide mediated polymerization NMP
[22,23] one can design very precisely the composition of the nal
hybrid particles. ATRP is one of the most often frequently used
techniques for the synthesis of such coreeshell materials. ATRP
provides a simple way to prepare many different types of polymers

http://dx.doi.org/10.1016/j.polymer.2015.03.044
0032-3861/ 2015 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Wodarczyk B, et al., Synthesis of hydroxyapatite particles with in situ immobilized ATRP initiator, Polymer
(2015), http://dx.doi.org/10.1016/j.polymer.2015.03.044

B. Wodarczyk et al. / Polymer xxx (2015) 1e8

with varied composition and topology [24] and has been used for
surface initiated polymerization from many particles [25e27] with
different chemical nature, shape and dimensions.
Grafting density is an essential parameter that determines the
properties of organic-inorganic hybrids [13,28,29]. Both high and
low grafting density brushes are useful in different application
areas. High-density polymer brushes for example may be used as a
barrier preventing adsorption on the surface [30]. While less
densely grafted systems more easily allow for post polymerization
modication along the polymer chain since the permeability of the
system is much higher than in the case of high-density brushes
[31]. Therefore one can tailor the nal properties of synthesized
materials simply by changing the grafting density and degree of
polymerization (DP) of polymer chains attached to the surface [32].
In the grafting from approach the variation of the polymer
grafting density results from the initial concentration of immobilized surface initiator. There are two strategies that can be used to
accomplish this objective. The rst one is based on control over the
amount of available initiator tethered to the surface, which can be
achieved by variation of the initiator concentration or the time of
initiator deposition on the modied surface [33,34]. A gradation of
grafting density can be obtained by controlling the intensity of the
stimulus or by application of a photomask [35e37]. However, the
main drawback of this approach is that most of these methods are
mostly limited to at surfaces.
The second strategy relies on the full coverage of functional sites
on the particle surface with a mixture of the active initiator and its
inert analog, also called a dummy initiator. This approach assumes the same reactivity of both molecules; therefore simply by
controlling their ratio in the reaction mixture one can control the
concentration of active species on the modied surface [27,38,39].
Unfortunately, in a case of any difference in the reactivity of real and
dummy initiators, different composition on the surface when
compared to the modifying mixture will be generated [40]. That can
make this technique to some extend tricky, as the concentration of
active species does not always scale with its concentration in the
reaction mixture.
Overall both of the mentioned approaches rely on the modication of already synthesized inorganic substrates, which means it
is a two-step procedure. Herein, a novel one step method for
incorporation of an ATRP initiator into hydroxyapatite particles
during their synthesis is described.
As reported in the literature a variety of synthetic techniques
can be used for the synthesis of hydroxyapatite. Synthesis of nano
and microcrystals of HAP with varied morphology, rods, spheres,
stars, and snowakes, has been widely reported. Techniques such
as solegel [41e43], co-precipitation, the so-called wet method
[38,44], hydrothermal synthesis [45] or the mechanochemical
method can be used [46]. The co-precipitation method is extremely
popular mainly because of its versatility and low cost of production.
Furthermore the size of the particles can be easily controlled. It has
been demonstrated that particle size can be tuned simply by
changing the reaction conditions, i.e. temperature, which also inuences the nucleation and growth of the crystals [47e49]. The
presence in the reaction mixture of such substances as citric acid
[50,51], amino acids [52], and ethylenediaminetetraacetic acid
(EDTA) [53] inuences the morphology of the growing particles.
Although the synthesis of HAP by the chemical precipitation
technique is simple, and provides HAP particles with a controlled
size and morphology, the obtained particles have a strong tendency
to agglomerate. Therefore there is a great motivation to modify its
surface properties during synthesis.
This manuscript describes a synthetic procedure for synthesis of
hydroxyapatite particles containing accessible ATRP initiators.

Since the amount of built-in initiator corresponds to its initial

concentration in the reaction mixture one can tailor the density of
initiating sites on the particle surface, and hence the grafting
density of attached polymer chains, which could effectively separate the single particles simply by varying the initiator
concentration.
The literature provides some examples for the HAP particles
modication by polymers obtained by ATRP, however according to
our knowledge no data on the described herein methodology for
initiator attachment could be found [54e56].
2. Experimental
2.1. Materials
Calcium nitrate tetrahydrate (Ca(NO3)2  4H20, 99%, Aldrich),
ammonium hydrogenphosphate ((NH4)2HPO4, 98%, Aldrich), 2bromo-2-methylpropionic acid (C4H7BrO2, 98%, Aldrich), aqueous
solution of ammonium hydroxide (NH4OH, 28%, Aldrich), 4,4dinonyl-2,2-dipyridyl (dNbpy, 97%, Aldrich), copper(I) bromide
(CuBr, 99.999%, Aldrich), copper(II) bromide (CuBr2, 99.999%,
Aldrich), methanol (99, Aldrich), dimethylformamide (99%,
Aldrich), aqueous solution of hydrochloric acid (HCl, 37%, Aldrich)
were used as received. Methyl methacrylate (MMA, 99%, Aldrich)
was distilled before the usage.
2.2. General procedure for the synthesis of hydroxyapatite particles
Entry 1, Table 1. A solution of Ca(NO3)2  4H20 (0.24 M, 5 g of
substance), in distilled water was added to a round bottom ask
equipped with magnetic stir bar and the ask was placed in an oil
bath at 25  C and sonicated for 5 min. Then a sonicated water
mixture of (NH4)2HPO4 (0.29 M, 2.4 g of substance) and C4H7BrO2
(0.8 M, 8.35 g of substance) was added drop wise to the ask and
the reaction mixture was vigorously stirred for 2 h. After that the
pH of the solution was adjusted to the desired value by adding an
ammonium hydroxide (NH4OH) solution. The reaction was run for
an additional 24 h and the precipitate was centrifuged and washed
a few times with distilled water. As a nal step, 70 mL of distilled
water was added to the wet hydroxyapatite and the mixture was
sonicated for 5 min before being frozen in liquid nitrogen and
lyophilized.
The synthetic procedure was the same for each entry in the
table while three parameters were varied: pH, initiator and
ammonium hydroxide concentration. In the series of examples
with constant initial initiator concentration (0.8 M) the pH was
adjusted to values from 7 to 12, as listed in Table 1. The initial
concentration of 2-bromo-2-methylpropionic acid added to the
reaction mixture was changed from 0.2 M to 1.4 M, while maintaining a pH 10, Table 2. Concentrated or diluted ammonium
hydroxide was used, namely 28% and 7%. The 7% was prepared by
diluting the 28% NH4OH with water at the ratio of NH4OH/H2O 1/
2, vol/vol, Table 2.
2.3. General procedure for surface initiated ATRP of methyl
methacrylate
Synthetic conditions: Table 3, Entry 1. HAP-g-Br (0.0843 g,
0.047 mmol Br), CuBr (0.0067 g, 0.047 mmol), CuBr2 (0.0026 g,
0.012 mmol), and dNbpy (0.0478 g, 0.117 mmol) were added to a
25 mL Schlenk ask equipped with a magnetic stir bar and purged
with argon for at least 40 min. Then the degassed MMA (5 mL,
0.047 mol) was added to the ask via syringe. The ask was placed
in preheated oil bath at 70  C and after a predetermined time (24 h),

Please cite this article in press as: Wodarczyk B, et al., Synthesis of hydroxyapatite particles with in situ immobilized ATRP initiator, Polymer
(2015), http://dx.doi.org/10.1016/j.polymer.2015.03.044

B. Wodarczyk et al. / Polymer xxx (2015) 1e8

Table 1
Size and bromine content of hydroxyapatite particles obtained at different pH values using 0.8 M of the initial ATRP initiator concentration.
Entry

pH [-]

Br content [%]

Radius in toluenea [nm]

PDI [-]

Radius in watera [nm]

PDI [-]

1950
2500
1140
780
750
780
1590
1090
1040
1220
1390
1020

1
1
1
0.50
0.66
0.63
1
0.67
0.80
0.91
0.75
0.94

520
370
900
1160
1270
1110
1160
1240
1140
930
1000
1330

1
0.78
1
0.34
0.17
0.42
0.29
0.08
0.33
0.26
0.23
0.28

2.94

10

7.38

11

5.99

12

2.83

PDI e polydispersity index of the particles size.

a
Three values relate to three measurements of one sample. Each sample was scanned ve times within one measurement.

Table 2
Size and bromine content of hydroxyapatite particles obtained at different initial ATRP initiator concentrations at pH 10.
Entry

Cinitiator [mol/dm3]

0.2

4.27

0.4

4.43

0.8

7.38

0.8a

6.31

0.8b

3.70

1.0

14.97

1.0a

7.00

1.4

14.27

Br content [%]

Radius in toluenec [nm]

PDI [-]

Radius in waterc [nm]

PDI [-]

2460
1660
1460
1970
2190
2720
780
750
780
980
860
910
370
520
340
610
660
530
1880
1370
1560
1580
1270
950

1
0.66
0.33
0.26
0.60
0.59
0.5
0.66
0.63
0.46
0.04
0.23
0.74
0.68
0.25
0.73
0.67
0.60
0.58
0.35
0.59
1
0.67
0.91

1030
1240
950
1290
1300
1320
1160
1270
1110
1030
1200
1150
1840
2150
2840
1900
2110
1810
600
800
770
660
520
670

0.35
0.29
0.46
0.29
0.30
0.35
0.34
0.17
0.42
0.14
0.15
0.27
0.59
0.32
1
0.46
0.46
0.35
1
1
1
0.66
0.48
0.51

PDI e polydispersity index of the particles size. For the Entries 1-3, 6 and 8, the concentration of used ammonium was 28%.
a
Ammonium concentration 7%.
b
pH of reaction mixture equals 7; ammonium concentration 7%.
c
Three values relate to three measurements of one sample. Each sample was scanned ve times within one measurement.

Table 3
TGA (mass loss) and GPC (Mn, Mw/Mn) data for poly(methyl methacrylate) synthetized from the surface of obtained hydroxyapatite particles.
Entry
a

1
2a
3a
4a
5a
6b
7b

Cinitiator [mol/dm3]

pH [-]

Br content [%]

Mass loss [%]

Mn [g/mol]

Mw/Mn [-]

0.4
0.8
1.0
1.4
0.8
1.0
1.4

10
10
10
10
10 (1:2)c
10 (1:2)c
10 (1:2)c

4.43
7.38
14.97
14.29
6.31
7.00
14.14

6.9
17.1
33.0
27.6
57.2
60.6
71.7

95,000
35,000
18,400
20,700
284,000
394,400
369,400

1.21
1.24
1.49
1.29
1.18
1.10
1.20

Polymerization conditions: MMA/Br/CuBr/CuBr2/dNbpy 1000/1/1/0.25/2.5 @

a
70  C.
b
50  C for 24 h.
c
The value 10 (1:2) relates to the use of 7% NH4OH, which was prepared by diluting of the 28% NH4OH with water in ratio of NH4OH/H2O 1/2, vol/vol), Table 2.

it was removed from the oil bath and opened to expose the catalyst
to air. The polymerization solution was added to methanol to precipitate the hybrid particle and the solvent was evaporated in the
vacuum oven.

2.4. Procedure for polymer detachment from particles

1 mL of a 35% HCl (aq.) solution was added to 1 mL of a solution
of the HAP polymer brush hybrid particles in tetrahydrofuran

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(~50 mg/mL), and the reaction was allowed to stir at room temperature overnight. The mixture was placed in an ice bath then
ammonium hydroxide solution (28.030.0% NH3 basis) was added
very slowly to the ask until the pH value reached and remained
above 8. The water part was separated from the organic part. The
cleaved polymer was recovered from organic part by evaporating
the tetrahydrofuran solution in air.
2.5. Characterization techniques
Elemental analysis was used to determine bromine content of
the synthesized hydroxyapatite particles. Bromine content analysis
} niger method. After mineralization on a
was performed by Scho
platinum catalyst in a ask with oxygen, resulting ions were
titrated against a standard solution of a suitable indicator. Thermogravimetric analysis (TGA) experiments were performed with
samples placed in aluminum pans, using a Mettler Tolledo Star,
operating in the 20e700  C temperature range, under nitrogen
atmosphere, at a heating rate 10 deg/minute. Average hydrodynamic diameters of particles and polydispersity index values were
determined by dynamic light scattering in water and toluene,
respectively, at a temperature of 25  C with Malvern Nanosizer S-90
instrument. The concentration of measured samples was 5 g/L.
Molecular weights and molecular weight distributions of PMMA
were determined with the dn/dc values of 0.083 using Wyatt
ASTRA software. The X-ray diffraction (XRD) patterns of the HAP
nanoparticles modied with ATRP initiator was obtained with CoKa
radiation (l 1.789 A) using Siemens D500 diffractometer. Transmission Electron Microscopic (TEM) images were obtained using a
JEOL JEM 2000 EX II. The 0.5 g/L solution of HAP were sonicated and
immediately drop cast onto a carbon coated copper grid.
3. Results and discussion
3.1. Synthesis of hydroxyapatite
3.1.1. Inuence of the pH of the reacting mixture
The initial series of experiments with the initiator concentration
equal 0.8 M were conducted at pH 7, 10, 11 or 12 respectively,
Entries 1-4; Table 1. There are two factors, which should be
considered when hydroxyapatite particles (HAP) synthesis is discussed within the current studies, namely the bromine (Br) content
and the particle size. It appears that HAP synthesis at the extreme
pH values, pH 7 and pH 12, are not the best conditions since the
lowest bromine weight contents were obtained in the nal materials, that is 2.94% and 2.83% respectively. Moreover it can be
assumed that the surface of the particles obtained at pH 7 was
clearly hydrophilic in its nature, and that was conrmed by DLS
analysis (plots are not shown), that this led to particle agglomeration in organic solvents. On the contrary the highest bromine
content 7.38%, was detected for particles obtained at a pH 10.
When the pH 11, the bromine content was slightly lower, at the
level of 6%. These numbers for the %Br are far below the maximum
value that could be obtained if 100% of reaction is assumed, the
value should be in the range of 17% by weight. This observation
could be attributed to variation in the solubility of the ATRP initiator in the reaction mixture under different pH values due to the
protonation of acidic groups or salt formation.
The size of the hydroxyapatite particles synthesized under the
controllable varied conditions was determined by dynamic light
scattering of lyophilized particles. Measurements were conducted
in two different solvents, namely in toluene and water. A comparison of the particles tendency towards agglomeration will provide
some information concerning the surface character of synthesized
particles. The results of those measurements are provided in

Table 1. The particles were relatively large (>1100 nm) and heterogeneous in size when the synthesis was carried out at pH 7
(PDI 1). The large particles were agglomerates rather than single
particles. The relatively smaller diameter of agglomerates, when
measured in water, means the hydroxyapatite surface is more hydrophilic than hydrophobic. That observation matches the results
of elemental analysis for the bromine content.
Larger particles were also obtained at higher pH, pH 11 and
pH 12. Particle radius was higher than 1000 nm and particles
were more homogenous in water. The opposite tendency was
observed in a case of synthesis carried out at pH 10. The tendency
towards agglomeration is lower in toluene, although as it can be
seen from the DLS plots that the samples were still not very uniform, Fig 1.
A more detailed analysis carried out by TEM conrmed that the
obtained particles are agglomerates rather than primary particles.
TEM analysis was conducted on layers obtained by casting toluene
or water dispersions. TEM images obtained for HAP synthesized at
pH 7 or pH 10 clearly showed particles in a shape of needles
whose size, only when individually isolated, would be in the
nanometer range (Fig. 2a, b). This is contrary to what was observed
by Palamivelu et al. who reported rod-like structures for particles
prepared at pH 7 while spherical particles were obtained at
pH 9. They assumed it was because of the different ionization of
substrates and different ions mobility rates [57].
3.1.2. Inuence of initiator concentration
It was expected that the concentration of ATRP initiators on the
particles surface could be easily tailored simply by changing the
initial concentration of initiators in the reaction mixture. As shown
in Table 2 the concentration of 2-bromo-2-methylpropionic acid
added to the reaction mixture was changed from 0.2 M to 1.4 M but
it appeared that at low initiator concentrations, namely 0.2 M and
0.4 M almost the same Br content was detected (~4%) but particles
size was signicantly smaller in water than toluene. This observation seems to be reasonable, since the concentration of organics on
the surface of the particles was still relatively low. A further increase in the amount of 2-bromo-2-methylpropionic acid, to 0.8 M
and 1.0 M, resulted in higher bromine content, up to ~15% in the
particles, and consequently decreased their size in toluene. In a case
of addition of 1.4 M of 2-bromo-2-methylpropionic acid, a reduced
solubility of initiator was observed which could explain why
bromine content almost remained constant. TEM images did not
demonstrate any signicant changes in the agglomerates of primary particles which when isolated were in nanometer size range.
There was no clear and direct correlation observed between the
initial concentration of initiator in the solution and within the
particles. It should be noted that the discussed bromine content
refers to the total amount of initiator incorporated into the particles
while the character of particles surface should mostly determine
the tendency of hydroxyapatite particles toward agglomeration.
One could conclude that as the concentration of initiator in the
solution increased the mechanism of Br incorporation as well as its
solubility in the reaction mixture changed, resulting in nonstraightforward correlations.
3.1.3. Inuence of the concentration of ammonium hydroxide on the
obtained particles
The inuence of the ammonium hydroxide concentration on the
properties of obtained particles, namely their size and incorporation of built-in initiator, was also investigated. Two different concentrations were examined, 28% and 7%, (NH4OH/H2O 1/2, vol/
vol). The concentration of the ATRP initiator was 0.8 M and 1.0 M,
and particle synthesis was carried out at pH values of 7 and 10 as
listed in Table 2. As the ammonium hydroxide is mainly responsible

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Fig. 1. Statistical particle size distribution for hydroxyapatite particles synthesized at pH 10, ATRP initiator 0.8 M, (Entry 2, Table 1), measured in a) toluene b) water dispersion.

Fig. 2. TEM images of hydroxyapatite particles synthesized at a) pH 7, b) pH 10,

ATRP initiator 0.8 M, (entry 1e2, Table 1), measured from toluene dispersion.

for the acceleration of the hydroxyapatite formation and its precipitation in the reaction mixture it was assumed that by using a
more dilute solution of NH4OH the process could be slightly slowed
down. Consequently that potentially could allow synthesis of particles with both a smaller and more homogeneous size. However, it
appeared that such a hypothesis was not fully correct.

When the particles synthesis was done at pH 10 the dilution of

the ammonium hydroxide solution resulted in a decrease in the
concentration of incorporated initiator. In the case when 0.8 M
initiator concentration was used the bromine content for HAP
decreased from 7.38% to 6.31% while the size of the particles in
toluene increased from the range 750e780 nm to 860e980 nm.
However, they became more uniform in size (PDI<0.46). At higher
initiator concentration (1.0 M), the same tendency was observed;
total Br content decreased and particles size in toluene was higher,
when compared to particle size measured in water. In water the
samples were very heterogeneous as was reected by PDI 1.
Different effects were observed for the particles obtained at
pH 7. First of all it should be noted the although bromine content
was lower when compared to the previously discussed case a slight
increase of Br content was observed when dilute ammonium hydroxide was used for the reaction. Furthermore looking at the DLS
data it can be concluded that under such conditions it was possible
to change the surface properties of the particles. That is from hydrophilic particles for HAP synthesized with 28% NH4OH solution to
hydrophobic ones obtained with a more diluted system (7%). Such a
signicant change in the nature of the surface properties reected
by differences in the size of particles in different solvents is very
surprising especially since the bromine content in the particles was
not signicantly affected and remained at a level of below 4% in

Please cite this article in press as: Wodarczyk B, et al., Synthesis of hydroxyapatite particles with in situ immobilized ATRP initiator, Polymer
(2015), http://dx.doi.org/10.1016/j.polymer.2015.03.044

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both cases. Therefore one can conclude that although lower pH

does not promote the bromine incorporation it is possible to
preferentially locate the initiators in the outer part of particles if
their synthesis is conducted with higher dilution of ammonium
hydroxide.
Overall it can be concluded that the concentration of ammonium hydroxide is very crucial from of point of view of ATRP
initiator incorporation. However it should be discussed individually
for different reaction conditions.
3.1.4. XRD and FT-IR analysis
The XRD and FT-IR analyses were carried out to conrm the
formation of hydroxyapatite particles. Fig. 3 shows the obtained
XRD patterns for arbitrarily chosen hydroxyapatite particles synthesized in the presence of varied concentrations of ATRP initiator
in the reaction mixture, that is 0.8 M; 1.0 M and 1.4 M. All detected
peaks (2 theta values) in the XRD patterns very clearly matched the
standard peaks of HAP in terms of the position of the lines. It can be
seen as the concentration of initiator increases the intensity of the
peaks decrease, which is a logic consequence of a change in the
composition of the particles because of incorporation of the initiator within the particles. As reported by Kumar lower intensity
could also be the result of a deciency of calcium and higher
crystalline size [57].
Fig. 4 shows FTIR spectra of the obtained HAP powders, containing varied bromine content. It can be seen that the character of
each plot is similar, demonstrating the same chemical composition.
The n3 vibration band of phosphate group could be identied in the
region between 1090 and 960 cm. The bands at 965e960 cm and
within 601e565 cm range correspond to n1 symmetric PeO
stretching vibrations of the phosphate ion. The broad absorption
bands around 3030 cm, 3000 cm or 3200 cm belong to the
stretching modes of hydroxyl groups (OH) in the HAP but could also
result from some water absorption. The absorption bands at the

range of around 1340, 1400 and 875 cm could correspond to the

bending methyl and carbonebromine bands.
3.2. ATRP of methyl methacrylate from the surface of
hydroxyapatite
To conrm the effectives of the presented approach and ability
to initiate polymerization from the particle surface a series of ATRP
polymerizations of methyl methacrylate initiated from the particles
surface were carried out. After the polymerization the molecular
weight of cleaved polymer and weight loss of the hybrid particles
were determined. The results are provided in Table 3.
It appeared in most cases that the polymerizations were
controlled, resulting in polymers with dispersity index Mw/
Mn < 1.3. The average molecular weight was in the range from
18,000 up to 400,000 Da. Although all polymerizations were
carried out under the same conditions; MMA/-Br 1000/1, the
concentration of initiators on the particles in the reaction mixtures varied because the different Br content. Therefore it can be
assumed the probability of the termination or gelation was not
the same. Moreover the particles size also varied for each sample. However what is most important is that it is not clear how
much of incorporated bromine was located directly on the surface of the particles and how uniform the incorporation was in
each case. The various values of weight loss are obviously not
only because of the different molecular weights Mn, but also due
to the different number of PMMA chains that were grown from
that specic HAP surface. It was not possible to calculate the
grafting density of chains in each of the obtained hybrid particles
since the particles were not uniform in size and as pointed above
aggregates rather than single particles should be discussed.
Further studies are under investigations to determine how
effectively the ATRP initiator was located on the particles
surface.

Fig. 3. X-Ray diffraction data for hydroxyapatite particles obtained at pH 10, 1:2 NH4OH:H2O, using the following initial concentration of ATRP initiator: 0.8 M; 1.0 M; 1.4 M.
Reference plot of the hydroxyapatite with low concentration of Br was marked as brown.

Please cite this article in press as: Wodarczyk B, et al., Synthesis of hydroxyapatite particles with in situ immobilized ATRP initiator, Polymer
(2015), http://dx.doi.org/10.1016/j.polymer.2015.03.044

B. Wodarczyk et al. / Polymer xxx (2015) 1e8

Fig. 4. FT-IR plots for hydroxyapatite particles obtained at pH 10, 1:2 NH4OH:H2O, using the following initial concentration of ATRP initiator: 0.8 M; 1.0 M; 1.4 M.

4. Conclusion
It was demonstrated an ATRP initiator could be directly incorporated into a HAP particle during HAP particle synthesis. The
amount of initiator incorporated into the nal particle can be varied
by its initial concentration added to the reaction mixture and by the
conditions employed for HAP particle synthesis. The size and homogeneity of the particles can be modied by the value of pH
reached during the synthesis and also by the concentration of the
ammonium hydroxide solution used to attain that pH. The obtained
particles could be used for polymer brushes synthesis. The ATRP
polymerization of methyl methacrylate initiated from the surface of
the HAP particles proceeded in a controlled manner. The proposed
strategy for in situ incorporation of an ATRP initiator into an inorganic particle prior to preparing organic-inorganic hybrids signicantly simplies the procedure for their synthesis.
Acknowledgments
The authors acknowledge the support from National Science
Center, Poland; grant UMO-2011/03/B/ST5/01084. MRB acknowledges support by the National Science Foundation (via grant CMMI1234263).
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.polymer.2015.03.044.
References
[1] Loo SCJ, Moore T, Banik B, Alexis F. Biomedical applications of hydroxyapatite
nanoparticles. Curr Pharm Biotechnol 2010;11:333e42.
[2] Moursi AM, Winnard AV, Winnard PL, Lannutti JJ, Seghi RR. Enhanced osteoblast response to a polymethylmethacrylate-hydroxyapatite composite.
Biomaterials 2001;23:133e44.
[3] Sakamoto K, Nakahira A, Okazaki M, Ichihara J, Inoue M, Yamaguchi S. Shapecontrolled synthesis of hydroxyapatite in organic media. Mater Res Soc Symp
Proc 1998;519:407e11.
[4] Luo P, Nieh TG. Preparing hydroxyapatite powders with controlled
morphology. Biomaterials 1996;17:1959e64.
[5] Dupraz AMP, van der Meer SAT, De Wijn JR, Goedemoed JH. Biocompatibility
screening of silane-treated hydroxyapatite powders, for use as ller in
resorbable composites. J Mater Sci Mater Med 1996;7:731e8.
[6] Bareiro FO, Dos Santos LA. Polydimethylsiloxane/calcium phosphates composites: effect of the llers modication on the cross-linking and surface
energy. Mater Sci Forum 2012;727e728:1175e80.
[7] Deng C, Xiao X, Yao N, Yang XB, Weng J. Effect of surface modication of nanohydroxyapatite particles on in vitro biocompatibility of poly (-Caprolactone)matrix composite biomaterials. Int J Polym Mater 2011;60:969e78.

[8] Fu G, Xia Z, Jiang J, Jing B, Zhang X. Fabrication and characterization of

nanocomposites with high-impact polystyrene and hydroxyapatite with
well-dened polystyrene via ATRP. J Reinf Plast Compos 2011;30:
1445e53.
[9] He J, Yang X, Mao J, Xu F, Cai Q. Hydroxyapatite-poly(l-lactide) nanohybrids
via surface-initiated ATRP for improving bone-like apatite-formation abilities.
Appl Surf Sci 2012;258:6823e30.
[10] Tsubokawa N. Modication of particle surfaces by grafting of functional
polymers. Spec Publ e R Soc Chem 1999;235:36e51.
[11] Yoshinaga K. Functionalization of inorganic colloidal particles by polymer
modication. Bull Chem Soc Jpn 2002;75:2349e58.
[12] Minko S. Grafting on Solid surfaces: Grafting to and Grafting from
methods. In: Stamm M, editor. Polymer surfaces and Interfaces. Springer
Berlin Heidelberg; 2008. p. 215e34.
[13] Tsujii Y, Ohno K, Yamamoto S, Goto A, Fukuda T. Structure and properties of
high-density polymer brushes prepared by surface-initiated living radical
polymerization. In: Jordan R, editor. Surface-initiated polymerization I.
Springer Berlin Heidelberg; 2006. p. 1e45.
[14] Cohen Y, Yoshida W, Nguyen V, Bei N, Jou J-D. Surface modication of inorganic oxide surfaces by graft polymerization. Surfactant Sci Ser 2001;103:
321e53.
[15] Olivier A, Meyer F, Raquez J-M, Damman P, Dubois P. Surface-initiated
controlled polymerization as a convenient method for designing functional
polymer brushes: from self-assembled monolayers to patterned surfaces. Prog
Polym Sci 2012;37:157e81.
[16] Wang JS, Matyjaszewski K. Controlled/living radical polymerization. Atom
transfer radical polymerization in the presence of transition-metal complexes.
JACS 1995;117:5614e5.
[17] Matyjaszewski K, Xia J. Atom transfer radical polymerization. Chem Rev
2001;101:2921e90.
[18] Braunecker WA, Matyjaszewski K. Controlled/living radical polymerization:
Features, developments, and perspectives. Prog Polym Sci 2007;32:93e146.
[19] Matyjaszewski K, Tsarevsky NV. Macromolecular engineering by atom
transfer radical polymerization. JACS 2014;136:6513e33.
[20] Boyer C, Whittaker MR, Luzon M, Davis TP. Design and synthesis of dual
thermoresponsive and antifouling hybrid polymer/gold nanoparticles. Macromolecules 2009;42:6917e26.
[21] Deng Y, Yang C, Yuan C, Xu Y, Bernard J, Dai L, et al. Hybrid organic-inorganic
block copolymer nano-objects from RAFT polymerization-induced self-assembly. J Polym Sci Part A: Polym Chem 2013;51:4558e64.
[22] Blas H, Save M, Boissiere C, Sanchez C, Charleux B. Surface-initiated nitroxidemediated polymerization from ordered mesoporous silica. Macromolecules
2011;44:2577e88.
[23] Chen F, Cai Z, Huang Y, Luo W, Chen J. Synthesis and characterization of
copolymer grafted magnetic nanoparticles via surface-initiated nitroxidemediated radical polymerization. Polym Eng Sci 2013;53:956e62.
[24] Matyjaszewski K. Atom transfer radical polymerization (ATRP): current Status
and Future perspectives. Macromolecules 2012;45:4015e39.
[25] Hui CM, Pietrasik J, Schmitt M, Mahoney C, Choi J, Bockstaller MR, et al.
Surface-initiated polymerization as an enabling tool for multifunctional
(Nano-)engineered hybrid materials. Chem Mater 2014;26:745e62.
[26] Wu D, Hui CM, Dong H, Pietrasik J, Ryu HJ, Li Z, et al. Nanoporous polystyrene
and carbon materials with core-shell nanosphere-interconnected network
structure. Macromolecules 2011;44:5846e9.
[27] Ojha S, Dang A, Hui CM, Mahoney C, Matyjaszewski K, Bockstaller MR. Strategies for the synthesis of thermoplastic polymer nanocomposite materials
with high inorganic lling fraction. Langmuir 2013;29:8989e96.
[28] Goel V, Pietrasik J, Dong H, Sharma J, Matyjaszewski K, Krishnamoorti R.
Structure of polymer tethered highly grafted nanoparticles. Macromolecules
2011;44:8129e35.

Please cite this article in press as: Wodarczyk B, et al., Synthesis of hydroxyapatite particles with in situ immobilized ATRP initiator, Polymer
(2015), http://dx.doi.org/10.1016/j.polymer.2015.03.044

B. Wodarczyk et al. / Polymer xxx (2015) 1e8

[29] Choi J, Hui CM, Pietrasik J, Dong H, Matyjaszewski K, Bockstaller MR.

Toughening fragile matter: mechanical properties of particle solids assembled
from polymer-grafted hybrid particles synthesized by ATRP. Soft Matter
2012;8:4072e82.
[30] Inoue Y, Ishihara K. Reduction of protein adsorption on well-characterized
polymer brush layers with varying chemical structures. Colloids Surf B
2010;81:350e7.
[31] Sugnaux C, Lavanant L, Klok H-A. Aqueous fabrication of pH-Gated, polymerbrush-modied alumina hybrid membranes. Langmuir 2013;29:7325e33.
[32] Mei Y, Wu T, Xu C, Langenbach KJ, Elliott JT, Vogt BD, et al. Tuning cell
adhesion on gradient poly(2-hydroxyethyl methacrylate)-grafted surfaces.
Langmuir 2005;21:12309e14.
[33] Binder WH, Zirbs R, Machl D, Gahleitner M. Grafting polyisobutylene from
nanoparticle surfaces: concentration and surface effects on livingness. Macromolecules 2009;42:7379e87.
[34] Lego B, Francois M, Skene WG, Giasson S. Polymer Brush Covalently attached
to OH-Functionalized Mica surface via surface-initiated ATRP: control of
grafting density and polymer chain Length. Langmuir 2009;25:5313e21.
[35] Li Y, Giesbers M, Gerth M, Zuilhof H. Generic top-functionalization of
patterned antifouling zwitterionic polymers on indium tin oxide. Langmuir
2012;28:12509e17.
[36] Nakai K, Morigaki K, Iwasaki Y. Molecular recognition on uidic lipid bilayer
microarray corralled by well-dened polymer brushes. Soft Matter 2010;6:
5937e43.
[37] Pranantyo D, Xu LQ, Neoh K-G, Kang E-T, Yang W, Lay-Ming Teo S. Photoinduced anchoring and micropatterning of macroinitiators on polyurethane
surfaces for graft polymerization of antifouling brush coatings. J Mater Chem B
2014;2:398e408.
[38] Liou SC, Chen SY, Lee HY, Bow JS. Structural characterization of nano-sized
calcium decient apatite powders. Biomaterials 2004;25:189e96.
[39] Pietrasik J, Bombalski L, Cusick B, Huang JY, Pyun J, Kowalewski T, et al.
Controlling polymer chain topology and architecture by ATRP from at surfaces. In: Urban MW, editor. Stimuli-responsive polymeric lms and coatings;
2005. p. 28e42.
[40] Ulman A. Formation and structure of self-assembled monolayers. Chem Rev
1996;96:1533e54.
[41] Padmanabhan SK, Balakrishnan A, Chu MC, Lee YJ, Kim TN, Cho SJ. Sol-gel
synthesis and characterization of hydroxyapatite nanorods. Particuology
2009;7:466e70.
[42] Ramanan SR, Venkatesh R. A study of hydroxyapatite bers prepared via solgel route. Mater Lett 2004;58:3320e3.
[43] Sanosh KP, Chu M-C, Balakrishnan A, Kim TN, Cho S-J. Preparation and
characterization of nano-hydroxyapatite powder using sol-gel technique. Bull
Mater Sci 2009;32:465e70.

[44] Wang F, Li MS, Lu YP, Qi YX, Liu YX. Synthesis and microstructure of hydroxyapatite nanobers synthesized at 37  c. Mater Chem Phys 2006;95:
145e9.
[45] Zhang G, Chen J, Yang S, Yu Q, Wang Z, Zhang Q. Preparation of amino-acidregulated hydroxyapatite particles by hydrothermal method. Mater Lett
2011;65:572e4.
[46] Nasiri-Tabrizi B, Honarmandi P, Ebrahimi-Kahrizsangi R, Honarmandi P.
Synthesis of nanosize single-crystal hydroxyapatite via mechanochemical
method. Mater Lett 2009;63:543e6.
[47] Cai Y, Mei D, Jiang T, Yao J. Synthesis of oriented hydroxyapatite crystals:
effect of reaction conditions in the presence or absence of silk sericin. Mater
Lett 2010;64:2676e8.
[48] Liu C, Huang Y, Shen W, Cui J. Kinetics of hydroxyapatite precipitation at pH
10 to 11. Biomaterials 2001;22:301e6.
[49] Wang P, Li C, Gong H, Jiang X, Wang H, Li K. Effects of synthesis conditions on
the morphology of hydroxyapatite nanoparticles produced by wet chemical
process. Powder Technol 2010;203:315e21.
[50] Jiang D, Li D, Xie J, Zhu J, Chen M, Lue X, et al. Shape-controlled synthesis of Fsubstituted hydroxyapatite microcrystals in the presence of Na(2)EDTA and
citric acid. J Colloid Interface Sci 2010;350:30e8.
[51] Santos C, Martins M, Almeida MM, Costa MEV. Inuence of the (citric acid/
calcium) ratio on Hap particles synthesis. Microsc Microanal 2009;15:
85e6.
[52] Tsetsekou A, Brasinika D, Vaou V, Chatzitheodoridis E. On the synthesis of
tailored biomimetic hydroxyapatite nanoplates through a bioinspired
approach in the presence of collagen or chitosan and L-arginine. Mat Sci Eng
C-Materials Biological Appl 2014;43:555e65.
[53] Xin R, Ren F, Leng Y. Synthesis and characterization of nano-crystalline calcium phosphates with EDTA-assisted hydrothermal method. Mater Des
2010;31:1691e4.
[54] Wang Y, Xiao Y, Huang X, Lang M. Preparation of poly(methyl methacrylate)
grafted hydroxyapatite nanoparticles via reverse ATRP. J Colloid Interface Sci
2011;360:415e21.
[55] Xiao Y, Lang S, Wang Y, Lang M. Effect of surface grafted hydroxyapatite on
the improved performance of hydroxyapatite/poly(-caprolactone) scaffold.
Curr Nanosci 2014;10:855e62.
[56] Zeng L, Wang H, Fu G, Jiang J, Zhang X. A new approach for synthesis of the
comb-shaped poly (-caprolactone) brushes on the surface of nanohydroxyapatite by combination of ATRP and ROP. J Colloid Interface Sci
2010;352:36e42.
[57] Palanivelu R, Saral AM, Kumar AR. Nanocrystalline hydroxyapatite prepared
under various pH conditions. Spectrochimica Acta Part a-Molecular Biomol
Spectrosc 2014;131:37e41.

Please cite this article in press as: Wodarczyk B, et al., Synthesis of hydroxyapatite particles with in situ immobilized ATRP initiator, Polymer
(2015), http://dx.doi.org/10.1016/j.polymer.2015.03.044