International Journal of Pharmaceutics 478 (2015) 187–192

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International Journal of Pharmaceutics

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

Pharmaceutical nanotechnology

Stability studies of As4S4 nanosuspension prepared by wet milling in

Poloxamer 407
 áková * , Erika Dutková, Matej Baláž, Erika Turianicová, Peter Baláž
Zdenka Bujn
Institute of Geotechnics, Slovak Academy of Sciences, Watsonova 45, Košice 040 01, Slovakia

A R T I C L E I N F O A B S T R A C T

Article history: In this paper the stability of the arsenic sulfide (As4S4) nanosuspension prepared by wet milling in a
Received 15 September 2014 circulation mill in the environment of copolymer Poloxamer 407 was studied. The obtained As4S4
Received in revised form 14 November 2014 particles in nanosuspension were of
100 nm in size. The influence of temperature and UV irradiation on
Accepted 17 November 2014
the changes in physical and/or chemical properties was followed. Long-term stability was observed via
Available online 18 November 2014
particle size distribution and zeta potential measurements. Influence of UV irradiation was studied via
UV–vis spectroscopy (UV–vis), photoluminicsence (PL) technique and Fourier transform infrared
Keywords:
spectroscopy (FTIR) measurements. The best stability of the nanosuspension (24 weeks) was achieved
Nanosuspension
Stability
when stored at 4  C and in the dark.
Nanomilling ã 2014 Elsevier B.V. All rights reserved.
Arsenic sulfide
Particle size

1. Introduction growth, a phenomenon known as Ostwald ripening (Boistelle and

The preparation of hydrophobic drugs in the form of nano-
suspensions represents a challenging and potentially useful
method for improving their bioavailability. Nanosuspensions are
defined as liquid sub-micron colloidal dispersions of nanosized
drug particles that are stabilized by a suitable polymer and/or
surfactant (Rabinov, 2004). Several technologies have been
developed for preparation of nanosuspensions (e.g., milling,
high-pressure homogenization, impinging jet, electro-spraying,
liquid-based methods and supercritical fluid processes) (Wang
et al., 2013). Among these, wet media milling is considered an
attractive approach that permits relatively easy scale-up with
respect to industrial pharmaceutical nanosuspension production
(Merisko-Liversidge and Liversidge, 2011). During the milling
process, the drug crystals break into smaller particles, and thus
fresh surfaces are continuously generated (Baláž, 2008).
In nanosuspensions, water-insoluble drugs become more
soluble because of increasing the saturation solubility and the
surface area available for dissolution. On the other hand, a
nanosuspension is a thermodynamically unstable colloid disper-
sion system. A high surface area associated with the small size of
the particles results in high interfacial tension, which in turn
results in an increase in the free energy of the system. The high
surface energy of nano-sized crystals results in particle size
* Corresponding author. Tel.: +421 557922607; fax: +421 557922604.
 áková).
E-mail address: bujnakova@saske.sk (Z. Bujn
Astier, 1988). During this process, coarse particles grow at the
expense of the redissolution of fine particles (Kim, 2004), which
are more soluble than large ones and mass transfer occurs from the
fine to coarse particles (Liu et al., 2011). To hinder this
phenomenon and consequently aggregation, the electrostatic or
steric stabilizers are needed.
Arsenic sulfide compounds have a long history of application in
a traditional medicine (Wang, 2001). In recent years, they have
been studied as promising drugs in cancer treatment (Wu and Ho,
2006; Ye et al., 2006; Baláž et al., 2009, 2012; Baláž and Sedlák,
2010; Wu et al., 2011; Yuan et al., 2013; Tian et al., 2014; Pastorek
et al., 2014). In this paper the stability of the arsenic sulfide
nanosuspension stabilized with Poloxamer 407 (PX407) was
studied. PX407 is a nonionic surfactant composed of polyoxy-
ethylene–polyoxypropylene triblock copolymers. It has a good
solubilizing capacity and is considered a good medium for drug
delivery systems with the least toxic properties of commercially
available copolymers (Gilbert et al., 1986; Escobar-Chávez et al.,
2006). It has been reported that both hydrophobic and hydrophilic
properties of stabilizers are required for ensuring the appropriate
stability. The driving force of the adsorption of stabilizers onto the
surface of a hydrophobic drug is the hydrophobic moieties of the
stabilizers. In the absence of adsorption, stabilization cannot occur,
and no dispersed nanosuspensions can be obtained (Wang et al.,
2013). The hydrophilic portion of the stabilizer tends to orient
toward water providing an effective steric stabilization. In the case
of PX407, central polyoxypropylene chain is hydrophobic (b)
flanked by two hydrophilic chains of polyoxyethylene (a) (Fig. 1).

http://dx.doi.org/10.1016/j.ijpharm.2014.11.043
0378-5173/ ã 2014 Elsevier B.V. All rights reserved.
188  áková et al. / International Journal of Pharmaceutics 478 (2015) 187–192
Z. Bujn
Fig. 1. Chemical structure of Poloxamer 407 (a-ethylene oxide portion, b-propylene
oxide portion).
Although, the arsenic sulfides seem to be very promising
anticancer agent, there is lack of information about stability of
these nanosuspensions in the literature. This is the main aim of
this study.
2. Materials and methods
2.1. Material
The investigation was carried out with arsenic sulfide (98%,
Sigma–Aldrich). Poloxamer 407 (Sigma–Aldrich) was applied as a
stabilizer.
2.2. Preparation of nanosuspensions
The nanosuspensions were prepared in a laboratory circulation
mill MiniCer (Netzsch, Germany). Five grams of arsenic sulfide
were subjected to milling in the presence of 300 ml PX407 water
solution (0.5%) for duration of 2 h at the milling speed 4000 rpm.
The mill was loaded with yttrium stabilized ZrO2 milling balls. The
resulting nanoparticle suspension was filtered through a 0.22 mm
sterile filter. After that, the nanosuspension was divided into two
parts and stored in dessicator at laboratory temperature (21 1  C)
or in refrigerator (4  C). The solid phase was subjected to
dissolution tests.
2.3. Dissolution experiments
The dissolution experiments were performed stirring 100 mg of
solid phase into 100 ml of water (final pH 5.3) at 4, 21 and 36.5  C
for 120 min. Aliquots of the solution were withdrawn at
appropriate time intervals to determine of the dissolved arsenic
content by atomic absorption spectroscopy (SPECTRAA L40/FS,
Varian, Australia).
2.4. UV irradiation
For UV irradiation, a 30 W mercury lamp emitting predomi-
nantly light at wavelength 254 nm was used. Six samples were
irradiated in 2 ml acrylic cuvettes. The total time of irradiation was
6 h. Every hour one sample was taken out and the particle size
distribution, FTIR, UV–vis and PL spectra were immediately
measured.
2.5. Characterization of nanosuspensions
2.5.1. Particle size analysis
The particle size distribution was measured by photon cross-
correlation spectroscopy using a Nanophox particle size analyzer
(Sympatec, Germany). A portion of each nanosuspension was
diluted with stabilizer containing solution to achieve a suitable
concentration for measurement. This analysis was performed
using a dispersant refractive index of 1.33. The measurements were
repeated 4 times for each sample.
2.5.2. Zeta potential measurement
The zeta potential was measured using a Zetasizer Nano ZS
(Malvern, Great Britain). The Zetasizer Nano measures the
electrophoretic mobility of the particles, which is converted into
the zeta potential by using the Helmholtz–Smoluchowski equation
built into the Malvern Zetasizer software. The zeta potential was
measured in the original dispersion medium. The measurements
were repeated 3 times and the mean values are reported.
2.5.3. UV–vis spectroscopy
The optical spectra were recorded using a UV–vis spectropho-
tometer Helios Gamma (Thermo Electron Corporation, Great
Britain) in the range 200–800 nm.
2.5.4. Photoluminiscence spectroscopy
The photoluminiscence (PL) spectra at room temperature were
acquired at right angle on a photon counting spectrofluorometer
PC1 (ISS, USA) with an excitation wavelength of 365 nm. A 300 W
xenon lamp was used as the excitation source. The emission was
collected in a 25 cm monochromator with a resolution of 0.1 nm
equipped with a photomultiplier.
2.5.5. FTIR spectroscopy
The FTIR spectra were recorded using a Tensor 29 infrared
spectrometer (Bruker, Germany) with TRANS and ATR method.
3. Results and discussion
3.1. Characterization of nanosuspension immediately after milling
As can be seen from Table 1 the average particle size (x50) of the
prepared nanosuspension was 115 nm and the largest particle size
(x99) did not exceed 170 nm. Zeta potential had the value 12.9 mV.
The pH of the suspension was 5.3.
For consideration of the interaction between As4S4 and PX407,
the FTIR spectroscopy was used. The characteristic peaks of
PX407 were found at 2889, 1348 and 1103 cm1 due to —C—H
stretch, in plane —O—H bend and —C—O stretch, respectively
(Fig. 2, black line). Since the —C—H bond stretching modes are
sensitive to environmental and conformational change (Guo et al.,
1999), the
2800–3000 cm1 region is employed to monitor
changes after the reaction. The shift of —C—H bond stretching
bands from 2889 cm1 to 2863 cm1 indicates more hydrophobic
microenvironment in polyoxypropylene centre block of PX407
(Storm et al., 1995; Chen et al., 2006; Liu et al., 2010; Shou et al.,
2011), resulted from the adsorption of PX407 molecules onto the
surface of hydrophobic As4S4 nanoparticles (Fig. 2, red line). This
mode of adsorption leaves the hydrophilic polyoxyethylene
side-arms in a mobile state because they extend outwards from
the particle surface. These side-arms provide stability to the
particle suspension by a repulsion effect through a steric
mechanism of stabilization, involving both enthalpic and entropic
contribution (Moghimi et al., 1993; Li and Caldwell, 1996; Moghimi
and Hunter, 2000). Besides the shift of —C—H stretch, no other
shifts were observed after the adsorption process.
Table 1
Peoperties of As4S4 nanosuspension after milling.
Compound Stabilizer Properties
Particle size (nm) Zeta potential (mV) pH
Arsenic sulfide 0.5% PX407 x50 = 115 12.9 5.3
x99 = 170
  áková et al. / International Journal of Pharmaceutics 478 (2015) 187–192
Z. Bujn
Fig. 2. FTIR spectra of pure PX407 and sample As4S4–PX407. (For interpretation of
the references to color in the text, the reader is referred to the web version of this
article.)
3.2. Effect of temperature on particle size
The influence of temperature on stability of nanosuspension
was followed via particle size distribution measurements. The
stability was monitored for 25 weeks. As shown in Fig. 3, no big
changes occurred after the four-week storage. After this time, the
temperature of storage started to have very significant impact on
stability of the nanosuspension. In the case of storage in desiccator
Fig. 3. Evolution of particle size distribution of As4S4 nanosuspension storage in 21  C and 4  C.
189
(21  C) the nanosuspension became unstable after 5 weeks. The
unimodal distribution changed into the polymodal. The formation
of hydrophobic interactions between the nanoparticles of As4S4
and stabilizer is a negative entropic process. Thus, the higher the
temperature of the nanosuspension, the stability of the system
becomes more thermodynamically unfavorable (Wang et al., 2013;
Deng et al., 2010). Moreover, higher temperature can alter the
solubility of the nanoparticles to a great extent and thus storage of
nanosuspension at laboratory temperature can lead to increase in
particle size. Subsequently, this increase can promote settling
which may further lead to aggregation and destabilization of the
nanosuspension (Pace et al., 1999).
The storage in refrigerator (4  C) has prolonged the stability of
the sample until 24 weeks. The particle size distribution started to
change after 25 weeks. Therefore, cool storage of As4S4 nano-
suspension prepared by milling in the environment of PX407 is
preferential to avoid thermal acceleration of decomposition. In this
case, good physical stability is related to the protection by the
stabilizer and homogeneous sizes of the nanoparticles. Long
swinging hydrophilic polyoxyethylene chains on the particle
surface provide an excellent steric hindrance, which prevents
the particles from aggregating. Moreover, the homogeneous As4S4
particles hinder both the dissolution of smaller particles and
growth of larger particles, i.e., Ostwald ripening.
The dependence of solubility of As4S4 nanoparticles on
temperature was followed to confirm the previous statements.
As can be seen from Fig. 4 the highest arsenic extraction was
achieved when the highest temperature (36.5  C) was applied. In
this case, 3.8% of arsenic was removed from As4S4 after 120 min of
190  áková et al. / International Journal of Pharmaceutics 478 (2015) 187–192
Z. Bujn
Fig. 4. Arsenic extraction from As4S4 sample at 4, 21 and 36.5  C.
leaching. At 21  C the amount of extracted arsenic was around 2.5%
and even less arsenic dissolution was achieved when 4  C was
applied (2%).
3.3. Effect of UV irradiation
Except temperature, light could also influence the stability of
the nanosuspension. The sample in desiccator was not protected
from the light, so the photolysis could play an important role. For
this reason it was necessary to test nanosuspension of As4S4 in
PX407 for its susceptibility to changes in its properties generated
by exposure to UV irradiation.
The interaction of arsenic sulfides with light is generally
complex process. The light can promote changes of electron
densities, but also induce chemical reactions among fragments
(Frumar et al., 1997a,b). It can also induce other photochemical
Fig. 5. (a) UV–vis absorption spectra, (b) particle size distribution, (c) PL spectra, and (d) FTIR spectra of As4S4 nanosuspension after UV irradiation at various times (up to 6 h).
reactions such as polymerization or depolymerization, photolysis,
changes of local structure, or changes of long-range order
(crystallization–amorphization). These processes can proceed
separately or simultaneously (Frumar et al., 1997a, 2001; Kotsalas
et al., 1998).
It was found, the properties of As4S4 nanosuspension after UV
irradiation have changed. The increased dissolution was also
denoted by the particle size distribution measurements. In all cases
not only the shift of right side of the Gaussian distribution
appeared, but also the broadening of left side and the overall
particle size distribution was observed at the lower values. The
most visible confirmation of the above mentioned assumptions
was that after 5 h of irradiation (UV5), the new distribution fraction
from 10 to approximately 100 nm appeared (Fig. 5b). This effect
was manifested in the UV–vis absorption spectra by a slight blue
shift (from 242 to 209 nm) with the increasing time of irradiation
(Fig. 5a). At the same time, there is a tendency of particles to
aggregate. As a result, the mean particle diameter (x50) continu-
ously shifted to the higher values (from 120 to 195 nm) during
irradiation. Moreover, the distribution of the sample irradiated for
6 h (UV6) changed from unimodal to polymodal. The particles
started to subject the process of Ostwald ripening and consequent
aggregation. A smooth decrease in intensity of absorption spectra
is due to disordering of nanosuspension stability.
In the case of photoluminescence study (Fig. 5c), As4S4
nanosuspension displays also smooth features associated with
UV irradiation. The maximum emission peak of non-treated
sample (UV0) was located at 534 nm (2.32 eV). With the increasing
time of irradiation the blue shift of the emission maxima to 528 nm
(2.35 eV) was observed. It is in a good accordance with the UV–vis
absorption spectra (Fig. 5a), though the intensity slightly increases.
The FTIR spectroscopy showed that during UV irradiation no
changes in PX407 molecule occur (Fig. 5d).
During UV irradiation the increase of As4S4 dissolution
occurs, which eventually means higher particle size, aggregation
  áková et al. / International Journal of Pharmaceutics 478 (2015) 187–192
Z. Bujn
and consequently destabilization of the nanosuspension, i.e.,
Ostwald ripening.
3.4. Effect of zeta potential
Zeta potential (ZP) measurement is another method for the
estimation of stability of prepared nanosuspension. It is an electric
potential at the hydrodynamic shear plane, which is the boundary
of the surrounding liquid layer attached to the moving particles in
the medium. The mobility depends on surface charge and the
electrolyte concentration of the employed stabilizers. In the case of
high molecular weight stabilizers, ZP values below 20 mV can
provide sufficient stabilization (Mishra et al., 2009). But for
sterically stabilized systems even lower values were observed
(down to 3 mV) (Müller, 1991). Adsorbed layers of polymers/large
molecules shift the plane of shear to increased distance from the
particle surface, thus reducing the measured ZP (Mishra et al.,
2009).
The ZP was measured in original dispersion medium of the
suspension. This was done in order to find out the thickness of the
diffuse layer. The higher absolute ZP value, the thicker the diffuse
layer and the more stable the suspension (Mishra et al., 2009). In
this case, the differences between the sample measured immedi-
ately after milling and the unstable one (stored in desiccator for
5-weeks) are obvious. The former exhibited high value of ZP
(12.9 mV), whereas in the case of the latter, the less satisfying
result was obtained (6.4 mV). After milling, adsorbed layer of
PX407 on the As4S4 nanoparticles is sufficiently thick to ensure the
long-term stability of the suspension (in our case 24 weeks). On the
other hand, under unfavorable conditions (high temperature and
light), the particles started to dissolve and the ionic strength of the
solution increased, resulting in the decrease of the absolute value
of ZP. Moreover, the decrease in pH from 5.3 to 3.7 was also noticed.
Arsenic is present in As4S4 in the form As(II). This form is unstable
in water and before dissolving into aqueous solutions is oxidized to
As(III) (Lengke and Tempel, 2005). This phenomenon and
subsequent dissolution is responsible for the observed pH changes.
The principle lies in the following Reaction (1):
AsS + 8H2O ! HAsO42 + 11e + 15H+ + SO42
4. Conclusions
Long-term stable nanosuspension of arsenic sulfide As4S4
was prepared by nanomilling in Poloxamer 407. The infrared
spectroscopy studies confirmed that hydrophobic polyoxypropy-
lene blocks from Poloxamer 407 were adsorbed on hydrophobic
As4S4 nanoparticles. It was observed, the laboratory temperature
has negative influence on storage of suspension. The unimodal
distribution changed into polymodal after destabilization. The
storage at low temperature (4  C) brought about prolonged
stability for 24 weeks. Also the sunlight is undesirable and the
physico-chemical properties are changed faster. After UV
irradiation of nanosuspension the blue shift of the UV–vis
absorption and PL spectra was observed. This blue shift is
probably related to the appearance of new size fraction in the
region from 10 to 100 nm. It seems that the increased solubility of
As4S4 nanoparticles is a key process causing the instability of the
system. It is recommended to store As4S4 nanosuspension in
PX407 in cold and dark place.
Acknowledgements
This work was supported by the projects APVV-0189-10, VEGA
2/0027/14, VEGA 2/0064/14, which are gratefully acknowledged.
191
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