J.

of Supercritical Fluids 77 (2013) 5262

Contents lists available at SciVerse ScienceDirect

The Journal of Supercritical Fluids

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

Formation of PLA particles incorporating 17-methyltestosterone by

supercritical uid technology
Priscila Soares Costa Sacchetin a , Ana Rita Morales a , ngela Maria Moraes a ,
Paulo de Tarso Vieira e Rosa b,
a
b

Department of Engineering of Materials and Bioprocesses, School of Chemical Engineering, State University of Campinas, 13083-852 Campinas, SP, Brazil
Department of Physical Chemistry, Institute of Chemistry, State University of Campinas, 13083-970 Campinas, SP, Brazil

a r t i c l e

i n f o

Article history:
Received 11 October 2012
Received in revised form 22 February 2013
Accepted 23 February 2013
Keywords:
Particles
PLA
SAS
Supercritical uids
17-methyltestosterone
Nile tilapia

a b s t r a c t
The use of polymeric particles as devices for the controlled release of active agents is attractive not only for
application in humans, but also in the veterinary eld. Studies on the production and characterization of
poly(lactic acid) (PLA) particles incorporating or not 17-methyltestosterone (MT), a hormone frequently
use for the sexual reversion of Nile tilapia raised in large scale in tanks are herein reported. The particles
were obtained employing a supercritical anti-solvent (SAS) method, using dichloromethane (DCM) as
solvent for the polymer and the hormone and CO2 as the supercritical uid. The inuence of the operating pressure, polymer solution concentration and ow rate on the size and morphology of particles was
evaluated, as well as the effects of the incorporation of the hormone at different MT/PLA mass ratios. The
results attained showed that the PLA particles presented appropriate DCM concentrations levels (below
600 ppm) and average diameters between 5.4 and 20.5 m when free of the hormone. The average diameters of the particles containing 17-methyltestosterone increased up to 4 times, and MT incorporation
efciencies up to 53% were achieved. A signicant decrease was noticed in particle crystallinity (from
26.52 to 1.72%) due to the addition of the hormone to the formulation. X-ray diffraction analysis showed
no separate diagrams of each substance as a result from the physical mixture of the polymer with the
hormone. The particles obtained had low zeta potential values, what indicates reasonable tendency to
agglomerate. MT release studies performed at different pH conditions (2.2, 5.0, 7.4 and 8.8) showed that
the particles presented appropriate stability for the intended use.
2013 Elsevier B.V. All rights reserved.

1. Introduction
The decrease of sea and freshwater shery stocks, together with
the continuous expansion of the global population and the increasing need for new protein sources, promoted, in the last 50 years, the
rapid growth of aquaculture (farming of sh, mollusks, crustaceans
and aquatic plants). Global aquaculture production numbers as
high as 68.3 million tones per year and worth over US$100 billion
are reported [1].
Among the cultivated species, Nile tilapia (Oreochromis niloticus) is distinguished as the second largest group of sh reared in
captivity (after carps), with an annual global production of approximately 2.3 million tons [1]. Tilapias have excellent organoleptic
characteristics [2] and part of the success of their growth when
conned is due to their tolerance to environmental differences and

Corresponding author. Tel.: +55 19 3521 2095; fax: +55 19 3521 3029.
E-mail addresses: prisoares@feq.unicamp.br (P.S.C. Sacchetin),
morales@feq.unicamp.br (A.R. Morales), ammoraes@feq.unicamp.br (.M. Moraes),
paulorosa@iqm.unicamp.br (P.d.T.V.e. Rosa).
0896-8446/$ see front matter 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.supu.2013.02.029

to high population densities [3]. In addition, tilapias readily accept

articial feed and present both high food conversion ratios, having also high fecundity, being easy to breed and resistant to several
diseases [35].
Despite all these characteristics, there are some disadvantages
that still need to be overcome regarding tilapia growth in captivity, such as the premature sexual maturity which leads to drastic
reduction in feeding activity for females (after spawning) and an
increase in the number of male social interactions, which can affect
their food intake [4].
Therefore, different alternatives have been considered for reproductive control of this species and the most common, practical,
effective and economically viable approach is through sex reversal by the use of masculinizing hormones, thus producing sh with
male phenotypes. Since males grow better than females, improved
mass productivity is attained as an added advantage [4].
According to Beardmore et al. [6], there are over 16 natural or
synthetic androgens that have been used in the sex reversal of
sh. The most commonly applied is 17-methyltestosterone (MT),
whose chemical structure is shown in Fig. 1A. This compound may
be administered by immersion or orally (by supplementing the sh

P.S.C. Sacchetin et al. / J. of Supercritical Fluids 77 (2013) 5262

Fig. 1. Chemical structures of 17-methyltestosterone (A) and poly(lactic acid) (B).

diet) [7]. Although high rates of sex reversal may be achieved by the
use of this compound [6], there are many concerns from both the
authorities and consumers regarding the use of hormones in the sex
reversal of sh. The main concerns relate to controlling the dosage
and the time of hormonal treatment [8], failures in the uniformity
of distribution of the hormone in the feed, thus causing difculty
in estimating the proper amount ingested by each animal [6] and
contamination of groundwater with residual hormone from the sex
reversal process. In addition, there are issues related to excessive
handling and continued exposure of the hormone to the sh tank
environment, which can result in synthetic androgens conversion
to feminizing estrogens, which might affect the overall sex reversal
procedure [9]. It should be noted also that the hormone has to be
administered to the animals several times a day for several weeks
to be effective. Such handling represents an additional risk to workers, who are exposed to the tumorigenic and teratogenic effects of
the compound [10].
Thus, an alternative approach that proves most attractive is
through the development of oral carriers able to release the hormone in a more controlled way, thus increasing safety for the
handler while minimizing the risks of premature compound degradation [11] in the environment or by sh metabolism through
exposure to the gastric pH (between 2.2 and 5.0) and intestinal
tract pH (from 6.8 and 8.8) [12]. In this context, polymer-based
particles have received particular attention, since they have high
versatility and can be used as drug delivery devices for different
routes, whether intravenous, subcutaneous, pulmonary or oral [11].
Among the biocompatible and bioabsorbable polymers that have
shown promise in the biomaterials eld is poly(lactic acid) or PLA
(Fig. 1B), an environmental friendly polymer produced from renewable carbon sources and chemically synthesized by condensation
polymerization of the l and d isomers of lactic acid [13].
PLA particles incorporating different types of compounds and
prepared by different methods are reported in the literature for
a wide range of applications, as reviewed by Anderson and Shive
[14], Mishima [15] and Wischkle and Schwendeman [16]. For the
specic desired application, i.e. the administration of MT orally for
the sex reversal of Nile tilapias, the particles should be resistant to
proteolysis and to the pH conditions both of the gastrointestinal
tract during the average food processing time (around 12 h) and
bloodstream (around 7.0 h) and to ideally have diameters below
50 m.

53

One of the strategies to produce polymer particles of the desired

size range involves particle nucleation and growth simultaneously
to the incorporation of an active agent, an approach centered on
technologies that employ supercritical uids such as CO2 [17]. Particle production through supercritical uid technologies has been
increasing lately, and many variations in procedure are found,
among them is the supercritical anti-solvent (SAS) technique,
which uses supercritical CO2 as an anti-solvent in the process. This
approach involves rapid mass transfer between the supercritical
uid and the polymer solution mixed with the active agent, resulting in the rapid expansion of the uid and consequently, in the
production of a supersaturated solution that easily precipitates. The
increasing use of the SAS technique is primarily due to its effective
control over reproducibility [18], as well as to the ability to produce
particles based on water-insoluble compounds [17]. More detailed
information about the formation of poly(-hydroxyesters) particles through the use of techniques based on supercritical uids can
be found elsewhere [19].
The aim of the present study was to analyze the inuence of
operating pressure, polymer solution concentration and ow rate
as well as hormone to polymer mass ratio on the morphological
and structural characteristics of the PLA particles produced by SAS,
contributing to the studies focusing the formation of particulate
systems applicable in the delivery of veterinary drugs. The effects of
using mild pressures (8 MPa) and relatively high concentrations of
polymer solutions (up to 1.5%, w/v) at high ow rates were investigated, since polymer particle production in these conditions is not
often reported in the literature. Success in the use of these conditions might contribute to the overall efciency of the process,
especially in regard to the amount of particles produced in a given
period, thereby reducing the number of batches when operating in
large scale.
2. Material and methods
2.1. Material
Poly(l-lactic acid) (Purasorb PL 18, Purac Biochem BV,
Netherlands), 17-methyltestosterone (MP Biomedicals, USA),
dichloromethane (JT Baker, USA) and commercial grade carbon
dioxide (99.5% purity, White Martins, Brazil) were used for particle production. For particle characterization, HPLC-grade methanol
and acetonitrile obtained from JT Baker (USA) and water puried
in a Milli-Q system (Millipore, Brazil) were used, as well as polyoxyethylene sorbitan monooleate (Tween 80), dimethylsulfoxide
and anhydrous sodium sulfate obtained from SigmaAldrich (USA)
and 2-propanol from Synth (Brazil).
2.2. Particle formation
The particles were obtained through the SAS supercritical uid
approach using solutions of variable concentrations of PLA in
dichloromethane (DCM) (from 0.5 to 1.5%, w/v) in the presence
or absence of MT (at concentrations from 0.19 to 1.13%, w/v). The
experimental apparatus shown schematically in Fig. 2 consists of
a CO2 supply system, a polymer injection unit, a high pressure
stainless steel column and a particle collecting ask.
Briey, the carbon dioxide was collected from the storage
tank using the high-pressure pump (model P50 TharSFC, Waters
Corp, USA) coupled with a digital thermocryostatic bath (model
MQBTC99-20, Microquimica, Brazil) and immediately cooled to
temperatures below 0 C to ensure the gas liquefaction. Then, the
CO2 was injected into the high pressure column (500 mL of internal
volume) at a ow rate of 18 g/min (manually controlled) with the
aid of a heated micrometer valve coupled to a rotameter used as a

54

P.S.C. Sacchetin et al. / J. of Supercritical Fluids 77 (2013) 5262

2.3.2. Mean diameter and size distribution

Particle mean diameters and their size distributions were
determined by laser scattering spectrometry (Mastersizer 2000,
Malvern, UK) [21], using 2-propanol as dispersant.

Fig. 2. Apparatus of particle formation system by SAS methodology.

gas ow meter. The internal temperature in the column was maintained at 40 C by using an ultrathermostatic cryostatic bath (model
521/D, Quimis, Brazil) and the system was operated at different
pressures, in the range from 8 to 16 MPa. After the temperature and
pressure were stabilized, the polymer solution was introduced into
the column using a HPLC pump (model SP930D, Allcrom, Korea)
at variable ow rates (from 0.5 to 2.5 mL/min) through a coaxial
nozzle with an internal diameter equal to 120 m. At the entrance
of the column already saturated with supercritical CO2 , rapid diffusion occurs at the interface between the polymer solution and
supercritical CO2 . Organic solvent evaporation from the surface
of the droplets increases polymer supersaturation, leading to the
beginning of the nucleation process which, in turn, leads to the precipitation of polymer particles on the internal surface of the high
pressure vessel and on the metallic lter at the bottom of the vessel.
After the complete injection of the polymer solution, the system
was maintained under the same temperature and pressure conditions for 30 min, with continuous ow of CO2 into the system to
remove the residual organic solvent from the particles. Afterwards,
the column was slowly depressurized and then the particles were
collected in glass bottles, sealed and stored under refrigeration.
The inuence of pressure, polymer solution concentration and
ow rate on particle characteristics was studied using a 23 factorial
experimental design with three repetitions in the central point and
MT incorporation effects were analyzed at the operating conditions
determined as the most adequate based on the results of the 23
factorial experiments (tests performed at least twice). The software
Statistica for Windows 7.0 was used for the statistical analysis of
the data obtained.
2.3. Particle characterization
The effects of process parameters on the characteristics of the
particles were observed by analysis of particle morphology, mean
diameter, size distribution, porosity, crystallinity degree, surface
charge, and the presence of residual organic solvent, as well as by
determining the efciency of hormone incorporation and particle
stability in simulated sh gastrointestinal conditions, as described
below.
2.3.1. Morphology
Particles were evaluated concerning their shape and surface
morphology by scanning electron microscopy (JSM-6360LV, JEOL,
USA) [20]. Before analysis, the samples were coated with gold and
palladium using an evaporator under vacuum.

2.3.3. Thermal behavior

Particles, isolated polymer, and hormone were evaluated
regarding variations in glass transition temperature [20,21] by differential scanning calorimetry (DSC2910, TA Instruments, USA).
Approximately 5 mg of samples pre-equilibrated in argon were
heated on a sealed aluminum plate containing nitrogen. Samples were heated from 25 C to 220 at a rate of 10 C/min to
determine the glass transition temperature (Tg ), crystallization
temperature (Tc ) and melting temperature (Tm ). The crystallinity
() of non-processed PLA and of the particles was calculated by
[(Hm Hc )/Hf ] 100%, where Hf is the theoretical heat of
fusion of 100% crystalline PLA [22,23] (equal to 93 J/g [24]), Hc is
the enthalpy of crystallization on heating, and Hm is the melting
enthalpy.
2.3.4. Crystallinity degree
Particles, isolated polymer and hormone were analyzed regarding their degree of crystallinity, which serves as an indication of
polymeric material degradation and allows prediction of the release
behavior of the encapsulated active agent. This analysis was performed using an X-ray diffractometer (XRD 7000, Shimadzu, Japan),
with an angular range from 10 to 40 with a step of 0.02 at 2.
2.3.5. Zeta potential
Since the zeta potential can be an indication of the electric
potential at the double layer of the particles, and thus, of their
potential stability in different media, this property was evaluated
after suspending the particles in a 1:1 (v/v) deionized water/ethanol
solution. Samples were placed in electrophoretic cells and analyzed
using a Zetasizer Nano S90 (Malvern, UK) coupled with Zetasizer
Series software (v. 6.32).
2.3.6. Presence of residual solvent
The analysis of residual DCM was performed as described by
the United States Pharmacopeia [25] through gas chromatography
GCMS (model Saturn 2100D, Varian) equipped with an ion-trap
detector, coupled to the software Saturn GCMS workstation v.
5.52, using the static headspace method.
Particle sample aliquots of 100 mg were weighed and transferred to a vial containing 5 mL dimethylsulfoxide (DMSO) and 1 g
anhydrous sodium sulfate. The vials were sealed and heated to 80 C
for 1 h and the headspace subsequently analyzed by gas chromatography using a 30 m 0.25 mm 0.25 m CP-Sil 8 CB-MS capillary
column (low bleed), with helium as carrier gas at about 35 cm/s.
The temperature was adjusted to 40 C for 4 min, increased rapidly
to 200 C and maintained at this temperature for 4 min. The detector was maintained at 260 C. The calibration curve was obtained
similarly, using DCM solutions at different concentrations in 5 mL
of DMSO also containing 1 g of anhydrous sodium sulfate.
2.3.7. Efciency of incorporation of 17-methyltestosterone in
the particles
The incorporation efciency of the hormone was determined
by HPLC analysis after dissolution of the particles as follows (basic
procedures adapted from United States Pharmacopeia [26,27] and
Giunghedi et al. [28], respectively). Samples of 515 mg of particles containing 17-methyltestosterone were dissolved in 5 mL of
dichloromethane and subsequently mixed with 10 mL of methanol.
Then, the samples were centrifuged for 20 min at 8000 rpm at 15 C.
The supernatant was separated from the precipitated polymeric

P.S.C. Sacchetin et al. / J. of Supercritical Fluids 77 (2013) 5262

55

Table 1
Results of the factorial design experiment used to study the effects of pressure, concentration and ow rate of polymer solution on PLA particle mean diameters and DCM
residual levels.
Assay

Pressure
(MPa)

Concentration of
polymer solution (%)

Flow rate of polymer

solution (mL/min)

Mean diameter
(m)

A1
A2
A3
A4
A5
A6
A7
A8
A9
A10
A11

8
8
16
16
8
8
16
16
12
12
12

0.5
0.5
0.5
0.5
1.5
1.5
1.5
1.5
1.0
1.0
1.0

0.5
2.5
0.5
2.5
0.5
2.5
0.5
2.5
1.5
1.5
1.5

6
21
5
18
7
12
7
11
10
6
9

material, ltered through a 0.45 m lter (Chromal, MachereyNagel, Germany) and, when necessary, stored in amber vials at 5 C
until further analysis.
The chromatographic analysis of 20 L samples was performed
using a liquid chromatograph (model 1525, Waters, USA) equipped
with a reversed-phase C18 (250 mm 4.6 mm) column (Symmetry,
Waters) with 5 m particles, a UV-visible detector (model 2487,
Waters) and the Breeze software (v. 3.2). A mobile phase consisting of a mixture of degassed and deionized water and acetonitrile
(45:55, v/v) was used in isocratic mode at a constant ow rate of
1.0 mL/min, and the detection was performed at 241 nm.
2.3.8. Determination of 17-methyltestosterone release kinetics
in simulated sh gastrointestinal conditions
The analysis of hormone release kinetics was conducted over
120 h at different pH conditions. Approximately 1025 mg of particles containing 17-methyltestosterone were placed in dialysis
bags from Inlab (Brazil) (pre-equilibrated with deionized water),
and immersed in about 200215 mL of buffering solutions with different pH values (2.2, 5.0, 7.4 and 8.8), each containing Tween 80
at approximately 0.0015 mg/mL to mimic the presence of surface
active molecules in the body (according to Wischke and Schwendeman [16]). The release tests were conducted at 26 C. Aliquots of
1 mL of each receiving solution were periodically collected, mixed
with 4 mL of methanol and stored at 5 C until further analysis by
HPLC, as described in Section 2.3.7.
3. Results and discussion
3.1. Effects of processing conditions on PLA particle morphology,
size distribution, mean diameter and residual DCM level
The results achieved through the 23 factorial design experiment
with three repetitions at the central point, performed to analyze
the inuence of pressure, concentration and ow rate of polymer
solution on PLA particles aspect, size and residual DCM content, are
shown in Figs. 3 and 4 and in Table 1.
As observed in Fig. 3, all tested conditions resulted in effective particle formation. The material obtained presented mostly
spherical or oval morphologies and reasonably smooth surfaces,
except for particles of experiment A4, which were less regular in
shape. A tendency of particle coalescence was noticed in all situations, but most intensively in the case of particles obtained in
the conditions of experiment A4. In this particular situation, polymer concentration was at the lowest value and both pressure and
polymer solution ow rate were at the highest levels. These conditions contributed to inadequate removal of the organic solvent by
the supercritical uid, possibly providing supersaturation of DCM
in the vessel, which in turn may have disturbed the processes of
regular particle nucleation and growth.

1
1
0
0
0
0
0
0
0
0
0

Residual
DCM (ppm)
790
1130
1280
6490
<600
5980
2040
2630
3400
4160
4670

Most of the particles were in the size range from 0.4 to less than
100 m (Fig. 4), presenting mean diameters from 5.4 to 20.5 m,
however this range was narrower in the case of particles obtained
in conditions A1 and A5. The statistical analysis of the mean diameter data showed that the variables with the most signicant effects
on particle size are the ow rate of polymer solution and its combination with the PLA concentration (Table 2). While increasing
the ow rate of PLA solution increases the mean diameter of the
particles, increases in the combination of ow rate and polymer
concentration result in size reduction. At high polymer solution
ow rates, the processing time to produce a certain xed quantity of particles is lower, turning the removal of DCM less efcient,
what is aggravated at low polymer concentration, as in conditions
A2 and A4. More intensive particle aggregation is then observed,
mostly if a too large amount of residual organic solvent is retained
in the system. Furthermore, the increase in ow rate of polymer
solution usually results in a better atomization process of the solution in the precipitation vessel, leading to a consequent decrease
in the average diameter of the particles formed [2931]. However,
in an opposite effect, when the ow rate of polymer solution is
increased, while maintaining low values of supercritical uid ow,
the fraction of the organic solvent in the precipitation vessel also
increases, thus contributing to more intensive particle aggregation
[3234]. These authors observed that nanoparticles can be efciently obtained when the conditions in the precipitation vessel
are maintained above the critical point of the mixture of antisolvent and organic solution. If a liquid phase or a combination of gas
and liquid phases is observed in the precipitation vessel, the formation of nano- and microparticles is noticed. If a gas phase is formed,
expanded particles with empty cores are often produced.
Effective removal of dichloromethane during particle production is of paramount importance, due to its inherent toxicity.
According to the United States Pharmacopeia [25], the concentration of residual DCM allowed in a drug product is 600 ppm,
therefore levels equal to or lower than that are aimed. The only processing conditions that resulted in DCM levels lower than 600 ppm
in the particles were those referring to experiment A5. Lower pressures, as used in A5, allowed a more efcient removal of organic
solvent since the density of the supercritical CO2 tends to decrease
linearly with pressure, resulting in higher mass transfer. The use of
increased ow rate (A2 and A6) had also positive effects on DCM
removal, since it allows a decrease in the driving force for mass
transfer of DCM in supercritical CO2 , as observed by Martin et al.
[35]. Moreover, it is noteworthy that although the increase in CO2
ow leads to a signicant reduction in the amount of DCM in the
particles, it is accompanied by a reduced amount of MT incorporated therein, thus resulting in lower encapsulation efciency of
the active agent.
In the case of run A6, an increase in mean diameter is observed,
as a consequence of increasing the polymer solution ow injected

56

P.S.C. Sacchetin et al. / J. of Supercritical Fluids 77 (2013) 5262

Fig. 3. Typical morphological features by scanning electron microscopy of the particles produced in the assays A1A11.

into the system. This led to the formation of wider size distribution
peaks, resulting in particles with diameters greater than 11 m. It
is observed that in the case of run A8, the increase in pressure to
16 MPa while maintaining xed the conditions of concentration
and ow rate of the polymer solution, (i.e., equal to those of run
A6), was not able to compensate the effect of the combination
concentration-ow rate in order to reduce the mean particle diameter. This is quite clear when observing experiment A7, in which
the ow rate was ve times lower, and 59% reduction in mean
particle diameter occurred. These data corroborate the results of
the statistical analysis of the factorial design, which shows that, at
a condence level of 95% (p < 0.05), only the ow rate of polymer
solution and its interaction with the polymer concentration were
able to produce signicant effects on particle diameter (Table 2).
Pressure and polymer concentration, as well as the interaction
between ow and pressure of the polymer solution and the interaction between pressure and polymer concentration did not have
statistically signicant effects on the diameters of the particles. It
was expected, therefore, that the increase in pressure had an effect

on the solubility of the organic solvent in the supercritical uid,

as the diffusion coefcient decreased accompanied by increased
viscosity of the medium. It is observed then that the effects of
increasing pressure depend on the value of these properties or
mixing pattern of the phases within the precipitation vessel.
Thus, an increase in the diameter of the particles would occur
[31,3638], but also the production of smaller particles [30,33].
Hence, if small non-aggregated particles with low DCM residual
content are desired, the system should be operated at low polymer solution ow rates and preferably using more concentrated
polymer solutions. As noted by Palakodaty and York [39], this phenomenon could be explained by the fact that, in such cases, the
nucleation rate depends on the saturation levels provided by the
rate of mass transfer of the supercritical uid in the drops, as a
function of the diffusion coefcient of the supercritical uid in the
solvent phase. Thus, when more concentrated solutions are used,
there is an increase in the concentration of solute in the droplet
which leads to a higher nucleation rate and, consequently, to less
agglomeration.

Table 2
Statistical analysis of the 23 factorial design experiment used to study the effects of pressure, concentration and ow rate of polymer solution on the mean diameter of PLA
particles.
Factor

Effects

Standard error

tCalc(4)

p Value

Average
Flow rate of polymer solution (F)a
Pressure (P)
Concentration of polymer solution (C)
FP
F Ca
PC

10.24300
9.22350
0.96050
3.22950
0.44100
4.66500
0.17400

0.662824
1.554460
1.554460
1.554460
1.554460
1.554460
1.554460

15.45358
5.93357
0.61790
2.07757
0.28370
3.00104
0.11194

0.000102
0.004044
0.570085
0.106315
0.790719
0.039901
0.916266

Variables with statistically signicant effect at p < 0.05.

P.S.C. Sacchetin et al. / J. of Supercritical Fluids 77 (2013) 5262

57

Fig. 4. Typical size distribution of the particles produced in the assays A1A11.

In this sense, the processing conditions used in experiment A5

seems to be the most appropriate to produce PLA particles with
the desired features, resulting in regular-shaped particles with a
narrow size distribution, mean diameter around 7 m and with
residual DCM levels inferior to 600 ppm. Thus, the subsequent
studies focusing hormone incorporation were performed in these
conditions.
3.2. Inuence of 17-methyltestosterone loading on the
characteristics of PLA particles
The addition of different concentrations of 17methyltestosterone during the production of the PLA particles,
carried out at 8 MPa and using polymer solution concentration
and ow rate of 1.5% and 0.5 mL/min, respectively, resulted in
signicant changes in the morphology and size distribution of
the particles, as shown in Figs. 5 and 6. Differences in the mean
diameters of the particles and in hormone loading efciencies were
also noticed (Table 3), but in all cases, as expected, the residual
DCM levels were below 600 ppm. This shows that, in spite of the
supposedly high afnity of the active agent toward the organic
phase, the hormone also contributed to improve the access of the
antisolvent CO2 to the interior of the particles.
A tendency to increase the size of the particles was observed
with the increase of the active agent concentration in solution. The
mean particle diameters ranged from 14 to 25 m (Table 3), and
this variation was more evident when compared with the size of
particles produced under the same conditions (experiment A5), but
without hormone addition (sizes equal to 7.4 m). It is also important to emphasize that all size distribution curves became broader
(Fig. 6), mostly because larger particles were produced and because
particle aggregates seem to have been more abundantly formed.

Variation in particle size distribution obtained with different

concentrations of active agent may be explained in terms of particle nucleation by saturation and precipitation processes, followed
by the growth step during the expansion of the droplets [40]. Thus,
in cases in which low drug concentrations were used, the process
of nucleation became the dominant mechanism, leading to the formation of small particles. When in the presence of larger quantities
of the active agent, the precipitation process occurred predominantly during the expansion process, leading simultaneously to
particle nucleation and growth, thus resulting in the formation of
larger particles. The occurrence of additional populations noticed
in the particle size distribution is probably the result of a competition between the growth and particle nucleation process [40].
Therefore, increasing drug concentration can positively inuence
the size of primary particles by increasing nucleation, as noticed in
run A24, and negatively inuence the further growth of secondary
ones (A21A23), as also shown by Reverchon [41].
Not only were the mean diameters of the particles affected by
variation in hormone concentration, but also their morphological
aspect. While particles not containing the hormone frequently had
oval shapes (Fig. 3A5), the particles obtained in the presence of MT
were more spherical (Fig. 5). Moreover, when the hormone was
added to the polymer solution, the roughness of the surface of the
particles seems to have increased. These changes in the characteristics of particle surfaces may be attributed to an increase in
porosity, since incrementing the hormone proportion in the polymer solution could disturb and even prevent the interactions of
the PLA chains. In this way, the formation of interphasic zones may
have occurred, resulting in a relatively heterogeneous distribution
of the active agent throughout the particles.
The drug loading of the resulting particles was low, between 22
and 54%. These results can be explained in terms of the solubility

58

P.S.C. Sacchetin et al. / J. of Supercritical Fluids 77 (2013) 5262

Fig. 5. Typical morphological features by scanning electron microscopy of the particles produced in the assays A21A24.

or partition of the drug in supercritical CO2 . MT solubility is probably high due to the presence of DCM, which contributes to increase
drug extraction from the particles during the entire process, especially at the drying step. Bodmeier et al. [22] also obtained similar
results. Despite the relatively low drug loading efciency obtained

in the present work, the particles appear to be appropriate for the

intended use, since very low daily amounts are enough to guarantee sex reversal of tilapias. Furthermore, the expected effects of
release over extended periods do not require the use of particles
containing high levels of hormone. Moreover, the hormone which

Fig. 6. Typical size distribution of the particles produced in the assays A21A24.

P.S.C. Sacchetin et al. / J. of Supercritical Fluids 77 (2013) 5262

59

Table 3
Characteristics of PLA particles obtained in the presence of different proportions of 17-methyltestosterone.
Assay

Initial MT to polymer
ratio (mg/mg)

Final MT to
polymer ratio (%)

Mean diameter
(m)

A21
A22
A23
A24

0.125
0.250
0.500
0.750

0.028
0.051
0.166
0.400

15
14
25
21

was not incorporated into the particles could be easily recycled and
used in further batches. Thus, the overall incorporation efciency
could easily reach 100%.
3.3. Thermal behavior of the particles
The ability of supercritical CO2 to produce changes in mechanical and physical characteristics of amorphous and semicrystalline
polymeric materials, such as the reduction of the glass transition
temperature in an effect similar to that of a plasticizer [42], is well
known. Additionally, a decrease of glass transition and melting
temperatures promotes agglomeration, causing other variations in
particle properties that can be easily observed for particles that stay
in contact with pressurized CO2 for a prolonged time [43].
Other polymer characteristics affected by CO2 are matrix
morphology and diffusivity of active agents. Depending on the
conditions, the contact of semicrystalline polymers such as PLA
with supercritical CO2 may increase the mobility of the polymer
chains, turning their rearrangement into kinetically-favored congurations easier and facilitating the occurrence of crystallization
processes [41,4446]. This effect is enhanced by the favorable
interactions promoted through carbonyl groups present in the
PLA chain and supercritical CO2 [47]. Enhancement of the diffusion of active agents through the polymeric matrix due to higher
polymer mobility [44] is frequently observed, being attributed to
increased interchain distance and increase of free volume with
consequent reduction of the interactions/entanglement between
polymer chains.
PLA particles containing or not MT were analyzed by DSC for verifying possible changes in the glass transition temperature and in
the crystallization of the polymeric matrix. The results of this analysis are given in Fig. 7, being summarized in Table 4. The increase
in pressure tends to promote polymer chain mobility, thus lowering the Tg of the matrix [48]. However, during particle production
under different conditions of pressure (8, 12 and 16 MPa) this effect
was not observed, since an increase in pressure resulted in a negligible effect in the decrease of Tg (T 0.7 C). A possible explanation

1
2
0
3

Hormone incorporation
efciency (%)
22
20
33
53

2
1
10
10

for such behavior in these conditions is that the compact crystallite structure restricted the solubility of CO2 in the crystal region,
thus decreasing the effect of CO2 pressure on the thermodynamics
of PLA crystallization [46]. Although the absorption of CO2 by the
polymer matrix is quite limited, with solubility around 0.04 wt% at
45 C and 30 MPa [49], it is responsible for increasing the free volume fraction. As a result, signicant swelling in PLA by plasticizing
the amorphous phase is observed, consequently inducing crystallization by reordering polymer chains at lower temperatures [47].
Zhai et al. [46] also reported similar results when using pressures
in the range from 3 to 15 MPa.
Although practically no changes were observed on the Tg and
TM of the particles not loaded with MT, variations in  were
noticed (Table 4) in addition to a decrease in the crystallization
temperature (TC ), as also reported by Liao et al. [47]. It was noted
that increasing the ow rate and its interaction with polymer
solution concentration probably facilitated the transformation
of the amorphous polymer phase into a crystalline structure of
lower free energy. The highest crystallinity (26.52%) was observed
for sample A5. In this condition, the removal of DCM seems to
be more efcient, preventing the adverse effect of the solvent
on PLA crystallinity [50]. The use of plasticizers such as CO2 is

Table 4
Summary of DSC curves for different PLA particles.
Run

Tg ( C)

Tc ( C)

Tm ( C)

 (%)

PLA
MT
A1
A2
A3
A4
A5
A6
A7
A8
A9
A10
A11
A21
A22
A23
A24

61.6

61.4
61.5
61.3
61.3
62.4
61.7
62.1
62.0
61.8
61.7
61.8
61.5
57.6
55.7
52.9

136.9
90.0
113.9
114.0
115.0
115.47
110
112.7
114.0
112.1
113.0
114.0
114.7
109.6
107.6
106.2
105.1

179.9
161.9
179.0
179.0
178.8
178.7
178.8
179.1
178.8
178.8
178.8
178.9
179.3
177.9
175.8
174.4
162.8

15.2

17.9
18.3
12.8
13.5
26.5
18.6
19.6
20.9
18.1
13.3
12.7
24.3
13.2
3.6
1.7

Fig. 7. DSC curves of PLA and particles not containing hormone (A) and the MT and
hormone-containing PLA particles (B).

60

P.S.C. Sacchetin et al. / J. of Supercritical Fluids 77 (2013) 5262

also reported to have a synergistic effect on PLA crystallization

[51]. In compressed CO2 processes, the nucleation process occurs
as a consequence of gas supersaturation, followed by a growth
mechanism [46]. The relatively low crystallization rate of PLA (due
to its high chain rigidity) [51] is then modied, especially due to
the positive role of CO2 in accelerating PLA crystallization, thus
leading to an increase in the crystallinity of PLA [46].
When compared to sample A5, hormone-loaded particles
obtained in equivalent conditions showed a signicant decrease
in  with the increase in MT proportion, from 26.52% to 1.72%.
Reductions in Tg and TC were also noticed. Probably the increase in
the amount of hormone in the polymer solution impaired the PLA
interchain interactions during the nucleation and particle growth
processes, thus preventing the formation of more rigid and compact structures, thereby reducing dramatically matrix crystallinity.
As already mentioned, these particles seem to be more porous, with
many amorphous regions, which might facilitate the controlled
release of the active agent despite the polymeric matrix, which is in
principle, formed by a material with a relatively high degradation
time (more than 1 year).
3.4. XRD analysis
Considering the pattern of particle crystallization produced
when applying the factorial design (Fig. 8), it is noted that for all
samples the principal crystallization peaks were, at 2, 16.66 and
19.02 , respectively, in agreement with results of Purnama and
Kim [52], who obtained peaks at 2 equal to 16.52 and 19.08 .
These results show that the tested experimental conditions within
the ranges analyzed do not affect the crystalline structure of the
polymer and the X-ray results obtained for the particles strongly
coincide with the major peaks obtained for PLA alone (16.68 and
18.96 ). The amount of amorphous particles is a result of conformational changes that occur during the PLA crystallization process
[53].
Similarly, the crystallization patterns of the samples containing
hormones were collected, and the principal characteristic patterns
of PLA peaks were noted for these samples. The main intense peak
had values between 16.38 and 16.68 and the second one had
values between 18.78 and 20.12 . This result conrms that the
MT was effectively incorporated into the polymeric matrix, since
the characteristic crystalline peaks of the active agent were not
observed for samples A21A23. However, for sample A24 the presence of a secondary peak at 14.42 is apparent. Its presence would
indicate a diagram superposition of each substance, which in this
instance would be equivalent to a physical mixture of particles from
both materials, in this case PLA and MT [43].
3.5. Zeta potential analysis
The determination of zeta potential () (net electric charge
density at the shear plane, which is an indicative of electrostatic
repulsion or attraction between particles) [54] in the study of particle formation is quite important, since it provides indications of
particle stability and dispersive capacity [55]. In ionic solution, PLA
particles have zeta potentials around 4050 mV [56], with predominantly negative surface charge due to the presence of PLA carboxyl
end groups [54].
In the present study, it was observed that the particles presented
absolute values of zeta potential lower than or up to 5 mV, for particles produced without hormone loading (Table 5). These results
demonstrate that the repulsive forces of the particles when they
approach each other are very low and insufcient to keep them
apart from each other, not allowing adequate particle dispersion in
aqueous media. Thus, as the repulsive forces are weak, the particles tend to remain agglomerated, resulting in the formation of an

Fig. 8. X-ray pattern of PLA and isolated MT (A), particles produced in the experimental design (B) and particles produced in the presence of MT (C).

Table 5
Zeta potential values of PLA particles loaded or not with 17-methyltestosterone.
Runs

Potential Zeta (mV)

A1A11
A21
A22
A23
A24

<5.0
20.0
39.8
67.7
13.9

P.S.C. Sacchetin et al. / J. of Supercritical Fluids 77 (2013) 5262

Fig. 9. Release kinetics of MT at different pH.

unstable suspension. Nevertheless, a gradual increase in the zeta

potential of the particles, and consequently, of their stability, was
noticed for initial hormone ratios up to 0.05 mg of MT/mg PLA. The
hormone-containing particles had higher dispersibility in aqueous
solutions when compared to the PLA particles not containing the
active agent, probably due to easier PLA chain unfolding in the
presence of MT. This potentially led to increased exposure of the
polymer carboxyl end groups, resulting in larger number of charges
exposed on the surface of the particles. The reduction of the zeta
potential of the particles prepared at the highest concentration of
hormone may probably be attributed to a possible higher amount
of residual DCM, thus decreasing the availability of surface charges
on the particles.
3.6. Hormone release studies in simulated sh gastrointestinal
conditions
The kinetic behavior observed in the hormone release studies
performed at different pH conditions with particles prepared at an
initial MT/PLA mass ratio of 0.40 is shown in Fig. 9.
During the rst 12 h, no release of MT at pH 2.2 was noted. This
observation is particularly important, indicating that the particles
are able to resist to strong acid pH and that they would probably
be effective in protecting the active agent from the adverse conditions of the tilapia gastric region. In this condition, it was noticed
that MT release initiated only 24 h after the beginning of the incubation period, and after 120 h, 40% of the active agent remained
incorporated into the particles at pH 2.2.
The sample incubated at pH 5.0 had a different behavior,
presenting much higher MT release in the early hours of incubation. Signicant MT release was already observed in the aqueous
medium during the rst 2 h, reaching about 26%. After 12 h, approximately 50% of the hormone was released, and at 72 h, only 10% of
active agent was still incorporated in the particles. In this condition, complete MT release was observed at approximately 120 h of
sample incubation.
At intermediate pH (7.4), it was noted that the release of the
active agent started after 6 h, with an initial release of less than 5%
of the active agent incorporated into the particles, reaching 60% of
release after 120 h. At pH 8.8, which simulates the tilapia intestinal
condition, it was observed that the particles were also quite stable.
In this situation, the release started only after 12 h of incubation
and approximately 30% of the active agent was still in the particles
after 120 h.
Considering the data obtained, it can be assumed that in the
early hours of incubation, the dominating mechanism for hormone

61

release is swelling followed by diffusion of the active agent through

the particles. This may be easily conrmed from the fact that PLA
is hydrolyzed by moisture and its water absorption may strongly
affect not only its own structure, but also other organic compounds,
by producing changes in their chemical and physical nature [57].
Ester groups as well as oxirane and hydroxyl groups present in the
PLA structure should be primarily responsible for the interaction of
the polymer with water molecules, thus increasing water uptake by
the matrix [57] and initiating polymer degradation. In this phase,
water diffusion into the amorphous and less organized polymer
regions is commonly observed, leading to conditions in which even
easier penetration of water is observed and to decrease in polymer
molar mass due to random chain scission [58]. After some time,
the rate of PLA degradation would increase as a result of the large
amount of water absorbed, and at the second stage of particles
degradation, the decrease in polymer molar mass followed by an
increase in mass loss (up to 50%) and by copolymer hydrolysis to
monomers would be observed, mainly due to the high degradation
rate of the amorphous polymer regions. In the nal phase, oligomer
hydrolysis to lactic glycolic acid would occur, with hydrolytic polymer attack taking place mostly from the outer regions to the center
of the polymer crystalline domain [58]. In these conditions, the
hydrolyzed products both at the center and on the matrix surface
probably would be dissolved in the aqueous medium, thus increasing particle degradation, resulting in increased release of the active
agent. Despite the base-sensitive character of polyesters such as
PLA [59], it is noted that, in acid pH, the acidic catalysis effect is
more effective. Nevertheless, it is expected that more advanced
stages of particle degradation should start only after several weeks
or months of sample incubation.
Based on these results, the particles obtained had a quite
attractive behavior. They were able to efciently protect the 17methyltestosterone both from the simulated extreme gastric and
intestinal pH conditions. Provided that the contact time of the
particles with pH conditions around 5.0 is short, potential particle absorption into the bloodstream through the intestinal mucosa
could occur, then allowing the controlled release of the active agent
for periods longer than 120 h. Therefore, the active agent could be
administered orally without the concern of major efciency loss
regarding the sex reversion of tilapia.
4. Conclusions
The present work describes the study of PLA particle formation by SAS. The effects of operating variables such as pressure,
ow rate and polymer solution concentration on particle characteristics were evaluated, simultaneously to the inuence of
17-methyltestosterone addition to polymer solution. Effective
hormone incorporation (up to 53%) into particles having adequate
mean diameters and size distribution was achieved. The particles
formed proved suitable for oral administration to sh, since hormone release in the simulated sh gastrointestinal conditions has
been delayed by the encapsulation in the polymeric matrix.
Acknowledgements
The authors are grateful to CNPq (proc. no. 473013/2009-6) for
nancial support. P.S.C. Sacchetin would also like to acknowledge
FAPESP for a PhD fellowship and nancial support. .M. Moraes
acknowledges CNPq for a fellowship.
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