Available online at www.sciencedirect.

com

Journal of Controlled Release 125 (2008) 96 106

www.elsevier.com/locate/jconrel

Supercritical antisolvent production of biodegradable micro- and

nanoparticles for controlled delivery of paclitaxel
Lai Yeng Lee a , Chi Hwa Wang a,b,, Kenneth A. Smith a,c
a

Molecular Engineering of Biological and Chemical Systems (MEBCS), Singapore-MIT Alliance, 4 Engineering Drive 3, 117576 Singapore
b
Department of Chemical and Biomolecular Engineering, National University of Singapore, 4 Engineering Drive 4, 117576 Singapore
Department of Chemical Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge MA 02139, United States
Received 2 April 2007; accepted 9 October 2007
Available online 13 October 2007

Abstract
Paclitaxel and poly (L-Lactic acid) (PLA) were co-precipitated to form micro and submicron particles in a manner similar to that used in the
supercritical antisolvent with enhanced mass transfer (SAS-EM) process. As compared with conventional processes, a major advantage of
supercritical CO2 as an antisolvent in the SAS-EM process is the effective removal of residual organic solvents. In this work, the organic phase was
sprayed into supercritical CO2 (for CO2, Tc = 31.1 C, Pc= 73.8 bar) from a 500 m ID capillary nozzle. Ultrasonic vibration with an amplitude of 0
to 120 m (from a 3/8 tip diameter titanium probe) was employed in the high pressure vessel during the antisolvent process to provide enhanced
mixing between the solvent and antisolvent phases. The role and effects of ultrasonication on the properties of the resulting particles were studied.
When no ultrasonication was applied, micrometer-sized particles were obtained. When ultrasonication was applied, more uniform particles in the
submicron size range were obtained. The size of the particles was found to vary with the ultrasonic vibration amplitude. Encapsulation efficiencies up
to 83.5% and controlled release of paclitaxel for more than 30 days were achieved with the particles fabricated in this study.
2007 Elsevier B.V. All rights reserved.
Keywords: Supercritical antisolvent; Paclitaxel; PLA; Biodegradable nanoparticles; Ultrasonication; Enhanced mixing

1. Introduction
Paclitaxel is a promising anticancer drug with efficacy
against a wide variety of carcinomas [14]. It works through the
inhibition of DNA synthesis by stabilizing microtubule
assembly [2]. However, its clinical application has been limited
due to its hydrophobic nature. Its current formulation requires
the use of an adjuvant, Cremophor EL, which has been
associated with several undesirable side effects [1,4]. One
method to overcome the problems brought about by Cremophor EL is to encapsulate paclitaxel in biodegradable
polymers such poly lactic acid (PLA) or poly lactic-co-glycolic
acid (PLGA). These biodegradable polymeric particles also
Corresponding author. Department of Chemical and Biomolecular Engineering, National University of Singapore, 4 Engineering Drive 4, 117576
Singapore. Tel.: +65 6516 5079; fax: +65 6779 1936.
E-mail address: chewch@nus.edu.sg (C.H. Wang).
0168-3659/$ - see front matter 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.jconrel.2007.10.002

have the advantage of providing sustained release of paclitaxel

for chemotherapy [24].
Well known conventional methods for encapsulating hydrophobic drugs include solvent evaporation [3] and spray drying
[3,4]. Electrohydrodynamic atomization (EHDA) [5] has also
been employed to produce monodisperse particles. In this study, a
modified supercritical antisolvent with enhanced mass transfer
(SAS-EM) [611] method was employed to produce submicron
particles of paclitaxel-loaded PLA particles without the use of
emulsifiers or surfactants.
Supercritical fluids provide several desirable features such as
low viscosity, and high solvent power for most organic solvents.
The most common supercritical fluid used in pharmaceutical
applications is CO2 due to its relatively accessible critical point,
abundance, and minimal toxicity. Several novel techniques of fine
particle formation using supercritical fluids have been developed
in the past two decades. These include rapid expansion of supercritical solutions (RESS) [1214], and antisolvent methods such

L.Y. Lee et al. / Journal of Controlled Release 125 (2008) 96106

as supercritical antisolvent (GAS/SAS) [1416], precipitation

with a compressed antisolvent (PCA) [17,18] and the aerosol
solvent extraction system (ASES) [19,20]. Most of these
processes employ CO2 as either the solvent or the antisolvent.
The RESS method represents one of the earliest attempts to
produce drug-loaded polymers by a supercritical fluid technique.
Debenedetti et al. [14] investigated the co-precipitation of
lovastatin in PLGA for sustained release purposes. Needles of
crystalline lovastatin in PLGA microspheres were obtained in
their studies. The RESS method has the advantage of low
processing temperatures and it does not require the use of any
organic solvent. Its main disadvantage, however, is the limited
solubility of high molecular weight polymers and pharmaceutical
compounds in pure supercritical CO2. The problem of low yield
and difficulty of scale up limits its applicability to expensive
pharmaceutical processing.
Other supercritical fluid techniques generally employ CO2 as
an antisolvent for particle fabrication. The antisolvent methods
have the advantage of utilizing the high miscibility of
supercritical fluids with organic solvents which have high
dissolving power for the substrates of interest. There has been
much research on the application of the SAS method for particle
fabrication of pharmaceutical products such as antibiotics [6,7]
and proteins [8,14]. The SAS process may also be used to
produce polymerically encapsulated versions of therapeutic
products in order to achieve controlled release [9,19].
The supercritical antisolvent with enhanced mass transfer
(SAS-EM) method was developed by Chattopadhyay and
Gupta [611] to produce smaller and more uniform particles
by introducing ultrasonic vibration during the SAS process. In
their study, it was shown that application of ultrasonic vibration
to the SAS process produced particles with a narrower size
distribution as compared to those produced by conventional
SAS methods. This is primarily due to the enhanced mixing and
turbulence generated by the ultrasonic vibration in the
supercritical medium. In the SAS-EM technique, organic

97

solution was introduced onto the vibrating surface of the

ultrasonic probe. It has been suggested that the role of ultrasonic
vibration in the SAS-EM process is liquid atomization [610]
and to enhance mixing within the vessel to promote better mass
transfer between the organic solvent and CO2 during the SAS
process. In this study, the need to spray the organic solution onto
the ultrasonic vibrating surface was evaluated, and a modified
SAS-EM setup was proposed to fabricate PLA particles in the
submicron size range.
One model [21,22] of ultrasonic liquid atomization assumes
that a liquid film covers the surface of the ultrasonic vibrating
surface. Square capillary waves are formed on the vibrating
surface and droplets are subsequently ejected from the tips of
the capillary waves as the waves grow in amplitude. The
behavior of a liquid free surface upon a surface vibrating normal
to itself has been investigated for more than a century. As far
back as the 1830s, Faraday [21] reported the formation of
surface waves in liquid films on vertically vibrating surfaces
and observed that the frequency of the surface waves is half the
frequency of the vibrating surface. Subsequently, observations
of both the half frequency [22,25,26] and the same frequency
[23,24] were reported. Benjamin and Ursell [23] later showed
that both responses are possible, depending on the operating
region in the stability diagram. In the context of liquid
atomization in the ultrasonic frequency regime, only half
frequency observations have been reported [25,26].
From the capillary wave breakup mechanism, the droplet
size is expected to be a function of the liquid surface tension and
density as described by Lang's correlation [26]. For organic
solventCO2 systems, surface tension is a strong function of
system pressure and temperature. Above the critical conditions
of the mixture, interfacial tension between organic solvent and
CO2 disappears [27] and the two phases are completely
miscible. In this case, it may be impossible to form capillary
waves on the vibrating surface, and the proposed liquid film
mechanism of ultrasonic liquid atomization in SAS-EM [610]

Fig. 1. Simplified experimental setup for SAS and SAS-EM production of micro and submicron particles for controlled delivery of paclitaxel. Different vessel
configurations used in this study were shown in the top left hand corner. A: conventional SAS process with no ultrasonication; B: modified SAS-EM process with
spray nozzle directed away from ultrasonic vibrating surface. C: SAS-EM process with spray nozzle directed at ultrasonic vibrating surface.

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L.Y. Lee et al. / Journal of Controlled Release 125 (2008) 96106

may be inoperative at conditions above the mixture critical

point.
This work has investigated the use of the SAS and SAS-EM
processes, with some modification, for production of paclitaxelloaded PLA particles for sustained delivery application. The role
of ultrasonic vibration intensity on particle size and morphology
was also studied. The performance of the controlled release
particles fabricated by this novel method was evaluated in terms
of particle size and morphology, encapsulation efficiency, and
the profiles of drug release vs. time.
2. Materials and methods
2.1. Materials
Poly ( L -lactic acid) (PLA, Product Number P1566,
MW = 85,000 160,000 Da) was purchased from Sigma
Aldrich. Paclitaxel was a generous gift from Bristol Myers
Squibb. Compressed CO2 (Air Liquide, Paris, France) was
purchased from Soxal (Singapore Oxygen Air Liquide Pte Ltd).
Dichloromethane (DCM) (DS1432, HPLC/Spectro Grade,
Tedia, Fairfield OH) and acetonitrile (ACN) (AS1122, HPLC/
Spectro Grade, Tedia, Fairfield OH) were purchased from
Tritech Scientific Pte Ltd, Singapore. Phosphate buffered saline
(PBS) was purchased from Sigma-Aldrich Pte. Ltd. (pH = 7.4).
Millipore water (Millipore Corporation, Billerica MA) was used
throughout the study.
2.2. Microparticle and nanoparticle preparation
The experimental equipment for the supercritical antisolvent
(SAS) and modified supercritical antisolvent with enhanced
mass transfer (SAS-EM) processes is shown in Fig. 1. Three

different modes of organic solvent delivery and subsequent

mixing during the antisolvent process were tested. Configuration A represents the conventional SAS process; configuration
B represents the modified SAS-EM process used in this study
and configuration C represents the SAS-EM process developed
by Chattopadhyay and Gupta.
For the preparation of drug-loaded particles, Paclitaxel and
PLA were first dissolved in DCM. Theoretical drug-to-polymer
loadings of 310% (w/w), and polymer-to-solvent loadings of
2% (w/v) were used for the experiments in this study. Liquefied
CO2 (C1, Polyscience refrigerating circulator) was introduced
into the high pressure vessel (HP, Jerguson 12-T-32, 70 cm3) by
means of a high pressure pump (P2, Jasco HPLC pump) to
attain the required supercritical pressure of 9 MPa. The
temperature in the vessel was controlled and maintained at
35 C by use of a circulating heated water bath (C2, Polyscience
712 circulator). The ultrasonic probe (Sonics & Materials Inc.,
Newton CT) with 3/8 in. tip diameter delivers a maximum
vibration amplitude of 300 m at 100% intensity with the
Branson 450 digital sonifier (U1, Branson Ultrasonics Corporation, Danbury CT) used in this study. Ultrasonic vibration
amplitudes of 0120 m (040% of maximum intensity) were
used in the experiments with the modified SASEM process. The
organic solution (consisting of dissolved paclitaxel and PLA)
was introduced into supercritical CO2 in the high pressure
vessel using a high pressure pump (P1, Eldex B-100-S) at a flow
rate of 2 ml/min through a capillary tubing with an ID of
500 m. Pressure and temperature in the high pressure vessel
were monitored using a pressure gauge (PI) and thermocouple
(TC) respectively.
At the conclusion of the antisolvent process, the DCM-CO2
mixture was vented to a fume cupboard. Fresh CO2 was
introduced into the vessel at 50 bars to provide three separate

Table 1
Paclitaxel-loaded PLA particles fabricated by modified SAS-EM method
Sample

UA

Measured properties of paclitaxel-loaded PLA particles

SEM
(nm)

B1
B2
B3
B4
B5
B6
B7
B8
C1

10
10
0
10
20
30
40
20
10

657 223
826 385

795 314
500 181
478 142
372 86

LLS (nm)

co,

D1,0

D4,3

D3,2

theoretical

723
804
2396
921
481
465
660

846

734
816
2432
935
488
472
670

859

731
812
2420
930
485
470
666

855

0.03
0.05
0.10
0.10
0.10
0.10
0.10
Blank
0.10

Parameters fitted to release profile using

diffusion/dissolution model
EE (%) a

co

cs

co cs

59.3 0.3
74.8 4.6
70.0 3.5
67.7 1.4
56.4 14.4

78.7 0.3

0.0198
0.0374
0.0700
0.0677
0.0564

0.0787

3.5 10 20
3.5 10 20

3.5 10 20

3 10 8

1 10 7

0.03
0.03

0.03

b0
N0

N0

UA is the ultrasonic vibration amplitude (defined as % of maximum amplitude) applied during the modified SASEM process.
co, theoretical is the theoretical loading of paclitaxel in the PLA micro- and nanoparticles.
co (mg/mg PLA) is the actual loading of paclitaxel in the PLA micro- and nanoparticles.
cs (mg/mg PLA) is the solubility of paclitaxel in PLA polymer matrix as defined in Eq. (1).
D (m2 s 1) is the diffusivity of paclitaxel in PLA polymer matrix as defined in Eq. (1).
k (s 1) is the dissolution constant of paclitaxel in PLA polymer matrix as defined in Eq. (1).
Actual loading of paclitaxel mg
a
Encapsulation efficiency, EE (%) is defined as Theoretical
loading of paclitaxel mg  100k.

L.Y. Lee et al. / Journal of Controlled Release 125 (2008) 96106

rinses to remove any residual DCM in the particles. Particles

were collected at the bottom of the vessel on a 0.22 m cellulose
acetate filter (F1) during the venting process.

99

weighted mean diameters of the measured particles are reported

in Table 1.
2.4. Thermogram properties analysis

2.3. Size and surface morphology analysis

Qualitative observation of the size and surface morphology of
the particles was first achieved by scanning electron microscopy
(SEM, JEOL JSM-5600 LV, Japan). Platinum coating (Autofine
Coater, JEOL JFC-1300, Japan) of the samples was required
before SEM analysis. A cryogenic microtome (Leica Model No.
CM3050S, Germany) was used to perform cross-sectioning of
paclitaxel-loaded PLA microparticles to examine the crosssection. Field emission scanning electron microscopy (FESEM,
JEOL, Japan) was used to study the cross-sections of the
microparticles at higher resolutions. The particle size of samples
obtained at varying ultrasonic intensities was measured using
SEM image analysis (SMILEVIEW software). Approximately
75 particles were measured for each sample and the number-

Phase behavior of the particles was studied by differential

scanning calorimetry (DSC, 2920 modulated, Universal V2.6D
TA instruments). Approximately 210 mg of particles was
loaded onto standard aluminum pans (40 mg) with lids. The
samples were purged with pure dry nitrogen at a flow rate of
5 ml/min. A blank aluminum pan was used as a reference in all
of the analyses. The analysis was carried out using a
temperature ramp of 10 C/min or 5 C/min from 20280 C.
2.5. Encapsulation efficiency and in vitro release profile
High performance liquid chromatography (HPLC, Agilent
HPLC Series 1100 with UVvisible detectors, CA, USA) was
used to determine the encapsulation efficiency and in vitro release

Fig. 2. A. Close-up of the organic solution jet issuing from the 500 m ID capillary nozzle during the conventional SAS process (configuration A). The snapshot on the
left shows the breakup of the horizontal jet upon entering the supercritical CO2 phase. The snapshot on the right shows that the dense organic phase later sinks toward
the bottom of the vessel. B. Close-up of the organic solution jet issuing from the 500 m ID capillary nozzle during the modified SAS-EM process (configuration B).
The snapshot on the left shows the breakup of the horizontal jet upon entering the supercritical CO2 phase. The snapshot on the right shows the later mixing of the
organic solution phase. C. Close-up of the SAS-EM process geometry (configuration C). The snapshot on the left shows the position of the capillary nozzle used for
introducing the organic solution into supercritical CO2. The snapshot in the center shows the later mixing of the solution phase. Agglomeration of solid particles was
observed at the capillary opening. The SEM image on the right shows the typical submicron particles achieved.

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L.Y. Lee et al. / Journal of Controlled Release 125 (2008) 96106

profile of the paclitaxel-loaded particles. An acetonitrile: water

(50:50 v/v) solution was used as the mobile phase for HPLC
analysis. Measurement of the encapsulation efficiency of each
paclitaxel-loaded sample was carried out in triplicate. Approximately 1 mg of sample was weighed and dissolved in 0.5 ml DCM
to extract the paclitaxel embedded in the polymer matrix. After all
of the DCM had evaporated, 5 ml of mobile phase was added and
placed in an ultrasonic water bath to dissolve the paclitaxel.
Subsequently, 2 ml of the solution was filtered into HPLC vials
using 0.22 m syringe filters for analysis.
The in vitro release profile was determined in triplicate for
up to 36 days. Approximately 5 mg of each sample was
weighed and suspended in 6 ml PBS and placed into a shaking
water bath (120 rpm) at 37 C to simulate physiological
conditions. At pre-determined time intervals, the solution was
removed from the shaking water bath and centrifuged at
9000 rpm for 45 min. After centrifugation, 5 ml of the
supernatant was removed and 5 ml of fresh PBS was added to
the particles to re-suspend them. The paclitaxel content in the
supernatant was subsequently extracted into 1 ml of DCM. The
DCM with the extracted paclitaxel was then collected and left to
evaporate before addition of 2 ml of the mobile phase.
3. Results and discussion
Vibration amplitude was varied from 0120 m (corresponding to 040% of maximum vibration intensity) to

determine the effect of ultrasonic vibration amplitude on

particle size and morphology. Sample B3 was fabricated using
the conventional SAS process. Samples B1B8 and C1 were
fabricated using SAS-EM process configurations B and C
respectively. SEM photos of representative particles from each
process configuration are shown in Figs. 2 and 3.
In configuration A, the organic solution was introduced into
the high pressure vessel horizontally through the 500 m ID
nozzle with no ultrasonication. This represents the conventional
SAS process without any external mixing. In configuration C,
the nozzle was placed very close to (or even on) the surface of
the ultrasonic probe and the organic solution was directed onto
the vibrating surface. Fig. 2C shows the introduction of the
organic solution into the vessel by configuration C during the
SAS-EM process.
In configuration B, the organic solution was introduced into
the high pressure vessel horizontally (as in configuration A), and
ultrasonication was applied to the supercritical CO2. The
capillary nozzle was placed approximately 1 cm away from
the ultrasonic vibrating surface (as shown in Fig. 2B). Fig. 2B
demonstrates the jet disintegration of the horizontal spray and
the subsequent mixing of the organic solution phase in the
supercritical CO2 by ultrasonication in this study. The possibility
of direct contact between the nozzle and the ultrasonic probe
surface was eliminated. The organic solution issued from a
horizontal capillary nozzle and ultrasonic liquid atomization on
the surface of the ultrasonic element was not possible. A

Fig. 3. SEM pictures taken of PLA particles fabricated using different ultrasonic vibration amplitude (A) Sample B3 (no ultrasonication); (B)(D) Samples B4, B5 and
B6 (ultrasonic vibration amplitude 30, 60 and 90 m respectively).

L.Y. Lee et al. / Journal of Controlled Release 125 (2008) 96106

comparison of the results obtained from configurations A, B and

C leads to a clearer understanding of the role of ultrasonication in
the SAS process.
3.1. Role of ultrasonic vibration in SAS
Different spray jet configurations were used in this study to
examine the effects of ultrasonication on the spray jet and
precipitation during the SAS process.
For configuration A, the precipitation of particles was carried
out with conventional SAS. Particles are formed as a result of
breakup of the solution jet upon entering the supercritical CO2
from the capillary nozzle and subsequent rapid mass transfer
between the DCM and CO2. During the precipitation process,
clouding was observed near the top of the vessel around the
organic solution spray. This clouding was not homogenous
throughout the entire vessel. This suggests a lack of mixing of
the CO2 and organic solvent phases during the process. Fig. 2A
shows the jet formation in configuration A and the subsequent
behavior of the spray in the vessel.
For configurations B and C, ultrasonication at 20 kHz was
used during the precipitation process. For configuration B, the
horizontal jet entering the vessel and the effect of ultrasonication on the jet is shown in Fig. 2B. During the modified SASEM process, we observed gradual clouding of the vessel and the
clouding was distributed throughout the entire vessel. Precipitated particles could be observed during the process and the
particles were agitated by the ultrasonic vibration in the vessel
regardless of their positions in the vessel. Similar phenomena
were observed for configuration C (SAS-EM setup) as shown in
Fig. 2C.
From the SEM pictures in Fig. 3A, it can be seen that, when
no ultrasonic vibration was applied, particle diameters ranged
from 1 to 10 m. When ultrasonic vibration was applied, much
smaller particles were obtained. Particles smaller than 1 m
were obtained regardless of whether the spray was directed at
the probe (Fig. 2C) or away from the probe (Fig. 3BC). This
implies that the formation of submicron particles in the SASEM method is not dependent upon atomization on the face of
the probe. In fact, it was observed that directing the spray at the
probe poses some problems in the particle fabrication process
due to agglomeration of particles at the probe tip and hindered
vibration of the titanium probe (due to possible contact between
the probe and the capillary tube).

101

ranging from 110 m are obtained. These particles are discrete

with a smooth surface morphology as shown in Fig. 3A. In the
modified SAS-EM process, submicro particles are obtained, as
can be observed from Fig. 3BD. The number-weighted mean
particle sizes of the samples are summarized in Table 1.
It is believed that the SEM image analysis of particle size
yields the size of the primary particles, without regard for any
possible agglomeration that might occur. The SEM data in Table 1
clearly show that the primary particle size monotonically decreases with increasing ultrasonic vibration amplitude. This presumably reflects enhanced mixing.
From these results, we may infer that ultrasonic vibration
intensity plays an important role in the mixing and mass transfer
between the organic and supercritical phases as well as in the
nucleation process. Moderate ultrasonic vibration intensity
(10 30%) was found to be useful in producing smaller
particles.
3.2.2. Thermal analysis
Thermal analysis is a useful tool for determining whether
solutes have been dispersed or dissolved in polymeric matrices

3.2. Effect of varying ultrasonic vibration intensity on particle

properties
The effect of ultrasonic vibration amplitude on the properties
of particles fabricated by the modified SAS-EM process was
also investigated.
3.2.1. Particle size and size distribution
Fig. 3A to D are representative SEM pictures of the paclitaxelloaded PLA samples at 10% drug loading. From Fig. 3A, it can
be clearly observed that, when no ultrasonication is applied to the
vessel during the SAS process, micrometer-sized particles

Fig. 4. A. Thermogram properties for blank PLA and paclitaxel loaded PLA
particles fabricated using SAS and SASEM methods. Placebo PLA was
fabricated using the modified SASEM process without any drug loading.
B. DSC thermogram of a physical mixture of 5% paclitaxel (crystalline)/ 95%
Placebo PLA and sample B3 before and after suspension in PBS solution.

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L.Y. Lee et al. / Journal of Controlled Release 125 (2008) 96106

[28,29]. The thermogram properties of the particles fabricated in

this study were analyzed by differential scanning calorimetry
(DSC). Pure crystalline paclitaxel has a characteristic endothermic melting peak at approximately 223.0 C [30]. The melting
peak for the pure PLA used in this study was 178.7 C. Fig. 4A
shows the exotherms for blank PLA (microparticles without
paclitaxel) and for drug-loaded PLA (microparticles with 10%
paclitaxel). The endothermic melting peak for the blank PLA
particles was 175.8 C which is approximately 3 C lower than
that for the untreated PLA pellets (as purchased from Sigma
Aldrich). It was also observed that, with paclitaxel-loaded
particles, an endothermic peak was obtained only in the vicinity
of the PLA melting temperature. Thus, the supercritical
antisolvent process with ultrasonication did not significantly
alter the polymeric structure of PLA. Furthermore, for all
paclitaxel-loaded samples, there was no obvious sign of phase
separation between PLA and paclitaxel.
The physical state of encapsulated paclitaxel in the drugloaded particles was also examined by using DSC at a more
sensitive heating rate of 5 C per min. The exotherms of a
physical mixture of 5% paclitaxel (crystalline) with 95% PLA
and of sample B3 before and after suspension in PBS (pH 7.4,
37 C) are shown in Fig. 4B. Here, it was observed that, after
suspension in PBS for more than 2 weeks, the particles showed
an endothermic peak at about the paclitaxel melting point. This
may be explained by the formation of small paclitaxel crystals
within the polymer matrix at high drug loadings following water
absorption by PLA, as this decreases the solubility of paclitaxel
in the PLA matrix [31].
3.3. Yield and encapsulation efficiency
The drug encapsulation efficiency for the formulations
fabricated with the modified SAS-EM processes are summarized
in Table 1. The recovery yield for the process ranged from 15%
to 57% which is comparable to spray drying methods for
fabricating similar controlled-release micro-particles. The
encapsulation efficiency of paclitaxel in the micro and submicron particles fabricated using the modified SAS-EM process
was in excess of 56%. An advantage of the supercritical antisolvent process over conventional spray drying is the rapid
removal of organic solvent from particles without the need for
high temperatures or extensive downstream purification processing, as well as possible size control of final particles if ultrasound
is employed.
Residual solvent content in particles fabricated using
supercritical CO2 as an antisolvent has also been reported to
be very low [32,33] and within the safety limits of less than
600 ppm for DCM as stated in the U.S. Pharmacopeia [34]. In
this study, the residual DCM content in sample B3 was
measured by Gas Chromatography (GC-MS with autosampler)
with N,N,Dimethylformamide (DMF) as the mobile phase. The
residual DCM content was determined to be 99.1 ppm which is
well within allowable limits. Hence, it is clear that the SAS
process produces particles with very low concentration of residual solvent without further downstream purification steps
such as freeze drying.

3.4. Controlled release of paclitaxel from PLA particles

In vitro release profiles of paclitaxel from PLA microparticles of different sizes are illustrated in Fig. 5. It was observed that, for larger particles (Sample B3), the release rate is
slower than that obtained with smaller particles (Sample B7). It
was also observed that, after a period of a month, only approximately half of the paclitaxel had been released. Similar release
patterns have been obtained for paclitaxel-loaded PLA particles
produced by other methods [3537]. For many drug-loaded
PLA or PLGA formulations, the drug release profile reaches a
plateau long before all of the drug is released [36,37], especially
in cases when the drug is hydrophobic. Liggins and Burt [36]
proposed a two compartment model of the drug release such
that only a fraction of the drug diffuses from the polymer
matrix. The remainder of the drug is non-diffusible. One reasonable explanation for the observed two compartment model
may the formation of drug crystals within the polymer matrix at
high drug loadings. Polakovic et al. [38] suggested that, for
hydrophobic drug systems at low drug loadings, the drug may
be homogeneously dissolved within the polymer matrix and
drug release is by simple diffusion of drug molecules. Conversely, at higher drug loadings, drug crystals may form inside
the polymer matrix if the solubility of the drug in the polymer is
exceeded. For a saturated drug-loaded system, dissolved drug
exist in equilibrium with drug crystals. In the work by Jasti et al.
[31], a method to determine the solubility concentration of
drugs in polymer matrices was developed.
In order to investigate the physical state of paclitaxel in PLA
microparticles, microparticles at higher drug loadings (5% and
10%) were frozen and sliced with a microtome. FESEM was
used to image the cross-section of the cut microparticles.
Fig. 6A and B show that particles before suspension in PBS
solution have very compact structures with no observable drug
crystals. The cross-section after 2 weeks suspension in PBS
solution (10% loading) is shown in Fig. 6C and D. Here, we
observed small particles in the cross sections of the microparticles as highlighted by the circle in Fig. 6D. These are likely
to be particles of paclitaxel crystallized from a supersaturated

Fig. 5. Effect of particle size on in vitro release of paclitaxel from PLA microparticles
(B3) and submicron particles (B4, B7).

L.Y. Lee et al. / Journal of Controlled Release 125 (2008) 96106

103

solution of paclitaxel in PLA. The observation from the FESEM

images supports the DSC results. A similar phenomenon was
also observed for microparticles at 5% loading.

result is:

2

Ac
A c 2 Ac
D

k cs  c
At
Ar2 r Ar

3.4.1. Diffusion and crystal dissolution model

A simple diffusion model is not sufficient to describe the
release mechanism of paclitaxel from PLA microparticles at
high drug loadings, as illustrated in Fig. 7 for 5% and 10%
loadings.
Kurnik and Potts [39] have developed a model for diffusion
and crystal dissolution. For a spherical particle and co N cs, the

where c is the concentration of dissolved drug in the polymer

matrix, co is the initial drug loading, cs is the solubility of the
drug in the polymer matrix and k is the dissolution constant for
the drug in the polymer. The first term on the right hand side of
Eq. (1) represents Fickian diffusion and the second term
represents dissolution of the drug into the polymer when the
concentration in the polymer matrix falls below the drug

Fig. 6. (A) and (B) FESEM image of cross-section of 10% loading (B3) samples before suspension in PBS solution; (C) and (D) FESEM image of cross-section of 10%
loading (B3) samples after 2 weeks suspension in PBS solution; (E) and (F) FESEM image of cross-section of 5% loading samples after 1 week suspension in PBS
solution.

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L.Y. Lee et al. / Journal of Controlled Release 125 (2008) 96106

solubility. When the initial drug loading (co) is less than the drug
solubility, crystals are absent. When initial drug loading (co) is
higher than the drug solubility (cs), it is assumed that the excess
forms crystallites which are dispersed in the polymer matrix.
In this model, the drug-loaded particles are assumed to be
spherical and non-porous. From the FESEM pictures in Fig. 6, it
was observed that the internal structure of the PLA microparticles
was fairly compact. The surface-volume mean diameter, D32, of

each sample was obtained from SEM experiments and was used
in the fitting of the experimental release profiles to the diffusion /
dissolution model. It is assumed that the volume of the drug
crystals is negligible compared to the overall matrix volume and
that the crystallites are small and uniformly distributed throughout the polymer matrix.
A solution to Eq. (1) may be obtained in non-dimensional
form by analogy to Harland's diffusion / dissolution solution
[40]:
w

Mt
4
cs 3 kR3
l
X



Di n2 k2 Dis n2 k2 1  exp Di n2 k2 s

n1

Di n2 k2 2
2

Where is the drug released relative to the amount of drug

initially dissolved
in

 the polymer matrix at the solubility concentration cs 43 kR3 .
Alternatively, we may represent the dimensionless release as:
w V


Mt
Mt
cs
4 3 w
MT co 3 kR
co

amount of drug initially loaded in the

where MT is the total

polymer co 43 kR3 :
The dimensionless groups Di and are defined by:
Di

Fig. 7. Effect of drug loading on in vitro release profiles of paclitaxel from

PLA microparticles. For 3% sample (top), release tends to unity after 3 weeks
and the release profile may be fit to a simple diffusion model. For 5% (middle)
and 10% samples (bottom), release profiles tend to more closely follow a
diffusion/dissolution model. Dimensionless drug release here is described
in Eq. (3).

kR2
Dt
;s 2
R
D

Fig. 7 shows the release profiles of paclitaxel from PLA

microparticles at 3%, 5% and 10% drug loadings respectively.
At low drug loadings (3%), the dimensionless drug release ()
profile tends to unity at about three weeks and the release curve
is in close agreement with a pure diffusion model. However, for
loadings of 5% and 10%, the release was only about 85% and
50%, respectively, at the same elapsed time. A diffusion/dissolution model provides a much better fit to the release profile in
these cases. The diffusion coefficient, dissolution constant and
drug solubility concentration used to fit the release profiles for
the 3%, 5% and 10% samples are summarized in Table 1.
The diffusion coefficient for paclitaxel in a PLA polymer
matrix was found to be approximately 5 10 20 m2/s. In the
studies by Polakovic et al. [38] for diffusion of lidocaine (a model
hydrophobic molecule) in PLA nanoparticles, the diffusion
coefficient was found to be 1.35 to 7.13 10 20 m2/s. Liggins
and Burt [36] obtained a diffusion coefficient of 10 15 m2/s for
diffusion of paclitaxel in PLA microspheres. However, the
molecular weight of the PLA used in their studies was 2 to
50 kDa and they have also shown that, with increasing molecular
weight of PLA, the diffusion coefficient of paclitaxel in the
polymer matrix tends to decrease. The molecular weight of the
PLA used in this study was 85 to 160 kDa.
The fitted values of the dissolution constant for paclitaxel in
PLA are 3 10 8 s 1 and 1 10 7 s 1 for the 5% (B2) and 10%

L.Y. Lee et al. / Journal of Controlled Release 125 (2008) 96106

(B4) samples, respectively. The dissolution process may be

modeled as corresponding to diffusion-limited transport from
the crystals into the matrix. Such a model suggests that the
dissolution constant should be given by:




D
k
5
 4kR2c  N
Rc
where Rc is the radius of the crystal and N is the number of crystals
per unit volume of the PLA polymer matrix. From Fig. 6(F)
and (D), the sizes of paclitaxel crystals for the 5% and 10% samples
are found to be approximately 275 nm and 203 nm, respectively.
This provides the necessary estimates of crystal size as required by
Eq. (5). The estimates are 4 10 8 s 1 and 3 10 7 s 1, for the 5%
and 10% loadings. These estimates compare favorably with the fit
values of 3 10 8 s 1 and 1 10 7 s 1.
4. Conclusions
A modified SAS-EM process was successfully employed to
fabricate controlled-release matrices for Paclitaxel. The results
suggest that the modified SAS-EM process developed in this
study allows fabrication of sub-micron particles without the
requirement of specialized nozzles or high temperatures. It was
shown that the application of ultrasonication to the SAS process
greatly enhances the mixing of the organic solventCO2 phases,
leading to significantly smaller particles. This technique may also
be applied to a spectrum of other hydrophobic drugs. In vitro
release studies showed that, at drug loadings of 3% or less, almost
all of the drug is released during a 1 month period. At higher drug
loadings (10%), approximately half the drug is released during a
1 month period and subsequent release is very slow. A similar
result is obtained at 5% loading. This is very likely due to the
formation of drug crystals dispersed within the polymer matrix.
The release profiles, and the ability to model them without
recourse to empiricism, suggest that a diffusion / dissolution
mechanism is present. These results provide important insights for
the design of paclitaxel-loaded PLA particles for chemotherapy.
Acknowledgements
This research was supported by Singapore MIT Alliance
(Grant no: C382-400-003-001) and Agency for Science,
Technology and Research (Grant no: R279-000-208-305). The
authors would like to thank Prof Yee C. Chiew and Dr Zhen
Huang for their technical support in this work. Special thanks are
due to Prof. Jefferson W. Tester for his advice and support in the
research. The authors would also like to thank Dr Jingwei Xie and
Dr Michael T. Timko for their helpful discussions in this study, as
well as Liang Kuang Lim for advice on high resolution imaging.
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