J.

of Supercritical Fluids 82 (2013) 3440

Contents lists available at ScienceDirect

The Journal of Supercritical Fluids

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

Lipid nanoparticles production by supercritical uid assisted

emulsiondiffusion
Roberta Campardelli a , Maxime Cherain b , Claire Perfetti b , Carlo Iorio b ,
Mariarosa Scognamiglio a , Ernesto Reverchon a , Giovanna Della Porta a,b,c,
a
Department of Industrial Engineering, Universit di Salerno, via Ponte don Melillo, 84084 Fisciano, SA, Italy
b
Microgravity Research Center, Chimie-Physique Lab., Universit Libre de Bruxelles, Avenue P. Heger, 1050 Bruxelles, Belgium
c
Research Centre for Nanomaterials and nanoTechnology (NANOMATES), Universit di Salerno, via Ponte don Melillo, 84084 Fisciano, SA, Italy

a r t i c l e i n f o a b s t r a c t

Article history: In this work a supercritical technology is proposed to improve the classical emulsication/diffusion tech-
Received 15 February 2013 nology for lipid nanoparticles (LNs) production. The process is based on the emulsion diffusion method
Received in revised form 25 May 2013 improved by the addition of a continuous supercritical uid processing step to eliminate the organic
Accepted 28 May 2013
solvent from the nanosuspension obtained.
Different emulsion/diffusion formulations for stearic acid nanoparticles production were tested and,
Keywords:
then, processed by supercritical continuous extraction at 80 bar and 45 C (liquid/gas ratio of 0.1) in
Lipid nanoparticles
a packed column, obtaining an efcient benzyl alcohol elimination. Solvent residues less than 100 ppm
Supercritical uid
Continuous processing
were measured. Stearic acid nanoparticles were not extracted or damaged by the supercritical processing
Emulsion/diffusion step, did not stick on the packing elements and showed mean diameters of 3050 nm; a value of one order
of magnitude smaller than the ones obtained by the conventional emulsion/diffusion technology with a
recovery efciency of 100%. Indeed, the fast and complete elimination of the benzyl alcohol around the
nanoparticles reduced the aggregation phenomena responsible of larger lipid particle sizes obtainable
by traditional process.
2013 Elsevier B.V. All rights reserved.

1. Introduction as lipid carriers due to their polymorphic behavior that improves

The production of drug nanocarriers able to overcome ther-
apy failures, such as, poor absorption or specic drug targeting,
is one of the challenges of the modern nanomedicine. Nanocarriers
can be emulsions, micelles, liposomes or polymer particles [1,2].
Recently, lipid nanoparticles (LNs) have been proposed as nano-
systems that may have the advantage of combining the properties
of polymer nanoparticles, for convenient drug sustained release
[35], fat based systems for low toxicity [6,7], and effective tar-
geting [8]. Moreover, the use of LNs for parental, topical and oral
administration has been proposed in very promising medical appli-
cations [911].
LNs suspension is formed as a rule by: a lipid matrix, a stabilizing
agent (emulsiers/surfactants), a continuous medium (water) and
can contain residues of the organic solvents used, depending on the
manufacture techniques adopted [12]. The term lipid is referred to
various kinds of molecules like: triglycerides, glycerides, fatty acids,
steroids and waxes. However, triacylglycerides are commonly used
Corresponding author at: Department of Industrial Engineering, University of
Salerno, I-84084 Fisciano, SA, Italy. Tel.: +39 089964104; fax: +39 089964056.
E-mail address: gdellaporta@unisa.it (G. Della Porta).
the stability of the nanoemulsion and allows a high drug loading
by the less ordinate structure of the lipid matrix. The choice of
the lipid to be used can be greatly inuenced by the drug to be
loaded and by the specic target required. Surfactants or surfac-
tant combinations could greatly inuence the particle size and the
stability of the dispersion, as well as, play an important role in con-
trolling the LNs crystallization process. Indeed, due to nanosize of
the particles, the number of lipid molecules that interacts with the
surfactant molecules can be large enough to inuence the crys-
tallization process and it has been demonstrated that a mixture
of nonionic (crystal modulation) and ionic surfactant (stabiliza-
tion) is recommended to fulll both criteria of dispersion stability
and crystal modulation [13]. In addition, the choice of the emul-
sier is largely dependent on the administration route, as well as,
the organic solvent used, which should be chosen, as non-toxic, as
possible.
Current methods to produce LNs include high-pressure
homogenization [14,15], microemulsion technique [16], sol-
vent emulsication/evaporation [14,17] and solvent emulsi-
cation/diffusion [18,19]. In the high pressure homogenization
technique, lipids are heated above their melting temperature;
then, poured into hot surfactant solution, homogenized and cooled
to crystallize the lipid droplets and obtain nanoparticles. The

0896-8446/$ see front matter 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.supu.2013.05.020
 R. Campardelli et al. / J. of Supercritical Fluids 82 (2013) 3440
microemulsion technique consists of the preparation of oil-in-
water emulsions at a temperature above the melting point of the
lipids with an adequate stirring that ensures the required droplet
size; then, the microemulsion is dispersed into a cold aqueous
medium, leading to a rapid crystallization of the oil droplets, form-
ing LNs. In the solvent emulsication/evaporation technique, the
lipophilic material is dissolved into an organic solvent, an oil-
in-water emulsion is produced and, then, evaporated with the
production of the LN dispersion. In the solvent diffusion technology
two saturated phases are used: an aqueous phase and an organic
phase. The drug and the lipids are often dissolved into the organic
phase. The mixing of the two phases generates an emulsion in
which the resulting droplets are converted into LNs by addition of a
large amount of water to induce solvent diffusion toward the con-
tinuous medium. Again, solvent and part of water are evaporated
under reduced pressure. For all technologies described, the average
particle size can be in the range of 100500 nm. However, high-
pressure homogenization is principally limited by the solubility of
the drug to be encapsulated in the lipid and by the high shear and/or
temperature used; whereas, the other methods often require long
processing times for solvents evaporation or extraction and high
solvents residues in the produced suspensions can be obtained. The
emulsion/diffusion technique is a very promising technique that
allows the easy and reproducible LNs preparation, but its applica-
tion is limited by the problematic removal of the organic solvents
used for the oil phase preparation. Indeed, the most used solvent
is benzyl alcohol (BA), because it has high solubility in water (i.e.,
fast diffusion rate) and is non-toxic; however, the main drawback
of this solvent, is its boiling point higher than water (Tb = 205 C)
that prevent the elimination by evaporation from a water based
suspension.
Supercritical uid based technologies have been frequently
proposed to overcome the traditional processes limitations, and
already used for emulsion processing or post-processing. For exam-
ple, Chattopadhyay et al. [20] proposed a Supercritical technology
for emulsion extraction applied to the production of micro and
nanoparticle suspensions taking the advantage of the enhanced
mass transfer of the supercritical extracting agent. In the process
solid particles are formed from droplets thanks to the supercrit-
ical extraction of the organic solvent of the oily phase of the
emulsion. A similar technology was also successfully described
by Della Porta et al. [21,22] in a continuous layout using a
high-pressure packed tower to contact the emulsion/supercritical
carbon dioxide in counter-current mode, developing a robust
and reproducible technology for micro and nanoparticles produc-
tion. Furthermore, the same apparatus has been also proposed as
a possible post-processing of nanoparticle suspensions obtained
by nanoprecipitation. In this case nanoparticle suspensions are
rst produced by conventional nanoprecipitation technique and,
then, the organic solvent removed using supercritical uids
[23].
Following this idea, the aim of this work concerns the opti-
mization of lipid nanoparticles production, based on a stearic
acid matrix, using the emulsion/diffusion method improved by
supercritical uid post-processing. In the rst process step,
the solid nanoparticles are produced by a classical solvent
emulsion/diffusion method; then, the organic solvent (BA) is con-
tinuously extracted in a countercurrent packed column taking
advantage of the high solubility of BA in supercritical carbon diox-
ide (SC-CO2 ). Different emulsion formulations, diffusion dilution
ratio, as well as, emulsion/diffusion operative temperatures have
been tested to obtain lipid nanoparticles. Nanosuspensions have
been then, processed by supercritical uid with the aim of: (1)
obtaining a fast elimination of the benzyl alcohol residue and (2)
reducing the aggregates due to the presence of solvent and obtain-
ing a more accurate control of the lipid nanoparticle size and
35
distribution. The produced SLNs are characterized by laser scat-
tering for the measurement of their particle size and distribution
and by and FE-SEM for the investigation of their morphology.
2. Materials and methods
2.1. Materials
Stearic acid (SA, purity: 98.5%, MW: 284.48, Aldrich Chemical
Co.) was used for SLN production. Polyoxyethylene (20) sorbitan
monooleate (Tween 80 , MW: 1310, Aldrich Chemical Co.) and
phosphatidylcholine-enriched fraction (Epikuron 200, purity: 92%,
Cargill Inc.) were used as surfactants. Benzyl alcohol (BA, purity:
>99.5%, Carlo Erba) was used as organic solvent. Carbon dioxide
(purity: 99.9%, SON, Naples, Italy) was used as extracting agent.
2.2. Emulsion preparation & diffusion step
The oil phase utilized in all the experiments is a water-saturated
benzyl alcohol solution. In detail, the oily phase was prepared by
dissolving 8.75% (w/w) of distilled water in benzyl alcohol at room
temperature; the solution was then stirred to reach the thermo-
dynamic equilibrium and the water in excess was removed by
decantation. Then, a xed amount of stearic acid and Epikuron
200 was added. Stearic acid solubility was measured as 15 g/L in
the oily phase at room temperature and this concentration was
used for all the present study. The water phase was prepared sat-
urating the water with BA. Tween 80 solubility in water saturated
phase was measured to be 2.1% (w/w), at ambient temperature. All
emulsions were prepared with a high-shear mixer (Silverson mod.
L4RT) operating at 7000 rpm/min. The aqueous phase was previ-
ously equilibrated and, then, the oil phase added progressively.
When all the oil phase was added, mixing was maintained for 4 min.
100 g of emulsion was prepared each time. Water was, then, added
to induce the diffusion of benzyl alcohol in water and consequently
the formation of stearic acid nanoparticles. Different dilution ratio
(emulsion/dilution water) were tested from 1/2 to 1/6.
2.3. Supercritical apparatus
The nanosuspension is continuously fed to the packed column
at a constant ow rate by a high pressure piston pump (mod. 305;
Gilson, Villiers le Bel, France). The high pressure column (i.d. 1.3 cm)
was formed by three stages of stainless steel cylindrical elements
of 30 cm height, connected by four way cross-unions and packed
with stainless steel packing (1889 m1 specic surface; 0.94 of
voidage; ProPak, Scientic Development Company, State College,
Pennsylvania). The apparatus was thermally insulated by ceramic
cloths and its temperature prole was controlled by six tempera-
ture controllers. SC-CO2 was fed at the bottom of the column by
a high-pressure diaphragm pump (model Milroyal B; Milton Roy,
Pont Saint-Pierre, France) at a constant ow rate. The supercritical
carbon dioxide (SC-CO2 ) was pumped from the bottom, to obtain a
counter current operation layout. A separator, located downstream
the top of the column, was used to recover the extracted oily phase.
Lipid nanosuspensions were continuously collected at the bottom
of the column by decompression, using a needle valve.
2.4. Analytical procedures
Emulsions and suspensions were observed using an optical
microscope (mod. BX 50 Olympus, Tokyo, Japan) equipped with a
phase contrast condenser. Samples were prepared by placing a drop
of the solution/suspension on a microscope glass slide. Samples
were analyzed quickly to avoid any heating due to the microscope
light.
36 R. Campardelli et al. / J. of Supercritical Fluids 82 (2013) 3440

Table 1
Effect of different oilwater ratios, surfactant concentrations and ratios on emulsion
stability.

Emulsion ratio Surfactant Ratio (Tween Results

(oil/water) concentration (w/w) 80/Epikuron 200)

20:80 1% 50:50 Not stable

20:80 2% 50:50 Not stable
20:80 2.5% 50:50 Stable
10:90 1% 100:0 Stable
10:90 1% 50:50 Stable
10:90 1% 75:25 Stable

LN samples were xed on an aluminum stub by drop-

evaporation; samples were, then, coated with chromium using a
sputter cold coater (mod. K575XD, Quorum Technology, Ashford,
UK) and analyzed by Scanning Electron Microscope (FE-SEM mod.
LEO 1525, Carl Zeiss SMT AG, Oberkochen, Germany) for morpho-
logical investigation.
A granulometer (Malvern Zetasizer mod. Nano ZS) was used for
the measurements of the LNs particle size distributions (PSDs) and
zeta potential. Samples were analyzed in cuvette at 25 C and all
results are based on an average of 3 measurements, which are cal-
culated on an average of 10 runs. The calculation of the particle size
is based on the calculation of the Brownian motion of the particles,
monitored thanks to the uctuation into the scattered light. Fig. 1. Schematic representation of the supercritical uid extraction process.

Benzyl alcohol content in the nal suspensions was analyzed, to

monitor the efciency of solvent removal from the emulsion dur-
ing SC-CO2 extraction. The solvent residue was measured using a
head space sampler (mod. 50 Scan, Hewlett & Packard, Palo Alto,
CA, USA) coupled to a gas chromatograph interfaced with a ame
ionization detector (GC-FID, mod. 6890 Agilent Series, Agilent Tech-
nologies Inc., Wilmington, DE). Benzyl alcohol was separated using
a fused-silica capillary column 30 m length, 0.25 mm internal diam-
eter, 0.25 m lm thickness (mod. DB-1, J&W, Folsom, CA, USA). GC
conditions were: oven temperature at 40 C for 8 min. The injector
was maintained at 250 C (split mode, ratio 1:1) and Helium was
used as the carrier gas (7 mL/min). Head space conditions were:
equilibration time 60 min at 230 C, pressurization time 2 min, loop

Fig. 2. (a and b) Detailed compositions and optical microscope images of two emulsions produced at different oil/water ratio; the overall amount of surfactants is 1% w/w
(Tween 80/Epikuron 200 ratio 50:50).
 R. Campardelli et al. / J. of Supercritical Fluids 82 (2013) 3440 37

Fig. 3. (ad) Effect of different diffusion ratios on benzyl alcohol extraction. Emulsion formulation: oilwater ratio of 10:90 with 1% w/w of surfactants (w/w); Tween 80 and
Epikuron 200 ratio xed at 50:50.

ll time 1 min. Head space samples were prepared in 10 mL vials
lled with 3 mL of suspension. Analyses were performed on each
sample in three replicates.
3. Results and discussion
3.1. Emulsion & diffusion optimization
Different emulsion ratios and different surfactant concentra-
tions were tested to nd the composition that can stabilize the
emulsion for the time required, before the diffusion step. The
results obtained in terms of emulsion stability are summarized
in Table 1. All the percentages were calculated as the mass frac-
tion of the aqueous phase and a stearic acid concentration of
15 g/L was always used in the oily phase. Two different oil/water
ratios were tested: 10/90 and 20/80; the emulsions were pre-
pared with a total concentration of 1% w/w of surfactants. Fig. 1a
and b report a comparison between the optical images of emul-
sions produced at the different oil/water ratios; larger droplet mean
diameter of 4.3 m (0.3 m) was measured in the case of the
emulsion obtained with oil/water ratio of 20/80. However, this
emulsion was not stable and showed large droplet coalescence
within the 2 h after its preparation; improved emulsion stability
was observed when an overall surfactant concentration of 2.5%
w/w was used (see Table 1). In the case of an oil/water ratio
of 10:90, a surfactant concentration of 1% was enough to sta-
bilize the emulsion and not coalescing droplets with a reduced
mean diameter of 1.9 m (0.2 m) were produced. For this rea-
son the 10/90 ratio was selected for the remaining part of the
work.
38 R. Campardelli et al. / J. of Supercritical Fluids 82 (2013) 3440
Fig. 4. (a and b) SLNs distribution curves produced by emulsion/diffusion at 80 C (a) and at 25 C (b) before supercritical uid processing.
Different Tween 80/Epikuron 200 ratio (100:0, 50:50, 75:25)
were also tested, obtaining always stable emulsions with non
coalescing droplets. In both cases, the change of surfactant ratio
did not affect the emulsion stability and droplet size distribution
and, therefore, a ratio of 50:50 Tween80/Epikuron200 was selected
(Fig. 2).
The emulsion with an oil/water ratio of 10/90 obtained with a 1%
of Tween80/Epikuron200 (ratio 50:50) was, then, used for the opti-
mization of the diffusion step, using different amounts of distilled
water. The goal was to nd the smallest emulsion/water ratio that
led to total diffusion of benzyl alcohol also producing the small-
est LNs; indeed, if benzyl alcohol diffusion was not fast enough or
incomplete, an over-growth of the stearic acid particles into resid-
ual benzyl alcohol drops was observed. All the diffusion tests were
performed at 25 C. A summary of the results obtained are reported
in Fig. 3bd, where optical images of the emulsion evolution during
the diffusion step are illustrated. An optical image of the emul-
sion before the diffusion step is also reported for comparison (see
Fig. 3a). From Fig. 3b it can be seen that the dilution ratio emul-
sion/water 1/2 was not enough to produce a complete diffusion of
benzyl alcohol in the continuous water phase; indeed after one hour
of diffusion, benzyl alcohol droplets were still present and visible
by optical microscope. Using the dilution ratio 1/4, after one hour
of mixing, all benzyl alcohol diffused into the continuous medium
(see Fig. 3c); however, due to a still slow diffusion kinetic, crys-
tallization of stearic acid with the formation of large crystals was
observed. A higher diffusion ratio (1/6) allowed the fast and com-
plete diffusion of benzyl alcohol in water (see Fig. 3d), where benzyl
alcohol droplets are no more visible, indicating that a complete dif-
fusion took place. In this case stearic acid crystal size was drastically
lowered. In all cases, due to the use of a dilute solution of stearic
acid in benzyl alcohol (15 g/L) the formation of several nanoparti-
cles inside a single droplet was observed. However, the selection
of an emulsion with a reduced droplets mean diameter of 1.9 m
(0.2 m) was useful to improve the diffusion process, increasing
the surface area available for diffusion. Indeed, interfacial diffusion
phenomena between the oil and water phases can have an inuence
on the size of the resulting lipid nanoparticles [24].
Different emulsication/diffusion temperatures were also
tested to change the diffusion and/or crystallization rate. A temper-
ature near to the melting temperature of stearic acid was tested,
to obtain a faster diffusion rate, because of the high solubility of
the benzyl alcohol into water at higher temperature and to main-
tain stearic acid in a molten state, preventing its crystallization.
Therefore, the emulsion step was performed at 80 C, pre-heating
both the oily and aqueous phase. For the diffusion step, two differ-
ent temperatures were tested; the rst test was performed adding
water at 80 C and progressively cooling the suspension at room
temperature; the second test was performed diluting with water at
room temperature (25 C). After one hour of mixing, both solutions
were stored at 4 C. In both cases no large crystals formation was
detected and the PSDs of the produced suspensions are reported
in Fig. 4a and b. LNs with mean diameters of 445.8 nm (120 nm)
and 374 nm (62 nm) were produced, respectively, and the same
distribution curves were also maintained after 2 week of storage
at 4 C. Zeta potentials of 3.4 and of 4.3 mV were measured for
the two LNs suspension, respectively. These zeta values are smaller
than 25 mV (region of suspension moderate stability) but, are rea-
sonable considering the nature of the surfactant used that are both
non-ionic, therefore able to stabilize the solution by steric interac-
tion and not by electrical repulsion.
3.2. Supercritical uid processing of LNs suspensions
Lipid nanoparticle suspensions produced with the previously
optimized emulsion/diffusion procedure were generated and
treated continuously with supercritical CO2 to benzyl alcohol
extraction. The operating conditions of the process were selected
from thermodynamic and uid dynamic considerations. In detail,
SEE-C operating conditions of pressure and temperature were cho-
sen to allow a selective extraction of the benzyl alcohols from
the nanosuspensions. Considering the high pressure vaporliquid
equilibrium diagram (VLE) of the system benzyl alcohol/CO2 [24],
the operating point was selected above the mixture critical point
(MCP); i.e., at a pressure of 80 bar for a temperature of 45 C. These
pressure and temperature conditions were convenient to maximize
the extraction of benzyl alcohol from water [25], and to avoid, at
the same time, the co-extraction of stearic acid that it is also sol-
uble in supercritical CO2 but at higher pressure [26]. Moreover
at 80 bar and 45 C, the solubility of the water external phase in
SC-CO2 is very limited. The liquid to gas ratio (L/G) was xed at
0.2 that is the value obtained in a previous optimization of the
tower uid dynamics, with an CO2 ow rate of 1.4 kg/h and an
emulsion ow rate of 4.7 mL/min [27]. The process performance
was always calculated after reaching the steady state conditions.
Indeed, an initial phase of the process is always requested to wet
the column packing with the surfactant, to favor LNs slipping on
the packing surface and to allow the mass transfer stabilization
between the liquid and the supercritical phase. PSDs of the pro-
duced nano-suspensions are reported in Fig. 5a and b; LNs with
mean sizes of 33 nm (6.5 nm) and 41 nm (7 nm), respectively,
were obtained. A mean size lowering of about one order of mag-
nitude was observed with respect to the results obtained using
traditional technique! Zeta potential between 7.2 and 8.3 mV
 R. Campardelli et al. / J. of Supercritical Fluids 82 (2013) 3440
Fig. 5. (a and b) SLNs distribution curves produced by emulsion/diffusion at 80 C (a) and at 25 C (b) and processed by supercritical uid at 80 bar and 45 C.
were also measured, indicating a moderate suspension stability.
Moreover, identical particle size distributions were monitored after
2 week of storage at 4 C, conrming the stability of the suspensions
produced.
Fig. 6. (a and b) SEM images of SLNs produced by emulsion/diffusion at 80 C: (a)
before the supercritical uid processing large aggregate are formed and (b) after
supercritical uid extraction at 80 bar and 45 C, SLNs are separated.
39
The reasons of the observed size lowering of the LNs can
be understood looking at SEM images reported in Fig. 6a and
b. LN aggregates are evident in the suspension treated by con-
ventional methods (see Fig. 6a) whereas, separated nanoparticles
were produced using the continuous diffusion step followed by
supercritical processing (see Fig. 6b). LN aggregates observed in
traditional suspension can be justied by the presence of ben-
zyl alcohol residues around the nanoparticles that favor their
adhesion into larger aggregate. When the nanosuspension is gen-
erated and treated with supercritical carbon dioxide, a fast and
complete elimination of the residual benzyl alcohol around the
particles will lead to their disaggregation and the formation of
nanoparticles with a strongly reduced mean diameter. The very
low solvent residues measured in the recovered suspensions
(less than 100 ppm) also conrmed the observed results. The
yield of the process was always 100%, suggesting that there was
no stearic acid co-extraction during the supercritical processing
step.
4. Conclusions and perspectives
The production of lipid nanoparticles of stearic acid by the
emulsion/diffusion technique plus supercritical processing is pro-
posed. The supercritical enhanced diffusion/extraction step allows
not only the elimination of benzyl alcohol from the suspen-
sions, but also the reduction of the LN sizes by eliminating
the aggregates formed during the traditional diffusion step.
Further process improvements may require a continuous emul-
sion/diffusion step by using a static mixer for the continuous
lipid nanosuspension generation and its direct processing by
supercritical uid for a continuous lipid nanoparticles produc-
tion.
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