Powder Technology 239 (2013) 183192

Contents lists available at SciVerse ScienceDirect

Powder Technology
journal homepage: www.elsevier.com/locate/powtec

Preparation and characterization of quercetin-loaded solid lipid microparticles for

pulmonary delivery
Luis Felipe Costa Silva a, Georgia Kasten a, Carlos Eduardo Maduro de Campos b,
Adilson Luiz Chinelatto c, Elenara Lemos-Senna a,
a
Departamento de Cincias Farmacuticas,Centro de Cincias da Sade, Universidade Federal de Santa Catarina, Florianpolis, SC, Brazil
b
Laboratrio de Difrao de Raios-X, Centro de Cincias Fsicas e Matemticas, Universidade Federal de Santa Catarina, Florianopolis, SC, Brazil
c
Laboratrio de Caracterizao Fsico-Qumica,Centro de Cincias Tecnolgicas, Universidade Estadual de Ponta Grossa, Ponta Grossa, PR, Brazil

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

Article history: The aim of this study was to develop quercetin-loaded solid lipid microparticles intended to be inhaled. The
Received 29 June 2012 microparticles were prepared from a mixture of glyceryl trimyristate and soy lecithin (MGTL), by the hot sol-
Received in revised form 22 December 2012 vent diffusion method, or from glyceryl behenate, in the presence (MGBL) or absence of soy lecithin (MB), by
Accepted 19 January 2013
the hot homogenization method. Quercetin was added to the formulations in a ratio of 1:25 or 1:50 (w/w),
Available online 1 February 2013
regarding the total lipid weight. Particles displaying spherical shape and rough surface were obtained as it
Keywords:
was visualized by scanning electron microscopy. The microparticles displayed porosity values varying from
Pulmonary delivery 68.83% to 85.94%, low bulk and tapped densities, and good to favorable to tolerable owability, according
Quercetin to Carr's index. Microparticles prepared from glyceryl trimyristate exhibited acceptable mass aerodynamic
Solid lipid microparticles diameter (MMAD) for pulmonary administration. However, those prepared from glyceryl behenate and soy
lecithin displayed only MMAD50% acceptable values, indicating that barely a powder fraction is able to
reach the deeper lung regions. The analyses of the lipid based microparticles and the raw materials evidenced
the presence of the and polymorphs for glyceryl trimyristate and glyceryl behenate, respectively, indicat-
ing that no polymorphic transition occurred by using both microparticle preparation techniques. In vitro re-
lease studies demonstrated that quercetin release rate is affected for both formulation composition and
particle size. The regional deposition of inhaled particles was evaluated using a multi-path model of particle
deposition (MPPD). The results indicated that MGTL would be predominantly deposited in the tracheobron-
chial and pulmonary regions, but a signicant fraction of MGBL also may reach the deeper regions of the lung.
2013 Elsevier B.V. All rights reserved.

1. Introduction inammatory properties. Nevertheless, this drug displays a very low

Asthma is a chronic inammatory lung disease characterized by
episodic symptoms and variable airow obstruction that occur ei-
ther spontaneously or in response to environmental exposures
[1]. Eosinophil recruitment, hypersecretion of the mucus, muscle
hypertrophy, and thickening of the lamina reticularis are charac-
teristic features of this disease, which is also accompanied by the
increase of intrapulmonary production of certain interleukins
(IL), especially IL-4, IL-5, and IL-13, by T helper cells [2]. Although
asthma is a well understood disease, few effective and safe drugs
are currently available for the disease management and most of
them exhibit severe adverse effects that preclude their long-term
use.
Quercetin is a widely distributed avonoid which is thought to
possess various biological effects, including antioxidant and anti-
Corresponding author. Tel.: +55 48 37215067.
E-mail address: lemos@ccs.ufsc.br (E. Lemos-Senna).
oral bioavailability due to its low water solubility and high metabo-
lism in the gut and liver [3]. In a previous study, we demonstrated
that a nanosized-emulsion formulation was able to increase the oral
bioavailability of quercetin, leading to a signicant reduction of the
most relevant phenotypes involved in the asthma process, namely
eosinophil recruitment, IL-4 and IL-5 levels in the broncho alveolar la-
vage uid, P-selectin expression, and mucus secretion in the lung,
when a dose of 10 mg/kg was given to mice immunized with ovalbu-
min [4]. These results suggest that quercetin might be a good candi-
date for direct delivery to the lungs for asthma treatment.
In spite of the advantages of the pulmonary drug delivery (i.e., reduc-
tion of the dose and reduction of the systemic absorption and side ef-
fects), the lung architecture limits the efciency of inhaled aerosols,
which is critically inuenced by the mean size and size distribution,
shape and density of the particles [5]. For an effective inhalation therapy,
optimal particle size should range from 1 to 5 m to reach the lower air-
ways. Particles larger than 5 m are likely to be deposited in the orophar-
ynx and the larynx, whereas those smaller than 1 m are more likely to
be exhaled [6,7]. On the other hand, phagocytic uptake of inhaled

0032-5910/$ see front matter 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.powtec.2013.01.037
184 L.F.C. Silva et al. / Powder Technology 239 (2013) 183192
particles is one of the main mechanisms for removing insoluble particles
from lung surfaces. Phagocytosis was shown to be dependent on particle
size; particulate drug delivery systems displaying the optimal particle size
for inhalation are also more likely to be phagocyted [8].
An approach that has recently been used to address the above
mentioned limitations of the inhalation therapy consists in obtaining
particles displaying larger geometric diameters but lower mass densi-
ties, so that the particle mean aerodynamic diameter ts into the
range of 1 to 5 m. This approach leads to more efcient aerosoliza-
tion in a given air eld as well as the reduction of the macrophage up-
take. Further, increase in the particle size results in a decreased
tendency of the particles to agglomerate due to the Van der Waals
and electrostatic forces [9]. In an endeavor to attain this goal, one ap-
proach aims to obtain porous microparticles that display particle
diameters higher than 5 m, but which have the ability to be deposit-
ed deep in the lungs. The systemic bioavailability of testosterone, for
instance, was largely increased after inhalation delivery of porous
particles with a mean diameter of 20 m, which is approximately 10
times that of conventional inhaled therapeutic particles [10]. Large
porous PLGA microparticles also exhibited desirable aerodynamic
properties, reduced macrophage uptake, and a high ability to control
the release of the encapsulated doxorubicin [6].
Another approach for obtaining low-density microparticles con-
sists of employing lipids as encapsulating materials. When compared
with polymeric carriers, lipid-based microparticles have received lit-
tle attention regarding pulmonary delivery, and only a few studies
have been reported in the literature [1113]. Lipid nano- or micro-
particles are well tolerated in living systems since they are made
from biocompatible materials for which metabolic pathways exist,
and display a high capacity to incorporate lipophilic drugs [14,15].
After a single intratracheal administration of solid lipid microparti-
cles made from glyceryl behenate in rats, no signicant inammato-
ry airway response was veried, evidencing the potential application
of lipid carriers to pulmonary drug delivery [11]. In order to impart a
sustained release prole to the bronchodilator drug salbutamol
acetonide, Jaspart et al. [12] prepared solid lipid microparticles by
the hot solvent emulsion technique using glyceryl behenate as lipid
material. In this study, the sustained release of the drug was
achieved, but only about 45% of particles were considered to display
an acceptable geometric diameter for pulmonary administration
(0.56 m). The potential of solid lipid microparticles as a means of
controlling the release of steroidal drugs in the respiratory tract
was investigated by Mezzena et al. [13]. Budesonide-loaded solid
lipid microparticles were obtained from glyceryl behenate by oil in
water emulsication via a phase inversion technique followed by
spray drying. Microparticles displaying median diameter of about
3.5 m exhibited an aerosol performance of about 20%. These results
were considered promising for pulmonary delivery.
Considering the above mentioned aspects, the aim of this study is
to develop quercetin-loaded solid lipid microparticles intended to be
inhaled. In this study, the effect of the particle composition on their
physicochemical properties and ability to load and release quercetin,
as well as on their aerosolization properties, is evaluated and compared.
2. Materials and methods
2.1. Materials
Quercetin was purchased from Sigma-Aldrich (USA) and hydroge-
nated soybean lecithin (Lipoid S100) was purchased from SP Pharma
(So Paulo, Brazil). Glyceryl trimyristate (Dynasan 114, TS) was kindly
donated by Sasol (Louisiana, USA). The fatty acid fraction was 99%
pure for this triglyceride quality and hydroxyl value below 5. Glyceryl
behenate (Compritol 888 ATO) was purchased from Brasquim (Brazil).
This excipient is comprised of a mixture of 1321% mono-, 4060% di-,
2135% tri-esters of glycerol and behenic acid (C22), and other fatty
acids than behenic acid (b20%). Polyoxypropylenepolyoxyethylene
block copolymer (Lutrol F-68 NF, called in this paper as Poloxamer
188), and polyglycol mono- and di-esters of 12-hydroxystearic acid
(Solutol HS-15, called in this paper as PEG 660-stearate) were kindly
donated by BASF (Trostberg, Germany). Ethanol, acetone, and other
chemicals used were of analytical grade.
2.2. Preparation of lipid microparticles
2.2.1. Glyceryl trimyristate microparticles
Glyceryl trimyristate/lecithin microparticles (MGTL) were prepared
by the hot solvent diffusion method [16]. Briey, 0.5 mg of soy lecithin
(Lipoid S100), 5 g of glyceryl trimyristate (Dynasan 114), and quercetin
were dissolved in ethanol at 65 C. The resulting organic solution was
quickly poured into 250 mL of an aqueous solution containing 1% (w/v)
PEG 660-stearate, maintained under magnetic stirring at 85 C. The
emulsion was sonicated for 30 s at 25% amplitude and 1.0 cycle with an
ultrasonic processor UP200S (Hielscher, Germany). The samples were
maintained under magnetic stirring until they reached room temperature
and, thereafter, they were centrifuged at 2500 rpm for one hour (Sigma
4 K15, USA), washed three times with distilled water, and freeze-dried
(Terroni Equipamentos Cientcos, Brazil). Unloaded-microparticles
were prepared using the same procedure. For drug-loaded microparti-
cles, the quercetin to lipid ratio in the formulations was 1:25 or 1:50.
All formulations were prepared in triplicate.
2.2.2. Glyceryl behenate microparticles
Glyceryl behenate microparticles were prepared by the hot emulsion
method [12]. Briey, 5 g of glyceryl behenate (MGB) or a mixture
consisting of 5 g of this lipid and 0.5 g of lecithin (MGBL) and quercetin
were heated up to 80 C. Afterwards, 250 mL of a 0.5% Poloxamer 188
(Pluronic F68 solution), previously heated up to the same tempera-
ture, was poured into the melted oil phase under magnetic stirring.
The resulting emulsion was sonicated for 30 s at 10% amplitude and
1.0 cycle using an ultrasonic processor Sonic Dismembrator Model 500
(Fisher Scientic, USA). The samples were maintained under magnetic
stirring until they reached room temperature and, thereafter, they were
centrifuged at 2500 rpm for 1 h (Sigma 4 K15, USA), washed three
times with distilled water, and freeze-dried (Terroni Equipamentos
Cientcos, Brazil). Unloaded-microparticles were prepared using the
same procedure. For drug-loaded microparticles, the quercetin to lipid
ratio in the formulations was 1:25 or 1:50. All formulations were prepared
in triplicate.
2.3. Powder characterization
2.3.1. Quercetin loading and entrapment efciency
About 50 mg of samples was exactly weighted into a 10 mL volu-
metric glass ask, and the volume was completed with methanol. The
samples were maintained under magnetic stirring for 2 h and were
then centrifuged (Sigma 4 K15, USA) at 2500 rpm for 10 min. The su-
pernatants were withdrawn and were analyzed by UV spectrophotom-
etry at 375 nm against methanol. From these results, entrapment
efciency (%) was estimated as being the percent ratio of drug
entrapped into the microparticles to the total drug added to the formu-
lations. Quercetin loading (wt.%) was expressed as being the percent
ratio of drug to the weight of microparticles.
2.3.2. Scanning electron microscopy
Microparticles were mounted onto aluminum pin stubs and were
coated with a thin layer of gold in a Leica EM SCD500 (Leica
Microsystems, USA) coating unit. The morphology of the lipid micro-
particles was examined using a Jeol JSM-6390LV (Jeol, USA) scanning
electron microscope. The SEM was operated at high vacuum with an
accelerating voltage of 10 kV. Micrographs were obtained at a magni-
cation of 400 and 800.
 L.F.C. Silva et al. / Powder Technology 239 (2013) 183192
2.3.3. Powder density and ow properties
Bulk density (bulk) of the microparticles was determined by
pouring a known amount of each sample under gravity into a gradu-
ated glass cylinder and recording the volume occupied by the pow-
der. Tapped density (tap) was subsequently determined by tapping
the samples 100 times, until no further volume change was observed
[17]. The static powder ow was characterized using Carr's compress-
ibility Index (CI), which was determined as the following equation:
! was placed into a dialysis bag (Spectra/Por CE MWCO 10000,
tap bulk
CI  100:
tap
On the basis of CI value, powder owability is dened as: 512%,
excellent; 1218%, good; 1821%, fair; 2125%, poor, uid; 2532%,
poor, cohesive; 3238%, very poor; and >38%, extremely poor.
2.3.4. Particle porosity
Particle porosity was evaluated using an AutoPore IV 9500 mercu-
ry intrusion porosimeter (Micromeritics, USA). About 0.2 g of each
sample, exactly weighted, was added into a 3 cm 3 powder penetrom-
eter with a 22,285 L/pF capacitance constant. The analyses were
conducted under low and high pressures. Curves of mercury intrusion
volume (mL) versus pressure (mPa) were obtained and the specic
surface area, mean pore size, porosity (), and skeletal density (dsk)
of the microparticles were determined using the AutoPore IV 9500
VI.05 software.
2.3.5. Geometric and aerodynamic particle size determination
Mean diameter and size distribution of microparticles were de-
termined by laser diffraction using a Mastersizer 2000 (Malvern In-
struments, UK). The samples were previously dispersed in a 0.15%
(w/v) Tween 80 aqueous solution and the analyses were carried
out at obscuration mode between 10% and 13%. The particle size dis-
tribution was estimated using the Fraunhofer diffraction model and
was expressed by equivalent volume diameters at 10% (d0.1), 50%
(d0.5), and 90% (d0.9) cumulative volume, respectively, as well as by
equivalent volume mean diameter (d4,3). The width of the size distri-
bution was expressed by Span, which was calculated as the following
equation:
d0:9 d0:1
Span : 2
d0:5
The theoretical mass aerodynamic diameter of microparticles was
estimated using the following equation [18]:
s encloses the formation of a hot oil-in-water (o/w) emulsion by
sk 1
MMAD d 
o  X
where d is the median (d0.5) or the mean diameter (d4,3) of micropar-
ticles, sk is the skeletal mass density of the particle, is the particle
porosity, 0 is a reference density of 1 g/mL, and X is the shape factor,
which is one for a sphere.
2.3.6. X-ray powder diffraction
The X-ray powder diffraction (XRPD) patterns of microparticles and
raw materials were recorded with a X'Pert PRO X-ray diffractometer
equipped with a X'Celerator detector (PANanalytical, Netherlands),
using a CuK radiation (45 kV voltage, 40 mA). Measurements were
carried out at 2 angles ranging from 3 to 60 with a scan step size of
0.033 and a scan step time of about 30 s.
2.3.7. Differential scanning calorimetry
Differential Scanning Calorimetry (DSC) analyses were carried out
using a DSC-50 instrument (Shimadzu, Japan). For the measurements,
185
about 5 to 15 mg of the microparticles or the bulk materials were ac-
curately weighed into aluminum pans, which were tightly sealed. The
samples were cooled down to 10 C and then heated to 220 C at a
scan rate of 5 C min 1. An empty pan was used as reference.
2.4. In vitro quercetin release studies
For the experiments, 300 mg of each microparticle formulation
USA). The dialysis bags were placed into a dissolution apparatus
1
Model 299 (Nova tica, Brazil) containing 300 mL of PEG 400 and dis-
tilled water (4:6, v/v; pH 4.0) solution. The release medium was
maintained at 37 C under mechanical stirring at 75 rpm. Aliquots
of the release medium were withdrawn at intervals during the fol-
lowing 24-hour period. The samples were analyzed by UV spectro-
photometry (Shimadzu, Japan) at 375 nm and the cumulative
amounts of quercetin released (%) were plotted against time (h).
The time in which 50% of the drug was released (t50%) and the disso-
lution efciency (DE%) were determined from quercetin release
curves. DE% was estimated from the area under dissolution curve
measured using the trapezoidal rule and expressed as a percent
area of the rectangle described by 100% dissolution at the same
time [19]. DE% values obtained for the different release proles
were statistically compared by ANOVA ( = 0.05).
2.5. Mathematical deposition model for inhaled solid lipid microparticles
In this study, the multiple-path model of particle deposition
(MPPD v2.11, Applied Research Associates, Inc., USA) was employed
to evaluate and compare the inhalation deposition performance of
the solid lipid microparticles, assuming the following conditions: av-
erage respiratory ow rate of 50 L/min, tidal volume of 700 cm 3,
breath frequency of 5 breaths/min, and breath pause of 10 s. Particles
were assumed to enter the lung via the mouth so that head deposi-
tion takes place only in the mouth cavity [20].
3. Results and discussion
3.1. Quercetin loading and microparticle morphology
In this study, two fatty acid esters of glycerol were chosen to pre-
pare solid lipid microparticles, glyceryl trimyristate and glyceryl
behenate; both lipids are recognized as Generally Recommended as
Safe (GRAS) and are included in the Food and Drug Administration's
(FDA) inactive ingredients guide. Glyceryl trimyristate microparticles
were prepared by the solvent diffusion emulsication method, which
adding a water miscible organic solution containing the drug, lipid,
3
and the lipophilic surfactant into an aqueous solution containing a
hydrophilic surfactant [16]. The organic solvent is then quickly re-
moved from the organic phase by diffusion toward the aqueous
phase and, after lipid solidication, it is eliminated from the formula-
tion by centrifugation and washing of the microparticles with water.
Due to the low solubility of glyceryl behenate in ethanol, the hot
melting dispersion method was used to prepare microparticles from
this lipid. This method is based on the formation of a hot o/w emul-
sion without the use of an organic solvent; in this case, the melted
lipid containing the drug was directly emulsied into a hot aqueous
surfactant solution [12]. Glyceryl trimyristate microparticles were
formulated only in the presence of lecithin, since the removal of this
hydrophobic surfactant, in this case, led to the precipitation of the
lipid without microparticle formation. On the other hand, glyceryl
behenate microparticles could be prepared with or without the addi-
tion of the lipophilic surfactant. It may be related to the difference in
the composition of the raw materials, which may have affected the
feasibility to obtain stable hot o/w emulsions from each lipid. While
186 L.F.C. Silva et al. / Powder Technology 239 (2013) 183192

Dynasan 114 is constituted by 99% of glyceryl trimyristate, Compritol

888 ATO is a mixture of mono-, di- and tri-esters of glycerol and
behenic acid and other fatty acids that may have contributed to the
interfacial tension reduction between the oil and aqueous phase,
forming a more stable emulsion. The physicochemical properties of
the lipids were, therefore, determining factors for selecting the micro-
particle preparation conditions.
Scanning electron micrographs of solid lipid microparticles pre-
pared from glyceryl trimyristate and glyceryl behenate are shown in
Figs. 1 and 2, respectively. The glyceryl trimyristate microparticles
display a spherical shape and rough surface. However, quercetin crys-
tals can be seen in the micrograph obtained from MGTL(1:25) (Fig. 1b),
evidencing that, at this drug to lipid ratio, there was an exceeding
drug amount that was not entrapped into the lipid matrix. Glyceryl
behenate microparticles displayed a rougher surface. It seems that
microparticles were damaged by the intense electron beam of the mi-
croscope, since its spherical shape was lost (Fig. 2af). This character-
istic was observed even when using a lower electron beam intensity.
The addition of soy lecithin to the formulations did not appear to
change the morphology of the particles (Fig. 2df). Furthermore, the
presence of quercetin crystals outside the particles was less evident
than for MGTL, evidencing the greater ability of glyceryl behenate mi-
croparticles in to load hydrophobic drugs as quercetin.
In this study, quercetin loading (QL%) and entrapment efciency
(EE%) varied from 0.52% to 3.84% and from 29.35% to 99.80%, respec-
tively, depending on the lipid microparticle formulation (Table 1). As
discussed above, ethanol (a water miscible-solvent) was employed to
prepare glyceryl trimyristate microparticles by the hot solvent diffu-
sion method. When the organic phase is poured into the aqueous
phase, ethanol quickly diffuses towards the latter, leading to a forma-
tion of a hot o/w emulsion. In this case, quercetin is most likely
partitioned between the lipid phase and aqueous phase containing
ethanol, being eliminated during the centrifugation and washing
steps, and resulting in a low EE% as demonstrated for MGTL(1:50).
When a greater quantity of drug is added to the formulation
(MGTL(1:25)), drug solubility in both the aqueous and lipid phase is
probably exceeded leading to drug precipitation, but in this case it
remained in the nal product as drug crystals outside of the particles,
providing high but unreal values of entrapment efciency. On the
other hand, when glyceryl behenate microparticles were prepared,
no solvent was added to the organic phase and, therefore, the drug
was probably partitioned in favor of the lipid phase. Besides, studies
have also demonstrated that microparticles prepared from partial
amorphous triglycerides, such as glyceryl behenate, exhibited a
higher ability to incorporate hydrophobic drugs between fatty acid
chains and in crystal imperfections [2123]. These are probably the
reasons why high EE% values were obtained for microparticles pre-
pared using glyceryl behenate. In this case, the higher values of
drug loading and entrapment efciency were veried when micro-
particles were prepared without lecithin and it may be related to
the drug solubility in the melted lipid.

Fig. 1. SEM images of glyceryl trimyristate microparticles: (a) MGTL, (b) MGTL(1:25), and
3.2. Powder density and ow properties (c) MGTL(1:50).

Many studies suggest that the powder aerosolization properties may

be improved by the reduction of the powder density [10,24,25]. Howev-
er, few of them emphasize that the lower density of solid lipid micropar-
ticles can be advantageous for lung deposition. The bulk and tapped
densities of the glyceryl trimyristate and glyceryl behenate microparti-
cles are shown in Table 2. The microparticles exhibited tapped density
values ranging from 0.22 to 0.27 g cm3, which may be considered fa-
vorable for aerosolization [24]. The samples exhibited relatively high CI,
varying from 14.93 (good owability) to 20.99 (fair owability),
suggesting that the addition of excipients should be tested to improve
powder owability for lung administration.
3.3. Porosity determination
The parameters describing the pore properties of solid lipid micro-
particles are depicted in Table 3. Lipid microparticles exhibited high
porosity values, which varied from 68.83% to 85.94%. Microparticles
prepared from glyceryl trimyristate exhibited higher porosity values
than those prepared from glyceryl behenate with or without the addi-
tion of lecithin. Still, these particles displayed lower median pore di-
ameter and higher total pore surface area, which is in agreement
 L.F.C. Silva et al. / Powder Technology 239 (2013) 183192 187

Fig. 2. SEM images of glyceryl behenate microparticles: (a) MGB, (b) MGB(1:25), (c) MGB(1:50), (d) MGBL, (e) MGBL(1:25), and (f) MGBL(1:50).

with the SEM micrographs obtained from MGTL samples, as is illus- but most of the available studies are concerned with the correlation
trated in Fig. 1. between pore properties and formulation parameters of polymeric
Porosity can be described as the ratio of the volume of voids be- microparticles [26]. However, some points can be highlighted to ex-
tween particles, plus the volume of pores, to the volume occupied plain the pore properties of lipid-based microparticles in this study.
by the powder; the higher the porosity, the higher the particle skele- The higher total pore surface and porosity, and lower median pore di-
tal density is. Several factors can affect the microparticles' porosity, ameter of glyceryl trimyristate-based microparticles could be related
to the preparation method used. Using the hot solvent diffusion
Table 1 method, ethanol diffuses instantly in the aqueous phase, forming an
Quercetin loading and entrapment efciency of solid lipid microparticles. o/w emulsion. The use of the organic solvent probably contributed
Formulation Quercetin loading (wt.%) Entrapment efciency % to the reduction of the oil droplet size of the emulsion and, then, sub-
sequently to the particle size, explaining to some extent to the higher
MGTL(1:25) 3.36 0.10 95.70 2.82
MGTL(1:50) 0.52 0.01 29.35 0.61 porosity obtained for these samples. On the other hand, glyceryl
MGB(1:25) 3.84 0.06 99.80 1.68 trimyristate recrystallization occurs at a slower rate than glyceryl
MGB(1:50) 1.95 0.05 99.57 2.50 behenate (from 85 C to 57 C and from 80 C to 72.2 C, respectively),
MGBL(1:25) 3.10 0.11 88.51 3.19 which may conduct to the formation of a more organized crystal lattice,
MGBL(1:50) 1.69 0.04 94.47 2.26
with reduced median pore diameter. Concerning the formation of
188 L.F.C. Silva et al. / Powder Technology 239 (2013) 183192

Table 2 MGBL exhibited aerodynamic diameters that are undesirable for pulmo-
Powder density values and ow properties of the solid lipid microparticles. nary administration. On the other hand, when the aerodynamic diameter
Samples Bulk density Tapped density Carr's index Flowability corresponding to 50% of the accumulated size distribution is considered,
(g cm3) (g cm3) (%) it is possible to verify that glyceryl behenate microparticles prepared in
MGTL 0.205 0.003 0.240 0.002 14.93 0.22 Good the presence of lecithin possess a 50% fraction that could reach the
MGTL(1:25) 0.239 0.001 0.294 0.003 18.49 0.22 Fair deep regions of the lungs. The difference found between MMAD and
MGTL(1:50) 0.186 0.003 0.220 0.003 15.79 0.30 Good MMAD50% for MGBL may be related to the high values of span obtained
MGB 0.189 0.003 0.227 0.007 16.89 0.55 Good
for these formulations.
MGB(1:25) 0.197 0.004 0.245 0.002 19.47 0.56 Fair
MGB(1:50) 0.191 0.003 0.236 0.003 18.80 0.38 Fair
MGBL 0.174 0.005 0.218 0.006 20.00 0.75 Fair
MGBL(1:25) 0.214 0.001 0.272 0.009 20.99 0.67 Fair 3.5. X-ray powder diffraction
MGBL(1:50) 0.223 0.005 0.265 0.021 15.87 1.30 Good

Lipids exhibit polymorphism due to the numerous lateral packing

possibilities of fatty acid chains specically designated as , sub ,
and , each of them corresponding to a particular lateral organization
glyceryl behenate microparticles, not only may have the fast recrystalli-
of the hydrocarbon chains. Briey, the most stable phase is the va-
zation rate contributed to the formation or a more disorganized lipid
riety for pure monoacid saturated triglyceride and phase for a mix-
structure displaying a higher pore diameter, but also the chemical com-
ture of fatty acids with long chains [27]. When melted, lipids undergo
position of this lipid, that is a mixture of mono-, di- and trialcylglycerols.
polymorphic transitions during the recrystallization process; the
-form has the tendency to be transformed quickly to a form with
3.4. Particle size properties better chain packing such as the -form and then to the most stable
-form. Therefore, the triglyceride transition pathway from liquid to
As it can be illustrated in Table 4, lipid microparticles prepared solid state occurs from -form to -form via form [28]. Lipid mi-
from glyceryl trimyristate exhibited volume median diameters (d0.5) croparticles are prepared from crystalline raw materials, with the
and volume mean diameters (d4,3) varying from 5.50 to 6.84 and preparation process including melting and recrystallization steps. It
6.67 to 8.43 m, respectively. When tryglyceryl behenate was has been demonstrated that the formulation composition and prepa-
employed as a solid lipid, not only were d0.5 (6.23 to 9.04 m) and ration technique, including the use of high shear dispersion tech-
d4,3 (18.16 to 26.71 m) values higher, but also the span values, indi- niques, may provoke changes in the solid-state properties of the
cating that more polydisperse particle populations were obtained. Be- matrix constituents [23], which, in turn, may affect microparticle char-
sides, when glyceryl behenate microparticles were prepared without acteristics such as entrapment efciency, drug release rate, stability, and
lecithin, an increase in the d0.5 (17.53 to 25.62 m) and d4,3 (23.11 to others [2931]. Then, the characterization of the solid-state of lipid mi-
34.30 m) was observed, with simultaneous reduction in the span croparticles is required to detect possible modications of the physico-
value. The difference in the particle size can be related to either the chemical properties of both drug and lipids.
presence of soy bean lecithin in the formulation or the preparation X-ray diffraction patterns of the supplied raw materials and solid
technique. Lecithin is a lipophilic surfactant, which promotes the sta- lipid microparticles are shown in Fig. 3. Glyceryl trimyristate and
bilization of the emulsion, reducing the droplet size during the micro- glyceryl behenate displayed peaks at 19.32, 23.17, and 24.0 2,
particle preparation process, and then, conducting the formation of and at 21.16, and 23.34 2, respectively, and they are attributable
reduced sized particles. On the other hand and in contrast with the to the and polymorphs of these lipids [28]. As can be seen in the
hot homogenization technique, the hot solvent diffusion technique X-ray patterns obtained from the solid lipid microparticles, both
appears to provide a more uniform distribution of lecithin in the oil lipids displayed the same crystalline structure than their correspond-
phase of the emulsion, contributing to the formation of monodisperse ing raw materials. Soy lecithin employed in this study exhibited a
particle populations. mixture of amorphous and crystalline phases and clearly did not af-
For deep lung deposition, dry powders are mainly required to exhibit fect the crystalline arrangement of the lipids in the microparticles.
adequate aerodynamic characteristics, i.e., the aerodynamic diameter Quercetin used as raw material exhibited the most intense peaks at
must range from 1 to 5 m [18]. In this study, the aerodynamic diameters 14.07, 26.51, and 27.38 2. The presence of quercetin in the micro-
of the different solid lipid microparticle formulations were evaluated by particles could be better observed at about 1015 and 27.3 2 in the
substituting both equivalent volume mean diameter (d4,3) and equiva- XRPD patterns obtained from MGTL(1:25), MGBL(1:25), and MGTL(1:50), in-
lent volume median diameter (d0.5) in Eq. (3). The results are shown in dicating, therefore, that this drug is at least partially in the crystalline
Table 4. MGTL exhibited the smallest aerodynamic diameters (up to state in these samples. Glyceryl behenate microparticles prepared
3.84 m), and MMAD and MMAD50% were quite similar, while MGB and without soy lecithin exhibited the same diffraction pattern of those
prepared in the presence of this surfactant and it was not shown in
Fig. 3.
In spite of lipid crystalline properties in the microparticles being
Table 3 the same as those of raw materials, the differences between the pack-
Pore properties of solid lipid microparticles. ing arrangements of lipid molecules in the particles probably contrib-
Sample Median pore Skeletal Porosity Total pore uted to the differences veried in the quercetin loading and EE%, as
diameter (nm) density (%) surface area described in Section 3.1. Glyceryl trimyristate is a pure triglyceride
(g/mL) (m2/g) in which the more stable form is prevailing, rendering a higher or-
MGTL 1602.3 1.404 83.83 9.220 dered crystal packing with lower ability to incorporate drugs. On
MGTL(1:25) 1988.7 1.480 85.94 8.309 other hand, glyceryl behenate is a mixture of mono-, di- and tri-
MGTL(1:50) 2246.0 1.469 84.86 6.797
behenate of glycerol (18%, 52%, and 28% in weight, respectively).
MGB 9487.0 0.828 71.28 1.265
MGB(1:25) 4599.4 0.839 68.83 2.290 The existence of diglycerides leads to a large number of lattice im-
MGB(1:50) 4365.7 0.835 69.70 2.189 perfections in the solid state, preventing the transformation of
MGBL 6762.9 0.811 73.43 2.014 to form [32]. Thus, the less ordered crystal lattice of glyceryl
MGBL(1:25) 4883.9 1.148 76.29 2.296 behenate microparticles may have favored quercetin inclusion in-
MGBL(1:50) 4177.5 0.820 69.07 2.724
side the matrix.
 L.F.C. Silva et al. / Powder Technology 239 (2013) 183192 189

Table 4
Size characteristics of unloaded and quercetin-loaded solid lipid microparticles.

Sample Cumulative percenta d4,3(m)b Span MMAD (m)c MMAD50% (m)d

d10% (m) d50% (m) d90% (m)

MGTL 1.41 0.52 6.84 0.92 18.99 0.65 6.67 2.56 2.56 1.04 3.18 1.22 3.26 0.44
MGTL(1:25) 1.22 0.37 6.33 1.65 18.36 1.58 8.43 1.84 2.71 0.99 3.84 0.84 2.89 0.75
MGTL(1:50) 1.21 0.40 5.59 1.31 14.91 3.48 6.96 3.80 2.45 1.75 3.28 1.79 2.64 0.62
MGB 2.04 0.40 25.62 1.30 77.38 2.66 34.30 1.84 2.94 0.90 16.72 0.90 12.49 0.63
MGB(1:25) 2.12 0.78 17.53 3.45 51.90 4.96 23.11 2.45 2.84 0.85 11.82 1.25 8.96 1.76
MGB(1:50) 2.03 1.03 20.72 3.22 66.22 3.69 30.61 1.25 3.20 1.83 15.40 0.63 10.52 1.62
MGBL 1.27 0.08 6.23 1.99 56.61 5.41 18.16 2.52 8.88 1.61 8.43 1.17 2.89 0.92
MGBL(1:25) 1.44 0.51 9.04 2.03 56.10 1.35 26.71 2.95 6.05 1.73 13.93 1.54 4.72 1.06
MGBL(1:50) 1.42 0.67 7.87 3.28 50.95 1.62 22.93 2.02 6.29 2.76 11.54 1.02 3.96 1.65
a
Equivalent volume diameters at 10 (d10%), 50 (d50%), and 90% (d90%) cumulative volume.
b
Volume mean diameter.
c
Mass mean aerodynamic diameter, calculated from volume mean diameter.
d
Mass median aerodynamic diameter, calculated from the volume diameter at 50% cumulative volume.

3.6. Differential scanning calorimetry could not be assessed by DSC, since when samples were heated to tem-
peratures above 300 C, an exothermic deviation from the baseline oc-
DSC curves obtained after analysis of supplied raw materials and solid curred, probably due to the lipid decomposition.
lipid microparticles are demonstrated in Fig. 4. Glyceryl trimyristate and
glyceryl behenate exhibited endothermic peaks at 57 C and 72.2 C,
which correspond to the melting of and forms, respectively [27,28].
DSC curve obtained from quercetin exhibited two endothermic peaks at
127.8 C and 327.2 C, and one exothermic peak at 351.1 C. The rst en-
dothermic peak corresponds to the release of water from the crystal
lattice; the high temperature being correlated to the strong hydrogen
bonds between water and quercetin. The second endothermic peak corre-
sponds to the melting point, and the third peak is related to the exother-
mic decomposition of this drug [29]. Soy lecithin did not exhibit any
peak from 0 to 150 C, evidencing the amorphous property of this sur-
factant. DSC curves obtained from microparticles did not display any
shifting in the endothermic peak of the lipids, indicating that they crystal-
lize in the same polymorphic forms as the raw materials and that querce-
tin had no effect on the crystalline structure of the lipid matrix. On the
other hand, the crystalline properties of quercetin in the microparticles

Fig. 3. X-ray powder diffraction patterns of supplied raw materials (upper) and
unloaded and quercetin-loaded solid lipid microparticles (lower): (a) glyceryl trimytistate, Fig. 4. DSC curves of supplied raw materials (upper) and quercetin-loaded solid lipid
(b) glyceryl behenate (c) quercetin, (d) soy lecithin, (e) MGTL, (d) MGTL(1:25), (e) MGTL(1:50), microparticles (lower): (a) glyceryl behenate, (b) glyceryl trimyristate, (c) quercetin
(f) MGBL, (g) MGBL(1:25), and (h) MGBL(1:50). (d) soy lecithin (e) MGTL(1:25), (f) MGB(1:25), and (g) MGBL(1:25).
190 L.F.C. Silva et al. / Powder Technology 239 (2013) 183192

3.7. In vitro release studies

The release rate of drugs from microparticles depends on several

factors, including the formulation composition and physicochemical
properties of particles, as size and morphology. Drug release is a com-
plex phenomenon which includes different types of mass transport
processes, in which, diffusional mass transport is the most important
mechanism [33]. However, to obtain reliable data from drug release
studies, the sink conditions should be respected. Sink conditions de-
scribe a dissolution system that is sufciently dilute so that the disso-
lution process is not hindered by approach to saturation of the
compound of interest. Since quercetin is a hydrophobic drug that ex-
hibits limited solubility in an aqueous medium, a mixture of polyeth-
ylene glycol and water (4:6, v/v) was used as a releasing medium to
attain the sink conditions. In order to evaluate the release proles of
quercetin from solid lipid microparticles, the dialysis bag method
was used, since it avoids further separation of particles from medium
after sampling and, besides to ensure that all microparticles are in
contact with the release medium, avoiding particles oating as ob-
served when conventional dissolution methods are used [34,35].
Also, the dialysis method is described to mimic in vivo conditions
where the particles are immobilized upon administration and surrounded
by a stagnant layer [34]. However, the membrane can act as a barrier for
the drug, leading to a misunderstanding of the release rate. Then, to
evaluate the effect of the dialysis membrane on quercetin release rate
from microparticles, a polyethylene glycol solution of this drug was
assayed and the release prole was compared with those obtained for
solid lipid microparticle formulations.
The in vitro release proles obtained for free drug and quercetin-
loaded solid lipid microparticles are shown in Fig. 5. The times re-
quired for 50% of quercetin dissolution/diffusion, and the dissolution
efciency values are listed in Table 5. Statistical analysis performed
using the dissolution efciency data obtained from release proles in-
dicated that both lipid composition and initial quercetin amount sig-
nicantly affected drug release rate (Fcal > Fcrit, = 0.05). The
slowest release rate of quercetin was obtained from MGTL(1:25) and it
may be related to the presence of a large amount of quercetin crystals
Fig. 5. In vitro release proles of quercetin from solid lipid microparticles prepared
in this sample, which, in turn, is related to the crystalline properties of from (a) glyceryl trimyristate/lecithin, (b) glyceryl behenate without lecithin, and
glyceryl trimyristate, as discussed above. In this case, the drug release (c) glyceryl behenate/lecithin. () Free quercetin; () microparticles prepared using
rate obtained for this sample is an end result of both diffusion of the a lipid to drug ratio of 1:25; () microparticles prepared using a lipid to drug ratio of
entrapped drug throughout the lipid matrix and dissolution of quer- 1:50.
cetin crystals. On the other hand, when MGTL(1:50) and MGBL(1:50) were
assayed, the release prole of quercetin was very similar to that
obtained for the free drug, evidencing that the control of the drug re- software. The model simulates the mass fraction of particles deposit-
lease rate was not achieved after encapsulation. Since these samples ed in different respiratory tract sites based on experimental micro-
contain lecithin, it is possible that most of the drug has been solubilized particles characteristics and on physiological conditions of the lungs
in the surfactant layer and, therefore, is preferentially located at the and the breathing. The results of the deposition fraction for solid
outer regions of the particles. It can be supported by the fact that lipid microparticles are shown in Fig. 6. In this study, we used two
when lecithin was removed from the formulation (MGB(1:50)) the time size parameters to estimate the aerodynamic diameter of the micro-
required for 50% quercetin release was increased by two times. On the particle formulations; the mean particle diameter and the median di-
other hand, glyceryl behenate microparticles prepared without lecithin ameter, the latter of which is the diameter obtained for 50% of the
displayed a more controlled release rate of quercetin, which was affect- particle population. When mathematical simulation was performed
ed by quercetin loading. In this case, it can be hypothesized that drug re- using the mean microparticle size, MGTL demonstrated the largest depo-
lease occurs by a diffusion mechanism and it can be supported by the sition fraction in the tracheobronchial and pulmonary regions (Fig. 6a),
aforementioned crystalline properties of glyceryl behenate. The less or-
dered lipid arrangement in the matrix allowed the incorporation of
higher amounts of quercetin inside the particles, which, in turn, was Table 5
slowly diffused through the particle towards the bulk release medium Times required for 50% of quercetin release (t50%) and dissolution efciency values (%).
[21,22] Still, the size of the particles could also have affected the rate Sample t50% (h) Dissolution efciency (%)
in which quercetin was released from microparticles.
Free quercetin 2.53 0.05
MGTL(1:25) 10.21 0.06 47.07 0.30
3.8. Estimation of regional deposition in human lungs MGTL(1:50) 2.54 0.05 76.04 2.35
MGB(1:25) 5.57 0.04 70.46 3.48
A comparative in silico deposition performance of solid lipid mi- MGB(1:50) 6.00 0.02 59.76 4.46
MGBL(1:25) 5.42 0.05 82.07 3.76
croparticles was carried out using a multiple-path model of particle
MGBL(1:50) 3.14 0.04 67.78 1.98
deposition (MPPD v2.11, Applied Research Associates, Inc., USA)
 L.F.C. Silva et al. / Powder Technology 239 (2013) 183192
Fig. 6. In silico predicted deposition patterns of solid lipid microparticles estimated from (a) MMAD, and (b) MMAD50%.
while MGB and MGBL were predominantly deposited in the head region,
with an insignicant amount in the pulmonary region. On the other
hand, MGB and MGBL displayed broader particle size distribution, as
can be veried by the higher span values. Then, in spite of larger values
of MMAD obtained for MGB and MGBL, glyceryl behenate microparticles
prepared in the presence of lecithin displayed satisfactory MMAD50%
values, indicating that at least 50% of the particle population can reach
the lungs, which, in turns, corresponds to the respirable aerosol fraction.
Respirable aerosol fraction (or alveolar fraction) is the sub fraction of
the inhaled particles [db 5 m] that penetrates into the alveolar region
of the lung. The respirable fraction of a therapeutic aerosol is often quot-
ed as the percentage of drug present in aerosol particles less than 5 m
in size [36]. Considering this concept, MGBL may represent a more ad-
vantageous formulation since it can carry a larger dose than MGTL,
even though a smaller fraction can reach the deep lungs.
4. Conclusions
This study has demonstrated the potential use of solid lipid micro-
particles to carry quercetin for pulmonary administration. Lipid mi-
croparticles may be associated with numerous advantages of the
use of lipids in the formulations (e.g., high tolerability, high ability
to incorporate hydrophobic drugs), with the possibility to obtain re-
spirable particles with higher geometric diameters, due to lower den-
sities of these materials. However, in this study, we demonstrated
that the formulation composition and preparation techniques affect
not only the physicochemical properties of the particles and the
drug release proles, but also the region in which particles will be de-
posited into the lungs. Even though many studies still need to be car-
ried out, including in vivo studies and formulation studies using
suitable excipients, solid lipid microparticles may be considered as
promising carriers to deliver drugs for local therapy to treat disorders
affecting the lungs.
Further works will be conducted with a perspective to decrease
the glyceryl behenate based microparticles MMAD, favoring their
aerosolization properties, as well as the use of the spray-dryer tech-
nique to produce powder with better ow properties.
191
Acknowledgments
We are grateful to Conselho Nacional de Desenvolvimento
Cientco e Tecnolgico (CNPq) for nancial support and LCME
(UFSC) for SEM analyses.
References
[1] P.A.B. Wark, P.G. Gibson, Asthma exacerbations 3: pathogenesis, Thorax 61
(2006) 909915.
[2] A.M. Singh, W.W. Busse, Asthma exacerbation 2: aetiology, Thorax 61 (2006)
809816.
[3] S. Bischoff, Quercetin: potentials in the prevention and therapy of disease, Cur-
rent Opinion in Clinical Nutrition and Metabolic Care 11 (2008) 733740.
[4] A.P. Rogrio, C.L. Dora, E.L. Andrade, J.S. Chaves, L.F.C. Silva, E. Lemos-Senna, J.B.
Calixto, Anti-inammatory effect of quercetin-loaded microemulsion in the air-
way allergic inammatory model in mice, Pharmaceutical Research 61 (2010)
288297.
[5] G. Taylor, I. Kellaway, Pulmonary drug delivery, in: A.M. Hilley, A.W. Lloyd, J.
Swarbuck (Eds.), Drug Delivery and Targeting for Pharmacists and Pharmaceuti-
cals Scientists, Taylor & Francis, London, 2001, pp. 269291.
[6] Y. Yang, N. Bajaj, P. Xu, K. Ohn, M.D. Tsifansku, Y. Yeo, Development of highly po-
rous large PLGA microparticles for pulmonary drug delivery, Biomaterials 30
(2009) 19471953.
[7] J.S. Patton, P.R. Byron, Inhaling medicines: delivering drugs to the body through
the lungs, Nature Reviews. Drug Discovery 6 (2007) 6774.
[8] M. Geiser, Update on macrophage clearance of inhaled micro and nanoparticles,
Journal of Aerosol Medicine and Pulmonary Drug Delivery 23 (2010) 210217.
[9] D.A. Edwards, J. Hanes, G. Caponetti, J. Hrkach, A. Benjebria, L.M. Eskew, J.
Mintzes, D. Deaver, N. Lotan, R. Langer, Large pourous particles for pulmonary
drug delivery, Science 276 (1997) 18681971.
[10] D.A. Edwards, A. Ben-Jebria, R. Langer, Recent advances in pulmonary drug deliv-
ery using large, porous inhaled particles, Journal of Applied Physiology 85 (1998)
379385.
[11] V. Sanna, N. Kirschvink, P. Gustin, E. Gavini, I. Reland, L. Delattre, B. Evrard, Prep-
aration and in vivo toxicity study of solid lipid microparticles as carrier for pul-
monary administration, AAPS PharmSciTech 5 (2004) 17.
[12] S. Jaspart, P. Bertholet, G. Piel, J.-M. Dogn, L. Delattre, B. Evrard, Solid lipid micro-
particles as a sustained release system for pulmonary drug delivery, European
Journal of Pharmaceutics and Biopharmaceutics 65 (2007) 4756.
[13] M. Mezzena, S. Scalia, P.M. Young, D. Traini, Solid lipid budesonide microparticles
for controlled release inhalation therapy, The AAPS Journal 11 (2009) 771778.
[14] R.H. Mller, K. Mder, S. Gohla, Solid lipid nanoparticles (SLN) for controlled drug
delivery a review of the state of the art, European Journal of Pharmaceutics and
Biopharmaceutics 50 (2000) 161177.
[15] W. Mehnert, K. Mader, Solid lipid nanoparticles: production, characterization and
Applications, Advanced Drug Delivery Reviews 47 (2001) 165196.
192 L.F.C. Silva et al. / Powder Technology 239 (2013) 183192
[16] C.L. Dora, L.F.C. Silva, M.P. Tagliari, M.A.S. Silva, E. Lemos-Senna, Formulation
study of quercetin-loaded lipid based nanocarriers obtained by hot solvent diffu-
sion method, Latin American Journal of Pharmacy 30 (2011) 289296.
[17] A.M. Healy, B.F. McDonald, L. Tajber, O.I. Corrigan, Characterisation of
excipient-free nanoporous microparticles (NPMPs) of bendroumethiazide,
European Journal of Pharmaceutics and Biopharmaceutics 69 (2008) 11821186.
[18] F. Ungaro, R. Bianca, C. Giovino, A. Miro, R. Sorrentino, F. Quaglia, M.I. La Rotonda,
Insulin-loaded PLGA/cyclodextrin large porous particles with improved aerosoli-
zation properties: in vivo deposition and hypoglycemic activity after delivery to
rats lungs, Journal of Controlled Release 135 (2009) 2534.
[19] U.V. Banakar, in: M. Dekker (Ed.), Pharmaceutical Dissolution Testing, Marcel
Dekker, New York, 1992, pp. 55100.
[20] Y.H. Kim, K.S. Shing, Supercritical uid-micronized ipratropium bromide for pul-
monary drug delivery, Powder Technology 182 (2008) 2532.
[21] D.Z. Hou, C.S. Xue, K.J. Huang, C.H. Zhu, The production and characterization of
solid lipid nanoparticles, Biomaterials 24 (2003) 17811785.
[22] S. Das, W.N. Ng, R.B.H. Tan, Are nanostructured lipid carriers (NLCs) better than
solid lipid nanoparticles (SLNs): development, characterizations and comparative
evaluations of clotrimazole-loaded SLNs and NLCs? European Journal of Pharma-
ceutical Sciences 47 (2012) 139151.
[23] S. Jaspart, G. Piel, L. Delatre, B. Evrard, Solid lipid microparticles: formulation,
preparation, characterization, drug release and applications, Expert Opinion 2
(2005) 7587.
[24] C. Bosquillon, C. Lombry, V. Prat, R. Vanbever, Inuence of formulation excipi-
ents and physical characteristics of inhalation dry powders on their aerosoliza-
tion performance, Journal of Controlled Release 70 (2007) 329339.
[25] R. Vanbever, J.D. Mintzes, J. Wang, J. Nice, D. Chen, R. Batycky, R. Langer, D.
Edwards, Formulation and physical characterization of large porous particles for
inhalation, Pharmaceutical Research 16 (1999) 17351741.
[26] K. Vay, S. Scheler, W. Frie, New insight into the pore structure of poly(D,
L-lactide-co-glycolide) microspheres, International Journal of Pharmaceutics 402
(2010) 2026.
[27] J.B. Brubach, V. Janin, B. Mahler, C. Bourgaux, P. Lessieur, P. Roy, M. Ollivon, Struc-
tural and thermal characterization of glyceryl behenate by X-ray diffraction
coupled to differential calorimetry and infrared spectroscopy, International Jour-
nal of Pharmaceutics 336 (2007) 248256.
[28] S. Bresson, M. El Marssi, B. Khelifa, Raman spectroscopy investigation of various
saturated monoacid triglycerides, Chemistry and Physics of Lipids 134 (2005)
119129.
[29] C. Freitas, R.H. Mller, Correlation between long-term stability of solid lipid
nanoparticles (SLN) and crystallinity of the lipid phase, European Journal of
Pharmaceutics and Biopharmaceutics 47 (1999) 125132.
[30] H. Ali, K. El-Sayed, P.W. Sylvester, S. Nazzal, Molecular interaction and localization
of tocotrienol-rich fraction (TRF) within the matrices of lipid nanoparticles: evi-
dence studies by Differential Scanning Calorimetry (DSC) and Proton Nuclear
Magnetic Resonance spectroscopy (1H NMR), Colloids and Surfaces. B,
Biointerfaces 77 (2010) 286297.
[31] A.A. Pawar, D.-R. Chen, C. Venkataraman, Inuence of precursor solvent proper-
ties on matrix crystallinity and drug release rates from nanoparticles aerosol
lipid matrices, International Journal of Pharmaceutics 430 (2012) 228237.
[32] V. Jenning, S. Gohla, Comparison of wax and glyceride solid lipid nanoparticles
(SLN), International Journal of Pharmaceutics 196 (2000) 219222.
[33] J. Siepmann, F. Siepmann, Mathematical modeling of drug delivery, International
Journal of Pharmaceutics 364 (2008) 328343.
[34] S.S. D'Souza, P.P. DeLuca, Methods to assess in vitro drug release from injectable
polymeric particulate systems, Pharmaceutical Research 23 (2006) 460474.
[35] M.M.A. Abdel-Mottaled, A. Lamprech, Standardized in vitro drug release test for
colloidal drug carriers using modied USP dissolution apparatus I, Drug Develop-
ment and Industrial Pharmacy 37 (2011) 178184.
[36] G. Taylor, I. Kellaway, in: A. Hillery, A.W. Lloyd, J. Swarbrick (Eds.), Pulmonary
Drug Delivery, Taylor & Francis, London, 2001, pp. 269272.