Journal of Drug Delivery Science and Technology 29 (2015) 152e158

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Journal of Drug Delivery Science and Technology

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

Research paper

Inhalable resveratrol microparticles produced by vibrational

atomization spray drying for treating pulmonary arterial hypertension
Frantiescoli A. Dimer a, *, Manoel Ortiz a, Adriana R. Pohlmann a, b, Silvia S. Guterres a
a
Programa de Po ~o em Ci^

s-Graduaça encias Farmac^
cia, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil
euticas, Faculdade de Farma
b
Departamento de Química Orga ^nica and Programa de Po

s-Graduaça~o em Química, Instituto de Química, Universidade Federal do Rio Grande do Sul, Porto
Alegre, RS, Brazil

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

Article history: The rare disorder pulmonary arterial hypertension is characterized by elevated pulmonary arterial
Received 30 May 2015 pressure and right heart failure. Currently, the treatments have improved, but a cure has not yet been
Received in revised form discovered. In this study, we developed a new formulation using an innovative controlled delivery
20 June 2015
system for resveratrol: s dry powder inhaler form designed for pulmonary administration. The particles
Accepted 14 July 2015
Available online 15 July 2015
were composed of resveratrol, as the therapeutic agent; poly(ε-caprolactone), for structuring the
controlled-release matrix; sodium deoxycholate, to prevent agglomeration; and trehalose, as drying
adjuvant, respectively. The particles were obtained using the piezoelectric atomization technique with a
Keywords:
Dry powder inhaler
Nano Spray Dryer B-90®. The fine particle fraction was approximately 50% and the theoretical aero-
Pulmonary arterial hypertension dynamic diameter was 2.32 mm. The process afforded excellent yields (approximately 80%) with low
Resveratrol powder moisture (less than 2.0%). Due to the low density and the increased flowability of the powder, the
Spray dryer spherical shape of the particles and an irregular surface, the microparticles possessed aerodynamic
Vibrational atomization properties suitable for drug deposition on the bronchial and alveolar regions of the lungs. The aero-
dynamic properties and in vitro sustained release profiles showed a great potential for the inhaled
administration of drugs such as resveratrol suitable for the treatment of pulmonary arterial hypertension.
© 2015 Elsevier B.V. All rights reserved.

1. Introduction effects, such as breathlessness, liver damage, nausea, diarrhea and

Pulmonary arterial hypertension (PAH) is characterized by the
pressure of the lungs exceeding 25 mm Hg at rest or 30 mm Hg
during exercise. The major symptoms of PAH are chest pain, fatigue
and shortness of breath. The development of this disorder is
associated with deregulation of the vasodilator prostaglandin
(PGI2) and the vasoconstrictor thromboxane (TXA2) [1,2]. The
treatment for PAHuses single or multidrug therapies, such as an-
ticoagulants, diuretics, calcium channel blockers and prostanoids
[3]. However, the use of these treatments does not cure of this
disease, but these treatments can retard its progression. Addition-
ally, this conventional treatment approach mainly uses mainly the
oral and intravenous routes and can induce important systemic side
* Corresponding author. Programa de Po
s-Graduaça~o em Cie
^ncias Farmace^uticas,
Faculdade de Farma
cia, Universidade Federal do Rio Grande do Sul, Av. Ipiranga
2752, Porto Alegre CEP 90610-000, RS, Brazil.
E-mail addresses: fdimer1@gmail.com (F.A. Dimer), manoel.ortiz@hotmail.com
(M. Ortiz), pohlmann@iq.ufrgs.br (A.R. Pohlmann), silvia.guterres@ufrgs.br
(S.S. Guterres).
pain, leading to limited treatment adherence by patients [2].
Alternatively, the administration of drugs through the inhalation
route is quite interesting because it is possible to obtain increased
bioavailability compared to oral administration. In addition, it is
patient friendly because it is painless and not as invasive as the
intravenous route [4]. The conventional treatments for PAH using
pulmonary administration have already been approved in some
countries with the use of Iloprost [5]. This drug can reduce the
mean pulmonary arterial pressure by up to 20% for less than 1 h.
Consequently, more than 10 inhalations per day are required to
maintain the effect of Iloprost using nebulizers systems [6].
A potential approach to improve the treatment is to administer
the drug loaded within a polymeric carrier to control the release of
the drug and to reduce the number of daily administrations [7].
These carriers, such as microparticles, can also impart the drugs
with great properties, such as increased bioavailability [8] and drug
stability [9], and they can deliver the drugs to specific targets [10].
Using a technology recently developed by Buchi Labortechnik, it is
possible to obtain dry powders with an aerodynamic diameter
between 1 and 5 mm to be efficiently deposited in the lower

http://dx.doi.org/10.1016/j.jddst.2015.07.008
1773-2247/© 2015 Elsevier B.V. All rights reserved.
 F.A. Dimer et al. / Journal of Drug Delivery Science and Technology 29 (2015) 152e158
respiratory tract [11,12]. The Nano Spray Dryer B-90® is a new
generation of laboratory-scale spray dryers, and it brings im-
provements compared to conventional spray dryers, such as better
yields and powders with reduced particle sizes [12e15].
The achievement of submicrometric and micrometric particles
using the Nano Spray Dryer B-90 was facilitated by the piezoelectric
atomization technique, which enables the production of small
particles directly from solutions or suspensions with high yields
when compared with the atomization process in the conventional
spray drying technique [12,16]. This piezoelectric system operates
by ultrasonically vibrating a membrane that contains tiny holes,
forming a mesh, at a frequency of 60 kHz. Moreover, a micro-pump
action is created inside the atomizer, due to the vibration of the
membrane, which generates millions of droplets with a narrow
particle size distribution [17].
Resveratrol (3,5,40-trans-trihydroxystilbene) is also currently
used to treat PAH because it can inhibit the production of monocyte
chemoattractant protein-1 (MCP-1) and its secretion in vascular
endothelial cells, which is a key intermediary factor that stimulates
the infiltration of inflammatory cells into the lungs [18,19]. In
addition, this drug which is present in grapes and red wine, has
attracted considerable interest from pharmaceutical researchers
because of ots excellent metabolic properties, such as its powerful
antioxidant, anti-inflammatory, lipid metabolism regulator activ-
ities and its ability to prevent cancers activities [20e22].
In this context, this work was focused on the development of
polymeric microparticles containing resveratrol intended for pul-
monary delivery using the Nano Spray Dryer aiming a controlled
released formulation with aerodynamic properties suitable for drug
deposition on the lungs.
2. Materials and methods
2.1. Materials
Resveratrol (RSV) was purchased from Deg (Sa ~o Paulo, Brazil),
poly(ε-caprolactone) (Mw 42.500 g mol
1) and sodium deoxy-
cholate were acquired from SigmaeAldrich (Steinheim, Germany).
Trehalose was kindly donated by Corn Products (Mogi Guaçu,
Brazil). The analytical-grade solvent acetone and the high-
performance liquid chromatography (HPLC)-grade solvent meth-
anol were purchased from F. Maia (Cotia, Brazil) and Tedia (Rio de
Janeiro, Brazil), respectively.
2.2. Preparation of microparticles
Initially, 0.1 g of poly(ε-caprolactone) and 0.1 g of RSV were
dissolved in 80 mL of acetone in a single flask at 40 ± 2  C with
controlled magnetic stirring at 300 rpm. Separately, 0.02 g of so-
dium deoxycholate and 0.1 g of trehalose were solubilized in 20 mL
of ultrapure water. After both phases were completely dissolved,
the aqueous phase was added to the organic phase under moderate
magnetic stirring. The suspension was loaded in a Nano Spray Dryer
B-90 (Büchi Labortechnik AG, Switzerland), using the following
drying parameters: pump mode 2 with 100% spray rate, air flow of
110 L/min, inlet temperature of 55  C and the spray mesh with a
pore size of 7.0 mm. The spray dryer was maintained in closed-mode
configuration with a residual oxygen level of less than 4%. Resver-
atrol microparticles (RSV-MP) were collected from the particle-
collecting electrode using a soft brush.
2.3. Spray-dried powder characterization
Particle size distribution of RSV-MP was determined using the
laser diffraction (LD) technique without any prior powder
153
treatment using Mastersizer 2000 (Malvern Instruments, Ger-
many) and the small volume wet dispersion unit. The particle size
of RSV-MP was measured immediately after the addition of the
particles with an obscuration level higher than 2%. The particle size
parameters were obtained directly from the software, a follows:
D4,3; d0,1; d0,5 and d0,9 representing the volume-weighted mean
and lower sizes of 10, 50 and 90% of the particles, respectively.
The process yield was calculated from the relation of the
resulting powder amount obtained by the initial concentration of
solids in the solution. The results were expressed in percentage.
Moreover, the residual moisture content of the powders was
analyzed using Karl Fischer titration (Mettler Toledo 37, Japan).
The bulk (rb) and tapped (rt) densities of the spray-dried
powders were determined in triplicate using an automatic tapper
(J. Engelshmann, Germany). After recording the bulk density, the
tapped density was determined by visual inspection after 1250
taps. This number of taps allowed the density to reach a plateau. An
indicator of the flowability of the samples, the Carr's index per-
centage (CI) was determined using the Equation (1). In addition, the
theoretical aerodynamic diameter (daero) of the particles was
calculated from the particle size determined by laser diffraction
(dgeo) and the tapped density (rt) using the Equation (2) [23].
rt
rb
Carr 0 s Indexð%Þ ¼  100 (1)
rb
sffiffiffiffiffiffiffiffiffiffiffiffi
 
rt
daero ¼ dgeo (2)
r0
where, r0 ¼ 1 g cm
3 (spheric shape).
Morphological analyses of the polymeric microparticles were
performed using a scanning electron microscope (SEM) (Zeiss EVO
HD15, Germany). Samples of raw resveratrol and RSV-MP were
added in aluminum stubs with carbon conductive double-sided
tape and sputter-coated with a 15e20 nm layer of gold. The ana-
lyses were conducted using an accelerating voltage of 5 kV at
magnifications of 2000 and 20,000.
The resveratrol content was assayed using high-performance
liquid chromatography (HPLC) according to a previously validated
method [9]. The chromatographic system consisted of a Discovery
C18, 5 mm, 4.6  150 mm column (Supelco, USA) and a Per-
kineElmer series 200 chromatograph (PerkineElmer, USA). The
isocratic mobile phase was composed of methanol:water (50:50, v/
v) at pH 3.0 adjusted with acetic acid, which was pumped at a flow
rate of 0.6 mL/min. The sample volume injected was 50 mL, and RSV
was detected using a wavelength of 305 nm. The HPLC showed
linearity in the range of 0.5e20 mg/mL (R2 ¼ 0.9845). The total
amount of RSV from the DPI was extracted from 20 mg of RSV-MP
with 10 mL of mobile phase of methanol:water (50:50, v/v) after
10 min of ultrasonic extraction and subsequent centrifugation at
1.500 g. In addition, 250 mL of the samples was diluted using the
mobile phase. The samples were filtered (Millipore 0.45 mm, USA)
and injected into the HPLC.
The crystallinity and the compatibility of resveratrol with other
components of the particles were characterized using a differential
scanning calorimeter (DSC e TA Instruments, model Q100, USA)
equipped with a refrigerated cooling accessory. Nitrogen was used
as the purge gas was used at a rate of 10 mL/min. The specimens
(3e7 mg) were packed in hermetic aluminum pans and heated
from 20 to 300  C at a heating rate of 10 ºC/min under a dry nitrogen
atmosphere. The DSC heating curves were analyzed using Universal
Analysis 2000 software (TA Instruments, USA).
154 F.A. Dimer et al. / Journal of Drug Delivery Science and Technology 29 (2015) 152e158
2.4. In vitro aerosol performance
The in vitro aerosol performance of RSV-MP was investigated
using the USP pharmacopeia apparatus 1, called the Andersen
cascade impactor (ACI, Type ACI-MDI 1000 Erweka, Germany) [24].
Initially, the inhalation rate generated by the vacuum pump (VP
1000, Erweka, Germany) was adjusted and calibrated to 28.3 ± 1 L/
min during 4 s after five inhalations of empty capsules. The analyses
(n ¼ 3) were performed using a gelatin capsule (size n 3) filled
with 20 mg of the RSV-MP powder. ACI consists of eight stages, and
it is composed of a first part called the throat and eight subsequent
stages (0e7). After inhalation using the Aerolizer® (Novartis,
Switzerland) as the inhaler device in the mouthpiece adaptor, the
powders were impacted into the different stages, according to their
aerodynamic diameters [24]. The particles that were retained in the
inhalation device, mouthpiece adaptor, throat device and each
stage from zero to seven were rinsed into volumetric flasks using
the HPLC mobile phase. The amount of RSV in each step was
determined using the previously validated HPLC method. The fine
particle fraction (FPF; less than 5.8 mm) was calculated from the
sum of the stages 2e7 of the ACI. In addition, the mass median
aerodynamic diameter (MMAD) was determined. MMAD is defined
as the diameter above and below where 50% of the mass of the
particles lie.
2.5. In vitro dissolution profile
The dissolution of free RSV and the drug release of RSV from
RSV-MP formulations (n ¼ 4) were performed using in a Vankel VK
7010 automatic sampling dissolution system (Vankel, United
Kingdom). Approximately 30 mg of the RSV-MP powder (contain-
ing approximately 9 mg of RSV) or the same amount (9 mg) of bulk
Resveratrol (RSV-FREE) was loaded in a gelatin capsule (size n 3).
Sink conditions were maintained following the conditions
employed: baskets, speed of 75 rpm, 900 mL of an aqueous medium
containing 1% of polysorbate 80 and controlled temperature of
37 ± 1  C. Samples (5 mL) were automatically withdrawn at pre-
determined time points of 5, 10, 15, 20, 25, 30, 45, 60, 120, 240, 720
and 1440 min and filtered through a 0.45 mm filter. Prior to HPLC
analysis, the samples were diluted with 5 mL of mobile phase. The
apparent kinetic rate constants for both formulations were ob-
tained from the release profiles fitted according to the first-order
(C ¼ C0$e
kt) mathematical model using the software Micro-
Math Scientist® 2.0 (Missouri, USA), where k and C0 are the
apparent kinetic rate constant and the initial drug concentration at
time t (h). Additionally, we used a model-independent model by
using the statistical moment of the mean dissolution time (MDT),
which is commonly used to describe the in vitro drug release profile
for the dissolution of controlled release products [25]. Statistical
analysis of the in vitro data was performed via t-test using Graph-
Pad Prism 5 software (GraphPad Software, USA) and a value of
p < 0.05 was considered significant.
3. Results and discussion
The delivery of drugs to the lungs using the inhalation therapy
for the local treatment of diseases could decrease the side effects
because lower systemic drug concentrations are reached compared
to oral delivery. In this work, microparticles were developed as dry
powder inhalers (DPI) systems intended for pulmonary application,
which were composed of PCL, sodium deoxycholate and trehalose
and loaded with Resveratrol using a Nano Spray Dryer B-90®. Using
this new formulation, the aim was to target RSV to the lungs,
leading to an increased bioavailability in the tissue, yield and FPF of
the particles. Thus, inhalable RSV-MP was prepared by means of
spray drying using the vibrational atomization technique.
One of the disadvantages of using Nano Spray Dryer B-90® is the
presence of incrustations at the nozzle, and thus, an optimization
process is always required [12]. Initially, the drying process used in
this work also presented this problem. In order to produce RSV-MP
powder formulation without incrustations, the process was opti-
mized by decreasing the inlet temperature from 70  C to 55  C,
reducing the solids contents of the feeding solution from 1.0% to
0.3%, and modifying the proportions acetone:water from 50:50 to
80:20 (v/v). After all changes, the RSV-MP powder formulation was
prepared without incrustations.
For the purposes of this study, a poly(ε-caprolactone) polymer
with a medium molecular weight (42.5 kDa) was used as a shell
structure to control the drug release rate. This water-insoluble
polymer is well known as a matrix for nano- or microparticle for-
mulations because of its biodegradability and biocompatibility
properties [26]. Acetone was selected as a solvent due to its high
capacity to dissolve both the polymer and the drug. The solvent is a
very important as process parameter because it can control the
process temperature, thereby influencing the drug loading. Sodium
deoxycholate was used as a surfactant to avoid particle agglomer-
ation and to increase the redispersion of the microparticles in the
airstream, which also increased the flowability because the liquid/
air interface of the droplets formed during the atomization process
prior to drying should preferably be occupied by a surfactant rather
than drug or polymers [27]. Trehalose was used as a drying adju-
vant. This carbohydrate is readily soluble in water and is widely
used in inhalation preparations [28,29]. In addition to the materials
used, the integrity of the polymeric particles is dependent of the
different steps of the spray drying process: formulation feed and
atomization, spray mixture with air drying, solvent evaporation,
and dry product separation [30].
One of the mainly problems in the spray drying process is the
high aggregation and fusion of the particles resulting from the
temperature used being considerably higher than the melting point
(Tm). However, the Nano Spray Dryer B-90 uses lower tempera-
tures compared to conventional spray dryers, facilitating the drying
process of particles [17]. The inlet temperature used was 55  C and
below the Tm of the polymer poly(ε-caprolactone) (60  C) [26].
During the few seconds of the drying step, the temperatures of the
atomized droplets and obtained powder remained lower than the
temperature of the circulating air. Therefore, it is possible to obtain
powders without directly exposing the materials to high temper-
atures, thereby allowing for the production of particles containing
sensitive materials without causing degradation [31]. Moreover,
formulation components self-assembled to form the structure of
the particles at this stage [32].
Most particles were obtained in the designed inhaled formula-
tion, with particle sizes ranging from 1 to 5 mm for their maximum
efficiency deposition in the deepest lung [11,33]. Although particles
larger than 5 mm would be deposited in the throat and easily
cleared, the smaller particles above 0.5 mm may not be effective due
to the Brownian motion in the airstream and can be exhaled during
breathing [2,24]. Using the laser diffraction technique, the volume-
weighted mean (D4,3) of RSV-MP was determined to be approxi-
mately 3.8 mm; additionally 10, 50 and 90% of the particles pre-
sented particle sizes of less than 0.7, 2.0 and 9.5 mm, respectively,
based on the volume distribution (Fig. 1A). When the number
distribution was used, 10, 50 and 90% of the particles presented
particle sizes lower than 0.4, 0.6 and 1.1 mm, respectively (Fig. 1B).
Therefore, the vast majority of the particles were in the respirable
range.
The bulk density, tapped density and CI of the RSV-MP spray-
dried powders are listed in Table 1. The obtained bulk and tapped
densities were 0.32 g/cm3 and 0.37 g/cm3, respectively. The
 F.A. Dimer et al. / Journal of Drug Delivery Science and Technology 29 (2015) 152e158
Fig. 1. Particle size distribution of RSV-MP as measured by laser diffractometry using volume (A) and number distributions (B).
Table 1
Technological parameters of spray-dried powder of resveratrol sub-
micrometric particles.
Technological parameter Value (±SD)
rb (g/cm3) 0.32 (0.01)
rt (g/cm3) 0.37 (0.02)
CI (%) 17.29 (5.41)
daero (mm) 2.32 (0.17)
Yield (%) 79.5 (8.3)
Residual moisture (%) 1.84 (0.25)
Drug content (%) 31.01 (0.12)
FPF (%) 46.48 (1.25)
MMAD (mm) 5.22 (0.3)
rb ¼ bulk density; rt ¼ tap density; CI ¼ Carr Index;
daero ¼ aerodynamic diameter; FPF ¼ fine particle fraction;
MMAD ¼ mass median aerodynamic diameter.
calculated aerodynamic diameter was approximately 2.3 mm. As
previously reported, spray-dried particles with densities of less
than 0.4 g/cm3 and with a mean volume diameter of less than 5 mm
are suitable for pulmonary administration and would be capable of
delivering drugs to the deep lung for providing a prolonged resi-
dence time in the alveolar region [34]. The powder flowability
directly affects directly the dry powder inhalation performance and
can be obtained from the CI value. CI values less than 25% indicates
a powder with good flowability, and values greater than 25% in-
dicates cohesive powder characteristics, corresponding to poor
flowability [13]. The CI value of RSV-MP was approximately 17%,
indicating a fluidity powder.
Yields of approximately 50% are typically achieved in spray
drying processes; however, a higher yield (approximately 80%) was
obtained for RSV-MP (Table 1). The increase in the yield with the
Nano Spray Dryer B-90 is due to the electrostatic particle collector
rather than to the cyclone collector in the conventional spray
drying technique [12]. The electrostatic collection separation
method consist of a stainless steel outer cylinder (anode) and a star-
shaped inner part (cathode). After a high voltage is passed between
the electrodes, an electrostatic potential difference is created,
which attracts the produced particles. In addition, our research
group previously demonstrated that the addition of surfactant fa-
cilitates the passage of liquid through the membrane pores and that
the use of ionic surfactants promotes a greater attraction of the
particles to the collector cylinder, and consequently increasing the
process yield [17].
The residual moisture content in RSV-MP was less than 2%,
indicating that the drying process was efficient [35], even at the low
drying temperature used (55  C). It is well-known that a high
moisture content or increased moisture sorption by powders can
affect the chemical, physico-chemical and microbial stability of
powders [36]. Note that these biodegradable microparticles must
155
be stored under lower moisture conditions to avoid potential
polymer degradation through hydrolysis and agglomeration during
storage [36]. The drug content determined by HPLC in the obtained
powder was 31.01 ± 0.12%. This value corresponds to 99.24% of the
theoretical drug content (312.5 mg/g).
Morphological analyses of the dry powders were performed
using SEM to verify their morphology and particle size. The inhal-
able microparticles consisted of mainly spherical particles with an
irregular surfaces (Fig. 2A and B), characteristic of amorphous
substances obtained by spray drying [37]. The surface asperities can
reduce the van der Waals forces between the particles, thereby
improving powder flow, fluidization, and dispersibility [38].
Regarding the irregular surface of the particles, this is an important
characteristic because it results in an increased superficial area
between the airstream and the particles surface, and it can also
improve the flowability as well. In addition, submicrometric par-
ticles sizes can be observed, supporting the data obtained from the
laser diffraction analysis. From the SEM micrograph, It is also
possible to observe that the particles are partially agglomerated
into a single structure, but these submicrometric particles can
readily redisperse in aqueous media.
The thermal properties of the submicrometric particles deter-
mined via DSC analysis are useful for verifying interactions be-
tween components within a range of temperatures. As observed
(Fig. 3), the resveratrol melting temperature (268.9  C) is similar to
that reported in a previous study (265.9  C) [39]. The DSC curve of
RSV-MP did not show characteristic peaks of the drug, polymer or
drying adjuvant, suggesting a complex interaction between the
components.
The aerodynamic behavior of the particles is the main param-
eter that influences their deposition in the lungs. Whereas the
larger particles are deposited mainly in the bronchial region, the
smaller particles can reach the deepest parts of the lung (the
alveolar region). The size of the microparticles is determined by the
formulation and the spray-drying process parameters, such as the
solvents used, the concentration of solids and mainly by the aper-
ture size of the spray mesh [14]. In the study of the aerosol prop-
erties, about 85% of the RSV in the RSV-MP was emitted from the
capsules (Fig. 4). This emitted dose met the European pharmaco-
poeia specification, which should be greater than 75% of the loaded
dose. In addition, the fraction of powder that can be deposited in
the lowers parts of the lungs (equivalent to the stages 2e7, FPF)
represented by the particle size of the RSV-MP less than 5.8 mm in
size (in these experimental parameters) was 46.48 ± 1.25. This FPF
value is in accordance with conventional dry powder inhaler for-
mulations [2,33]. Moreover, the MMAD of the RSV-MP obtained
using the ACI was 5.22 ± 0.3 mm. This value is slightly greater than
the daero possibly due to some individual particles behaving as
aggregates after the aerosolization. This specific MMAD is in the
ideal range for PAH treatment using the inhalation therapy because
156 F.A. Dimer et al. / Journal of Drug Delivery Science and Technology 29 (2015) 152e158
Fig. 2. Scanning electron microscopy images of resveratrol submicrometric particles at 2000 (A) and 20,000 (B).
Fig. 3. DSC heating curves of: Resveratrol, PCL (poly(ε-caprolactone)), trehalose and
RSV-MP (resveratrol microparticles).
the small diameter of the particles ensure the alveolar deposition
[3]. Therefore, the microparticles loaded in hard gelatin capsules
are suitable for use in commercially available DPIs, such as Aerol-
izer®, allowing targeted pulmonary administration of resveratrol,
and they could represent an effective treatment for PAH. The
spherical shape of these inhalable particles is appropriate for
Fig. 4. In vitro inhalation performance of resveratrol microparticles using Andersen
cascade impactor. C, T and FPF represent the capsule stage, throat stage and the sum of
stages 2e7, respectively. Each point represents the mean ± S.D. (n ¼ 3).
pulmonary delivery because it can improve the impaction and
diffusion phenomena's in the respiratory tract. In addition to the
particle morphology, the irregular surface of RSV-MP is responsible
for the improved aerosol performance of the particles when
compared to particles with a smooth surface. With all of these
important characteristics associated with the very small and
controlled aerodynamic particle size (3 mm) and high FPF, these
particles can reach in the deepest part of the lungs, thereby
improving the PAH treatment [38].
To evaluate the changes in the solubility and dissolution rate of
RSV-MP obtained through the use of the Nano Spray Dryer B-90®
compared to the RSV raw material in bulk form, dissolution in vitro
studies were conducted using a dissolution system with a basket
apparatus (Fig. 5). After 30 min, more than 80% of RSV-FREE had
already solubilized in the medium. Moreover, RSV-MP released
only 25% in the same time period. The release was controlled up to
720 min, and more than 85% of RSV was released in this period. This
drug release of RSV from the microparticles is consistent with other
spray-dryed powders for different drugs [21,26]. An empirical first-
order model was used to fit the drug release data to compare both
formulations. In this model, the amounts of drug released are
directly proportional to the amount remaining in the dosage form
following a concentration gradient pattern based on Fick's law [40].
Fig. 5. The in vitro cumulative release profiles of resveratrol microparticles (RSV-MP)
and the free drug (RSV-FREE) (n ¼ 4).
 F.A. Dimer et al. / Journal of Drug Delivery Science and Technology 29 (2015) 152e158
By applying this model, the first-order rate constant (K) and the
time at which 50% of the drug is dissolved (T50) were obtained. For
RSV-FREE, the values of K and T50 were 3.67 ± 0.67 h
1 and
0.19 ± 0.04 h, respectively, whereas for RSV-MP, the values of K and
T50 were 0.30 ± 0.04 h
1 and 2.34 ± 0.35 h, respectively. Addi-
tionally, the MDT values were 0.214 ± 0.03 h and 3.41 ± 0.4 h for
RSV-FREE and RSV-MP, respectively. By assessing the release
through both dependent models as with the independent model, a
significant controlled release of Resveratrol from the microparticles
was obtained. The controlled release of RSV-MP represented a
suitable dose of the drug at the site of action because the first times
with the slight burst released resveratrol, and a sustained dose was
released during the evaluated period. Despite the need for further
studies on the drug release, it could be possible to administer RSV-
MP using inhalation once to three times per day for the treatment
of PAH, which is considerably much less than the actual treatment.
Although the formulation presented many qualities and advantages
in vitro, further in vivo studies should be performed to confirm the
potential of the developed formulation.
4. Conclusions
In this paper, we produced a microtechnology-based powder by
vibrational atomization spray-drying capable to control the
resveratrol release from the microparticles. This new dry inhalable
formulation containing resveratrol was produced with aero-
dynamic particle properties suitable to deposition in the deep lung.
The results open the perspective to further investigation towards
the biological evaluation of the new formulation, as well as to apply
this microtechnology as a platform to produce other drug-loaded
powders intended for pulmonary administration.
Conflict of interest
The authors declare no competing financial interest.
Acknowledgments
These studies were supported by the following Brazilian
agencies: Conselho Nacional de Desenvolvimento Científico e Tec-
nolo ~o de Aperfeiçoamento de Pessoal de Nível

gico (CNPq), Coordenaça
Superior (Capes) and Fundaça~o de Amparo a Pesquisa do Estado do
Rio Grande do Sul (FAPERGS).
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