Photostability and skin penetration of different E-resveratrol-loaded supramolecular structures

Article abstract of DOI:10.1111/j.1751-1097.2012.01147.x

It is desirable and challenging to prevent E-resveratrol (E-RSV) from photoisomerizing to its Z-configuration to preserve its biological and pharmacological activities. The aim of this research was to evaluate the photostability of E-RSV-loaded supramolec

Full text of DOI:10.1111/j.1751-1097.2012.01147.x

Photochemistry and Photobiology, 2012, 88: 913–921
Photostability and Skin Penetration of Different E-Resveratrol-Loaded
Supramolecular Structures
Cassia Britto Detoni1, Gabriele Dadalt Souto2, Ana Luiza Maurer da Silva1, Adriana Raffin Pohlmann1,3
and Silvia Stanisuaski Guterres*1,2
1
´
ˆ
ˆ
Programa de Pos-graduac¸a˜o em Ciencias Farmaceuticas, Universidade Federal do Rio Grande do Sul,
Porto Alegre, RS, Brazil
2
´
Programa de Pos-Graduac¸a˜o em Nanotecnologia Farmaceutica, Universidade Federal do Rio Grande do
Sul, Porto Alegre, RS, Brazil
ˆ
3
´
ˆ
Departamento de Quımica Organica, Instituto de Quimica, Universidade Federal do Rio Grande do Sul,
Porto Alegre, RS, Brazil
Received 16 November 2011, accepted 15 March 2012, DOI: 10.1111/j.1751-1097.2012.01147.x
indicated that E-RSV is capable of treating melanoma by
inducing cellular apoptosis (7).
ABSTRACT
It is desirable and challenging to prevent E-resveratrol (E-RSV)
from photoisomerizing to its Z-configuration to preserve its
biological and pharmacological activities. The aim of this
research was to evaluate the photostability of E-RSV-loaded
supramolecular structures and the skin penetration profile of
chemically and physically stable nanoestructured formulations.
Different supramolecular structures were developed to act as
carriers for E-RSV, that is, liposomes, polymeric lipid-core
nanocapsules and nanospheres and solid lipid nanoparticles. The
degrees of photostability of these formulations were compared
with that of an ethanolic solution of E-RSV. The skin
penetration profiles of the stable formulations were obtained
using vertical diffusion cells (protected from light and under
UVA radiation) with porcine skin as the membrane, followed by
tape stripping and separation of the viable epidermis and dermis
in a heated water bath. Photoisomerization was significantly
delayed by the association of resveratrol with the nanocarriers
independently of the supramolecular structure. Liposomes were
the particles capable of maintaining E-RSV concentration for
the longest time. On the other hand, E-RSV-loaded liposomes
reduced in size showing low physical stability under UVA
radiation. In the dark, the skin penetration profiles were very
similar, but under UVA radiation the E-RSV-loaded nano-
carriers showed increasing amounts in the total epidermis.
Topical application is a route of administration that greatly
exposes the formulation to sunlight and unfortunately E-RSV
is susceptible to UV-induced isomerization, and it is almost
completely converted to the less active Z conformation by
UVA radiation (1). Studies on the structure-activity relation-
ship of E-RSV show that the presence of a hydroxyl group at
position 4 (4–OH), as well as the E stereoisomer configuration,
are required for the inhibition of cell proliferation and a high
antioxidant activity (2). Thus, it is worthwhile and challenging
to prevent RSV from isomerizing to preserve its biological and
pharmacological activities.
Recently, nanocapsules have been proposed as a strategy
to overcome inactivation and ⁄ or degradation of photosensi-
tive drugs (8–11). The materials and designs used to build
nanostructures capable of protecting biologically active mol-
ecules from light vary immensely. Among the possibilities for
nanostructured carriers there is
a preference for those
prepared with biodegradable materials such as lipids or
biodegradable polymers. The supramolecular structures of
nanoparticles may vary, having a compartmented structure
(core and wall) or a matrix structure (12). Of the biodegrad-
able nanostructured carriers described to date, the most
studied supramolecular structures are the polymeric nano-
particles (nanocapsules and nanospheres), lipid nanoparticles
and liposomes (13,14).
Polymeric nanoparticles are known to stabilize photolabile
drugs by providing physical protection from UV radiation (8).
The particles containing a mixture of medium-chain triglyce-
rides and sorbitan monostearate in their core, named lipid-core
nanocapsules, have exhibited the properties of great stability
and high drug association efficiency for different drugs
intended for administration by various routes (15). Indeed,
lipid-core nanocapsules have exhibited a good photostabiliza-
tion capacity for the sunscreens octyl methoxycinnamate and
benzophenone-3 as well as the polyphenol quercetin (8,16).
Furthermore, the proposed E-RSV lipid-core nanocapsules
have already proven their pharmacological potential, by
exhibiting higher gastrointestinal safety and better tissue
distribution after oral administration than free E-RSV (17).
INTRODUCTION
Resveratrol (RSV; 3,5,4-trihydroxystilbene) is a naturally
occurring nonflavonoid polyphenolic compound presenting
Z- and E-isomeric forms (1,2). This substance has received
considerable interest from the scientific community in recent
years as a potential chemopreventive agent for skin cancer (3).
Studies have shown that topical application of E-resveratrol
(E-RSV) can prevent in vivo UV-induced skin damage and
chemical-induced skin cancer (4–6). An in vitro study also
*Corresponding author email: silvia.guterres@ufrgs.br (Silvia Stanisuaski
Guterres)
ꢀ 2012 Wiley Periodicals, Inc.
Photochemistry and Photobiology ꢀ 2012 The American Society of Photobiology 0031-8655/12
913

914 Cassia Britto Detoni et al.
Other lipid-based nanoparticles are also capable of photo-
stabilizing drugs. For instance, liposomes are an extensively
studied photoprotective carrier system and successful results
have been reported in many studies (11,18). Phosphatidyl-
profile of a chemically and physically stable E-RSV-loaded
colloidal formulation was evaluated and compared with that
of the free molecule, aiming at its retention in the upper layers
of the skin.
choline liposomes have been applied as
a vehicle for
tretinoin, increasing its photostability and reducing its
photoisomerization. The results indicated that higher drug
inclusion in liposomes leads to a higher photostabilization
(11). Solid lipid nanoparticles and nanostructured lipid
carriers have appeared as an alternative to liposomes due
to improved physical stability and low cost compared with
phospholipids (19) and have also been successfully used to
photostabilize tretinoin (20). Lipid-based nanoparticles may
be prepared using the high-pressure homogenization tech-
nique, which allows the pharmaceutical industry to obtain
liposomes and nanostructured lipid carriers in large-scale
production (19,21).
MATERIALS AND METHODS
Solvents and reagents. Saturated soy phosphatidylcholine S 75-3 was
donated by Lipoid (Ludwigshafen, Germany). Lipid glyceryl dibehe-
nate (Compritol 888ꢁ) was purchased from Brasquim (Porto Alegre,
Brazil); coconut fat, medium-chain triglycerides and E-RSV were from
Cosmetrade (Porto Alegre, Brazil); sorbitan monooleate from Oxiteno
(Maua, SP, Brazil) and polysorbate 80 from Vetec (Rio de Janeiro,
Brazil). Poly (e-caprolactone; Mw = 65 000 gÆmol)1) and sorbitan
monoestearate were acquired from Sigma–Aldrich (Steinheim, Ger-
many). The analytical grade solvent chloroform was purchased from
Labsynth (Diadema, Brazil) and acetone from F. Maia (Cotia, Brazil).
The high-performance liquid chromatography (HPLC) grade solvents
acetonitrile and ethanol were obtained from Tedia (Rio de Jeneiro,
Brazil).
Besides maintaining the chemical stability of E-RSV in the
formulation, nanostructured formulations intended for cuta-
neous administration should guarantee its physical stability
following UVA radiation, in terms of mean particle size and
size distribution. Among the chosen carriers, liposomes present
the most dynamic behavior and therefore they are, theoreti-
cally, the least physically stable. On the other hand, polymeric
nanoparticles are known for their high structural stability (22),
and these carriers are frequently preferred due to this
characteristic. Solid lipid nanoparticles and nanostructured
lipid carriers have been developed aiming to aggregate the
benefits of a lipid structure with higher physical stability (19).
Another characteristic that leads to difficulties in relation to
the topical application of E-RSV is that in its nonionized form
(pH 5.0–8.0) this drug has a high permeation and penetration
capacity, although this facilitates its transdermal application
(23). By increasing the retention of E-RSV in the upper layers
of the skin it is possible to increase its topical activity, reducing
loss to the systemic circulation. In this regard, nanocarriers
may improve the skin retention of substances that are designed
to act at the upper layers of the skin, such as sunscreens,
antioxidants and repellents (24,25). A topical chemopreventive
active compound should mostly act in the various layers of the
epidermis where UVA absorption is higher and where most
mutagenesis occurs (26).
Resveratrol skin administration has been tested in different
hidrogel matrices at a concentration of 5% and the highest
permeability coefficient was observed using hydroxyethyl
cellulose (23). The topical administration of RSV in nanocar-
riers has been previously proposed in liposomes and solid lipid
nanocarriers viewing improved chemotherapeutic and chemo-
preventive activities (27,28).
Even though polymer based and lipid nanoparticles have
the potential to protect drugs from photoreactions it is
unknown which kind of supramolecular structure protects
sensitive drugs the most. Taking these considerations into
account, the objective of this study was to prepare different
E-RSV-loaded supramolecular structures with a variety of
materials and architectures, to compare the photostabilizing
capacity of the different structures. Thus, two lipid-based
carriers, liposomes and nanostructured lipid carriers (NLC),
as well as two polymer based structures, lipid-core nanocap-
sules and nanospheres, were studied as potential photostabil-
ity enhancers for E-RSV. In addition, the skin penetration
Particle preparation. Initially, lipid-core nanocapsules, nanospheres,
liposomes and NLC containing E-RSV were prepared. The polymeric
particles were obtained through the precipitation of a preformed
polymer. Briefly, a water miscible organic solvent, in this case acetone,
containing the hydrophobic components was slowly injected into the
aqueous phase containing a hydrophilic surfactant, followed by
solvent elimination under vacuum evaporation at moderate rotation
(Buchi R 114). The volumes of the two phases were 67.5 mL of organic
phase and 132.5 mL of aqueous phase. The final volume, after
evaporation, was 25 mL. The formulation contained the hydrophobic
components; poly(e-caprolactone; 10 mgÆmL)1), sorbitan monoestea-
rate (3.8 mgÆmL)1), E-RSV (1 mgÆmL)1) and, in the case of the lipid-
core nanocapsules, medium-chain triglycerides (33 mgÆmL)1). The
aqueous phase of the formulation contained polysorbate 80
(3.8 mgÆmL)1).
The lipid-core nanocapsule formulation using poly (e-caprolactone)
was based on a previews study (17). To prepare nanosphere the same
materials and proportion were used, except for the liquid oil (medium-
chain triglycerides) that is absent. In addition, these compositions were
equal or very similar to those described in various previews article (8–
10,12,13,15,16).
Liposomes were prepared by the thin lipid film hydration method
followed by high-pressure homogenization (Panda 2K NS1001L, GEA
Niro Soavi). Saturated soy phosphatidylcholine was dissolved with
E-RSV (1 mgÆmL)1) in a mixture of chloroform:methanol (75:25) and
evaporated under reduced pressure. The lipid film was hydrated with
distilled water under sonication. The suspension was then homoge-
nized by high-pressure homogenization. Variations of phosphatidyl-
choline concentration (1.6 and 3.2 mgÆmL)1), homogenization pressure
(180 and 250 bar) and number of cycles (6 and 10) were tested to
obtain a nanosized formulation. The final formulation was chosen
based on the particle size distribution.
The NLC were also prepared by high-pressure homogenization
(Panda 2K NS1001L, GEA Niro Soavi) that followed a pre-emulsion
process at high temperature (70ꢂC). In the hydrophobic phase, the
solid lipids Compritol 888ꢁ (Brasquim; 10 mgÆmL)1) and coconut fat
(0 and 6 mgÆmL)1) were used. Medium-chain triglycerides (26 and
20 mgÆmL)1), sorbitan monooleate (3.8 mgÆmL)1
) and E-RSV
(1.0 mgÆmL)1) were added to the hydrophobic phase which was
melted at 70ꢂC. The aqueous phase contained 3.8 mgÆmL)1 of
polysorbate 80 and was added to the hydrophobic phase to form a
pre-emulsion that was prepared with an UltraTurraxꢁ (24 000 rpm;
IKAꢁ Works, Inc. Sa
˜
o Paulo, Brazil) prior to the high-pressure
homogenization. Variations of coconut oil and medium-chain trigly-
cerides concentration as well as number of cycles (three and six) were
used as preliminary tests to produce a nanosized formulation.
Drug content and association efficiency. The drug content and
association efficiency were determined by a validated HPLC. The
developed method was based on a method previously used by Sapino
et al. (1). A solution of methanol:water (1:1, vol ⁄ vol) at pH 3.0,
adjusted with acetic acid, was used as mobile phase. The column used
˚
was a Luna 5 l C18 (2) 100 A with size of 150 · 4.60 mm (Phenom-
enexꢁ, Torrance, CA). The mobile phase flow was 0.6 mLÆmin)1
,

Photochemistry and Photobiology, 2012, 88 915
wavelength 305 nm and the volume of sample injected was 20 lL. The
method had a squared correlation coefficient (R2) of 0.999 in the range
of 1–20 lgÆmL)1, was precise (with relative standard deviations [SDs]
under 4.0% for all formulations) and accurate (with a recovery of 95–
105% for all formulations). This method is capable of separating E-
and Z-isomers and the retention times are 6.2 and 9.5 min, respec-
tively. The drug content was determined by adding E-RSV loaded
formulations to methanol followed by 5 min of ultrasonication,
filtration and quantitation. To determine the association efficiency,
free E-RSV was separated from the particles by ultrafiltration
centrifugation (Ultrafree-MC 10 000 MW, Merk Millipore, Darm-
stadt, Germany) at 5000 rpm for 10 min at 20ꢂC and the free E-RSV
was quantified.
Particle size distribution. The particle z-average (diameter) and
polydispersity index were determined by dynamic light scattering
(Zetasizer Nanoseries; Malvern Instruments, Worcestershire, UK) at
an angle of 173º. Twenty microliters of the samples was added to
10 mL of Milli Qꢁ water and transferred to polystyrene cuvettes for
each analysis.
%A ꢁ eE ꢁ Co
eZ
¼
ð3Þ
CZ
where eE and ez are the molar extinction coefficients of E- and Z-RSV,
respectivly, %A is the percentual area represented by Z-RSV in the
cromatogram, CZ is the complementary concentration of E-RSV ob-
tained by the HPLC method (Cz = Co ) CE) and Co is the initial
concentration of E-RSV.
The degradation profile of the E-RSV concentration was plotted as
a function of the exposure time, and modulated using first kinetic
models (Eq. [4]; software MicroMath Scientistꢁ, St. Louis, MO).
lnðCÞ ¼ lnðCoÞ ꢀ kt
ð4Þ
Physical stability of nanostructures. The physical stability of the
particles was evaluated by measuring the particle size by dynamic light
scattering (Zetasizer Nanoseries; Malvern Instruments) after UVA
radiation under the same conditions as the chemical stability assay for
0, 120 and 720 min.
Transmission electron microscopy. The morphological aspect of the
particles was evaluated by transmission electron microscopy (TEM;
Jeol, JEM 1200 Exll, Centro de Microscopia Eletroˆnica-UFRGS)
operating at 120 kV. The diluted suspensions were deposited on
formvar-carbon supported on copper grids (Electron Microscopy
Sciences). The samples were negatively stained with a uranyl acetate
solution (2% m ⁄ vol).
Particle concentration. The number of particles per milliliter was
estimated by turbidimetry according to the method described by
Zattoni et al. (29). Briefly, the samples were diluted with MilliQꢁ water
to give five different concentrations that presented absorbance values
between 0.2 and 1.0 and analyzed at the wavelength 590 nm (where
there is no absorption). The number of particles was calculated from
the absorbance, diameter and density values.
E-RSV photostability assay. The photostability assay was performed
by irradiating the samples in a mirrored chamber with a UVA light
source (<1 mWÆcm)2). Samples were placed in polystyrene cuvettes
with a 20 cm distance from the light source and the experiment was
carried out in triplicate for each sample (liposomes, lipid-core nanocap-
sules, nanospheres and NLC). All formulations were aqueous suspen-
sion and the control (free E-RSV) was an ethanol solution. This solvent
was chosen because E-RSV presents high solubility in ethanol. Fur-
thermore, ethanol presents a protective effect on the photodegradation
of E-RSV if compared with water (medium of the tested formulations),
as cited by Sapino et al. (1), assuring that the solvent is not responsible
for the extent of degradation of the free E-RSV. Degradation was
verified by UV spectroscopy following the method described by
Camont et al. (30), this method was compared to the HPLC method
described earlier in this article. In addition, none of the formulations’
components, except RSV, absorb between 250 and 400 nm.
Skin penetration. A penetration assay using automated Franz cells
(MicroettePlus Multi Groupꢁ Hanson Research Corporation) with a
modified liquid formulation apparatus and porcine skin as a mem-
brane was performed with the particles that presented improved
chemical and physical stability, lipid-core nanocapsules and NLC. As a
control a water:ethanol (6:4, vol ⁄ vol) solution of free E-RSV with a
concentration of 1 mgÆmL)1 was used. A volume of 0.2 mL was
applied on the skin surface for all samples. The receptor medium was
water with 2% of polysorbate 80, ensuring sink condition during the
entire experiment, at a temperature of 32
at 400 rpm. The porcine skin, obtained from
(Frigorıfico Porto Ijuı, Brazil), was comprised of total epidermis and
dermis. The skin was reduced to a width of 2.0 0.15 mm, cleaned
2ꢂC and constant stirring
a
slaughterhouse
with sodium lauryl sulfate aqueous solution (1%) on the internal
surface and with ether on the external surface. The area of the exposed
skin was 1.76 cm2.
After 8 h the experiment was ended, the stratum corneum was
separated by the tape stripping technique with 18 tapes (Scotch tape
3Mꢁ, Sumare, Brazil) and the viable epidermis and dermis were
separated by heating the skin in a water bath at 60ºC for 45 s and
separating the epidermis with a spatula. E-RSV was extracted from the
skin layers with acetonitrile:water (3:1, vol ⁄ vol) and 1 h of sonication.
The extraction recovery from skin samples using this method was
85.3
3.7%. E-RSV was quantified by the HPLC method described
elsewhere. This procedure was carried out in sextuplicate, in the dark
and under UVA exposure.
Statistical analysis. Data were evaluated by two-way analysis of
variance (ANOVA) followed by Tukey’s test (BioEstat 5.0ꢁ, Instituto
de Desenvolvimento Sustentavel Mamiraua, Belem, Para, Brazil). The
significance level (alpha) applied was 0.05. The values are expressed as
At determined time intervals, aliquots of 50 lL were taken from
each sample and diluted to 5 mL of ethanol, homogenized, centri-
fuged, placed in quartz cuvettes and analyzed in a spectrophotometer.
The E-RSV concentration was calculated at each time interval using
Eq. (1) as follows:
the mean
standard deviation.
RESULTS
ꢀ
ꢁ
Nanocarrier preparation and characterization
1
R ꢁ A
l ꢁ e
CE
¼
ꢁ
ꢀ Co
ð1Þ
Both polymeric particles had a nanometric and monomodal
size distribution (266 nm for nanocapsules, 207 nm for nan-
ospheres and PI values of 0.16 and 0.16, respectively). To
prepare an adequate nanosized liposome (189 nm and PI of
0.29), phosphatidylcholine concentration was 1.6 mgÆmL)1
and the process parameters were 10 cycles of homogenization
at a pressure of 250 bars. The nanostructured lipid carrier
formulation without coconut fat did not present a monomodal
size distribution showing two particle populations (137 nm
and 531 nm). The addition of coconut fat (6 mgÆmL)1) and the
reduction of medium-chain triglycerides from 26 to
20 mgÆmL)1 resulted in a formulation with a monomodal size
distribution (176 nm and PI 0.12).
R ꢀ 1
where R = eE,305 nm ⁄ eZ,305 nm = 3.60 at 305 nm, CE is the concen-
trations of E-RSV (lgÆmL)1) at the time of the analysis, e is the molar
extinction coefficient of E-RSV (lgÆmL)1), Co is the initial concen-
tration of E-RSV (before exposure), A is the measured absorbance and
l is the optical path length (cm).
The molar extinction coefficient of E-RSV was calculated for the
wavelength 305 nm and the optical path length of 1 cm, using the
Lambert–Beer law (Eq. [2]),
A ¼ eE ꢁ l ꢁ C
ð2Þ
where A is the absorbance, l is the optical path length, C is the con-
centration and e is the molar extinction coefficient. The molar
extinction coefficient of Z-RSV measured after 60 min of irradiation of
a E-RSV ethanolic solution, which was analyzed by HPLC and a UV
spectrophotometer. The molar extinction coefficient of Z-RSV was
calculated using Eq. (3):
A comparison among lipid-core nanocapsules (266.2 nm),
nanospheres (207.1 nm) and liposomes (189.4 nm) showed

916 Cassia Britto Detoni et al.
statistically similar values in terms of particle size (P > 0.05).
The smallest z-average size was observed for the NLC
(176.0 nm). The polydispersity indexes (PI) were below 0.3
for all nanostructured carriers suggesting that each prepara-
tion method led to controlled size distributions. The pH for all
aqueous suspensions ranged from 5.4 to 5.9. E-RSV was
successfully loaded in all particles and presented association
efficiency of 98–100%, which is expected for hydrophobic
molecules such as E-RSV (Table 1). TEM showed that all
particles had almost spherical shapes and confirmed their
nanometric size (Fig. 1).
experimental conditions applied, a E-RSV solution was
exposed to UVA radiation and quantified simultaneously by
the validated HPLC method and the spectrophotometric
method first described by Camont et al. (30) with four
different time intervals. The results for the E-RSV concentra-
tion provided by the two methods were similar (Fig. 2),
confirming that the methods are interchangeable.
The photodegradation profile studies showed a 90.4%
decrease in the E-RSV concentration of the ethanolic solution
in 4 h (240 min) of experiment. This photoreaction was
significantly slowed down by the association of E-RSV with
all four types of nanocarriers (Fig. 3). The liposome particles
maintained the E-RSV concentration for the longest time, the
initial concentration decreasing by only 29.3% after 4 h.
Concentrations of E-RSV reduced to 67.2%, 77.8% and
70.0% of the initial concentration within 4 h in the case of the
lipid-core nanocapsules, nanospheres and NLC, respectively.
The degradation profiles were modeled considering first-
and second-order kinetics (Table 2).The first-order model was
chosen as the best fit for all formulations and for the ethanolic
For the lipid-core nanocapsules the results of the turbidimetry
assays indicated an estimated concentration of 4.7
particles per mL, nanospheres of 7.6
0.2 · 1012 particles per
mL, liposomes of 6.1
0.2 · 1010 particles per mL and NLC of
1.4
0.05 · 1011 particles per mL.
0.2 · 1012
E-RSV photostability assay
For E-RSV, the molar extinction coefficient at the wavelength
of 305 nm was 27 775 LÆmol)1Æcm)1 and for Z-RSV it was
7703 M)1Æcm)1. To verify that Eq. (1) was applicable under the
Table 1. Physical–chemical characteristics of the E-RSV loaded nano-
structures. Data are presented as means
SD.
z-average
(nm)
E-RSV Association
content (%) efficiency (%) pH
Particles
Nanocapsules
Nanospheres
Liposomes
Nanostructured 176.0
lipid carriers
266.2
207.1
189.4
0.72
98.1
0.19
0.32
0.12
0.12
99.6
98.0
99.1
98.4
5.85
5.50
5.47
5.89
8.11 102.1
14.1 100.9
5.06 102.4
Figure 2. Degradation profile of E-RSV ethanol solution analyzed
simultaneously by the validated HPLC method and by the spectro-
photometric method first suggested by Camont et al. (30).
Figure 1. Transmission electron micrographs of lipid-core nanocap-
sules (a), nanospheres (b), lioposomes (c) and nanostructured lipid
carriers (d).
Figure 3. The photodegradation profile of E-RSV in ethanol solution
and encapsulated in lipid-core nanocapsules, nanospheres, nanostruc-
tured lipid carriers and liposomes in aqueous suspensions.

Photochemistry and Photobiology, 2012, 88 917
Table 2. Kinetic parameters (coefficient R2, kinetic constant k, model selection criterion MSC and half-live t1 ⁄ 2) of E-RSV photodegradation
during UVA exposure for 720 min (n = 3). Data are presented as mean
SD.
First order
k (min)1
Formulation
R2
)
MSC
2.879
2.194
2.487
1.094
2.954
t1 ⁄ 2 (min)
Ethanol solution
Nanocapsules
Nanospheres
Liposomes
Nanostructured lipid carrier
0.974
0.977
0.975
0.988
0.986
0.012
0.033
0.008
0.003
0.002
0.0306
0.0057
0.0104
0.00130
0.0051
0.0070
0.431
0.104
0.388
0.301
0.174
19.5
122.6
67.0
3.7
16.3
5.2
9.6
5.7
0.00070
0.00080
0.00002
0.00020
592.2
137.0
Table 3. Z-average diameter (SD) and polydispersity index (SD) of the nanostructured carriers after UVA radiation.
Irradiation time (UVA)
0 min
Diameter (nm)
120 min
Diameter (nm)
720 min
Diameter (nm)
Formulation
PI
PI
PI
LNC
Nanosphere
Liposomes*
Nanostructured lipid carriers
266.2
207.1
189.3
176.0
4.1
8.1
14.2
5.1
0.16
0.16
0.29
0.12
0.02
0.02
0.05
0.02
317.7
206.6
216.2
184.5
23.3
8.8
25.4
6.7
0.15
0.14
0.43
0.12
0.05
0.03
0.02†
0.06
309.3
211.6
188.6
188.7
9.9
3.0
14.5
2.9
0.13
0.12
0.41
0.11
0.02
0.02
0.03†
0.01
*bimodal size distribution profile.
solution according to the MSC and R2-values. All of the
nanocarriers were capable of reducing the isomerization rate
(K; P < 0.5) of E-RSV. The lipid-core nanocapsules and NLC
had the same degradation rate (K; P < 0.5) and maintained
the E-RSV concentration for a longer period of time than the
nanospheres.
Physical stability of nanostructures
The z-average sizes of the lipid-core nanocapsules and nano-
spheres slightly increased upon UVA exposure, from
266.2
211.6
4.1 to 309.3
3.0 nm, respectively. Although the lipid-core nano-
9.9 nm and from 207.1
8.1 to
capsules presented a greater increase in size than the nano-
spheres, no statistical differences between the z-average size at
time zero and at 12 h (P > 0.05) were observed for any of the
particles. The polydispersity indexes (PI) of the polymeric
formulations did not vary under UVA radiation maintaining
values of between 0.12 and 0.16.
Figure 4. Particle size distributions obtained by PCS according to the
intensity of E-RSV-loaded liposomes before UVA radiation and after
720 min of UVA radiation.
After 720 min of UVA exposure the liposomes exhibited
considerable fluctuations in the z-average (189.3
14.2,
216.2 25.4 and 188.6 14.5 nm) and a notable increase
in the PI (from 0.29 to 0.41). NLC presented a behavior similar
to the lipid-core nanocapsules and nanospheres, maintaining
their original size (from 176.0 to 188.7 nm) and degree of
dispersion (PI of 0.12 and 0.11; Table 3).
The liposomes assumed a dynamic and bimodal profile while
all other particles remained monomodal after UVA radiation.
As a consequence, the appearance of a new population of
particles was observed. The mean peak diameter of the second
particle population of liposomes was 86 nm (Fig. 4).
UVA radiation the blank liposomes presented a second
population of particles with larger z-average diameters than
the particles before irradiation.
Skin penetration
In the case of the particles, which presented improved E-RSV
photostability and a constant size distribution after UVA
radiation, the skin penetration was evaluated in the dark and
during exposure to UVA radiation (Fig. 6).
When protected from the light, larger quantities of the free
E-RSV penetrated into the upper layers of the skin (11.8 lg in
the stratum corneum and 23.2 lg in the viable epidermis)
To verify the influence of E-RSV on the physical instability
of the liposomes, liposomes formulated without E-RSV were
evaluated under the same conditions (Fig. 5). After 8 h of

918 Cassia Britto Detoni et al.
compared to the E-RSV-loaded carriers (9.5 and 7.3 lg in the
stratum corneum, 15.7 and 8.1 lg in the epidermis for lipid-
core nanocapsules and nanostructured lipid carries, respec-
tively). Lipid-core nanocapsules showed a tendency to prevent
E-RSV reaching the dermis (26.5 lg) when compared to the
free E-RSV (36.8 lg) and E-RSV-loaded nanostructured lipid
carries (41.1 lg), although no statistically significant difference
was observed.
When exposed to UVA radiation the quantities of E-RSV in
the total epidermis (stratum corneum plus viable epidermis)
were larger for lipid-core nanocapsules (2.7 lg) and for NLC
(3.3 lg). In the dermis, the quantity of E-RSV was larger for
the free molecule (17.7 lg), followed by the NLC (10.5 lg) and
then lipid-core nanocapsules (7.8 lg). After 8 h of experiment
none of the samples showed a quantifiable concentration of
E-RSV in the receptor compartment.
Figure 5. Particle size distribution obtained by PCS according to
intensity of blank liposomes before UVA radiation and after 720 min
of UVA radiation.
DISCUSSION
Particle preparation and characterization
All formulations showed particle sizes consistent with the type,
raw materials and preparation methods used. The nanocap-
sules had a z-average of 266 nm and nanospheres of 207 nm,
consistent with the expected size range (200–500 nm) for
polymeric formulations prepared by the method of preformed
polymer precipitation (12). The liposomes had a z-average
diameter of 189 nm, which is in the reported size range (80–
200 nm) of nanoliposomes (31). NLC had a z-average of
176 nm in agreement with the size range (<500 nm) described
for similar formulations (32). All formulations had narrow
dispersity according to the polydispersity indexes (PI), which
varied from 0.11 to 0.29 (33), and these particles presented a
controlled size distribution. In nanostructured systems, PI is a
value used to describe the homogeneity of the particle
distribution and serves as a quality control parameter. The
PI of liposomes (0.29) was the highest of the formulations
prepared, and for the other three types of particles this value
was between 0.11 and 0.16.
(a)
The physicochemical characteristics of the particles are
strongly determined by the solids concentration and volumet-
ric fraction. As the formulations prepared required different
materials and proportions, they differed in terms of particle
(b)
concentration (nanocapsules 4.7
0.2 · 1012, nanospheres
7.6
1.4
0.2 · 1012, liposome 6.1
0.2 · 1010 and NLC
0.05 · 1011 particles per mL). This parameter did not
appear to have a direct influence on the photostability or the
isomerization rate (k) of the E-RSV-loaded nanocarriers, as
seen in Tables 1 and 2.
The pH for all particles ranged between 5.0 and 6.0, which is
acceptable considering that E-RSV is more photostable in the
range of pH 5.0–8.0 (1). E-RSV presents higher skin perme-
ation at pH values close to 6.0 (23).
Photostability assays
The association of E-RSV with supramolecular structures
enhanced its photostability under UVA radiation, this may be
observed by the increase of t1 ⁄ 2 from 19.5 min to at least
67 min (Table 2). The observed differences between the
associated and free RSV are probably higher than reported,
Figure 6. Skin penetration profiles of free E-RSV, E-RSV-loaded
lipid-core nanocapsules and nanostructured lipid carrier in the dark (a)
and exposed to UVA radiation (b). *Statistically different to the free
molecule (P < 0.05).

Photochemistry and Photobiology, 2012, 88 919
considering that ethanol may exhibit a protective effect on E-
RSV when compared with an aqueous medium (1).
The different designs of polymeric particles resulted in
nanocarriers with different photostabilizing capacities. The
vesicular polymeric system of lipid-core nanocapsules was able
to photostabalize E-RSV for a longer period of time than the
nanospheres. The t1 ⁄ 2 was almost twice as long for the
nanocapsules (122.6 16.3 min) compared with the nano-
spheres (67.0 5.2 min).
The lipid-core nanocapsules and nanospheres were physi-
cally stable for 12 h (720 min) under UVA exposure. In a
different study, a similar result was obtained with polymeric
nanocapsules containing OMC and quercetin after exposure
for 15 days (8). Therefore, E-RSV association did not influ-
ence the physical stability of the particles, which maintained PI
values of 0.15 and 0.13 after 120 and 720 min, respectively.
The system in which the E-RSV had the highest photo-
Figure 7. Illustration of resveratrol E–Z isomerization in the bilayer
membrane leading to fragmentation of the vesicle.
stability was the liposome structure, with
592.2 9.6 min. In our study, the lipophilic characteristic
a
t1 ⁄ 2 of
of E-RSV and the high association efficiency of the E-RSV
liposomes indicate that the molecules are probably imbibed in
the phospholipid bilayer, leading to a high photostabilization.
Phosphatidylcholine degradation may lead to UVA absorp-
tion (34), which would result in erroneous results for the
determination of the E-RSV concentration by the method
used herein. For this reason, the UV absorption of a blank
formulation was monitored during UVA exposure for the
experimental period and no absorption was observed at
between 250 and 400 nm.
Liposomes were not physically stable during exposure to
UVA radiation, in the case of both drug loaded and blank
vesicles. Liposomes assumed a dynamic and bimodal profile. A
second population of larger particles appeared in the blank
formulation, probably as a result of the phenomena of fusion
and ⁄ or agglomeration due to the high energy ‘‘input’’ during
UVA radiation. For the liposomes containing E-RSV, a
second population of smaller particles appeared after 8 h of
UVA radiation. The appearance of smaller particles was
attributed to a disruption of the vesicle into smaller fragments
due to the E–Z isomerization of the RSV imbibed in the
bilayer. The disruption of the vesicle into smaller fragments is
well described for phosphatidylcholines with azobenzone
groups (35). When comparing the molecular structure of
azobenzone and RSV we noted that the basic skeleton of the
molecules is the same, and that RSV has a hydroxyl group on
each aromatic ring. It is possible that the linear structure of
E-RSV favored the liposome bilayer stability, and that on
isomerizing to Z-RSV the molecule loses its linear configura-
tion and the vesicle breaks (Fig. 7).
to the lipid nature, this nanostructure may present morpho-
logical variations after an increase in temperature or a high
energy input, exposure of solid lipid nanoparticles to light may
cause particle interaction and consequent gel network forma-
tion (36). In our particular case, the mixture of a lipid of high
melting point (Compritol 888ꢁ) with one of low melting point
(coconut fat) resulted in particles that were resistant on
exposure to UVA at a temperature of 27–30ºC. The use of
Compritol 888ꢁ was based on its high melting point and
chemical inertness, but the use of this solid lipid alone led to an
unsuccessful E-RSV association. Thus, coconut fat was chosen
as a solid lipid of lower melting point to be mixed with
Compritol 888ꢁ because of earlier reports of its affinity with
E-RSV (37).
Skin penetration
A combination of improved chemical stability of E-RSV and
physical stability of the nanostructured system was used as
the selection criteria. Lipid-core nanocapsules and NLC did
not differ in terms of these two aspects and for this reason
both particles were assayed to investigate E-RSV skin
penetration.
The E-RSV was mostly concentrated in the dermis in all
tests, confirming the high penetration capacity as described in
a previous study (23). In the dark, the E-RSV penetration
profile for lipid-core nanocapsules was similar to that for the
free E-RSV, but in the case of the former there was a slight
tendency toward lower drug concentrations in the viable
epidermis and dermis. The NLC exhibited a lower concentra-
tion of E-RSV in the viable epidermis when compared to the
free molecules in the dark. Under dark conditions, the
association to a nanocarrier has little influence on the retention
of E-RSV in the skin layers, resulting in no statistical
differences. The maintenance of the penetration profiles of
E-RSV from lipid-core nanocapsules and nanostructures’ lipid
carriers indicates that these formulations act as carriers and
E-RSV is released from the structure and then penetrates the
skin.
The NLC had statistically the same isomerization rate (K)
and t1 ⁄ 2 as the lipid-core nanocapsules, with t1 ⁄ 2 values of
122.6
16.3 and 137.0
5.7 min, respectively. These two
types of particles were formulated to have similar solids
concentrations, 0.041 gÆmL)1 for NLC and 0.048 gÆmL)1 for
lipid-core nanocapsules, and the liquid lipid used was also the
same (medium-chain triglycerides). The similarities in terms of
composition may have contributed to similarities in the
photoisomerization profile.
Nanostructured lipid carriers presented physical stability
after UVA exposure. The monomodal profile with a polydi-
The use of a hydroalcoholic solution (40% vol ⁄ vol) was
necessary to dissolve E-RSV at 1 mgÆmL)1. Even though
spersion index of 0.12
0.02 was maintained over 12 h. Due

920 Cassia Britto Detoni et al.
Acknowledgements—This work was supported by the following
Brazilian government agencies: Conselho Nacional de Desenvolvi-
ethanol is a permeability enhancer an decrease in skin
deposition was not observed in dark conditions when com-
pared the nanocarriers. A similar result was observed by Hung
et al. (37), a 25% (vol ⁄ vol) hydroalcoholic solution had similar
skin deposition value when compared with a pH 6 aqueous
buffer.
mento Cientifico e Tecnologico (CNPq), Coordenaca
mento de Pessoal de Nıvel Superior (CAPES), Fundaca
Pesquisa do Estado do Rio Grande do Sul (FAPERGS) and
Programa de Apoio a Nucleos de Excele
˜
o de Aperfeicoa-
˜
o de Amparo a
ˆ
ncia (PRONEX-CNPq ⁄ FA-
PERGS).
When exposed to UVA radiation, application of the free
molecule leads to higher quantities of E-RSV in the dermis and
viable epidermis and lower quantities in the stratum corneum
when compared with both nanostructures (lipid-core nano-
capsules and NLC). Considering the total epidermis, the main
site of action of topically applied antioxidants and chemopre-
ventives, the nanostructures were capable of retaining the E-
RSV.
The horny layer functions as a reservoir for topically
applied drug, for this reason the interaction between the
substance and the stratum corneum is very relevant for topical
activity (38). The stratum corneum serves as the principal
barrier for many molecules, but not for nonionic E-RSV (23).
Although Hung et al. (37) observed that the stratum corneum
did not serve as a barrier for RSV, under UVA radiation we
demonstrataed that lipid-core nanocapsules and nanostructur-
ted lipid carriers enhance the retention of RSV in the stratum
corneum.
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