Improvements of cellular stress response on resveratrol in liposomes.

Abstract

Resveratrol (RSV) has proven potential in prophylaxis and treatment of various disorders mediated by free radicals and oxidative stress. RSV solubility, stability, and cytotoxicity must be regulated for satisfactory bioavailability. Here, RSV was loaded into liposomes, characterized by PCS and TEM and evaluated on HEK293 cell line by metabolic activity assay, electron paramagnetic resonance, and fluorescence microscopy. RSV at 10 microM induced changes in cell metabolic activity and significantly improved antioxidative capacity. At 100 microM it showed concentration-dependent cytotoxicity. Oligolamellar liposomes with mean diameter 84 nm, polydispersity index 0.2, and zeta potential -40 mV showed high entrapment of RSV and rapid cellular internalization. Cell stress caused by UV-B irradiation diminished cell metabolic activity by 50%. RSV loaded into them showed no cytotoxicity at 100 microM and stimulated cellular metabolic and antioxidant activity levels to eliminate the harmful effect of the stress. Localization of RSV within liposomal bilayer is crucial for stimulation of cell-defense system, prevention of RSV cytotoxicity, and its long-term stability. In summary, evidence of different metabolic activity using free RSV and LIP-RSV is presented indicating that liposome-mediated uptake of RSV is more effective for improvement of the cell-stress response.

Full text

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Research paper
Improvements of cellular stress response on resveratrol in liposomes
Julijana Kristla,*, Karmen Teskac ˇa, Carla Caddeob, Zrinka Abramovic ´c, Marjeta Šentjurcc
aDepartment of Pharmacy, University of Ljubljana, Ljubljana, Slovenia
bDepartment Technological Chemical Drug, University of Cagliari, Cagliari, Italy
cJoz ˇef Stefan Institute, Ljubljana, Slovenia
a r t i c l ei n f o
Article history:
Received 13 May 2009
Accepted in revised form 9 June 2009
Available online 13 June 2009
Keywords:
Antioxidant
Liposome
Cytotoxicity
Free radicals
EPR
Cell proliferation
Stability
a b s t r a c t
Resveratrol (RSV) has proven potential in prophylaxis and treatment of various disorders mediated by
free radicals and oxidative stress. RSV solubility, stability, and cytotoxicity must be regulated for satisfac-
tory bioavailability. Here, RSV was loaded into liposomes, characterized by PCS and TEM and evaluated on
HEK293 cell line by metabolic activity assay, electron paramagnetic resonance, and fluorescence micros-
copy.
RSV at 10 lM induced changes in cell metabolic activity and significantly improved antioxidative
capacity. At 100 lM it showed concentration-dependent cytotoxicity. Oligolamellar liposomes with mean
diameter 84 nm, polydispersity index 0.2, and zeta potential ?40 mV showed high entrapment of RSV
and rapid cellular internalization. Cell stress caused by UV-B irradiation diminished cell metabolic activ-
ity by 50%. RSV loaded into them showed no cytotoxicity at 100 lM and stimulated cellular metabolic
and antioxidant activity levels to eliminate the harmful effect of the stress. Localization of RSV within
liposomal bilayer is crucial for stimulation of cell-defense system, prevention of RSV cytotoxicity, and
its long-term stability. In summary, evidence of different metabolic activity using free RSV and LIP–
RSV is presented indicating that liposome-mediated uptake of RSV is more effective for improvement
of the cell-stress response.
? 2009 Elsevier B.V. All rights reserved.
1. Introduction
Antioxidants have received a great deal of attention in recent
years for their prophylactic and therapeutic potential for diseases
in which the cell defense against reactive oxygen species (ROS) is
compromised [1]. Externally supplied antioxidants neutralize
ROS before they react with biological targets to reduce oxidative
stress [2]. The emerging view is that many antioxidants are likely
to exert additional beneficial cellular effects, through modulation
processes at different biochemical levels including protein synthe-
sis and signaling pathways [3]. Natural and synthetic sources are
therefore being screened for novel antioxidants [4].
The polyphenolic compound resveratrol (trans-3,5,40-trihydr-
oxystilbene; CAS 501-36-0; RSV) has been found in more than 70
plant species, including human foods such as grapes, peanuts,
and various berries and herbs. It is thought to serve as a phyto-
alexin, protecting plants against environmental stress and patho-
genic attack [5]. Its relatively simple molecular structure enables
free radicals overproduced in disease conditions to be scavenged
and the redox signaling pathways of the cells to be regulated.
RSV was shown to affect mitochondrial function and metabolic
homeostasis through induction of genes for oxidative phosphoryla-
tion and mitochondrial biogenesis [6]. The chemopreventive effect
of RSV is thought to be due to inhibition of quinone reductase 2
activity, which in turn, up-regulates the expression of cellular anti-
oxidant and detoxification enzymes (such as catalase, quinone
reductase 1, glutathione-S-transferase) to improve cellular resis-
tance to oxidative stress [7]. Additionally, its ability to interact
with receptors and enzymes gives rise to other biological effects
such as suppression of growth, induction of differentiation, cell-cy-
cle regulation, down-regulation of pro-inflammatory mediators,
regulation of gene expression by affecting transcription factor
activity, and up-regulation of death-inducing factors. Recently,
studies on a variety of species showed that RSV increased activity
of SIRT (a member of the sirtuin family of nicotinamide adenine
dinucleotide-dependent deacetylase) which resulted in improved
cellular stress resistance and longevity [8]. Moreover, RSV is struc-
turally similar to diethylstilbestrol which broadens its activity on
estrogenic area. Thus, through its phytoestrogenic properties it
can regulate the expression of hormone-dependent genes, such
as the oncosuppressor BRCA1, in breast cells [9,10]. All of these ac-
tions are dependent on cell conditions and RSV concentration [5].
These potential therapeutic and prophylactic applications are,
however, restricted by the low bioavailability caused by its physi-
cal properties. Polyphenols typically are largely metabolized and
consequently have oral bioavailability of <10% [11]. Additionally,
0939-6411/$ - see front matter ? 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.ejpb.2009.06.006
* Corresponding author. University of Ljubljana, Faculty of Pharmacy, Aškerc ˇeva
7, 1000 Ljubljana, Slovenia, Tel.: +386 1 4769500; fax: +386 1 4258031.
E-mail address: julijana.kristl@ffa.uni-lj.si (J. Kristl).
European Journal of Pharmaceutics and Biopharmaceutics 73 (2009) 253–259
Contents lists available at ScienceDirect
European Journal of Pharmaceutics and Biopharmaceutics
journal homepage: www.elsevier.com/locate/ejpb

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RSV has low water solubility and stability making its clinical suc-
cess a formidable technological and medical challenge [12].
Liposomal formulations have been proposed as a means of
improving the therapeutic efficacy of poorly bioavailable drugs
[13], and examples of formulation containing antioxidants, such
as Cu/Zn superoxide dismutase, vitamins C and E, N-acetyl cys-
teine, and glutathione, have shown promise for treatment of dis-
eases involving oxidative stress [14]. Although the effects of RSV
on the molecular biology of cells have been well documented, only
our previous report [15] has described its formulation in lipo-
somes. This showed that stimulation of cell activity was inversely
proportional to the RSV dose. Fluorescence images of the treated
cells demonstrated effective antioxidant activity of RSV at 10 lM,
but toxicity seen as changes in cell shape, detachment, and apop-
totic features at higher concentrations was observed [15].
Our hypothesis is that liposomes increase RSV biological activ-
ity and improve its protective effect on stressed cells. This study
compares the effects of RSV-loaded liposomes (LIP–RSVs) to free
RSV on cell metabolic activity and cell-redox system as well. Ef-
fects of RSV on metabolic activity were quantified by MTS assay,
while electron paramagnetic resonance (EPR) method was used
to define activity of cell-redox system. Internalization of liposomes
within cells was visualized by fluorescent microscopy. Evidence of
different mitochondrial activity using free RSV and LIP–RSV is pre-
sented indicating that liposome-mediated uptake of RSV is more
effective for improvement the cell-stress response.
2. Materials and methods
2.1. Materials
Resveratrol (RSV; >99% pure), cholesterol, and dicetyl phosphate
(DCP) were obtained from Sigma (Germany); ATX Tris buffer from
Fluka(Germany);andenrichedsoyphosphatidylcholine(Phosphol-
ipon 90G, P90G) from Natterman Phospholipids (Germany).
2.2. Methods
2.2.1. Solubility testing
Twelve different amounts of RSV were dispersed in water (ob-
tained concentrations varied from 5 to 70 mg/l) and stirred for
24 h. The concentration of RSV in the supernatant was determined
by UV–VIS spectrophotometry at 306 nm (Hewlett Packard, 8453,
Germany). The maximal concentration gave the solubility of RSV
in water.
2.2.2. RSV sample preparation
Stock solution of 100 mM of RSV in ethanol was stored at
?20 ?C. Final working concentrations were prepared by diluting
with culture medium. The highest concentration of ethanol used
on cell culture was 1.1% v/v, which preliminary experiments had
verified to have no effect on viability.
2.2.3. Liposome preparation
Empty liposomes (e-LIP) were prepared by suspending P90G
(129 lmoles), DCP (36 lmoles), and cholesterol (26 lmoles) in
5 ml of Tris–HCl buffer, adjusting the pH to 7.4, then sonicating
with a High Intensity Ultrasonic Processor equipped with a tapered
microtip (Cole-Parmer, USA), until an opalescent dispersion was
obtained. The pulse function of sonicating was used to inhibit heat
build-up in the sample. RSV was loaded into liposomes (LIP–RSV)
by adding RSV (6.5 lmoles) to the suspension mixture. Liposomal
dispersions were first extruded through 100 nm filter pore size in
a LiposoFastTMextruder (Avestin, Canada) to produce small oligola-
mellar vesicles, and then dialyzed against water using Spectra/Por?
membranes (Spectrum Laboratories, Inc. USA) to separate the free
RSV.
Fluorescently-labeled liposomes were prepared by adding cou-
marin-6 dye (3 lmoles) (Sigma, Germany) to the lipophilic compo-
nents before sonication.
2.2.4. Liposome characterization
Size and polydispersity index (PI) of liposomes were deter-
mined by dynamic light scattering (DLS), while zeta potential
(ZP) was measured by laser Doppler electrophoresis (Zetasizer
nano-ZS; Malvern Instrument, UK), taking the mean of five mea-
surements [15]. For determination of liposomes stability, the size,
PI, and ZP of prepared liposomes were accompanied during their
storage for at least two months. Vesicle formation and the mor-
phology of liposomes were observed by transmission electron
microscopy (TEM) (Philips CM 100 microscope; Amsterdam, the
Netherlands).
The amount of RSV loaded into liposomes (Encapsulation effi-
ciency) was determined by high performance liquid chromatogra-
phy. Possibilityof
cis/trans
Separation was achieved on an XBridge C18 (4.6 ? 150 mm,
5 lm) column by (Waters, USA) run on an Agilent 1100 Series HPLC
System (Germany). RSV was separated using a mobile phase of 75%
v/v methanol, 22.5% v/v acetonitrile, 2.4% v/v water, and 0.1% v/v
acetic acid at 0.8 ml/min. Absorbance at 306 nm was recorded by
a diode array detector.
conformationwasconsidered.
2.2.5. Cell culture and treatments
Human-derived renal epithelial cells (HEK293 cells, American
type culture collection ATCC, Manassas, VA, USA) were selected
as model cells, since they can be easily manipulated. They were
grown as adherent cultures in growth medium consisting of Dul-
becco’s modified Eagle medium (DMEM; Sigma, Germany) supple-
mented with 10% fetal bovine serum (FBS; Gibco?– Invitrogen,
USA), 1.0% 200 mM L-glutamine, and 1.0% antibiotic/antimycotic
(Sigma, Germany) at 37 ?C in a humidified atmosphere of 5% CO2
in air.
HEK293 cells were seeded at a density of 2 ? 105cells/ml over
an appropriate growing area. After one day (attachment phase),
the cells were treated for 24 h with (a) RSV at 10 lM or 100 lM,
(b) empty liposomes (e-LIP), or (c) resveratrol-loaded liposomes
(LIP–RSV) with concentration of RSV at 100 lM. Control cells re-
ceived culture medium alone.
Some samples were exposed to UV-B radiation (UV lamp, Spec-
tronics, New York, USA) at 280–320 nm for 1 or 3 h. Non-irradiated
cells were kept in the dark in the incubator.
2.2.6. Metabolic activity of cells
Cell metabolic activity was evaluated using the MTS assay (Cell
titer 96?Aqueous One Solution Cell Proliferation Assay; Promega,
Madison, WI). It is based on the capacity to metabolize of a yellow
tetrazolium salt (MTS) to the dark blue formazan product by mito-
chondrial dehydrogenase. Briefly, after 24 h treating of cells, MTS
reagent was added for 3 h and the finally the absorbance at
492 nm was measured. Metabolic activity of treated cells was ex-
pressed as percentage of untreated cells [15].
2.2.7. Morphological examination of cells
Cell growth and morphology were observed using an inverted
phase-contrast microscope (Olympus CKX41, Tokyo, Japan). To
establish fixed-slide preparations, HEK293 cells (2 ? 105cells/ml)
were plated on square glass cover slips and incubated in six-well
plates overnight. To visualize liposomes, the cells were incubated
with fluorescently labeled liposomes. Following incubation with
different test dispersions, the cells were fixed with ice-cold 4%
paraformaldehyde in PBS (pH 7.4) for 10 min and permeabilized
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for 10 min in 0.1% Triton X-100 (Sigma, Chemical Co., Saint Luis,
ZDA). To visualize cells, the actinic fibres were stained with
green-fluorescent dye Phalloidin–Fluorescein isothiocyanate or
red-fluorescent Phalloidin–Tetramethylrhodamine B isothiocya-
nate (both from Sigma) according to the manufacturer’s instruc-
tions. The red dye was used when fluorescent liposomes were
added to the cells. Cell nuclei were then stained with DNA inter-
chelating dye Hoechst 33342 (Riedel de Haen, Germany) (5 lg/
ml) for 30 min in the dark. After staining, the cover slips were
mounted on a slide and transmission micrographs and fluores-
cence images were collected simultaneously with the same focus
settings (60- and 20-fold objective magnification) on an Olympus
IX 81 fluorescence microscope and merged with Cell^R Software
(Olympus).
2.2.8. EPR experiments
EPR spectroscopy enables the detection of cell-redox activity by
the reduction kinetics of the nitroxide moiety of spin-label MeFASL
(10, 3) (5-doxyl-methyl palmitate) that is incorporated in cell
membrane [16] (Fig. 2). Cell-redox systems reduce nitroxide to
non-paramagnetic hydroxylamine, which is not detectable by
EPR. Thus, in metabolically active cells the EPR signal gradually de-
creases as reported by Swartz et al. [17].
Cells were treated as described in 2.2.5. After 24 h of incubation
with 10 or 100 lM RSV, followed by UV-B irradiation for 1 h the
cells were detached by thorough pipetting to yield a cell suspen-
sion. The suspension density was adjusted to 1 ? 106cells/ml
and 3 ml was spin labeled with 75 lL 10?4M of lipophilic spin
probe MeFASL (10, 3) as follows: a thin film of MeFASL (10, 3)
was spread on the wall of a glass tube by rotary evaporation of eth-
anolic solution. The cell suspension was added and the tube was
gently shaken by hand for 10 min, centrifuged at 120g for 2 min,
and the supernatant was carefully removed. For EPR measure-
ments, the remaining cell pellet was drawn into a standard quartz
capillary (1 mm diameter) and spectra were recorded using an X-
band EPR spectrometer (Bruker ESP 300). Settings were microwave
power 10 mW, modulation frequency 100 kHz, modulation ampli-
tude 0.2 mT, centre field 340 mT, and microwave frequency
9.59 GHz.
The influence of RSV on the redox activity of the cells after 24-h
treatment by RSV and then by UV-B irradiation was determined via
reduction kinetics of the nitroxide group of the spin label. EPR
spectra intensity (I) was measured with time after incubation of
cells with the spin label. Signal intensity of the spin label is propor-
tional to the number of the nitroxide groups. It can be calculated as
the double integral of the EPR spectra and is approximated by Eq.
2:
I ¼ h0DH2
where DH0and h0are the width and amplitude, respectively, of the
middle line of the EPR spectrum (Fig. 2C).
0
ð1Þ
2.2.9. Statistical analysis
The values reported are means and standard deviations of
experimental done in triplicate at least three times. Data were ana-
lyzed using one-way analysis of variance (ANOVA) and p < 0.05
was considered significant.
3. Results
The development of delivery systems that improve the biologi-
cal profile of active ingredient is of utmost importance. This study
reports how incorporation into liposomes improved the efficacy of
RSV for prophylaxis or therapeutics. MTS colorimetric assay mea-
sures mitochondrial dehydrogenase activity in metabolically active
cells, while the EPR method assesses the reduction of nitroxide-de-
rived lipophilic radicals which have been implicated in the mito-
chondrial electron transport chain [18].
3.1. Influence of RSV on cell metabolism
Mitochondrial dehydrogenase activity is a measure of cell via-
bility, where a decrease indicates an arrest of cell proliferation
[19]. Thus, the MTS assay evaluates the dose-dependent effect of
RSV on cell activity under UV-stress conditions (Fig. 1).
RSV at 10 lM had no effect, while at 100 lM halved mitochon-
dria activity. A similar decrease was caused by stress, triggered
with UV-B irradiation. RSV at 10 lM partially protected against
UV damage, but at100 lM there was no protection. These results
suggest that RSV at higher concentration has a cytotoxic mecha-
nism different from the UV-stress mechanism.
CONTROL
CONTROL
RSV 10 µM
RSV 10 µM
RSV 100 µM
RSV 100 µM
A
A
F
F
E
E
D
D
C
C
B
B
0.0
0.5
1.0
1.5
2.0
no UVUV 1 h
Metabolic activity
(fraction of non-irradiated control)
Control
RSV 10 µM
RSV 100 µM
**
* ** *
*
Fig. 1. Metabolic activity and morphology of HEK293 cells after treatment with
resveratrol (RSV) and exposure to the stress condition. (Above) Bar charts show
effects on metabolic activity after 24-h incubation of cells with medium (control),
RSV at 10 or 100 lM, and further non-irradiated (no UV) or irradiated with UV-B
irradiation for 1 h (UV 1 h). Calculations were made from the absorbance measured
after addition of MTS reagent for 3 h relative to non-radiated control (mean ± sd;
N = 3;*p < 0.05; and**p < 0.001). (Below) Fluorescent-transmission micrographs of
cells (untreated (control) or treated with RSV at 10 or 100 lM) show green-stained
actin fibres embedded by single cytoplasmic membrane. First column are non-
radiated cells, while second column are irradiated cells. Bar is 50 lm.
J. Kristl et al./European Journal of Pharmaceutics and Biopharmaceutics 73 (2009) 253–259
255

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Changes in metabolic activity are reflected also in cell growth
and morphology, because all growth and divisions are enzyme con-
trolled. Changes in these enzyme functions can be quickly reflected
in cell confluence and shape. The empty areas in Fig. 1B show
reduction of cell proliferation following UV-B radiation. RSV at
10 lM maintained the confluence under UV stress and preserved
the shape typical of adherent cells (Fig. 1C and D). RSV at
100 lM reduced confluence and altered cell shape (Fig. 1E and F).
The rounded and detached cells, which are indicative for necrosis
or apoptosis, suggest cytotoxicity of RSV at 100 lM. This supports
our previous findings, where fluorescence microscopy showed
morphology changes and disintegrated actinic fibres after 24 h
incubation with RSV at 100 lM, in both radiated and non-irradi-
ated cells [15].
3.2. Influence of RSV on the cell-redox system
The protective effect of RSV on the cell defense mechanism was
investigated. Influence of RSV and the effects of UV radiation on the
cell-redox system were assessed by EPR using spin-label MeFASL
(10, 3). The lipophilic nature of MeFASL (10, 3) localizes it in the
cell membrane (Fig. 2A) [16], where it is bioreduced (Fig. 2B) and
gives characteristic EPR spectra (Fig. 2C).
The ability of HEK293 cells to reduce MeFASL (10, 3) to the non-
detectable hydroxylamine analogue (10, 3) after RSV treatment is
shown in Fig. 3. EPR signal intensity was normalized relative to
the intensity measured 4 min after cells exposure to MeFASL
(10, 3). First order reduction kinetic characteristics of MeFASL
(10, 3) are listed in Fig. 3C.
The reduction of radicals by the redox system of HEK293 cells is
shown as a fall in signal intensity. Contact of cells with 10 lM RSV
for 24 h stimulates the redox system which reduces free radicals.
The gain for 10 lM RSV (k = 6.26 s?1; control = 4.94 s?1; p = 0.095)
issmallinnon-irradiatedcells,but10 lMRSVshowssignificantpro-
tectiontoirradiation(k = 5.25 s?1;control = 2.64 s?1;p = 0.01).Rad-
ical elimination is even slower in the presence of 100 lM RSV,
showing inhibition of the cell-redox system. The rate constants are
presented in the Fig. 3C.
Redox activity was even more inhibited by UV radiation. Rate
constants were lowered to 53% and 59% of non-irradiated but un-
treated and 100 lM RSV-treated cells, respectively. Student’s T-test
showsbotharesignificantatp < 0.05.With10 lMRSVthesmallde-
creaseto84%wassignificantonlyatp = 0.12,suggestingstimulation
of the redox system at low concentration. The sequence of the rate
constants in irradiated and non-irradiated groups was consistent
with fastest reduction in RSV at 10 lM and slowest at 100 lM.
3.3. Comparison of metabolic activity determined by MTS and EPR
assays
Results are expressed relative to those from non-treated or non-
irradiated cells as appropriate (Table 1). Values from both, MTS and
EPR results, are similar (lowest p value 0.24), and the trends are
preserved throughout. Irradiated cells show a fall in activity to
56% in the MTS assay and to 53% in the nitroxide reduction. In
non-irradiated samples both methods show an increase in cell
metabolic activity in 10 lM and a decrease in 100 lM RSV.
Earlier studies on different cell types using lipophilic nitroxide
spin labels revealed that they are not only localized in the plasma
membrane, but are in dynamic equilibrium with other membranes
[18]. Close agreement between the MTS and EPR assays points to
an important involvement of the mitochondria in the changes pro-
voked by RSV and UV radiation.
Our previous research has already shown the cytotoxic effect of
100 lM RSV as increased sub-G1 phase (apoptotic cells) [19]. Thus,
RSV is a potent active substance with multiple antioxidative mech-
anisms that should be delivered to the cells in low dose. Ideally,
RSV should be released slowly over a prolonged period from an
appropriate carrier to avoid triggering cytotoxicity. This requires
thatliposomesneedtocontainanamountofRSVthatwouldbetoxic
in its unloaded state to maintain therapeutic-free concentration.
3.4. Amelioration of biological effects of RSV by liposomes
Liposomes loaded with RSV (LIP–RSV) were prepared in order to
improve RSV biological activity and stability and to diminish cyto-
Fig. 2. Schematic representations of: (A) – distribution of spin-label MeFASL (10, 3) in the cell membrane; (B) – molecular structure of MeFASL and its non-paramagnetic
hydroxylamine reduction product; (C) – diminution of EPR spectrum intensity of MeFASL during reduction by cytoplasmatic redox system of HEK293 cells.
256
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toxicity at higher total dose. Composition of liposomes enables to
incorporate RSV, which expresses the lipophilic nature (log p of
3.1, water solubility <30 mg/l). Therefore, the entrapment effi-
ciency was >70% and prepared liposomes were stable on storage.
TEM photomicrographs and dynamic light scattering showed small
oligolamellar vesicles with mean diameter of 84 nm and the poly-
dispersity index of 0.2. The zeta potential was approximately –
40 mV.
Metabolic activity and morphology of HEK293 cells after treat-
ment with free RSV alone were presented in Fig. 1. Fig. 4 shows the
corresponding results for high concentration of RSV delivered by
liposomes and illustrates their potential as carrier for RSV stimu-
lating the intracellular enzymatic systems. A significant increase
(p < 0.05) in metabolic activity was seen in cells treated with e-
LIP and LIP–RSV at 100 lM, which is in complete contrast to the
drastic lowering caused by free RSV at this concentration (Fig. 1),
showing that incorporation within liposomal bilayers suppresses
RSV cytotoxicity at 100 lM.
MTS results revealed that ‘‘empty” liposomes, e-LIP, had no pro-
tective effect against UV-irradiation-inhibited metabolism com-
paring to non-irradiated samples (Fig. 4). LIP–RSV 100 lM fully
protected the cells, since their metabolic activities were compara-
ble to those of the non-irradiated controls. Furthermore, un-
changed metabolic activity after more than 3 h of UV-irradiation
demonstrated the possibility of prolonged protection (data not
shown).
Unloaded liposomes gave no protection against UV-irradiation
(Fig. 4D), and the unaltered cell morphology confirmed their bio-
compatibility (Fig. 4A and C). Treatment with LIP–RSV at 10 lM
was unsuccessful (data not shown), but at 100 lM the growth area
and cell morphology were unchanged (Fig. 4E and F), probably as a
result of the slow, sustained release of RSV. Therefore, liposomes
act as an appropriate, inert carrier system.
The last but not the least question of our research was if RSV
diffuses through the cell membrane alone or it is carried into the
cells by liposome uptake. Matthaus et al. used Raman microspec-
troscopy to show that liposomes enter cells by endocytotic pro-
cesses [20]. In our case, localization of the liposomes was
confirmed by fluorescently labeling liposomes which appeared as
green dots dispersed throughout the cytoplasm around blue nuclei
and embedded with red-stained actinic fibres (Fig. 5; e-LIP and
LIP–RSV 100 lM). The cell morphology was unchanged illustrating
their biocompatibility. On the other hand, actin fibres were de-
graded and hardly seen after addition of RSV 100 lM which
pointed on its harmful effect (Fig. 5; RSV 100 lM). The results
are supported with figures of cell population obtained by using in-
vert microscope (Fig. 5; first column). It is shown, that cell conflu-
ence was similar between control cells and those treated with e-LIP
and LIP–RSV 100 lM, while the cell growing area became rarely
covered when RSV at 100 lM was presented in cell culture for
24 h.
4. Discussion
The influence of RSV delivered by liposomes on cell metabolic
and antioxidant activity and its stress protective effect has been
compared with free RSV. Liposomes clearly enable the carrier-
mediated uptake of RSV by the cells and thus influence its intracel-
lular fate.
Free radicals are ubiquitous in cells being generated by normal
physiological processes, and their production is enhanced by UV-
irradiation [21]. Because free radicals can cause cellular damage,
organisms have evolved several defense lines. The most important
Table 1
Cell metabolic and redox activities determined by MTS assay and EPR without (no UV)
or with UV-irradiation for 1 h (UV). Cells were treated with medium only (control),
RSV solution at 10 lM or 100 lM for 24 h. Values are reported relative to the result of
non-treated and non-radiated control cells as mean ± SD; N = 3.
ConditionNo UVUV
Metabolic-redox activityMTSEPRMTSEPR
Control
RSV 10 lM
RSV 100 lM
1.00 ± 0.00
1.11 ± 0.009
0.57 ± 0.01
1.00 ± 0.31
1.27 ± 0.23
0.59 ± 0.22
0.56 ± 0.00
0.84 ± 0.12
0.50 ± 0.07
0.53 ± 0.27
1.06 ± 0.23
0.35 ± 0.18
1.74 ± 0.331.74 ± 0.332.93 ± 0.542.93 ± 0.54
RSV 100 µM
5.25 ± 0.565.25 ± 0.566.26 ± 0.576.26 ± 0.57
RSV 10 µM
2.64 ± 0.572.64 ± 0.574.94 ± 0.764.94 ± 0.76
Control
UV-B IrradiatedNon Irradiated
Rate constant (k) [(1/s) *10-2]
Rate constant (k) [(1/s) *10-2]
C
0.4
0.5
0.6
0.7
0.8
0.9
1
1.1
036912151821
no UV
I/Io
RSV 100 µM
RSV 10 µM
Control
Measurement time (min)
A
0.4
0.5
0.6
0.7
0.8
0.9
1
1.1
036912 1518 21
UV
I/Io
RSV 100 µM
RSV 10 µM
Control
Measurement time (min)
B
Fig. 3. First order kinetics of RSV-mediated EPR signal reduction of spin-label MeFASL (10, 3) in HEK293 cells that were (A) – non-irradiated or (B) – UV irradiated. (C) – Table
shows the rate constant (1/S) of nitroxide reduction curves. Cells were treated with medium only (control), RSV at 10 or 100 lM for 24 h, exposed to non- or UV-irradiation
and spin labeled by adding 2.5 nmol of MeFASL (10, 3) per 106cells. Subsequently, EPR spectra were recorded at the given time points. Reduction curves were normalized to
the initial signal intensity.
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are enzymatic (e.g. superoxide dismutase, catalase) and non-enzy-
matic (e.g. glutathione, vitamins E and C, ubiquinone) antioxidant
systems [22]. An external supply of RSV is beneficial in overpro-
duction scavenging directly and indirectly, influencing the cell-re-
dox-signaling pathways via regulation of enzymes activity. All
these processes are modulated via mitochondria [6].
Mitochondrial activity after RSV treatment and radiation-in-
duced stress was assessed by EPR, for activity of the cell-redox sys-
tem, and MTS assay, for mitochondrial dehydrogenase activity.
These methods gave very similar results showing a dose-depen-
dent effect of RSV on mitochondrial activity. Larger amounts of
RSV triggered a cytotoxic effect, seen as lowered activity of dehy-
drogenase and the cell-redox system (Figs. 1 and 3).
RSV is known to upregulate endogenous antioxidant enzymes
and other enzymes involved in protein replication and cell division
[10]. RSV at 25 and 50 lM inhibited cell proliferation by cell cycle
arrest in the G1/S phase [23]. The effect of RSV on growth and cell
activity varied with cell type and depended on RSV concentration
used and external conditions [10,24–28]. Interestingly, Stojanovic ´
et al. [29] observed that RSV is a better radical scavenger than vita-
min E.
The effectiveness of antioxidants incorporated into liposomes
has already been investigated by other researchers and shown to
be appropriate delivery system for RSV for several reasons
[14,30]. It is readily incorporated into their lipophilic bilayers be-
cause of its physical–chemical characteristics and transfer from
them into the lipid domains of biological membranes. The amphi-
philic properties of cell phospholipids enable further delivery to
multiple intracellular sites as is shown in Fig. 5 and reported by
Fang et al. [31]. Similar passive distribution of spin labels between
the membranes and the uptake of the carrier into leukocytes was
reported by Kristl et al. [32].
Localization of RSV into liposomal bilayers prevents transfor-
mation of the active trans conformation into inactive cis form
(shown by HPLC analysis, 2.2.4). The –OH groups responsible for
antioxidant activity are disposed at the liposomal surface for scav-
enging of radicals. Description of molecular orientation on the
hydrophilic–lipophilic interface with respect to the chemical struc-
ture of compound can be found in the literature [33].
Finally, the liposomal bilayers store RSV and thus preventing
overloading of the cell membranes. Its lipophilicity ensures that re-
CONTROL
e-LIP
LIP-RSV
100 µM
A
FE
DC
B
0.0
0.5
1.0
1.5
2.0
no UVUV 1 h
Metabolic activity
(fraction of non-irradiated control)
Control
e-LIP
LIP-RSV 100 µM
**
*
*
**
Fig. 4. Effects of liposomal preparations on metabolic activity and morphology of
HEK293 cells treated with liposomal formulations and exposed to UV stress.
(Above) Bar charts show effects on metabolic activity after 24-h incubation of cells
with medium (control), empty liposomes (e-LIP) or liposomes loaded with RSV at
100 lM (LIP–RSV 100 lM) and further non-irradiated (no UV) or irradiated with
UV-B irradiation for 1 h (UV 1 h). Calculations were made from the absorbance
measured after addition of MTS reagent for 3 h relative to non-radiated control
(mean ± sd; N = 3;
micrographs of cells (untreated (control) or treated with e-LIP or LIP–RSV 100 lM)
show green-stained actin fibres embedded by single cytoplasmic membrane. First
column is non-radiated cells, while second column is irradiated cells. Bar is 50 lm.
*p < 0.05; and
**p < 0.001). (below) Fluorescent-transmission
Fig. 5. Internalization of liposomes in HEK293 and their morphology after
treatment for 24 h with medium (control), free resveratrol (RSV 100 lM), empty
liposomes (e-LIP), or with liposomes loaded with resveratrol (LIP–RSV 100 lM).
First column presents treated cells, pictured with an inverted phase-contrast
microscope (Olympus CKX41, Tokyo, Japan), while pictures in second column were
taken using the 60-fold objective on an Olympus IX 81 fluorescence microscope. On
the pictures with ‘‘blue–green” fluorescence cells ‘‘nuclei-actin” are indicated, while
with the ‘‘blue–red–green” fluorescence cells ‘‘nuclei–actin–liposomes” are stained.
Bar is 10 lm.
258
J. Kristl et al./European Journal of Pharmaceutics and Biopharmaceutics 73 (2009) 253–259

Page 7

lease of RSV into the cytosol is slow, which consecutively reduces
its cytotoxicity. Liposomal bilayers offer the environment for RSV
storage and thus cell membranes are prevented against high RSV
loading, which caused deteriorative effects on the cells.
This study shows that RSV increases cell antioxidant capacity
that can be consequences of direct scavenging action or the activa-
tion of the cellular pathways, which upregulate endogenous anti-
oxidant enzymes. Nevertheless, liposomal formulation enables
prolonged intracellular delivery of RSV with no cytotoxic response.
5. Conclusions
RSV is effective in protecting cells from free radical damage only
when it is loaded into liposomes. Characteristics of RSV prefer its
localization at the liposome surface, where it remains in the bio-
logically effective trans conformation. Suchlike delivery system
prevents the cells against cytotoxicity of RSV for long-term at
100 lM, controls its release and stimulates cell metabolic and anti-
oxidant activities, which obviates the harmful effect of stress.
Acknowledgements
We acknowledge Dr. John Pugh for proof reading the manu-
script. This work was funded by EU under the Marie Curie Early
Stage Scholarship Program, Project name: Towards a Euro-PhD in
advanced drug delivery, Contract No.: MEST-CT-2004-504992
and by the Ministry of Higher Education, Science and Technology
of the Republic of Slovenia, research group no. 0787-001.
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