International Journal of Pharmaceutics 538 (2018) 40–47

Contents lists available at ScienceDirect

International Journal of Pharmaceutics

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

Stability, biocompatibility and antioxidant activity of PEG-modified T

liposomes containing resveratrol
⁎
Carla Caddeoa, , Laura Puccib, Morena Gabrieleb, Claudia Carbonec,
Xavier Fernàndez-Busquetsd,e,f, Donatella Valentia, Ramon Ponsg, Antonio Vassalloh,
Anna Maria Faddaa, Maria Manconia
a
Dept. of Scienze della Vita e dell’Ambiente, Sezione di Scienze del Farmaco, University of Cagliari, Via Ospedale 72, 09124, Cagliari, Italy
b
Institute of Agricultural Biology and Biotechnology, CNR Pisa, Via Moruzzi 1, 56124, Pisa, Italy
c
Dept. of Scienze del Farmaco, University of Catania, Viale A. Doria 6, 95125, Catania, Italy
d
Nanomalaria Group, Institute for Bioengineering of Catalonia (IBEC), The Barcelona Institute of Science and Technology, Baldiri Reixac 10-12, Barcelona, E08028,
Spain
e
Barcelona Institute for Global Health (ISGlobal), Barcelona Centre for International Health Research (CRESIB, Hospital Clínic-Universitat de Barcelona), Rosselló 149-
153, Barcelona E08036, Spain
f
Nanoscience and Nanotechnology Institute (IN2UB), University of Barcelona, Martí i Franquès 1, ES-08028 Barcelona, Spain
g
Dept. of Tecnologia Química i de Tensioactius, Institut de Química Avançada de Catalunya (IQAC-CSIC), Jordi Girona 18-26, 08034 Barcelona, Spain
h
Dept. of Science, University of Basilicata, Viale dell’Ateneo Lucano 10, 85100 Potenza, Italy

A R T I C L E I N F O A B S T R A C T

Keywords: The present investigation reports the development of PEG-modified liposomes for the delivery of naturally oc-
Resveratrol curring resveratrol. PEG-modified liposomes were prepared by direct sonication of the phospholipid aqueous
Antioxidant dispersion, in the presence of two PEG-surfactants. Small, spherical, unilamellar vesicles were produced, as
PEG-surfactants demonstrated by light scattering, cryo-TEM, and SAXS. The aging of the vesicles was assessed by using the
PEG-modified liposomes
Turbiscan® technology, and their physical stability was evaluated in vitro in simulated body fluids, results
Human erythrocytes
showing that the key features of the liposomes were preserved. The biocompatibility of the formulations was
demonstrated in an ex vivo model of hemolysis in human erythrocytes. Further, the incorporation of resveratrol
in PEG-modified liposomes did not affect its intrinsic antioxidant activity, as DPPH radical was almost com-
pletely inhibited, and the vesicles were also able to ensure an optimal protection against oxidative stress in an ex
vivo human erythrocytes-based model. Therefore, the proposed PEG-modified liposomes, which were prepared
by a simple and reliable method, represent an interesting approach to safely deliver resveratrol, ensuring the
preservation of the carrier structural integrity in the biological fluids, and the antioxidant efficacy of the
polyphenol to be exploited against oxidative stress associated with cancer.

1. Introduction metabolism, which lead to subtherapeutic levels in vivo and require

Polyphenolic compounds, such as resveratrol, have gained great
interest in the pharmaceutical research area due to antioxidant, anti-
inflammatory, anticarcinogenic, antibacterial, and antiviral properties
(Caddeo et al., 2016; Lund and Pantuso, 2014). This wide spectrum of
therapeutic activities, coupled with the safety profile (GRAS status) and
natural origin, make resveratrol an attractive candidate for the devel-
opment of novel pharmaceutical products (Augustin et al., 2013; Shih
et al., 2013). However, the potential health benefits of resveratrol are
limited by poor aqueous solubility (50 μg/ml) (Robinson et al., 2015),
instability (e.g., temperature, light, pH; Zupančič et al, 2015), and rapid
higher or more frequent doses (Cadena et al., 2013). In this framework,
liposomes have been proven to be effective in protecting, controlling
the release, and enhancing the action of bioactive compounds (Sper
Simão et al., 2015). However, liposomes circulating in the blood stream
are rapidly captured and removed by the mononuclear phagocyte
system, if not properly designed. Liposome surface modification by
using hydrophilic polyethylene glycol (PEG) has been shown to prolong
circulation time in the blood, thanks to the shielding effect of PEG
against opsonin recognition and consequent liposome phagocytosis
upon binding (Liang et al., 2005). Various surface modification ap-
proaches have been investigated, including the use of phospholipid-PEG

⁎
Corresponding author.
E-mail address: caddeoc@unica.it (C. Caddeo).

https://doi.org/10.1016/j.ijpharm.2017.12.047
Received 19 September 2017; Received in revised form 27 December 2017; Accepted 29 December 2017
Available online 30 December 2017
0378-5173/ © 2017 Elsevier B.V. All rights reserved.
C. Caddeo et al.
conjugates or PEG-containing block copolymers (Pluronics): the lipid/
hydrophobic part is anchored into the liposome bilayer, and the PEG
chains surround the surface providing an effective steric stabilization
(Oberoi et al., 2016; Pitto-Barry and Barry, 2014; Wang et al., 2013).
While synthetic PEGylated phospholipids are expensive and have raised
immunogenicity and accelerated blood clearance concerns, Pluronic
copolymers are inexpensive, biocompatible, and FDA-approved for
parenteral injection (Wang et al., 2013; Ishida and Kiwada, 2008). In
addition, they are commercially available in a number of different block
lengths, and since the end groups consist of hydroxyl groups, they
provide a platform, similar to the PEGylated phospholipids, for con-
jugation of various small molecule-based targeting agents, such as an-
tibodies, peptides, and vitamins.
Herein, we describe the development and optimization of liposomes
modified with two PEG-surfactants, Pluronic® L64 (poly(ethylene
glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol); PEG-
PPG-PEG) and tocopherol PEG succinate (TPGS), for the delivery of
antioxidant resveratrol by evaluating the improvement of liposome
stability and safety in the biological milieu. Further, the current study
was carried out to determine whether liposomes would increase re-
sveratrol bioactivity in coping with oxidative stress in vitro and ex vivo.
The formulation is expected to have superior efficacy over the free
agent, owing to the advantages offered by the nanovector, specifically
tailored for systemic delivery.
It was previously reported that the intravenous administration of
resveratrol resulted in a short t1/2 ranging from 7.8 to 33 min (Colom
et al., 2011; Marier et al., 2002). Das et al. (2008) prepared intravenous
formulations of resveratrol with HP- and RM-β-cyclodextrins, and stu-
died the impact of the latter on the pharmacokinetics of the polyphenol
in healthy rats. Although complexation with cyclodextrins was able to
largely increase resveratrol solubility, the pharmacokinetic profile
showed no change as compared to unformulated resveratrol. The in-
travenous injection of 5 mg/kg body weight of liposomal resveratrol in
mice with subcutaneous head and neck squamous cell carcinoma led to
a significant reduction (∼70%) in tumor volume (Coimbra et al.,
2011). In an attempt to prolong the systemic circulation of resveratrol,
enhance its biological half-life and passive brain targeting, solid lipid
nanoparticles (SLN) coated with TPGS were developed (Vijayakumar
et al., 2016). After intravenous administration in rats, resveratrol TPGS-
SLN showed ∼11 and 9 times higher area under the curve and plasma
half-life than resveratrol solution. Moreover, brain distribution of re-
sveratrol TPGS-SLN was found to be ∼9 times higher in comparison
with that of unformulated resveratrol. To the best of our knowledge, the
possibility of overcoming the physicochemical, pharmacokinetic and
metabolic limitations of resveratrol through the incorporation in lipo-
somes modified with PEG-surfactants that allow protection, controlled
release, and targeting functionalities, has not been explored so far.
2. Materials and methods
2.1. Materials
Phospholipon 90G (P90G) was purchased from Lipoid GmbH
(Ludwigshafen, Germany). resveratrol (RSV, trans-3,5,4’-trihydrox-
ystilbene; > 99% pure), poly(ethylene glycol)-block-poly(propylene
glycol)-block-poly(ethylene glycol) (PEG-PPG-PEG; Pluronic® L64;
average Mw ∼2900), D-α-tocopherol polyethylene glycol 1000 succi-
nate (TPGS; average Mw ∼1513), and all other reagents, if not other-
wise specified, were purchased from Sigma-Aldrich (Milan, Italy).
2.2. Vesicle preparation and characterization
P90G, PEG-PPG-PEG, TPGS, and resveratrol were weighed in a glass
vial and left hydrating overnight in water (Table 1). To obtain PEG-
modified liposomes, the dispersion was sonicated (2 s on and 1 s off, 30
cycles; 13 μm of probe amplitude) in an ice bath with a Soniprep 150
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International Journal of Pharmaceutics 538 (2018) 40–47
Table 1
Composition of the vesicular formulations.
P90G PEG-PPG-PEG TPGS RSV H2 O
RSV PEG-modified liposomes 90 mg 9 mg 5 mg 5 mg 1 ml
Empty PEG-modified liposomes 90 mg 9 mg 5 mg 1 ml
RSV liposomes 90 mg 5 mg 1 ml
Empty liposomes 90 mg 1 ml
P90G, phosphatidylcholine.
PEG-PPG-PEG, poly(ethylene glycol)-poly(propylene glycol)-poly(ethylene glycol) tri-
block copolymer.
TPGS, tocopherol PEG1000 succinate.
RSV, resveratrol.
(MSE Crowley, London, UK). Empty PEG-modified liposomes and con-
ventional liposomes (both empty and loaded with resveratrol, without
PEG-PPG-PEG and TPGS) were also prepared (Table 1). All the samples
were prepared and kept in the dark during the experimental time.
Vesicle formation and morphology were examined by cryogenic
transmission electron microscopy (cryo-TEM). After glow-discharge to
make the carbon film on a Holey Carbon 400-mesh copper grid hy-
drophilic, a 3 μl drop of the sample was deposited onto it. The grid was
mounted on a plunger (Leica EM GP) and blotted with a Whatman No.1
filter paper. The vesicle dispersion was immediately vitrified by rapid
immersion in liquid ethane. The grid with the vitrified sample was
mounted on a Gatan 626 cryo-transfer system and inserted into a Jeol
JEM 2011 cryo-electron microscope operated at 200 kV, under low-
dose conditions, and using different degrees of defocus (500–900 nm) to
obtain an adequate phase contrast. Images were recorded on a Gatan
Ultrascan US1000 CCD camera and analyzed with the Digital
Micrograph 1.8 software.
The average diameter (i.e., the intensity weighted mean hydro-
dynamic size), polydispersity index (P.I., a dimensionless measure of
the broadness of the size distribution), and zeta potential of vesicles
were determined by Dynamic and Electrophoretic Light Scattering
using a Zetasizer nano-ZS (Malvern Instruments, Worcestershire, UK).
Samples (n = 6) were diluted with bidistilled water (1:100) and ana-
lysed at 25 °C.
Vesicles were purified from the non-incorporated drug by dialysis.
Each sample (2 ml) was loaded into Spectra/Por® tubing (12–14 kDa
MW cut-off; Spectrum Laboratories Inc., DG Breda, The Netherlands)
and dialyzed against bidistilled water (1 l) for 2 h, at room temperature.
The entrapment efficiency (EE) was calculated by using the following
formula:
amount ofRSV in purified vesicles ⎞
EE = ⎜⎛ ⎟ x 100
⎝ amount ofRSV in unpurified vesicles ⎠ (1)
and drug loading (DL) was calculated by using the following for-
mula:
amount ofRSV in purified vesicles
DL = ⎜⎛ ⎞
⎟ x100
⎝ amount ofphospholipid in purified vesicles ⎠ (2)
after disruption of unpurified and purified vesicles with methanol.
The resveratrol content was assayed by HPLC (Alliance 2690,
Waters, Milan, Italy) using a XSelect C18 column (3.5 μ, 4.6 × 150 mm,
Waters), with a mobile phase made of methanol, acetonitrile, water,
and acetic acid (65:31.5:3.38:0.12, v/v), and a flow of 0.8 ml/min. A306
was measured for resveratrol (tR = 1.3 min).
The phospholipid content was assessed by the Stewart assay, as
previously reported (Caddeo et al., 2015). An aliquot of the liposome
formulations (1:1000) was added to a biphasic mixture of aqueous
ammonium ferrothiocyanate solution (0.1 N; 2 ml) and chloroform
(2 ml). The concentration of P90G, in both unpurified and purified
samples, was obtained by measuring the absorbance at 485 nm into the
organic phase. A calibration curve was built by using different volumes
C. Caddeo et al.
(0–500 μl) of a P90G stock solution (1 mg/ml in chloroform), adding
chloroform to make the final volume of the organic phase 2 ml, and
mixing with 2 ml of the aqueous ammonium ferrothiocyanate solution.
2.3. Small-angle X-ray scattering
Small-Angle X-ray Scattering (SAXS) measurements were carried
out using a S3-MICRO (Hecus X-ray systems GMBH Graz, Austria)
coupled to a GENIX-Fox 3D X-ray source (Xenocs, Grenoble), which
provides a detector focussed X-ray beam with λ = 0.1542 nm Cu Kα-
line with more than 97% purity and less than 0.3% Kβ. Transmitted
scattering was detected by using a PSD 50 Hecus. Temperature was
controlled by means of a Peltier TCCS-3 Hecus. The samples were in-
serted in a flow-through glass capillary of 1 mm diameter and 10 μm
wall thickness. The SAXS scattering curves are shown as a function of
the scattering vector modulus:
q = (4π/λ)·sin(θ/2) (3)
where θ is the scattering angle. The q values with this setup ranged
from 0.1 to 6.0 nm−1. The scattering vector was calibrated by mea-
suring a standard silver behenate sample. Because of the use of a de-
tector focussed small beam (300 × 400 μm full width at half maximum)
the scattering curves are mainly smeared by the detector width and
detector depth. This smearing mainly produces a widening of the peaks
without noticeable effect on the peak position in the small angle re-
gime. The scattering curves have been background subtracted taking
into account the concentration of solvent and surfactant in the samples.
The instrumentally smeared experimental SAXS curves were fitted to
numerically smeared models for beam size, detector width and detector
depth effects. A least squares routine based on the Levenberg-
Marquardt scheme was used. A model of the scattering by liposomes
was used, based on the description of the bilayer electronic density
profile using a Gaussian for each polar head and a Gaussian for the
description of the methyl dip at the centre of the bilayer, and an error
function to describe the methylene contribution. The stacking of the
lamellae in the multilamellar liposomes is based on the Caillé descrip-
tion. For more details, see for instance (Caddeo et al., 2017).
2.4. Physical stability of the formulations
The stability of resveratrol PEG-modified liposomes and conven-
tional liposomes was evaluated by standard long-term stability tests, i.e.
analysing vesicle mean size, P.I. and zeta potential over 2 months at
4–8 °C, and by accelerated stability tests using the Turbiscan® AG
Station (Formulaction, l’Union, France). The apparatus consists of the
Turbiscan Lab Expert, which measures the multiple dispersion of light
by the samples, and an Aging Station constituted by a robot with three
thermoregulated blocks for sample storage. Each sample (20 ml) was
placed in a glass cell in the Turbiscan block and kept at 25 and 40 °C.
The detection head, which is composed of a pulsed near-infrared light
source (λ = 850 nm), scans the height of the sample cell and acquires
transmitted (T) and backscattered (BS) light every 40 μm. The
Turbiscan® makes scans at various pre-programmed times and overlays
the profiles on one graph, showing the presence of destabilization
phenomena, such as flocculation, coalescence, creaming, and sedi-
mentation. In our experiments, the stability of the samples was eval-
uated on the basis of the variation of backscattering (ΔBS).
Backscattering variations are shown in ordinate, and the height of the
cell in abscissa. The first profile is displayed in blue (day 0), and the last
one in red (day 21). Further, the Turbiscan Stability Index (TSI) was
used for a comparative evaluation between the different samples,
especially focusing on the improvement of the stability of the vesicles
by the surface modification (Carbone et al., 2016; Carbone et al., 2014).
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International Journal of Pharmaceutics 538 (2018) 40–47
2.5. In vitro drug release studies
The in vitro release of resveratrol from the vesicular formulations
was assessed in saline with 1% Tween80, a surfactant commonly added
to dissolution media in case of sparingly water soluble active molecules.
The formulations (2 ml) were loaded in dialysis tubes (see Section 2.2)
immersed in the dissolution medium (500 ml) and thermostated at
37.0 ± 0.5 °C by using a USP basket dissolution apparatus. At sched-
uled time intervals up to 24 h, an aliquot of the formulations was
withdrawn, and the medium was refreshed to ensure sink conditions.
The resveratrol content was assayed by HPLC (see Section 2.2). The
experiments were performed in triplicate.
2.6. Vesicle behavior in simulated biological fluids
The behavior of the vesicles in biological fluids was evaluated by
incubating the formulations with Hank's balanced salt solution (pH
7.4 ± 0.2), which was prepared according to the composition reported
by Yang et al. (2007), or in fetal bovine serum (Gibco™, Fisher Scien-
tific, Milan, Italy). Hanks' solution is commonly used to simulate body
fluids, as it contains inorganic salts and glucose that confer a similar
osmotic pressure as that of the intercellular fluids (Witecka et al.,
2016). The average diameter and polydispersity index of the vesicles
were measured immediately after dilution (1:100 v:v) of the vesicles
with Hank’s solution or serum, and after 24 h of incubation at 37 °C.
2.7. Biocompatibility of the formulation: ex vivo hemolytic activity
Erythrocytes were collected from healthy blood donors upon in-
formed consent for the use of residual blood for research purposes,
according to the Italian regulations and, in particular, the regulations of
“Fondazione G. Monasterio CNR-Regione Toscana”. The blood samples
were collected in EDTA-treated tubes, and centrifuged (2300×g) for
10 min at 4 °C. Plasma and buffy coat were discarded. Erythrocyte he-
molysis was measured as previously described by Frassinetti et al.
(2015), with some modifications. Briefly, an erythrocyte suspension at
5% hematocrit was incubated for 18 or 24 h with PBS (spontaneous
hemolysis), or resveratrol in ethanolic solution or in liposomes, in order
to evaluate their biocompatibility and exclude hemolytic effects. Total
erythrocyte hemolysis was evaluated by exposure of 5% erythrocyte
suspension to 50 mM AAPH (2,2’-azobis (2-amidinopropane) dihy-
drochloride), a peroxyl radical generator used as a positive control (the
extent of hemolysis is proportional to the amount of free radicals
formed). After incubation, the samples were centrifuged for 5 min at
1000× g. After a 10-fold dilution in PBS, the hemolysis was spectro-
photometrically evaluated at 540 nm as hemoglobin released in su-
pernatants. Data were expressed as the percentage of hemolysis relative
to spontaneous hemolysis.
2.8. In vitro antioxidant activity: DPPH assay
The antioxidant activity of resveratrol was assessed by evaluating its
ability to scavenge 2,2-diphenyl-1-picrylhydrazyl (DPPH), a stable ni-
trogen-centered free radical. DPPH was dissolved in methanol to yield a
25 μM concentration, and 980 μl of this solution was mixed either with
20 μl of resveratrol in methanolic solution or in liposomes, or with
empty liposomes. All the samples were stored at room temperature for
30 min, in the dark. Thereafter, the absorbance was measured at
517 nm against blank (methanol/water). The percent antioxidant ac-
tivity (or free radical scavenging activity) was calculated according to
the following formula, where A is the absorbance:
ADPPH − Asample ⎞
Antioxidantactivity = ⎛⎜ x 100
⎟
⎝ ADPPH ⎠ (4)
All the experiments were performed in sextuplicate.
C. Caddeo et al.
Table 2
Characteristics of PEG-modified liposomes and conventional liposomes in the absence or presence of resveratrol: mean diameter (MD), polydispersity index (P.I.), zeta potential (ZP),
entrapment efficiency (EE), and drug loading (DL). Each value represents the mean ± SD, n = 6 at least.
MD nm ± SD
RSV PEG-modified liposomes 86 ± 2.7
Empty PEG-modified liposomes 84 ± 3.4
RSV liposomes 171 ± 27.8
Empty liposomes 163 ± 23.6
2.9. Ex vivo cellular antioxidant activity (CAA) in human erythrocytes
The antioxidant activity of the resveratrol formulations was eval-
uated in an ex vivo erythrocyte model, as described by Frassinetti et al.
(2015) with some modifications.
Erythrocytes were collected as reported above (Section 2.7), diluted
with PBS (1:100) to have a final number of about 105 cells/ml, and
incubated for 2 or 4 h at 37 °C with resveratrol in ethanolic solution or
in liposomes (1:100, 1:500, and 1:2000), or Trolox (6-hydroxy-2,5,7,8-
tetramethylchroman-2-carboxylic acid; 500 μM), used as a reference
antioxidant standard, or PBS used as a blank and control sample. An
oxidant-sensing fluorescent probe, 2′,7′-dichlorofluorescein diacetate
(DCFH-DA), was added to all the samples to a final concentration of
15 μM. After incubation, the erythrocytes were washed twice to remove
excess samples, and re-suspended in cold PBS. 180 μl of erythrocyte
suspension was transferred to a 96-well microplate and 20 μl of AAPH
(peroxyl radical generator) was added to a final concentration of
1.2 mM. The fluorescence generated upon oxidation of DCFH-DA to
highly fluorescent DCF by intracellular radicals, and its reduction in
intensity as the radicals are scavenged by the antioxidant samples, was
read at 485 nm excitation and 535 nm emission by using a VictorTM X3
Multilabel Plate Reader (Perkin Elmer, Waltham, MA). Each value was
expressed as cellular antioxidant activity (CAA) units, according to
Wolfe and Liu (2007), as follows:
CAA unit = 100 − (∫ SA ⁄ ∫ CA) × (100)
where ∫ SA is the integrated area from the sample curve, and ∫ CA is the
integrated area from the control curve (AAPH only treated cells).
2.10. Statistical analysis of data
Results are expressed as the mean ± standard deviation (SD) or
standard error of mean (SEM). Data were analyzed statistically by
ANOVA and Tukey’s test to substantiate statistical differences between
groups, while Student’s t-test was used to compare two samples.
Significant differences (p < 0.05) are denoted by different letters.
3. Results and discussion
3.1. Vesicle design and characterization
The present investigation was carried out to examine the possibility
of developing liposomes modified with PEG-surfactants as a surface
modification strategy to improve their stability, and to ascertain whe-
ther such system would protect and deliver resveratrol, without af-
fecting its antioxidant power.
The surface modifiers used in this study were PEG-PPG-PEG, a
water-soluble block copolymer non-ionic surfactant (HLB 12–18), and
TPGS, a water-soluble non-ionic surfactant (HLB 13.2) formed by the
esterification of vitamin E succinate with PEG 1000, both FDA-ap-
proved for pharmaceutical use (Guo et al., 2013; Pitto-Barry and Barry,
2014; Zhang et al., 2012). The assembling of PEG on the liposome bi-
layer was achieved by allowing the two PEG-surfactants to participate
in the vesicle formation process, so that the hydrophobic portions (the
PPG block for PEG-PPG-PEG and the alkyl chain for TPGS) become an
International Journal of Pharmaceutics 538 (2018) 40–47
P.I. ± SD ZP mV ± SD EE % ± SD DL % ± SD
0.20 ± 0.03 −21 ± 4.1 94.9 ± 2.9 5.3 ± 0.2
0.27 ± 0.01 −15 ± 1.6
0.41 ± 0.07 −25 ± 1.9 87.9 ± 6.7 4.9 ± 0.4
0.42 ± 0.09 −19 ± 1.5
integral part of the bilayer, and the hydrophilic portions (the PEG
chains) are projected on one or both sides of the bilayer. This process
provides strong anchoring of PEG onto the liposomes, thus preventing
desorption during particle collision (Kostarelos et al., 1999).
PEG-modified liposomes were prepared by a simple method invol-
ving the sonication of the phospholipid (P90G), PEG-PPG-PEG, TPGS,
and resveratrol in water. To evaluate the effect of the two PEG-sur-
factants and the loading of the polyphenol on the vesicle arrangement,
conventional liposomes were also prepared and characterized, along
with empty systems (Table 1). As shown by Dynamic Light Scattering
results summarized in Table 2, PEG-modified liposomes displayed small
size (∼ 85 nm), good homogeneity (P.I. −0.2) and negative zeta po-
tential (−20 mV), regardless of the presence of resveratrol. On the
other hand, based on the comparison with the results from conventional
liposomes, the effect of PEG-PPG-PEG and TPGS was dramatic. Con-
ventional liposomes exhibited a diameter twice as large as PEG-mod-
ified liposomes, with broad size distribution (−170 nm, P.I. −0.4;
Table 2). As the PEG-surfactants participate in the vesicle formation,
their hydrophobic portions arrange along with the phospholipid as in-
tegral parts of the bilayer, altering the geometric packing and la-
mellarity, and thus the vesicle diameter, while the PEG chains extend
on one side of the bilayer or on either side of it. Hence, it is evident that
the addition of the PEG-PPG-PEG and TPGS was crucial to produce
vesicles with optimum size and homogeneity, while the presence of
resveratrol had a negligible effect on the vesicle assembly (Table 2).
The entrapment efficiency was approximately 95% for PEG-mod-
ified liposomes (Table 2), and the amount of loaded resveratrol did not
diminish significantly during storage over the course of 2 months, with
no sign of degradation of the polyphenol. The entrapment efficiency of
conventional liposomes was lower, even if not statistically different
(88%; p > 0.05). It is reasonable to presume that the PEG-surfactants
facilitated the solubilisation of lipophilic resveratrol and thus its
loading within the vesicles (reflected in higher entrapment efficiency),
besides favouring the arrangement of the phospholipid molecules in
small, uniformly-sized structures. Drug loading, calculated as a function
of the weight of the phospholipid, was 5.3 and 4.9% for PEG-modified
liposomes and conventional liposomes, respectively (Table 2).
The vesicles were observed under cryo-TEM, which allowed the
accurate determination of their morphology and lamellar structure
(Fig. 1). In agreement with size measurements, it was found that PEG-
modified liposomes were small in size (< 100 nm), and displayed fairly
spherical shape with predominant unilamellar morphology (Fig. 1A).
On the other hand, larger, oligo/multilamellar vesicles were detected in
conventional liposomes (Fig. 1B). These findings point to a reduction in
size and lamellarity induced by the participation of the two PEG-sur-
factants in the vesicle formation.
The SAXS patterns of resveratrol PEG-modified liposomes and
conventional liposomes are shown in Fig. 2, together with the fits of the
lamellar model. The conventional liposomes where fitted allowing for
all the parameters to be optimized. This produced a very good fit,
showing the presence of oligolamellar vesicles, as the scattering curve
featured two bumps (4–5 correlated lamellae). PEG-modified liposomes
were fitted fixing the parameters corresponding to the electronic den-
sity profile of the liposome fit, and fixing also the number of correlated
lamellae to no correlation. Therefore, only the intensity and flat
43
C. Caddeo et al.
Fig. 2. SAXS patterns of resveratrol (RSV) PEG-modified liposomes (open symbols) and
conventional liposomes (closed symbols) together with model best fits (lines). The scat-
tered intensity is shown as a function of the scattering q vector, at 25 °C.
background correction was used for this fit. The quality of the fit was
very good, showing the presence of unilamellar vesicles, as indicated by
the broad symmetric band (Fig. 2). These results are in perfect agree-
ment with structural data from cryo-TEM images.
According to the composition of PEG-modified liposomes, we were
able to calculate a maximum PEG chain density possibly adsorbed onto
the vesicles. This would correspond to about one molecule of PEG every
6 nm2. If we consider an adsorbed layer thickness of 2.6 nm, calculated
as 0.2 nm per ethylene oxide group in a meander configuration (Pons
et al., 1995), the contrast provided by this layer should amount to a
maximum of 0.01 e/Å2, which would be undetectable as compared to
the contrast provided by the phospholipid.
Although from SAXS results we cannot demonstrate directly the
adsorption of the PEG-surfactants on the external surface of liposomes,
there is a clear evidence that the PEG-surfactants interacted with the
bilayer causing an alteration of the phospholipid arrangement and
leading to the formation of unilamellar vesicles. This means that the
PEG-surfactants are not localized solely in the bulk dispersion (where
they would not have any influence on lamellarity), but they must be
significantly adsorbed on the bilayer surfaces, with the thermodinamic
consequence of preventing the lamellar phase from any significant
stacking and stabilizing the bilayer.
The release profiles of resveratrol from PEG-modified liposomes and
conventional liposomes are reported as a function of time in Fig. 3. In
the first 8 h, the release of resveratrol from PEG-modified liposomes
was constant and slower than from conventional liposomes (20% vs.
45% after 8 h), to achieve the same extent of polyphenol released after
24 h (∼70%). Therefore, it can be concluded that PEG-modified lipo-
somes modulated the release in a time-dependent fashion, and that the
PEG-surfactants played an important role in controlling the release rate.
The sustained release is expected to maintain the effective
44
International Journal of Pharmaceutics 538 (2018) 40–47
Fig. 1. Cryo-TEM images of resveratrol PEG-modified liposomes (A)
and resveratrol conventional liposomes (B). Scale bar represents
200 nm.
Fig. 3. Resveratrol (RSV) release from PEG-modified liposomes and conventional lipo-
somes over 24 h in saline with 1% Tween80, at 37 °C. Error bars represent standard de-
viation, n = 3.
concentration of resveratrol in plasma for a long time. Moreover, the
poor bioavailability of resveratrol due to its rapid metabolism and
elimination are prevented by the nanoincorporation, thus prolonging its
biological half-time in vivo (Peng et al., 2010).
3.2. Stability of the vesicles
For safe and effective use of liposomes, it is critical to establish their
stability, which is dependent on both formulation and manufacturing
method parameters. The prepared formulations were monitored in
terms of size distribution and polydispersity. PEG-modified liposomes
showed no sign of physical alteration, as demonstrated by the fact that
their size were basically unchanged after 2 months of storage (MD
91.6 ± 3.2 nm, P.I. 0.19 ± 0.02), whereas conventional liposomes
lacking PEG-surfactants showed progressive aggregation and pre-
cipitation, with the mean diameter exceeding 800 nm and P.I. > 0.9.
To corroborate the crucial role of PEG-PPG-PEG and TPGS for li-
posome stabilization, the Turbiscan® technology was used to build up
accelerated stability profiles, allowing us to identify any destabilization
processes occurring in the vesicle dispersions. As previously reported,
the presence of important variations at the bottom or the top of the
Turbiscan® spectra, in terms of transmission or backscattering, high-
lights the occurrence of migration phenomena (clarification, sedi-
mentation or creaming), while variations in the middle of the spectra
are related to particle aggregation (Carbone et al., 2014).
As depicted in Fig. 4A, the backscattering profile of resveratrol li-
posomes stored at 25 °C shows the presence of instability phenomena
related both to vesicle aggregation and migration, with the formation of
a compact white sediment at the bottom of the test cuvette (Supple-
mentary Video 1). However, the sediment could be easily re-dispersed
by gentle shaking. On the other hand, resveratrol PEG-modified lipo-
somes showed a greater stability, since no vesicle migration was ob-
served and aggregation phenomena were not significant (ΔBS < 10%)
(Fig. 4B, Supplementary Video 2). The higher storage temperature
C. Caddeo et al. International Journal of Pharmaceutics 538 (2018) 40–47

Fig. 4. Turbiscan backscattering profiles (ΔBS) of resveratrol

(RSV) PEG-modified liposomes (A) and conventional lipo-
somes (B) stored at 25 and 40 °C. Data are shown as sample
height (0–20 mm) as a function of time (0-21 days). The sense
of analysis time is indicated by the arrow. The Turbiscan
Stability Index (TSI) is also reported for the samples (C).

tested (40 °C) reduced the stability of the vesicle dispersions, particu-
larly in the case of conventional liposomes, which underwent phase
separation with the formation of a sediment at the bottom of the test
cuvette (Supplementary Fig. 1). TSI index profiles (Fig. 4C) clearly
show that the modification of liposomes with PEG-PPG-PEG and TPGS
led to a significant improvement of the vesicles’ stability at both tem-
peratures, as also confirmed by the visual observation of the samples at
the end of the stability study (Supplementary Fig. 1).
In addition, the stability of the vesicles was evaluated under con-
ditions mimicking the body fluids. PEG-modified liposomes remained
stable after 24 h of incubation with Hank’s solution and serum, as the
size and P.I. did not change from the initial values of ∼94 nm and
∼0.20, respectively (Table 3), which indicates that their structure was
preserved despite osmotic stress and protein adsorption. It is note-
worthy that, under such conditions, the presence of the PEG-surfactants
was not essential to vesicle stability in both media, since conventional
liposomes were also unchanged (Table 3).
human erythrocytes with the liposomal formulations, no significant
effect was observed (data not shown). After 18 h of incubation, a slight
raise of hemolysis was observed in samples diluted 1:100 with respect
to samples diluted 1:500 (Fig. 5A). In particular, empty and resveratrol
loaded conventional liposomes diluted 1:100 induced a greater hemo-
lysis on erythrocytes, but to a much lower extent in comparison with
the control obtained by treating the erythrocytes with AAPH, a radical
generator capable of inducing total oxidative hemolysis.
As shown in Fig. 5B, the hemolysis levels were slightly higher after
24 h of incubation with samples diluted 1:500, and no significant dif-
ferences were observed between the formulations. Once again, con-
ventional liposomes diluted 1:100 induced a greater erythrocytes he-
molysis than PEG-modified liposomes, which confirms the
biocompatibility of the latter.
3.4. Antioxidant activity of the formulations

Antioxidant power plays a major role in resveratrol biological ac-

3.3. Biocompatibility of the formulations
tivity. Therefore, it is crucial to verify that the vesicle system does not
interfere with its inherent activity. First, the antioxidant activity was
Since the prepared liposomal formulations are expected to come
determined in vitro by the DPPH assay. This test exploits the reduction
into direct contact with blood once administered, a hemolysis test was
in concentration of the DPPH radical in the presence of an antioxidant
performed to determine their possible hemolytic effect. Free resveratrol
molecule that captures the odd electron becoming paired with hy-
in ethanolic solution caused erythrocyte hemolysis under the experi-
drogen, the solution discolors, and the degree of this discoloration,
mental conditions tested. On the contrary, after 6 h of incubation of
corresponding to a decrease or loss of absorbance, indicates the
scavenging efficiency of the tested sample. From data listed in Table 4,
Table 3
Mean diameter (MD) and polydispersity index (P.I.) of PEG-modified liposomes and it is evident that resveratrol possesses a potent antioxidant activity, as
conventional liposomes diluted and incubated with a biomimic body fluid (Hank’s solu- expected, since the DPPH radical was almost completely inhibited
tion, pH 7.4) or fetal bovine serum for 24 h, at 37 °C. The measurements were carried out (∼93%). The empty vesicles exhibited antioxidant activity too, due to
immediately after the dilution (t0) and after 24 h (t24). Mean values ± SD are reported active soy phosphatidylcholine (∼25%). Most importantly, the results
(n = 6).
showed that the liposomal formulation did not affect the antioxidant
Time h MD nm ± SD P.I. ± SD activity of the polyphenol (> 90%, p > 0.05 vs. resveratrol methanolic
solution), regardless of the presence of the PEG-surfactants.
RSV PEG-modified t0 94 ± 3.7 0.25 ± 0.04 In addition, the antioxidant activity of resveratrol formulations was
Hank’s liposomes t24 96 ± 4.2 0.23 ± 0.03
solution
assessed ex vivo in human erythrocytes by the CAA test. Erythrocytes
RSV liposomes t0 180 ± 15.7 0.48 ± 0.05 represent the best cell model for antioxidant screening, since they have
t24 166 ± 19.5 0.34 ± 0.07 neither nucleus nor mitochondria, and they play a crucial role in an-
RSV PEG-modified t0 95 ± 5.3 0.20 ± 0.04 tioxidant and anti-inflammatory protection of the body. In this study,
Serum liposomes t24 88 ± 8.1 0.22 ± 0.04
human erythrocytes, under a slight oxidative insult induced by AAPH,
RSV liposomes t0 170 ± 16.3 0.45 ± 0.05
t24 163 ± 24.8 0.32 ± 0.01 were incubated with three different dilutions of liposomal formulations.
After 2 h of incubation, empty liposomes caused erythrocyte hemolysis

45
C. Caddeo et al. International Journal of Pharmaceutics 538 (2018) 40–47

Fig. 5. Effect of PEG-modified liposomes and con-

ventional liposomes, empty or loaded with resvera-
trol (RSV), on the spontaneous hemolysis of human
erythrocytes following 18 h- (A) or 24 h- (B) ex-
posure. The peroxyl radical generator AAPH was
used to induce total oxidative hemolysis. Assays
were carried out in triplicate for two independent
experiments: the results are expressed as mean
values ± SEM. Different letters indicate statistical
differences among samples (p < 0.05) analyzed by
one-way ANOVA with Tukey’s test.

Table 4 exerted an antioxidant activity on erythrocytes, while conventional li-

Antioxidant activity (% AA) of PEG-modified liposomes and conventional li- posomes induced hemolysis during the test, irrespective of the presence
posomes in the presence or absence of resveratrol (RSV), in comparison with a
of resveratrol. These results suggest an important dose-dependent
methanolic solution of the polyphenol, calculated as the inhibition percentage
of DPPH radical. Data are expressed as mean ± SD (n = 6). scavenging effect of resveratrol PEG-modified liposomes on oxidative
stress induced by AAPH on human red blood cells, at all the tested
% AA concentrations.
RSV in solution 92.9 ± 1.7
RSV PEG-modified liposomes 93.1 ± 1.4
Empty PEG-modified liposomes 23.7 ± 2.7 4. Conclusions
RSV liposomes 90.5 ± 1.0
Empty liposomes 26.7 ± 5.3 The results of the present work suggest that the prepared PEG-
modified liposomes represent a promising carrier system for the de-
livery of bioactive resveratrol, in terms of long-term stability and bio-
at the highest tested concentrations (1:100 and 1:500 dilutions; data
compatibility. The potential of the proposed system for therapeutic
not shown). Conversely, a dose-dependent antioxidant effect was ob-
applications against oxidative stress was demonstrated, thus offering
served for PEG-modified liposomes and resveratrol conventional lipo-
good perspectives for the use of resveratrol PEG-modified liposomes in
somes, with no significant differences among CAA values (Fig. 6A). As
vivo, with a special focus on the extension of half-life in the blood cir-
shown in Fig. 6B, after 4 h of incubation, PEG-modified liposomes
culation.

46
C. Caddeo et al.
Acknowledgments
This research was partially supported by grants BIO2014-52872-R
(Ministerio de Economía y Competitividad, Spain), which included
FEDER funds, and 2014-SGR-938 (Generalitat de Catalunya, Spain).
ISGlobal and IBEC are members of the CERCA Programme, Generalitat
de Catalunya. The authors would like to acknowledge Jaume Caelles
from the SAXS-WAXS service at IQAC for the SAXS measurements.
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.ijpharm.2017.12.047.
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