Biophysical Chemistry 233 (2018) 55–63

Contents lists available at ScienceDirect

Biophysical Chemistry
journal homepage: www.elsevier.com/locate/biophyschem

Protective effect of quercetin and rutin encapsulated liposomes on induced T

oxidative stress
⁎ ⁎
Claudia Bonechia,b, , Alessandro Donatia,b, , Gabriella Tamasia,b, Gemma Leonea,c,
⁎
Marco Consumia,c, Claudio Rossia,b, Stefania Lamponia,c, Agnese Magnania,c,
a
Department of Biotechnologies, Chemistry and Pharmacy, University of Siena, Via Aldo Moro 2, 53100 Siena, Italy
b
Centre for Colloid and Surface Science (CSGI), University of Florence, Via della Lastruccia 3, 50019 Sesto Fiorentino, FI, Italy
c
National Interuniversity Consortium of Materials Science and Technology (INSTM), Via G. Giusti 9, 50121 Firenze. Italy

H I G H L I G H T S G RA P H I C A L AB S T R A C T

• face
Biological response to different sur-
liposome charge
• Better cell internalization of anti-
oxidant molecules
• Cell protection from oxidative stress
processes

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

Keywords: Natural antioxidants show many pharmacological properties, but poor solubility and inability to cross cell
Liposome membrane. Liposomes are biocompatible and phospholipid vesicles able to carry hydrophilic, hydrophobic, and
Quercetin amphiphilic molecules. This paper focus on the synthesis of anionic, cationic and zwitterionic liposomes, loaded
Rutin with quercetin or rutin, and on the evaluation of their cytotoxicity and protective effects against oxidative stress.
Oxidative stress
Chemical characterization was obtained by dynamic light scattering and z-potential experiments. In vitro cell
Cell viability
behavior was evaluated by Neutral Red Uptake test.
All liposomes, empty and loaded with antioxidants, are stable. The cytotoxicity of both quercetin and rutin
encapsulated in zwitterionic and anionic liposomes is higher than that of their solutions. Quercetin and rutin
loaded in cationic liposomes are able to inhibit the toxic effect of empty liposomes. The encapsulation of rutin at
5.0 × 10− 5 and 5.0 × 10− 4 M, in zwitterionic and anionic liposomes, protects fibroblasts by H2O2 treatment,
while the loading with quercetin does not have effect on improving cell viability.
All data suggest that the tested liposomes are stable and able to include quercetin and rutin. The liposomes
encapsulation of antioxidants makes easier their internalization by cells. Moreover, zwitterionic and anionic
liposomes loaded with rutin protect cells by oxidative stress. Liposomes stability together with their good in vitro
cytocompatibility, both empty and loaded with antioxidant molecules, makes these systems suitable candidates
as drug delivery systems. Moreover, the encapsulation of rutin, is able to protect cells by oxidative stress.

⁎
Corresponding authors at: Dept. Biotechnology, Chemistry and Pharmacy, University of Siena, Via Aldo Moro 2, 53100 Siena, Italy.
E-mail addresses: claudia.bonechi@unisi.it (C. Bonechi), alessandro.donati@unisi.it (A. Donati), agnese.magnani@unisi.it (A. Magnani).

https://doi.org/10.1016/j.bpc.2017.11.003
Received 28 August 2017; Received in revised form 14 November 2017; Accepted 20 November 2017
Available online 22 November 2017
0301-4622/ © 2017 Elsevier B.V. All rights reserved.
C. Bonechi et al.
1. Introduction
Antioxidants are biomolecules having the ability to protect organ-
isms from diseases associated with oxidative stress, including cardio-
vascular diseases, cancer, inflammation, and other degenerative dis-
orders [1]. Flavonoids, a group of naturally occurring benzo-γ-pyrone
derivatives, have a large number of biological effects and particularly
strong antioxidants and free radical scavengers [2,3]. Flavonoids may
help to prevent and/or reduce oxidative-inflammatory status related to
obesity through the regulation of different molecular pathways, such as
mitogen-activated protein kinase (MAPK), adenosine monophosphate-
activated protein kinase (AMPK), peroxisome proliferator-activated
receptor-α and sterol regulatory element-binding protein-1c pathways
[4].
Rutin (2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-3-[(2S,3R,4S,5S,6R)
-3,4,5-trihydroxy-6-[[(2R,3R,4R,5R,6S)-3,4,5-trihydroxy-6-methyloxan
-2-yl]oxymethyl]oxan-2-yl]oxychromen-4-one, R), is a flavonol glyco-
side, that shows various pharmaceutical proprieties, like anti-in-
flammatory, anti-hypertensive, anti-hemorrhagic, that have been at-
tributed to its ability as free radicals scavenger [5]. It has been reported
that such activity of polyphenols is highly sensitive to the chemical
properties like solvent polarity, micellar solution, etc. [6].
Quercetin (2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxychromen-4-
one, Q) is a lipophilic compound, of great interest for human nutrition
and functional foods [7,8] and it is the most abundant flavonoid in
human dietary sources [9]. The anti-obesity effect of quercetin is
mediated by the AMPK and MAPK signaling pathways [10,11]. Apart
from its antioxidant capacity quercetin is also associated with anti-viral,
anti-inflammatory, anti-bacterial and muscle-relaxing properties [12].
Jan et al. (2012) also reports substantial evidence related to the
chemo-preventive properties of quercetin against certain types of can-
cers (including bladder, prostate, esophagus and stomach), as oxidative
free radicals are considered to be one of the main causes of cancers
formation, as well as tumor proliferation [12]. Many studies have de-
monstrated that levels of low molecular weight compounds in foods,
such as beneficial secondary metabolites, can be affected by heat
treatments that food undergo prior to consumption. Moreover, post-
harvest processing can seriously reduce the level of phytochemicals in
plant materials, by as much as 70% in some instances [13].
Quercetin and rutin protect against ultraviolet radiation damage,
and both has been reported to have many biological activities such as
the reduction of age-associated cell damage induced by cellular pro-
duction of Reactive Oxygen Species (ROS) [14,15].
Due to their poor solubility in hydrophilic solutions (rutin: water
solubility 0.125 mg/dm3, 0.2 mM, and quercetin: water solubility
60.0 mg/dm3, 0.2 mM at 16 °C), the production of carrier systems can
help to overcome this drawback by encapsulation of these compounds
in lipid-based systems (emulsions, nanoparticles, micelles or liposomes)
[16,17,18]. Liposomes are suitable for use as delivery systems in bio-
logical systems and to ensure a good cellular uptake [19,20,21,22].
The phospholipid bilayers of liposomes surround an aqueous en-
vironment suitable for drugs loading in order to have a targeted release.
Liposomes composition, charge and size are important factors for both
efficacy and adverse effects [23,24,25,26,27]. The study of liposomes
with different superficial charge allows to evaluating chemical and
physical properties that can affect both encapsulation and release
processes of the two antioxidants. In vitro study, the liposome surface
charge influence many activities such as the binding and endocytosis of
interactions [28]. Cationic liposomes induced apoptosis in mouse
splenic macrophages and macrophage-like cell [29,30], whereas the use
of anionic liposomes has been fairly restricted to the delivery of specific
therapeutic macromolecules [31] influencing many aggregation or ab-
sorption phenomena [32].
On the basis of these reasoning a series of liposomes encapsulating
quercetin and rutin, have been synthesized, chemically characterized
and evaluated as regards the correlations between chemical properties
56
Biophysical Chemistry 233 (2018) 55–63
and biological responses.
Two important points were considered in this paper: (1) the choice
of two natural active compounds having a similar chemical structure
(quercetin and rutin) whose physic-chemical characteristics may affect
the biological response; (2) the synthesis and therefore the use, of dif-
ferent surface charged liposomes (anionic, cationic and zwitterionic or
non-ionic) as preliminar step for setting down the best drug delivery
system for natural products. The different surface charge may be
strongly correlated to toxicity, cell growth, etc.
Three different lipid compositions, varying the surface charge
(zwitterionic, cationic and anionic) have been used to synthesize lipo-
somes, that are then evaluated for in vitro cytotoxicity towards fibro-
blasts NIH3T3. Moreover, the liposomes, loaded with both quercetin
and rutin, have been tested for the ability to protects cells against hy-
drogen peroxide-induces oxidative, in comparison with free rutin and
quercetin solution.
The main purpose was to evaluate the protective effects on oxidative
stress phenomena that different natural products loaded into liposomes
could have on stabilized cell lines.
2. Material and methods
2.1. Materials
Quercetin (Q) and rutin (R) were purchased from Sigma-Aldrich
Chemie GmbH (Buchs, Switzerland) and used without further pur-
ification. Stock solutions in ethanol were stored at −20 °C. DOPC (1,2-
dioleoyl-sn-glycero-3-phosphocholine), DOPE (1,2-di-(9Z-octadece-
noyl)-sn-glycero-3-phosphoethanolamine), DOPA (1,2-dioleoyl-sn-gly-
cero-3-phosphate) and DOTAP (1,2-dioleoyl-3-trimethylammonium-
propane) were purchased from Avanti Polar Lipids Inc. (Alabaster, AL,
USA). Medium 131 was obtained from Invitrogen (USA). Dulbecco's
Modified Eagle's Medium (DMEM), trypsin solution, and all the solvents
used for cell culture were purchased from Lonza (Belgium). Mouse
immortalized fibroblasts NIH3T3 were from American Type Culture
Collection.
2.2. Quercetin and rutin loaded liposomes preparation
DOPC/DOPE, DOPE/DOPA, and DOTAP/DOPE were prepared at
1:1 M ratio with a final total lipid concentration of 1.0 × 10− 2 M.
Quercetin and rutin loaded liposomes were prepared to obtain a final
1:1 M ratio between total lipids and antioxidants.
Liposomes were prepared in a round bottom vial by mixing appro-
priate amounts of stock solutions, which were 4.0 × 10− 2 M in
chloroform for lipids, and 2.0 × 10− 2 M in ethanol for Q and R. A dry
lipid film (with and without antioxidants) was obtained by evaporating
the solvent under vacuum overnight. Rehydrating with Milli-Q grade
H2O yielded multilamellar dispersion.
Upon vortexing, multilamellar vesicles were obtained, which were
then submitted to eight freeze/thaw cycles. This method improved the
homogeneity of the size distribution in the final suspension. Liposomes
were subsequently reduced in size and converted to unilamellar vesicles
by extrusion through 100 nm polycarbonate membranes. Twenty-seven
extrusions were performed with the LiposoFast apparatus (Avestin,
Ottawa, Canada). All liposomes were stored at 4 °C.
2.3. Size and surface charge of liposomes
Dynamic Light Scattering (DLS) and ζ–potential measurements were
performed on a Zeta-sizer Instrument Nano ZS90 (Malvern Instrument
Ltd., UK).
ζ-potential values were measured at 25 °C. As the radii of liposomes
were always large enough compared with the Debye–Huckel para-
meters, the ζ-potentials were calculated directly from the
Helmoholtz–Smolowkovski equation (by the zetasizer) [33]. Sizes were
C. Bonechi et al. Biophysical Chemistry 233 (2018) 55–63

also calculated in the same experiments, according to the procedure viability was evaluated after 24 h incubation at 37 °C in 5% CO2 at-
described by Langley [34]. mosphere.

2.4. Encapsulation efficiency (EE) 2.6. Evaluation of NIH3T3 viability

The EE data was obtained by UV–visible spectra, recorded at 25 °C The NIH3T3 viability was determined as follows. First, the following
with a Perkin-Elmer Lamda 25 spectrophotometer (10 mm quartz solutions were prepared in order to determine the percentage of viable
cuvettes). cells: Neutral Red (NR) stock solution (0.33 g NR dye powder in 100 mL
Prior to spectra recording, liposome disruption was carried out in sterile H2O); NR medium (1.0 mL NR stock solution in 99 mL of routine
order get rid of the scattering background (scaling as λ− 4), due to large culture medium pre-warmed to 37 °C); NR desorb solution (1:50:49
aggregates in solution, which can affect precise intensity evaluation. To solution of glacial acetic acid:ethanol:H2O).
disrupt liposomes and release the entrapped antioxidants, samples have At the end of incubation, the routine culture medium was removed
undergone several cycles of freezing (− 32 °C). A calibration curve was from each plate and lid. The cells were carefully rinsed with 1 mL pre-
built by measuring the absorbance of solutions with known Q and R warmed D-PBS. Plates and lids were then gently blotted with paper
concentrations at 375 and 373 nm, respectively. towels. An aliquot of 1.0 mL NR medium was added to each dish and lid
The encapsulation efficiency of liposomes was calculated by the and further incubated at 37 °C, 95% humidity, 5.0% CO2 for 3 h. The
following equation: cells were checked during incubation for NR crystal formation. After
incubation, the NR medium was removed and the cells were carefully
EE (%) = [C/T] × 100
rinsed with 1 mL pre-warmed D-PBS. PBS was decanted and blotted
where T is the total amount of Q or R, and C is the amount of Q or R from the dishes and lids and exactly 1 mL NR desorb solution was added
determined by UV–visible experiments. to each sample. Plates were placed on a shaker for 20–45 min to extract
NR from the cells and form a homogeneous solution. During this step
2.5. Cytotoxicity and hydrogen peroxide treatment the samples were covered to protect them from light. Five min after
removal from the shaker, absorbance was read at 540 nm, by using UV/
2.5.1. Cell cultures and cytotoxicity test visible spectrophotometer.
To evaluate the in vitro cytotoxicity of products, the direct contact
tests, proposed by ISO 10995-5:2009 [35] was used. This test is suitable 3. Results and discussion
for sample with various shapes, sizes or physical status (i.e. liquid or
solid). 3.1. Size and surface charge of liposomes
Fibroblasts NIH3T3 were used for the experiments. Cells were
propagated in DMEM at 37 °C in a humidified atmosphere containing Liposome characteristics are shown in Table 1. The DLS results of
5% CO2. The culture medium was supplemented with 10% fetal calf liposome of different formulations with and without antioxidants are
serum, 1% L-glutamine-penicillin-streptomycin solution, and 1% MEM shown in Fig. 1. To test whether the charge on the liposome surface is
non-essential amino acid solution. Once at confluence, the cells were influencing the release of loaded compounds, liposomes having three
washed with PBS 0.1 M, separated with trypsin-EDTA solution and different lipid compositions were synthesized, varying the liposomal
centrifuged at 1000 rpm for 5 min. The pellet was re-suspended in surface net charge. DOPC liposomes exhibited a low negative charge,
complete medium (dilution 1:15). 1.5 × 103 cells suspended in 1 mL of whereas DOPE/DOPA liposomes exhibit a distinguished negative zeta
complete medium were seeded in each well of a 24 well round multi- potential and DOPE/DOTAP liposomes a highly positive charge, re-
wells and incubated at 37 °C in humidified atmosphere of 5% CO2. Once spectively (Table 1). The incorporation of Q into the liposomes resulted
cells reached the 50% of confluence (i.e. after 24 h of culture) the in the changes of zeta potential of the negatively charged liposomes and
culture medium was discharged and the test compounds, properly di- a similar behavior was shown in Rutin loaded liposomes (all types)
luted in completed medium, were added to each well. All samples were changing the zeta potential towards more negative values (Table 1). It
set up in triplicate. Complete medium was used as negative control. appears that both antioxidants significantly reduced the negative sur-
After 24 h of incubation, cell viability was evaluated by Neutral Red face charge of DOPE/DOPA liposomes, which can be explained by their
uptake (Sigma-Aldrich, Switzerland). negative charge.
The in vitro cytotoxicity assay was performed on empty liposomes Mean sizes and distribution of quercetin and rutin loaded liposomes
and liposomes loaded with Q and R at different concentrations. The were also measured (Table 1). The Q and R loaded liposome showed a
liposomes concentrations tested were 1.0 × 10− 5 M, 1.0 × 10− 4 M size increasing compared with pure liposomes and both sizes and dis-
and 5.0 × 10− 4 M, containing, respectively, the following quercetin tribution of all Q and R, in loaded liposomes showed no significant
and rutin concentrations: 5.0 × 10− 5 M, 5.0 × 10− 4 M and differences to each other. These chemical data confirm the correct in-
2.5 × 10− 3 M. The in vitro cytotoxicity of R and Q free solution in clusion of antioxidants in the vesicles. Furthermore, all liposomal
ethanol was also determined, for comparison reasons, using the fol-
lowing range of concentrations: 1.0 × 10− 5–5.0 × 10− 4 M and Table 1
4.0 × 10− 5–4.0 × 10− 3 M, for R and Q, respectively. These values Mean particle size (Size, mean ± S.D.; nm), polydispersity index (P.I.), and surface
have been chosen according to the antioxidants concentrations de- charge (ζ potential, mean ± S.D.; mV) of pure, quercetin and rutin loaded liposomes
obtained by extrusion through 100 nm polycarbonate membranes.
termined as encapsulated into the liposomes.
Cell morphology of treated and not treated fibroblasts was eval- Liposome composition Size ± S.D. (nm) P.I. ζ potential ± S.D. (mV)
uated by inverted optical microscope analysis (Axiovert 200 M, Zeiss,
Germany). DOPC/DOPE 107 ± 15 0.16 − 17 ± 3
DOPC/DOPE + Q 119 ± 10 0.20 − 19 ± 7
DOPC/DOPE + R 129 ± 13 0.19 − 21 ± 3
2.5.2. Hydrogen peroxide treatment DOPE/DOPA 114 ± 13 0.17 − 32 ± 2
To determine the protective effect of quercetin, rutin, loaded lipo- DOPE/DOPA + Q 125 ± 12 0.21 − 37 ± 5
somes, against oxidative stress, fibroblasts were pre-incubated with DOPE/DOPA + R 139 ± 10 0.21 − 46 ± 4
DOPE/DOTAP 112 ± 12 0.15 42 ± 5
different hydrogen peroxide concentrations (1, 5, 10, 25, 30, 50, 75,
DOPE/DOTAP + Q 134 ± 11 0.19 37 ± 4
100 μM) for 15 min. Then, cells were washed out and cultured in DOPE/DOTAP + R 129 ± 13 0.16 35 ± 3
medium with different concentrations of the tested compounds. Cell

57
C. Bonechi et al. Biophysical Chemistry 233 (2018) 55–63

Fig. 1. Dynamic light scattering results for (a) DOPC/DOPE liposomes plain and loaded with quercetin and rutin; (b) DOPE/DOPA liposomes plain and loaded with quercetin and rutin;
(c) DOPE/DOTAP liposomes plain and loaded with quercetin and rutin.

preparations showed a homogeneous size distribution, as indicated by a Table 3

low polydispersity index (P.I.), that also underlined that the synthesized Encapsulation efficiency of quercetin (Q) in liposomal formulations (mean ± S.D.)
liposomes are stable and not aggregation processes in solution occur.
Liposome composition Encapsulation efficiency (EE) %
The P.I. was used as a measure of the width of the size distribution. P.I.
less than 0.2 indicates a homogenous and monodisperse population. All DOPC/DOPE + Q 18.9 ± 5.3
liposomes (empty or Q or R loaded) are stable and do not exhibit the DOPE/DOPA + Q 14.2 ± 1.9
aggregation process, especially the anionic and cationic liposomes DOPE/DOTAP + Q 15.6 ± 1.5

which show absolute values of zeta potential greater than 30 mV.

Table 4
Encapsulation efficiency of rutin (R) in liposomal formulations (mean ± S.D.)
3.2. Stability of liposome
Liposome composition Encapsulation efficiency (EE) %
To define the stability of liposome preparations, size analyses on
DOPC/DOPE + R 12.9 ± 5.2
pure and loaded liposomes were performed at different storage time (0, DOPE/DOPA + R 12.3 ± 6.1
1, 3 months, Table 2). The experimental data showed that all liposome DOPE/DOTAP + R 12.9 ± 4.7
systems are stable, as the size distribution, as well as the P.I. values did
not revealed relevant variations up to 3 months storage (at 4 ± 1 °C).
This indicates that the liposomes do not form aggregates. Therefore, we processes over the 3 months study periods.
concluded that liposomes loaded with quercetin or rutin were stable,
because there were negligible changes in particle size and aggregation 3.3. Encapsulation efficiency

Table 2 Tables 3 and 4 show that the liposomal encapsulation efficiencies

Mean particle size (Size, mean ± S.D.; nm) of pure, quercetin and rutin loaded liposomes
from the two different antioxidants were remarkably similar, indicating
versus time (t = 0, 1, 2 and 3 months).
that the chemical differences between Q and R do not affect very much,
Liposome t=0 t = 1 month t = 2 months t = 3 months their ability to be loaded into liposomes.
Composition The rutin, despite hydrophilic and hydrophobic properties attrib-
Size ± S.D. Size ± S.D. Size ± S.D. Size ± S.D. uted to its disaccharide content or aglycone structure, not present more
(nm) (nm) (nm) (nm)
effective penetration between the lipid chain than quercetin. The en-
DOPC/DOPE 107 ± 15 110 ± 13 111 ± 12 117 ± 10 capsulation efficiency was also very similar changing the lipids for-
DOPC/DOPE + Q 119 ± 10 123 ± 11 123 ± 11 129 ± 10 mulation and the values of EE% demonstrate that the Q and R were
DOPC/DOPE + R 129 ± 13 129 ± 15 130 ± 10 135 ± 9 loaded in zwitterionic, anionic and cationic liposomes with low effi-
DOPE/DOPA 114 ± 13 115 ± 15 116 ± 11 119 ± 10
DOPE/DOPA + Q 125 ± 12 129 ± 11 128 ± 9 130 ± 5
ciency.
DOPE/DOPA + R 139 ± 10 139 ± 12 139 ± 11 139 ± 12
DOPE/DOTAP 112 ± 12 117 ± 12 118 ± 9 119 ± 9
3.4. Cell cytotoxicity
DOPE/DOTAP + Q 134 ± 11 137 ± 10 137 ± 7 137 ± 9
DOPE/DOTAP + R 129 ± 13 130 ± 11 130 ± 11 132 ± 9
Assessment of cytotoxicity was based on the amount of neutral red

58
C. Bonechi et al.
Fig. 2. Fibroblasts viability percentage after 24 h of contact with different concentrations
of quercetin. The concentration of 3.0 × 10− 4 M reduced cell viability by 50%.
incorporation by fibroblasts NIH3T3 after 24 h of contact with different
concentrations of quercetin and rutin. Fig. 2 shows the effect of quer-
cetin concentration ranging from 1.0 × 10− 5 up to 5.0 × 10− 4 M,
with respect to the100% viability of untreated control samples
(medium; reported as first point in the graph in Fig. 2). A strong re-
duction in cell viability was observed for concentrations higher than
5.0 × 10− 5 M and the 50% cell viability reduction was estimated at a
quercetin concentration by 3.0 × 10− 4 M.
Similarly the effect of rutin is shown in Fig. 3, that shows the per-
centage of cell viability decreasing with increasing the antioxidant
molecule concentration with respect to the untreated control samples.
The concentration of 2.0 × 10− 3 M reduced cell viability by 50% and
an almost complete cell death was observed at 4.0 × 10− 3 M treatment
dose.
The cytotoxicity of the three liposome systems, empty and loaded
with quercetin and rutin, was also evaluated. The empty liposomes
were tested a three different concentration of total lipids: 1.0 × 10− 5,
1.0 × 10− 4 and 5.0 × 10− 4 M, whereas the antioxidants were:
5.0 × 10− 5, 5.0 × 10− 4, and 2.5 × 10− 3 M. The results were re-
ported in Fig. 4.
DOPC/DOPE liposomes resulted not cytotoxic to any of the three
tested concentrations. The presence of quercetin imparts a cytotoxic
effect to the liposomal system that increases by increasing the anti-
oxidant molecule concentration. Such effect is greater than that shown
Fig. 3. Fibroblasts viability percentage after 24 h of contact with different concentrations
of rutin. The concentration of 2.0 × 10− 3 M reduced cell viability by 50% and at
4.0 × 10− 3 M an almost complete cell death was observed.
59
Biophysical Chemistry 233 (2018) 55–63
by the free molecule, not loaded in liposomal systems (Fig. 2). Cell
viability after contact with the liposomes DOPC/DOPE loaded with
rutin is comparable to that of the control at the lowest tested con-
centration of antioxidant. By increasing the rutin concentration, the
cytotoxic effect increases and is greater than that shown by the free
antioxidant molecule (Fig. 3).
The liposomes DOPE/DOPA reduced cell viability only at the
highest tested concentration (total lipids 5.0 × 10− 4 M). The presence
of quercetin did not affect the cell viability at low concentration
(5.0 × 10− 5 M), but induced a severe decrease at higher antioxidant
concentrations. Similarly to the previous liposomal system, also in this
case the molecule loaded into liposomes has a greater cytotoxic effect
when compared with not loaded molecule. In the presence of rutin, cell
viability follows the same trend observed for the DOPC/DOPE + R
vesicles, And the cytotoxicity of rutin loaded into liposomal system was
greater than that shown by the free antioxidant molecule.
Finally, the liposomes DOTAP/DOPE resulted extremely cytotoxic at
all the tested concentrations, and the loading of both antioxidant mo-
lecules, showed a reduction the toxic effect at 5.0 × 10− 5 and
5.0 × 10− 4 M concentrations. On the contrary, at the higher con-
centration of antioxidants (2.5 × 10− 3 M), both vesicles, DOPE/
DOTAP + Q and DOPE/DOTAP + R, show a marked cytotoxicity.
However, as occurred for the other two liposomal systems, also in this
case the antioxidants alone are less effective to influence cell viability
compared to when the same molecules are loaded on liposomal sys-
tems.
It can be hypothesized that the greater cytotoxicity degree shown by
quercetin and rutin loaded into liposomes may be due to their easier
internalization process by the cells. It is well known that free quercetin
and rutin, as almost all the flavonoids, are not water miscible and
probably poor bioavailable at cellular level [36] and this behavior may
explain their low cytotoxicity. The use of liposome as molecular carriers
is in agreement with the wish to let the antioxidants to be better in-
ternalized in the cells [37].
Cell morphology and density confirmed the NRU results. (Fig. 5).
Fibroblasts in contact with liposomes DOPC/DOPE at the highest con-
centration showed the same density and morphology as well as not
treated cells. On the contrary, DOPE/DOPA treated cells at the highest
concentration showed a lower density in comparison to untreated fi-
broblasts but preserved their morphology. Cells in contact with lipo-
somes DOPE/DOTAP at concentration of 5.0 × 10–4 M showed a very
low density and modified morphology demonstrating the high cyto-
toxicity power of these liposomes.
3.5. Cell viability after hydrogen peroxide treatment
Concentration-effect relationship for the cytotoxic effects of H2O2
on NIH3T3 fibroblasts were obtained by testing solution of hydrogen
peroxide ranging in concentration between 1 and 100 μM. Cytotoxicity
was determined by revealing the decreasing of cell viability after
treatment with H2O2 followed by contact with different concentrations
of free quercetin or rutin solutions and empty and loaded liposomes.
Fig. 6 shows that cell viability decreases by increasing hydrogen
peroxide concentration. The same trend is observed for rutin
5.0 × 10− 5 M. Quercetin 5.0 × 10− 5 M is able to increase cell viabi-
lity at H2O2 concentrations of 1, 5, 10, 25 and 30 μM but not at higher
values. Quercetin 5.0 × 10− 4 M shows the highest cytotoxic effect at
all the H2O2 concentrations tested. On the contrary, rutin
5.0 × 10− 4 M interferes with the H2O2 toxicity also at the highest
concentrations, demonstrating a good ability to protect cells and to
reduce cell death.
The presence of liposomes DOPC/DOPE, at any of the tested con-
centrations, both empty and loaded with quercetin, do not affect the
toxic effect of hydrogen peroxide towards NIH3T3 viability (Fig. 1S).
On the contrary, solutions of rutin 5.0 × 10− 5 and 5.0 × 10− 4 M
protect cells by the toxic effect of hydrogen peroxide treatment (Fig. 7)
C. Bonechi et al.
but a rutin concentration by 2.5 × 10− 3 M, revealed a trend of cell
viability similar to that observed after treatment with just H2O2. This
latter effect is, probably, due to the rutin cytotoxic effect just above
reported (Fig. 3).
Also liposomes DOPE/DOPA, at all the tested concentrations, are
not able to protect cells by H2O2 treatment, and the presence of quer-
cetin does not improve cell viability (Fig. 2S).
When liposomes DOPE/DOPA are loaded with rutin, an increase in
cell viability is observed at the concentration values of 5.0 × 10− 5 and
5.0 × 10− 4 M but not at 2.5 × 10− 3 M at which the percentage of
Fig. 5. Optical microscope images of: a) untreated fibroblasts; b) fibroblasts treated with DOPC/DOPE at concentration of 5.0 × 10− 4 M; c) fibroblasts treated with DOPE/DOPA at
concentration of 5.0 × 10− 4 M; d) fibroblasts treated with DOPE/DOTAP at concentration of 5.0 × 10− 4 M.
60
Biophysical Chemistry 233 (2018) 55–63
Fig. 4. Fibroblasts viability after 24 h of contact with dif-
ferent concentrations of empty liposomes and liposomes
loaded with antioxidant (A), quercetin and rutin (total
phospholipids, L 1.0 × 10− 5, 1.0 × 10− 4, and
5.0 × 10− 4 M; antioxidants, A 5.0 × 10− 5, 5.0 × 10− 4,
−3
and 2.5 × 10 M, respectively).
viable cells is the same of cell-treated hydrogen peroxide (Fig. 8).
Liposomes DOPE/DOTAP, at all the three tested concentrations,
induce complete cell death and show a toxicity level higher in com-
parison with cell treated just with H2O2 (Fig. 9a).
The loading with quercetin and rutin, slightly reduces cell toxicity
just for low level of ossidative stress (H2O2 treatment by 1 and 5 μM)
(Fig. 9b and c). However, by the comparison of these data with those
shown in Fig. 4, it is evident that the toxic effect of and DOPE/DOTAP,
both empty and loaded with antioxidant molecules, seems to be in-
dependent by hydrogen peroxide treatment.
C. Bonechi et al. Biophysical Chemistry 233 (2018) 55–63

Fig. 6. Influence of H2O2 concentration on cell viability. Cells

were incubated with H2O2 for 15 min, than washed and in-
cubated for 24 h with two different concentrations of quercetin
(5.0 × 10− 5 and 5.0 × 10− 4 M) and rutin (5.0 × 10− 5 and
5.0 × 10− 4 M).

Fig. 7. Influence of H2O2 concentration on cell viability. Cells

were incubated with H2O2 for 15 min, than washed and in-
cubated for 24 h with three different concentrations of DOPC/
DOPE (1.0 × 10− 5, 1.0 × 10− 4, and 5.0 × 10− 4 M) loaded
with rutin (5.0 × 10− 5, 5.0 × 10− 4, and 2.5 × 10− 3 M).

Fig. 8. Influence of H2O2 concentration on cell viability. Cells

were incubated with different concentrations of H2O2 for 15 min,
than washed and incubated for 24 h with three different con-
centrations of DOPE/DOPA (1.0 × 10− 5, 1.0 × 10− 4, and
5.0 × 10− 4 M) loaded with rutin (5.0 × 10− 5, 5.0 × 10− 4, and
2.5 × 10− 3 M).

61
C. Bonechi et al.
Fig. 9. Influence of H2O2 concentration on cell viability. Cells were incubated with H2O2
for 15 min, than washed and incubated for 24 with three different concentrations of: (a)
DOPE/DOTAP (1.0 × 10− 5, 1.0 × 10− 4, and 5.0 × 10− 4 M); (b) DOPE/DOTAP
(1.0 × 10− 5, 1.0 × 10− 4, and 5.0 × 10− 4 M) loaded with quercetin (5.0 × 10− 5,
5.0 × 10− 4, and 2.5 × 10− 3 M); and (c) DOPE/DOTAP (1.0 × 10− 5, 1.0 × 10− 4, and
5.0 × 10− 4 M) loaded with rutin (5.0 × 10− 5, 5.0 × 10− 4, and 2.5 × 10− 3 M).
4. Conclusion
All experimental chemical data suggest that the liposomes studied in
this work (zwitterionic, anionic and cationic) are stable and are able to
include the two antioxidants: quercetin and rutin. In fact, the size and ζ-
potential values showed modifications according to chemical properties
of Q and R. Liposomes are stable and not present aggregation processes
versus conservation time, at least up to 3 months. The Q and R loaded
liposomes show a fairly low encapsulation efficiency which does not
negative influence the biological data. This result highlights that lipo-
somes are excellent drug delivery systems for quercetin and rutin,
which are protected from chemical and physical degradation processes.
Moreover, the zwitterionic liposomes show chemical and biological
properties better than the anionic and cationic vesicles. The anionic
62
Biophysical Chemistry 233 (2018) 55–63
liposomes can be excellent drug delivery systems for specific compound
for specific lipid concentration.
Biological results confirm that the use of liposomes as carriers, may
help cell internalization of the studied flavonoids, which better inter-
fere with cell at lower concentrations, with respect when they are free
in solution. Moreover, in agreement with the evidence of neuropro-
tective effects of rutin against H2O2 induced oxidative stress in cerebral
glioma cell line [38], rutin both in solution and liposomes loaded, in-
terferes with the hydrogen peroxide induced toxicity, showing a good
ability as cells protector. So, the loading of liposome with antioxidant
molecules can be considered a good method to improve cell inter-
nalization of molecules characterized by low solubility in water media.
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bpc.2017.11.003.
Acknowledgements
The authors thank the University of Siena.
Declarations of interest
The authors report no declarations of interest.
Conflict of interest
The authors declare that they have no conflicts of interest.
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