Colloids and Surfaces B: Biointerfaces 112 (2013) 548–553

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Colloids and Surfaces B: Biointerfaces

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

Anticancer activity of liposomal bergamot essential oil (BEO)

on human neuroblastoma cells
Christian Celia a,b,1 , Elena Trapasso c,1 , Marcello Locatelli a , Michele Navarra d ,
Cinzia Anna Ventura d , Joy Wolfram b,e , Maria Carafa f , Valeria Maria Morittu c ,
Domenico Britti c , Luisa Di Marzio a,∗,2 , Donatella Paolino c,∗
a
Department of Pharmacy, University “G. d’Annunzio” of Chieti—Pescara, Via dei Vestini 31, 66013 Chieti, Italy
b
Department of Nanomedicine, The Methodist Hospital Research Institute, 6670 Bertner Ave., Houston, TX 77030, USA
c
Department of Health Sciences, University “Magna Graecia” of Catanzaro, University Campus “S. Venuta”, Building of BioSciences, V.le “S. Venuta” 88100
Germaneto, Catanzaro, Italy
d
Department of Drug Science and Health Products, University of Messina, Viale Annunziata, 98168 Messina, Italy
e
CAS Key Laboratory for Biomedical Effects of Nanomaterials & Nanosafety, National Center for Nanoscience & Technology of China, Beijing 100190, China
f
Department of Drug Chemistry and Technologies, University “Sapienza” of Rome, Piazzale Aldo Moro 5, 00185 Rome, Italy

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

Article history: Citrus extracts, particularly bergamot essential oil (BEO) and its fractions, have been found to exhibit
Received 5 July 2013 anticancer efficacy. However, the poor water solubility, low stability and limited bioavailability have
Received in revised form 13 August 2013 prevented the use of BEO in cancer therapy. To overcome such drawbacks, we formulated BEO liposomes
Accepted 7 September 2013
that improved the water solubility of the phytocomponents and increased their anticancer activity in vitro
Available online 17 September 2013
against human SH-SY5Y neuroblastoma cells. The results warrant further investigation of BEO liposomes
for in vivo applications.
Keywords:
© 2013 Published by Elsevier B.V.
Natural products
Liposomes
Bergamot essential oil (BEO)
Citrus extracts
Cancer
SH-SY5Y human neuroblastoma cells

1. Introduction
Natural compounds have been used extensively in medic-
inal chemistry. Several drugs derived from natural products
have already received clinical approval and many are currently
enrolled in clinical trials [1,2]. Natural compounds often exhibit
anti-inflammatory, antioxidant, antibacterial, antiviral, anticancer
and/or tissue regenerative activity [3–6]. In fact, some natural
products contain several bioactive molecules that synergistically
provide therapeutic efficacy [7,8]. For instance, bergamot essential
oil (BEO) derived from Citrus bergamia Risso et Poiteau consists of
more than 345 compounds, which can be divided into two frac-
tions [9–11]. The volatile fraction (93–96%) contains monoterpene
and sesquiterpene, such as limonene, ␣- and ␤-pinene, ␤-myrcene,
∗ Corresponding authors. Tel.: +39 0961 369 4211; fax: +39 0961 369 4237.
E-mail addresses: l.dimarzio@unich.it (L. Di Marzio),
paolino@unicz.it (D. Paolino).
1
These authors contributed equally.
2
Tel.: +39 0871 3554705; fax: +39 0871 3554911.
the ␥-terpinene, linalool and linalyl acetate, while the non-volatile
fraction (4–7%) has polymethoxylated flavones, coumarins and pso-
ralens, such as bergamottin and bergaptene.
A major focus within the field of herbal medicine is the use
of natural compounds for cancer therapy [12,13]. Particularly,
flavonoids and monoterpens that are found in fruits and vegeta-
bles exhibit anticancer activity in vitro and in vivo [14–16]. During
the last decade, the use of Citrus plants as a source of natural
therapy against cancer has gained interest within the scientific
community [17,18]. BEO was recently shown to have antiprolifer-
ative activity against SH-SY5Y human neuroblastoma cells in vitro
[19]. BEO derived compounds, such as limonene, limonene-related
monoterpenes, perillyl alcohol and perillic acid have also been
demonstrated to inhibit cell proliferation of breast cancer cells and
provide a chemopreventive and chemotherapeutic effect in mam-
mary tumor models [20].
Although BEO has shown promising anticancer activity, one of
the major limitations with this product is poor water solubility.
The hydrophobic nature of the oil requires the use of solubilizing
agents, such as dimethyl sulfoxide (DMSO), which can elicit cell
toxicity. Indeed, DMSO was previously shown to be harmful for

0927-7765/$ – see front matter © 2013 Published by Elsevier B.V.

http://dx.doi.org/10.1016/j.colsurfb.2013.09.017
 C. Celia et al. / Colloids and Surfaces B: Biointerfaces 112 (2013) 548–553
primates after prolonged exposure [21,22]. To avoid the use of
toxic solubilizers, hydrophobic agents can be loaded within bio-
compatible liposomes [23–25]. The features of liposomes can be
tailored by modifying the phospholipid composition and prepara-
tion procedures [26]. Moreover, surface modification of liposomes
with polyethylene glycol (PEG) provides increased protection from
recognition and uptake by the immune system [27].
In addition to providing a means to deliver hydrophobic agents,
liposomes can also improve the biopharmaceutical properties of
the encapsulated drug by increasing the circulation time, reducing
clearance and enhancing tumor accumulation [28,29]. Liposomal
encapsulation can also protect the contents from enzymatic degra-
dation, which is common for natural compounds that undergo rapid
degradation or metabolism in vivo [30,31]. Furthermore, the com-
position and architecture of liposomes can facilitate the delivery
of bioactive compounds across the blood-brain barrier, providing a
means to treat neurological disorders [32,33].
Recently, our group designed lipid-based derivatives to deliver
natural products with the goal of improving therapeutic effi-
cacy [34–36]. For instance, liposomal Aloe vera was shown to
have regenerative properties, causing the increased proliferation
of human keratinocytes in vitro [37]. Lipophilic fatty acid esters of
hydroxytyrosol were also synthesized and evaluated as skin perme-
ation agents against transdermal inflammation [38]. In this study,
SH-SY5Y neuroblastoma cells were used to investigate the antipro-
liferative activity of BEO and bergapten-free BEO (BEO-BF) loaded
in pegylated liposomes. The results demonstrate that the liposomal
formulations have increased anticancer activity when compared to
the free compound.
2. Materials and methods
2.1. Materials
Cholesterol (Chol), phosphate buffered saline (PBS), trypan
blue buffer solution (10×), 3-[4,5-dimethylthiazol-2-yl]-3,5
diphenyltetrazolium bromide (MTT) dye (TLC purity ≥97.5%),
dimethyl sulphoxide (DMSO) and amphotericin B solution
(250 ␮g/ml) were obtained from Sigma-Aldrich (Milan, Italy). 1,2-
dipalmitoyl-sn-glycero-3-phospocholine monohydrate (DPPC) and
N-(carbonyl-methoxypolyethylene glycol-2000)-1,2-distearoyl-
sn-glycero-3-phosphoethanolamine (DSPE-mPEG2000) were
purchased from Genzyme (Suffolk, UK). RPMI1640 culture medium,
heat-inactivated fetal bovine serum (FBS), penicillin/streptomycin
solution, sodium pyruvate and glutamine were obtained from
GIBCO (Invitrogen Corporation, Giuliano Milanese (Mi), Italy).
SH-SY5Y human neuroblastoma cells were kindly provided by
Prof. Michele Navarra, Pharmaco-Biological Department, Faculty
of Pharmacy, University of Messina, Messina, Italy and purchased
from ICLC-IST (Genoa, Italy). BEO and BEO-BF were kindly provided
by the company “Simone Gatto” (San Pier Niceto (ME), Italy). The
analytical certificate was provided by “Stazione Sperimentale
per le Industrie delle Essenze e dei Derivati dagli Agrumi” (SSEA,
Reggio Calabria, Italy) and further characterized as previously
reported [39]. Double-distilled pyrogen-free water and sterile
saline solution were Sifra S.p.A. (Verona, Italy) and Fresenius
Kabi Potenza S.r.l. (Verona, Italy) products, respectively. Chemical
reagents were of analytical grade and they were used without
further purification (Carlo Erba, Milan, Italy).
2.2. High performance liquid chromatography (HPLC) of BEO and
BEO-BF
The physicochemical characterization of BEO and BEO-BF was
carried out using an HPLC apparatus, consisting of a P2000
549
Spectra System pump with a SN4000 controller, a photodiode
array detector (UV6000LP) and a SCM1000 degasser (Thermo
Scientific Inc, Finnigan division, Philadelphia, PA, USA). A Grace
Smart RP18 column (Grace, Deerfield, IL, USA), with a size of
250 × 4.6 mm and an internal diameter (i.d.) of 5 ␮m, was used
during analysis. The procedure was performed at 22 ± 1 ◦ C. The
mobile phase consisted of water (A) and acetonitrile (B) and a
gradient was used to separate BEO and BEO-BF: 0–1 min, 30% B;
1–5 min, 60% B; 5–8 min, 60% B; 8–30 min, 100% B; 30–35 min,
30% B; 35–40 min, 30% B. Water was obtained using a Millipore
Milli-Q Plus water apparatus (Millipore Bedford Corp., Bedford, MA,
USA). 20 ␮l of sample was injected before analysis and the HPLC
was set at 1 ml/min. BEO and BEO-BF were detected using a diode
array detector (DAD) with a wavelength range of 190–500 nm.
The obtained data was analyzed using Excalibur 2.0 SUR1 soft-
ware (Thermo Scientific Inc, Finnigan division, Philadelphia, PA,
USA).
2.3. Liposome preparation
Liposomes were made up using DPPC/Chol/DSPE-mPEG2000
(6:3:1 molar ratio), as previous reported [40]. Briefly, phospho-
lipids (20 mg/ml) were dissolved in a round-bottom flask with
a chloroform/methanol mixture (3:1 v/v). Organic solvents were
evaporated using a Rotavapor® R-210 (Büchi-Italia S.r.l., Assago
(MI), Italy) under vacuum to obtain a thin lipid film. Resid-
ual organic solvent was removed under vacuum overnight at
room temperature, using a T51 glass oven dryer (Büchi-Italia
S.r.l., Assago (MI), Italy). The lipid film was then hydrated with
double-distilled pyrogen-free water (1 ml) or sterile saline solu-
tion (1 ml) by using a vortex-mixer (700 rpm for 3 min) and a
water bath (42 ± 1 ◦ C for 3 min). Multilamellar liposomes were
then extruded at 42 ◦ C through a stainless steel extrusion device
(Lipex Biomembranes, Northern Lipids Inc., Vancouver, BC, Canada)
using two stacked polycarbonate membrane filters (Costar, Corn-
ing, Inc., NY, USA) of 800, 400 and 200 (10 passages for each
filters).
2.4. Physicochemical characterization of liposomes
The average size, polydispersity index (PDI) and zeta potential
of liposomes were measured using a Zetasizer Nano ZS (Malvern
Instruments Ltd., Worcestershire, UK). Dynamic light scattering
analysis and a third-order cumulant fitting correlation function
were used to evaluate the average size and PDI. The light source
for analysis was set to 670 nm and a laser diode of 4.5 mW was
used during analysis. Liposomes were detected at 173◦ in backscat-
tering. The real and imaginary refractive indexes were 1.59 and
0.0. The medium refractive index was set at 1.330, the medium
viscosity was set at 1.0 mPa s and dielectric constant was 80.4.
Quartz cuvettes were used to run each samples. Liposomes were
diluted before analysis by using isotonic and inert buffer (pH
7.4) filtered through polypropylene membranes (0.22 mm) (What-
man Inc., Clifton, NJ, USA). Zetasizer Nano ZS was further used to
measure the zeta-potential of liposomes. The measurement was
performed by using a Smoluchowsky constant F (Ka) of 1.5 as a
function of the electrophoretic mobility. Measurements represent
the average of three batches (10 runs per measurement) ±standard
deviation.
The measurement of the entrapment efficiency of BEO and BEO-
BF was carried out as previously reported [41]. Briefly, the liposomal
formulations were collected into polycarbonate tubes and then
centrifuged at 100,000 × g for 1 h at 4 ◦ C using a Beckman OptimaTM
Ultracentrifuge equipped with a TL S55 fixed angle rotor (Beckman
Coulter Inc., Fullerton, CA, USA). Supernatants were separated from
the pellets and the amount of unentrapped drug was immediately
550 C. Celia et al. / Colloids and Surfaces B: Biointerfaces 112 (2013) 548–553
Table 1
Physicochemical characterization of BEO and BEO-BF loaded liposomes. Data is presented as mean ± standard deviation.
Formulations Average size (nm)
Empty-liposomes 177.91 ± 2.89
BEOb -liposomes 188.25 ± 2.19
BEO-BFc -liposomes 185.14 ± 1.97
a
Polidispersity index.
b
Bergamot essential oil.
c
Bergamot essential oil bergapten-free.
measured by HPLC. The r2 values for BEO and BEO-BF were 0.9999
and 0.9998, respectively. BEO and BEO-BF demonstrated linear-
ity in the concentration range of 2.25–600 ␮g/ml. The entrapment
efficiency (EE) was measured using the following equation:
EE (%) = [(DrugT − DrugS )/DrugT ] × 100,
where DrugT is the total amount of BEO and BEO-BF added to lipo-
somes during the preparation of the lipid film and DrugS is the
amount of BEO and BEO-BF measured in the supernatant.
2.5. Cell culture
SH-SY5Y human neuroblastoma cells were seeded in RPMI
1640 supplemented with 0.407 mM of MgSO4 ·7 H2 O, FBS (10%
v/v), amphotericin B solution (250 ␮g/ml) (1% v/v), penicillin (100
UI/ml)-streptomycin (100 ␮g/ml) solution (1% v/v), 1 mM sodium
pyruvate and 2 mM glutamine at 37 ◦ C in 5% CO2 .
2.6. Cytotoxicity studies
SH-SY5Y cells were grown in 25 cm2 /cell culture flasks
(70% confluence) before being seeded into 96-well plates
(1.56 × 104 cells/cm2 ) and 6-well plates (2.08 × 104 cells/cm2 ).
After 24 h, cells were treated using varying concentrations of BEO
and BEO-BF (0.01%, 0.02%, 0.03%, 0.04% and 0.05% v/v). Untreated
cells were used as the control. Different time points (24, 48 and 72 h)
were evaluated during the experiment. Cytotoxicity was carried
out using the trypan blue dye exclusion assay (cell death) and MTT
assay (cell viability). The trypan blue dye exclusion assay was per-
formed by mixing cells with trypan blue solution (0.4% v/v) (100 ␮l)
and counting the number of dead cells (blue color) with a hemato-
cytometer and an optical microscope (Labophot-2, Nikon, Japan).
The percentage of dead cells was measured with the following
equation:
CD
Dead cells (%) = × 100
CT
where CD is the number of dead cells and CT is the total number
of cells. The cell viability was assessed using an MTT assay. MTT
solution (5 mg/ml in PBS buffer, 10 ␮l) was added to each well and
incubated for 3 h at 37 ◦ C. After 3 h the medium was removed and
replaced with ethanol and DMSO solution (1:1 v/v, 100 ␮l) to dis-
solve crystal formazan that had precipitated inside the cells. Plates
were then gently shaken at 230 rpm (IKA® KS 130 Control, IKA®
WERKE GMBH & Co, Staufen, Germany) for 20 min. The sample
absorbance was measured using an ELISA microplate reader (Lab-
systems mod. Multiskan MS, Midland, ON, Canada) at a wavelength
of 570 nm. The percentage of viable cells was measured using the
following equation:
AbsT
Cell viability (%) = × 100
AbsC
where, AbsT is the absorbance of the treated cells and AbsC is the
absorbance of control (untreated) cells.
PDIa Zeta potential (mV) Entrapment efficiency (%)
0.220 ± 0.089 −4.57 ± 1.76 –
0.230 ± 0.059 −2.95 ± 1.29 75.0 ± 2.32
0.228 ± 0.062 −2.57 ± 1.38 77.0 ± 1.94
2.7. Statistical analysis
A one-way ANOVA was used for statistical analysis. A poste-
riori Bonferroni t-test was carried out to evaluate the statistical
significance of the ANOVA test. A p value <0.05 was considered
statistically significant. Results are the average of three experi-
ments ± standard deviation.
3. Results and discussion
BEO and BEO-BF were characterized using high-performance
liquid chromatography (HPLC) (Supplementary Fig. 1). HPLC analy-
sis showed specific peaks with retention times at 7.77 and 7.98 min
for BEO distilled essential oil (Supplementary Fig. 2). Both of these
peaks were absent from the BEO and BEO-BF samples (Supple-
mentary Figs. 3 and 4). Specific retention times at 8.90, 9.50
and 9.88 min were poorly detected for BEO distilled essential
oil and BEO-BF (Supplementary Figs. 2–4), although they were
most prominent in the BEO group (Supplementary Fig. 4). The
UV/Vis spectra obtained using DAD demonstrated difference in
the intensity of signal and retention times for BEO and BEO-BF
(Supplementary Figs. 5–8). The obtained chromatograms showed
similar profiles to what has already been reported in the literature
[39].
The entrapment efficacy, average size, polydispersity index and
zeta potential of the pegylated liposomes were measured. The main
physicochemical parameters are displayed in Table 1. The entrap-
ment efficacy was 75 ± 2.32% and 77 ± 1.94% for BEO and BEO-BF,
respectively. The liposomal size increased upon loading with BEO
(188.25 nm) and BEO-BF (185.14 nm) as compared to empty vesi-
cles (177.91 nm). This increase in size is likely due to intercalation
of BEO and BEO-BF in the bilayer, which may reduce the cohe-
sion between phospholipids, a phenomenon already demonstrated
for liposomes containing the hydrophobic compound fisetin [42].
The addition of BEO and BEO-BF also increased the PDI and led to
the formation of polydisperse formulations with a size distribu-
tion of over 0.2 (Table 1). The size difference between the empty
and loaded liposomes was statistically significant (p value <0.05),
while the difference in PDI was not. These results demonstrate
that BEO derivates can perturb the structure of liposomes due
to the presence of multiple components, which assemble into a
phytocomplex. The zeta potential value for the control liposomes
was −4.57 mV, which is consistent with previous reports, showing
that liposomes with PEG moieties have an anionic surface charge
[29,30,40]. The zeta potential values increased to −2.95 mV for BEO
liposomes and −2.57 mV for BEO-BF liposomes (Table 1), although
this increase was not statistically significant. The slight change in
the zeta potential could depend on the partial distribution of BEO
and BEO-BF in the external layer of the pegylated liposomes. In
the outer liposomal layer the functional groups of components in
the BEO phytocomplex may interact with the acylic chains on PEG
phospholipids, thus changing the surface charge. This phenomenon
has previously been reported for paclitaxel liposomes, where
 C. Celia et al. / Colloids and Surfaces B: Biointerfaces 112 (2013) 548–553
Fig. 1. Dose-dependent cytotoxic effect of (A): BEO and (B): BEO-loaded liposomes
against SH-SY5Y cells at different incubation times: 24 h (䊉), 48 h () and 72 h ().
Anticancer activity is expressed as the percentage of viable cells measured by an
MTT-assay. Untreated cells and cells exposed to empty liposomes were used as con-
trols. Error bars, if not shown, are within the symbols. Results are presented as the
average of three different experiments ± standard deviation. Results are statistically
significant (p < 0.05).
drug-loading caused an increase in the surface charge, suggesting
that the hydrophilic groups of the paclitaxel molecule interact with
PEG [35].
The anticancer activity of the BEO and BEO-BF as free com-
pounds or liposomal formulations was evaluated in vitro using
SH-SY5Y cells. Cell viability was determined using an MTT assay,
while cell mortality was evaluated using trypan blue staining. Via-
bility and mortality were measured at different time-points (24, 48
or 72 h) with varying concentrations of BEO and BEO-BF (from 0.01%
to 0.05% v/v). The results demonstrate that BEO and BEO-BF lipo-
somes outperform their non-liposomal counterparts in anticancer
activity in vitro. At various concentrations and time points, the lipo-
somal formulations showed a greater reduction in cell viability
(Figs. 1 and 2) and a greater increase in cell mortality (Figs. 3 and 4),
both of which where statistically significant (p value <0.05). In
particular, after 24 h the liposomes showed efficacy at low con-
centrations (0.01 and 0.02%v/v), while the free compounds had no
effect. At higher concentrations (0.04 and 0.05% v/v) the differ-
ence in the performance of liposomes and free components was
551
Fig. 2. Dose-dependent cytotoxic effect of (A): BEO-BF and (B): BEO-BF-loaded lipo-
somes against SH-SY5Y cells at different incubation times: 24 h (䊉), 48 h () and 72 h
(). Anticancer activity is expressed as the percentage of viable cells measured by an
MTT-assay. Untreated cells and cells exposed to empty liposomes were used as con-
trols. Error bars, if not shown are within the symbols. Results are presented as the
average of three different experiments ± standard deviation. Results are statistically
significant (p < 0.05).
less evident (Figs. 1–4). A possible explanation for the enhance-
ment of anticancer efficacy with the liposomal delivery system is
the increased intracellular uptake of the liposomes in comparison
to the free phytocomplex. Indeed, several studies have demon-
strated the improved intracellular accumulation of a compound
when delivered with liposomes [26,28].
BEO-BF proved to be more efficacious than BEO when adminis-
tered as either a free compound or encapsulated within a liposome.
For instance, after 72 h at a concentration of 0.01% v/v, the BEO-BF
liposomes reduced the cell viability to ∼20% (Fig. 2b), as compared
to BEO liposome that decreased the viability to ∼60% under the
same conditions (Fig. 1b). This observation suggests that bergapten
is not the main component responsible for BEO-induced cell death.
Indeed, it was previously found that none of the individual compo-
nents of BEO are able to induce a loss of cell viability [43]. However,
when the main products of BEO were administered in various com-
binations, only the combination of limonene and linalyl acetate
stood out as the contributing factor for neuroblastoma cell death.
These results are consistent with the ones obtained in this study,
552 C. Celia et al. / Colloids and Surfaces B: Biointerfaces 112 (2013) 548–553
Fig. 3. Dose-dependent cytotoxic effect of (A): BEO and (B): BEO-loaded liposomes
against SH-SY5Y cells at different incubation times: 24 h (䊉), 48 h () and 72 h ().
Anticancer activity is expressed as the percentage of dead cells measured using the
trypan blue dye exclusion assay. Untreated cells and cells exposed to empty lipo-
somes were used as controls. Error bars, if not shown, are within the symbols. Results
are presented as the average of three different experiments ± standard deviation.
Results are statistically significant (p < 0.05).
suggesting that BEO-BF holds greater potential than BEO for the
treatment of neuroblastoma.
4. Conclusions
BEO was recently shown to have anticancer activity against neu-
roblastoma cells. However, the poor solubility of this compound
has limited its use as a therapeutic agent. In this study, the encap-
sulation of BEO within liposomes eliminated the need for toxic
solubilizing agents and allowed for increased anticancer efficacy
in vitro. Liposomal BEO and BEO-BF were able to reduce the via-
bility of SH-SY5Y cells at far lower concentrations than their free
drug counterparts, highlighting the importance of a liposomal drug
delivery system for the future development of BEO as an anticancer
agent.
Competing financial interest
The authors declare no competing financial interest.
Fig. 4. Dose-dependent cytotoxic effect of (A) BEO-BF and (B) BEO-BF-loaded lipo-
somes against SH-SY5Y cells different incubation times: 24 h (䊉), 48 h () and 72 h
(). Anticancer activity is expressed as the percentage of dead cells measured using
the trypan blue dye exclusion assay. Untreated cells and cells exposed to empty lipo-
somes were used as controls. Error bars, if not shown, are within the symbols. Results
are presented as the average of three different experiments ± standard deviation.
Results are statistically significant (p < 0.05).
Acknowledgments
This manuscript was supported by a grant from PONa3 00359
Inter-Regional Research Center for Food Safety and Health.
Appendix A. Supplementary data
Supplementary material related to this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.colsurfb.
2013.09.017.
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