Novel Tween 20 derivatives enable the formation of efficient pH-sensitive drug delivery vehicles for human hepatoblastoma

Article abstract of DOI:10.1016/j.bmcl.2010.04.010

We describe the synthesis, the physicochemical characterization and the biological evaluation of three novel pH-sensitive systems prepared derivatizing polysorbate 20 (Tween 20) with glycine, N-methyl-glycine and N,N-dimethyl-glycine (TW20-GLY, TW20-MMG and TW20-DMG). These derivatives form pH-sensitive vesicles and translocate small molecules into cells. The reported systems are efficient drug delivery systems for human hepatoblastoma cells.

Full text of DOI:10.1016/j.bmcl.2010.04.010

Bioorganic & Medicinal Chemistry Letters 20 (2010) 3021–3025
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Ò
Novel Tween 20 derivatives enable the formation of efficient pH-sensitive
drug delivery vehicles for human hepatoblastoma
a, ,
b,
c
d
d
d
*
Andrea Masotti b, Paola Vicennati , Anna Alisi , Carlotta Marianecci , Federica Rinaldi , Maria Carafa ,
Giancarlo Ortaggi
a
Gene Expression—Microarrays Laboratory, IRCCS-Children’s Hospital Bambino Gesù, P.za S. Onofrio 4, 00165 Rome, Italy
Department of Chemistry, SAPIENZA University of Rome, P.le A. Moro 5, 00185 Rome, Italy
Liver Unit, IRCCS-Children’s Hospital Bambino Gesù, P.za S. Onofrio 4, 00165 Rome, Italy
Department of Chimica e Tecnologie del Farmaco, Faculty of Pharmacy, SAPIENZA University of Rome, P.le A. Moro 5, 00185 Rome, Italy
b
c
d
a r t i c l e i n f o
a b s t r a c t
Article history:
We describe the synthesis, the physicochemical characterization and the biological evaluation of three
Received 1 February 2010
Revised 2 April 2010
Accepted 6 April 2010
Available online 9 April 2010
novel pH-sensitive systems prepared derivatizing polysorbate 20 (TweenÒ 20) with glycine, N-methyl-
glycine and N,N-dimethyl-glycine (TW20-GLY, TW20-MMG and TW20-DMG). These derivatives form
pH-sensitive vesicles and translocate small molecules into cells. The reported systems are efficient drug
delivery systems for human hepatoblastoma cells.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Tween 20
Ò
pH-sensitive vesicles
Drug delivery systems
Hepatoblastoma
Polysorbate 20 (TweenÒ 20) is a stable non-ionic and non-toxic
surfactant widely used in a variety of scientific, pharmaceutical
and cosmetic applications. Tween 20 is a polyoxyethylene deriv-
ative of sorbitan monolaurate, distinguished from the other
Tween-molecules by the length of the fatty acid ester moiety
lated material into the cell cytoplasm.7,8 However, in these systems
the pH-sensitivity is achieved with the aid of CHEMS, a pH-reactive
molecule employed together with TweenÒ 20 and cholesterol.
Therefore, with the aim of integrating niosomes’ delivery proper-
ties and pH-sensitivity within the same moiety, we synthesized a
novel class of compounds based on TweenÒ 20 surfactant bearing
different pH-sensitive head groups (i.e., glycine, N-methyl-glycine,
N,N-dimethyl-glycine). To the best of our knowledge, these pH-
sensitive niosomes have never been reported in the literature to
date. These novel compounds form pH-sensitive vesicles and we
assessed their delivery properties on a human hepatoblastoma
(HepG2) cell line. Hepatoblastoma is the most common liver can-
1
Ò
(Fig. 1).
Non-ionic surfactants are able to form vesicles, called ‘nio-
2
somes’. Niosomes have a closed bilayer structure analogous to
phospholipid vesicles (liposomes). Niosomes share with liposomes
the ability to encapsulate hydrophilic as well as lipophilic mole-
cules and therefore they act as drug carriers. In recent years, nio-
Ò
somes made of equimolar amounts of Tween 20 and cholesterol
have been prepared and characterized.3 Despite this, the prepara-
,4
cer in children. To date, surgical resection represents the best-
9
tion of charged (pH-sensitive) vesicles is highly desirable for bio-
5
,6
medical applications. To couple the versatility of niosomes and
the properties of pH-sensitive vesicles, two recent papers dealed
with the physicochemical and biological characterization of
O
O
OH
HO
Ò
x
charged niosomes based on Tween 20 and their interaction with
w
7,8
Raw 264.7 (mouse monocyte macrophage). These vesicles were
O
O
OH
Ò
O
prepared using Tween 20, cholesterol and cholesteryl hemisucci-
y
nate (CHEMS). This formulation efficiently interacts with target
membranes (i.e., endosomal membrane) and releases the encapsu-
O
C11H23
x+y+z+w=20
z
O
®
TWEEN 20
*
Corresponding author. Tel.: +39 06 6859 2650; fax: +39 06 233234321.
E-mail addresses: andrea.masotti@opbg.net, andrea_maso@yahoo.it (A. Masotti).
These two authors equally contributed to the work.
Figure 1. Molecular formula of polyoxyethylene-sorbitan monolaurate (TweenÒ
20).
0
960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.04.010

3
022
A. Masotti et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3021–3025
choice therapy. In advanced cancer stages with poor prognoses,
adjuvant and neoadjuvant chemotherapy have contributed to im-
Table 1
Ò
Dynamic light scattering (DLS) and f potential measurements of the novel Tween 20
derivatives at pH 5.5 and pH 7.4
10
prove resectability and long-term survival probability. To im-
prove the patient outcome, systems like liposomes or
macromolecular drugs have been used for the delivery of small
Samples
Mean diameter (nm)
f Potential (mV)
pH 5.5
pH 7.4
pH 5.5
pH 7.4
1
1,12
molecules.
For their peculiar physicochemical characteristics
TW20-GLY/Chol (1:1)
247 ± 7
247 ± 8
271 ± 6
—
232 ± 18
246 ± 13
262 ± 18
313 ± 16
263 ± 9
ꢁ4.5 ± 0.7 ꢁ40.3 ± 0.1
ꢁ5.5 ± 0.2 ꢁ39.6 ± 0.7
ꢁ4.9 ± 0.4 ꢁ39.7 ± 0.4
(
dimension and permeability), the novel derivatives prepared in
TW20-GLY/Chol (1:0.75)
TW20-GLY/Chol (1:0.5)
TW20-MMG/Chol (1:1)
TW20-MMG/Chol (1:0.75)
TW20-MMG/Chol (1:0.5)
TW20-DMG/Chol (1:1)
TW20-DMG/Chol (1:0.75)
TW20-DMG/Chol (1:0.5)
this work could represent efficient delivery systems for hepato-
blastoma cells. In fact, due to their pH-sensitivity these novel sys-
tems may undergo protonation once penetrated into cells. These
vesicular structures may destabilize the endosomal membrane as
a consequence of the protonation of the numerous amino groups
present on the surface (‘proton sponge’ effect). This effect, even if
more evident in polymeric delivery systems,13 is also active in
these novel systems and it may facilitate the release of their inner
cargo into the cytoplasm.
—
—
—
ꢁ35.4 ± 0.7
ꢁ31.9 ± 0.2
ꢁ34.0 ± 1.4
—
—
289 ± 18
365 ± 19
176 ± 8
500 ± 38
394 ± 38
560 ± 35
ꢁ7.2 ± 0.3 ꢁ49.6 ± 0.2
ꢁ9.6 ± 1.2 ꢁ46.7 ± 0.2
ꢁ7.1 ± 0.2 ꢁ49.2 ± 1.4
443 ± 1
Dimensional and f potential data for TW20-MMG/Chol are not reported (—) due to
vesicles’ structure disruption at pH 5.5.
To obtain pH-sensitive TweenÒ 20 derivatives with differently
substituted head groups, we decided to functionalize the terminal
hydroxyl groups with glycine (GLY), N-methyl-glycine (MMG) and
N,N-dimethyl-glycine (DMG).
4
4
3
50
00
50
TW20-GLY
TW20-MMG
TW20-DMG
14
We synthesized DMG following the Eschweiler–Clarke reac-
tion,1
5,16
which is commonly used to obtain methyl derivatives of
primary or secondary amines (Fig. 2). The crude product was con-
verted into the corresponding hydrochloride form (DMGꢀHCl) and
used for the following esterification reaction. The procedure to ob-
300
Ò
17
18
tain Tween 20 derivatives (TW20-GLY,
TW20-MMG,
and
1
9
TW20-DMG ) is schematized in Figure 3.
Based on potentiometric measurements, the dissociation con-
stant of TW20-GLY, TW20-MMG and TW20-DMG were pK = 8.26
±0.04), pK = 8.52 (±0.02) and pK = 8.39 (±0.05), respectively. All
pK values were lower than the corresponding pK of glycine
= 10.05) and dimethyl-glycine
= 9.80) in the same experimental conditions. This is consis-
2
50
00
a
2
(
a
a
a
a
2
0
20
150
(
(
pK
pK
a
a
= 9.60), methyl-glycine (pK
a
5
.5
6.0
6.5
7.0
7.5
2
0
pH
tent with the evidence that methyl- or ethyl-esters of glycine (and/
or their amino derivatives such as MMG and DMG) have lower pK
a
Figure 4. Particle size (nm) variation for TW20-GLY/Chol, TW20-MMG/Chol and
TW20-DMG/Chol niosomes at different pH values (5.5 < pH < 7.4). Interpolated
lines are a guide for the eyes.
20
values with respect to unmodified glycine.
All the derivatives are able to form vesicle suspensions. Unila-
mellar pH-sensitive vesicles were prepared separately from a fixed
amount (15 mM) of TW20-GLY, TW20-MMG and TW20-DMG and
cholesterol (Chol) in 1:1, 1:0.75 and 1:0.5 molar ratios by means
more clearly observed at pH 7.4, although we noted few excep-
tions. In fact, the sterically hindered head group of TW20-DMG
may lead to a more expanded vesicular structure with respect to
those formed with TW20-GLY or TW20-MMG. Moreover, for the
TW20-DMG/Chol system at a cholesterol molar ratio of 0.75, we
observed the formation of a vesicle population with the smallest
4
,21
of the ‘film’ method.
Vesicles made of TW20-DMG are generally bigger than those
obtained with TW20-GLY and TW20-MMG. This phenomenon is
HCOOH (99%)/
HCHO (37%) (1:1)
O
O
H3C
H
O
HCl conc.
Cl-
H N
N
N
+
2
reflux 3h, RT 12h
OH
Dimethylglycine
DMG)
Acetone/Ethanol
precipitation
OH
OH
H C
3
Glycine (GLY)
DMG . HCl
(
Figure 2. Reaction scheme for the synthesis of dimethyl-glycine (DMG).
R1
N
O
O
R1
N
O
R1
N
O
®
O
TWEEN 20
R2
R1
O
R2
O
R2
OH
O
x
H SO conc.
0ºC, 12h
2
4
w
O
9
1
2
1
2
GLY, R =R =H
TW20-GLY, R =R =H
TW20-MMG, R =Me, R =H
TW20-DMG, R =R =Me
O
O
O
N
R2
1
2
1
2
MMG, R =Me, R =H
DMG, R =R =Me
y
1
2
1
2
O
C11H23
z
O
Figure 3. Reaction scheme for the synthesis of pH-sensitive TweenÒ 20 derivatives TW20-GLY, TW20-MMG and TW20-DMG.

A. Masotti et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3021–3025
3023
Table 2
Samples
Mean diameter (nm)
f Potential (mV)
pH 5.5
pH 6.0
pH 6.5
pH 7.0
pH 7.4
pH 5.5
pH 6.0
pH 6.5
pH 7.0
pH 7.4
TW20-GLY/Chol (1:0.75)
TW20-MMG/Chol (1:0.75)
TW20-DMG/Chol (1:0.75)
247 ± 8
—
394 ± 38
246 ± 7
320 ± 30
250 ± 26
246 ± 5
280 ± 12
225 ± 23
245 ± 15
265 ± 9
200 ± 13
246 ± 13
263 ± 9
176 ± 8
ꢁ5.0 ± 0.2
—
ꢁ9.0 ± 1.2
ꢁ25.5 ± 0.3
ꢁ10.1 ± 0.4
ꢁ25.0 ± 1.3
ꢁ31.2 ± 1.4
ꢁ13.7 ± 0.4
ꢁ23.6 ± 0.5
ꢁ34.6 ± 1.5
ꢁ19.0 ± 0.9
ꢁ32.2 ± 0.6
ꢁ39.6 ± 0.7
ꢁ34.9 ± 0.7
ꢁ46.7 ± 0.2
diameter (176 ± 8 nm) and a low polydispersity index (PDI = 0.29).
For TW20-GLY/Chol we detected a slight increase in vesicle dimen-
sions as the concentration of cholesterol decreased. It has been
previously demonstrated that a decrease in cholesterol content
leads to higher bilayer flexibility and, as a consequence, to in-
creased vesicle dimensions.7
At more acidic pH values both TW20-MMG/Chol and TW20-
DMG/Chol systems displayed a gradual increase of particle size.
Again, TW20-GLY/Chol niosomes displayed no significant varia-
tion. In addition, the f potential measurements emphasize the pH
dependence: at acidic pH (pH 5.5) the f potential ranges from
ꢁ4.5 (±0.7) mV to ꢁ9.6 (±1.2) mV while at neutral pH (pH 7.4) it
ranges from ꢁ31.9 (±0.2) mV to ꢁ49.7 (±0.2) mV. As a comparison,
vesicles formed by TweenÒ 20 (a non-pH-sensitive system) dis-
played an almost constant f potential (from ꢁ35.7 (±0.8) mV at
,22
At acidic pH (pH 5.5), the amino (or methylamino) groups are
protonated and this generally lead to an increase of niosomes’
vesicle size like in the case of TW20-DMG/Chol. This may be
due to the minimization of electrostatic repulsion on the outer
surface and/or to an increase in the vesicle coalescence and fu-
sion phenomenon. The only exception is represented by TW20-
GLY/Chol vesicles that do not display a significant size increase
at this pH value. For TW20-MMG/Chol system we were not able
to record statistically significant high-quality data at this pH for
all the three surfactant/cholesterol ratios (Table 1). This was due
to the disruption of vesicular structures at this acidic pH. To
study in details this phenomenon, we prepared TW20-GLY/Chol,
TW20-MMG/Chol and TW20-DMG/Chol niosomes at a (1:0.75)
molar ratio and we determined their size and f potential over
a gradual incremental change in pH (5.5 < pH < 7.4) (Fig. 4 and
Table 2).
7
pH 5.5 to ꢁ38.7 (±2.5) mV at pH 7.4). The f potential measure-
ments confirm the niosomes’ pH-sensitivity and support the
hypothesis that the fusogenic tendency observed at pH 5.5 is prob-
ably due to the bilayer phase transition from a lamellar phase (pH
8
7.4) to a hexagonal phase (pH 5.5).
Taken together, all these physicochemical data suggest that
these novel pH-sensitive niosomes should display good endosomal
escape properties in vivo.
To assess the delivery efficiency of pH-sensitive niosomes, cell
interaction studies were performed employing TW20-GLY/Chol
2
3
derivatives on hepatoblastoma cells. For comparison purpose, a
solution of sodium calcein and calcein-loaded neutral niosomes
made of equimolar (15 mM) amounts of TweenÒ 20 and Chol
ꢁ
2
Figure 5. (A) Phase contrast (visible) images of hepatoblastoma (HepG2) cells. (B) Fluorescence image of HepG2 incubated with sodium calcein (10 M) for 15 min. (C)
Fluorescence image of HepG2 incubated with calcein-loaded TW20/Chol vesicles for 15 min. (D) Fluorescence image of HepG2 incubated with calcein-loaded TW20-GLY
vesicles for 15 min. In panels B, C and D nuclei are visualized in blue after DAPI staining. Calcein intracellular delivery (green fluorescence) is visible only in panel D. Scale
bar = 10 lm.

3
024
A. Masotti et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3021–3025
Figure 6. Fluorescence images of HepG2 incubated with calcein-loaded TW20-GLY/Chol vesicles at (A) 0 min, (B) 5 min, (C) 10 min, (D) 15 min (E) 30 min and (F) 2.5 h.
Nuclei are stained with DAPI and visualized in blue while calcein fluorescence is in green. Fluorescence images were merged.
(
TW20/Chol) were also employed. The entrapped calcein concen-
References and notes
ꢁ5
ꢁ5
tration resulted 3.5 ꢂ 10 M for TW20/Chol and 2.9 ꢂ 10
M
1.
2.
3.
Kerwin, B. A. J. Pharm Sci. 2008, 97, 2924.
Uchegbu, I. F.; Vyas, S. P. Int. J. Pharm. 1998, 172, 33.
Santucci, E.; Carafa, M.; Coviello, T.; Murtas, E.; Riccieri, F. M.; Alhaique, F.;
Modesti, A.; Modica, A. STP Pharma Sci. 1996, 6, 29.
for TW20-GLY/Chol while the structured surfactant concentration
was ꢃ1 mM for both preparations.
Calcein is a carboxylic fluorescent dye unable to enter the cells
4
.
.
Carafa, M.; Santucci, E.; Alhaique, F.; Coviello, T.; Murtas, E.; Riccieri, F. M.;
Lucania, G.; Torrisi, M. R. Int. J. Pharm. 1998, 160, 51.
Momekova, D.; Rangelov, S.; Lambov, N. Methods Mol. Biol. 2010, 605, 527.
(Fig. 5B) even after a prolonged incubation (data not shown). Sim-
ilar results were also obtained for the neutral vesicle formulation
TW20/Chol in the same conditions (Fig. 5C). The intracellular cal-
cein fluorescence shown in Figure 5D, indicates that TW20-GLY/
Chol vesicles rapidly and efficiently enter the hepatoblastoma cells
in 15 min at 37 °C. A time-course fluorescence study indicated that
the internalization process occurs between 10 and 15 min (Fig. 6C
and D). After 30 min, the fluorescence signal remains constant up
to 2.5 h (Fig. 6E and F). All the other derivatives reported in this
work displayed good internalization properties and are compara-
ble to the TW20-GLY/Chol system. The reported niosomes dis-
played also a low toxicity in the experimental condition used
5
6. Karanth, H.; Murthy, R. S. J. Pharm. Pharmacol. 2007, 59, 469.
7. Carafa, M.; Di Marzio, L.; Marianecci, C.; Cinque, B.; Lucania, G.; Kajiwara, K.;
Cifone, M. G.; Santucci, E. Eur. J. Pharm. Sci. 2006, 28, 385.
8.
Di Marzio, L.; Marianecci, C.; Cinque, B.; Nazzarri, M.; Cimini, A. C.; Cristiano, L.;
Cifone, M. G.; Alhaique, F.; Carafa, M. BBA Biomembranes 2008, 1778, 2749.
9. Litten, J. B.; Tomlinson, G. E. Oncologist 2008, 13, 812.
10. Finegold, M. J.; Egler, R. A.; Goss, J. A.; Guillerman, R. P.; Karpen, S. J.;
Krishnamurthy, R.; O’Mahony, C. A. Liver Transpl. 2008, 14, 1545.
11. Gabizon, A. A.; Shmeeda, H.; Zalipsky, S. J. Liposome Res. 2006, 16, 175.
12. Maeda, H.; Bharate, G. Y.; Daruwalla, J. Eur J. Pharm. Biopharm. 2009, 71, 409.
13. Masotti, A.; Moretti, F.; Mancini, F.; Russo, G.; Di Lauro, N.; Checchia, P.;
Marianecci, C.; Carafa, M.; Santucci, E.; Ortaggi, G. Bioorg. Med. Chem. 2007, 15,
1504.
(
cell viability >93%).
14. DMG: The reaction consisted in reacting glycine in a (1:1) mixture of formic
As expected, all TweenÒ 20 derivatives synthesized in this work
acid (99%) and formaldehyde (37%) at reflux for 3 h. The one-pot reaction
ꢁ
1
afforded DMG in quite good yield (>70%). Mp 182–183 °C;
m(KBr) 1631 cm ;
+
are pH-sensitive. Based on their peculiar acid-base properties, we
hypothesize that these systems are able to interact with DNA
and other biologically relevant molecules like oligonucleotides,
RNA and other charged molecules with therapeutic properties even
at physiological pH. The pH dependence of TW20-GLY, TW20-
MMG and TW20-DMG is an extremely important property because
it has been successfully demonstrated that similar systems are fus-
1
2
H NMR (300 MHZ, D O):d (ppm) 3.75 (s, 2H), 2.95 (s, 6H); m/c 104 (MH) .
15. Eschweiler, W. Ber. 1905, 38, 880.
1
1
6. Clarke, H. T.; Gillespie, H. B.; Weisshauss, S. Z. J. Am. Chem. Soc. 1933, 55, 4571.
7. TW20-GLY: The esterification reaction between Tween 20 (1 g, mol wt
Ò
ꢃ1228 Da, ꢃ0.82 mmol) and GLY (185 mg, 2.47 mmol) was carried out in the
presence of an equimolar amount of H SO 98% (2.47 mmol, ꢃ200 ll) at 90 °C
2
4
for 12 h. 1H NMR (300 MHz, CDCl
(
): d (ppm) 0.84 (t), 1.22 (br), 1.58 (m), 2.06
3
13
s), 2.29 (t), 2.91 (s), 2.99 (s), 3.40 (br), 3.62 (br), 4.18 (m), 7.82 (br). C NMR
300 MHz, CDCl ): d (ppm) 14.00, 22.57, 24.83, 29.05, 29.18, 29.22, 29.37,
9.50, 31.81, 61.57, 63.25, 68.54 (br, several signals), 72.57, 78.83, 80.18, 80.64,
82.56, 84.82, 86.22, 167.49.
(
3
4
ogenic and able to enter cells rapidly. The novelty of these deriv-
2
atives resides in the fact that the vesicle pH-sensitivity has been
achieved without the addition of pH-sensitive molecules to
TweenÒ 20 formulations, but with a simple functionalization of
the surfactant used for vesicle formation. Herein, we demonstrated
the usefulness of the novel pH-sensitive Tween 20 derivatives as
delivery vehicles for hepatoblastoma cells. These systems have un-
ique physicochemical and delivery characteristics that make them
versatile systems for drug delivery to different tumours.
Ò
1
8. TW20-MMG: The esterification reaction between Tween 20 (1 g, mol wt
ꢃ1228 Da, ꢃ0.82 mmol) and MMG (220 mg, 2.47 mmol) was carried out in the
presence of an equimolar amount of H
2
SO
4
98% (2.47 mmol, ꢃ200 ll) at 90 °C
for 12 h. 1H NMR (300 MHz, CDCl
s), 2.31 (t), 3.42 (br), 3.60 (br), 4.20 (m), 4.82 (br). C NMR (300 MHz, CDCl
d (ppm) 14.10, 22.77, 24.80, 29.22, 29.24, 29.27, 29.43, 29.46, 31.94, 61.67,
3.38, 68.55 (br, several signals), 72.65, 78.91 (several signals), 86.12, 166.93.
3
): d (ppm) 0.85 (t), 1.23 (br), 1.56 (m), 2.15
13
(
3
):
6
Ò
19. TW20-DMG: The esterification reaction between Tween 20 (1 g, mol wt
ꢃ1228 Da, ꢃ0.82 mmol) and DMGꢀHCl (346 mg, 2.47 mmol) was carried out in
the presence of an equimolar amount of H
2
SO
4
98% (2.47 mmol, ꢃ200 ll) at
1
9
0 °C for 12 h. H NMR (300 MHz, CDCl
3
): d (ppm) 0.83 (t), 1.22 (br), 1.58 (m),
13
Acknowledgements
2.30 (t), 3.06 (br), 3.62 (br), 4.19 (m), 4.86 (br). C NMR (300 MHz, CDCl ): d
3
(
7
ppm) 14.02, 22.59, 24.83, 29.38 (br, several signals), 31.82, 34.13, 44.06, 63.26,
0.46 (br, several signals), 165.44.
Ministero della Salute and Ministero dell’Istruzione, dell’Uni-
versità e della Ricerca (MIUR) are gratefully acknowledged for
financial funding (PRIN 2005-prot. No. 2005039758).
2
0. IUPAC evaluation (25 °C, KCl 0.10 M).
21. Dried films were hydrated with a 10ꢁ2 M solution of sodium calcein (HEPES,
pH 7.4) sonicated (V.C.X 400-Sonics) and purified by size exclusion

A. Masotti et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3021–3025
3025
chromatography on a Sephadex G75 column. Dynamic light scattering and f
potential measurements have been carried out using a Malvern Zetasizer Nano
ZS90 (Malvern, UK).
After incubation at 37 °C for 15 min, cells were washed three times with cold
phosphate buffered saline (PBS) and fixed by incubation with a methanol/
acetone (2:1) solution for 10 min at ꢁ20 °C. After three PBS washes, nuclei
0
2
2
2. Uchegbu, I. F.; Florence, A. T. Adv. Colloid. Interface Sci. 1995, 58, 1.
3. HepG2 cells were seeded onto sterile glass coverslips and placed in 24-well
plates. Cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM)
were stained with 100
l
l of a solution of 4 ,6-diamidino-2-phenylindole (DAPI)
dye (300 nM in PBS) and washed again. Fixed cells were mounted with a
glycerol/PBS solution (3:1) to prevent dye photobleaching before fluorescence
image acquisition. Images were acquired with a Nikon Eclipse E600 microscope
(Nikon, Italy) equipped with epifluorescence optics and a Nikon digital camera
DXM1200. Fluorescence images were processed with the dedicated Nikon AC-1
software.
supplemented with 10% foetal bovine serum (FBS) at 37 °C under 5% CO
2
and
1
00% humidity. After reaching 40–50% confluence, cells were incubated with a
solution of sodium calcein alone or loaded inside TW20/Chol and TW20-GLY/
Chol vesicles. All preparations were suspended in complete culture medium.