Structurally related glucosylated liposomes: Correlation of physicochemical and biological features

Article abstract of DOI:10.1016/j.bbamem.2019.06.003

Liposomes functionalized on their surface with carbohydrates (glycoliposomes) represent an optimal approach for targeting of drugs to diseased tissues in vivo, thanks to biocompatibility, low toxicity and easy manufacturing of these lipid nanoparticles. Here we report on the study of liposomes including a novel glycosylated amphiphile and on the comparison of their features with those of glycosylated analogues described previously. Further, the capability of the different glucosylated formulations to interact with three breast cancer cell lines was investigated. Our results show that the hydrophobic portion of the lipid bilayer strongly influences both the properties and the internalization of glycosylated liposomes.

Full text of DOI:10.1016/j.bbamem.2019.06.003

Accepted Manuscript
Structurally related glucosylated liposomes: Correlation of
physicochemical and biological features
Alessandro Mauceri, Luisa Giansanti, Giuseppina Bozzuto, Maria
Condello, Agnese Molinari, Luciano Galantini, Antonella Piozzi,
Giovanna Mancini
PII:
S0005-2736(19)30135-X
DOI:
https://doi.org/10.1016/j.bbamem.2019.06.003
BBAMEM 83004
Reference:
To appear in:
BBA - Biomembranes
Received date:
Revised date:
Accepted date:
25 January 2019
29 May 2019
7 June 2019
Please cite this article as: A. Mauceri, L. Giansanti, G. Bozzuto, et al., Structurally related
glucosylated liposomes: Correlation of physicochemical and biological features, BBA -
Biomembranes, https://doi.org/10.1016/j.bbamem.2019.06.003
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ACCEPTED MANUSCRIPT
Structurally related glucosylated liposomes:
correlation of physicochemical and biological
features
§
# ‡
†
†
Alessandro Mauceri, Luisa Giansanti, Giuseppina Bozzuto, Maria Condello, Agnese
†
§
§
#
Molinari, Luciano Galantini, Antonella Piozzi and Giovanna Mancini *
§
Dipartimento di Chimica Università degli Studi di Roma “Sapienza”, P.le A. Moro 5, 00185
‡
Roma (Italy). Dipartimento di Scienze Fisiche e Chimiche, Università degli Studi dell’Aquila,
#
Via Vetoio, 67010 Coppito, Italy. CNR–Istituto per i Sistemi Biologici, Via Salaria km 29.300,
†
0
0016 Monterotondo Scalo (RM), Italy. Centro Nazionale per la Ricerca e la Valutazione
preclinica e clinica dei Farmaci, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161
Roma, Italy.
KEYWORDS: lectins, agglutination, glucosylated liposomes, active drug delivery, human breast
cancer cells.
1

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ABSTRACT
Liposomes functionalized on their surface with carbohydrates (glycoliposomes) represent an
optimal approach for targeting of drugs to diseased tissues in vivo, thanks to biocompatibility,
low toxicity and easy manufacturing of these lipid nanoparticles. Here we report on the study of
liposomes including a novel glycosylated amphiphile and on the comparison of their features
with those of glycosylated analogues described previously. Further, the capability of the different
glucosylated formulations to interact with three breast cancer cell lines was investigated. Our
results show that the hydrophobic portion of the lipid bilayer strongly influences both the
properties and the internalization of glycosylated liposomes.
INTRODUCTION
Glycolipids are amphiphilic molecules contains a saccharidic moiety as hydrophilic
headgroup. They are in general biocompatible, show good solubility and are largely investigated
as potential components of thermotropic and lyotropic liquid crystals, pharmaceutical products,
or lubricants. Many biological processes involving cell–cell interactions such as signaling,
recognition and adhesion are initiated, maintained and supported by carbohydrate–protein
1
interactions and are modulated by glycans or glycolipids located on cell surface. Therefore,
glycolipids are considered bioadhesives and have been included in many potential drug delivery
2
3-5
systems such as niosomes, liposomes and lipid nanoparticles to increase their specificity
towards specific target cells and tissues. Galactosylated and mannosylated liposomes are largely
investigated for targeting drug delivery; the former were used as selective targeting agents via
6
-9
specific receptor-mediated uptake in cancer cells, whereas the latter were studied for
2

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macrophage and/or dendritic cells selective targeting via mannose receptor-mediated uptake in
1
0-14
various tissues.
The functionalization of liposomes with glycosylated amphiphiles might increase their
specificity toward lectins, a class of non-enzymatic proteins, mostly located on cell membrane,
that recognize and bind selectively with carbohydrates. The high specificity of carbohydrate–
lectin interactions has already been studied as a strategy for drug delivery by using either
carbohydrates or lectins as homing devices to target specific diseased cells or tissues. For
example, the elevated levels of some lectins, such as galectin-3, on the surface of metastatic
murine and human cancer cells suggest the attractive possibility to exploit these proteins as
1
5-18
targets of glycosylated carriers in anticancer therapies.
A huge number of synthetic
glycosylated ligands differing for the saccharidic residues and the lipophilic segment have been
1
9,20
prepared and explored,
In fact many parameters such as the configuration and the
conformation of the carbohydrate moiety and its proper exposure on the surface of the lipid
nanocarrier, together with water molecules bound on liposome surface, counterions and
2
1
nanoparticle dynamics control the recognition process.
We previously demonstrated by agglutination experiments the capability of liposomes
composed of 1,2-dimistroyl-sn-glycero-phosphocholine (DMPC) and any of the two synthetic
glucosylated amphiphiles, 1 and 2 (Chart 1) to interact with plant lectin Concanavalin A (Con
2
2,23
A).
Herein we report on the synthesis and physicochemical characterization of the new
glucosylated amphiphile 3 (Chart 1) that, due to the presence of two aliphatic chains in the
hydrophobic portion, interacts differently with liposome membranes with respect to amphiphiles
1
and 2 and, most importantly, exerts a minor detergent effect thus allowing being included in
3

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larger amounts in liposome formulations. Liposomes composed of amphiphile 3, as single
component and in formulation with DMPC at different molar ratios, were investigated. The
binding of 3 was investigated by the displacement of glycogen from Con A fluorescently labeled
with fluorescein-isothiocyanate (FITC-Con A); displacement experiments were carried out on 3
as a monomer (i.e. as a pure component, below its cmc) and when included in DMPC liposomes,
by fluorescence measurements, and by agglutination experiments, respectively. Furthermore, cell
uptake of glucosylated liposomes DMPC/1, DMPC/2 and DMPC/3, labeled with fluorescent 1,2-
dimyristoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl)
(ammonium salt) (NBD-PE), was evaluated by laser scanning confocal microscopy and by flow
cytometry on three human breast cancer cell lines (MCF7, MDA-MB-231 and SKBR3)
expressing a glucose receptor.
Chart 1. Liposome components.
METHODS
Instrumentation
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1
13
A Bruker 300 Avance spectrometer (operating at 300 MHz for H and 75 MHz for C) and a
1
13
Bruker 400 Avance spectrometer (operating at 400 MHz for H and 100 MHz for C) were used
1
to record NMR spectra. H resonances of deuterated solvents were used as internal standards.
A LTQ Orbitrap XL instrument was used to record HRMS-ESI spectrum.
A Fluoromax-4 Horiba-JobinYvon spectrofluorimeter was used to carry out steady-state
fluorescence experiments.
A Cary 300 UV–vis double beam spectrophotometer (Varian Australia PTY Ltd., Mulgrave,
Vic., Australia) was used to carry out OD and UV measurements.
A Malvern Nano-ZetaSizer spectrometer, equipped with a 5 mW HeNe laser (λexc = 632.8 nm)
and a digital logarithmic correlator was used to perform DLS measurements. The normalized
intensity autocorrelation functions were measured at an angle of 173° at 25.0 ± 0.1 °C. The
autocorrelation functions were analyzed by using the cumulant fit. The first cumulant was used
to obtain the apparent diffusion coefficients D of the particles, further converted into apparent
hydrodynamic diameters, d , by using the Stokes–Einstein relationship d = k T/3πηD, where
h
h
B
kBT is the thermal energy and η is the solvent viscosity.
A METTLER TA 3000 calorimeter provided with a TC 10 A processor by keeping the cell
(
DSC30) under N flow was used to perform DSC measurements.
2
Materials
DMPC and NBD-PE were purchased from Avanti Polar Lipids (Alabaster, AL, USA). FITC-
Con A was purchased from Invitrogen. Laurdan (6-Dodecanoyl-N,N-dimethyl-2-naphthylamine)
and 4-heptadecyl-7-hydroxycoumarin (HC) were purchased from Fluka. Con A, from Canavalia
ensiformis (Jack bean), glycogen, from bovine liver, phosphate-buffered saline (PBS; Aldrich;
0
.01 M phosphate buffer; 0.0027 M KCl; 0.137 M NaCl; pH 7.4), and all reagents employed for
5

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the synthesis of amphiphile 3 were purchased from Sigma-Aldrich. Amphiphiles 1 and 2 were
2
2,23
prepared as previously described.
Synthetic procedures to prepare amphiphile 3
Preparation of N,N-didodecylmethylamine. 3.6 mL (14 mmol) of N-dodecylmethylamine and
.4 mL (27 mmol) of 1-bromododecane were dissolved in 30 mL of absolute EtOH. The mixture
6
was heated to reflux for two days. The solvent was removed under reduced pressure, the residue
was dissolved in CHCl and washed three times with a saturated aqueous solution of NaHCO .
3
3
The organic layer was dried over anhydrous Na SO , filtered and the solvent was removed under
2
4
reduced pressure. The oily residue was purified by chromatography on silica gel (eluent CHCl3)
1
to afford 2.89 g of N,N-didodecylmethylamine (yield 55%). H-NMR (δ CDCl , 300 MHz) ppm:
3
3
0
.84 (t, J = 9.18 Hz, 6H, CH CH ); 1.16-1.29 (m, 36H, NCH CH (CH ) CH ); 1.54 (m, 4H,
HH
2
3
2
2
2 9
3
13
NCH CH ); 2.34 (s, 3H, NCH ); 2.49 (m, 4H, NCH ). C-NMR (δ CDCl , 75 MHz) ppm:
2
2
3
2
3
1
4.11; 22.69; 27.28; 27.63; 29.36; 29.64; 31.92; 42.31; 57.91.
Preparation of amphiphile 3. (Scheme 1) 300 mg (0.60 mmol) of 4 (prepared as described
2
2
previously) and 239 mg (0.65 mmol) of N,N-didodecylmethylamine were suspended in 5 mL of
anhydrous CH CN. The mixture was heated to reflux for one week. The solvent was removed
3
under reduced pressure and the oily residue was purified by chromatography on silica gel (eluent
from CHCl 100% to CHCl /MeOH 80:20) to afford 0.62 g of the product 3 (yield 56%) as a
3
3
1
3
pale yellow oil. H-NMR (δ CD OD, 400 MHz) ppm: 0.85 (t, J = 7.88 Hz, 6H, CH CH );
3
HH
2
3
+
+
+
1
3
.20-1.40 (m, 36H, N CH CH (CH ) CH ); 1.71 (m, 4H, N CH CH CH ); 3.06 (s, 3H, N CH );
2
2
2 9
3
2
2
2
3
3
+
.19 (t, J = 7.96 Hz, 1H, H-2); 3.25-3.46 (m, 7H, H-3, H-4, H-5, N CH CH CH ); 3.52-3.69
HH
2
2
2
+
(
m, 11H, H-6(1H), OCH CH O, OCH CH N ); 3.83-3.92 (m, 5H, H-6(1H), C=CNCH CH O,
2 2 2 2 2 2
+
3
3
OCH CH N ); 4.41 (d, J = 7.95 Hz, 1H, H-1); 4.58 (t, J = 6.40 Hz, 2H, C=CNCH CH O);
2
2
HH
HH
2
2
6

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1
3
4
1
6
1
.76-4.97 (m, 2H, OCH N); 8.15 (s, 1H, s, 1H, HC=C). C-NMR (δ CD OD, 100 MHz) ppm:
2 3
4.44; 23.25; 23.69; 27.34; 30.19; 30.44; 30.51; 30.62; 30.72; 30.73; 33.03; 51.39; 62.20; 62.71;
3.00; 63.72; 65.59; 70.31; 71.35; 71.40; 71.49; 71.59; 75.02; 77.96; 78.01; 103.59; 126.04;
-
+
45.49. HRMS: calculated for C H N O [M-Br ] : 787.6160; found: 787.6185. Elemental
4
2
83
4
9
analysis for C, H, N was within the theoretic value.
Determination of critical micellar concentration (cmc) of amphiphile 3
The cmc of amphiphile 3 was measured at 25 °C according to a reported procedure that
exploits the variation in the intensity of the vibronic fine structure of pyrene monomer
2
4
fluorescence upon association with micellar/vesicular aggregates. Aqueous solutions (3 mL) of
amphiphile 3 at concentrations between 10 μM and 80 M were added to a defined amount of
pyrene in order to obtain ~ 0.5 μM final concentration of pyrene (prepared from 7 μL of a 160
μM stock solution of pyrene in absolute ethanol dried by a nitrogen flux). The solutions were
kept above 37 °C, under stirring, for 12 h. Emission spectra of the solutions were acquired in the
range 350–450 nm (λexc = 335 nm). A sharp increase of the ratio of the third and first vibronic
bands of pyrene I /I (I at 380 nm, I at 370 nm) occurs at cmc.
3
1
3
1
Binding of amphiphile 3 with FITC-Con A
The capability of amphiphile 3 to interact with FITC-Con A was investigated at 25 °C by
steady-state fluorescence experiments (at 525 nm; exc = 488 nm) according to a reported
2
5
procedure. The variation of fluorescence intensity I of 2.5 mL of a solution containing 2.4
μg/mL of FITC-Con A and 3.2 μg/mL of glycogen in PBS upon the addition of small volumes of
a 14.2 mM aqueous solution of amphiphile 3 in PBS was monitored. In a blank experiment the
variation of fluorescence intensity I of 2.5 mL of a solution containing 2.4 μg/mL of FITC-Con
B
A (in the absence of glycogen) upon the addition of small volumes of the aqueous solution of
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amphiphile 3 in PBS (to obtain concentrations of 3 analogous to those of the previous
experiment) was monitored. The fluorescence spectra were recorded after equilibrium state was
reached. All fluorescence intensities were normalized according to the dilution factor. The
following equation was thus applied to evaluate the percentage of fluorescence increase due to
displacement of glycogen I(%):

I(%) = I(%)–I (%) = [(I–I )/ I –(I –I )/ I ]100 (1)
B
0
0
B
B0
B0
where I and I are the fluorescence intensities of the FITC-Con A/glycogen conjugate and of
0
B0
FITC-Con A, respectively, both in the absence of amphiphile 3.
Liposomes preparation
Lipid films were prepared by evaporation of solutions containing the proper amount of DMPC
(
dissolved in CHCl ) and 3 (dissolved in MeOH) to obtain the desired molar percentage mixture.
3
For Tm determination of DMPC/3 liposomes, Laurdan (dissolved in CHCl ) was included in the
3
lipid films. For liposome uptake by human breast cancer cells, NBD-PE (dissolved in CHCl3)
was added to the components of the lipid films. The lipid films were kept overnight under
reduced pressure (0.4 mbar), then PBS was added to obtain a lipid dispersion of the desired
concentration; liposome solutions were vortex-mixed and multilamellar vesicles (MLV) were
obtained. MLV were freeze-thawed six times from liquid nitrogen to 50 °C and then extruded
(10 times) through a 100 nm polycarbonate membrane.
Determination of aggregate size by DLS
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DLS measurements were carried out on aqueous solutions of amphiphile 3 (80 mM) and
suspensions of DMPC/3 liposomes at molar ratios 95:5, 80:20, 70:30 (1.25 mM total lipids in
PBS) soon after preparation and after 48 hours.
Tm determination by fluorescence experiments
Tm of DMPC/3 (95:5, 80:20, 70:30) liposome formulations was investigated by fluorescence
experiment applying an indirect method described in the literature that exploits Laurdan as
2
6
fluorescent probe (DMPC/3/Laurdan, 95:5:0.1). Emission spectra (λ = 360 nm) of liposome
exc
suspension (1 mM total concentration) in PBS were acquired varying the temperature in the
range 15-35 °C. The temperature of the sample was measured by a thermocouple. Equation 2
provides the generalized polarization parameter (GP):
GP = (I –I )/(I +I ) (2)
B
R
B
B
where I (450 nm) and I (490 nm) are the fluorescence emission intensities corresponding to
B
R
the emission maxima of Laurdan in the gel and in the liquid crystalline phase, respectively. The
Tm value corresponds to the inflection point of the sigmoidal curve obtained by plotting the GP
versus the temperature of the sample. Error in determination is of ± 0.3 °C.
Determination of thermotropic properties of DMPC/3 liposomes
DSC measurements were carried out on 30 μL of MLV (1 mg/10 μL, 148 mM total lipids).
Two heating scans were recorded at the rate of 5 °C/min and two subsequent heating scans were
recorded at the rate 1 °C/min. Under the experimental conditions, reproducible thermal
recordings were obtained. Uncertainty on temperatures was determined to be 0.1 °C.
Determination of surface potential of glucosylated liposomes
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Surface potential (ψ°) of DMPC/3 liposomes was determined by an indirect method using HC,
2
7
a pH-sensitive fluorescent probe. Excitation spectra of 5 mM liposome suspension between 300
and 400 nm (emission wavelength = 450 nm) were recorded. Fluorescence measurements of
DMPC/3/HC (95:5:0.3) in PBS were performed by scanning the excitation wavelength upon
variation of the pH of solution in the 2-12 range (obtained by addition of aqueous sodium
hydroxide or hydrochloric acid). The extent of dissociation of HC included in the lipid bilayer
was monitored by the ratio of the excitation fluorescence intensities at 380 and 330 nm (pH-
independent isosbestic point). pKa of HC associated with cationic lipid bilayer (pKa charged)
corresponds to the inflection point of the plot of I380/I330 ratio versus pH. The ψ° was obtained
by a conversion and rearrangement of the Boltzmann equation:
charged
neutral
ψ° = (pKa
- pKa
)k T ln 10/e
B
where k is the Boltzmann constant, T is the absolute temperature, e is the electron charge, and
B
pK neutral is the pK of HC associated with neutral lipid bilayers (DMPC).
a
a
Agglutination of glucosylated liposomes by Con A monitored by OD measurements
The variation of specific turbidity of a 3 mL sample of 0.83 mM DMPC/3 (95:5, 80:20 and
0:30 molar ratios) liposomes in PBS upon the addition of Con A (0.33 mg/mL) was recorded
7
over 1000 minutes. Scans were carried out on liposome PBS suspension in a 1 cm quartz cell
upon addition of Con A, at 525 nm, immediately after mixing and every minute.
Cell culture
Human breast cancer cell lines MCF7, MDA-MB-231 and SKBR3 were grown as monolayer
in DMEM supplemented with 10% FBS, 1% penicillin (50 IU/mL) and streptomycin (50 IU/mL)
in a humidified atmosphere of 5% CO in a water-jacketed incubator at 37 °C.
2
Flow Cytometry
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The uptake of liposomes, fluorescently labeled with N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)
tagged lipid NBD-PE, was evaluated by flow cytometry after 30 min, 1h, 4h and 18 h of
treatment with 20 M suspensions of the following liposome formulations: (i) DMPC/1 95:5, (ii)
DMPC/2 95:5, (iii) DMPC/3 95:5 and (iv) DMPC/3 7:3. After treatment, cells were washed with
ice-cold PBS, detached with EDTA and trypsin, resuspended in ice cold PBS and immediately
analyzed. Fluorescence signals were analyzed with a FACScan flow cytometer (Becton
Dickinson, Mountain View, CA) equipped with a 15 mW, 488 nm, and air-cooled argon ion
laser. The NBD-PE fluorescent emission was collected through a 530 nm band pass filter. At
least 10,000 events were analyzed. Liposome uptake was evaluated as fluorescence intensity and
expressed in arbitrary units (AU), calculated as the ratio between the mean fluorescence channel
(MFC) of treated cells and the MFC of untreated cells. The analysis was performed by CellQuest
software (Becton Dickinson).
Laser Scanning Confocal Microscopy
Cells, grown on 12 mm glass coverslips, were inoculated with liposome formulations.
Intracellular distribution of fluorescently labeled liposomes was analyzed by Laser Scanning
Confocal Microscopy (LSCM) after 18 h of treatment. After treatment cells were fixed in 3.7%
paraformaldehyde in PBS, for 10 minutes at room temperature. Observations were performed by
a Leica TCS SP2 laser scanning confocal microscopy (Leica, Microsystems, Mannheim,
Germany) equipped with an Ar/Kr laser.
1
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RESULTS
Amphiphile 3 properties
Krafft point and Krafft temperature of 3 were evaluated as <4 °C because a 20 mM aqueous
solution of 3 (above cmc) is completely transparent and stable at 4 °C. The cmc of amphiphile 3
in deionized water at 25 °C, determined by a fluorimetric method that exploits pyrene as
-5
fluorescent probe, was found to be 2.5∙10 M.
Interaction of amphiphile 3 with FITC-Con A
It is known that the fluorescence of FITC-Con A is quenched upon complexation with
multibranched polysaccharides of D-glucose, such as glycogen, and can be restored by addition
2
5
of monomeric D-glucose. The fluorescence of FITC-Con A was restored upon the addition of 3
to a PBS solution containing the FITC-Con A/glycogen conjugate because the glycosylated lipid
displaced glycogen from the binding sites of FITC-Con A (Figure SI 1).
Size and size distribution of 3 and of DMPC/3 liposomes
DLS measurements on solution of pure 3 showed that this amphiphile forms aggregates of ~6
nm diameter. The DLS analysis of DMPC/3 liposomes at 95:5, 80:20 and 70:30 molar ratios
were performed soon after extrusion and after 48 hours; results indicated the presence of stable
monomodal distributions of liposomes featuring a hydrodynamic diameter of ~120, 105 and 165
nm, respectively (PDI lower than 0.2).
Tm of DMPC/3 liposomes and relative transition enthalpies
The values of Tm (obtained by a fluorimetric method and by DSC) and relative transition
enthalpies (obtained by DSC) for DMPC/3 formulations are reported in Table 1.
1
2

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Table 1. Tm values and relative transition enthalpies for DMPC and DMPC/3
liposomes at molar ratios 95:5, 80: 20 and 70:30. Uncertainty in determination is
±
0.5°C, ±0.1°C and ±0.5 kJ/mol for a, b, and c entries, respectively.
c
-1
Formulation
Tm / °C
Laurdan]
Tm / °C
[DSC]
H / kJ mol
a
b
[
DMPC
24.1
23.8
22.5
-
28.9
27.8
8.5
-
DMPC/3 (95:5)
DMPC/3 (80:20)
DMPC/3 (70:30)
24.6
21.8
22.2
Thermotropic properties of DMPC/3 liposomes
Thermograms and Tm and H values obtained by DSC measurements are reported in Figure 1
and Table 1, respectively. The value of Tm of DMPC/3 liposomes at 95:5 molar ratio is very
close to the Tm of liposomes of mere DMPC whereas the corresponding peak on the thermogram
is considerably broader. When 3 is included in DMPC liposomes at 20% molar percentage the
peak corresponding to the main transition is broader with respect to DMPC liposomes and both
Tm and H values are shifted to lower values whereas when 3 is included at 30% molar
percentage the transition is not observable.
1
3

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Figure 1. Thermograms relative to various mole ratios DMPC/3 MLVs organized according to
increasing percentages of GA 3, the first thermogram from the bottom being relative to vesicles
formulated only with DMPC. Scan rate is 1 °C/min.
Surface potential of DMPC/3 liposomes
DMPC/3 liposomes at molar ratios 95:5, 80: 20 and 70:30 feature, as expected, a positive
surface potential whose value increases at increasing percentage of 3 in the formulation, 26 mV,
5
6 mV, and 95 mV, respectively.
Agglutination of DMPC/3 liposomes by Con A monitored by OD measurements
The addition of Con A to DMPC/3 formulations 95:5 and 80:20 yielded an increase of OD of
the suspensions (Figure 2), whereas in the case of DMPC/3 70:30 agglutination was not
observed. Figure 2 shows that agglutination of DMPC/3 at 80:20 lipid ratio occurs faster and to a
higher extent with respect to 95:5. In the case of DMPC/3 95:5 a decrease of turbidity was
observed after ~ 600 minutes, due to precipitation of agglutinated particles; note that in the
1
4

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absence of Con A or in the case of not functionalized DMPC vesicles, no significant OD changes
were detected within 1000 minutes.
Figure 2. Effect of the addition of Con A on the optical density, measured as absorbance at 525
nm, of a suspension of DMPC/3 liposomes in PBS at lipid ratios of 95:5, 80:20 and 70:30. Con
A concentration 0.33 mg/mL; total lipids concentration 0.83 mM.
Cell uptake association of glycosilated liposomes
Liposome-cell association was evaluated by flow cytometry at 30 min, 1h, 4h and 18 hr. No
significant differences were observed between liposome formulations after 30 min and 1 h (data
not shown). Flow cytometry analysis, reported in Figure 3, shows the association (expressed as
arbitrary units, A.U.) of liposomes with breast cancer cells. After 4 h of treatment, MFC values
revealed in all cell lines incubated with DMPC/1 95:5, DMPC/2 95:5 and DMPC/3 95:5
liposomes did not significantly differ from those of cells treated with DMPC liposomes. On the
other hand, a significant increase of MFC values was detected in MDA and SKBR3 cell lines
treated with DMPC/3 liposomes at 70:30 molar ratio.
The analysis performed after 18 h of treatment showed an increase of A.U. values in cells
treated with DMPC/1 95:5 and DMPC/2 95:5 formulations when compared to cells treated with
1
5

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DMPC liposomes. No significant differences were observed between A.U. values of samples
treated with DMPC/3 95:5 formulation and those of cells treated with DMPC liposomes. On the
other hand, A.U. values of cells significantly increased after the incubation with DMPC/3 70:30
liposomes in all three breast cancer cell lines.
Figure 3. Association of different glucosylated liposomes with three different human breast
cancer cell lines.
Intracellular distribution of glycosilated liposomes
The observations performed by LSCM after 18h of treatment showed different cell
distributions of the evaluated liposome formulations (green signal, Figure 4). Generally, in
agreement with flow cytometry data, MDA-MB-231 cells resulted the most efficient in the
internalization of liposomes. In all cell lines DMPC, DMPC/1 95:5 and DMPC/2 95:5 liposomes
appeared to be distributed as fluorescent spots, very likely in cytoplasmic vesicles localized in
the perinuclear region. On the other hand, DMPC/3 70:30 liposomes appeared strongly clustered
in the proximity of the plasma membrane.
1
6

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Figure 4. Images obtained by LSCM analysis after 18h of treatment on three cells lines with
different DMPC/GA liposomes at 95:5 molar ratio and DMPC/3 at 70:30 molar ratio.
DISCUSSION
Physicochemical characterization of amphiphile 3
Amphiphile 3 was obtained by alkylation of N,N-didodecylmethylamine with compound 4 in
anhydrous CH CN (Scheme 1). The preparation of the hydrophilic alkylating residue 4 was
3
2
2
previously described, whereas N,N-didodecymethylamine was obtained by alkylation of N-
dodecylmethylamine with 1-bromododecane in absolute EtOH.
1
7

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Scheme 1. Synthesis of amphiphile 3.
The hydrophobic portion of 3 is considerably larger with respect to the two analogues 1 and 2
2
8
investigated previously. This structural difference can influence its aggregation features;
actually the aim of this variation in the molecular structure of the glycosylated lipid component
was reducing the detergent capability of the amphiphile in order to increase its amount in the
formulation with phospholipids glycosylated component in mixed liposomes. In fact in the case
of 1 and 2 their amount in formulation with DMPC can not exceed 5% due to their detergent
2
2,23
properties.
Further, the different hydrophobic portion might affects other properties of lipid
membrane, such as lipid packing, fluidity, permeability and, as a consequence, their interaction
29
with cells. The DLS analysis of aggregates of pure 3 suggested that this amphiphile forms
micelles. This behavior was unexpected, because in general twin type amphiphiles form
2
8
vesicles, however it can be explained considering that the polar headgroup is quite large and the
balance between the hydrophobic and hydrophilic region of the amphiphile, hence the packing
parameter, addresses aggregation towards micelles rather than vesicles. In the case of DMPC/3
liposomes the large headgroup of glycolipid is counterbalanced by the smaller headgroup of
DMPC and the formation of liposomes is not hampered. All the investigated mixed formulations
showed a diameter in good agreement with the dimension imposed by the extrusion protocols.
Differently from what observed in the case of DMPC/1 and DMPC/2 liposomes that were
2
2,23
monomodal and stable only at 95:5 molar ratio,
3 can be included up to 30% in DMPC
1
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ACCEPTED MANUSCRIPT
liposomes due to the low detergent effect attributed by the presence of twin tails. These results
indicate that, though 3 is a micelle-forming amphiphile, its twin tails confer it a less detergent
effect with respect to its structural single tail analogues 1 and 2.
The results obtained by DSC measurements clearly indicate that the presence of amphiphile 3
in DMPC formulations, even at very low percentages, significantly loosens lipid packing. Yet at
5
% of 3 in the formulation Tm and H are shifted to lower values and the cooperativity of the
transitions is sensibly reduced (as reflected by the width of the peak) in analogy with DMPC/1
2
3
and DMPC/2 liposomes at the same molar ratio. The disturbing effect of 3 on lipid packing
increases as a function of its amount in the formulation. In fact, in the case of 80:20 DMPC/3 the
transition is still observable, but the peak is significantly broadened and both Tm and H values
decrease. The fact that the peak relative to the main transition is practically absent in the case of
7
0:30 DMPC/3 liposomes indicates that lipid packing is slackened and lipid bilayer is probably
very leaky. However, despite its destabilizing action, these results confirm that the detergent
effect of 3 is lower with respect to that of 1 and 2. This is certainly due to the higher number of
van der Waals contacts of its large hydrophobic portion.
The capability of 3 (as monomer or included in mixed liposome) to interact with lectins was
investigated using as a model Con A, a homotetrameric protein that bears four binding sites for
glucose. Fluorescence experiments indicated the capability of 3 to bind with Con A due to its
glucose moiety because the addition of 3 to a PBS solution of FITC-Con A/glycogen conjugate
displaced glycogen thus restoring the fluorescence of FITC-ConA (Figure SI 1), similarly to
23
what reported in the case of 1 or 2. However, in the experiments concerning 1 and 2 the
complete restoration of fluorescence signal was observed at lower concentration of the
glucosylated component (≈ 1mM vs 5 mM). This suggests that the bulkiness of the hydrophobic
1
9

ACCEPTED MANUSCRIPT
portion of 3 might hamper the interaction of the sugar moiety with the binding sites of the lectin.
In fact in this experiment the glucosylated amphiphile should displace glycogen, a bulky
multibranched polysaccharide, conjugate with FITC-Con A. It is thus reasonable that steric
hindrance of 3 might slow down the completion of the process.
In the case of DMPC/3 liposomes the binding of glucose moiety with Con A was evaluated by
following by OD the eventual agglutination upon the addition of the protein to the liposome
suspension: the increase of the OD of DMPC/3 liposome suspensions (at 95:5, 80:20 and 70:30
molar ratios) indicates the increase of particle size due to agglutination. The decrease of OD
observed in the case of 95:5 DMPC/3 liposomes at ~ 600 minutes clearly indicates particle
precipitation. Results clearly indicate that the extent of agglutination decrease by increasing the
amount of GA. This apparent incongruity can be explained considering that the presence of a
high amount of 3 in formulation disturbs lipid packing, as indicated by DSC results, and that the
defects formed on the surface of vesicles might accommodate the sugar moiety. Analogous
30
observation was reported in the case of a pH sensitive glucosylated amphiphile. The role of
lipid compaction on the exposure of glucose moiety in the bulk is supported by the comparison
of agglutination and DSC experiment results obtained in the case of 95:5 DMPC/3 liposomes
with the results of analogous agglutination and DSC experiments reported previously concerning
9
5:5 DMPC/1 and DMPC/2 liposomes. In fact it was observed that 95:5 DMPC/3 liposomes
feature the slowest and lowest extent of OD increase (Figure 5A), in correspondence with the
2
3
lowest compaction of lipid bilayer as shown by thermograms (Figure 5B). Further, a previous
investigation on the influence of the state of phase of lipid membrane on the exposure of glucose
residues on the surface of liposomes also showed the crucial role of lipid packing on the control
3
1
of the interaction with lectins, due to the housing of sugar moieties in the voids formed by loose
2
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lipid packing. Therefore, proper surface functionalization (ligand connected to a proper
hydrophilic spacer) might not guarantee targeting, without a control on the state of phase and on
the organization of lipid membrane.
Figure 5. A) Effect of the addition of Con A on the optical density, measured as absorbance @
5
25 nm, of suspensions of DMPC/1, DMPC/2 and DMPC/3 liposomes in PBS at lipid ratio 95:5.
ConA concentration 0.33 mg/mL; total lipids concentration 0.83 mM. B) Thermograms relative
to DMPC/1 (top), DMPC/2 (middle), DMPC/3 (bottom) MLV. Scan rate is 1◦C/min. Data for
DMPC/1 and DMPC/2 liposomes are taken from ref. 23.
Biological investigation
The targeting efficacy of glucosylated liposomes DMPC/1, DMPC/2 and DMPC/3 was evaluated
3
2
on three human breast cancer cell lines, which express glucose receptor, namely GLUT1.
GLUT1 is present at variable levels in many tissues and is believed to be responsible for basal
3
3,34
glucose uptake.
Elevated GLUT1 expression has been described in many cancers, including
hepatic, pancreatic, esophageal, brain, renal, lung, cutaneous, colorectal, endometrial, ovarian,
3
5,36
and breast carcinoma.
GLUT1 expression in breast cancer has been extensively studied also
3
7
in patients and has been correlated with poor prognosis. Thus, breast cancer cells represent an
2
1

ACCEPTED MANUSCRIPT
optimal model to investigate the targeting features of glucosylated liposomes. The expression of
GLUT1 in the selected cell lines was checked before the investigation (data not shown).
Flow cytometry experiments (cell-liposome association) and LSCM observations (liposome
internalization) clearly indicated that (i) the three cell lines feature different cell-association and
cell-internalization, and (ii) DMPC/3 liposomes show a different behaviour with respect to
DMPC/1 and DMPC/2 liposomes.
The differences in the cellular association among the three breast cancer cell lines are very likely
due to the different composition of their plasma membrane (i.e. lipid composition, proteins such
3
8
as glucose receptors, etc). The different behaviour of the formulations in the interaction with
each cell line is mainly controlled by the hydrophobic portion of the glucosylated component
and, as a consequence, by lipid organization. In fact, in the case of 95:5 formulations, those
containing 1 and 2 interact more efficiently with cells with respect to liposomes containing 3.
Though liposome-cell association significantly increases in the case of 70:30 DMPC/3
liposomes, it is worth of note that the intracellular distribution of DMPC/3 liposomes is in both
cases (95:5 and 70:30) dramatically different from that of DMPC/1 and DMPC/2 ones. In fact
DMPC/3 liposomes result mainly localized in the proximity of cell membrane, whereas DMPC/1
and DMPC/2 ones appear distributed in intracytoplasmic vesicles. The different uptake of
DMPC/3 liposomes could be ascribed to their reduced lipid packing with respect to DMPC/1 and
DMPC/2 liposomes (as suggested by DSC results) that probably involves a different exposure of
the glucose moiety and/or its inclusion in the voids present in lipid bilayer. Another possible
reason of the different behavior of liposomes containing 3 with respect to those containing 1 or
2
could be the different fluidity/elasticity of lipid bilayer, parameter that can affect cell
internalization of liposomes.
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CONCLUSIONS
The correlation of some physico-chemical properties of different mixed DMPC/GA liposomes
with their ability to influence the uptake and the intracellular distribution in three breast cancer
cell lines pointed out that the functionalization of liposomes with glycolipids can improve the
interaction with cells. The modality and the extent of such interaction strongly depend on the
molecular structure and/or the amount of the glucosylated component. The hydrophobic region
of the lipid bilayer, in particular lipid packing, seems to play a pivotal role in the interaction of
glycosylated liposomes with sugar receptors on target cells. As a whole, besides confirming that
the decoration of liposomes surface with sugar residues has very good potential in active
targeting, this investigation put in evidence how a proper design of the glycosylated component,
even in the hydrophobic portion, is crucial to ensure the highest delivery efficiency.
AUTHOR INFORMATION
Corresponding Author
*
giovanna.mancini@uniroma1.it
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Conflict of interest
The authors certify that they have no affiliations with or involvement in any organization or
entity with any financial interest or non-financial interest in the subject matter or materials
discussed in this manuscript.
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