Recognition of concanavalin a by cationic glucosylated liposomes

Article abstract of DOI:10.1021/la502946t

The specificity of carbohydrate-lectin interaction has been reported as an attractive strategy for drug delivery in cancer therapy because of the high levels of lectins in several human malignancies. A novel cationic glucosylated amphiphile was therefore

Full text of DOI:10.1021/la502946t

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Article
Recognition of Concanavalin A by cationic glucosylated liposomes
Alessandro Mauceri, Stefano Borocci, Luciano Galantini, Luisa Giansanti,
Giovanna Mancini, Antonio Martino, Livia Salvati Manni, and Claudio Sperduto
Langmuir, Just Accepted Manuscript • DOI: 10.1021/la502946t • Publication Date (Web): 03 Sep 2014
Downloaded from http://pubs.acs.org on September 11, 2014
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Recognition of Concanavalin A by cationic
glucosylated liposomes
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§
‡
§
†
Alessandro Mauceri, Stefano Borocci, Luciano Galantini, Luisa Giansanti, Giovanna
#*
§
§
#
Mancini, Antonio Martino, Livia Salvati Manni, and Claudio Sperduto
§
Dipartimento di Chimica Università degli Studi di Roma “Sapienza”, P.le A. Moro 5, 00185 Roma
‡
(Italy). Dipartimento di Scienze Ambientali, Università della Tuscia, Largo dell’Università, 01100
†
Viterbo (Italy). Dipartimento di Scienze Fisiche e Chimiche, Università degli Studi dell’Aquila,
#
Via Vetoio, 67010 Coppito (Italy). CNR, Istituto di Metodologie Chimiche ꢀ Sez. Meccanismi di
reazione and Dipartimento di Chimica Università degli Studi di Roma “Sapienza”, P.le A. Moro 5,
00185 Roma (Italy).
ABSTRACT: The specificity of carbohydrateꢀlectin interaction has been reported as an attractive
strategy for drug delivery in cancer therapy because of the high levels of lectins in several human
malignancies. A novel cationic glucosylated amphiphile was therefore synthesized, as a model
system, to attribute specificity toward Dꢀglucose receptors to liposome formulations. Fluorescence
experiments demonstrated that the monomeric glucosylated amphiphile is capable of interacting
with fluorescently labeled Concanavalin A, a Dꢀglucose specific plant lectin. The interaction of
Concanavalin A with liposomes composed of a phospholipid and the glucosylated amphiphile was
demonstrated by agglutination observed by optical density and dynamic laser light scattering
measurements, thus paving the way to the preparation of other glycosilated amphiphiles differing
for the length of polyoxyethylenic spacer, the sugar moieties and/or the length of the hydrophobic
chain.
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INTRODUCTION
The efficiency of existing and newlyꢀdeveloped therapeutics is often hampered by the absence of a proper control
over biodistribution because they rely on nonꢀtargeted compounds. The possibility of directing a drug toward the
diseased sites without changing its structure and hence its biological activity is fundamental for its therapeutic
application. Encapsulation of drugs into targeted liposomes constitutes an optimum strategy for drug delivery because
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of the biocompatibility, low toxicity and easy of manufacturing of these lipid nanoparticles. In fact the encapsulation of
drugs into liposomes can improve their solubility in aqueous media, protect them, when necessary, from the action of
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lytic enzymes and facilitate their delivery inside the cells. The specificity toward a given target can be obtained by
decorating the liposome surface with proper homing devices, such as receptor ligands or antibodies, capable of
controlling the biodistribution of the drug so that a large part of the dose reaches the target cells, thus reducing side
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effects.
The functionalization of vesicles with natural or synthetic glycolipids might ascribe them specificity toward lectins, a
class of nonꢀenzymatic sugarꢀbinding proteins whose most important function appears to be in cellular recognition and
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adhesion, including the infectivity of pathogens, immune response and reproduction. Mannosylated liposomes, for
example, were shown to preferentially target immune cells such as alveolar and peritoneal macrophages that
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ꢀ9
overexpress a Cꢀtype lectin on their surface, attaining enhanced cellular uptake both in vitro and in vivo.
Glycoliposomes displaying galactose residues on their surface were effectively recognized by the galactose particle
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receptor (lectin) on the Kupffer cells.
The specificity of carbohydrateꢀlectin interaction may indeed offer an attractive strategy for drug delivery in cancer
1
2ꢀ15
therapy because the high levels of lectins such as galectinꢀ3 in several human malignancies
convey glycosylated liposomes into these tumor cells.
can be exploited to
Herein it is reported on the synthesis and the physicochemical characterization of the new glucosylated synthetic
amphiphile 1 (Figure 1) and on the aggregates that it forms as a pure component and in formulation with dimyristoylꢀ
snꢀglyceroꢀphosphocholine (DMPC). The binding of the monomeric glucosylated amphiphile with Concanavalin A
(
Con A), a soy bean lectin, was investigated by fluorescence experiments on Con A fluorescently labeled with
fluoresceinisothiocyanate (FITCꢀCon A), whereas the binding of glucosylated liposomes DMPC/1 with the protein was
investigated by agglutination monitored by optical density (OD) and dynamic laser light scattering (DLS)
measurements.
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OH
Br
N
O
HO
HO
O
O
N
N N
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OH
3
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Figure 1. Structure of the synthetic glucosylated amphiphile 1.
EXPERIMENTAL SECTION
Instrumentation. NMR spectra were recorded on a Bruker 300 Avance spectrometer (operating
1
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at 300 for H and 75 MHz for C) and on a Bruker 400 Avance spectrometer (operating at 400
1
13
MHz for H and 100 MHz for C). Deuterated solvents were used as internal standards.
HRMSꢀESI spectrum was recorded on a LTQ Orbitrap XL instrument.
Steadyꢀstate fluorescence experiments were carried out on a Fluoromaxꢀ4 HoribaꢀJobin Yvon
spectrofluorimeter.
The optical density (OD) and UV measurements were carried out on Cary 300 UV–vis double
beam spectrophotometer (Varian Australia PTY Ltd., Mulgrave, Vic., Australia).
Conductivity measurements were carried out on a Hanna conductimeter, HIꢀ9932, equipped with
a thermostating apparatus, in the temperature range 4–60 °C. All measurements were carried out in
a jacketed cell that was maintained at the appropriate temperature (± 0.1 °C).
Dynamic light scattering (DLS) measurements were performed with a Malvern NanoꢀZetaSizer,
equipped with a 5 mW HeNe laser (wavelength = 632.8 nm) and a digital logarithmic correlator.
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 k T is
the thermal energy and the solvent viscosity. The inverse Laplace transform (CONTIN) was also
h
h
B
B
η
used to resolve correlation functions of multimodal system.
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Materials. 1,2ꢀdimyristoylꢀsnꢀglyceroꢀ3ꢀphosphocholine (DMPC) was purchased from Avanti
Polar Lipids (Alabaster, AL). Fluoresceinisothiocyanate labeled Concanavalin A (FITCꢀCon A)
was purchased from Invitrogen. Concanavalin A (Con A), from Canavalina ensiformis (Jack bean),
glycogen, from bovine liver, PBS (Aldrich, 0.01 M phosphate buffer 0.0027 M KCl, 0.137 M NaCl,
pH 7.4) and all reagents employed for the synthesis of 1 were purchased from SigmaꢀAldrich.
Preparation of compound 3. 20 mL of aqueous KOH 1M were added to a solution of 1.00 g of 2
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(2.6 mmol) in 60 mL of EtOH. The mixture was stirred at room temperature for 1.5 hours and then
neutralized by the addition of Amberlite IRꢀ120. After filtration the solvent was removed under
reduced pressure and the residue was purified by cromatography on silica gel (eluent
CH2Cl2/MeOH=8/2) to give 0.33 g of 3 (yield 58%) as a white solid. 1HꢀNMR (δ CD3OD, 300
MHz) ppm: 2.86 (t, 4JHH = 2.88 Hz, C≡CH); 3.20 (t, 4JHH = 8.33 Hz, 1H, Hꢀ2); 3.26ꢀ3.41 (m, 3H,
Hꢀ3, Hꢀ4, Hꢀ5); 3.63ꢀ3.69 (m, 1H, Hꢀ6); 3.84ꢀ3.89 (m, 1H, Hꢀ6); 4.41ꢀ4.45 (m, 1H , CH2C≡C);
13
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.48 (d, 3JHH = 8.33 Hz, 1H, Hꢀ1). CꢀNMR (δ CD
3
OD, 75 MHz) ppm: 55.2; 61.2; 70.0; 72.9;
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4.2; 76.4; 76.5; 79.5; 100.6.
Preparation of compound 4. 10 mL of aqueous NaOH 5 M and 45 mL of tetraethylene glycol
0.26 mol) were added to 10 mL of THF at 0 °C. A solution of 5.0 g of tosyl chloride (TsCl) (26
(
mmol) in 30 mL of THF was added dropwise at 0 °C and the reaction mixture was stirred until
complete addition of TsCl. After diluting the mixture by adding 200 mL of THF, 800 mL of water
and NaCl were added until complete phases separation. The organic layer was separated, the
solvent was removed under reduced pressure and the residue dissolved in CH
2 2
Cl . The organic
solution was washed with water (10 x 150 mL) and dried over anhydrous Na SO . After filtration
2
4
the solvent was removed under reduced pressure to afford 7.8 g of 4 (yield 85%) as a pale yellow
1
oil. HꢀNMR (δ CDCl
3
, 300 MHz) ppm: 2.43 (s, 3H, CH
3
); 2.65 (s, 1H, OH); 3.59ꢀ3.68 (m, 14H,
3
13
CH
CDCl
40.8.
2
OCH
2
, CH
2
OH); 4.15 (t, JHH = 6.50 Hz, 2H, CH
2
OS); 7.32ꢀ7.75 (m, 4H, CH). CꢀNMR (δ
3
, 75 MHz) ppm: 22.3; 61.7; 68.8; 69.3; 70.3; 70.4; 70.7; 70.8; 72.4; 128.0; 129.9; 138.2;
1
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Preparation of compound 5. 1.98 g of 4 (5.7 mmol) and 0.50 g of NaN (7.6 mmol) were
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suspended in 10 mL of dry DMF under nitrogen. The mixture was stirred and heated at 70 °C for 14
hours. The solvent was removed under high vacuum and the oily residue was dissolved in CH
2 2
Cl .
After filtration the solvent was removed under reduced pressure to afford 0.96 g of 5 (yield 77%) as
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1
3
a yellow oil. HꢀNMR (δ CDCl
3
, 300 MHz) ppm: 2.81 (s, 1H, OH); 3.38 (t, JHH = 6.02 Hz, 2H,
1
3
CH N ); 3.61ꢀ3.70 (m, 14H, CH O). CꢀNMR (δ CDCl , 75 MHz) ppm: 50.7; 61.7; 70.0; 70.4;
2
3
2
3
7
0.6; 70.7; 72.5.
Preparation of compound 6. 0.96 g of 5 (4.4 mmol) were dissolved in 15 mL of dry CH
nitrogen. The mixture was cooled to 0 °C and a solution of 0.13 mL of PBr (1.5 mmol) in 10 mL of
dry CH Cl was added dropwise. A saturated aqueous solution of NaHCO was added after two
hours. The aqueous phase was washed with CH Cl (2 x 20 mL), the organic layers were combined
2
2
Cl under
3
2
2
3
2
2
and dried over anhydrous Na SO . After filtration, the solvent was removed under reduced pressure
2
4
3
and the oily residue was purified by chromatography on silica gel (eluent CHCl ) to afford 0.49 g of
1
3
6
(yield 40%). HꢀNMR (δ CDCl
3
2 3
, 300 MHz) ppm: 3.37 (t, JHH = 6.04 Hz, 2H, CH N ); 3.47 (t,
3
3
J
HH = 6.65 Hz, 2H, CH
2
Br); 3.65ꢀ3.70 (m, 10H, OCH
2
CH
2
O, CH
2
CH
2
N
3
); 3.82 (t, JHH= 6.65 Hz,
1
3
2
H, CH CH Br). CꢀNMR (δ CDCl , 75 MHz) ppm: 30.3; 50.5; 69.9; 70.4; 70.5; 70.6; 71.0.
2 2 3
Preparation of compound 7. 0.32 g of 3 (1.5 mmol) and 0.42 g of 6 (1.5 mmol) were suspended
in 6 mL of H O/tꢀBuOH (1:1). 150 ꢀl of a freshly prepared aqueous solution of ascorbic acid 1 M
and 50 ꢀl of an aqueous solution of CuSO ꢁ5H O 0.3 M were then added to the mixture that was
2
4
2
stirred at room temperature for 24 hours. The reaction mixture was then cooled to 0 °C and
neutralized by an aqueous saturated solution of NaHCO ; the solvent mixture was then removed
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under reduced pressure. The residue was dissolved in MeOH, and after filtration, the solvent was
removed under reduced pressure. The residue was purified by cromatography on silica gel (eluent
1
CHCl
3
/MeOH=85/15) to give 0.64 g of 7 (yield 86%) as a pale yellow oil. HꢀNMR (δ CD
3
OD, 300
3
3
MHz) ppm: 3.20 (t, J = 8.69 Hz, 1H, Hꢀ2); 3.29ꢀ3.35 (m, 3H, Hꢀ3, Hꢀ4, Hꢀ5); 3.51 (t, JHH = 6.52
HH
3
Hz, 2H, CH Br); 3.58ꢀ3.70 (m, 9H, Hꢀ6(1H), OCH CH O); 3.79 (t, J = 6.52 Hz, 2H,
2
2
2
HH
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CH CH Br); 3.87ꢀ3.91 (m, 3H, Hꢀ6(1H), NCH CH ); 4.39 (d, J = 8.69 Hz, 1H, Hꢀ1); 4.53 (t,
2
2
2
2
HH
3
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JHH = 6.25 Hz , 2H, NCH ); 4.76ꢀ5.02 (m, 2H, OCH C=C); 8.15 (s, 1H, C=CH). CꢀNMR (δ
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2
3
CD OD, 75 MHz) ppm: 30.2; 49.9; 61.5; 61.7; 69.0; 70.0; 70.1; 70.3; 71.0; 73.7; 76.6; 76.7; 102.2;
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Preparation of amphiphile 1. 0.65 mL (1.95 mmol) of N,NꢀdimethylꢀNꢀhexadecylamine and 0.65
g of 7 (1.3 mmol) were suspended in 15 mL of CH CN. The mixture was heated to reflux for one
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week. The solvent was removed under reduced pressure and the residue was washed several times
2
with Et O to give 0.62 g of the product 1 (yield 56%) as a pale yellow oil that resulted pure by
1
3
elemental analysis. HꢀNMR (δ CD
3
OD, 400 MHz) ppm: 0.81 (t, JHH = 7.66 Hz, 3H, CH
2
3
CH );
+
+
1.22ꢀ1.42 (m, 26H, N CH
2
CH
2
(CH
2
)
13CH
3
); 1.79 (m, 2H, N CH
2
CH
2
CH
2
); 3.18 (s, 6H,
+
3
+
CH N CH ); 3.21 (t, J = 7.96 Hz, 1H, Hꢀ2); 3.29ꢀ3.47 (m, 5H, Hꢀ3, Hꢀ4, Hꢀ5, N CH CH CH );
3
3
HH
2
2
2
+
3
.56ꢀ3.79 (m, 11H, Hꢀ6(1H), OCH CH O, OCH CH N ); 3.83ꢀ3.92 (m, 3H, Hꢀ6(1H),
2 2 2 2
3
3
C=CNCH
2
CH
2
O); 4.41 (d, JHH = 7.96 Hz, 1H, Hꢀ1); 4.59 (t, JHH = 6.38 Hz, 2H, C=CNCH
2
CH
OD, 100 MHz) ppm: 13.2; 21.7;
22.4; 22.5; 26.1; 28.9; 29.1; 29.2; 29.3; 29.4; 31.7; 50.1; 51.0; 61.3; 61.7; 63.1; 64.4; 65.6; 69.0;
2
O);
13
4
2 3
.76ꢀ5.00 m, 2H, OCH N); 8.11 (s, 1H, Hꢀ15). CꢀNMR (δ CD
ꢀ
+
7
0.0; 70.2; 70.3; 74.0; 76.6; 76.6; 102.2; 124.7; 144.1. HRMS: calculated for C H N O [MꢀBr ] :
35 69 4 9
6
89.5065; found: 689.5086 [α]
D 3
(CH OH, C= 0.81) = ꢀ10.35°. Elemental analysis: calc. for
C
35
H
69
N
4
O
9
∙
5H O: C, 48.89; H, 9.26; N, 6.52. Found: C, 49.01; H, 9.30; N, 6.49.
2
Methods. Determination of the critical micellar concentration (cmc) of amphiphile 1 by conductivity. Conductivity
measurements were carried out at 25 °C by adding known volumes of a surfactant stock solution (0.176 M) to 30 mL of
deionized water. Conductivity was measured after thorough mixing and temperature equilibration after each addition.
The error in the conductivity measurements was within ± 0.1%. The plot of specific conductivity, K, versus the
ꢀ3
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concentration of amphiphile 1 shows two linear trends whose intersection at 1.0ꢀ10 M defines the cmc.
Binding of amphiphile 1 with FITCꢁCon A. The affinity of amphiphile 1 with FITCꢀCon A was investigated at 25 °C
1
8
by steadyꢀstate fluorescence experiments (at 525 nm;
λex= 488 nm) according to a reported procedure. The
fluorescence intensity I (corrected according to the dilution factor) of 2.5 mL of a solution containing 2.4 ꢀg/mL of
FITCꢀCon A and 3.2 ꢀg/mL of glycogen in PBS was investigated as a function of the addition of small volumes of a
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1
B
8.5 mM aqueous solution of amphiphile 1 in PBS. In a blank experiment the corrected fluorescence intensity I of 2.5
mL of a solution containing 2.4 ꢀg/mL of FITCꢀCon A (in the absence of glycogen) was investigated as a function of
the addition of small volumes of the aqueous solution of amphiphile 1 in PBS in order to obtain the same concentrations
of amphiphile 1 of the previous experiment. The fluorescence increase percentage due to displacement of glycogen was
thus evaluated by calculating ꢁꢁI(%) as defined in equation 1
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ꢁ
ꢁI(%)=ꢁI(%)–ꢁI
B
(%)=[(I–I
0
)/ I
0
–(I
B
–IB0)/ IB0]100
1
0
where I and IB0 are the fluorescence intensities of the FITCꢀCon A/glycogen conjugate and of
FITCꢀCon A, respectively, both in the absence of amphiphile 1.
Liposome preparation. Aqueous dispersions of DMPC/1 liposomes were prepared according to
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the procedure described by Hope et al. Briefly, lipid films were prepared on the inside wall of a
roundꢀbottom flask by evaporation of solutions containing the proper amount of DMPC (dissolved
in CHCl
experiments relative to Tm and surface potential determination of DMPC/1 liposomes, the
preparation of lipid films included Laurdan (dissolved in CHCl ) and 4ꢀheptadecylꢀ7ꢀ
3
) and 1 (dissolved in MeOH) to obtain the desired molar percentage mixture. For
3
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hydroxycoumarin (HC) (dissolved in THF), respectively.
The obtained lipid films were kept
overnight under reduced pressure (0.4 mbar); then 3 mL of PBS were added to obtain a lipid
dispersion of the desired concentration. The aqueous suspensions were vortexꢀmixed and then
freezeꢀthawed six times from liquid nitrogen to 40 °C. Lipid suspensions were then extruded (10
times) through a 100 nm polycarbonate membrane.
Determination of aggregate size by DLS. A solution of amphiphile 1 above cmc (50 mM) in
filtered bidistilled water and suspensions of DMPC/1 (95:5) and DMPC/1 (90:10) liposomes (1.25
mM total lipids in PBS) were analyzed by DLS measurements soon after preparation and after 48
hours.
Tm determination by OD measurements. Thermal phase transition of mixed liposomes was
20
determined from the temperatureꢀdependent changes of optical density (OD) at 260 nm.
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Measurements were made at 2.5 mM total lipid concentration in PBS. The temperature scan, in the
2
0ꢀ35 °C range, was carried out a 1 °C/min rate. Results were expressed in turbidity (OD) as a
function of temperature; in the obtained plot the Tm of the liposome formulation corresponds to the
inflection point.
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Tm determination by fluorescence experiments. Tm was also determined by an indirect method
21
described in literature that exploits Laurdan as a fluorescent probe. Emission spectra (λ = 360
ex
nm) of liposome suspension (1 mM total concentration) of DMPC/1/Laurdan (95:5:0.1) in PBS
were acquired as a function of temperature in the range 20ꢀ35 °C. Temperature was controlled by a
circulating bath and the actual temperature was measured in the sample cuvette by a thermocouple.
The generalized polarization parameter (GP) was calculated by equation 2
GP = (I –I )/(I +I )
2
B
R
B
B
B R
where I (450 nm) and I (490 nm) are the fluorescence emission intensities corresponding to the
emission maxima of Laurdan in the gel and in the liquid crystalline phase, respectively. The
transition temperature corresponds to the inflection point in the GP versus temperature plot.
Determination of surface potential (ψ°) of DMPC/1 (95:5) liposomes. Surface potential was
determined by an indirect method described in the literature that exploits the pHꢀsensitive
22
fluorophore HC. Fluorescence measurements of liposome suspension (5 mM total concentration)
of DMPC/1/HC (95:5:0.3) in PBS were performed by scanning the excitation wavelength between
3
00 and 400 nm, at an emission wavelength of 450 nm, varying the pH of solution between 2 and
2 by addition of concentrated aqueous sodium hydroxide or hydrochloric acid. The extent of
1
dissociation of liposome included HC was monitored by the ratio of the excitation fluorescence
a
intensities at 380 nm and 330 nm (pH independent isosbestic point). pK of HC associated with the
charged
cationic liposome bilayer (pK
a
) corresponds to the inflection point of the plot of I380/I330 ratio
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as a function of pH. The surface potential (ψ°) was obtained by a conversion and rearrangement of
Boltzmann equation 3
ꢀ1
charged
neutral
ψ° = ꢀe (pK
a
ꢀ pK
a
)k
B
Tln10
3
10
11
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where k is the Boltzmann constant, T is the absolute temperature, e is the electron charge and
B
neutral
and pKa
is the pK of HC associated with neutral lipid bilayers (DMPC).
a
Agglutination of glucosylated liposomes by Con A investigated by OD measurements. The
agglutination of DMPC/1 (95:5) liposomes in the presence of Con A was determined from the timeꢀ
dependent changes of specific turbidity of a 3 mL sample of 0.83 mM DMPC/1 (95:5) liposomes in
PBS in a 1ꢀcm quartz cell upon addition of Con A (0.33 mg/mL final concentration) in PBS. Scans
were carried out at 525 nm immediately after mixing and every minute for 200 minutes.
Agglutination of glucosylated liposomes by Con A investigated by DLS measurements. The
agglutination of functionalized liposomes in the presence of Con A in the same experimental
conditions of OD measurements, described above, was determined from the timeꢀdependent change
of the apparent hydrodynamic diameter of particles in suspension, evaluated by DLS measurements.
Scans were carried immediately after mixing and at selected times in the time range of 2 hours.
RESULTS AND DISCUSSION
Synthesis and characterization of amphiphile 1. The amphiphilic properties of 1 result from
the presence of a hexadecyl hydrophobic chain linked to a polar portion, i.e. a quaternary charged
nitrogen linked to a glucose moiety (i.e. the glucose receptor recognition functionality) by a
hydrophilic spacer that should guarantee a good exposure of the sugar on the liposome surface.
Amphiphile 1 was synthesized according to the synthetic pathway reported in Scheme 1.
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2
2
2
2
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3
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3
3
3
3
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5
5
5
5
5
5
5
5
5
6
O
TsO
OH
3
4
NaN
3
(77%)
OAc
DMF dry
70°C
O
AcO
AcO
O
OAc
O
N
3
OH
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
3
KOH 1M
EtOH
r.t.
(
62%)
5
PBr
CH
3
(
40%)
OH
2
Cl
2
dry
0°C
O
HO
HO
O
O
N3
Br
OH
3
3
6
H
2
O/t-BuOH=1/1 r.t.
CuSO 0.3M
(
86%)
4
ascorbic a. 1M
OH
O
HO
HO
O
N
O
Br
OH
N
N
3
7
(
56%)
N
1
5
CH
3
CN, ref.
OH
Br
O
HO
HO
O
O
N
N
N
1
5
OH
N
3
1
Scheme 1. Synthesis of glucosylated amphiphile 1.
The key step of the convergent synthesis is the Cu(I)ꢀcatalyzed “click” reaction between the
azidoꢀcompound 6 and the terminal alkyne 3 to obtain, in high yield and under mild reaction
conditions, the bromoꢀintermediate 7 containing the glucose moiety and the hydrophilic spacer. The
1
click adduct 7, confirmed by the presence of the triazolic signal at 8.15 ppm in the H NMR
spectrum, was obtained in relative high yield (86%), though lower than yields generally obtained in
click reactions, probably because the presence of the glucose moiety involves a loss of product in
the purification on silica gel. The 1,4ꢀdisubstitution of the triazolic ring was confirmed by the
presence of the correlation peaks between the signal due to the triazolic proton and the two signals
1
1
due to the methylenic protons at 4.53 and 4.76ꢀ5.02 ppm in the Hꢀ H NMR NOESY spectrum.
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The azidoꢀcompound 6 was obtained starting from tetraethylene glycol by simple synthetic steps
only one of which, i.e. the bromination of 5 to obtain 6, required purification by chromatography on
silica gel. Alkyne 3 was obtained by deacetylation of commercially available glucopyranoside 2
under alkaline conditions. Amphiphile 1 was finally obtained by alkylation of N,Nꢀ
dimethylhexadecylamine with compund 7.
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Krafft point and Krafft temperature of 1 were evaluated as <4 °C because the 20 mM aqueous
solution of 1 is completely transparent and stable at 4 °C.
The cmc of amphiphile 1 was determined by conductivity measurements in deionized water at
ꢀ
3
2
2
5°C, and was found to be 1.0∙10 M.
The DLS analysis of a solution of amphiphile 1 above its cmc (50 mM in deionized water) at
5°C indicated the presence of micelles characterized by hydrodynamic diameter of about 6 nm.
Interaction of monomeric amphiphile 1 with Con A. The binding affinity of amphiphile 1 with
Con A was investigated by fluorescence experiments exploiting fluorescently labeled Con A, FITCꢀ
Con A. The fluorescence of FITCꢀCon A is quenched upon complexation with multibranched
polysaccharides of Dꢀglucose such as glycogen and is restored by addition of monomeric Dꢀ
18
glucose. It was thus expected that glucosylatedamphiphile 1 could displace glycogen from the
binding sites of FITCꢀCon A, thus dequenching its fluorescence, similarly to Dꢀglucose. Therefore,
we investigated the fluorescence emission spectrum of a FITCꢀCon A/glycogen conjugate solution
upon the addition of amphiphile 1. As expected, the addition of 1 to a PBS solution containing the
FITCꢀCon A/glycogen conjugate restored the fluorescence of the protein as shown in Figure 2. This
indicates that the glucose moiety of amphiphile 1 is able to displace glycogen and to bind with Con
A.
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9
0
0
0,2
0,4
0,6
0,8
1
1,2
[
1] mM
Figure 2. Effect of the addition of an aqueous solution of amphiphile 1 on the fluorescence
intensity of a solution of FITC/Con A/glycogen conjugate in PBS. FITC/Con A initial concentration
2
.4 ꢂg/mL; glycogen initial concentration 3.2 ꢂg/mL. The given values are calculated as the mean
and standard deviation of three samples.
Preparation and characterization of mixed liposomes DMPC/1. Mixed DMPC/1 liposomes
were prepared at 95:5 and 90:10 lipid ratios. The DLS analysis, performed soon after extrusion and
after 48 hours, indicated the presence of a stable monomodal distribution of liposomes
characterized by a hydrodynamic diameter of ~120 nm only in the case of the 95:5 formulation,
whereas a multimodal distribution was found in the case of the 90:10 formulation. Thus further
investigations were carried out only on the 95:5 DMPC/1 formulation.
Transition Temperature. The transition temperature of the 95:5 DMPC/1 liposome formulation
was measured by both turbidimetric and fluorescence experiments. Both procedures indicated that
the presence of 5% of 1 in the formulation does not induce any significant change in the
organization of the lipid bilayer with respect to DMPC. In fact the values of Tm obtained by
turbidimetric and fluorescence method were 24.2 °C and 24.5 °C (Figure 3), respectively (the Tm of
DMPC being 24.1 °C).
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,1
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94
9
2
0
9
88
86
-0,1
20
22
24
26
28
30
T (°C)
Figure 3. Percentage variation of optical density OD (
•) at 260 nm reported as percentage, and
generalized polarization parameter, GP ( ), of a DMPC/1 (95:5) liposome suspension (1 mM and 5
▲
mM in total lipids for OD and GP experiment, respectively) plotted as a function of temperature.
The inflection points define the Tm. The given values are calculated as the mean of three samples.
Surface potential. The surface potential (ψ°) of DMPC/1 (95:5) liposomes was measured
according to a described procedure that exploits the spectrofluorimetric determination of the K of
a
HC at the surface of liposomes as shown in Figure 4 and calculates the ψ° by including the pK
a
22
value in eq. 3. DMPC/1 (95:5) liposomes are characterized, as expected, by a positive value of
potential surface ψ° (23 ± 3 mV).
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9
0
,5
1
,5
0
2
4
6
8
10
12
14
pH
Figure 4. Titration curve relative to HC included in DPPC/1 (95:5) liposomes; the inflection
charged
point defines the pK
a
. The given values are calculated as the mean of three samples.
Interaction of DMPC/1 (95:5) liposomes with Con A. Next we investigated if the inclusion of 1
in the DMPC lipid bilayer actually attributes it specificity toward a glucose receptor. Con A is a
tetrameric protein with four binding sites for glucose. Because of the presence of multiple binding
sites, the interaction of Con A with glucose decorated vesicles should result in the aggregation of
vesicles, known as agglutination. Agglutination involves an increase of the dimension of particles in
solution that can be detected by OD and DLS measurements.
The increment of OD of the liposome suspension observed upon the addition of Con A indicated
the increase of particle size due to agglutination of vesicles; further, after ~260 minutes a strong
decrease of OD, due to particle precipitation, was observed (Figure 5). In the absence of 1, the
agglutination of DMPC liposomes in the presence of Con A also occurred, due to non specific
interaction of the protein with the DMPC lipid bilayer, however it occurred over a longer length of
time with respect to glucosylated liposomes. Actually in this case (DMPC liposomes devoid of 1)
OD changes were not detected whithin 4 hours and precipitation was observed only after 24 hours.
In the absence of Con A, neither OD increase nor precipitation was observed.
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,4
,3
,2
,1
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60
0
50
100
150
200
250
300
350
400
time (min)
Figure 5. Effect of the addition of Con A on the optical density, OD, of a suspension of 95:5
DMPC/1 liposomes in PBS, over time. OD increases due to the agglutination process; after ~260
minutes liposome precipitation involves a decrease of OD. Con A concentration 0.33 mg/mL; total
lipids concentration 0.83 mM. Similar results are obtained replicating the experiment on three
different samples.
The results obtained by OD were confirmed by DLS analysis of the agglutination experiment
carried out in the same conditions. In fact the apparent hydrodynamic diameter increased within
four hours, from 120 nm to 180 nm, upon the addition of Con A to the liposome suspension of
DMPC/1 (95:5) (Figure 6). Also in this case after ~260 minutes the precipitation of vesicles was
observed.
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1
2
3
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5
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8
9
0
0
50
100
150
200
250
300
time (min)
Figure 6. Effect of the addition of Con A on the apparent hydrodynamic diameter (d ) of a
h
suspension of DMPC/1 (95:5) liposomes in PBS. Con A concentration 0.33 mg/mL; total lipids
concentration 0.83 mM. Similar results are obtained replicating the experiment on three different
samples.
CONCLUSIONS
We synthesized a novel glucosylated amphiphile by a synthetic strategy that exploits a “click
chemistry” reaction. The results of the investigation indicated that the glucosylated amphiphile is
capable of interacting specifically with Con A both as a monomer and when included in DMPC
liposomes. The designed molecular backbone is therefore suitable for the development of
glycosilated liposomes and an analogous synthetic pattern can be applied to other carbohydrates
such as galactose or lactose to obtain lipid components specific toward human lectins.
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(
(
(
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(22) Zuidan, N. J.; Barenholz, Y. Electrostatic parameters of cationic liposomes commonly used
for gene delivery as determined by 4ꢀheptadecylꢀ7ꢀhydroxycoumarin. Biochim. Biophys. Acta,
Biomembr. 1997, 1329, 211–222
.
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OH
Br
HO
HO
O
O
=
OH
O
N
NN
3
N
15
Glucosylated
amphiphile 1
agglutination
=
Concanavalin A
precipitation
agglutination
Glucosylated liposome
DMPC/1 (95:5)
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