Molecular recognition induced aggregation and fusion between vesicles containing lipids bearing complementary hydrogen bonding head-groups

Article abstract of DOI:10.1039/a606340c

Equimolar mixtures of large unilamellar vesicles, obtained from mixtures of egg lecithin and lipids containing complementary hydrogen bonding head-groups (barbituric acid and triaminopyrimidine), aggregate and fuse generating much larger vesicles.

Full text of DOI:10.1039/a606340c

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Molecular recognition induced aggregation and fusion between vesicles
containing lipids bearing complementary hydrogen bonding head-groups
Vale´rie Marchi-Artzner,a Ludovic Jullien,a† Thadde´e Gulik-Krzywickib and Jean-Marie Lehn*a
a Laboratoire de Chimie des Interactions Mole´culaires, UPR285, Colle`ge de France, 11, Place Marcelin Berthelot, F-75005
Paris, France
b Centre de Ge´ne´tique Mole´culaire, CNRS, 91190 Gif-sur-Yvette, France
Equimolar mixtures of large unilamellar vesicles, obtained
from mixtures of egg lecithin and lipids containing com-
plementary hydrogen bonding head-groups (barbituric acid
and triaminopyrimidine), aggregate and fuse generating
much larger vesicles.
experiments, the size polydispersity of vesicles prepared by
extrusion through Nucleopore filters was low, making them
suitable to analyse the evolution of the vesicle solution by these
techniques. Whereas aqueous suspensions of pure 5, 6 and 4b
exhibited a sharp signal at 41 ± 1 °C by differential scanning
calorimetry (DSC), the 1:9 mixtures with EPC did not display
any transition, thus suggesting the absence of an extensive lipid
segregation within the bilayer.
When identical amounts of weakly light scattering vesicular
preparations of 5-EPC 1:9 and 6-EPC 1:9 were mixed, the
solution became turbid after a few minutes whereas the
appearance of individual preparations remained stable for a
week. This observation was quantified by dynamic light
scattering measurements (Table 1). The diameters of the
starting vesicular suspensions were in the 150 nm range
whereras the multimodal mathematical treatment of the data
from the final state of the mixture provided an apparent
diameter equal to 2000 nm. Considering the relation of size to
scattered light intensity, this value can be reasonably assumed to
represent the diameter of the largest objects.
In order to define more precisely the nature of the events
taking place in these solutions, electron microscopy pictures
were recorded at different times after mixing complementary
vesicles. First, a rapid aggregation leading to the formation of
large clusters of vesicles was observed [Fig. 1(B)]. After 15
min, two different populations of vesicles were visible
[Fig. 1(C)]. A few very large vesicles (micrometer range) were
surrounded by many smaller ones exhibiting size and mor-
phology close to that of starting preparations. Support for the
identification of vesicle fusion as the phenomenon triggering
vesicle growth was provided by the observation of singular
Molecular recognition research has focussed especially on the
selective formation of discrete supermolecules from com-
plementary receptor–substrate pairs.1 Extension to poly-
molecular assemblies should allow the recognition-directed
generation of well-defined, extended supramolecular entities
via the introduction of suitable recognition features into the
molecular components.
Thus, in previous work from our group, the self-assembly of
units bearing complementary hydrogen bonding patterns has
been used for generating liquid crystalline phases2,3 and
supramolecular polymers.3,4 On the other hand, selective
interactions take place at molecular layers into which recogni-
tion groups have been introduced.5–7 Further on, molecular
recognition may direct processes occuring between discrete
polymolecular bodies such as liposomes. Indeed, surface
recognition is a basic process controlling the interactions
between biological cells.8 Endowing artificial vesicles with
recognition features may provide a means of provoking their
interaction and for inducing processes such as aggregation and
fusion. In the longer term it should permit the generation of
polyvesicular architectures in a controlled fashion, a prerequi-
site for constructing organized assemblies of vesicles, of
interest both as artificial functional nanoarchitectures and as
models of biological tissue.
We report here some of our first results on the study of
vesicles containing complementary lipids bearing as head
groups the barbituric acid (BAR) and triaminopyrimidine
(TAP) units which not only interact through three hydrogen
bonds, but also, due to their double-faced nature, form self-
assembled, extended ribbons.9 the polymeric character of which
is expected to enhance the global adhesion between com-
plementary surfaces. We have previously studied the behavior
of electrostatically complementary vesicles bearing opposite
charges,10 and recently the interaction between vesicles con-
taining a biotinylated lipid and streptavidin in solution has been
investigated.11 For this work, we selected the complementary
amphiphiles 5 and 6; they were synthesized according to
Scheme 1.‡ In view of the supramolecular organization
imposed by hydrogen bonding interaction, a polyoxyethylenic
spacer has been introduced in order to provide sufficient
orientational freedom to the recognition sites.
Electron microscopy showed that 4b and 1:9 mixtures of 5 or
6 with 4b form lamellar phases in water under all conditions of
vesicle preparation that have been investigated in the present
study (detergent removal, sonication or lipid swelling in
presence of an alternative electrical field). Large unilamellar
vesicles were prepared from 1:9 mixtures of 5, 6 or 4b and egg
lecithin (EPC) either by detergent dialysis15 or extrusion16
methods. As indicated by freeze-fracture electron microscopy
[Fig. 1(a)] and by quasistatic and dynamic light scattering
Scheme 1 Reagents and conditions: i, N2CHCO2Bn, BF3(OEt2), CH2Cl2,
room temp., 60%; ii, TsCl, pyridine, 0 °C, 69%; iii, Ac2O, pyridine, room
temp., 80%; iv, (a): H2, Pd/C, CH2Cl2, (b) N-hydroxysuccinimide, DCC,
DMAP, (c) HN(C18H37)2, 30 °C; v, NaI, butanone reflux, 93%; vi, NaOH,
EtOH, 96%; vii, CH2(CO2Me)2, NaOMe MeOH, reflux, 85%; viii,
OC(NH2)2, NaOMe, MeOH, reflux, 72%; ix, CH2(CN)2, NaH, Me2SO,
60%; x, HNC(NH2)2, NaOMe, MeOH, reflux, 70%
Chem. Commun., 1997
117

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patterns plays an important role in the events observed. Studies
in progress should provide more information about the nature of
the interaction between complementary vesicles and about its
role in the vesicle fusion event. The intervesicular processes
described here represent a first step towards the controlled,
recognition-directed construction of organized, functionally
integrated assemblies of vesicles leading towards ‘tissue-like’
artificial entities.
events such as the formation of necks between adhering vesicles
and budding on large vesicles [Fig. 1(D)].
In the present system, different interactions may contribute to
the observed coalescence phenomena. In view of the acidobasic
properties of the BAR and TAP lipids leading to the formation
of species of opposite charge, an attractive electrostatic
interaction may also play a role as observed previously.10
Furthermore, ion-pairing reinforced hydrogen bonding between
BAR and TAP units has been observed.17 Since poly-
oxyethyleneglycol is known to promote vesicle fusion, we
established that the spacer itself did not do so, since vesicles
prepared from 1:9 4b-EPC mixtures were stable at least for
several days. The present experiments consequently suggest
that surface recognition via complementary hydrogen bonding
We thank L’OREAL for a graduate fellowship to V. M. A.
Footnotes
† Present address: Laboratoire de Chimie URA 1679, Ecole Normale
Supe´rieure, 24, rue Lhomond, F. 75231 Paris Cedex 05, France
‡
The reaction of an excess of tetraethyleneglycol with benzyldi-
azoacetate.12 followed by tosylation of the remaining alcohol group yielded
compound 2a which, via hydrogenolysis over Pd/C in CH2Cl2 followed by
coupling with dioctadecylamine gave the amide 3a which was converted to
4a. This led to the barbituric acid derivative 5 by monoalkylation of
dimethyl malonate followed by condensation with urea.13 The substituted
triaminopyrimidine 6 was obtained in a similar way via monoalkylation of
malonodinitrile by 4a and then condensation with guanidine.14 The alcohol
4b was synthesized by a modification of the pathway via 2b and 3b.
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Fig. 1 Freeze-fracture electron micrographs of the vesicles prepared from
9:1 mixtures of egg lecithin and lipids 5 or 6 containing either barbituric
acid (BAR) or triaminopyrimidine (TAP) head-groups. (A) Typical image
of vesicle preparations containing either BAR or TAP lipids. (B) Equimolar
mixture of BAR and TAP vesicles immediately after mixing. (C), (D): the
same as (B) but after more than 15 min incubation. Notice the aggregation
of vesicles after mixing and the appearance of much larger vesicles after
incubation, some of which show vesicle fusion [arrows in (D)]. All
photographs are at the same scale; the bar on (D) represents 500 nm.
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Table 1 Diameters (in nm) of large unilamellar vesicles determined by
dynamic light scattering (± 10nm)a
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6-EPC
Angle(°) vesicles
5-EPC
vesicles
1:1 Vesicle
mixture
90
70
50
137
144
155
135
153
156
—
—
2000
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a
Vesicles prepared by extrusion from 1:9 mixtures of 5 or 6 and EPC at
35 °C in 20 mm glucose; vesicle concentration: 0.5 mg dm23
Received, 16th September 1996; Com. 6/06340C
118
Chem. Commun., 1997