Fig. 2 (A) Transmission electron microscopy image of the glycolipid
1 vesicles (0.1 mM) stained with uranyl acetate. (B) Particle size
histogram of the glycolipid 1 vesicles (0.1 mM) by TEM. (C) Histogram
analysis of the DLS measurements of the glycolipid 1 vesicles (0.1 mM)
in water. The solution was filtered through a 0.45 mm syringe filter
(Toyo Roshi, Japan) prior to use, and then the DLS measurement of the
sample was performed at a fixed scattering angle of 901 at 25 1C.
Fig. 3 ESI-MS spectra of the glycolipid 1 vesicle encapsulating azido
compound 3 after addition of azido compound 2, CuSO4Á5H2O, and
sodium ascorbate at room temperature.
with propargyl bromide.9 The monosaccharide glycolipid
was synthesized from D-glucose and 2-decyl-1-tetradecanol.10
Unilamellar vesicles of glycolipid 1 were prepared by extrusion
of the vesicles through a 0.45 mm syringe filter after sonication,
according to the methods reported previously.11,12 The size of
the glycolipid 1 vesicles was measured by transmission electron
microscopy (TEM) and dynamic light scattering (DLS). The
TEM image of the vesicles showed circles with a diameter of
approximately 100 nm, indicating the formation of vesicles using
glycolipid 1. DLS measurement showed that the average particle
size of the vesicles in water was 93.8 Æ 21.6 nm. These observa-
tions suggest the formation of unilamellar vesicles (Fig. 2).
The modification of the glycolipid 1 vesicle surface by click
chemistry was carried out using two azido compounds (2, 3)
based on oligo (ethylene glycol) with different molecular
weights.13 To modify the outer surface of the vesicles, the
copper(I) catalyst and azido compound 2 were added to a
vesicle solution. When the resulting mixture was measured by
electrospray ionization mass spectrometry, the molecular
weights of glycolipid 1 and the click reaction product were
observed (Fig. S1, ESIw). The average particle size of the
glycolipid 1 vesicle after surface modification was 82.7 Æ 16.1 nm
as determined by DLS and TEM measurements (Fig. S2
and S3, ESIw), indicating that the particle size did not change
by the surface modification. These results indicate that the
vesicles maintain their structure by the modification with the
azido compound 2 using the click reaction, and the outer
surface of the vesicle may be selectively modified.
outside of the vesicle, overnight. ESI-MS measurement of
this resulting mixture showed the signal for the molecular
weight of the click reaction product of glycolipid 1 and azido
compound 2, while the product of glycolipid 1 and azido
compound 3 was not observed. This result indicates that the
selective modification of the outer surface of the glycolipid
1 vesicle can be achieved using this method. Furthermore, this
result also suggests that the azido compounds and the copper
ions do not translocate across the bilayer membrane of
glycolipid 1 at room temperature (Fig. 3).
To carry out the selective modification of the inner surface of
the vesicles, it is necessary to perform the click reaction after
formation of the vesicle. If a free copper ion is added to the
solution during vesicle formation, the click reaction could occur
before formation of the vesicle. To solve this problem, the
photoresponsive copper complex [Cu(OH2)(cage)], which releases
a copper ion by ultraviolet (UV) light irradiation, was used.14
When using [Cu(OH2)(cage)] instead of a free copper ion, the
click reaction does not occur in the dark. Photolysis with 350 nm
UV light cleaves the ligand backbone of [Cu(OH2)(cage)],
releasing copper ions so that the click reaction can proceed.
A vesicle thus prepared using 1 mM glycolipid 1, 5 mM
azido compound 3, 2 mM [Cu(OH2)(cage)], and 0.5 mM
sodium ascorbate contains [Cu(OH2)(cage)], azido compound
3, and sodium ascorbate in the internal aqueous phase. The
azido compound 2 was then added to this vesicle solution to
give a final concentration of 0.02 mM. The resulting vesicle
solution contained azido compound 2 in the solution outside
of the vesicle, and azido compound 3 in the inside of
the vesicle. [Cu(OH2)(cage)] and ascorbate were also located
inside the vesicle, so the click reaction could be initiated by
irradiation with UV light. After irradiation for one hour, the
modification by click reaction was carried out overnight at
room temperature in the dark. ESI-MS measurement of
the resulting mixture showed the signal for the molecular
weight of the click reaction product of glycolipid 1 and azido
compound 3, but not for that of glycolipid 1 and azido
compound 2. This result suggests that the inner surface of
the vesicle was selectively modified (Fig. 4).
In order to confirm the specific modification of the vesicle
surface, a vesicle solution containing two azido compounds,
one located in the internal aqueous phase and the other one
outside of the vesicle, was prepared, and modification of the
outer surface was carried out by adding the copper(I) catalyst
to the solution outside of the vesicle.
The vesicles encapsulating the azido compound were
obtained by dispersing a dried glycolipid 1 thin film in water
(1 mM) containing azido compound 3 (5 mM) with vortexing,
followed by sonication. The resulting vesicles were then frozen
in liquid N2 and thawed. This freeze–thaw cycle was repeated
at least three times. Excess azido compound 3 in the solution
was removed by gel filtration chromatography using a HiTrap
Desalting column (GE Healthcare). The outer surface modification
of the vesicle encapsulating azido compound 3 was then carried out
in the presence of 0.1 mM azido compound 2, 0.02 mM copper
sulfate, and 0.05 mM sodium ascorbate, which were in the solution
In summary, we have synthesized a novel glycolipid with a
terminal acetylene and prepared unilamellar vesicles from it.
A convenient method for the specific inner surface modification
of the vesicle was developed using the photoresponsive copper
complex [Cu(OH2)(cage)] as the catalyst for a click reaction.
The inner surface modification is achieved via the encapsulation
c
This journal is The Royal Society of Chemistry 2012
Chem. Commun., 2012, 48, 5650–5652 5651
Ito, Hidehiro
