Temporal emergence of giant vesicles accompanied by hydrolysis of ammonium amphiphiles with a Schiff-base segment

Article abstract of DOI:10.1246/cl.2004.1442

An aqueous dispersion of the ammonium amphiphile with a Schiff-base segment exhibited the formation and decomposition of giant vesicles with diameters of several micrometers 2-4 h after the preparation. The time course trace of the morphological changes of the self-aggregates by means of light microscopy, electron microscopy and ¹HNMR revealed that the temporal emergence of the giant vesicles was dependent on the progress of hydrolysis of the Schiff-base segment.

Full text of DOI:10.1246/cl.2004.1442

1442
Chemistry Letters Vol.33, No.11 (2004)
Temporal Emergence of Giant Vesicles Accompanied by Hydrolysis
of Ammonium Amphiphiles with a Schiff-base Segment
Taro Toyota, Katsuto Takakura, and Tadashi Sugawaraꢀ
Department of Basic Science, Graduate School of Arts and Sciences, The University of Tokyo,
3-8-1 Komaba, Meguro, Tokyo 153-8902
(Received July 28, 2004; CL-040888)
An aqueous dispersion of the ammonium amphiphile with a
of the relative amount of substrate 1 and the hydrolized products
2, 3 as shown in Figure 1a.
Schiff-base segment exhibited the formation and decomposition
of giant vesicles with diameters of several micrometers 2–4 h af-
ter the preparation. The time course trace of the morphological
changes of the self-aggregates by means of light microscopy,
electron microscopy and 1H NMR revealed that the temporal
emergence of the giant vesicles was dependent on the progress
of hydrolysis of the Schiff-base segment.
N-[4-[2-(trimetylammonio)ethoxy]benzylidene]-4-octylani-
line bromide 1 was synthesized by the dehydrocondensation of
N-octylaniline 2 with 2-(4-formylphenoxy)ethyltrimethylammo-
nium bromide 3 as described in the previous papers.2f,2g,3 Am-
phiphile 1 was dissolved in distilled water and sonicated for
30 s, affording a 10 mM aqueous dispersion. To discern the na-
ture of the self-aggregates in the early stage of hydrolysis, the
dispersion was examined with the aid of negative-stain transmis-
sion electron microscopy (TEM, JEM-1230, JEOL, Japan). The
TEM sample was prepared by air-drying the suspension of 1 on
the carbon-deposited Collodion membrane sustained over cop-
per grids. Figure 1b (left) shows the TEM image of spherical ag-
gregates with diameters in the range of 50–300 nm. Dynamic
light scattering (DLS) measurement (Microtrac UPA150,
NIKKISO, Japan) indicated that self-aggregates with the size
of 89 nm (standard deviation = 76 nm) at t ¼ 10 min were
formed, supporting the above result. Accordingly, these aggre-
gates can be assigned to small vesicles (SVs).3
Figure 1b also shows the photographs of the 10 mM disper-
sion of 1 observed under a differential interference contrast
(DIC) microscope (BX51, Olympus, Japan) at 25 ꢁ 1 ꢂC (Figure
1b, middle and right). The photographs showed that GVs
emerged and elongated up to ca. 5 mm in the second stage ca.
200 min after the mixing. After ca. 400 min, the emergence of
oil droplets on the membrane of GV in the specimen was recog-
nized, and eventually all the GVs were converted to brilliant
spots which were identified as oil droplets (Figure 1b right).4
Morphological changes of giant vesicles (GVs) have drawn
much attention from the aspect of cooperative dynamics of bio-
molecular membranes composed of amphiphilic molecules.1
Such morphological changes are induced by the application of
electric field, osmotic pressure, addition of electrolytes etc.,
and the real time observation by light microscopy enables us
to reveal their mechanism of membrane dynamics.1 Recently it
was found that chemical formation of membrane molecules in-
side the vesicular membrane caused transformation of GVs.2
Here we prepared amphiphile 1 in which the Schiff-base
segment was incorporated into the hydrophobic chain close to
the trimethylammonium polar head (Figure 1a). Although am-
phiphile 1 formed only a small vesicle, it was found that the dis-
persion of amphiphile 1 exhibited time-dependent morphologi-
cal changes of the self-aggregates (small vesicle–giant vesicle–
oil droplet), associated with the progress of the hydrolysis of
the imine part of amphiphile 1 (Figure 1b). This is the first dem-
onstration that the morphological changes of the self-aggregate
in the dispersion of amphiphile 1 was induced by the change
Figure 1. (a) Schematic illustration of amphiphile 1, hydrophobe 2, and hydrophile 3. Black-filled blocks represent the imine
part which separates into a hydrophobic amine part and formyl part. The white-filled part is a hydrophilic unit. Illustrated scheme
of morphologies (SV; small vesicle, GV; giant vesicle, Oil; oil droplet) composed of 1, 2, and 3 with the ratio of [1] / [2] / [3] deter-
mined by 1H NMR analysis is also shown. (b) TEM image of the first stage of morphologies in the 10 mM dispersion of 1 (1st stage).
Phase contrast micrographs of giant vesicles (2nd stage) and oil droplets (3rd stage) in the dispersion containing 1.
Copyright ꢀ 2004 The Chemical Society of Japan

Chemistry Letters Vol.33, No.11 (2004)
1443
This morphological changes correspond to the third stage. The
time dependence of the size population of self-aggregates in
the dispersion of 1 was monitored by DLS. The average diame-
ters of the self-aggregates were 610 ꢁ 410 nm at the second
stage and 640 ꢁ 250 nm at the third stage, respectively.
In order to confirm that the morphological change of the
self-aggregates was induced by the chemical transformation of
amphiphile 1 to 2 and 3, 1H NMR of the dispersion was recorded
on the same specimen as that of the light microscopic observa-
tion. The conversion of amphiphile 1 was monitored by a singlet
signal at ꢀ ¼ 8:4 ppm and ꢀ ¼ 9:8 ppm which were assigned as
the imine proton of ampphiphile 1 and the formyl proton of hy-
drophile 3, respectively.2f,2g The conversion of 1 to 3 was deter-
mined to be 13, 46, and 88% (ꢁ3%) at the first, second and third
stage, respectively. The pH value of the 10 mM aqueous disper-
sion of 1 remained in the range of 4.8–5.4 over all through
stages.
On the basis of the above results, we may argue that the for-
mation of GVs in the dispersion of 1 was induced by the gener-
ation of hydophobe 2 and hydrophile 3 produced by the hydrol-
ysis of amphiphile 1. The hydrolysis of 1 is expected to proceed
because the reactive part (the imine part) is located in the vicin-
ity of the surface of GV and the hydrophile 3 can easily diffuse
into water region from the intramenbrane phase.2h,6 Since a mix-
ture of 2 and 3 ([2]/[3] = 10/10 (mM)) forms only micrometer-
sized oil droplets and no structured aggregates, we prepared a
mixture of 1, 2, and 3 ([1]/[2]/[3] = 5/5/5, 5/5/0 (mM)) and
confirmed the generation of giant vesicles in the dispersion.
The morphology of the dispersion of the mixture corresponds
to that in the second stage.
Figure 2. Schematic illustration of fusion of small vesicles
through the formation of inverted-micellar structure (right) be-
tween the contacting membranes. Transformation from small
vesicles to GVs is thus thought to occur.
is incorporated in amphiphile 1 and it is regenerated through
the dehydrocondensation between hydrolyzed products 2 and
3. It may be stated that the Schiff base segment which is buried
in the self-aggregates operates as a clock to regulate the stages of
the morphological changes.
This work was supported by Grant-in-Aid for Center of
Excellence (No. 11CE2006) from the Ministry of Education,
Culture, Sports, Science and Technology. T. T. is supported by
Research Fellowships of the Japan Society for the Promotion
of Science for Young Scientists (No. 08289).
References and Notes
1
‘‘Giant Vesicles,’’ ed. by P. L. Luisi and P. Walde, John
Wiley & Sons, Ltd., NY (2000) and references cited
therein.
2
a) R. Wick, P. Walde, and P. L. Luisi, J. Am. Chem. Soc.,
117, 1435 (1995). b) D. A. Jaeger, C. L. Schilling, III,
A. K. Zelenin, B. Li, and E. Kubicz-Loring, Langmuir, 15,
7180 (1999). c) J. M. Holopainen, M. I. Angelova, and
P. K. J. Kinnunen, Biophys. J., 78, 830 (2000). d) D. A.
Jeager and T. Clark, Jr., Langmuir, 18, 3495 (2002). e)
The reversibility of the morphological change coupled with
the hydrolysis of amphiphile 1 with the imine segment was
confirmed by the following experiment. When the dispersion
of 2 and 3 ([2]/[3] = 100/100 (mM)) was prepared at 25 ꢁ
1 ꢂC, GVs were generated from the surface of oil droplets in
J. M. Holopainen, M. I. Angelova, T. Soderlund, and
¨
P. K. J. Kinnunen, Biophys. J., 83, 932 (2002). f) K.
Takakura, T. Toyota, K. Yamada, M. Ishimaru, K. Yasuda,
and T. Sugawara, Chem. Lett., 2002, 404. g) K. Takakura,
T. Toyota, and T. Sugawara, J. Am. Chem. Soc., 125, 8134
(2003). h) K. Takakura and T. Sugawara, Langmuir, 20,
3832 (2004).
1
30 min. The time course of H NMR of the 200 mM dispersion
of 2 and 3 showed that the conversion ratio of 1 was 49%
(ꢁ2%) in the dispersion 24 h after the preparation.5 The forma-
tion of GVs from oil droplets was also recognized when the
10 mM oil emulsion, which was obtained by the completely
hydrolyzed dispersion of 1 (24 h after preparation), was concen-
trated by 10 times.
The mechanism of the transient generation of GVs in this
vesicular system is interpreted as follows. The generation of hy-
drophile 3 in bulk water through the hydrolysis of 1 flocculates
vesicles of 1 due to the decrease of ‘‘thickness’’ (Debye length)
of the electronic double layer on the vesicular surface.7 Simulta-
neously, hydrophobe 2 assists a fusion of small vesicles to
GV (Figure 2) by forming an inverted-micellar structure within
the membranes at the contacting surfaces of small vesicles
(Figure 2, inset), since the polar head of 2 is smaller than that
of 1.7–9 As the ratio of 2 to 1 increases accompanied by the
progress of hydrolysis, the decomposition of GVs proceeds
and eventually the oil droplets were formed.
3
a) T. Kunitake, Y. Okahata, M. Shimomura, S. Yasunami,
and K. Takarabe, J. Am. Chem. Soc., 103, 5401 (1981). b)
Y. Liang, L. Wu, Y. Tian, Z. Zhang, and H. Chen, J. Colloid
Interface Sci., 178, 703 (1996).
4
5
The shining spots are undoubtedly assigned to oil droplets on
the basis of optical characteristics.
The sample for the 1H NMR analysis was prepared by
neutralization and lyophilization. Measuring the residues in
DMSO-d6.
6
a) M. T. A. Behme and E. H. Cordes, J. Am. Chem. Soc., 87,
260 (1964). b) Y. Okahata, R. Ando, and T. Kunitake, Bull.
Chem. Soc. Jpn., 56, 802 (1983).
7
8
J. N. Israelachivili, ‘‘Intermolecular and Surface Forces,’’
Academic Press, LTD., London (1992).
a) S. M. Gruner, Proc. Natl. Acad. Sci. U.S.A., 82, 3665
(1985). b) L. Chernomordik, Chem. Phys. Lipids, 81, 203
(1996). c) K. Morigaki and P. Walde, Langmuir, 18, 10509
(2002).
Although the precise structure and mechanism of self-aggre-
gates at each stage is still veiled, it should be noted that this ve-
sicular system shows temporal generation of GV composed by
two simple molecules which do not form GV by itself. More-
over, we demonstrated for the first time that GV is formed ac-
companied by the hydrolysis of the Schiff base segment which
9
W. Helfrich, Prog. Colloid Polym. Sci., 95, 7 (1994).
10 We thank Mr. Yasuhiko Saito (JEOL, Japan) for his skillful
measurement in terms of transmission electron microscopy.
Published on the web (Advance View) October 2, 2004; DOI 10.1246/cl.2004.1442