Preparation and characterization of phase-segregated vesicles of photopolymerizable diacetylene mixed with nonpolymerizable amphiphiles

Article abstract of DOI:10.1246/bcsj.20100192

A mixture of sodium 1,2-di(hexadecyloxycarbonyl)ethanesulfonate (2C16S) with photopolymerizable 1,2-di(10,12- tricosadiynoyl)-sn-glycerol 3-phosphocholine (DTPC) in a 2:100 ratio was treated by modified thin-film hydration to give an aggregate which became polymerized giant vesicles (GVs) under irradiation at 254 nm. The autofluorescence of the GVs was analyzed with a confocal laser scanning microscope at the cross section, revealing a 3.8-μm diameter ring shape and the presence of a dark part of ca. 1 μm in the ring. When octadecylrhodamine B (RhB) as an amphiphilic fluorescence probe was added to the GV, the fluorescence of RhB was emitted from the whole ring. Therefore, phase segregation of 2C16S from DTPC was confirmed. Similarly, mixed vesicles of N,N-di(2-hexadecanoyloxyethyl)dimethylammonium iodide with DTPC were found to be 3.7-μm diameter phase-segregated vesicles with a dark portion of ca. 1 μm on the ring in the cross sectional image. On the other hand, DTPC vesicles mixed with 1,2-di(dodecyloxycarbonyl)ethanesulfonate, N,N-di(2- dodecanoyloxyethyl)dimethylammonium iodide, and N,N-di(2-tetradecanoyloxyethyl) dimethylammonium iodide formed sphere structures filling the inside of the vesicles. The segregation mechanism was explained by the difference in the main phase transition temperature of each amphiphile.

Full text of DOI:10.1246/bcsj.20100192

© 2010 The Chemical Society of Japan
Bull. Chem. Soc. Jpn. Vol. 83, No. 12, 1551–1557 (2010) 1551
Preparation and Characterization of Phase-Segregated Vesicles
of Photopolymerizable Diacetylene Mixed with
Nonpolymerizable Amphiphiles
Jin Matsumoto,* Koshiro Yoneda, Jun Tasaka, Tsutomu Shiragami, and Masahide Yasuda
Department of Applied Chemistry, Faculty of Engineering, University of Miyazaki,
Gakuen-Kibanadai, Miyazaki 889-2192
Received July 9, 2010; E-mail: jmatsu@cc.miyazaki-u.ac.jp
A mixture of sodium 1,2-di(hexadecyloxycarbonyl)ethanesulfonate (2C16S) with photopolymerizable 1,2-di(10,12-
tricosadiynoyl)-sn-glycerol 3-phosphocholine (DTPC) in a 2:100 ratio was treated by modified thin-film hydration to give
an aggregate which became polymerized giant vesicles (GVs) under irradiation at 254 nm. The autofluorescence of the
GVs was analyzed with a confocal laser scanning microscope at the cross section, revealing a 3.8-¯m diameter ring shape
and the presence of a dark part of ca. 1 ¯m in the ring. When octadecylrhodamine B (RhB) as an amphiphilic fluorescence
probe was added to the GV, the fluorescence of RhB was emitted from the whole ring. Therefore, phase segregation of
2C16S from DTPC was confirmed. Similarly, mixed vesicles of N,N-di(2-hexadecanoyloxyethyl)dimethylammonium
iodide with DTPC were found to be 3.7-¯m diameter phase-segregated vesicles with a dark portion of ca. 1 ¯m on the ring
in the cross sectional image. On the other hand, DTPC vesicles mixed with 1,2-di(dodecyloxycarbonyl)ethanesulfonate,
N,N-di(2-dodecanoyloxyethyl)dimethylammonium iodide, and N,N-di(2-tetradecanoyloxyethyl)dimethylammonium
iodide formed sphere structures filling the inside of the vesicles. The segregation mechanism was explained by the
difference in the main phase transition temperature of each amphiphile.
Lipids or amphiphilic compounds can form highly ordered
aggregates with unique shapes and functions by self-assembly
in aqueous solution.1,2 A vesicle, which is an egg-shaped lipid
aggregate (liposome), is a typical lipid assembly. A variety of
vesicles of natural lipids or synthetic-amphiphilic compounds
have been characterized from the standpoints of the mimicking
of biomembranes and the development of liposome drugs such
as for gene delivery and cancer therapy.3 Their performance
has been modified by the following methods. The control of
stability and release of drug has been modified by polymer-
ization of vesicles using photopolymerizable diacetylenic
amphiphiles which can form self-assembled structures in
aqueous solution.4,5 The resulting polydiacetylene has been
applied as a carrier of drugs6-8 and various colorimetric
biosensors.9,10 Moreover, the combination of the liposome and
the additional components modified the surface properties
of liposome such as charge or specific binding using self-
assembly and chemical transformation.3,11
Among the modification of functionalized vesicles, the
phase segregation of vesicles has received much attention in
order to elucidate the functions of signal transduction and
membrane transport of the vesicles.12 Phase segregation of
vesicles has been usually examined by calorimetric analysis
and fluorescence microscopy using phase-selective fluores-
cence probes.13 Diacetylenic phosphatidylcholine, 1,2-di-
(10,12-tricosadiynoyl)-sn-glycerol 3-phosphocholine (DTPC),
is known to have high affinity for a variety of lipid molecules
because DTPC contains the hydrophilic head group occurring
in natural phospholipids (Scheme 1).7,14-16 Okazaki and co-
workers have prepared substrate-supported planar lipid bilayers
composed of polymeric DTPC and fluid-nonpolymerizable
phospholipids under lithographically controlled UV irradia-
O
O
O
O
SO3Na
NMe2 I
P
2C12S; n = 12
2C16S; n = 16
H(CH2)n
H(CH2)n
O
O
H(CH2)10
H(CH2)10
(CH2)8
O
O
O
H
(CH2)8
O
O
O
NMe3
H(CH2)n−1
DTPC
O
C
O
2C12N; n = 12
2C14N; n = 14
2C16N; n = 16
H(CH2)n−1 C O
O
Scheme 1. The polymerizable DTPC, anionic 2CnS, and cationic 2CnN amphiphiles.
Published on the web December 2, 2010; doi:10.1246/bcsj.20100192

1552 Bull. Chem. Soc. Jpn. Vol. 83, No. 12 (2010)
Phase-Segregated Vesicles of Polydiacetylene
tion.17 The bilayers obtained were characterized by atomic
force microscopy, fluorescence microscopy, and ellipsometry.
On the other hand, photopolymerized DTPC molecules emit
autofluorescence at 550 nm,18,19 similar to other diacetylenic
compounds. The autofluorescence of polydiacetylene is useful
to distinguish it from nonpolymerizable amphiphiles in fluores-
cence microscopy without addition of any fluorescence probes.
Therefore, our attention was focused on the preparation of
phase-segregated vesicles between nonpolymerizable amphi-
philes and polymerizable DTPC which are large enough to
analyze with a confocal laser scanning fluorescence microscope
(CLSM). Here, it will be examined whether the small amount of
nonpolymerizable amphiphiles could change the phase segre-
gation and the morphology of the mixed vesicles by changing
the alkyl-chain length of the nonpolymerizable amphiphiles.
ing residue was dissolved in hot methanol and filtered. After
evaporation of the filtrate, the crude product was washed with
hexane and water and purified by recrystallization from hexane.
The identification was performed by the comparison of spectral
data with authentic samples.20
General Procedure for the Synthesis of Cationic Amphi-
philes (2CnN). The esterification of N-methyldiethanolamine
was performed by the reaction of N-methyldiethanolamine
(5 mmol) and fatty acid (10.6 mmol) in the presence of 4-
dimethylaminopyridine (3.2 mmol) and N,N¤-dicyclohexyl-
carbodiimide (10.6 mmol) in CHCl3 (30 cm3). The mixture
was cooled in an ice-water bath for 1 h and then stirred at room
temperature for 1 week. After filtration to remove precipitated
1,3-dicyclohexylurea, the solution was washed with HCl
(0.01 mol dm¹3) and a saturated aqueous NaCl solution, and
then dried with Na2SO4. The solution was evaporated to yield
crude N,N-di(2-alkanoyloxyethyl)methylamine, which was
subjected to purification by column chromatography. The
quaternization of N,N-di(2-alkanoyloxyethyl)methylamine
(1 mmol) by methyl iodide (50 mmol) was performed in dry
MeCN (10 cm3). After stirring for 1 h under a nitrogen
atmosphere, the mixture was evaporated to yield a crude
product which was purified by recrystallization from methanol.
Experimental
Materials.
Water was purified by a Millipore Elix III
(USA). Glassware, such as flasks and glass plates, was washed
with concentrated nitric acid at 90 °C for 2 h and rinsed with
pure water for use in the preparation and observation of vesicle
solutions. The washed glassware was immersed in an aqueous
KOH solution (1 mol dm¹3) for 12 h, followed by sonication in
pure water. The cleaned glassware was stored in pure water.
The DTPC was purchased from Avanti Polar Lipids, Inc.
(USA) and was used without further purification. As a
lipophilic fluorescence probe, octadecylrhodamine B (RhB,
emission maximum at 585 nm) was purchased from Invitrogen
N,N-Di(2-dodecanoyloxyethyl)dimethylammonium
Io-
dide (2C12N): 1H NMR: ¤ 0.88 (t, J = 7.0 Hz, 6H), 1.26-
1.30 (m, 32H), 1.62 (quint, J = 7.7 Hz, 4H), 2.38 (t, J = 7.7
Hz, 4H), 3.56 (s, 6H), 4.16-4.18 (m, 4H), 4.60-4.62 (m, 4H).
13C NMR: ¤ 14.08, 22.65, 24.63, 29.10, 29.25, 29.31, 29.45,
29.58, 26.58, 31.87, 34.07, 52.93, 57.41, 64.01, 172.71; Exact
MS (MALDI-MS) Calcd for C30H60NO4: 498.4522, Found
498.4506.
(USA).
2-[4-(2-Hydroxyethyl)-1-piperazinyl]ethanesulfonic
acid (0.01 mol dm¹3, HEPES, Dojindo, Japan) was dissolved
in pure water and pH was adjusted to 7.0 with aqueous NaOH
solution. The resulting HEPES buffer solution was filtered
through a 1 ¯m pore size membrane and bubbled with argon
gas before use.
N,N-Di(2-tetradecanoyloxyethyl)dimethylammonium Io-
dide (2C14N): 1H NMR: ¤ 0.88 (t, J = 6.8 Hz, 6H), 1.26-
1.33 (m, 40H), 1.58-1.65 (m, 4H), 2.38 (t, J = 7.7 Hz, 4H),
3.57 (s, 6H), 4.15-4.18 (m, 4H), 4.60-4.62 (m, 4H). 13C NMR:
¤ 14.08, 22.66, 24.64, 29.11, 29.26, 29.33, 29.47, 29.60, 29.63,
29.63, 29.67, 31.89, 34.07, 52.94, 57.42, 64.02, 172.71; Exact
MS (MALDI-MS) Calcd for C34H68NO4: 554.5148, Found
554.5163.
N,N-Di(2-hexadecanoyloxyethyl)dimethylammonium Io-
dide (2C16N): 1H NMR: ¤ 0.88 (t, J = 7.0 Hz, 6H), 1.26-
1.30 (m, 48H), 1.62 (quint, J = 7.8 Hz, 4H), 2.38 (t,
J = 7.8 Hz, 4H), 3.57 (s, 6H), 4.16-4.18 (m, 4H), 4.60-4.62
(m, 4H). 13C NMR: ¤ 14.08, 22.66, 24.64, 29.11, 29.26, 29.34,
29.48, 29.61, 29.64, 29.65, 29.65, 29.65, 29.65, 31.90, 34.07,
52.92, 57.40, 64.01, 172.71; Exact MS (MALDI-MS) Calcd for
C38H76NO4: 610.5774, Found 610.5782.
Instruments.
1H NMR (400 MHz) and 13C NMR (100
MHz) spectra were taken on a Bruker AV 400M spectrometer
(Germany) for CDCl3 and CD3OD solutions using SiMe4 as an
internal standard. Matrix-assisted laser desorption/ionization
mass spectra (MALDI-MS) were measured on a Bruker
Daltonics Autoflex III TOF/TOF (Germany) in the positive
ion mode. Calibration of the mass number was performed with
an internal standard using poly(ethylene glycol). UV-vis
spectra of the solutions were obtained with a JASCO V-550
spectrophotometer (Japan). Fluorescence spectra were mea-
sured on a Shimadzu FR-5300PC fluorometer (Japan).
General Procedure for the Synthesis of Anionic Amphi-
philes (2CnS). Double-chain anionic amphiphiles without the
polymerizable group, sodium 1,2-di(alkyloxycarbonyl)ethane-
sulfonate (2CnS: n = 12, alkyl = dodecyl and n = 16, alkyl =
hexadecyl), were synthesized according to the literature.20
1-Alkanol (3.3 mmol) was refluxed with maleic anhydride
(1.5 mmol) in the presence of concentrated sulfuric acid
(0.10 cm3) in toluene (50 cm3) at 110 °C in a Dean-Stark
apparatus. After the neutralization with aqueous NaHCO3
solution, the crude dialkyl maleate was extracted with toluene
and purified by recrystallization from hexane. The dialkyl
maleate (1 mmol) was refluxed in an aqueous solution
(100 cm3) of NaHSO3 (35 g) for 3 h under bubbling with air.
After neutralization with NaHCO3 and evaporation, the result-
Fluorescence Imaging.
The CLSM analysis was per-
formed with an Olympus FV-300 (confocal aperture: 150 ¯m,
Japan) equipped with an objective lens (©100 oil, numerical
aperture: 1.3). A vesicle solution was placed in the hole of a
silicon spacer (1 cm © 1 cm, 50-¯m thickness) on a slide glass
and was enclosed with a cover glass. The objective lens was
moved downward to obtain cross-sectional images of the
vesicles. The moving distance (z) was defined as the distance
from the uppermost surface of the vesicle (z = 0). The CLSM
had a positional resolution of «0.1 ¯m in the vertical direction
and «0.1 ¯m in the horizontal direction. Fluorescence images
of the polymerized DTPC were measured by passing through

J. Matsumoto et al.
Bull. Chem. Soc. Jpn. Vol. 83, No. 12 (2010) 1553
(B)
(A)
-2
0
2
-5
0
5
Distance/µm
Distance/µm
(C)
-4
-2
0
2
4
Distance/µm
Figure 1. Distribution of the fluorescence intensity (>565 nm) along a line on cross-sectional CLSM images (inset) of 1 at
z = 3.6 ¯m (A) and 3 at z = 2.3 ¯m under 543-nm laser excitation (B); and 3¤ at z = 2.8 ¯m under 488-nm laser excitation (C). The
scale bar in inset is in 3 ¯m units.
cutoff filter (>565 nm) under the excitation by 488-nm laser
irradiation. The nonirradiated DTPC vesicles and nonpolymer-
izable amphiphiles vesicles were observed using the addition of
1 mol % RhB relative to the amount of the amphiphiles under
543-nm laser irradiation.
Measurement of Phase Transition Temperatures of
2CnN. Differential scanning calorimetric (DSC) measure-
ments were carried out with a Rigaku Thermo Plus 8240
differential scanning calorimeter (Japan). Vesicle solution was
prepared by TFH (described below) of 2CnN (1.67 ¯mol cm¹3).
Vesicles were concentrated by centrifugation of the solution
and the removal of supernatant. The collected vesicle solution
was resuspended in water to give a solution (10 ¯mol cm¹3).
The vesicle solution (10 © 10¹6 dm3) was placed in a hermeti-
The solvents were evaporated gently to produce a thin film.
After the thin film was dried under a vacuum at 75 °C for 3 h,
the dried film was hydrated in HEPES buffer solution at 80 °C
under sonication. The solution was left to stand at 22.5 °C for 3
days during the formation of an aggregate. After RhB acting as
fluorophore was added to the aggregates, the fluorescence
image coming from RhB was analyzed on the CLSM
(Figure 1A). A vesicle structure with a ring-shaped image
and a dark area inside the ring were observed at every cross
section of the selected aggregate. When the fluorescence
intensities were mapped in the cross section at a 3.6 ¯m depth
(z) from the uppermost of the aggregate, two intensity maxima
appeared 7.1 ¯m apart from each other which was defined as
the diameter of the vesicle. So we called it a giant vesicle (GV)
of 2C12S (1). The TFH method was successfully applied to
sodium 1,2-di(hexadecyloxycarbonyl)ethanesulfonate (2C16S)
which formed the GV of 2C16S (2) with a 3.4 ¯m diameter.
The peak width at the half height of fluorescence intensity was
about 0.5 ¯m. This size was not comparable to the molecular
size of the bilayer of 2CnS, because the resolution limit of the
fluorescence microscopy was near 0.5 ¯m.
cally sealed aluminum pan. DSC measurement was performed
¹1
at a heating rate of 2 °C min
.
Results and Discussion
Preparation of Vesicles by Thin-Film Hydration. Our
first effort was paid to prepare vesicles of a large enough size
to analyze by CLSM. It has been reported that sodium 1,2-
di(dodecyloxycarbonyl)ethanesulfonate (2C12S) treated by the
usual direct sonication method, forms vesicles at submicro-
meter scale which is too small to be analyzed by optical
microscopy.20 Therefore, we performed the preparation of
micrometer size vesicles of 2C12S by modified thin-film
hydration (TFH) as follows. The 2C12S was dissolved in
In general, DTPC forms aggregated structures with supra-
molecular morphologies such as vesicles15 and helical or
tubular structures in aqueous solution,21,22 depending on the
preparation conditions. Here, the TFH of DTPC was proceeded,
giving GV 3 whose diameter was determined to be 4.5 ¯m by
CLSM analysis using RhB (Figure 1B). It is well known that a
diacetylene moiety of DTPC was photopolymerized topotacti-
¹3
CHCl3-MeOH (10:1) to give a 1.0 © 10¹4 mol dm solution.

1554 Bull. Chem. Soc. Jpn. Vol. 83, No. 12 (2010)
Phase-Segregated Vesicles of Polydiacetylene
cally to give the ene-yne backbone. Therefore, the solution of
3 was irradiated at 254 nm for 60 min at 4 °C to give the
polymerized GV 3¤. The diameter of 3¤ was measured to be
5.5 ¯m from autofluorescence images (Figure 1C). Thus, single
GVs 1, 2, 3, and 3¤ were successfully prepared from 2C12S,
2C16S, and DTPC by TFH, respectively (Table 1).
with a diameter of 4.0 ¯m, revealing that 5 was not a vesicle
structure but a filled sphere (Figure 2B).
Mixed Vesicles of DTPC with 2CnN. It is expected that
DTPC forms the mixed vesicles with not only the anionic 2CnS
but also the cationic surfactants, since DTPC is a zwitterion. As
cationic surfactants therefore, N,N-di(2-alkanoyloxyethyl)di-
methylammonium iodide (2CnN: n = 12 (dodecanoyl), n = 14
(tetradecanoyl), and n = 16 (hexadecanoyl); Scheme 1) were
prepared. The molar ratio of 2CnN and DTPC was optimized to
be 2:100, judging from F values in UV-spectra described below
(Figure S1 in Supporting Information). The mixture of 2C16N
and DTPC in a molar ratio of 2:100 was subjected to TFH and
UV irradiation for 60 min to give aggregate 6. The selected 6
was analyzed by the fluorescence intensity at cross sections
(Figure 3A). The diameter of the ring was measured to be
3.7 ¯m and a dark part 1 ¯m in length was observed in the cross
section in the range of z = 1.4-2.6 ¯m. Detailed images in
the cross section are shown in the Supporting Information
Figure S2. When RhB was added to 6, the emission was
coming from the whole ring. Therefore, the dark part was
composed of 2C16N. Based on the observed diameter (3.7 ¯m),
the surface area of the nonfluorescent 2C16N part and the
polymerized DTPC part of 6 were calculated to be 0.79 and
43 ¯m2, respectively. The surface ratio of 2C16N and DTPC
was 1.8:100, a ratio which is in good agreement with the
mixing ratio of 2C16N and DTPC (2:100) in the preparation.
Therefore, DTPC and 2C16N were completely segregated into
two phases to give s-GV.
Mixed Vesicles of DTPC with 2CnS. The mixed vesicles
of DTPC were prepared by mixing 2C16S with DTPC in a
molar ratio of 2:100 followed by TFH. Aqueous solution of
the resulting mixed aggregates was immediately irradiated at
254 nm for 60 min to give the polymerized aggregate 4. The
fluorescence intensity of the selected 4 was analyzed at the
cross section z = 2.0 ¯m, revealing the 3.8-¯m diameter ring
shape and the presence of a dark part of ca. 1 ¯m in the ring
(Figure 2A). When RhB was added to 4, fluorescence of RhB
appeared in the whole ring of 4, but no fluorescence came
from inside of GV. Therefore, the dark part on the ring was
composed of 2C16S. Thus, the formation of the phase-
segregated GV (s-GV) between 2C16S and DTPC was proven.
A similar treatment was applied to a mixture 2C12S with
DTPC. However, the resulting aggregate 5 showed almost
uniform fluorescence coming from the whole circular plane
Table 1. The Characterization of Single Vesicles 1-3 and
Mixed Vesicles 4-8
Main
Other
Diametera)
/¯m
F
/%c)
Aggregates
Structureb)
amphiphile amphiphile
1
2
3
3¤ d)
2C12S
2C16S
DTPC
DTPC
®
®
®
®
7.1
3.4
4.5
5.5
GV
GV
GV
GV
®
®
®
24
It was confirmed that the TFH treatment of 2C14N and
2C12N gave single GVs of 4.6 and 10.6 ¯m diameters,
respectively (See Supporting Information Figure S3). A mixed
solution of 2C12N or 2C14N and DTPC at a molar ratio of
2:100 was treated by TFH and polymerized by UV irradiation.
However, the fluorescence from DTPC was emitted from the
whole circular plane at all z positions, resulting in the filled
aggregates 7 and 8 (Figure 3B and Figures S4 and S5 in
Supporting Information).
4d)
5d)
DTPC
DTPC
2C16S
2C12S
3.8 (1.0)e) s-GV
4.0 filled
26
17
6d)
7d)
8d)
DTPC
DTPC
DTPC
2C16N 3.7 (1.0)e) s-GV
25
13
16
2C14N 7.5
2C12N 9.0
filled
filled
Structures of the Polymerized DTPC in Vesicles. It is
known that DTPC is polymerized in the solid phase, depending
on the vesicle structure.23 The color changes of polydiacetylene
are attributed to the length of the overlapping ³-orbitals which
can be affected by conformational change in the polymer
backbone.24 In the absorption spectra of 3¤, the absorption
a) Diameter was measured from the intensity maxima at the
cross section of the aggregate. b) The structure of the
aggregates: GV: giant vesicle, s-GV: phase-segregated GV,
filled: aggregate filled inside. c) F values after 60-min
irradiation. d) Photopolymerized vesicles. e) The values of
parenthesis are a length of the dark region in ¯m on the ring.
X
Y
(B)
(A)
X
Y
-3
-2
-1
0
1
2
3
-2
0
2
Distance/µm
Distance/µm
Figure 2. Distribution of the fluorescence intensity (>565 nm) along a line on cross-sectional CLSM images (inset) of 4 at
z = 2.0 ¯m (A) and 5 at z = 2.2 ¯m (B) under 488-nm laser excitation. The scale bar in inset is in 3 ¯m units.

J. Matsumoto et al.
Bull. Chem. Soc. Jpn. Vol. 83, No. 12 (2010) 1555
X
Y
(A)
(B)
X
Y
-2
-1
0
1
2
-5
0
5
Distance/µm
Distance/µm
Figure 3. Distribution of the fluorescence intensity (>565 nm) along a line on cross-sectional CLSM images (inset) of 6 at
z = 2.2 ¯m (A) and 8 at z = 4.5 ¯m (B) under 488-nm laser excitation. The scale bar is in 3 ¯m units.
the alkyl segment would mismatch with the proximal segment
of DTPC to induce conformational change of polymer back-
bone of polymerized DTPC. As a result, the additive
amphiphiles caused formation of filled aggregates with low F
0.06
values.
0.04
Mechanism of Phase Segregation and Morphological
Change. The morphology of 4-5 and 6-8 vesicles depends on
the carbon number, n, of 2CnN and 2CnS. The mechanism of
the morphological change can be surmised from the fluidity of
0.02
each of the amphiphiles. The main phase transition temperature
(Tc) from the liquid crystalline phase to the solid-analogous
phase of DTPC, 2C16S, and 2C12S were reported to be 38.5,14
61, and 28 °C,30 respectively. During cooling from 80 to
22.5 °C in the preparation of 4, the 2C16S components lost
fluidity to form a solid domain, but the DTPC still remained
fluid up to the Tc of DTPC. This caused the phase segregation
of 2C16S from the DTPC component. On the other hand, in the
preparation of 5, DTPC was first crystallized whereas 2C12S
remained fluid at the Tc of DTPC. As a result, the fluid 2C12S
was blended into the DTPC phase to change the aggregated
structure of DTPC molecules from that of pure DTPC.
Therefore, Tc values of DTPC and 2CnS determined whether
the morphology of the mixed vesicles changed or not.
Similarly, the Tc values of 2C16N and 2C14N were determined
to be 43 and 35 °C by DSC, respectively (Figure 6). The Tc
peak of 2C12N did not appear. It was expected Tc of 2C12N
was lower than that of 2C14N. The Tc value of 2C16N, which
segregated from the DTPC phase, was larger than that of
DTPC, whereas the Tc values of 2C14N and 2C12N, which
mixed with the DTPC, were lower than that of DTPC. The
dependence of the F values and the morphological change of
6-8 on the carbon number, n, of 2CnN were similar to that of
the mixed vesicles of DTPC and 2CnS. Thus, the fluidity of
2CnN and DTPC greatly affected the formation of the mixed
vesicles. A similar phase segregation mechanism has been
proposed by O’Brien and co-workers for the vesicles of DTPC
and nonpolymerizable phosphatidylcholine based on Tc deter-
mined by DSC.16
0
400
500
600
700
Wavelength/nm
Figure 4. Absorption spectral change of the solution of
DTPC under UV irradiation for 5 (dotted line), 15 (broken
line), 30 (dashed line), and 60 min (solid line).
maxima at 600 and 485 nm are assigned to be long and short
conjugation lengths, respectively (Figure 4).25-27 Therefore, the
polymerization behavior can be evaluated by absorption spectra
of the polymerized DTPC. The contribution of a long
conjugation length to short conjugation length was evaluated
by the ratio (F) of absorbance at 600 nm (A600) to that at 485 nm
(A485): F (%) = 100 © A600/A485. The F value of 3¤ was
determined to be 24% in a steady state, as shown in time-course
plots of the F values (Figure 5A). The F values of s-GV 4 and
6 were 26% and 25%, which are similar to that of 3¤. On the
other hand, the F values of the mixed vesicles of 5, 7, and 8
were 17%, 17%, and 13%, respectively, under UV irradiation
for 60 min, although the F values at the beginning of the UV
irradiation were higher than 20% (Figure 5B).
The additive effects of other surfactants on DTPC have been
precisely investigated.7,28,29 Singh et al. have found that DTPC
was polymerized to give highly conjugated polymer by
addition of saturated short-chain phosphatidylcholines such as
1,2-dinonanoyl-sn-glycerol 3-phosphocholine.28 They have
proposed that the short-chain saturated phosphatidylcholines
match the size of proximal segment between the head group
and the diacetylene of DTPC, resulting in the formation of
tubular assemblies. In the cases of 2C12S, 2C12N, and 2C14N,
Conclusion
Phase segregation in vesicles has been previously found in
vesicles formed from DTPC and nonpolymerizable phospha-

1556 Bull. Chem. Soc. Jpn. Vol. 83, No. 12 (2010)
Phase-Segregated Vesicles of Polydiacetylene
(A)
(B)
30
30
20
10
20
10
0
15
30
45
60
0
15
30
45
60
UV irradiation time/min
UV irradiation time/min
Figure 5. Time course plot of F for (A) the 3 ( ), 4 ( ), and 6 ( ) and for (B) 5 ( ), 7 ( ), and 8 ( ) under UV irradiation.
excitation (Figure S4). CLSM images of 7 and 8 obtained at
the specified z under 488-nm laser excitation (Figure S5). This
material is available free of charge on the web at http://
www.csj.jp/journals/bcsj/.
0.1 mW
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