pH-Tolerant giant vesicles composed of cationic lipids with imine linkages and oleic acids

Article abstract of DOI:10.1039/d0ra06822e

Giant vesicles (GVs) have attracted attention as functional materials because they can encapsulate both hydrophilic and hydrophobic compounds. For next generation functional GVs, both tolerance and stimuli-sensitivity are needed. So far, vesicles tolerant to acidic or basic conditions were generated using a mixture of cationic lipids and fatty acids. Here, to create functional GVs that are tolerant to a wide pH range but sensitively respond at below a specific pH, the behaviour of GVs composed of a cationic lipid with an imine bond and oleic acid was investigated. Even though the GVs prepared by the film swelling method were tolerant to strongly acidic conditions, GVs without oleic acid gradually shrank, accompanied by the generation of oil droplets at the same pH. 1H NMR analysis revealed that during hydration of the film, the imine bond hydrolysed to provide a cationic surfactant and an oil component in the presence of oleic acid due to its own Lewis basicity, suggesting the dissociation of oleic acid. The results of fluorescence spectroscopy using an environment-responsive probe and IR spectroscopy indicated that the GV tolerance originated from the intermolecular interactions of cationic lipids and anionic oleate. This journal is

Full text of DOI:10.1039/d0ra06822e

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pH-Tolerant giant vesicles composed of cationic
lipids with imine linkages and oleic acids†
Cite this: RSC Adv., 2020, 10, 34247
*
Daichi Sawada, Ayana Hirono, Kouichi Asakura and Taisuke Banno
Giant vesicles (GVs) have attracted attention as functional materials because they can encapsulate both
hydrophilic and hydrophobic compounds. For next generation functional GVs, both tolerance and
stimuli-sensitivity are needed. So far, vesicles tolerant to acidic or basic conditions were generated using
a mixture of cationic lipids and fatty acids. Here, to create functional GVs that are tolerant to a wide pH
range but sensitively respond at below a specific pH, the behaviour of GVs composed of a cationic lipid
with an imine bond and oleic acid was investigated. Even though the GVs prepared by the film swelling
method were tolerant to strongly acidic conditions, GVs without oleic acid gradually shrank,
accompanied by the generation of oil droplets at the same pH. 1H NMR analysis revealed that during
hydration of the film, the imine bond hydrolysed to provide a cationic surfactant and an oil component
in the presence of oleic acid due to its own Lewis basicity, suggesting the dissociation of oleic acid. The
results of fluorescence spectroscopy using an environment-responsive probe and IR spectroscopy
indicated that the GV tolerance originated from the intermolecular interactions of cationic lipids and
anionic oleate.
Received 7th August 2020
Accepted 8th September 2020
DOI: 10.1039/d0ra06822e
rsc.li/rsc-advances
responsiveness to stimuli is relatively slow. Moreover, vesicles
composed of low-molecular-weight amphiphiles sensitively
Introduction
respond to stimuli. However, they are fragile and rapidly
collapse even under relatively mild conditions.
Amphiphilic molecules form various molecular aggregates in
water depending on their hydrophilicity and hydrophobicity.
Among them, vesicles in which the bilayer membranes are
closed like a bag can encapsulate both hydrophilic and hydro-
phobic compounds because of their characteristic structure.
Directed toward functional materials, many stimuli-responsive
vesicles were reported using precisely designed amphiphilic
molecules. For example, the introduction of transmembrane
protein-like functional molecules into the vesicular membrane
provides notable properties, such as releasing1–5 and uptaking
compounds from the external and internal phases, respectively,
under specic conditions.6–8 Vesicles are also useful as micro
reaction elds for analysis in biological systems and as tissue
markers.9–12 In addition, it is well known that polymersomes of
amphipathic polymers are more stable than vesicles composed
of low-molecular-weight amphiphiles under various conditions.
The shape of nanometer-sized polymersomes differs according
to the composition of the polymer chain, and the affinity with
cells depends on their shape; therefore, polymersomes are
useful for nanomedicines.13–15 The structural durability of vesi-
cles is one of the important factors for functional material
applications. Although polymersomes are generally stable, their
To resolve such a dilemma between stability and stimuli-
responsiveness, functional vesicles composed of mixtures of
cationic and anionic surfactants and other compounds were
recently reported.16–19 They exhibit stable macroscopic struc-
tures but the size decreases in response to stimuli, such as ion
addition, pH change, and temperature. In addition, it is known
that anionic surfactants self-assemble in the presence of
cationic surfactants to form a unique molecular assembly.20,21
Fatty acids are also key compounds creating stable vesicles.
Suga et al. reported that, although oleic acid forms vesicles at
a pH of 8.0–9.8, the stable pH range extended to 10.6 by adding
dodecyldimethylammonium bromide.22 However, the creation
of tolerant vesicles under both acidic and basic conditions is
still challenging.
Herein, we report catanionic giant vesicles (GVs) that are
tolerant to a wide pH range but fragile, rapidly collapsing at
a specic pH. We have previously reported that the morphology
of GVs composed of amphiphiles having polymerisable and
hydrolysable groups depends on the lipid composition,
including polymers and hydrolysates.23,24 These results suggest
that the membrane structures that induce a drastic change in
GVs are affected by molecular packing based on intermolecular
interactions between lipid building blocks. Thus, a cationic
lipid having an imine bond (Im), which is hydrolysed under
acidic conditions to produce the oil component HBA and the
Department of Applied Chemistry, Faculty of Science and Technology, Keio University,
3-14-1 Hiyoshi, Kohoku-ku, Yokohama 223-8522, Japan. E-mail: banno@applc.keio.
ac.jp
† Electronic supplementary information (ESI) available: Synthetic procedures of
the compounds, Fig. S1–S10, and Table S1. See DOI: 10.1039/d0ra06822e
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a
confocal laser microscope (FV10i-DOC, Olympus). The
concentration of all uorescent dyes was 1 mM.
Time course measurement of the Im hydrolysis
A 7.5 mM dispersion of Im or 10 mM of Im/oleic acid ¼ 75/25
(mol%) was prepared similar to the procedure for microscopy
observations using D2O. To adjust the pH of the dispersions,
HCl or NaOH aqueous solution was added to be 10ꢂ4 to 10ꢂ2 M.
The total volume of each sample dispersion was 100 mL. Aer
the samples were kept at room temperature (23–25 ꢀC) for 24 h,
650 mL of CD3OD-d4 containing DMF (36.5 mM) was added as
a standard compound to homogenise the samples. The molec-
Fig. 1 Hydrolysis of Im to generate oil component HBA and cationic
surfactant Am under acidic conditions.
cationic surfactant Am, was designed (Fig. 1). By coexisting Im
with oleic acid, which affords vesicles under weak basic condi-
tions,25,26 not only the hydrolysis ratio of Im but also the acid
dissociation degree of oleic acid could change depending on
pH.27–29 GVs that are tolerant from basic to acidic conditions but
rapidly collapse below a certain acidic pH are created because of
the different molecular packing of the vesicular membrane.
1
ular conversion of Im was analysed by H NMR spectroscopy.
Hydrolytic degradation of the imine was calculated by inte-
grating the imine proton peak at d 8.38 ppm of Im and the
aldehyde proton at d 9.78 ppm of HBA. The measurements were
performed in triplicate.
Materials and methods
General
Fluorescence emission
Initially, 20 mM and 100 mM chloroform solutions of lipids and
Laurdan, respectively, were prepared. A mixture of 200 mL of
lipid solution, 40 mL of Laurdan solution, and 1.8 mL of chlo-
roform was dried under reduced pressure at room temperature
(23–25 ꢀC) for 2 h or more to form thin lms. Four millilitres of
distilled water was gently added to swell the lm at room
temperature for 2 h. The nal lipid and Laurdan concentrations
were 1 mM and 1 mM, respectively. Then, the samples were
mixed using a vortex mixer for 10 s, and the uorescence spectra
of Laurdan were recorded using a uorometer (RF-6000, Shi-
madzu Co., Ltd., Kyoto, Japan) in the uorescence wavelength
range of 380 nm to 600 nm, while the excitation wavelength was
set at 340 nm. The spectra of the vesicle dispersion containing
hydrolysates of Im and 1,6-diphenyl-1,3,5-hexatriene (DPH)
were also recorded by setting excitation and emission at 360 and
430 nm, respectively.
Commercial reagents and solvents were purchased from Tokyo
Chemical Industry Co., Ltd. (Tokyo, Japan), Wako Chemical
Industry Co., Ltd. (Osaka, Japan), and Kanto Chemical Co., Ltd.
(Tokyo, Japan). They were used without further purication.
Synthetic procedures for the compounds are described in the
ESI.† The compounds were identied by 1H NMR and mass
1
spectrometry. The H NMR apparatus was an ECA-400 Fourier
transform spectrometer (JEOL Ltd., Tokyo, Japan), and the
chemical shi was based on tetramethylsilane (0 ppm). Mass
spectrometry was performed by electrospray ionisation using an
LCT-Premier (Waters Corp., Milford, MA, USA).
Methods
Preparation of GVs by thin-lm swelling method and
microscopic observation
Aliquots of 20 mM Im and 20 mM oleic acid chloroform solu-
tions were prepared at room temperature (23–25 C). A 2 mM
lipid solution (2 mL) was formulated and dried under reduced
Results and discussion
Microscopic observations of GVs under various pH conditions
ꢀ
pressure for 2 h or more to form a thin lm. A volume of 4 First, the GVs composed of hydrolysable Im that were prepared
millilitres of distilled water was gently added to swell the lm at according to a thin lm swelling method were obserꢀved under
room temperature (23–25 ꢀC) for 2 h. The nal lipid concen- various pH conditions at room temperature (23–25 C). Using
tration was 1 mM of Im/oleic acid ¼ 100/0, 75/25, 50/50, 25/75, either a uorescent lipid, Texas Red-1,2-dihexadecanoyl-sn-
and 0/100 (mol%). Aer stirring for 10 s using a vortex mixer glycero-3-phosphoethanolamine trimethylammonium salt
(TM-1, AS ONE, Osaka), the desired amount of 1 M HCl or 1 M (DHPE), or Uranine, a water-soluble uorescent compound, GVs
NaOH solution was added to the sample dispersion to adjust were observed in the dispersion without HCl and NaOH under
the pH. Then, the sample (75 mL) was sealed in a glass chamber a confocal laser microscope. Under the basic conditions
(15 ꢁ 15 ꢁ 0.28 mm; frame seal chamber, MJ Research Inc., prepared using a 1 M NaOH solution, the GVs were stable in the
Waltham) and observed under an optical microscope (BX51, microscopic eld for 24 h. In the dispersion of pH 2 prepared
Olympus, Tokyo, Japan) equipped with a CCD camera (DP22, using HCl, no GVs were observed immediately aer the obser-
Olympus, Tokyo, Japan). Fluorescence microscopy observations vation started, indicating that GVs rapidly collapsed when the
of GVs containing Texas Red-DHPE, uranine, or Laurdan were HCl solution was added. At pH 3, the GVs gradually shrunk,
captured using a mercury lamp excitation source and lters for accompanied by the generation of droplets at the membrane
excitation and emission (lex ¼ 473 nm and lem ¼ 490–590 nm within 30 min (Fig. 2). Similar shrinkage dynamics were
for Texas Red-DHPE, lex ¼ 592 nm and lem ¼ 612 nm for ura- observed under an optical microscope for more than 20 GVs
nine, and lex ¼ 359 nm and lem ¼ 461 nm for Laurdan) under with diameters in the range of 4–17 mm in six independent
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was carried out to trace the hydrolysis of Im in D2O with/without
HCl and NaOH from the lm swelling process. Amphiphilic
compounds, such as Im and oleic acid, are mainly concentrated in
the vesicle membrane rather than monodisperse in the bulk water
phase due to their hydrophobicity. Because the Im hydrolysis
should occur around the membrane, a higher concentration was
used compared to the microscopic observations. The Im hydro-
lysis was traced integrating the imine proton (d 8.38 ppm) in Im
and aldehyde proton (d 9.78 ppm) in HBA aer homogenisation
by adding CD3OD-d4 containing DMF (d 7.99 ppm) as an internal
standard (Fig. S2†). In the absence of oleic acid, the Im hydrolysis
hardly progressed in the lm swelling process (Fig. 3a). In addi-
tion, no signicant hydrolysis occurred in the dispersion with/
without 10ꢂ4 M HCl. Under the condition of 10ꢂ3 M HCl, the
reaction proceeded immediately aer adding HCl, and the
hydrolysis ratio of Im was around 30%. In a more concentrated
10ꢂ2 M HCl solution, the hydrolysis ratio exceeded 80% within
15 min. Moreover, in the presence of oleic acid, the Im hydrolysis
accelerated during the lm swelling process, and the hydrolysis
ratio reached approximately 25% before the addition of HCl or
NaOH (Fig. 3b). This ratio was comparable with the initial
composition of oleic acid, suggesting that the imine group of the
Lewis base withdrew the proton of oleic acid, and thus the acti-
vated imine was rapidly hydrolysed. Indeed, the Im hydrolysis
accelerated depending on the amount of oleic acid (Fig. S3†). The
Fig. 2 Confocal laser microscope bright field (top) and fluorescence
(bottom) images of shrinkage of GVs composed of Im. Droplets indi-
cated by red arrows generated at the membrane. Scale bar: 10 mm.
experiments. Shrinkage of GVs with diameters of <2 mm was not
distinguishable due to the resolution of the microscope. The
vesicle size did not affect the shrinkage dynamics of the
observable GVs. Based on previous studies,30 it was more
appropriate that the GVs were multilamellar rather than uni-
lamellar because the GV dispersions were prepared by the thin
lm method. Although it is possible that the observed
shrinkage dynamics were associated with the lamellarity of the
vesicle membrane, estimation of their relationship was difficult
because of the limited spatial resolution of the microscope
image. A similar shrinkage of GVs forming droplets was also
induced by domain formation through the enzymatic hydrolysis
of phospholipids to generate diacylglycerols.31 Thus, the current
observed collapse of GVs could be associated with the compo-
sitional change of lipids. In the dispersion containing 10ꢂ3
M
NaCl, the GVs composed of Im hardly shrunk within 30 min. In
addition, the GVs were stable at 10ꢂ4 M HCl. These results
claried that the hydrolysis of Im under stronger acidic condi-
tions affects GV durability, which induces shrinkage. Next,
observations of GVs composed of Im and oleic acid at the ratio
of 75/25, 50/50, 25/75, and 0/100 (mol%) were carried out under
a confocal laser microscope. The total lipid concentration was
1 mM. When the composition of Im/oleic acid was 75/25 or 50/
50 (mol%), GVs were observed. In the case of 25/75 and 0/100
(mol%), no GVs were observed. GVs containing 25 mol% oleic
acid (Im/oleic acid ¼ 75/25 (mol%)) were tolerant from the basic
condition of 10ꢂ3 M NaOH to an acidic condition of 10ꢂ3 M HCl.
Oleic acid did not form GVs at the tested pH conditions. In
addition, when non-reactive N-dodecyl-N-hexadecyl-N,N-dime-
thylammonium bromide (C16–C12) was used instead of Im, size
changes and collapse dynamics of GVs were not observed at the
tested pH conditions despite the presence and absence of oleic
acid (Fig. S1†). Therefore, it was implied that the combination
of Im and oleic acid with the appropriate ratio is associated with
the durability of GVs over a wide pH range.
Fig. 3 Time course of the hydrolysis ratio of Im in the dispersion of Im
(a) and Im/oleic acid (b) calculated by 1H NMR. Each measurement was
performed in triplicate. Water (black triangle); 10ꢂ2 M HCl (red filled
circle); 10ꢂ3 M HCl (pink circle); 10ꢂ4 M HCl (yellow filled circle);
Time course of the Im hydrolysis
To elucidate the effect of changes in lipid composition on the
durability of GVs under certain pH conditions, H NMR analysis 10ꢂ3 M NaOH (blue filled circle).
1
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reaction mechanism thus indicated the dissociation of oleic acid measurements using infrared and uorescence were carried
to generate oleate (Fig. 4). Due to the ion-pairing between the out. IR measurements were performed using the attenuated
generated anionic oleate and cationic Im and Am in the vesicular total reection method (Fig. 5). The peak intensities of the
membrane, the molecular packing increased. Under conditions of carbonyl and hydroxyl groups in oleic acid at 1707 cmꢂ1 and
10ꢂ4 and 10ꢂ3 M HCl and 10ꢂ3 M NaOH, the reaction ratio was 2550–3250 cmꢂ1, respectively, weakened in the presence of Im.
almost constant. It was considered that the acidic catalyst did not This suggests an ion–dipole interaction between the quaternary
penetrate into the vesicular membrane because ion-pairing was ammonium group in Im and the carbonyl group in oleic acid
maintained at low electrolyte concentrations; therefore, no further (and oleate), indicating intermolecular interactions of the
hydrolysis of Im occurred. However, under the condition of cationic compounds, Im and Am, and oleic acid.
10ꢂ2 M HCl, the reaction advanced to about 70% within 15 min.
This was probably due to the dissociation of the ion-pairing to from uorescence spectra. The environment-responsive uo-
regenerate oleic acid because of the 100 times higher concentra- rescent dye N,N-dimethyl-6-dodecanoyl-2-naphthylamine
Next, the vesicular membrane properties were estimated
tion of HCl relative to the lipid components. It was therefore (Laurdan) is widely used to investigate the uidity of vesicle
concluded that the signicant compositional change was related membranes from uorescence spectra and microscope
to rapid vesicle shrinkage.
imaging. It is empirically known that the emission spectrum of
To clarify the inuence of the lipid composition on the Laurdan in a phospholipid membrane depends on the phase
vesicle durability, observations of dispersions containing state of the membrane.32,33 In the dispersion of Im in the
50 mol% hydrolytic products of Im, HBA, and Am were carried presence and absence of 10ꢂ3 M NaOH 2 h aer preparation,
out. The size reduction in GVs containing 50 mol% hydrolytes the uorescence spectra were almost the same (Fig. 6). However,
(Im/HBA/Am ¼ 0.5 mM/0.5 mM/0.5 mM) was more frequently the acidic condition of the 10ꢂ4 M HCl concentration provided
observed in 10ꢂ3 M HCl dispersions when compared to those a different spectrum; that is, the uorescence intensity of the
composed of 1 mM Im for 30 min (Fig. S4†). To analyse the size dispersion in the presence of oleic acid was signicantly higher
reduction in GVs, the time until the size stabilized aer the than that in the absence. This indicated that the membrane
addition of HCl (T) was measured. As a result of the statistical structures of vesicles containing oleic acid were harder than
analysis, T of GVs composed of Im (1 mM) was shorter than those without oleic acid. Moreover, using 10ꢂ3 M HCl, GVs
those of Im/HBA/Am (0.5 mM/0.5 mM/0.5 mM), which was containing oleic acid were conrmed under a confocal micro-
independent of the GV size (Fig. S5†). This result clearly indi- scope immediately aer preparation of the samples (Fig. S7†),
cated that the lipid composition strongly affected the durability but the uorescence intensity in the spectrum was signicantly
of GVs. In addition, to estimate the relationship between the low (Fig. 6b). Similar spectra were also obtained at pH 2 in the
composition of compounds and GV formation, dispersions presence and absence of oleic acid. These uorescence spectra
without electrolytes were observed (Table S1†). When Im was were probably due to the concentration quenching of Laurdan
present, GVs were noticed among the tested compositions. Even accompanied by a change in the membrane structures. When
though a mixture of HBA and oleic acid (0.75 mM/0.25 mM) non-reactive C16–C12 was used instead of Im, no signicant
afforded droplets, GVs were formed using Am and oleic acid differences in the uorescence spectra were observed depend-
(0.75 mM/0.25 mM, Fig. S6†). At 1 mM Am, no micrometer-scale ing on the HCl concentration despite the presence and absence
structures were observed. These results suggest the intermo- of oleic acid (Fig. S8†). Even though the uorescence spectra
lecular interaction between the cationic compounds, Im and using Laurdan did not completely reect the microscopic
Am, and oleic acid, which contributes to the stronger molecular observation, these results qualitatively suggested that the
packing might be an important factor to form stable GVs.
membrane structure of GVs composed of Im and oleic acid was
more tolerant to a stronger acidic condition.
Estimation of vesicle membrane structure
To estimate the vesicle membrane structure based on the lipid
composition at the tested pH conditions, two spectroscopic
Fig. 5 IR spectra of Im/oleic acid ¼ 100/0 (solid blue), 75/25 (solid
Fig. 4 Estimated reaction mechanism of Im hydrolysis in the presence green), 50/50 (dashed green), 25/75 (dashed red), and 0/100 (solid red)
of oleic acid during the film swelling process.
(mol%).
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Fig. 7 Internal membrane fluidity of Im (blue) and Im/oleic acid (red)
vesicles with hydrolysis ratio of Im containing DPH. Im/HBA/Am (blue);
Im/HBA/Am/oleic acid (red).
Based on these results, the difference in the durability of Im
vesicles with/without oleic acid was interpreted as follows. In the
absence of oleic acid, hydrolysis of Im did not proceed during the
lm swelling process. In addition, to adjust the pH, HCl and
NaOH aqueous solutions were added to the dispersion aer the
vesicle preparation. Thus, the electrolyte concentration in the
outer water phase was higher than that in the inner water pool of
the GVs. GVs were stable at the low electrolyte concentration of
10ꢂ4 M HCl, in which hydrolysis of Im did not proceed. Due to
the weak molecular packing of the vesicular membrane, Im was
rapidly hydrolysed to generate Am and HBA under the more
concentrated HCl conditions. In the dispersion containing
10ꢂ3 M HCl, where Im hydrolysis moderately occurred, the GVs
gradually shrank because of the osmotic pressure caused by the
difference in the electrolyte concentration between the inner and
outer water phases (Fig. 8a). Most Im was hydrolysed in the
presence of 10ꢂ2 M HCl, and thus, the GVs were fragile and
rapidly collapsed. Mixing oleic acid with Im GVs generated
different vesicular membrane structures (Fig. 8b). During the
swelling process of the lipid lm, Im hydrolysis proceeded due to
the Lewis-basic imine group. This afforded a specic membrane
structure with strong molecular packing based on the ion pair
between cationic compounds, Im and Am, and anionic oleate
(Fig. 4). Even when HCl was added to the GV dispersion at
10ꢂ3 M, no further hydrolysis of Im occurred; therefore, GVs were
tolerant to relatively strong acidic conditions. The molecular
system composed of both the imine lipid and oleic acid, where
the vesicular membrane structure changes, provides a strategy to
construct tolerant vesicles with a wider pH range. In addition, the
current model may be linked to the emergence of life. Since the
pH in plausible circumstances of early earth is estimated from 2
to 11,36,37 the durability of structures under a wider pH would
have advantages for selection. The imine bond is one of the
dynamic covalent bonds that can be hydrolysed and generated
under mild conditions and has drawn attention to construct
prebiotic supramolecular self-assemblies.38,39 Oleic acid/oleate
also represents a good model system for the evolution of life
because fatty acids are considered prebiotic molecules.40 There-
fore, it is considered that the designed molecular system affords
Fig. 6 Fluorescence spectra of dispersion with (a) Im and (b) Im/oleic
acid ¼ 75/25 (mol%) containing Laurdan. Water (black); 10ꢂ2 M HCl
(red); 10ꢂ3 M HCl (pink); 10ꢂ4 M HCl (yellow); 10ꢂ3 M NaOH (blue).
Furthermore, using a hydrophobic compound that exhibits
uorescence in the vesicular membrane, 1,6-diphenyl-1,3,5-
hexatriene (DPH), the uorescence anisotropy in the
membrane was estimated with respect to the lipid composition
containing the hydrolysates of Im. Since rod-shaped DPH has
strong polarisation properties, the uidity of the lipid bilayer
can be estimated from depolarisation in a membrane.22,34 The
uidity parameter of the vesicular membrane, 1/P, is calculated
from P ¼ (I ꢂ GIt)/(I + GIt), G ¼ it/i , where It, I , it, and i
k
k
k
k
k
are emission intensities for perpendicular and parallel to the
vertically and horizontally polarised light, respectively, and G is
the correction factor. The larger the 1/P value, the higher the
uidity of the vesicular membrane. The uidity of the vesicular
membrane was maximised at a composition containing around
60% hydrolysates of Im, and decreased at 80% despite the
presence and absence of oleic acid (Fig. 7). This indicated that
the membrane structure was not a bilayer but similar to
a monolayer35 due to the oil component HBA. In the absence of
oleic acid, the uidity increased with a higher composition of
hydrolysates. The decrease in uidity of the membrane con-
taining 80% hydrolysates suggested a drastic change in struc-
tures, such as phase separation, that is related to the formation
of domains in the membrane.30 Since there was a difference in
the 1/P value in the presence and absence of oleic acid, it was
considered that the membrane structure was different in the
presence of oleic acid.
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Acknowledgements
Prof. Taro Toyota (Univ. of Tokyo) is acknowledged for the
fruitful discussion. We would like to thank Editage
(www.editage.com) for English language editing. This work was
partially supported by JSPS KAKENHI grant number 18K05066
for T. B.
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Conclusions
In this study, pH-responsive GVs were investigated using
a mixture of cationic lipids having an imine bond and oleic acid.
From the 1H NMR, IR, and uorescence spectra, it was revealed
that the durability of GVs containing oleic acid under a wide pH
range from basic to acidic conditions was related to the strong
molecular packing, which was based on the formation of ion-
pairing between cationic lipids and oleate. This enhancement
of membrane structures occurred by hydrolysis of the imine
lipid to promote dissociation of oleic acid during the lm
swelling process. GVs were fragile and rapidly collapsed under
more acidic conditions due to dissociation of the ion-pairing
followed by further hydrolysis of the imine lipid caused by the
ion strength. The current molecular system can not only provide
the methodology to generate tolerant vesicles for a wide pH
range from the viewpoint of supramolecular chemistry but also
show a possibility in the chemical evolution of protocells,
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Conflicts of interest
The authors declare no conict of interest.
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