Metal Complex Lipids for Fluid–Fluid Phase Separation in Coassembled Phospholipid Membranes

Article abstract of DOI:10.1002/anie.202102774

We demonstrate a fluid–fluid phase separation in 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) membranes using a metal complex lipid of type [Mn(L1)] (1; HL1=1-(2-hydroxybenzamide)-2-(2-hydroxy-3-formyl-5-hexadecyloxybenzylideneamino)ethane). Small a

Full text of DOI:10.1002/anie.202102774

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Supramolecular Chemistry
Metal Complex Lipids for Fluid–Fluid Phase Separation in
Coassembled Phospholipid Membranes
Ryo Ohtani,* Yuka Anegawa, Hikaru Watanabe, Yutaro Tajima, Masanao Kinoshita,*
Nobuaki Matsumori, Kenichi Kawano, Saeko Yanaka, Koichi Kato, Masaaki Nakamura,
Masaaki Ohba, and Shinya Hayami*
Abstract: We demonstrate a fluid–fluid phase separation in
1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) mem-
branes using a metal complex lipid of type [Mn(L1)] (1;
HL1 = 1-(2-hydroxybenzamide)-2-(2-hydroxy-3-formyl-5-
hexadecyloxybenzylideneamino)ethane). Small amount of
1 produces two separated domains in DMPC, whose phase
transition temperatures of lipids (Tc) are both lower than that
of the pristine DMPC. Variable temperature fluorescent
microscopy for giant-unilamellar vesicles of DMPC/1 hybrids
demonstrates that visible phase separations remain in fluid
phases up to 378C, which is clearly over the Tc of DMPC. This
provides a new dimension for the application of metal complex
lipids toward controlling lipid distributions in fluid mem-
branes.
understand functionalities and formation mechanisms of
coassemblies.[4] Notably, cell membranes form fluids. How-
ever, “heterogeneous” coassembled platforms with fluid–
fluid phase separation systems formed by thousands of lipids
allow laterally separated domains to precisely manipulate
biofunctions.[2a,5,6] On the other hand, the chemical control of
phase separation in fluid coassemblies of lipids is still difficult
owing to the uncontrollable and high miscibility of lipid
molecules.
Metal complex lipids are versatile materials. Their hydro-
philic domains consist of metal complex moieties with
catalytic, fluorescent, and transportation properties.[1b,7]
Such metal complex heads exhibit stronger electronic inter-
action with natural lipids than organic molecules; thus, they
can be used to impact and modulate some properties of
multilipid coassemblies such as miscibility and phase tran-
sition. Based on this, synthetic approaches for generating
artificially separated “rigid” regions with regular structures by
metal complex lipids in 1,2-dimyristoyl-sn-glycero-3-phos-
phocholine (DMPC) and cell membranes have been demon-
strated.[8] However, the development of artificial lipids those
can produce stable “fluid” domains separated by other fluid
regions in coassemblies remains largely unexplored.
Herein, we demonstrate the first example of a fluid–fluid
phase separation in natural lipid membranes of DMPC by
coassembling single-chain metal complex lipids of type [Mn-
(L1)] (1; HL1 = 1-(2-hydroxybenzamide)-2-(2-hydroxy-3-
formyl-5-hexadecyloxybenzylideneamino)ethane). By the
introduction of 1 to DMPC membranes, two fluid domains
formed separately; the separation was maintained up to 378C
in the fluid membranes. We discovered that the intermolec-
ular interaction of the metal complex cores with the aldehyde
groups of 1 and phosphate groups in DMPC gave rise to phase
separations.
A
mphiphilic molecules form high-order structures via self-
assemblies in solutions.[1] Nature utilizes amphiphiles in
constructing multicomponent coassembly systems in various
fields.[2] Coassembling is a ubiquitous strategy to improve
functionalities. It has also been employed in material designs
to achieve morphology control, tunable energy transport
processes, high charge carrier mobility, and long phosphor-
escence with high stability.[3] Recently, “phase separation” in
such coassembled systems has been proposed as a key to
[*] Prof. R. Ohtani, H. Watanabe, Y. Tajima, Dr. M. Kinoshita,
Prof. N. Matsumori, Prof. M. Ohba
Department of Chemistry, Faculty of Science, Kyushu University
744 Motooka, Nishi-ku, Fukuoka 819-0395 (Japan)
E-mail: ohtani@chem.kyushu-univ.jp
kinoshi@chem.kyushu-univ.jp
Y. Anegawa, Prof. M. Nakamura, Prof. S. Hayami
Department of Chemistry, Graduate School of Science
Kumamoto University
2-39-1 Kurokami, Chuo-ku, Kumamoto 860-8555 (Japan)
E-mail: hayami@kumamoto-u.ac.jp
Two amphiphilic ligands HL1 and 1-(2-hydroxybenza-
mide)-2-(2-hydroxy-5-hexadecyloxybenzylideneamino)-
ethane (HL2) were synthesized by a stepwise method (see the
experimental section in supporting information). The metal
complex lipids 1 and [Mn(L2)] (2) were synthesized by mixing
the corresponding ligands with Mn(OAc)2·4H2O, which
yielded their brown powders. Single crystals of 1·3H2O and
2·2MeOH were obtained through their recrystallizations
using suitable alcohols. 1·3H2O and 2·2MeOH crystalized in
Dr. K. Kawano
Institute for Chemical Research, Kyoto University
Uji, Kyoto 611-0011 (Japan)
Dr. S. Yanaka, Prof. K. Kato
Exploratory Research Center on Life and Living Systems (ExCELLS)
and Institute for Molecular Science (IMS)
National Institutes of Natural Sciences
5-1 Higashiyama, Myodaiji, Okazaki, 444-8787 (Japan)
and
ꢀ
triclinic P1 and monoclinic P21/c, respectively (Figure 1 and
Graduate School of Pharmaceutical Sciences, Nagoya City University
3-1 Tanabe-dori, Mizuho-ku, Nagoya, Aichi, 467-8603 (Japan)
Table S1). X-ray structural analyses revealed structural differ-
ences in manganese complex cores of the metal complex
lipids in which 1 incorporates L1 with an aldehyde substitu-
ent, whereas, 2 consists of a similar ligand L2 but without
Supporting information and the ORCID identification number(s) for
the author(s) of this article can be found under:
https://doi.org/10.1002/anie.202102774.
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Figure 2. a) Differential scanning calorimetry results for 1,2-dimyris-
toyl-sn-glycero-3-phosphocholine (DMPC)/1(x) hybrids and variable
temperature wide-angle X-ray scattering results for b) DMPC and
c) DMPC/1(0.13).
DMPC/1(x) hybrids (x = 0.063, 0.083, 0.13, and 0.25) were
studied by differential scanning calorimetry (DSC) (Fig-
ure 2a). The lipid bilayers of DMPC exhibited a phase
transition from the low-temperature gel phase to a high-
temperature fluid phase at the Tc of 23.48C (x = 0 in
Figure 2a). In the case of the hybrid liposomes of DMPC/
1 (0.063) and DMPC/1 (0.083), two peaks were observed,
which demonstrate that introducing small amount of 1 into
the DMPC induced phase separations. DMPC/1 (0.063)
showed a large peak at 22.68C and a small shoulder with
a peak top at 22.08C (Figure 2a). Similarly, DMPC/1 (0.083)
showed two peaks at 22.38C and 21.78C. In contrast, DMPC/
1 (0.13) and DMPC/1 (0.25) showed a single and a broad peak
at TC = 22.08C and 21.98C, respectively. Total enthalpy of the
phase transitions in DMPC/1(x) decreased gradually as the
ratio of 1 increased (Table S3). The resultant decrease in
transition temperature and enthalpy suggest that 1 is hybrid-
ized with DMPC, disturbing their lipid packings. These DSC
results demonstrate that DMPC/1 (0.063) and DMPC/
1 (0.083) consist of separated domains: one at a low Tc,
which incorporates highly concentrated 1 (H domain), and
the other at a high Tc, which incorporates less concentrated
1 (L domain). On the other hand, DMPC/1 (0.13) and DMPC/
1 (0.25) were formed by the respective single H domains. We
confirmed the H domain exhibited its phase transition
between the gel and fluid phases with a lower Tc than
DMPC by variable temperature wide-angle X-ray scattering
(VT-WAXS) from 168C to 308C for multilamella vesicles of
the pristine DMPC and DMPC/1 (0.13) (Figure 2b,c and
Figure S4). The broad diffraction peaks at higher temper-
atures above Tc indicate that fluid phases form, and their
Figure 1. Crystal structures of a) 1·3H2O and b) 2·2MeOH-color code:
purple (Mn), gray (C), blue (N), and red (O). H atoms are omitted for
clarity.[17]
aldehyde substituents. The two lipids consist of amphiphilic
molecules packed separately between hydrophilic and hydro-
phobic domains: manganese complex cores incorporating the
respective N2O2 tetradendate ligands form hydrophilic
domains, and alkyl chains with sixteen carbon atoms form
hydrophobic (lipophilic) domains (Figure 1). Lattice and
coordinating solvent molecules were present in the hydro-
philic domains, which were confirmed by thermogravimetry
and the elemental analyses of 1·3H2O and 2·2MeOH (Fig-
ure S1 and Figure S2). Before preparing hybrid liposomes of
DMPC combined with 1 and 2, we treated the crystals at
1008C to remove the lattice and coordinating solvents.
We prepared the hybrids of DMPC with 1 and 2, during
which we found that DMPC/1 hybrids produce corresponding
liposomes and DMPC/2 does not disperse in water but
precipitates as aggregations (Figure S3). Dynamic light scat-
tering measurements of the DMPC/1(x) hybrids at molar
ratios x = 0.063, 0.13, and 0.25 revealed that DMPC/1(x) are
liposomes, whose sizes decrease with increasing x, which
indicates that the critical packing parameter of 1 involves
large head groups (Table S2).[3d] To investigate the miscibility
of 1 in DMPC bilayers, the phase transition behavior of the
2
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disordered structures are attributed to the melting of carbon
lex = 470 nm) assembling in fluid areas was used.[11] Further-
chains. It is noted that no tilted carbon chains were present in
the gel phase of DMPC/1 (0.13) as opposed to that of pristine
DMPC.[9] This structural difference would impact membrane
thickness of DMPC/1 (0.13). The small-angle X-ray scattering
(SAXS) results for DMPC and DMPC/1 (0.13) demonstrate
slightly different peak positions at room temperature, indi-
cating that DMPC/1 (0.13) has thicker membranes than
DMPC (Figure S5).
In multiple-lipid coassemblies, strong immiscibility
between lipids gives separated regions such as gel-gel and
gel-fluid phases at temperatures below the Tc.[10] On the other
hand, the separated domains with the phases usually disap-
pear immediately above the Tc; thus, both regions become
miscible fluids with a single phase of blended lipids.[10] Thus, to
visually investigate the phase behavior in the DMPC/
1 (0.063), we carried out fluorescent microscopy for its
giant-unilamellar vesicles (GUVs). As a fluorescent dye, a b-
BODIPYTM FL C12-HPC with green emission (bodipy-PC;
more, to identify the position of 1 in the GUVs, we
synthesized a luminescent derivative metal complex lipid of
594neg-1 using a red emission probe (594neg; lex = 603 nm)
through a click reaction using 594neg-N3 having an azide
group[12] and HCC-1 having an ethynyl group at the terminal
position of 1 (Scheme 1 and Figure S6). GUVs of DMPC/
1 (0.063) containing both bodipy-PC and 594neg-1 at 0.2%
molar ratios were prepared by an electroformation method.
Note that the respective bodipy-PC and 594neg-1, and their
coexistence were found to have no effect on domain
formation in the GUVs without 1 (Figure S7).
The GUV images of DMPC/1 (0.063) at 308C show green
fluorescent regions and dark regions are separated (Figures 3
(left) and Figure S8). Moreover, areas showing red emissions
of 594neg-1 correspond to the green fluorescent regions in the
overlay image (Figure 3 (middle and right)). Thus, the
separated green regions incorporating bodipy-PC are attrib-
utable to the H domain with a high concentration of 1,
Scheme 1. Syntheses of a) HCC-1 and b) 594neg-1.
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domains remained separated up to a temperature of at least
158C above the Tc (Figure 4b). Moreover, we confirmed that
the separated domains formed again by cooling, resulting in
the reversible transition between the “fluid–fluid phase
separation” and fluid uniform phases (Figure 4c and Fig-
ure S10).
Infrared (IR) spectroscopy revealed that the fluid–fluid
phase separation phenomenon in DMPC/1(x) is attributed to
the intermolecular interaction between DMPC and 1. The
pristine DMPC liposomes exhibited a stretching mode of the
phosphate group (nO O) at 1232 cmÀ1 (Figure S11).[14] This
Figure 3. GUV images of DMPC/1(0.063) under excitation by 470 (left)
and 560 nm (middle) and their overlay (right) at 308C. GUVs at the
upper right and lower corners show the corresponding surface images
while one at the left shows the sliced image.
= =
P
mode was observed at the same wavenumber in the aggre-
gations of DMPC/2 (0.13), whereas DMPC/1 (0.13) displayed
whereas, the dark regions with lower fluidity than occuring an
accumulation of bodipy-PC are the L domain with a low
concentration of 1. Since 308C is above the TC of the H and L
domains, the whole GUVs are in the fluid phase with
disordered structures as demonstrated by VT-WAXS. More-
over, the fluid phase of both domains separated in the GUVs
were proved by the dissolution phenomenon with a detergent,
Triton X-100 (Figure S9).[13] Therefore, we conclude that the
observed phase separations in the GUVs are rarely visible
“fluid–fluid” phase separations between the H and L
domains.
Furthermore, variable temperature fluorescent microsco-
py measurements demonstrated that visible fluid–fluid phase
separations remained in the temperature range between 308C
and 378C (Figure 4a). With increase in temperature, the
shape of the separated domains and GUVs changed, and
mobility of the lipids increased. On the other hand, the phase
separations were maintained up to 378C. Around 378C, the
whole surface of the GUVs shows uniform emissions without
phase separations (Figure 4a). These results reveal that both
a peak of nO
at 1228 cmÀ1 (Figure S11). Therefore, this
=
P
=
O
shift involves the aldehyde group of 1 interacting with DMPC
via its phosphate groups in the hydrophilic parts of mem-
branes. Such intermolecular interaction leads to changing
membrane properties such as stiffness and thickness. Lower
Tc of the H domain than that of the L domain and the
assembly nature of bodipy-PC on fluid regions[11] indicate that
the H domain is more fluid than the L domain. It was reported
that the thickness mismatches between the separated domains
notably stabilized the respective domains and impacted the
domain sizes.[6b] In this context, the difference in thickness
between the H and L domains in the DMPC/1 hybrids
indicated by SAXS results could account for the resultant
phase separations.
To the best of our knowledge, only three cases of similar
fluid–fluid phase separation in binary lipid bilayer mem-
branes have been reported thus far: 1,2-dipalmitoyl-sn-
glycero-3-phosphoethanolamine (DPPE) and 1,2-dierucoyl-
sn-glycero-3-phosphocholine membranes,[15a–c] dihydrosphin-
gomyelin (DHSM) and 1,2-dioleoyl-sn-glycero-3-phospho-
choline membranes,[15d] and phosphatidylcholine and phos-
phatidylserine (PS) with Ca2+ ion systems.[15e–g] In the former
two cases, the hydrogen bonds between DPPE or DHSM
lipids predominate to form the respective DPPE- and DHSM-
rich domains separated from other lipid species. In the latter
case, Ca2+ interacts with the head groups of PS lipids, allowing
the formation of PS-rich domains as Ca(PS)2 against the PS-
PS electrostatic repulsion, which tends to stabilize the mixed-
lipids phase. These three cases provide “more rigid” regions
formed by the aggregation of single lipid species than the
other surrounding lipid regions. In contrast, 1 demonstrates
the ability to produce separated “soft domains,” wherein it
works as a “dopant” coassembled with the DMPC molecules,
resulting in characteristic fluid–fluid phase separations. It is
well-known that surfactants with a single hydrocarbon chain
destabilize and break lipid packing structures.[13,16] This is
consistent with the effects of 2 on the DMPC membranes,
yielding aggregations rather than bilayers (Figure S3, Fig-
ure S5 and Figure S12). A similar effect might exist in the
hydrophobic part of 1 systems as indicated by the lower
transition temperature and enthalpy of the H domains than
those of the DMPC membranes. However, the intermolecular
interaction of the metal complex cores with the aldehydes of
1 in the hydrophilic parts is key to preventing the break of
membranes and keep up the characteristic fluid regions.
Figure 4. a) VT-fluorescent microscopy images of DMPC/1(0.063)
during the heating process. The upper images display the surface of
GUVs and the lower show the sliced images. b) Phase diagram of
DMPC/1(0.063), and c) schematic representation of the phase separa-
tion. Blue and red amphiphiles indicate DMPC and 1, respectively.
4
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Yang, P. F. Duan, Y. Q. Jiang, M. H. Liu, Angew. Chem. Int. Ed.
We synthesized metal complex lipid 1, which produced
separated domains with their respective fluid phases in
DMPC membranes above the main transition temperature.
This is the first demonstration of fluid–fluid phase separation
of lipid membranes using a metal complex lipid. The phase
separations were retained up to 378C, which is a very high
temperature taking into account the main transition temper-
ature of DMPC (238C). The fluorescent probe was attached
with the metal complex cores via the click reaction. We
speculate that HCC-1 will also be applicable for constructing
artificially separated platforms, assembling other functional
molecules in lipid bilayers. This study provides new insights
into the design of molecular probes for the chemical control
of lipid distributions and assemblies in fluid membranes.
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Acknowledgements
This work was supported by JSPS Grant-in-Aid for Young
Scientists JP18K14245 and JSPS Grant-in-Aid for Scientific
Research on Innovative Areas, Mixed Anion JP19H04701.
This work was also supported by KAKENHI Grant-in-Aid
for Scientific Research (A) JP17H01200. This work was
partially supported by the Cooperative Research Program of
“Network Joint Research Centre for Materials and Devices”,
and Kyushu Synchrotron Light Research Center (SAGA-LS)
1807056F. This research was supported by Joint Research by
Institute for Molecular Science (IMS) (IMS program No.101,
102, and 20–101). We thank Prof. Hayato Ishikawa and Mr.
Kenta Rakumitsu (Kumamoto university) for the synthesis of
HCC-L1 and writing of the organic synthesis section.
Conflict of interest
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[17] Deposition Numbers 2033868 and 2033869 (for 1·3H2O and 2·
2MeOH) contain the supplementary crystallographic data for
this paper. These data are provided free of charge by the joint
Cambridge Crystallographic Data Centre and Fachinforma-
Manuscript received: February 24, 2021
Accepted manuscript online: March 15, 2021
Version of record online: && &&, &&&&
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Communications
Supramolecular Chemistry
Coassembly of metal complex lipids with
DMPC demonstrates a very rare fluid–
R. Ohtani,* Y. Anegawa, H. Watanabe,
Y. Tajima, M. Kinoshita,* N. Matsumori,
K. Kawano, S. Yanaka, K. Kato,
M. Nakamura, M. Ohba,
fluid phase separation up to 378C.
S. Hayami*
&&&& — &&&&
Metal Complex Lipids for Fluid–Fluid
Phase Separation in Coassembled
Phospholipid Membranes
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