Preparation and characterization of a novel organic-inorganic nanohybrid "cerasome" formed with a liposomal membrane and silicate surface

Article abstract of DOI:10.1002/chem.200700175

A novel class of organic-inorganic hybrids, the so-called cerasomes, which have a bilayer vesicular structure and a silicate surface, has been synthesized by combination of sol-gel reaction and self-assembly of organoalkoxysilanes with a molecular structure analogous to lipids. We have synthesized two cerasome-forming organoalkoxysilanes, N-[N-(3-triethoxysilyl)propylsuccinamoyl] dihexadecylamine (1) and N,N-dihexadecyl-;N"-[6-[(3-triethoxysilyl) propyldimethylammonio]hexanoyl]glycinamide bromide (2), and investigated the synthetic conditions of the cerasomes and their structural characteristics. For the proamphi philic 1, the cerasome was obtained under restricted pH conditions where acid-catalyzed hydrolysis of the triethoxysilyl moiety proceeded without disturbing the vesicle formation. In contrast, the amphiphilic 2, additionally having a hydrophilic quaternary ammonium group, formed stable dispersions of the cerasome in a wide pH range. The hydrolysis behavior of the triethoxysilyl groups was monitored by ¹H NMR spectroscopy. Morphology of the cerasomes having the liposomal vesicular structure was confirmed by TEM observations. Extent of the development of siloxane networks through condensation among the silanol groups on the cerasome surface was evaluated by using MALDI-TOF-MS spectrometry. Formation of oligomers of the cerasome-forming lipids in the vesicle was clearly confirmed. Due to the siloxane network formation, the cerasome showed remarkably high morphological stability compared with a reference liposome, as evaluated by surfactant dissolution measurements.

Full text of DOI:10.1002/chem.200700175

DOI: 10.1002/chem.200700175
Preparation and Characterization of a Novel Organic–Inorganic Nanohybrid
“Cerasome” Formed with a Liposomal Membrane and Silicate Surface
Kiyofumi Katagiri,*[a, b] Mineo Hashizume,[a] Katsuhiko Ariga,[a, c]
Takashi Terashima,[a] and Jun-ichi Kikuchi*[a]
Abstract: A novel class of organic–in-
organic hybrids, the so-called cera-
somes, which have a bilayer vesicular
structure and a silicate surface, has
been synthesized by combination of
sol–gel reaction and self-assembly of
organoalkoxysilanes with a molecular
structure analogous to lipids. We have
synthesized two cerasome-forming or-
ganoalkoxysilanes, N-[N-(3-triethoxysi-
lyl)propylsuccinamoyl]dihexadecyla-
mine (1) and N,N-dihexadecyl-Na-[6-
[(3-triethoxysilyl)propyldimethylammo-
nio]hexanoyl]glycinamide bromide (2),
and investigated the synthetic condi-
tions of the cerasomes and their struc-
tural characteristics. For the proamphi-
philic 1, the cerasome was obtained
under restricted pH conditions where
acid-catalyzed hydrolysis of the tri-
the cerasomes having the liposomal ve-
sicular structure was confirmed by
TEM observations. Extent of the devel-
opment of siloxane networks through
condensation among the silanol groups
on the cerasome surface was evaluated
by using MALDI-TOF-MS spectrome-
try. Formation of oligomers of the cera-
some-forming lipids in the vesicle was
clearly confirmed. Due to the siloxane
networkformation, the cerasome
showed remarkably high morphological
stability compared with a reference lip-
osome, as evaluated by surfactant dis-
solution measurements.
A
disturbing the vesicle formation. In
contrast, the amphiphilic 2, additionally
having a hydrophilic quaternary ammo-
nium group, formed stable dispersions
of the cerasome in a wide pH range.
The hydrolysis behavior of the tri-
A
1H NMR spectroscopy. Morphology of
Keywords: lipid bilayers · organic–
inorganic hybrid composites · self-
assembly · sol–gel processes
Introduction
due to their great potential for use in a wide variety of ap-
plications through fusion of individual organic and inorganic
properties.[1–4] Up to the present time, various kinds of mate-
rial combinations and synthetic strategies have been devel-
oped. The sol–gel method is one of the most powerful tech-
niques to prepare hybrid materials and has provided moder-
ate preparative conditions for construction of inorganic
oxide frameworks that are derived from hybrid materials.[5]
Especially preparation of novel materials with organized
nanostructures is a fascinating research subject in the field
of hybrid materials.[6–12] These approaches are generally
based on the sol–gel technique in the presence of molecular
assemblies as the templates, such as rod-like micelles,[13–15]
block-copolymers,[16–18] microemulsions,[19,20] organogels,[21]
cast films of bilayer membranes[22] and bilayer vesicles.[23]
Thus the properties of nanohybrids depend on their nano-
structures, especially the structure at the interface between
the inorganic and organic components. Pre-organization em-
ploying electrostatic and hydrogen-bonding interactions be-
tween an organic template and an inorganic precursor is re-
alized to be very important. However, the interface between
Organic–inorganic hybrid materials have attracted much at-
tention from material scientists and chemists in recent years,
[a] Dr. K. Katagiri, Dr. M. Hashizume, Dr. K. Ariga, T. Terashima,
Prof. J.-i. Kikuchi
Graduate School of Materials Science
Nara Institute of Science and Technology (NAIST)
8916-5 Takayama, Ikoma, Nara 630-0192 (Japan)
E-mail: katagiri@apchem.nagoya-u.ac.jp
jkikuchi@ms.naist.jp
[b] Dr. K. Katagiri
Present address:
Graduate School of Engineering
Nagoya University, Furo-cho, Chikusa-ku
Nagoya 464-8603 (Japan)
Fax : (+81)52-789-3201
[c] Dr. K. Ariga
Present address
Supermolecules Group
National Institute for Materials Science (NIMS)
1-1 Namiki, Tsukuba, Ibaraki 305-0044 (Japan)
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FULL PAPER
the organic and inorganic components in these hybrids
seems to be structurally ambiguous, and difficult to control
at the molecular level compared with the individual compo-
nent structures. In addition, most of these materials are fi-
nally composed of inorganic components alone and the or-
ganic parts are simply employed as templates. In contrast, a
novel class of layered organic–inorganic nanocomposites,
composed of amphiphilic molecules with a covalent bond
between the silicate and the surfactant, have been devel-
oped in recent years.[24–28] These materials would offer much
potential, since the hybrid precursors can form three-dimen-
sional networks during the self-assembling process whereby
inorganic layers and organic moieties are covalently linked
models but also for functional nanocapsules, their morpho-
logical instability is always one of the serious problems for
practical applications. The cerasome was expected to over-
come this problem by the existence of a silicate layer on its
surface. In addition, we can say that the cerasome is a novel
organic–inorganic nanohybrid having precisely designed
nanostructure. That is, the thickness of both organic and in-
organic layer of the cerasome is attributed to the molecular
structure of the cerasome-forming lipid, and the vesicular
size of the cerasome is basically controllable, by applying
conventional methodologies for preparing monodispersed
liposomes.
In this article, we report in detail the preparation and
characterization of cerasomes derived from the lipidic orga-
noalkoxysilanes 1 and 2. We investigated in participation of
the head group of the lipid molecules during the hydrolysis
in the cerasome formation, especially focusing on the corre-
lations between the sol–gel process and pH conditions. In
addition, characterization of the cerasomes was performed
by various physical measurements in order to clarify the re-
lationships between the molecular structure of the cera-
some-forming lipids and the resulting morphology and phys-
icochemical properties.
ꢀ
with stable Si C bonds.
We have recently developed a novel type of the organic–
inorganic nanohybrids “cerasome” in aqueous media
through a combination of sol–gel reaction and self-assem-
bling of lipidic organotrialkoxysilanes to form bilayer vesi-
cles covered with a silicate surface.[29a] We have first pre-
pared a proamphiphilic organoalkoxysilane (1) with a tri-
ethoxysilyl head moiety, a hydrophobic double-chain seg-
ment and a connector unit between them. Upon applying
the sol–gel technique to compound 1, the resulting amphi-
philes with a silanol head aggregated to form morphologi-
cally stable bilayer vesicles. The cerasome is composed of a
spherical lipid-bilayer membrane having an internal aqueous
compartment, like a so-called liposome,[30–35] and additional-
Results and Discussion
ly covered with
(Figure 1). We have also designed another cerasome-form-
a
silicate frameworkon its surface
Molecular design of the cerasome-forming lipid: The basic
idea for designing a bilayer-forming lipid can be referred to
the concept of the critical packing parameters for lipid as-
semblies.[36] In addition, we considered the importance of
the connector part,[37] which can form intermolecular hydro-
gen bonding to add morphological stability to the lipid as-
sembly, between the hydrophobic alkyl chains and the hy-
drophilic part in the lipid molecule. We previously revealed
the effect of the connector part by systematic investigations
of so-called peptide lipids, which have amino acid or oligo-
peptide moieties between the hydrophobic and the hydro-
philic part.[34] Our first design of the cerasome-forming lipid
tookinto account these points, and also the simplicity of
synthesis. In addition, the silanol group was capped by
ethoxy groups because of its instability under air. Lipidic or-
ganoalkoxysilane 1 can be synthesized by simple condensa-
tion reactions between the three molecular units, dihexade-
cyamine, succinic anhydride, and 3-aminopropyltriethoxylsi-
lane. After hydrolyzing the triethoxysiliyl group, 1 is expect-
ed to become suitable structure to form lipid bilayer. Anoth-
er lipid, that is, lipid 2 used the same frameworkof the
typical peptide lipid,[34] in which the dihexadecylamine unit
and quaternary ammonium group sandwiched glycine unit
and pentamethylene chain, and one of the methyl group of
the trimethylammonium group was replaced by 3-triethoxy-
propyl group. Contrary to lipid 1, lipid 2 can act as an am-
phiphilic molecule even if its silanol group is capped by
ethoxy groups. Lipid 2 was expected to show similar physical
parameters such as phase-transition temperature as those of
the corresponding peptide lipid.
Figure 1. Molecular structures of the cerasome-forming lipids (1 and 2)
and schematic drawing of the cerasome.
ing lipid (2) having a quaternary ammonium group at the
head moiety in addition to the triethoxysilyl part. While lip-
osomes are widely used as not only for biomembrane
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K. Katagiri et al.
pH Dependence for hydrolysis of the triethoxysilyl moiety:
As for the lipidic organoalkoxysilane 1, hydrolysis of the
triethoxysilyl head group converts the proamphiphile into
the corresponding amphiphilic lipid to form a self-assembly
of the liposome-like bilayer membrane. In addition, conden-
sation among the silanol groups on the relatively hydropho-
bic vesicular surface would proceed to develop a silica-like
inorganic framework, or a siloxane network. However, if the
hydrolysis and subsequent condensation occur much quicker
prior to the self-assembling, formation of the bilayer struc-
ture may be disturbed. Therefore, control of the reaction
rate in the sol–gel process, especially the hydrolysis rate,
seems to be important for the cerasome formation.
1 does not molecularly change to an amphiphilic form and
remains as oil droplets at neutral pH. It is probably due to
the extremely slow hydrolysis of the head group. On the
other hand, a translucent solution characteristic of a liposo-
mal dispersion is obtained under moderate acidic conditions
at pH 3 where hydrolysis of the ethoxysilyl groups is gently
preceded by acid catalysis. Under stronger acidic conditions
(pH 1), however, a stable dispersion is not obtained and pre-
cipitation is observed immediately. In this case, the hydroly-
sis and subsequent condensation reaction were so fast that a
vesicular structure could not be formed. At pH 12, the re-
sulting solution was translucent but oil droplets still re-
mained. Thus, the hydrolysis seemed to proceed heterogene-
ously under the basic conditions.
The different behavior observed under the present pH
conditions can be explained on the basis of the generally ac-
cepted mechanism for hydrolysis of alkoxysilane compounds
in the sol–gel process.[5] Under acidic conditions, hydrolysis
of the triethoxysilyl group proceeds equally for each of the
molecules in a one-by-one manner. This process would pro-
vide a suitable condition for the preparation of the bilayer
assembly from lipid 1. On the other hand, under basic con-
ditions, particular molecules are preferentially hydrolyzed
while the other molecules remain as unreacted species. This
process leads to heterogeneous hydrolysis and various reac-
tion stages of lipid 1 are observed. Thus, basic conditions
seem to be unsuitable for the preparation of cerasomes.
In the case of the p–A isotherms for lipid 2 (Figure 2b),
transition from an expanded phase to a condensed phase
was clearly observed and the collapsed pressure was suffi-
ciently high under all pH conditions examined. These results
are different from those of lipid 1, presumably reflecting
that lipid 2 bearing a quaternary ammonium group is capa-
ble of forming monolayer membrane regardless of the hy-
drolysis states of the triethoxysilyl moiety. As for the cera-
some derived from 2, homogeneous aqueous dispersions
were obtained under all pH conditions examined. The re-
sulting dispersions were so stable that the turbidity did not
change over 12 h. Thus monolayer properties of the cera-
some-forming lipids at the air–water interface are useful to
evaluate appropriate preparation conditions for the cera-
some formation in aqueous media.
At first, we investigated the monolayer property of lipid 1
and 2 on water at various pH conditions by measurements
of the surface pressure (p)–molecular area (A) isotherms
(Figure 2a). In the case of lipid 1, the p–A isotherm on pure
Figure 2. p–A Isotherms of a) 1 and b) 2 on aqueous H2SO4 (pH 2, c),
pure water (pH 6, a) and aqueous NH4OH (pH 12, g) at 208C.
water at pH 6 gave a presence of an expanded phase and
collapsed at low pressure (27 mNmꢀ1) before reaching a less
compressible region, similar to observations at pH 12. On
the other hand, in the case of the p–A isotherm at pH 2,
transition from an expanded phase to a condensed phase
was clearly observed and the collapsed pressure was found
to be much higher. The limiting area evaluated from the
latter isotherm was 0.46 nm2, indicating a well-packed state
of the dialkyl chains. These results imply that the acid-cata-
lyzed hydrolysis of the proamphiphilic compound 1 and fol-
lowed condensation proceeds to give a well-packed amphi-
philic monolayer around pH 2. Therefore, the acid catalysis
seems to be suitable for the conversion of compound 1 to
the corresponding amphiphilic form being capable of self-as-
sembly. Our previous results for the dispersion states of
lipid 1 after vortex mixing in aqueous media show that lipid
The hydrolysis behavior of lipids 1 and 2 was monitored
1
1
by H NMR spectroscopy. H NMR spectra of the aqueous
dispersion of 1 at pD 7 showed a broad quartet signal at d
3.75 ppm corresponding to the methylene protons in the
ethoxy group of the lipid ((CH3CH2O)3Si-). No signal as-
signed to hydrolyzed product was detected even after a 12 h
incubation period at 258C. On the other hand, at pD 3 the
broad signal for the methylene protons in the ethoxy group
of the lipid gradually decreased to be replaced by a sharp
quartet signal at d 3.65 ppm, which is assigned to the meth-
ylene protons of ethanol as a product of the hydrolysis reac-
tion. Time course for the hydrolysis of 1, as evaluated from
the peakarea changes in the methylene proton signal of the
produced ethanol, are shown in Figure 2. While the hydroly-
sis hardly proceeded at pD 7 (Figure 3a), slow hydrolysis
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Organic–Inorganic Nanohybrid “Cerasome”
FULL PAPER
Figure 3. Time courses for the cerasome formation process as evaluated
from the hydrolysis of the lipids (1.0 mmoldmꢀ3) in D2O at pD 3 and
~
*
*
258C. 1 at pD 7 ( ); 1 at pD 3 ( ); 2 at pD 3 ( ).
was observed at pD 3 in the initial stage of the reaction fol-
lowed by a gradual rate acceleration showing a sigmoidal
curve to complete after 8 h (Figure 3b). Such behavior is
due to a change in the reaction system from a heterogene-
ous to a homogeneous state with time, since lipid 1 is less
soluble in water and is initially present as oil droplets. On
the other hand, hydrolysis of lipid 2 proceeded rapidly to
complete within 2 h under the similar pD conditions (Fig-
ure 3c). The results imply that amphiphilic lipid 2 spontane-
ously forms bilayer vesicles prior to hydrolysis and that the
hydrolysis of the triethoxysilyl head efficiently occurs on the
vesicular surface.
Figure 4. TEM images of the cerasome of 2 prepared by a) vortex mixing
and followed by b) ultrasonication. The cerasome dispersions were pre-
pared in the presence of the negative staining agent.
Morphology of the cerasome: The aggregate structures of
the cerasomes were observed by transmission electron mi-
croscopy (TEM). The cerasomes were prepared in aqueous
HCl at pH 3.0 at a lipid concentration of 0.5 mmoldmꢀ3.
Formation of the multilamellar vesicles (MLVs) with a lipid
bilayer thickness of about 4 nm and a vesicular diameter of
200 nm in the slightly turbid aqueous dispersion of 1 under
vortex mixing was clearly confirmed by the TEM images,
similar as our previous report.[29a] Ultrasonication of the
aqueous dispersion with a probe-type sonicator for 20 min
at 30 W gave a clear solution with low-surface tension. The
electron micrographs showed the presence of cerasomes
with a diameter range from 150 to 300 nm, which sizes cor-
respond to the hydrodynamic diameter (214 nm) as evaluat-
ed by dynamic light scattering (DLS) measurements. Unlike
conventional liposomes, ultrasonication did not alter the
structure of the cerasome 1 from MLVs to single unilamellar
vesicles (SUVs).
The TEM images of the cerasome prepared from lipid 2
are shown in Figure 4. Cerasome 2 is directly prepared in
the presence of a staining agent. The vesicles in a diameter
range from 50 to 100 nm were observed for cerasome 2 pre-
pared by vortex mixing (Figure 4a). An internal view of the
MLVs with a bilayer thickness of about 5 nm was clearly
confirmed. Upon sonication of the dispersion sample with a
probe-type sonicator for 10 min at 30 W, smaller particles
with a diameter of 20–40 nm were observed (Figure 4b),
which suggests formation of SUVs of the cerasome. The ve-
sicular size was well consistent with the hydrodynamic diam-
eter (30 nm) as evaluated by DLS measurements.
The formation of siloxane bonds on the cerasome surface
was examined by FT-IR spectroscopy. Stretching bands as-
signed to the Si-O-Si and Si-OH groups were observed
around n˜ 1100 and 950 cmꢀ1, respectively.[38] The former
peakintensity was much weaker than the latter in cera-
somes both derived from 1 and 2 in the aqueous dispersion
state. Thus it is suggested that the cerasomes have a silica-
like surface with siloxane frameworks but the degree of
polymerization is not so high. The detectable species of the
lipid oligomers in cerasome 1 as evaluated by MALDI-
TOF-MS spectra were listed in Table 1. Trimethylsilylation
was performed for the aqueous dispersion samples of the ce-
rasome prepared after 15 min and 10 h. While the monomer,
dimer and trimer species were detected in the sample pre-
pared after 15 min, oligomers with higher molecular weight
such as tetramers and pentamers were additionally detected
for the sample during prolonged incubation. It seems that
the siloxane networkgrew with increasing incubation time.
From cryoscopic measurements, the number-average molec-
ular weight, Mn, was determined to be 1300 for the aqueous
dispersion of the cerasome 1 incubated for 10 h. This value
corresponds to the molecular weight of the dimer species.
On the other hand, the size of the cerasomes did not practi-
cally change after enough incubation time as confirmed by
TEM and DLS measurements. Accordingly, the siloxane
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K. Katagiri et al.
Table 1. Detectable species of lipid oligomers for the cerasome of 1 as
evaluated by MALDI-TOF-MS spectra.
equivalents of TX-100. Such surprising morphological stabil-
ity of cerasome 1 was also confirmed by the DLS measure-
ments. Morphological stability of the present cerasome
seems to be superior to that of an excellent example of the
polymerized liposomes recently developed by OꢁBrien
et al.[39] It is noteworthy that the resistance of cerasome 1
toward TX-100 was not sufficient immediately after prepara-
tion. Judging from the results in Table 1 and Figure 5, it is
clear that morphological stability of the cerasome comes
from development of the siloxane networkon the vesicular
surface. As for cerasomes prepared from lipid 2, the resist-
ance against TX-100 is comparable to that of the conven-
tional liposome even after prolonged incubation. It seems
that the quaternary ammonium group of lipid 2 strongly in-
teracts with oligo(ethyleneglycol) group of TX-100 to desta-
bilize the vesicular structure. It is supported by the fact we
found quite recently that cerasome 2 has a high morphologi-
cal stability against other kinds of surfactants, such as cetyl-
trimethylammonium bromide (CTAB), which completely
dissolved DMPC liposomes.[40] Accordingly, we can control
the morphological stability of the bilayer vesicles through
modification of molecular design of the cerasome-forming
lipids.
Oligomer
species
Calcd
Detected molecular weight[a]
Mw
Incubation
for 15 min
Incubation
for 10 h
monomer
dimer
trimer (cyclic)
trimer (linear)
tetramer
900.7
901.7
1640.4
ud
2380.3
ud
901.7
1640.4
ud
2380.3
2957.3
1641.0
2217.9
2380.3
2957.3
(cyclic or branched)
tetramer (linear)
pentamer
3119.6
3696.7
ud
ud
3117.4
3695.3
(cyclic or branched)
pentamer (linear)
3859.0
ud
ud
[a] ud: undetectable.
networkis not so highly developed on the cerasome surface.
These observations are also supported by the computer-
aided molecular model study since the length of the Si-O-Si
bond is much shorter than the diameter of the cross-section
of the dialkyl tail.
Surfactant dissolution of the cerasomes: Surfactant solubili-
sation is a useful method to evaluate morphological stability
of liposomes in aqueous media.[35c,39] For example, Regen
et al. reported that polymerized liposomes were morphologi-
cally highly stable against unpolymerised liposomes towards
lysing agents such as ethanol and surfactants.[35a,c] Figure 5
Phase-transition behavior of the cerasomes: Phase-transition
parameters (enthalpy change from gel to liquid-crystalline
state, DH, and temperature at the peakmaximum, Tm) for
the cerasomes were measured by DSC. The concentration of
the cerasome-forming lipid for the DSC measurements was
fixed to 2.0 mmoldmꢀ3. DH and Tm for the aqueous disper-
sion of the cerasome prepared from lipid
1 were
47.5 kJmolꢀ1 and 10.58C, respectively. Upon ultrasonication
of cerasomes with a probe-type sonicator for 10 min at
30 W, the DH value decreased to 11.5 kJmolꢀ1 whereas the
Tm value did not change. For cerasome 2 in the aqueous dis-
persion state, the DH and Tm values were 33.3 kJmolꢀ1 and
25.78C, respectively. These phase-transition parameters
were comparable to those for peptide lipids previously re-
ported.[34] Upon ultrasonication of cerasome 2 with a probe-
type sonicator for 10 min at 30 W, the endothermic peakfor
phase transition apparently disappeared. We have previously
clarified that the transformation of MLV to the correspond-
ing single unilamellar vesicle is reflected in the decrease of
both the DH and Tm values.[34] In addition, DH is more sen-
sitive than Tm to such morphological changes. Since it is well
known that MLVs formed with the conventional liposomes
generally transform to SUVs under the ultrasonication con-
ditions employed in this study, cerasome 1 is more tolerant
towards morphological changes than the liposome-forming
lipids. Formation of the siloxane networkon the vesicular
surface would prevent such morphological transformations.
Figure 5. Light scattering intensities of the cerasomes prepared from 1 or
2, and DMPC-liposome as a function of added equivalents of TX-100 at
*
258C: cerasome of 1 incubated for 24 h ( ); cerasome as-prepared from
~
!
*
1 ( ); cerasome of 2 incubated for 24 h ( ); DMPC-liposome ( ).
shows the resistance of cerasomes against a nonionic surfac-
tant TX-100 as determined by the light scattering intensity
of the vesicles. Liposomal membrane formed with dimyris-
toylphosphatidylcholine (DMPC) was used as a reference.
When three equivalents of TX-100 were added to the
DMPC liposome, the light scattering intensity was drastical-
ly decreased indicating a collapse of the vesicle. In contrast
to the DMPC liposome, the cerasome prepared from lipid 1
exhibited remarkable morphological resistance toward TX-
100. The light scattering intensity of the cerasome incubated
for 24 h did not changed at all even in the presence of 36
Zeta-potential of the cerasomes: The pH dependences of
surface charges of cerasomes 1 and 2 were evaluated by
zeta-potential measurements (Figure 6). The cerasomes
were prepared in aqueous NaCl (10 mmoldmꢀ3) at pH 3.0
with a lipid concentration of 1.0 mmoldmꢀ3 and followed
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Organic–Inorganic Nanohybrid “Cerasome”
FULL PAPER
ultrasonication with a probe-type sonicator for 10 min at
30 W. The zeta-potential of cerasome 1 changed from +10
to ꢀ70 mV depending on the medium pH (Figure 6a). The
isoelectric point (IEP) of the cerasomes was found to be 4.3.
Cerasome 1 had large negative charges under neutral and
basic conditions, reflecting deprotonation of the silanol
groups on the cerasome surface. It is well known that the
IEP values for the typical silica particles derived from the
sol–gel method lie in the region of 2–3 and have a zeta-po-
tential ranging from +20 to ꢀ80 mV in the analogous pH
region.[41,42] The results imply that the surface electrical state
of cerasome 1 resembles that of the silica particles. In addi-
tion, the IEP value for the cerasome was somewhat larger
than those for the silica particles, presumably due to the
electron-donating character of the alkyl group bound to the
silicon atom in the former. On the other hand, the IEP
value shifted to 12.0 for the cerasome prepared from lipid 2
(Figure 6b). In a pH range lower than 12.0, the zeta-poten-
tial of the cerasome increased with a decrease in pH to
reach +70 mV at pH 6. This value was considerably higher
than the maximal zeta-potential of cerasome 1. Such differ-
ence is attributable to the existence of a quaternary ammo-
nium group in lipid 2. At neutral pH, cerasome 1 acts as a
polyanionic vesicular particle, whereas cerasome 2 is poly-
cationic. Thus we can control the IEP value of the cerasome
to a desired value between 4 and 12 by mixing lipids 1 and 2
in an appropriate ratio.
Figure 7. pH Dependence of hydrodynamic diameter for the cerasomes
of 1 prepared upon sonication of the aqueous dispersion at pH 3. The
measurements were done immediately after pH adjustment.
around the IEP value, however, the hydrodynamic diameter
was increased drastically to form the low dispersive vesicu-
lar aggregates due to the loss of charges on the cerasome
surface.
Conclusion
In this article, we have demonstrated that novel organic–in-
organic hybrid materials, the so-called cerasomes, which
consist of a liposomal bilayer structure and a silicate frame-
workon their surfaces, and can be prepared by applying the
sol–gel method for two lipidic organoalkoxysilanes. Charac-
teristic vesicle parameters for the cerasomes are listed in
Table 2. The cerasome formation is dependent on the molec-
Table 2. Characteristic vesicle parameters for the cerasomes prepared
from the lipids 1 and 2
Parameter entity
Cerasome of Cerasome of
1
2
available pH region for preparation
reaction time for hydrolysis at pH 3 h
phase transition temperature[a]/8C
around 3
10
10.5
2–12
2
25.7
33.3
phase transition enthalpy change[a]
kJmolꢀ1
/
47.5
hydrodynamic diameter[b]/nm
isoelectric point[b]
214
4.3
30
12.0
[a] Aqueous dispersion state. [b] Ultrasonicated sample with a probe-
type sonicator for 10 min at 30 W.
Figure 6. pH Dependence of zeta-potential for the cerasomes: cerasome
*
*
of 1 ( ); cerasome of 2 ( ).
ular structure of the organoalkoxysilanes, in particular on
the head group of the lipids. For a proamphiphilic 1 the
preparation of the cerasome was limited to moderate acidic
conditions. In contrast, the cerasome was readily prepared
over a wide pH range for amphiphilic 2 having a quaternary
ammonium moiety, since the hydrolysis and self-assembling
processes proceeded independently. The results indicate that
structural differences in the head moiety of the lipids have a
significant influence on the cerasome-formation process,
particularly with regards to the hydrolysis step. The vesicu-
lar form of the cerasome was observed by TEM images. The
cerasomes prepared by vortex mixing of the aqueous disper-
The hydrodynamic diameters for the cerasome 1 at vari-
ous pH values were evaluated by DLS measurements
(Figure 7). The cerasome was prepared in aqueous HCl at
pH 3.0 with a lipid concentration of 1.0 mmoldmꢀ3 and fol-
lowed ultrasonication with a probe-type sonicator for 10 min
at 30 W. The hydrodynamic diameter and polydispersity
index of the cerasome at pH 3 was 214 nm and 0.155, re-
spectively. The hydrodynamic diameter did not change in
the regions below pH 4 and above pH 6. In a pH region
Chem. Eur. J. 2007, 13, 5272 – 5281
ꢀ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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5277

K. Katagiri et al.
drying overnight with DRIERITE. 1H and 13C NMR Spectra were re-
corded on a JEOL JNM-LA400 or JNM-EX270 spectrometer in CDCl3
with tetramethylsilane (TMS) as an internal reference. Melting points
were recorded on a Yanaco MP-500D melting point apparatus (hot-plate
type) with a filter for polarized light. Column chromatography was per-
formed using silica gel (Wako gel C-300, Wako Pure Chemical Industries,
Ltd.); thin-layer chromatography on Wako silica gel 70FM plates.
sion have a multi-walled bilayer structure and, as for cera-
some 2, can be further transformed into a single-walled vesi-
cle upon ultrasonication. Formation of the lipid oligomers in
the cerasome was confirmed by FT-IR spectroscopy and
MALDI-TOF-MS spectrometry. Morphological stability of
the cerasome is much superior than that of conventional lip-
osomes; this mainly results from the siloxane network
formed on the vesicular surface. DSC measurements re-
vealed that the cerasomes possess phase transitional behav-
ior from gel to liquid-crystalline states, in analogous to the
liposomes. This means that the cerasome is a unique nano-
hybrid which has both highly stable entire structure and
mobile inside nanostructure. In addition, the zeta-potential
and isoelectric point of the cerasome can be controlled
easily by changing the molecular design of the lipids. We be-
lieve that the cerasome can
Synthesis of the cerasome-forming organotriethoxysilanes: Synthetic
routes for lipids 1 and 2 are shown in Schemes 1 and 2, respectively. The
route for 1 was referred to that for the lipid having analogous frame-
work.[43]
N,N-Dihexadecylsuccinamic acid (3): Dihexadecylamine (3.00 g,
6.44 mmol) and succinic anhydride (1.29 g, 12.9 mmol) were added to dry
THF (50 mL) and dissolved upon heating. The solution was stirred for
24 h at room temperature. The solvent was evaporated in vacuo and the
crude product was dissolved in dichloromethane (50 mL). The solution
was then washed with 10% aqueous citric acid and saturated aqueous
open a new field in constructing
organic–inorganic nanohybrid
materials. On the basis of these
findings, optimization of molec-
ular design for a super-stable
cerasome with a well-developed
silicate surface and its function-
alisation are in progress in our
laboratory.
Experimental Section
Scheme 1.
Materials: Unless otherwise stated all
reagents and chemicals were obtained
commercially and used without further
purification. Succinic anhydride, N,N’-
dicyclohexylcabodiimide (DCC) and
50% dimethylamine solution were ob-
tained from Wako Pure Chemical In-
dustries, Ltd. 3-Aminopropyltriethoxy-
silane,
3-bromopropyltriethoxysilane
and trimethylchlorosilane were pur-
chased from Shin-Etsu Chemical Co.,
Ltd.
N-(tert-Butoxycarbonyl)glycine
and 6-bromohexanoyl chloride were
obtained from Peptide Institute, Inc.,
and Aldrich Chemical Company, Inc.,
respectively. Dihexadecylamine were
prepared by reaction of hexadecyla-
mine with 1-bromohexadecane in the
presence of sodium carbonate and pu-
rified by recrystallisation from ethanol.
For the cerasome preparation, distilled
and deionised water was used and pre-
pared using an Autostill WS33
(Yamato Scientific) and Milli-Q Labo
(Nihon Millipore), respectively.
Dry solvents for syntheses were puri-
fied as follows: Dichloromethane was
distilled on calcium hydride. Tetrahy-
drofuran (THF) was distilled after re-
fluxing with sodium and benzophe-
none. N,N-Dimethylformamide (DMF)
was distilled at 50–608C under a re-
duced pressure of 20–30 mmHg after
Scheme 2.
5278
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ꢀ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2007, 13, 5272 – 5281

Organic–Inorganic Nanohybrid “Cerasome”
FULL PAPER
sodium chloride in this sequence. After removing residual water using
phase separation filter paper, the solvent was evaporated in vacuo. Sub-
sequent recrystallization from acetonitrile gave a white solid (2.87 g,
78.8%). M.p. 58.1–58.48C; TLC: Rf =0.50 (hexane/ethyl acetate/acetic
acid 90:30:1); 1H NMR (400 MHz, CDCl3, 258C, TMS): d=0.88 (t, J=
N,N-Dihexadecyl-Na-(6-bromohexanoyl)glycinamide (6): Triethylamine
(2.86 g, 28.3 mmol) and 5 (3.00 g, 4.71 mmol) were dissolved in dry di-
chloromethane (30 mL) and the solution was cooled to 08C. 6-Bromo-
hexanoyl chloride (2.01 g, 9.42 mmol) dissolved in dry dichloromethane
(20 mL) was added dropwise to the solution at 08C with stirring. The
mixture was stirred for 1 h at 08C and then for 16 h at room temperature.
Then the solution was washed with 10% aqueous citric acid, saturated
aqueous sodium chloride, 4% aqueous sodium hydrogen carbonate and
saturated aqueous sodium chloride in this sequence. After residual water
was removed using phase separation filter paper, the solvent was evapo-
rated in vacuo to give a solid, which was subsequently purified on a
column of silica gel with ethyl acetate/hexane 1:1. Recrystallization from
methanol gave a white solid (2.77 g; 83.9%). M.p. 51.0–51.78C; TLC:
Rf =0.78 (ethyl acetate/hexane 1:1); 1H NMR (400 MHz, CDCl3, 258C,
6.6 Hz, 6H, NCH2CH2
(CH2)13CH3), 1.54 (m, 4H, NCH2CH2
(CH2)2CON), 3.15 (t, 2H, J=7.8 Hz, NCH2CH2
2H, J=7.8 Hz, NCH2CH2(CH2)13CH3).
ACHTREUNG
A
ACHTREUNG
N
ACHTREUNG
AHCTREUNG
N-[N-(3-Triethoxysilyl)propylsuccinamoyl]dihexadecylamine (1): DCC
(1.01 g, 4.90 mmol) was added with stirring at 08C to a solution of 3
(2.40 g, 4.24 mmol) in dry dichloromethane (50 mL). After 15 min of stir-
ring, 3-aminopropyltriethoxysilane (1.22 g, 5.51 mmol) was added to the
solution and the mixture was stirred for 4 h at 08C and subsequently for
a further 12 h at room temperature. Precipitates (N,N’-dicyclohexylurea)
were removed by filtration. The solvent was evaporated in vacuo and re-
sidual oil was dissolved in ethyl acetate (50 mL). The solution was cooled
overnight at 48C and the further precipitated N,N’-dicyclohexylurea was
removed by filtration. The mixture was evaporated in vacuo and the
crude product was purified by column chromatography. Impurities were
eluted with ethyl acetate/chloroform 1:9; subsequent elution with 100%
ethyl acetate gave a colorless oil (1.70 g, 52.1%). TLC: Rf =0.28 (chloro-
form/ethyl acetate 20:1); 1H NMR (400 MHz, CDCl3, 258C, TMS): d=
0.62 (t, J=8.4 Hz, 2H, SiCH2), 0.88 (t, J=6.6 Hz, 6H, NCH2CH2-
TMS): d = 0.88 (t, J=6.8 Hz, 6H, (NCH2CH2
ACHTREUNG
52H, NCH2CH2(CH2)13CH3), 1.46–1.60 (m, 6H, NCH2CH2
A
ACHTREUNG
BrCH2CH2CH2CH2CH2CO), 1.69 (m, 2H, BrCH2CH2CH2CH2CH2CO),
1.88 (m, 2H, BrCH2CH2CH2CH2CH2CO), 2.26 (t, J=7.3 Hz, 2H,
BrCH2CH2CH2CH2CH2CO), 3.15 (t, J=7.8 Hz, 2H, NCH2CH2-
A
N
6.8 Hz, 2H, BrCH2CH2), 4.02 (d, J=5.9 Hz, 2H, NHCH2CO), 6.63 ppm
(br, 1H, (CH3)3CONH).
N,N-Dihexadecyl-Na-[6-(dimethylamino)hexanoyl]glycinamide (7): Dry
dimethylamine gas was introduced into dry THF (100 mL) to saturate. To
the dimethylamine saturated THF solution, 6 (1.40 g, 2.00 mmol) was
added and the mixture was subsequently stirred at room temperature for
46 h. Then air was bubbled into the mixture in order to remove excess di-
methylamine. The solvent was evaporated in vacuo and the residual solid
was dissolved in chloroform (50 mL). The solution was then washed with
saturated aqueous sodium chloride, 4% aqueous sodium hydrogen car-
bonate and saturated aqueous sodium chloride in this sequence. After re-
moving residual water using phase separation filter paper, the solvent
was evaporated in vacuo. The residue was purified by recrystallization
from acetonitrile to give a white solid (1.14 g, 85.7%). M.p. 37.5–38.18C;
TLC: Rf =0.45 (chloroform/methanol 4:1); 1H NMR (400 MHz, CDCl3,
ACHTREUNG
A
ACHTREUNG
AHCTREUNG
=
171.39, 58.45, 48.03, 46.33, 42.00, 31.97, 31.89, 29.75, 29.71, 29.68, 29.62,
29.49, 29.41, 28.99, 27.85, 27.15, 27.01, 22.96, 22.73, 18.32, 14.14,
7.76 ppm; HRMS (FAB+): m/z: calcd for C45H93N2O5Si: 769.6854; found:
769.6857 [M+H]+; elemental analysis calcd (%) for C45H92N2O5Si: C
70.31, H 12.05, N 3.65; found: C 70.26, H 12.05, N 3.64.
258C, TMS): d = 0.88 (t, J=6.8 Hz, 6H, NCH2CH2
1.29 (m, 54H, NCH2CH2(CH2)13CH3, (CH3)2NCH2CH2CH2CH2), 1.44–
1.59 (m, 6H, NCH2CH2(CH2)13CH3, (CH3)2NCH2CH2CH2CH2CH2CO),
A
N,N-Dihexadecyl-Na-(tert-butoxycarbonyl)glycinamide (4): DCC (5.98 g,
29.0 mmol) was added with stirring at 08C to a solution of N-(tert-butoxy-
carbonyl)glycine (4.40 g, 25.2 mmol) in dry dichloromethane (160 mL).
After 15 min, N,N-dihexadecylamine (11.7 g, 25.2 mmol) was added to
the solution and the mixture was stirred for 4 h at 08C and for a further
12 h at room temperature. Precipitates (N,N’-dicyclohexylurea) were re-
moved by filtration. The solvent was evaporated in vacuo and the residu-
al oil was dissolved in ethyl acetate (160 mL). The solution was cooled
overnight at 48C and the further precipitated N,N’-dicyclohexylurea was
removed by filtration. The solution was then washed with 10% aqueous
citric acid, saturated aqueous sodium chloride, 4% aqueous sodium hy-
drogen carbonate and saturated aqueous sodium chloride in this se-
quence. After removing residual water by using phase separation filter
paper, the solvent was evaporated in vacuo. The residue was purified on
a column of silica gel with ethyl acetate/chloroform 1:9 to give a colorless
oil (9.70 g, 61.8%). TLC: Rf =0.60 (hexane/ethyl acetate 3:1); 1H NMR
(400 MHz, CDCl3, 258C, TMS): d = 0.88 (t, J=6.8 Hz, 6H, NCH2CH2-
AHCTREUNG
AHCTREUNG
1.63–1.69 (m, 2H, (CH3)2NCH2CH2CH2CH2CH2CO), 2.20 (s, 6H,
(CH3)2NCH2CH2), 2.23–2.27 (m, 4H, (CH3)2NCH2CH2CH2CH2CH2CO),
3.15 (t, J=7.8 Hz, 2H, NCH2CH2
A
NCH2CH2(CH2)13CH3), 4.03 (d, J=5.9 Hz, 2H, NHCH2CO), 6.64 ppm
AHCTREUNG
(br, 1H, (CH3)3CONH); elemental analysis calcd (%) for C42H85N3O2: C
75.95, H 12.90, N 6.33; found: C 75.67, H 12.80, N 6.30.
N,N-Dihexadecyl-Na-[6-[(3-triethoxysilyl)propyldimethylammonio]hexa-
noyl]glycinamide bromide (2): Compound 7 (0.300 g, 0.451 mmol) was
added under a nitrogen atmosphere to a solution of 3-bromopropyltrie-
thoxysilane (0.645 g, 3.61 mmol) in dry DMF (25 mL), and the mixture
was stirred for 120 h. The solvent was evaporated in vacuo and the resi-
due was purified by gel filtration chromatography (Sephadex LH-20, eth-
anol as the eluent) to afford a colorless viscous oil (0.417 g, 97.7%).
TLC: Rf =0.49 (chloroform/ethanol 1:1); 1H NMR (400 MHz, CDCl3,
258C, TMS): d=0.66 (t, J=7.8 Hz, 2H, SiCH2), 0.88 (t, J=6.6 Hz, 6H,
A
N
NCH2CH2
CH2CH2CH2CH2CH2CO), 1.22 (t, J=7.0 Hz, 9H, CH3CH2OSi), 1.52–1.98
(m, 10H, NCH2CH2
(CH2)13CH3, CH2CH2CH2N+CH2CH2CH2CH2CH2-
CO), 2.33 (t, J=7.0 Hz, 2H, N+CH2CH2CH2
CH2CH2CO), 3.16 (t, J=
7.8 Hz, 2H, NCH2CH2(CH2)13CH3), 3.27–3.33 (m, 2H, NCH2CH2-
(CH2)13CH3,), 3.34 (s, 6H, CH2N+
(CH3)2CH2), 3.46–3.56 (m, 4H,
A
G
AHCTREUNG
A
ACHTREUNG
AHCTREUNG
3.94 (d, J=5.9 Hz, 2H, NHCH2CO), 5.56 ppm (br, 1H, (CH3)3CONH).
A
ACHTREUNG
N,N-Dihexadecylglycinamide trifluoroacetic acid salt (5): Trifluoroacetic
acid (10.0 g, 87.7 mmol) was added to 4 (6.50 g, 10.4 mmol) and the mix-
ture was stirred for 4 h at room temperature. Evaporation of an excess
amount of trifluoroacetic acid in vacuo below 408C gave colorless oil.
The crude product was purified by recrystallization from acetonitrile to
AHCTREUNG
A
ACHTREUNG
CH2CH2N+CH2CH2), 3.83 (q, J=7.0 Hz, 6H, CH3CH2OSi), 4.01 (d, J=
6.2 Hz, 2H, NHCH2CO), 6.95 ppm (br, 1H,CH2CONHCH2); 13C NMR
(100 MHz, CDCl3, 258C, TMS): d = 172.73, 167.62, 70.69, 65.87, 64.20,
58.77, 51.34, 47.13, 46.33, 41.06, 35.66,35.37, 31.98, 29.75, 29.42, 28.81,
27.71, 27.12, 26.98, 25.47, 24.67, 22.74, 22.28, 19.42, 18.36, 16.73, 14.15,
13.96, 6.91 ppm. HRMS (FAB+): m/z: calcd for C51H106N3O5Si: 868.7901;
found: 868.7606 [MꢀBr]+; elemental analysis calcd (%) for
C51H106N BrO Si·1= H O: C 63.91, H 11.25, N 4.38; found: C 63.98, H
give
(400 MHz, CDCl3, 258C, TMS): d=0.88 (t, J=6.8 Hz, 6H, NCH2CH2-
(CH2)13CH3), 1.24–1.29 (m, 52H, NCH2CH2(CH2)13CH3), 1.53 (m, 4H,
NCH2CH2(CH2)13CH3), 3.15 (t, J=7.8 Hz, 2H, NCH2CH2(CH2)13CH3),
(CH2)13CH3), 3.94 ppm (d, J=5.9 Hz,
a
white powder (6.01 g, 90.8%). M.p. 50.2–50.78C; 1H NMR
A
ACHTREUNG
A
ACHTREUNG
3.30 (t, J=7.8 Hz, 2H, NCH2CH2
2H, NHCH2CO).
ACHTREUNG
3
5
2
2
11.29, N 4.40.
Chem. Eur. J. 2007, 13, 5272 – 5281
ꢀ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
5279

K. Katagiri et al.
p–A Isotherm measurements: Surface pressure–molecular area (p–A)
isotherms were measured using a computer controlled FSD-300 film bal-
ance system (USI System). Spectral grade benzene was used as the
spreading solvent. The starting trough area was 150”463 mm2 and
around 100 mL of the lipid solution was spread. Compression at a rate of
0.2 mmsꢀ1 started about 10 min after spreading. The subphase tempera-
ture was kept at 20.0ꢁ0.28C. The surface pressure was measured with a
Wilhelmy plate, which had been calibrated using the transition pressure
of an octadecanoic acid monolayer. The pH conditions of the subphase
were controlled using H2SO4 and NH4OH.
with an acceleration voltage of 300 kV. The images were obtained as
zero-loss image through an in-column energy filter.
Surfactant dissolution of the cerasomes: The cerasomes were prepared
by sonication of the dispersion sample using a probe-type sonicator for
20 min at 30 W and mixing in aqueous HCl (pH 3). For comparison con-
ventional liposomes, derived from dimyristoylphosphatidylcholine
(DMPC), were prepared using similar sonication conditions in pure
water. The cerasomes and the DMPC liposomes obtained were character-
ized by light scattering intensity employing a fluorescence spectropho-
tometer (F-4500, HITACHI) for a 2 mL sample with a lipid concentra-
tion of 0.10 mmoldmꢀ3. The light scattering intensities were determined
at a 908 angle. Aliquots of 50 mm Triton X-100 (TX-100) solution were
added until the liposomes dissolved or 36 equivalents for the lipid.
Preparation of the cerasomes: In the sol–gel process, a mutual solvent
such as alcohol (for example, ethanol) is usually added to give a homo-
genized solution. In the present case, however, a mutual solvent should
not be used as it would prevent disruption of the bilayer structure. Thus,
the cerasomes were prepared using the following procedure, unless other-
wise stated: 1–10 mg of 1 or 2 was suspended in 2–5 mL of H2O under
various pH conditions and dispersed by shaking with a vortex mixer until
the oil droplets disappeared completely to give a translucent solution.
Differential scanning calorimetry (DSC): The phase-transition tempera-
ture (Tm, temperature at a peakmaximum of the DSC thermogram) of
the cerasomes was measured with a differential scanning calorimeter
(DSC-6100, Seiko Instruments). The aqueous dispersion (2 mmoldmꢀ3
)
was weighed and sealed in a silver capsule. The enthalpy change for the
phase transition (DH) was determined by measuring the peakarea of the
DSC thermogram.
The concentration of the lipid was controlled in
a range of 1.0–
5.0 mmoldmꢀ3. In some cases, the aqueous dispersion was ultrasonicated
with a probe-type sonicator (Sonifier 250D, BRANSON).
Zeta-potential measurements: Zeta-potentials of the cerasomes were
evaluated by using an instrument for electrophoretic light scattering with
a laser Doppler system (ELS-6000, Otsuka Electronics). For the zeta-po-
tential measurements, the cerasome was prepared in aqueous NaCl
(10 mmoldmꢀ3) upon sonication of the dispersion sample with a probe-
type sonicator for 20 min at 30 W. The pH of the solution was adjusted
by addition of HCl or NaOH.
1
1H NMR spectroscopy: H NMR spectra of the lipids dispersed in a deu-
terated solvent were measured by a JEOL JNM-LA400 NMR or JNM-
EX270 spectrometer (400 and 270 MHz, respectively); sodium 3-(trime-
thylsilyl)propionate in D2O was used as an internal reference. The pD
value was adjusted by using aqueous deuterium chloride.
Mass spectrometry: For MS data of the cerasomes, free silanol and unhy-
drolyzed alkoxysilyl groups on the surface should be end-blocked by tri-
methylsilyl groups to avoid changes in the polymerization state during
the measurements. Trimethylchlorosilane ((CH3)3SiCl) was employed for
trimethylsilylation of the cerasome. Silanol and alkoxysilyl groups were
capped with (CH3)3SiOH, generated by hydrolysis of (CH3)3SiCl. It is
known that the original condensed form of silicates is maintained at 80–
90% by this reaction.[44] All MS spectra were taken by using a Voyager
DE-STR matrix-assisted laser desorption ionization time-of-flight
(MALDI-TOF) mass spectrometer (PerSeptive Biosystems) operated at
25 kV accelerating voltage in linear mode with positive ionization. Di-
thranol was used as a matrix material.
Dynamic light scattering (DLS): The hydrodynamic diameter and its
polydispersity index of the cerasomes were measured by a dynamic light
scattering spectrophotometer (DLS-6000HL, Otsuka Electronics). The
instrument consisted of a He/Ne laser, which was operated at 633 nm and
10 mW. The data obtained was analyzed using the cumulant method.
Acknowledgements
Cryoscopic measurements: Cryoscopic determinations of the molecular
weights of the cerasomes were carried out using trimethylsilylated sam-
ples in spectral grade benzene. The difference between the freezing point
of the solution containing the trimethylsilylated sample and that of ben-
zene itself, the freezing point depression, DTf, was measured. The
number-average molecular weight, Mn, was calculated from the following
relationship, where g is the weight of the sample and G is the weight of
benzene:
This workwas supported by the Japan Society for the Promotion of Sci-
ence, Grant-in-Aid for Scientific Research. The authors deeply thankMr.
K. Furusho, Graduate School of Materials Science, NAIST, for TEM
measurements.
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Kf  1000  g
Mn
¼
ð1Þ
ðG Â DTfÞ
The constant, Kf, of the benzene was determined with benzophenone as a
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were carried out for a series of solutions at various concentrations in 1.0–
5.0% (w/w). The number-average molecular weight of the sample was es-
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Transmission electron microscopy (TEM): The aggregate structure of the
cerasomes prepared from 1 and 2 were examined by TEM. In the case of
cerasome 1, the cerasome (0.5 mm) was prepared in aqueous HCl (pH 3)
by vortex mixing and it was mixed with equal volume of 4 wt% of uranyl
acetate and left for min. In the case of cerasome 2, the cerasome (2.5 or
5 mm) was prepared directly in 2wt% uranyl acetate solution by vortex
mixing for 3 min. In some cases, the cerasome dispersions were ultrasoni-
cated. An aliquot of these dispersions was cast on an ultrathin carbon-de-
posited Cu grid (Cu200, JEOL DATUM Ltd) and dried in a desiccator
overnight followed by in vacuo for 1 h. TEM observations were carried
out by using a JEOL JEM-3100FEF field emission electron microscope
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Chem. Eur. J. 2007, 13, 5272 – 5281

Organic–Inorganic Nanohybrid “Cerasome”
FULL PAPER
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Received: February 1, 2007
Published online: April 3, 2007
Chem. Eur. J. 2007, 13, 5272 – 5281
ꢀ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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