langmur monolayers of cerebroside with different head groups originated from sea cucumber: binary...
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Colloids and Surfaces B: Biointerfaces 72 (2009) 272–283
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Colloids and Surfaces B: Biointerfaces
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angmur monolayers of cerebroside with different head groups originated fromea cucumber: Binary systems with dipalmitoylphosphatidylcholine (DPPC)
uriko Ikedaa, Masanori Inagakib,c, Koji Yamadad, Tomofumi Miyamotob,yuichi Higuchib, Osamu Shibataa,∗
Department of Biophysical Chemistry, Faculty of Pharmaceutical Sciences, Nagasaki International University, 2825-7 Huis Ten Bosch, Sasebo, Nagasaki 859-3298, JapanDivision of Natural Products Chemistry, Graduate School of Pharmaceutical Sciences, Kyushu University, 3–1–1 Maidashi, Higashi-ku, Fukuoka 812-8582, JapanFaculty of Pharmaceutical Sciences, Yasuda Women’s University, 6-13-1 Asaminami-ku, Hiroshima 731-0153, JapanLaboratory in Medical Plants Garden, Graduate School of Biomedical Sciences, Nagasaki University, 1-14 Bunkyomachi, Nagasaki 852-8521, Japan
r t i c l e i n f o
rticle history:eceived 22 February 2009ccepted 19 April 2009vailable online 24 April 2009
eywords:angmuir monolayererebrosidePPCrewster angle microscopy (BAM)luorescence microscopy (FM)tomic force microscopy (AFM)
a b s t r a c t
Surface properties (Langmuir monolayer) of two different cerebrosides which are extracted from the seacucumber (Bohadschia argus) were investigated. A main difference in chemical structure of cerebrosidebetween BAC-2a and BAC-4 is their head groups (glucose and galactose, respectively). Furthermore, mis-cibility and interaction between dipalmitoylphosphatidylcholine (DPPC) and cerebrosides (BAC-2a andBAC-4) in the monolayer have been systematically examined. The surface pressure (�)−area (A), the sur-face potential (�V)−A, and the dipole moment (�⊥)−A isotherms for monolayers of DPPC, cerebrosides,and their binary combinations have been measured using the Wilhelmy method and the ionizing elec-trode method. BAC-4 forms a stable liquid-expanded (LE) monolayer, whereas BAC-2a has a first-orderphase transition from the LE phase to the liquid-condensed (LC) state on 0.15 M NaCl at 298.2 K. The funda-mental properties for each cerebroside monolayer were elucidated in terms of the surface dipole momentbased on the three-layer model [R.J. Demchak, T. Fort Jr., J. Colloid Interface Sci. 46 (1974) 191–202] forboth cerebrosides and the apparent molar quantity change (�s� , �h� , and �u� ) for BAC-2a. In addition,their miscibility with DPPC was examined by the variation of the molecular areas and the surface poten-tials as a function of cerebroside mole fractions, the additivity rule. The miscibility was also confirmedby constructing the two-dimensional phase diagrams. The phase diagrams for the both binary systems
were of negative azeotropic type. That is, the two-component DPPC/BAC-2a and DPPC/BAC-4 monolayersare miscible. Furthermore, the Joos equation for the analysis of the collapse pressure of binary monolay-ers allowed calculation of the interaction parameter and the interaction energy between the DPPC andcerebroside monolayers. The miscibility in the monolayer state was also confirmed by the morphologi-cal observation with Brewster angle microscopy (BAM), fluorescence microscopy (FM), and atomic forcemicroscopy (AFM).. Introduction
One of the most intriguing questions about cell membraness why these supramolecular emsemble contain so many differ-nt types and species of lipids. After all, the chief purpose of
embranes is to compartmentalize cells while serving as selectiveermeability barriers in the export and import of various cellularaterials, and then only a handful of lipids should be necessary.lycosphingolipids (GSLs) are the one of important components of
∗ Corresponding author.E-mail addresses: [email protected], [email protected] (O. Shibata).URL: http://www.niu.ac.jp/∼;pharm1/lab/physchem/indexenglish.html
O. Shibata).
927-7765/$ – see front matter © 2009 Elsevier B.V. All rights reserved.oi:10.1016/j.colsurfb.2009.04.015
© 2009 Elsevier B.V. All rights reserved.
membrane constituents. They are primarily located on the outerleaflet of the cellular membranes, especially animal cell plasmamembranes, where they participate in the assembly of signalingmolecules, modulation of cell adhesion, and cellular differentia-tion. Intracellular GSLs are also important for membrane proteintrafficking.
Glycosphingolipids (GSLs), which consist of sugar chain andceramide moiety, are thought to play a role in a number of cellularfunctions such as cell recognition [1,2], cell differentiation [3–5],signal transduction [6,7], apoptosis [8], and receptors for virus [9].
They may act to protect the membrane from harsh conditions like alow pH or degradative enzymes [10]. Recent cell biological studiesshow that GSLs in plasma membranes form cluster, so called a raft,with cholesterol and are relatively less in content of phospholipidsthan other parts of plasma membrane. GSLs can mediate the sig-
ces B: Biointerfaces 72 (2009) 272–283 273
nplSdG
ccp(eswtfdtnbcmmspatcywtwobtc
o[miftmwr[tnfm
itsotrsamsa
gs
Table 1Acyl chain and LCB compositions of BAC-2a and BAC-4.a.
Composition (%)
BAC-2a BAC-4
Acyl chain Ch22:0 29.7 29.9Ch23:0 25.2 25.9Ch24:1 29.7 25.0Ch24:0 15.5 19.2
LCB Cd16:1 15.7 24.4Cd17:1 iso 61.9 59.5
Y. Ikeda et al. / Colloids and Surfa
al transduction pathway through interaction with these signalingroteins, circulate between the plasma membrane and intracellu-
ar organs, and also move laterally over the exoplasmic membrane.uch migration could be conducted by the raft [11,12]. A detailedescription of the chemical, structural, and functional properties ofSLs has been summarized in a review article by Maggio [13].
GSLs are classified into gangliosides, globosides, sulfatides,eramides dihexosides (lactosides), and cerebrosides based ononstituent sugar chain types. Gangliosides are amphiphilic com-ounds containing sugar chains with one or more N-acetylneuramicsialic) acid residues and ceramide moieties. In addition, they arespecially enriched in the brain and nervous tissues and con-titute ∼5−10% of the total lipid mass in nerve cells [14]. GSLsith N-acetylgalactosamine as the terminal sequence and tri- or
etrasaccharide containing head group, known as globosides, areound in the erythrocyte membrane [15–17]. Sulfatides (sulfatederivatives of galactocerebrosides) is concerned with the HIV infec-ion as a receptor [18]. Lactosylceramides are present in matureeutrophils as major components and serve as a receptor foracteria and fungi [19–21]. Cerebrosides possess saccharide anderamide moiety and are ubiquitous components of the plasmaembrane of all eukaryotic cells [22,23]. Galactocerebrosides areajor components of the myelin sheath [24–28]. Glucocerebro-
ides occur in all mammalian cells like sphingomyelins and arerecursor of complex GSLs; for example, gangliosides, globosides,nd lactosylceramide [29]. GSLs show heterogeneity not only inheir head group but also in their ceramide moieties. The biologi-al significance of ceramide heterogeneity is not understood well,et. However, the structure of fatty acid moieties in ceramidesas found to influence the localization and function of GSLs in
he plasma membrane, which is probably due to direct interactionith cholesterol, phospholipids, and the transmembrane domains
f receptor proteins [30–33]. GSLs exist not only in the vertebrateut also in the mollusk [34,35], the plant [36–40], porifers [41–43],he echinoderm [44–50] and so on; for example, GSLs from the seaucumbers [44,47–49].
The interfacial behavior of GSLs has been investigated by meansf a Langmuir monolayer [51–58], bilayer [59], and liposome60,61,62]. Langmuir monolayer is particularly used as a simplest
odel of biomembrane and can provide not only exact and detailednformation on surface behavior but also interaction between film-orming materials. In addition, the phase behavior of monolayers athe air/water interface under lateral compression can be clarified
orphologically as well as thermodynamically. The data obtainedith optical techniques such as microscopy (BAM) [63–65], fluo-
escence microscopy (FM) [66–68], and atomic force microscopy69–71] are commonly combined with the isothermal data in ordero elucidate the monolayer behavior. Thus, the monolayer tech-ique has been employed for studies on biological and biophysical
unctions at the interface, which are exerted by rafts [72], pul-onary surfactants [73], GSLs [57–60], and so on.Cerebrosides are especially the simplest class of GSLs and are
mportant surface-lying molecules found virtually in all cells. Galac-ocerebrosides and their metabolites have been shown to possessignificant functions on promoting the regulation of nerve cell [74],n regulating protein kinase C activities [75], and on modulatinghe function of hormone receptors [76]. Glucocerebrosides have aelation with the precursor of gangliosides or the accumulated sub-trate of Gaucher disease. They have several activities; for example,nti-ulcerogenic activity [77], anti-tumor activity [35,78], and anti-icrobial activity [36]. Therefore, it is expected that cerebrosides
ynthesized and/or extracted from natural products are utilized asnew medical resource.
In the previous studies, we have reported the surface behavior oflucocerebrosides (LMC-1, LMC-2, and LLC-2) [79–81] and ganglio-ides (DSG-1) [82]. The glucocerebrosides were completely miscible
Cd17:1 normal 9.2 4.1Cd18:1 13.3 12.0
a Ch: 2-hydroxyl fatty acid, Cd: sphingosine type (1,3-dihydroxy) long chain base.
with dipalmitoylphosphatidylcholine (DPPC) within a monolayerstate. However, the binary monolayers of the cerebrosides anddipalmitoylphosphatidylethanolamine (DPPE) or cholesterol (Ch)indicated immiscible or phase-separated behavior. The miscibil-ity of GSLs with lipid components in biomembrane is considerablyimportant for understanding the various functions in raft clusters.In order to know the detailed interactions of sphingolipids andtheir roles in the cell membrane, it is necessary to collect moreinformation on their dependence on difference in the molecularstructure; i.e. the number, location, and orientation of hydroxylgroups attached to acyl chains and so on. Also, it is expected thatsphingolipids is utilized as a new medical resource from naturalproducts.
The Langmuir monolayer properties of glycocerebroside werereported from 1970s, whereas the molecular mechanisms underly-ing the cerebrosides from marine invertebrates have not been madeclear yet. Here, we focus our attention on two different polar headgroups of cerebrosides [glucocerebroside (BAC-2a) and galactocere-broside (BAC-4)] isolated from sea cucumber (Bohadschia argus)[83]. BAC-2 and BAC-4 are molecular species, however, possessingalmost same ceramide moiety. Comparing with BAC-2 and BAC-4, we are able to see whether the saccharide part is playing anydifference sort of a role on the membrane surface.
Surface pressure (�)–molecular area (A), surface potential(�V)–A, and surface dipole moment (�⊥)–A isotherms were mea-sured for DPPC, cerebrosides (BAC-2a and BAC-4), and their binarycombinations. The mutual interaction of the two-componentmonolayers was analyzed in terms of additivity of molecular sur-face areas and surface potentials. An interaction parameter and aninteraction energy between the two were evaluated from the Joosequation [84]. Furthermore, the phase behavior of the Langmiurmonolayer states was confirmed by the morphological observationwith BAM, FM, and AFM.
2. Experimental
2.1. Materials
The sea cucumber B. argus (Janomenamako in Japanese) belongsto the kingdom Animalia, the phylum Echinodermata, the classHolothuroidea, and the family Holothuriidae. It was collected in thesea near Zanpamisaki in Okinawa (Japan) in 1999. The isolated cere-brosides (BAC-2a and BAC-4) from the sea cucumber were checkedby 1H and 13C NMR, FAB-MS and GC–MS spectra after purificationby column chromatography. The compositions of the hydrophobicacyl chain and long chain base (LCB) for BAC-2a and BAC-4 are given
in Table 1. BAC-2a and BAC-4 are molecular species for hydropho-bic parts. The chemical structures (Fig. 1) of BAC-2a and BAC-4 are1-O-ˇ-d-glucopyranosyl-ceramide and 1-O-ˇ-d-galactopyranosyl-ceramide, respectively. That is, the structural difference betweenthe two is mainly the species of their sugar moieties. More detail
274 Y. Ikeda et al. / Colloids and Surfaces B:
ip
(z(Au(T
ws2sst
2
apoetbo0tt∼
2
taet
Fig. 1. Chemical structures of BAC-2a (a) and BAC-4 (b).
nformation and structural determination for them were reportedreviously [82].l-�-1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine
DPPC; purity >99%) and 1-palmitoyl-2-[6-[(7-nitro-2-1,3-ben-oxadiazoi-4-yl)amino]hexanoyl]-sn -glycero-3-phosphocholineNBD-PC; purity >99%) as a fluorescent probe were purchased fromvanti Polar Lipids, Inc. (Alabaster, AL, USA). These lipids weresed without further purification. n-Hexane (98.5%) and ethanol99.5%) used as spreading solvents come from Cica-Merck (Uvasol,okyo, Japan) and nacalai tesque (Kyoto, Japan), respectively.
Stock solutions of cerebrosides (0.5 mM) and DPPC (1.0 mM)ere prepared with n-hexane/ethanol (7/3, v/v) mixture. The
odium chloride (nacalai tesque) was roasted at 1023K for4 h to remove any surface active organic impurity. The sub-trate solution was prepared using thrice distilled water (theurface tension = 71.96 mN m−1 at 298.2 K and the electrical resis-ivity = 18 M� cm).
.2. Surface pressure (�)−area (A) isotherms
The surface pressure (�) of monolayers was measured usingn automated homemade Wilhelmy film balance. The surfaceressure balance (Mettler Toledo, AG-245) had a resolutionf 0.01 mN m−1. The surface pressure-measuring system wasquipped with filter paper (Whatman 541, periphery = 4.0 cm). Therough (effective area = 720 cm2) was made from Teflon-coatedrass. The �−A isotherms were recorded mainly at 298.2 ± 0.1 Kn 0.15 M NaCl. The monolayer was compressed at a speed of.12 nm2 molecule−1 min−1. The spreading solvents were allowedo evaporate for 15 min prior to compression. The standard devia-ions for molecular area and surface pressure were ∼0.01 nm2 and0.1 mN m−1, respectively.
.3. Surface potential (�V) measurements
The surface potential (�V) was simultaneously recorded withhe surface pressure while the monolayer was compressed at their/water interface. It was monitored by using an ionizing 241Amlectrode at 1–2 mm above the interface while a reference elec-rode was dipped in the subphase. The electrometer (Keithley 614
Biointerfaces 72 (2009) 272–283
and 6517) was used to measure the surface potential. The standarddeviation for the surface potential was ∼5 mV.
2.4. Brewster angle microscopy (BAM)
The monolayer was directly visualized by a Brewster anglemicroscope (KSV Optrel BAM 300, KSV Instruments Ltd., Finland)coupled to a commercially available film balance systems (KSVMinitrough, KSV Instruments Ltd., Finland). The application of a20 mW He–Ne laser emitting p-polarized light of 632.8 nm wave-length and a 10× objective lens allowed a lateral resolution of∼2 �m. The angle of the incident beam to the air/water interfacewas fixed to the Brewster angle (53.1◦) at 298.2 K. The reflectedbeam was recorded with a high grade charge coupled device (CCD)camera (EHDkamPro02, EHD Imaging GmbH, Germany), and thenthe BAM images were digitally saved to the computer hard disk.
2.5. Fluorescence microscopy (FM)
The film balance system (KSV Minitrough) was mounted onto the stage of the Olympus microscope BX51WI (Tokyo, Japan)equipped with 100 W mercury lamp (USH-1030L), an objective lens(SLMPlan50×, working distance = 15 mm), and a 3CCD camera witha camera control unit (IKTU51CU, Toshiba, Japan). The z-directionalfocus on the monolayer was exactly adjusted using an automationcontroller (MAC 5000, Ludl Electronic Products Ltd., NY, USA). Aspreading solution of the samples was prepared as mixed solutiondoped with 1 mol% of a fluorescence probe (NBD-PC). The excita-tion (460 nm) and emission (534 nm) were selected by a mirrorunit (U-MWIBA3). FM images were directly recorded with the harddisk via an online image processor (DVgate Plus, Sony Corp., Japan)connected to the microscope. Image processing and analysis werecarrying out by using the software, Scion Image Beta 4.02 for Win-dows (Scion Corp., Frederick, MD, USA). The total amount of ordereddomains was evaluated and expressed as a percentage per frame bydividing the respective frame into dark and blight regions.
2.6. Atomic force microscopy (AFM)
Langmuir–Blodgett (LB) film preparations were carried out witha KSV Minitrough. Freshly cleaved mica (Okenshoji Co., Tokyo,Japan) was used as a supporting solid substrate for the filmdeposition. At selected surface pressures, a transfer velocity of5 mm min−1 was used for film-forming materials on a 0.15 M NaClat 298.2 K. LB films with deposition rate of ∼1 were used in theexperiments. AFM images were obtained using an SPA 400 instru-ment (Seiko Instruments Co., Chiba, Japan) at room temperature ina tapping mode, which provided both a topographical image anda phase contrast one. The tapping mode images (512 points perline) were collected with scan rates of 0.2–1.0 Hz, using silicon tips(Olympus Co.) with nominal spring constant of 1.8 N m−1 under theambient conditions. The lateral and vertical resolutions were 0.2and 0.1 nm, respectively. The transferred samples were checked forpossible tri-induced deformation by zooming out after a region hadbeen scanned.
3. Results and discussion
3.1. Surface behavior of pure DPPC and cerebrosides
3.1.1. Monolayer stability
The surface pressure (�)–time (t) isotherms for DPPC and cere-brosides were measured on 0.15 M NaCl at 298.2 K to check thestability of these Langmuir monolayers. The monolayers were com-pressed up to 35 mN m−1 and then the Teflon-barrier was stopped,after which relaxation of surface pressure was traced against time
Y. Ikeda et al. / Colloids and Surfaces B: Biointerfaces 72 (2009) 272–283 275
F43t
(d∼ot(li
3
mBttfmHribaoBta∼p
gaopaoS�mT�dtii
ig. 2. Relaxation of the surface pressure (�) for pure DPPC, BAC-2a, and BAC-monolayers as a function of time (t). The monolayers were compressed up to
5 mN m−1 on 0.15 M NaCl at 298.2 K. Then, the �−t measurement was started, wherehe moving barrier is stopped.
Fig. 2). For pure DPPC monolayers, the surface pressure of DPPCecreased during the first 20 min and then remained constant at33 mN m−1 up to 120 min as reported previously [67,82]. On thether hand, both cerebrosides indicated slow relaxation. However,hey reached almost plateau values of 33 (BAC-2a) and 31 mN m−1
BAC-4), respectively. It suggests that cerebrosides used here areess soluble in the subphase and are possible to be studied as annsoluble monolayer at the air/water interface.
.1.2. �–A, �V−A and �⊥–A isothermsFig. 3 shows the �–A isotherms of DPPC, BAC-2a, and BAC-4
onolayers spread on 0.15 M NaCl at 298.2 K. The �–A curve ofAC-2a showed a transition pressure (�eq) of 30 mN m−1, wherehe monolayer state changes from the liquid-expanded (LE) tohe liquid-condensed (LC) phase. On further compression, the sur-ace pressure increased to reach ∼43 mN m−1 (0.34 nm2) in the
onolayer state. The extrapolated area of BAC-2a was ∼0.69 nm2.owever, judging from the cross-sectional area of one satu-
ated hydrocarbon chain (∼0.20 nm2), its area is too large. Thiss attributable to the larger cross-section of the unsaturated andranched acyl chains. As for BAC-4, on the other hand, it formedtypical LE monolayer up to 45 mN m−1 (0.37 nm2). Its extrap-
lated area is 0.72 nm2, which is almost the same value as theAC-4 one. The similarity at the close-packed state reveals thathe compositions of hydrophobic chains for both cerebrosides arelmost identical. A DPPC monolayer has the transition pressure of12 mN m−1 and collapses at ∼55 mN m−1, which was reportedreviously [79].
The surface potential (�V) is a measure of the electrostatic fieldradient perpendicular to the surface and thus varies consider-bly with the molecular surface density. It generally reflects anrientational and conformational change of monolayers upon com-ression. The important feature of the surface potential is that itllows the Langmuir monolayer to be probed at much earlier stagesf monolayer compression in comparison with pi–A isotherms [85].uch behavior is very clear in the case of DPPC. The behavior ofV–A isotherms for cerebrosides corresponds to the change ofolecular orientation upon compression as shown in Fig. 3(b).
he �V value of DPPC and cerebrosides is always positive. The
V−A isotherm of DPPC monolayer showed large variations withecreasing molecular area. As a result, the value reached ∼550 mVhrough the some transition states. On the contrary, the changen �V value for BAC-2a and BAC-4 is rather small in spite of thencrease in surface pressure. Such phenomenon is often observed
Fig. 3. The �−A (a), �V−A (b), and �⊥−A (c) isotherms of pure DPPC, BAC-2a, andBAC-4 monolayers on 0.15 M NaCl at 298.2K.
in the monolayers of molecular species like cerebrosides and gan-gliosides [79,81,82]. The maximum �V value of BAC-2a (285 mV)is larger than that of BAC-4 (250 mV) due to the formation of LCmonolayer for BAC-2a.
The vertical component of surface dipole moment (�⊥) wascalculated from the measured �V values using the Helmholtz equa-tion,
�V = �⊥/ε0εA, (1)
where ε0 is the permittivity of vacuum, ε is the mean permittiv-ity of monolayer (which assumed to be 1), and A is the surfacearea occupied by the molecule. The �V values involve the resultantdipole moments accompanied by the subphase, the head group,the hydrophobic chain. Contrary to the �V behavior, the �⊥ valueof BAC-2a and BAC-4 monolayers steeply decreased from ∼550
(A = 1.0 nm2) to ∼200 mD (at closed-packed state) on compression(Fig. 3(c)). Analyses of the molecular behavior suggested that the �⊥value is more sensitive to molecular orientation and conformationthan the �V value.
2 ces B:
3
tppOMpFhmtBas
pe
�
wtac(c
F4b
76 Y. Ikeda et al. / Colloids and Surfa
.1.3. Surface dipole moments (�⊥) of cerebrosidesWe analyzed the surface potentials of the monolayers using the
hree-layer model proposed by Demchak and Fort [86]. This modelostulates independent contribution of the subphase (layer 1), theolar head group (layer 2), and the hydrophobic chain (layer 3).ther models such as the Helmholtz model and the Vogel andöbius model are also available [85(b)]. The �⊥ estimation of
olar head groups and hydrocarbon chains using the Demchak andort model assumes a condensed Langmuir monolayer, where itsydrophobic chains are closely packed [84,87]. Application of thisodel to the LE monolayer of BAC-4 may lead to a rough estima-
ion. However, if this model is applied to the resultant value of theAC-4 monolayer in the close-packed state, it is possible to induceuseful estimation, which can provide qualitative explanation of
urface dipole moment behavior.Thus we have substituted the experimental �⊥ values of close-
acked monolayers for the �⊥calc values of the three-layer modelquation,
⊥ calc = �1
ε1+ �2
ε2+ �3
ε3, (2)
here �1/ε1, �2/ε2, and �3/ε3 are the contribution of the subphase,
he polar head group and the hydrophobic chain, respectively. Theim of the following analysis is to understand the difference in theontribution of the polar head group between BAC-2a and BAC-4glucose and galactose). First, the contribution of the hydrophobichains needs to be determined.ig. 4. AFM topographic images of LB films of BAC-2a (A) and BAC-4 (B) monolayers on0 mN m−1. The scan area is 3 × 3 �m and the scale bar in the lower-right corner represenelow each row. In (A) at 30 mN m−1, white line circle means circular LC domain.
Biointerfaces 72 (2009) 272–283
The initial set of values proposed by Demchak and Fort(�1/ε1 = 0.040 D, ε2 = 7.6 and ε3 = 5.3) were determined for mono-layers made from terphenyl derivatives and octadecyl nitrileon 4 M NaCl [86]. The other set was determined by Petrov etal. (�1/ε1 = 0.025 D, ε2 = 7.6 and ε3 = 4.2) for n-heptanol and 16-bromohexadecanol monolayer on 0.1 M phosphate buffer [88].We have used a set of values introduced by Taylar and Olivia(�1/ε1 = −0.065 D, ε2 = 6.4 and ε3 = 2.8) for monolayers of ω-halogenated fatty acids and amines on water [89]. Furthermore, wehave changed and utilized a combination of values (�1/ε1 = 0.025 D,ε2 = 7.6 and ε3 = 2.8) for BAC-2a and BAC-4 monolayers on 0.15 MNaCl due to the similarity of the subphase condition. The validity ofthe set was previously confirmed by applying to standard samplessuch as stearic acid and DPPC [79,81].
Here, we evaluate the contribution of the hydrophobic tail-endgroup for BAC-2a and BAC-4 monolayers. Although the hydrophobicmoiety of BAC-2a is slightly different from that of the cerebrosidesreported previously [74], the polar head group is the same as theprevious cerebrosides. Therefore, we can evaluate the contributionof hydrophobic tail-end part using the value of 0.63 D for �2
Glc inEq. (3).
�⊥(BAC − 2a) = �1
ε+ �Glc
2ε
+ �3
ε= 0.26 D (3)
1 2 3
From the above equation, we obtained �3 = 0.43 D.Second, in order to evaluate the contribution of the hydrophilic
group of BAC-4 (galactose), we assumed the contribution ofhydrophobic tail-end group (�3 = 0.43 D) to be the same for BAC-2a
0.15 M NaCl at 298.2 K. The monolayers were transferred onto mica at 20, 30, andts 1 �m. The cross-sectional profiles along the scanning line (white line) are drawn
Y. Ikeda et al. / Colloids and Surfaces B: Biointerfaces 72 (2009) 272–283 277
Table 2Surface potential data for dipole moment evaluation.
BB
a
�
Tsbs
3
fBaos�t∼mh3dhfdtTcthsd
Adabhrdp
3
tralctcattt
t
where �s� is an apparent molar entropy change, a˛ and aˇ aremolecular area (in square nanometers, the superscript ˛ and ˇ referto phase states), �eq the transition pressure from the ˛ phase to theˇ phase, and the �0 the surface tension of the substrate. a˛ andaˇ are estimated as follows. a˛ is the area at the point where the
Area (nm2) � (mN m−1) �V (mV) �⊥ (D) �2 (D) �3 (D)
AC-2a 0.360 40 273 0.26 0.63 (Glc) 0.43AC-4 0.399 40 249 0.26 0.65 (Gal) 0.43
nd BAC-4 and assigned this value to Eq. (4).
⊥(BAC − 4) = �1
ε1+ �Gal
2ε2
+ �3
ε3= 0.26 D (4)
The contribution of the head group for BAC-4 is 0.65 D (Table 2).he �2 value of BAC-2a is almost the same as that of BAC-4. It isuggested therefore that the structural difference in head groupetween glucose and galactose does not contribute much to theurface dipole moment at the close-packed state.
.1.4. Atomic force microscopy (AFM)AFM has been performed for Langmuir–Blodgett (LB) film trans-
erred onto mica to investigate the phase behavior of BAC-2a andAC-4 on the nanometer level. The films transferred at 20, 30,nd 40 mN m−1 were examined in the tapping mode. In the casef BAC-2a monolayers (Fig. 4(A)), the AFM images at 20 mN m−1
hows a coexistence of two different phases. Judging from the–A isotherm as well as BAM and FM micrographs (latter sec-
ions) for BAC-2a, the higher regions with a height difference of1 nm from the surroundings are not LC domains but LE domainsade of BAC-2a components with longer hydrophobic chains. The
igher LE domains shrink in size by a surface pressure increase to0 mN m−1 which corresponds to the transition pressure to the LComain. Nevertheless, there is no height difference between theigher LC domains and the circular LC domain. When the sur-
ace pressure increases further, the circular domains are finelyecreased to become homogeneous LC phase (40 mN m−1). Thus,he dispersed LC domains could not be observed with BAM and FM.his phenomenon of LC domains for pure components on lateralompression is not a common case. That is, it might be possiblehat the phenomenon is associated with the distribution of theydrophobic-chain species for BAC-2a. Therefore, unsaturation andteric hindrance of the hydrophobic chains may disperse the LComains at high surface pressures.
As for BAC-4 monolayer (Fig. 4(B)), on the other hand, theFM height image at 20 mN m−1 shows bright domains and darkomains. Considering the results of the �–A isotherm, BAM images,nd FM images, both of the domains indicate the LE phase. Theright LE domains are made of BAC-4 components with higherydrophobic chains and better orientation compared with the sur-ounding LE phases. These AFM images indicate that the structuralifference between BAC-2a and BAC-4 has an influence on theirhase behavior, but not on the surface dipole moment.
.1.5. Apparent molar quantity changes on the phase transitionWe measured the temperature dependence of the phase
ransition pressure for BAC-2a monolayers. Fig. 5(a) shows the rep-esentative �–A isotherms of BAC-2a on 0.15 M NaCl at 288.2, 298.2,nd 310.2 K. Monolayer compressibility, Cs for BAC-2a was calcu-ated from �–A isotherms using Cs = (−1/A) (dA/d�). The inverseompressibility, Cs−1 is expressed against A (Fig. 5(b)) [90]. All ofhe curves have the first-order phase transitions, which are indi-ated by arrows (Fig. 5(b)). Shown in Fig. 6 are the plots of �eq
nd �c against temperature. The transition pressures increased and
he collapse pressure decreased with increasing temperature. Theemperature-dependent behavior of �eq and �c is very similar tohat for standard samples such as myristic acid and DPPC [81,91].The change of thermodynamic quantity on the phase transi-ion of monolayers was calculated using the previous method [68],
Fig. 5. The temperature dependence of �−A (a) and Cs−1−A (b) isotherms of BAC-2amonolayers on 0.15 M NaCl at typical temperatures.
which takes the contribution of the substrate to monolayers intoaccount. The apparent molar entropy change (�s� ) of the phasetransition was evaluated by following equation [92],
�s� (˛, ˇ) = (aˇ − a˛)
[(∂�eq
∂T
)P
−(
∂�0
∂T
)P
](5)
Fig. 6. Change of the transition pressures (�eq) and collapse pressures (�c) for BAC-2a monolayers on 0.15 M NaCl as a function of temperature.
278 Y. Ikeda et al. / Colloids and Surfaces B:
Table 3Apparent molar quantity change (�s� , �h� , and �u�) on the phase transition at298.2 K on 0.15 M NaCl.
�s� (J K−1 mol−1) �h� (kJ mol−1) �u� (kJ mol−1)
BL
fidpesia[a
Fr
AC-2a −11 −3.1 −4.1LC-2-15a −70 −21 −22
a LLC-2-15: A pure cerebroside isolated from starfish Linckia laevigata [71].
lm starts to transform from the ˛ to the ˇ state. The aˇ value isetermined following manner; when the point (a˛, �eq) is movedarallel to the area axis to zero area, it comes into contact with thelongated line of the �–A isotherms of the solid state to the lower
ˇ
urface pressure. The intersection point gives the a value. Thenclinations of �eq and �0 against temperature are ∂�eq/∂T = 0.27nd ∂�0/∂T = −0.16 mN m−1 K−1 (the value is quoted from the refs.93–95]). Furthermore, the apparent molar enthalpy change (�h� )nd energy change (�u� ) of the phase transition are evaluated byig. 7. The �−A, �V−A and �⊥−A isotherms of the binary DPPC/BAC-2a (A) and DPPC/BAespectively) on 0.15 M NaCl at 298.2 K.
Biointerfaces 72 (2009) 272–283
using Eqs. (6) and (7), respectively [92].
�h� (˛, ˇ) = T�s� (˛, ˇ) (6)
�u� (˛, ˇ) = −(�eq − �0)(aˇ − a˛) + T�s� (˛, ˇ) (7)
The apparent molar quantity changes (�s� , �h� , and �u� ) ofthe phase transition for BAC-2a monolayers at 298.2 K on 0.15 MNaCl are shown in Table 3. The apparent molar enthalpy shows anegative value. Therefore, the transition from the LE phase to the LCone is exothermic. As for the apparent molar entropy, the value is
also negative as expected. The apparent molar changes for BAC-2aare considerably smaller compared with those for a single cere-broside such as LLC-2-15 [81]. It means that the molecular speciesin hydrophobic chains exert the terminal resistance for the phasetransition.C-4 (B) monolayers as a function of cerebroside mole fractions (XBAC-2a and XBAC-4,
Y. Ikeda et al. / Colloids and Surfaces B: Biointerfaces 72 (2009) 272–283 279
Fig. 8. Mean molecular areas (A) of binary DPPC/BAC-2a (A) and DPPC/BAC-4 (B) sys-tlm
3
3
btsDatniisib�
gaamiFsp
pression, the LC domain (bright contrast) appears at 12 mN m ,
ems as a function of cerebroside mole fractions at different pressures. The dashedines were calculated by the additivity rule, and the solid circles represent experi-
ental values.
.2. Two-component monolayers of DPPC and cerebrosides
.2.1. Compression isothermsThe two-component monolayers composed of DPPC and cere-
rosides (BAC-2a and BAC-4) have been studied in order to clarifyhe interaction and the miscibility between them in the monolayertate. The �–A, �V–A, and �⊥–A isotherms of DPPC/BAC-2a andPPC/BAC-4 systems were systematically measured on 0.15 M NaClt 298.2 K. For the �–A isotherms of DPPC/BAC-2a (Fig. 7(A)), all ofhe curves existed between those of the respective pure compo-ents. The transition pressure increases and becomes unclear with
ncreasing XBAC-2a. Likewise, the �V–A and �⊥–A isotherms shiftn between pure components with XBAC-2a. As for the DPPC/BAC-4ystem, the similar isothermal variation and behavior are observedn Fig. 7(B), too. These results suggest that DPPC is miscible withoth BAC-2a and BAC-4 in the monolayer state from the change ineq and �c against the mole fraction (Xcerebrosides).
The interaction between DPPC and cerebrosides were investi-ated by examining whether the variation of the mean molecularreas and surface potentials as function of Xcerebrosides satisfied thedditivity rule [96,97]. A comparison between the experimentalean molecular areas and the molecular areas based on ideal mix-
ng at four surface pressures (5, 15, 25, and 35 mN m−1) are shown inig. 8. For the DPPC/BAC-2a system (Fig. 8(A)), the A–XBAC-2a curveshow a positive deviation from the additivity line at all surfaceressures except for 5 mN m−1, indicating repulsive interactions
Fig. 9. Surface potential (�V) of the binary DPPC/BAC-2a (A) and DPPC/BAC-4 (B)systems as a function of cerebroside mole fractions at four different pressures. Thedashed lines were calculated by assuming additivity rule, and the solid circles rep-resent experimental values.
between DPPC and BAC-2a. At 5 mN m−1, the area values agree withideal ones in 0 ≤ XBAC-2a ≤ 0.5 and positively deviate from the idealline in 0.5 ≤ XBAC-2a ≤ 1. These positive deviations result from thesteric hindrance of BAC-2a hydrophobic chains. In the case of theDPPC/BAC-4 system, the similar behavior (positive deviations) isalso observed in Fig. 8(B).
Analysis of the surface potentials of the binary monolayersbased on additivity rule is also performed in Fig. 9. Both of theDPPC/BAC-2a and DPPC/BAC-4 monolayers indicate a good agree-ment with ideal line at 5 mN m−1 and negative deviations at 15, 25,and 35 mN m−1. The negative deviations can be attributed to lessordered orientation induced by the steric hindrance of hydrophobicchains in cerebrosides.
3.2.2. Brewster angle microscopy (BAM)FM measurements need an adequate amount of probes in the
monolayer. There is a possibility that the dye molecule affectsthe original monolayer behavior, though the additional amountof FM probe is quit small (1 mol%). Thus, the phase behavior ofthe binary monolayers has been examined by BAM, which pro-vided us an in situ direct monolayer image without FM probes.The representative BAM images are shown in Fig. 10. For the BAMimages of DPPC monolayers (Fig. 10(a)), the image at 10 mN m−1
shows the homogeneous LE phase (dark contrast). On further com-−1
where the monolayer transfers from the LE phase to the LC one(data not shown). With an increase in surface pressure from 12to 20 mN m−1, the LC domains grow in size. Beyond 20 mN m−1,the BAM image becomes homogeneous bright (data not shown).
280 Y. Ikeda et al. / Colloids and Surfaces B: Biointerfaces 72 (2009) 272–283
F XBAC-4 = 0.1) (c) monolayers observed on 0.15 M NaCl at 298.2 K. The scale bar represents5
Tp
mt(abs
3
tFpiLtFpsdT[
2toeohpa
dDo
ig. 10. BAM images of DPPC (a), DPPC/BAC-2a (XBAC-2a = 0.1) (b), and DPPC/BAC-4 (0 �m.
he phase variation for DPPC monolayers is the same as reportedreviously [98].
When a small amount of BAC-2a (XBAC-2a = 0.1) is added to DPPConolayers (Fig. 10(b)), the LC domains become smaller in size and
heir shape also changes. Likewise, the addition of the equal amountXBAC-4 = 0.1) exerts the similar phase variation (Fig. 10(c)). The vari-tions of the domain shape and the transition pressure inducedy the blend of cerebrosides support the miscibility of the binaryystems within a monolayer.
.2.3. Fluorescence microscopy (FM)FM has higher resolution and magnification than BAM. Thus,
he more detail phase behavior can be understood using FM. TheM contrast is generated due to the difference in solubility of FMrobes between LE and LC phases. Shown in Fig. 11(A) are FM
mages for the DPPC/BAC-2a system. It is noticed that shape of DPPCC domains observed in the FM images (Fig. 11) is the same ashat observed in the BAM images (Fig. 10(a)). That is, the 1 mol%M probes have no influence on the original phase behavior. Forure DPPC, the LC domains grow up in size on compression asimilarly seen in the BAM images (Fig. 10(a)). The growth of theomains is also supported by the increase in percentage of them.he FM images for DPPC monolayers agree with the reported data71].
For the DPPC/BAC-2a system (Fig. 11(A)), as the amount of BAC-a in monolayer increases, the transition pressure increases andhe LC domains become smaller in size. Correspondingly, the ratiof the LC domains reduces. As mentioned above, BAC-2a monolay-rs indicate the LE/LC transition at 30 mN m−1. As for the FM imagesf pure BAC-2a (right column), the images at 15 and 20 mN m−1 areomogeneously bright, indicating the LE phase. On further com-ression beyond the transition pressure, quite small LC domains
ppear in the FM images at 30 mN m−1 (see inset).In the case of the DPPC/BAC-4 systems (Fig. 11(B)), the LComains become smaller with increasing XBAC-4 similarly to thePPC/BAC-2a system. The both systems being compared, the ratiof LC domains of the latter for X = 0.1 and 0.3 is smaller by ∼10%
Fig. 11. FM images of the binary DPPC/BAC-2a (A) and DPPC/BAC-4 (B) monolayerson 0.15 M NaCl at 298.2 K as functions of cerebroside mole fractions and surfacepressures. The monolayers contained 1 mol% of the fluorescent probe (NBD-PC). Thepercentage (%) refers to the LC phase in the micrograph. The scale bar represents50 �m. (Inset) The zoomed-in FM domains at 30 mN m−1. The scale bar represents10 �m.
Y. Ikeda et al. / Colloids and Surfaces B: Biointerfaces 72 (2009) 272–283 281
F pse pD Eq. (8a notes
aii(to
3
tftwtfc
mbpt
1
wm�2ai�tc
ig. 12. Phase diagrams from the variation of the transition pressure (�eq) and collaPPC/BAC-2a (A) and DPPC/BAC-4 (B). The dashed lines were calculated according tomixed monolayer formed by DPPC, BAC-2a, and BAC-4 species, whereas “Balk” de
t 40 mN m−1 than that for the former. It suggests that BAC-4 flu-dized the LC domains to more extent at high surface pressures. Thiss supported by the fact that pure BAC-4 forms only a LE monolayerright column). These results, the increase of �eq and the variation ofhe domain shape against the mole fractions, provide an evidencef the miscibility of the both binary systems in the monolayer state.
.2.4. Two-dimensional phase diagramsTwo-dimensional phase diagrams were constructed plotting the
ransition pressures and collapse pressures as a function of moleraction of BAC-2a and BAC-4 in Fig. 12(A) and (B), respectively. Theransition pressures for the binary systems change almost linearlyith the mole fractions. Judging from the continuous change of
he transition pressure, two components are miscible at all moleractions. This behavior is an evidence of the miscibility of the twoomponents in the monolayer state.
Assuming that the binary monolayers behave as a regular surfaceixture with a hexagonal lattice, the coexistence phase boundary
etween the ordered monolayer phase (2D phase) and the bulkhase (3D phase) can be theoretically simulated by the Joos equa-ion [84],
= xs1exp {(�c
m − �c1)ω1/kT}exp {�(xs
2)2}
+xs2exp {(�c
m − �c2)ω2/kT}exp {�(xs
1)2} (8)
here xs1 and xs
2 denote the mole fraction in the two-componentonolayer of the components 1 and 2, respectively, and �c
1 andc2 are the corresponding collapse pressures of components 1 and, �c
m is the collapse pressure of the two-component monolayer
t given composition of xs1 and xs2. ω1 and ω2 are the correspond-
ng limiting molecular surface areas at the collapse points. �1 and2 are the surface activity coefficients at the collapse point, � is
he interaction parameter, and kT is the product of the Boltzmannonstant and the Kelvin temperature. The solid curve obtained by
ressure (�c) on 0.15 M NaCl at 298.2 K as a function of cerebrosides mole fractions:) for � = 0. The solid symbols represent experimental values. “Monolayer” indicates
a solid phase of DPPC, BAC-2a, and BAC-4.
adjusting the interaction parameter in the above equation coincideswith the experimental values. It is worth noting that the both sys-tems exhibit a positive interaction parameter, although the otherDPPC/cerebrosides systems reported previously result in a nega-tive interaction parameter [79,81]. The new finders here are thatthe DPPC/BAC-2a system shows � = 0.25 and the DPPC/BAC-4 sys-tem indicated � = 0.90. The positive interaction parameter meansthat an interaction energy between different molecules is smallerin magnitude than the mean energy of interaction. The interac-tion energies (−�ε = −�RT/6) were calculated to be −103 J mol−1
(for DPPC/BAC-2a) and −372 J mol−1 (for DPPC/BAC-4). As a result,the DPPC/BAC-2a and DPPC/BAC-4 systems are likely to be of thenegative azeotropic type.
The values of interaction parameters and interaction energiesfor DPPC/BAC-2a and DPPC/BAC-4 are completely opposite in signto those of the other GSLs [79,81,82]. The steric hindrance ofhydrophobic chains (cis-type unsaturation and branch) in cere-brosides investigated in the present study may lead to such theinteractions with DPPC.
4. Conclusions
The two different cerebrosides (BAC-2a and BAC-4) isolatedfrom sea cucumber can form a stable monolayer on 0.15 M NaClat 298.2 K. BAC-4 forms a typical LE monolayer, whereas BAC-2amonolayer has the LE/LC transition pressure. The Demchak andFort model was applied to analyze the surface potential of thecerebrosides. The contribution of galactose head group to the sur-face dipole moment was determined to be 0.65 D using the value
(�2 = 0.63 D). The apparent molar thermodynamics changes (�s� ,�h� , and �u� ) of the phase transition for BAC-2a monolayersat 298.2 K were evaluated. It suggested that molecular speciesin hydrophobic chains lead to thermal resistance for the phasetransition. The binary DPPC/BAC-2a and DPPC/BAC-4 monolayer
2 ces B:
bibstattbtte
A
2
R
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82 Y. Ikeda et al. / Colloids and Surfa
ehavior was investigated to clarify the mutual miscibility andnteraction. In terms of isothermal aspects, the miscibility for theinary systems was supported from the changes in transition pres-ure and collapse pressure of compression isotherms. In addition,he miscibility was morphologically confirmed also from the vari-tions in domain shape and transition state with BAM and FM. Thewo-dimensional phase diagrams and the Joos equation yieldedhe interaction parameter (�) and the interaction energy (−�ε)etween DPPC and cerebrosides. The negative azeotropic types ofwo-dimensional phase diagrams were obtained, where an interac-ion energy between different molecules is smaller than the meannergy of interaction.
cknowledgements
This work was supported by a Giant-in-Aid for Science Research0500414 from the Japan Society for the Promotion of Science.
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