morphological manipulation of bolaamphiphilic polydiacetylene assemblies by controlled lipid doping

Chemistry and Physics of Lipids

114 (2002) 203–214

Morphological manipulation of bolaamphiphilicpolydiacetylene assemblies by controlled lipid doping

Jie Song a, Quan Cheng a,*, Raymond C. Stevens a,b,*a Materials Sciences Di�ision, Lawrence Berkeley National Laboratory, Uni�ersity of California, Berkeley, CA 94720, USA

b Department of Chemistry and Molecular Biology, The Scripps Research Institute, La Jolla, CA 92037, USA

Received 8 August 2001; received in revised form 23 January 2002; accepted 28 January 2002

Abstract

Morphological transformations of bolaamphiphilic polydiacetylene (L-Glu-Bis-3) lipid assemblies from helicalribbons to vesicles and flat sheets through controlled doping are described, and the role of specific lipid dopants inthese processes is discussed. Upon doping with cell surface receptor GM1 ganglioside, fluid vesicular structures startto emerge, coexisting with the micro-crystalline helical ribbons. The vesicle formation is further facilitated andstabilized by the introduction of cholesterol into the system, presumably through surface curvature variation inducedby inhomogeneous distribution and dynamic clustering of GM1 and cholesterol within the doped assemblies. Extendedhelical ribbons are ‘truncated’ into patches of flat sheets when a sufficient amount of Bis-1, a structurally compatiblesymmetric bolaamphiphilic diacetylene lipid, is doped. The results reaffirm the important roles of packing geometryand headgroup chirality in the formation of extended helical ribbon structures. The doped assemblies of bolaam-phiphiles allow for capture of intermediate structures of morphological transformation using transmission electronmicroscopy (TEM). A vesicle-to-ribbon transformation mechanism via lateral reorganization within relatively fluidvesicular microstructures has been suggested. Understanding of the doping-induced transformation process providesuseful information for the design of advanced materials where the microscopic morphology of material is crucial toits function. © 2002 Elsevier Science Ireland Ltd. All rights reserved.

Keywords: Bolaamphiphilic polydiacetylene; Lipid doping; Helical ribbons

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1. Introduction

We reported recently the synthesis and charac-terization of a bolaamphiphilic polydiacetylenematerial (poly-L-Glu-Bis-3, Fig. 1) that is capableof a pH-induced and color-coded morphologicaltransformation from blue helical ribbons to rednanofibers (Song et al., 2001). The unique opticalproperty and defined microstructural morphologyobserved for poly-L-Glu-Bis-3 offers great poten-

* Corresponding authors. Present address: Department ofChemistry, University of California, Riverside, CA 92521,USA. Tel.: +1-909-787-2702; fax: +1-909-787-4713 (Q.C.);Tel.: +1-858-784-9416; fax: +1-858-784-9483..

E-mail addresses: [email protected] (Q. Cheng),[email protected] (R.C. Stevens).

0009-3084/02/$ - see front matter © 2002 Elsevier Science Ireland Ltd. All rights reserved.

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J. Song et al. / Chemistry and Physics of Lipids 114 (2002) 203–214204

tial to the design and fabrication of advancednanomaterials (Whitesides et al., 1991) and bio-in-spired materials that have found extensive appli-cations as membrane mimics (Fendler, 1982;Ringsdorf et al., 1988), supramolecular immuno-gens (Reichel et al., 1999) and biosensors (Song etal., 1998a,b).

Previous colorimetric sensors were constructedusing polydiacetylene Langmuir–Blodgett filmsincorporating cell surface receptors (sialo-lipid orGM1 ganglioside) for the detection of influenzavirus (Charych et al., 1993) and a number ofbacterial toxins (Charych et al., 1996). These sen-sors integrated molecular recognition and signaltransduction into one supramolecular assembly.The conjugated polymer backbone provided thesignal transduction pathway and responded tobinding events by a straightforward color change.The unique device-free detection feature of these

colorimetric sensors allows for on-site detection ofbiological hazards and offers great potential for avariety of household medical or diagnostic appli-cations. There are, however, limitations to the useof these amphiphilic lipid-based sensors. For ex-ample, their fabrication requires the Langmuir–Blodgett technique, and the detection sensitivitiesobtained are yet to be further improved. Wecontinue our efforts in designing new materials tobe used for the development of more sensitive anddurable colorimetric biosensors that can be conve-niently fabricated. Bolaamphiphilic diacetylenelipid L-Glu-Bis-3 is a promising candidate mate-rial for the development of future generations ofsensitive colorimetric sensors because it sponta-neously self-assembles under mild conditions inan aqueous environment, adopts a rigid packingarrangement, and displays defined optical andmorphological properties (Song et al., 2001).

Fig. 1. Structures of constituent lipids.

J. Song et al. / Chemistry and Physics of Lipids 114 (2002) 203–214 205

One fundamental consideration in designingbiosensors is to establish an effective signal trans-duction pathway upon the interaction of incorpo-rated receptors with analytes at the detectioninterface. For self-assembling polydiacetylenelipid-based biosensors, this requirement trans-forms into a balance between the rigidity andflexibility of the sensor scaffold. Often times thisbalance is reflected in the microscopic morphol-ogy and the extent of polymerization (which isdirectly influenced by molecular packing) of thesensor material. In nature, membrane-spanningbipolar lipids provide extraordinary stability toarchaebacteria, a class of microorganisms thatresist extreme environmental conditions such aslow pH, high temperature, and high salt (Lang-worthy, 1982).

We are exploring the use of membrane-span-ning diacetylene lipids such as L-Glu-Bis-3 as amatrix lipid during construction of polydi-acetylene-based colorimetric sensors as a directapproach to rigidify the sensor scaffolds. How-ever, a certain degree of flexibility of the sensorscaffold must be maintained so that receptor con-formation at the membrane surface can beadapted to allow initiation of effective bindingevents. Such a balance in biosensor fluidity maybe achieved by lipid doping. While the incorpora-tion of lipid-associated receptors provides bindingspecificity to the sensor, it also introduces a spe-cies that could alter the desirable morphologicalproperties of the biosensor material. From thispoint of view, the lipid-associated receptors alsofunction as structural dopants of the biosensorscaffold. Controlled lipid doping, either with thereceptor as the only additive or by incorporatingadditional dopants, provides abundant possibili-ties to fine-tune the flexibility and morphologicalproperties of the biosensor system.

In this paper, transmission electron microscopy(TEM) is used to investigate the morphologicalproperties of supramolecular assemblies formedby the membrane spanning diacetylene lipid L-Glu-Bis-3 in the presence of GM1 ganglioside andother lipid dopants. Particular emphasis is placedon determining the doping-induced morphologicaltransitions from one microstructure to another,and the role that specific lipid dopants play in

such transitions. The overall aim of this work is togain insight into how changes in chemical compo-sition of the supramolecular assemblies affect themorphological changes observed. By understand-ing the relationship between assembly composi-tion and morphology, in combination withdetermining likely morphological transformationmechanisms, efforts in rational design of effectivebiosensing materials can be facilitated.

2. Experimental section

2.1. Materials

10,12-Docosadiynedioic acid (Bis-1) was ob-tained in 95% purity from Lancaster (Windham,NH), and was further purified by dissolving intetrahydrofuran (THF) with passage through ashort silica pad to remove polymerized impurities.Lipid L-Glu-Bis-3 was synthesized by couplingL-glutamic acid with one end of Bis-1 through anamide linkage. The synthetic details and charac-terization of L-Glu-Bis-3 was described previously(Song et al., 2001). Cholesterol (5-cholesten-3-�-ol) was purchased from Sigma (St. Louis, MO) in99+% purity and GM1 ganglioside was obtainedfrom Matreya, Inc. (Pleasant Gap, PA) in 98+%purity. Both were used without furtherpurification.

2.2. Supramolecular assembly preparation

Three milliliters of 0.1 N sodium chlorideaqueous solution was added to 0.9 mg of totallipids, which was a mixture of L-Glu-Bis-3 and5% GM1 ganglioside with 0–20% cholesterol or0–40% Bis-1 (molar ratios). To obtain stablesupramolecular assemblies, three alternativepreparation methods were used and compared:vortexing, probe sonication, and heating. In thevortexing procedure, the mixture was vortexed for2–3 min until a clear, colorless solution was ob-tained. For mixtures with high cholesterol con-tents (10 and 20%), turbid suspensions wereobtained. The assemblies were then incubated at4 °C for 30 min. For the probe sonication ap-proach, the lipid suspension was sonicated for 20


Fig. 2. Transmission electron micrographs of undoped L-Glu-Bis-3 assembly. Note the right-handed helical ribbon morphology.

min with a 40 W probe sonicator. The resultingclear solution was cooled to room temperaturebefore incubation at 4 °C for 30–60 min. Theheating method involved heating the mixture at80 °C for 5 min followed by the same incubationprocedure that was used during the probe sonica-tion method. Although, all three preparations ledto readily polymerizable materials upon UV irra-diation, the vortexing method appeared to yieldthe most homogeneous assemblies, and therefore,was chosen as the method of choice for the prepa-ration of all doped assemblies generated in thisstudy.

2.3. Crosslinking of the supramolecular assemblies

Freshly prepared supramolecular assemblieswere pipetted into 96-well polystyrene microtiterplates and irradiated with UV light (254 nm, CL1000 Ultraviolet Crosslinker) until a stable bluecolor developed. The exposure time for varioussamples ranged from 12 to 60 s. Longer irradia-tion time was required for assemblies with highcholesterol content.

2.4. Transmission electron microscopy (TEM)

TEM images were obtained for both polymer-ized and un-polymerized forms of the supramolec-ular assemblies using a Zeiss electron microscopeoperating at 80 kV. Samples were deposited oncarbon film coated Cu grids. Microstructures ofdiacetylene lipids were readily visualizable owingto their high electron density, but negative stain-

ing with 0.5% uranyl acetate was performed toenhance image quality.

3. Results and discussion

3.1. Microstructures of undoped L-Glu-Bis-3assemblies

Lipid L-Glu-Bis-3 is a wedge-shaped bolaam-phiphile conveniently synthesized from symmetriclipid Bis-1 (Fig. 1). Undoped L-Glu-Bis-3 readilyassembles into stable helical ribbons with variousdegrees of right-handed helicity in an aqueousenvironment under mild conditions (Fig. 2). Incontrast to probe sonication and subsequent lowtemperature incubation methods that are com-monly used for the preparation of mono-func-tional lipid assemblies, vortexing and roomtemperature incubation is sufficient to ensure for-mation of stable supramolecular assemblies of thisbis-functional lipid. Ultraviolet (UV) irradiationof these assemblies results in rapid (within sec-onds) polymerization of L-Glu-Bis-3, affordingthe material a dark blue color. This rapid poly-merization indicates high order structure of theassemblies and good alignment of the diacetyleneunits. Under TEM, the polymerized material ap-pears to adopt the same microstructural morphol-ogy as unpolymerized L-Glu-Bis-3. These ribbonsare up to tens of microns in length. Ribbonthickness is between 5 and 10 nm, suggestingmonolayer or double layer packing arrangementsat various regions of the microstructures. The


widths of the ribbon structures vary from tens tohundreds nanometers, with typically less than a100 nm width for highly twisted ribbons (Fig. 2B).

3.2. Structure and function of lipid dopants

To probe the effect of lipid doping on themicrostructural morphology of L-Glu-Bis-3 as-semblies, three structurally different lipids, GM1

ganglioside, cholesterol and Bis-1, were chosen aslipid dopants. The structures of these lipids areshown in Fig. 1. Gangliosides are a family ofglycosphingolipids localized to the outer leaflet ofthe plasma membrane of vertebrate cells. They areenriched in neuronal membranes, particularly insynapses (Svennerholm, 1994). When inserted intoartificial membranes, the oligosaccharide motif ofgangliosides is exposed at the membrane surfaceand functions as recognition groups for a numberof bacterial toxins (Song et al., 1998a; Charych etal., 1996). Ganglioside GM1, a known receptor ofcholera toxin (Holmgren et al., 1975), was chosenin this work to be incorporated into the assemblyof L-Glu-Bis-3. Structurally alien polycyclic lipidcholesterol was selected as a dopant because it isan essential component of biological membranesand also a natural target of streptolysin O, atransmembrane pore-forming toxin (Alouf andGeoffroy, 1991; Bhakdi et al., 1985) that is an-other potential detection target of interest. Lowconcentrations of GM1 or cholesterol were shownto modulate domain structure and phase separa-tion in model membrane systems (Hwang et al.,1995). Bis-1 is a symmetric achiral lipid struc-turally similar to L-Glu-Bis-3, and was used toprobe the effect of the geometry and chirality ofconstituent lipids on microstructure formation.

3.3. Incorporation of GM1 ganglioside as dopant

The effect of incorporating GM1 ganglioside onthe morphology of L-Glu-Bis-3 assemblies wasinvestigated. Gangliosides consist of a ceramidebackbone and a rather bulky oligosaccharideheadgroup. They display unique physical proper-ties in aqueous solution, falling in the transitionregion between micelle- and bilayer-forming am-phiphiles (Raudino et al., 2000). The size of the

ganglioside headgroup is known to dramaticallyinfluence the formation of supramolecular assem-blies in single lipid formulations. For example,GM3 ganglioside, which contains a relatively smalltrisaccharide headgroup, was shown to undergospontaneous formation of vesicles with very lowbending rigidity in addition to forming lamellarfragments in aqueous solution (Cantu et al.,1994). For GM1 ganglioside, which bears a largerpentasaccharide headgroup, micelle formationwas observed at low concentrations (Orthaberand Glatter, 1998) and cubic ordering was seen athigh concentrations (Boretta et al., 1997). In re-cent years, increasing experimental evidence sug-gests that the biological activity of gangliosides ismodulated not only by their individual chemicalstructures, but also by their lateral distributionwithin the lipid membrane, both in model systems(Vie et al., 1998; De Luca et al., 1999) and in cellmembranes (Simons and Ikonen, 1997). There-fore, it is important to determine the influence ofGM1 as a co-lipid on the shape and morphologyof lipid-based self-assembling materials, as relatedto the construction of membrane mimics and thedesign of bio-inspired materials. Such investiga-tions are also crucial to the success of fabricatingeffective biosensors where GM1 ganglioside func-tions as the indispensable recognition unit forvarious biological interactions at the sensinginterface.

Earlier investigation showed 5% GM1 (molarratio) added to polydiacetylene films was an ap-propriate amount to be incorporated into theselipid-based sensors for detection of a variety oftoxins (Charych et al., 1996). We therefore, fixedthe GM1 content at this level in all the dopedsystems throughout this study. When 5% GM1

ganglioside was introduced into the L-Glu-Bis-3system, a fair amount of vesicles were formedalong with ribbons, as evidenced by the TEMmicrographs (Fig. 3). The size of these vesiclesvaried from �100 to �500 nm in diameter. Asignificant number of vesicles appeared to be at-tached to the ribbon structures, often at the junc-tion of several entangled ribbons (Fig. 3B).

A question that arises from these TEM obser-vations is whether the distribution of GM1 is equaland homogeneous between these two different


forms of microstructures. Based on the structuralfeatures of GM1 and the matrix lipid L-Glu-Bis-3,as well as the distribution of sphingolipids foundin biological membranes, GM1 is likely to predom-inantly reside in vesicles in an aggregated form.The presence of GM1 in ribbon structures, if at all,should be minimal. This is because the molecularpacking of the ceramide tails of GM1 with thekinked diacetylene lipids within the semi-crys-talline ribbon structures would be unfavorable.Instead, effective mixing of these two lipidsshould result in rather fluid microstructures. Inaddition, without inclusion of GM1, L-Glu-Bis-3does not form vesicles, establishing an indis-putable role that the GM1 must play in promotingvesicle formation in the doped system.

3.4. Incorporation of both GM1 ganglioside andcholesterol as dopants

To further investigate how the chemical compo-sition and structural compatibility of lipid do-pants influence the microstructural morphology ofL-Glu-Bis-3 assemblies containing GM1 gan-glioside, we introduced a third lipid componentinto the system. Incorporation of cholesterol, astructurally very different lipid, was studied up toa 20% molar concentration. At low cholesterolconcentrations (5%), a uniform blue color wasstill attainable for the ternary assembly upon UVirradiation. However, with high cholesterol con-tent (20%), only turbid suspensions could be ob-tained, even after prolonged vortexing or probe

sonication, and polymerization became difficult.TEM micrographs of lipid systems doped with 5and 10% cholesterol revealed that with increasedcholesterol content, more vesicles were formedwith continued coexistence of the ribbon struc-tures (Fig. 4). Apparently, addition of cholesterolfurther facilitates and stabilizes the formation ofvesicles. There were a significant amount of vesi-cles attached to ribbons (Fig. 4B) or nested on theframework of entangled ribbons (Fig. 4A). Bud-ding and fission of vesicles were also observed(Fig. 4C).

Cholesterol has long been known to stabilizemembrane structures. It has been suggested thatdynamic clustering of sphingolipids and choles-terol in cell membranes results in the formation ofrafts which function as platforms for the attach-ment of proteins when membranes are traffickedin cells as well as during signal transduction pro-cesses (De Luca et al., 1999). It is thought thatsphingolipids associate laterally with one anotherthrough interactions between their headgroups,whereas cholesterol molecules function as spacers,filling the voids at the hydrophobic regions be-tween associating sphingolipids. Such preferentialpacking was believed to lead to the formation ofrafts within the membrane bilayers. The proposedrole of cholesterol in facilitating vesicle formationin synthetic membranes has also been well docu-mented. Ringsdorf and coworker studied the roleof cholesterol in assisting vesicle formation usingsingle chain symmetric diacetylene diphosphatelipids (Bader and Ringsdorf, 1982). They showed

Fig. 3. Transmission electron micrographs of L-Glu-Bis-3 doped with 5% GM1. Note the appearance of vesicles along with helicalribbons.


Fig. 4. Transmission electron micrographs of L-Glu-Bis-3 doped with GM1 and cholesterol. (A) L-Glu-Bis-3 doped with 5% GM1 and5% cholesterol; (B and C) L-Glu-Bis-3 doped with 5% GM1 and 10% cholesterol. Note the vesicles fused to or sitting on the networkof ribbons (A and B) and the budding and fission of vesicles (B and C).

that without cholesterol in the membrane, thediphosphate lipid did not assemble into vesicles,due to its symmetric geometry (Bader and Rings-dorf, 1982). However, a recent study using a seriesof double-chain bolaamphiphiles claimed that thestructural mismatch of lipid dopants, specificallythe presence of cholesterol in the matrix, couldblock the insertion process and curb vesicle for-mation (Eguchi et al., 2000). The authors con-cluded that vesicle formation occurred only whenthe added molecules were closely compatible withthe current constituents of the lipid layer. Ourresults here clearly contradict any generalizationssuggested in their study. In both systems studiedhere (the binary system doped with GM1 and theternary systems doped with both GM1 and choles-terol), the dopants (such as glycosphingolipid andpolycyclic cholesterol) are structurally very differ-ent from the bolaamphiphilic matrix lipid, and yetthey were found to facilitate vesicle formationsignificantly. In the three-component systems, wespeculate that cholesterol molecules are inserted inthe outer-surface of the vesicles, filling the voidsat the hydrophobic region between aggregatedgangliosides and membrane spanning lipids.Given the fact that neither GM1 ganglioside,cholesterol, nor membrane spanning matrix lipidL-Glu-Bis-3 by themselves assemble to form vesi-cles, it is apparent that inserting the proper do-pants between membrane spanning lipids isessential to inducing surface curvature and vesicleformation.

An interesting morphological detail of the vesi-cle formation observed here was the presence ofapparent budding and fission of vesicles (Fig. 4Band C). Various studies have suggested that thephysical nature of the lipid matrix plays a domi-nant role in the budding and fission process(Ringsdorf et al., 1988; Dobereiner et al., 1993).The multiple lipid components used here, andtheir inhomogeneous distribution within the sys-tems observed, lead to the formation of phaseswith varied local lipid composition and surfacecurvature. When the surface curvature differencesbetween various regions exceed a threshold, vesi-cle budding occurred. The budding process gener-ates an energetically unfavorable line tensionbetween neighboring domains that tends to dimin-ish, leading to the eventual detachment of vesiclesfrom their parental clusters.

3.5. Doping effect of a structurally compatiblebolaamphiphile

Achiral and symmetric bolaamphiphile Bis-1shares a structurally identical lipophilic core withL-Glu-Bis-3. When Bis-1 was introduced as adopant, a different trend in morphological changewas observed. With the incorporation of smallamounts of Bis-1 (5 and 10%) in combinationwith 5% GM1 ganglioside into the L-Glu-Bis-3assembly, the microstructure remained predomi-nantly a mixture of ribbons and vesicles, but withthe emergence of a small amount of fragmentedsheets (Fig. 5A). When the Bis-1 content was


increased to 40%, the extended helical ribbonsand vesicles disappeared completely, and werereplaced with patches of flat, rectangular sheets(Fig. 5B).

The morphological outcome of doping Bis-1 isdirectly associated with the structural features ofBis-1 as compared to matrix lipid L-Glu-Bis-3.The structural similarity in the lipophilic coresegment of the two lipids underlines the highmiscibility of the lipids and establishes the role ofBis-1 as a structural ‘diluting’ agent. However, thedifference in size, charge and chirality of theheadgroups gives rise to the difference in thegeometry of the lipid, the ability of forming H-bonding network at the surface, the chirality ofthe assembly at the supramolecular level, andeventually the morphological outcome of themixed system. We believe that doping a highpercentage (40%) of miscible lipid Bis-1 leads tointerruption of the tight, crystalline chiral packinginduced by the wedge-shaped bolaamphiphile (L-Glu-Bis-3) (Song et al., 2001), and ultimately pro-hibits the formation of extended helical ribbons.It is known that both general thermodynamicconstraints and the geometry of each amphiphilicmolecule present in a lipid matrix are crucialfactors in determining the final shape and mor-phology of the aggregates formed (Israelachvili etal., 1976). Specifically, chirality and the appropri-ate geometry of constituent lipids are crucial de-terminants for the chiral packing of

self-assembling materials, which has been consid-ered by many as the driving force for tubular andhelical microstructure formations (Thomas et al.,1999; Schnur, 1993; Eckhardt et al., 1993;Viswanathan et al., 1994; Selinger et al., 1996;Oda et al., 1999). The dramatic morphologicalchange observed here in the membrane spanningsystem doped with a high content of Bis-1 sup-ports the important role of geometry and contin-ued chiral packing of the constituent lipids in theformation of extended helical ribbon structures.

3.6. Morphological transformation between�esicles and ribbons

In this study, some intriguing morphologicaldetails of doped L-Glu-Bis-3 assemblies were cap-tured by TEM, providing an opportunity to ex-amine intermediate states of microstructuraltransformation. A number of polystyrene blockcopolymers have been studied by TEM for theirrod-to-vesicle and vesicle-to-rod transitions in-duced by solvents and dilution (Yu et al., 1999;Chen et al., 1999). Shear flow-induced, surfactant-based vesicle-to-wormlike micelle and micelle-to-vesicle transitions have been studied by usingsmall angle neutron scattering and TEM (Zhenget al., 2000; Oberdisse et al., 1998; Mendes et al.,1997; Escalante et al., 2000). Lipid doping effectson microstructure transitions, however, are rela-tively less explored (Schroder and Schurholz,

Fig. 5. Transmission electron micrographs of L-Glu-Bis-3 doped with GM1 and structurally similar bolaamphiphilic lipid Bis-1. (A)L-Glu-Bis-3 doped with 5% Bis-1 and 5% GM1. Note the appearance of small amount of fragmented flat sheets. (B) L-Glu-Bis-3doped with 40% Bis-1 and 5% GM1. Note the exclusive formation of patches of flat sheets with (0.1–0.4)× (0.1–0.4) �m2

dimensions.


Fig. 6. Transmission electron micrographs of the morphological details of vesicle domain separations (A) and typical vesicle-to-rib-bon transformations (B and C) observed with doped L-Glu-Bis-3 systems ((A) doped with 5% GM1 and 5% cholesterol; (B) dopedwith 5% GM1; (C) doped with 5% GM1 and 10% cholesterol). Note the ribbon outlining the periphery of large vesicles in micrographB and the origination of ribbons along domain edges within a large vesicle in micrograph C.

1996), especially for polymerizable bolaam-phiphilic self-assembling systems. Fig. 6 highlightsthe morphological details of some distinctive mi-crostructures observed for the doped L-Glu-Bis-3assemblies. It is clear from these micrographs thatribbons (relatively rigid) and vesicles (relativelyfluid) were physically interrelated during the for-mation of different assemblies.

Heterogeneity in lipid composition and distri-bution was first reflected by domain separationsthat were observed with large size vesicular struc-tures. For large vesicles formed with multi-com-ponent lipids, especially for mixtures ofpolymerizable and unpolymerizable lipids, do-main separation is expected (Gaub et al., 1984).This is most clearly shown in Fig. 6A, wherecircular domains up to 100 nm in diameter arescattered throughout a large vesicle. Formation ofthese domains suggests the fluid nature of vesicu-lar microstructures, where lateral diffusion andreorganization readily occurs. Indeed, domainseparation was predominantly observed with largevesicles rather than with extended helical ribbonstructures, arguably as a result of the higher con-centrations of unpolymerizable GM1 and choles-terol, and therefore higher fluidity within thesevesicles.

A vesicle-to-ribbon transition mechanism at theperiphery of some of the vesicles was suggestedbased on the observed morphological details ofinterconnected microstructures shown in Fig. 6B(doped with 5% GM1) and Fig. 6C (doped with

5% GM1 and 10% cholesterol). The edges of vesi-cles or vesicle domains were outlined in the shapeof ribbons. Such morphological details were ob-served for all mixed systems studied. The growthof a ribbon and its extension away from a vesicu-lar microstructure is most clearly seen in theimage shown in Fig. 6C. A vesicle-to-ribbon tran-sition is a probable process during domain reorga-nizations within less crosslinked and more fluidmicrostructures. Lateral reorganization of lipidswithin these areas may have resulted in phases ordomains with particularly low dopant concentra-tions, thus having a higher continuity of chirallypacked matrix lipid L-Glu-Bis-3. Apparently, it isat the edge of such domains that the transforma-tion into more rigidly packed helical ribbonsoccurs.

It is worth noting that these inter-connectedvesicle-ribbon structures are stable over time. Weobserved similar morphologies of samples storedat 4 °C for over a week. Further kinetic studiesinvolving trapping at various intermediate stagesof the microstructure transformation betweenvesicles and ribbons will be helpful for under-standing of the process on a much shorter timescale.

4. Conclusions

Morphological transformation of bolaam-phiphilic polydiacetylene assemblies from helical


ribbons to various microstructures can beachieved by controlled lipid doping. Vesicles, rib-bons and patches of flat sheets were obtained as aresult of binary or ternary doping of the L-Glu-Bis-3 assembly with structurally similar or aliendopants. GM1 ganglioside, cholesterol, and mem-brane spanning lipid Bis-1 have been shown to beeffective in this process. The resulting microstruc-tures are found to be dependent on both the typeand amount of dopants used.

Inhomogeneous and clustered distribution ofGM1 ganglioside in the doped assemblies results inincrease of regional surface curvature, and is be-lieved to be responsible for the generation ofvesicular microstructures. The apparent promo-tion and stabilization of vesicle formation by dop-ing cholesterol is rationalized as the result ofpreferential packing of cholesterol in the outer-half of vesicles, where it fills voids between thehydrophobic spaces between GM1 ceramide tailsand kinked membrane spanning matrix lipid L-Glu-Bis-3.

What appears to be of particular significance tous is that controlled doping allows observation ofintermediate states in microstructure transitionfrom one form to another. Morphological detailsof microstructures in doped systems, includingdomain separation within fluid vesicles, vesicle-to-ribbon transition at the edges of vesicle domains,and the budding and fission of vesicles were allvisualized by TEM. The structural influence of thenon-polymeric lipid dopants on packing, surfacecurvature, and fluidity of the assemblies werereflected in domain separation and lateral reorga-nization that was observed within fluid vesicles.The unique structural and conformational proper-ties of GM1, due to the bulky pentasaccharideheadgroup and ceramide tail that it has, may havecontributed to the structural versatility it inducedin these microstructures.

The transformation from extended helical rib-bons to fragmented flat sheets induced by dopingthe achiral symmetric lipid Bis-1 into the systemsuggests a crucial role that the geometry andchirality of constituent lipids plays in the forma-tion of extended helical ribbon structures. Such adramatic influence of lipid doping on the mor-phology of the material obtained demonstrates

the importance of fine-tuning the specific receptorconcentration used in biosensor formulations.These lipid doping results also demonstrate theneed for careful selection of co-lipids with struc-tural similarities to the matrix lipid when design-ing new generations of biosensing materials,where the microscopic morphology of the biosen-sor material has been found to directly affect thesignal transduction efficiency. Continued studiesof doping-induced morphological transition pro-cesses will also be useful for the rational design ofadvanced nanomaterials that require defined mor-phological templates.

Acknowledgements

This work is supported by the Director, Officeof Nonproliferation and National Security, Officeof Research and Development of the U.S. Depart-ment of Energy under Contract No. DE-AC03-76SF00098. The authors thank Dr RichardBruehl for critical reading of the manuscript andDr Mark Alper, Program Director of the Centerfor Advanced Materials, Biomolecular MaterialsProgram, for continued encouragement and sup-port of this research program.

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