Kotula, P. G.

Last, J. A.

Sasaki, D. Y.

Waggoner, T. A.

UNT Digital Library

.‘ {Self-Assembled Columns of Stacked LipidBilayers Mediated by Metal Ion Recognition~:$&J:ggoner+, Julie A. Last’, Pad G. Kotula*,and Darryl.Sandia National Laboratories‘Bimolecular Materials and Inte~aces Dept., MS 1413‘Materials Characterization Dept., MS 1405Albuquerque, NM 87185.Received Date (will be automatically inserted ajier manuscriptis accepted).Molecular self-assembly can generate elaborate two- andthree-dimensional supramolecular structures through thecomplementary interaction of attractive and repulsive forcesbetween molecular components and their environment.’Synthetic self-assembling systems are currently limited to theone-step creation of suprarnolecular structures from molecularcomponents, unlike biological systems which can organizesupramolecular assemblies into higher ordered structures tocreate tissue and functional materials of specific dimensions.zHerein, we report the first observation of a syntheticallyproduced higher ordered self-assembled structure of lipidbilayers with self-limiting dimensions mediated by chemicalrecognition at the membrane surface. Transmission electronmicroscopic (TIM) images of the self-assembled materialreveal a columnar structure of stacked lipid bilayers uniform inwidth with lengths spanning dozens of bilayer thickness.Previous work found that liposomes composed of a pyrene-Iabeled synthetic receptor lipid mixed into adistearylphosphatidylcholine (DSPC) matrix performed ashighly selective optical sensors for heavy metal ions?d6 Inthe absence of metal ions, the liquid-phase receptor lipidseparates from the solid-phase DSPC matrix, producingfluorescence spectra with large excimer emission (A= = 470nm) and relatively small monomer emission (AM = 375 rim).Addition of di- or trivalent metal ions causes an inversion ofthe fluorescence emission peaks (excimer emission attenuatesas the monomer emission intensifies), revealing dkpersion ofthe receptor lipids into the DSPC matrix upon the metrd ionrecognition. Complete reversibility of the process waspossible by removal of the metal ions with EDTA.PSIDAFigure 1. MatrixlipidDSPCand pyrene-labeledlipidPSIDA.(1) (a) Lehn, J.-M. Science 1993,260, 1762. (b) Whhesides,G. M.;Mathias,J. P.; Seto,C. T. Science 1991,254,1312.(2) Alberts,B.; Bray,D.; Lewis, J.; Raff, M.; Roberts,K.; Watson, J.D. Molecular Biology of The Ce[k third cd.; Garland publishing NewYork 1994.(3) Sasaki, D. Y.; ShneQ D. W, Pack, D. W.; Arnold, F. H. Arrgew.Chem., W. Ed. Engl. 1995,34,905.(4) Sasti: D. Y.;Padilla,B. E. CherruConrrn.1998,1581.(5) Sasaln,D. Y.; Waggoner, T. A. Proc. SPIE-Int. Sot. Opt. Eng.19!)9,3606,46.Addition of CU2+ to lipid bilayers composed of 570PSIDA/DSPC (Figure 1) at 0.1 mM lipid concentration causedthe solution to become turbid, indicative of vesicleaggregation. The turbidity was, like the metal ion response,reversible upon the addition of EDTA. ‘Ihrbid solutions werenot observed with other receptor lipids frmctionalized withdithioamide or a crown ether that were selective for Hg2+ orPb2+,respectively, nor with the 5% PSIWVDSPCliposomes inthe presence of other divalent metrd ions, such as Mn2+or Ca2+.In an effort to characterize the aggregate shuctures formedupon metal ion recognition, the bilayers were imaged byelectron microscopy before and after addition of CU2+. TheIiposomes were prepared as described previously andnegatively stained using a standard TEM preparationprotocolf TEM images were taken on a Philips CM-30operated at 300kV. Figure 2 is an image of small unilaroellmvesicles (SW) of 5% PSIDA/DSPC bilayers formed in MOPSbuffered water (0.02 M MOPS, 0.10 M NaCl) at pH 7.4. Twofree-floating liposome structures are observed in the middle ofthe image. Liposome sizes ranged between 400 – 700 nm indiameter. In addition, various forms of unstructured bilayeraggregates can be seen on the top and bottom of the image,accounting for approximately 5070 of the observed features.Figure 2. TEM imageof 5% PSIDAAXPC bilayers in MOPSbufferedwaterat pH 7.4 (ammoniummolybdatestain).Upon addition of CU2+(1.0 I.LM)to this solution a remarkableself-assembly of the bilayers into columnar structures results.A representative image is shown in Figure 3, with more imagesavailable in the Supporting Information. Approximately 15 –20% of the observed bilayer structures on the TEM grid were inthe form of these columns, the rest as aggregates and freeliposomes. These unique structures have been reproduciblyprepared.The bilayer stacks exhibit uniform width throughout most ofthe structure. Other self-organizing lipid bilayer structuresformed through bilayer-DNA7 or bilayer-actirf complexation,(6) New, R R C. Liposorrw Oxford University press New York,1990(7) (a) l-Uidler,J. O.; Koltover,L; SaIdittjT.; Satinya, C. R Science1997, 275, 810. (b)Artzner, F.; Z.anti,R, Rapp, G.; Riidler,J. O. Plzys.Rev. Left. 1998,81,5015.----.. —. ..——- ,. . . - -.--. --v-+.-.--., ---- ..-, ?,-, .-DISCLAIMERThis repo~ was.prepared as an account of work sponsoredby an agency of the United States Government. Neitherthe United States Government nor any agency thereof, norany of their employees, make any warranty, express orimplied, or assumes any legal liability or responsibility forthe accuracy, completeness, or usefulness of anYinformation, apparatus, product, or process disclosed, orrepresents that its use would not infringe privately ownedrights. Reference herein to any specific commercialproduct, process, or service by trade name, trademark,manufacturer, or otherwise does not necessarily constituteor imply its endorsement, recommendation, or favoring bythe United States Government or any agency thereof. Theviews and opinions of authors expressed herein do notnecessarily state or reflect those of the United StatesGovernment or any agency thereof.DISCLAIMERPortions of this document may be illegiblein electronic image products. Images areproduced from the best available originaldocument.I.. ./ .7Z- CT.,-.. .G.r ..- ,, . . >. .-,.-, ,. . ., , .. , :.-,, .,m:-~.. . . . ...+ ,> , .,~y+-~<.-.-.m...,~..... . ,, 7 . ;~-; --- -—. . ..e, imetal ion coordination? or biotin-streptavidln molecularrecognitionlodo notexhibitsuch self-limiting dimensions.These columnar structures varied in width from 600 to 900 ~with lengths between several (300 ~) to -45 (3300 & bilayerthickness. The structure appears to be composed of individuallipid bilayers -40 ~ tldck with -30 ~ spacing between eachlayer. It does not appear that these are flattened Iiposomes forthe edges of each bilayer in the stack are discreet with noconnectivity with its adjacent neighbor. One end of each stackthere is a short, poorly organized stack of bilayers that isapproximately half the diameter of the column. At the otherend the stacks tend to taper off in size in the finrd few bilayers,sometimes leading off to another poorly organized stack.Figure 3. TEM image of self-assembled, columnar structured lipidbilayer stack that forms after CuC~ (1.0 pM) addition to liposomes of5% PSIDNDSPC.The mechanism of formation of these self-assembledstructures with uniform width is difficult to imagineconsidering that the initial liposome sizes are polydisperse.From the fluorescence data, we do know that Cuz+binding tothe iminodiacetic acid (IDA) receptor induces a dkpersion ofPSJDA molecules into the lipid matrix. Coordination of CU2+to the IDA headgroup leaves open an equivalent coordinationsite for another lDA to bind. The fluorescence data indicatesthat intra-liposome 2:1 coordination of IDA to CU2+does notoccur, most probably due to geometric constraints of the two-dimensional membrane surface. Orientation of the CU2+-IDAcomplex perpendicular to the surface would, however, allowcomplexation with an IDA from another liposome. Throughthis metal ion-coordination-mediated event, numerousadhesion points between the two liposomes would rapidlypropagate resulting in a flattening of the opposing bilayers, asillustrated in Figure 4. The dkpersion of receptors due to metalion binding should aid in creating a uniform adhesion betweenthe bilayer surfaces. At some point, the liposome ruptures as aresult of deformation from the adhesion process, allowing CU2+(8) Wong, G. C. L.; Tang, J. X.; Lin, A.; Li, Y.; Jamney, P. A.;Safinya, C. R. Science 2000,288,2035.(9) Constable, E. C.; Meier, W.; Nardin, C.; Mundwiler, S. Clretn.Comm. 1999, 1483.(10) Chlmvolu, S.; Walker, S.; Israelachvili, J.; SchmiL F.-J.;Leckband, D.; Zasa&mski, J. A. Science 1994,264, 1753.to reach receptors in the liposome’s interior. Although wehave no confirming images at this time, the bilayers are mostlikely in the shape of a disc, as one might think of a flattenedsphere. The f~st one or two bilayers formed may serve as atemplate for successive stacking of bilayers, regulating thewidth through the adhesion points between bilayers. The self-organizing nature of lipid assemblies may allow the bilayers torestructure, either recruiting or displacing lipids from adjacentbilayers to form a contiguous bilayer that matches the templatesize.Cu(ll)-bis[lDA] complexFigure 4. proposed mechanism of lipid bilayer stack formation. Topleft structure represents the initial liposome. Following metal ionaddition, Iiposomes adhere to each other through Cu2+-bis[IDA]complexation (right) causing membrane flattening. Subsequent adhesionand liposome lysis leads to bilayer stacking (bottom left).Investigations into the mechanism of formation of thesebilayer stacks are currently underway. Such structurallydefined, asymmetric materials may have potentird use asscaffoldhg in nanoarchitecture or as structures with uniquechemical or optical function.Acknowledgement. Sandia is a multiprogmm laboratory operated bySandia Corporation, a Lockheed Martin Company, for the United StatesDepartment of Energy under Contract DE-AC04-94AL85000.Supporting Information Available. Additional TEM images of lipidbilayer stacks.8 ISupporting InformationTEM images of columnar lipid bilayer stacks of 5% PSIDAIDSPC (1.0 mM total lipid)composition formed through the introduction of CUCIZ(1 jJM) followed by incubation atroom temperature for 12 hours (ammonium molybdate stain).-.,--n-” ,.- r T-—’y —.,. ,,. , \ .. ’,...:. -x<.-, . :. ... .. -..,,.,r, .. m,v.:-’> .- ... : .f -m.:.-.#P.-. .- ,-. :!., ..,+~~~-f- -— >. ’..

Self-Assembled Columns of Stacked Lipid Bilayers Mediated by Metal Ion Recognition

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Self-Assembled Columns of Stacked Lipid Bilayers Mediated by Metal Ion Recognition

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