Controlling the Morphology of Chiral Lipid Tubules Mark S. Spector,* Jonathan V. Selinger, Alok Singh, Jose M. Rodriguez, † Ronald R. Price, and Joel M. Schnur Center for Bio/Molecular Science and Engineering, Code 6900 Naval Research Laboratory, Washington, D.C. 20375-5348 Received January 23, 1998. In Final Form: March 25, 1998 Several techniques for controlling the morphology of self-assembled lipid tubules are investigated by using circular dichroism spectroscopy and electron microscopy. These studies show that variations in the molecular structure of the diacetylenic phospholipid, lipid concentration, and solution conditions allow for control of the number of bilayers in the tubule walls, but not their diameter. Tubules formed in water and mixtures of alcohols adopt interesting morphologies and allow for further control of tubule structure. In addition, studies of lipids with different acyl chains show that tubule morphology is sensitive to the degree of order within the chains. Because of the chiral molecular architecture in lipid tubules, intense peaks in their circular dichroism spectra are observed. These peaks can be monitored to obtain information on the tubule morphology. This information is correlated to direct observations made using electron microscopy. Results of these studies have led to the optimization of large scale preparations of tubules for technological applications. Introduction Phospholipids self-organize in solution due to their amphiphilic structure. For diacyl phospholipids in excess water, the most common structure found is a spherical, bilayer aggregate known as a liposome. 1 In addition to being amphiphilic, glycerophospholipids are chiral, with the L-enantiomer predominant in nature. While the enantiomeric excess of L-R-amino acids and D-sugars in nature affects the structure and biological activity of proteins and nucleic acids, the chiral asymmetry of phospholipids does not typically manifest itself in the supramolecular structure of lipid biomembranes. One exception is the helical structures observed in saturated bile solutions, where lipids are apparently an essential ingredient. 2 Another exception is the unusual supra- molecular aggregates formed from bilayers of synthetic phospholipids with diacetylenes in the acyl chains. These lipids are found to self-assemble into hollow, cylindrical structures, known as tubules, when the lipids are cooled into the gel phase. 3 Tubules differ from cochleate cylinders formed from anionic lipid bilayers because tubules are hollow while cochleate cylinders are filled with a rolled- up bilayer. 4 While formation of cochleate cylinders is induced by electrostatic interactions, formation of tubules is apparently induced by chiral interactions. 5 This chirality can be seen in the helical markings that are often observed when these tubules are decorated or stained. 6,7 The most commonly studied diacetylenic phospholipid is 1,2-bis(tricosa-10,12-diynoyl)-sn-glycero-3-phosphocho- line. This lipid, shown in Figure 1a, is designated DC 8,9 - PC because it has eight methylenes between the ester and diacetylene group and nine methylenes between the diacetylene and the terminal methyl group. It forms tubules with a characteristic diameter of 0.5 μm and a characteristic length of 50-200 μm. The molecular architecture of tubules has been explained by theories based on molecular chirality. 5,8,9 In these theories, chiral interactions cause molecules to pack at a nonzero angle with respect to their nearest neighbors, as shown in Figure 1b. This chiral packing contributes to the twisting of the bilayer, which leads to the formation of a cylinder. Phospholipid tubules have been extensively studied for use in technological applications, such as electroactive composites and controlled-release systems. 10 Those ap- plications depend on coating tubules with a thin film of metal or ceramic to make them more rugged. 11 Figure 2 shows a scanning electron micrograph of a copper-plated lipid tubule. The tubules are observed to be hollow and contain spiral wrappings. To optimize tubules for ap- plications, one may wish to manipulate the tubule morphology, e.g., the tubule length, diameter, and wall thickness. Control of length and diameter is important because those parameters determine the release rate in controlled-release systems and the electromagnetic prop- erties of metallized cylinders in electroactive composites. Control of tubule wall thickness is important because the thickness determines whether the tubules can be coated. If the tubule walls are only one bilayer thick, then they are too fragile to be coated. On the other hand, tubule walls that are many bilayers thick make inefficient use * To whom correspondence should be addressed. † Present address: Texas A&M University, College Station, TX 77840. (1) Lasic, D. D. Liposomes: From Physics to Applications; Plenum: New York, 1993. (2) Chung, D. S.; Benedek, G. B.; Konikoff, F. M.; Donovan J. M. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 11341-11345. (3) Yager, P.; Schoen P. E. Mol. Cryst. Liq. Cryst. 1984, 106, 371- 381. (4) Papahadjopoulos, D.; Vail, W. J.; Jacobson, K.; Poste, G. Biochim. Biophys. Acta 1975, 394, 483-491. (5) Selinger, J. V.; Schnur, J. M. Phys. Rev. Lett. 1993, 71, 4091- 4094. Selinger, J. V.; MacKintosh, F. C.; Schnur, J. M. Phys. Rev. E 1996, 53, 3804-3818. (6) Singh, A.; Burke, T. G.; Calvert, J. M.; Georger, J. H.; Herendeen, B.; Price, R. R.; Schoen, P. E.; Yager, P. Chem. Phys. Lipids 1988, 47, 135-148. (7) Burkett, S. L.; Mann, S. J. Chem. Soc., Chem. Commun. 1996, 321-322. (8) Helfrich, W.; Prost, J. Phys. Rev. A 1988, 38, 3065-3068. Zhong- can, O.-Y.; Ji-xing, L. Phys. Rev. A 1991, 43, 6826-6836. (9) Nelson, P.; Powers T. J. Phys. II (Fr.) 1993, 3, 1535-1569. (10) Schnur, J. M. Science 1993, 262, 1669-1676. (11) Schnur, J. M.; Price, R.; Schoen, P.; Yager, P.; Calvert, J. M.; Georger, J.; Singh, A. Thin Solid Films 1987, 152, 181-206. 3493 Langmuir 1998, 14, 3493-3500 S0743-7463(98)00086-9 This article not subject to U.S. Copyright. Published 1998 by the American Chemical Society Published on Web 05/29/1998