ก Chirality Control on Lipid Nanotubule Morphology Investigated by Circular Dichroism 1, 1, 2, 2,,

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1 ก Chirality Control on Lipid Nanotubule Morphology Investigated by Circular Dichroism 1, 1, 2, 2,, ก 3, 1 Yuwathida Jantippana 1, Weerawat Intaratat 1, Wisit Singhsomroje 2, Sujint Wangsuya 2, Piboon Pantu 3, Jumras Limtrakul 3, Nattaporn Chattham 1 ก กก กก 1,2-bis(tricosa-10,12-diynoyl)-sn-glycero-3- phosphocholine (DC 8,9 PC) ก ก DC 8,9 PC : [75:25 (v:v)] ก 50 C ก ก ก ก ก ก ก ก ก ก กก กก (AFM) ก ก ก ก ก ก กก ก ก ก กก กก ก ก ก 532 nm,632 nm 810 nm กก ก (Circular Dichroism) ก ก กก ก ก ก ก ก ก ก ก ก ก Abstract Self-assembled cylindrical tubules of chiral 1,2-bis(tricosa-10,12-diynoyl)-sn-glycero-3- phosphocholine (DC 8,9 PC) formed under controlled cooling process. A mixture of DC 8,9 PC in ethanol : water [75:25 (v:v)], was heated to 50 C to dissolve the lipid, and then was allowed to cool to room temperature with controlled cooling rate. Ribbons of lipid bilayers self assembled into tight helical structure forming hollow cylindrical nanotubules during the cooling process. Images from Atomic Force Microscopy (AFM) revealed strong dependence of helical pitches on cooling rates. The helical pitches increase with decreasing cooling rate. We report experimental measurement on absorption of two orthogonal circularly polarized light by chiral lipid nanotubules of different helical pitches. The optical experiment is carried out with 532 nm, 632 nm and 810 nm lasers. The preference of absorption of one handed of circularly polarized light is revealed through circular dichroism study along with an evidence of absorption strength increasing with the length of lipid tubule helical pitches. Thus, other than molecular chirality, chirality of lipid tubules can be enhanced through controlled cooling process during the lipid tubule formation.

2 Key Words: Self-assembled, chirality, ;lipid tubules, nanotubules, circular dichroism Y Jantippana: g @ku.ac.th 1 ก ก ก Department of Physics, Faculty of Science, Kasetsart University, Bangkok Thailand 2 ก ก Department of Physics, Faculty of Science, Mahidol University, Bangkok Thailand 3 ก ก Department of Chemistry, Faculty of Science, Kasetsart University, Bangkok Thailand Introduction Chirality has been significantly employed in many areas of chemistry and physics. When one chiral molecule is substituted by its mirror image, the interactions between molecules change dramatically. This spectacular property leads them to be the basis of many fields, for example stereoisomerism, chiral drug industry and liquid crystals. In liquid crystals, molecular chirality controls to the formation of phases with long-range helical modulations, such as the cholesteric phase used in regular liquid crystal displays. Chirality also plays a major role in generating dipoles in tilted liquid crystal phases, leading to ferroelectric liquid crystals for display applications (Clark and Langerwall 1980). Self-assembly has shown promising potential for improved fabrication of micro and nanostructures. In the past decades, lipid molecules have been reported to self-assemble into micro and nanostructures of different morphologies. Lipid molecules self assemble by aggregation in aqueous solution arranging themselves such that the hydrophilic headgroups of the molecules are exposed to water and the hydrophobic tails are shielded from water normally in the form of bilayers. The lowest-energy state of the bilayers is in general expected to be flat, or to be large spherical vesicles with the minimum curvature needed to shield the hydrophobic region from the water. However, bilayers of many chiral molecules instead form long, narrow cylinders of micro or nanometer ranged diameter, known as tubules (Yager and Schoen 1984; Georger et al. 1987; Schnur 1993). The tubules are formed by the ribbons of chiral diacetylenic phosphocholine molecules detach from lipid bilayer films and wrap themselves into cylinders with diameter 500 nm and typical length of µm. This high aspect ratio makes them attractive for both scientific research and various applications. Helical winding of bilayer ribbons into lipid tubules suggests the mechanism of the tubule formation majorly controlled by molecular chirality (Selinger and Schnur 1993). Attempt has been made in controlling the length of lipid tubules by controlled cooling rate during the tubule formation. It was found that the slower the cooling rate the longer the tubule length (Thomas et al. 1995). The question must then be discussed since the length can be varied by

3 controlling the cooling rate then the cooling process must effect the organization of the molecules into lipid tubules. We investigate this hypothesis by means of optical experiment examining circular dichroism (CD) property caused by chirality of lipid tubules. CD measures differences in the absorption of left-handed circularly polarized light versus right-handed circularly polarized light which arise due to structural asymmetry of materials leading to their optically active behavior. CD of lipid tubules was reported for fixed cooling rate on various wall thicknesses of lipid tubules(spector, Price, and Schnur 1999). We report the similar approach to understand the dependence of CD signal on cooling rates during the process of tubule formation. Experiment Details Lipid tubules were prepared by controlling the cooling process of R-enantiomer 1,2-bis- (10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine (DC 8,9 PC) (Avanti Polar Lipids, Alabaster, AL) at a concentration of = 1 mg/ml dissolved in ethanol/water (75:25, v:v). The solution were cooled at cooling rates (R) 0.6 C/hr, 1.2 C/hr, 1.8 C/hr, 4.8 C/hr and 10.0 C/hr from 60 C to room temperature obtaining lipid tubules suspended in the mixture. 254 nm-uv-polymerization was carried out for 20 minutes at room temperature. Lipid tubules were stored at 4 C until removed for characterization. AFM imaging of dried lipid tubules was performed in tapping mode to probe the physical structure of the tubules. Angle β between the helical stripes and tubule length normal was measured to understand the relationship between the helical pitch and the cooling rate. We then characterized the property of lipid tubules from each cooling rates further by investigating the circular dichroism property of the tubes. Lipid tubule suspension was diluted to the concentration of 0.33 mg/ml so that the mixture is transparent enough for the transmission of the laser light. It was filled in the 18 mm x 18 mm x 3mm glass box made of microscope glass coverslips for optical investigation. The optical setup for circular dichroism study is shown in Figure 1. Polarized lasers of wavelengths 532 nm, 633 nm and 810 nm were employed as light sources. Polarized light was directed to a quarter waveplate obtaining either right circularly polarized light or left circularly polarized light depending on the orientation +45 or -45 of the quarter waveplate with respect to the polarization axis of the laser. The circularly polarized laser was directed to the sample box placed in an optical path with transmission light detected by the PicoHarp 300 single photon counter from PicoQuant. The data collected by the single photon counter were analyzed to understand the chiral property of lipid tubules.

4 Figure 1 Experimental setup for circular dichroism study Experimental Results AFM images of chiral lipid tubules from different cooling rates were obtained. We measured the angle β of helical winding with respect to tubule length normal shown as white line drawn on an AFM image of lipid tubules cooled at 1.2 C/hr in Figure 2. The measurements were performed for 100 repeats on each cooling rate attaining a plot of average angle β measured versus inverted cooling rate (1/R) together with a guassian fitting curve of angle β illustrated in Figure 3. With lower cooling rates, β increases which can be concluded geometrically that the pitch of helical winding must increase as well. 1 µm Figure 2 An AFM image of lipid tubules cooled at 1.2 C/hr along with the drawing of helical angle measurement with respect to tubule length normal.

5 Figure 3 plot of average angle measured with respect to tubule length normal versus inverted cooling rate (1/R). Transmission intensity I was averaged from collected transmission signal by a single photon counter shown in Figure 1. The signal was detected at 100 ms time interval for 30 seconds on each sample prepared at different cooling rates. Transmission intensity through an empty glass box was obtained as reference intensity I o. Absorbance A of the sample is calculated from A log I I = for the data collected with left circularly polarized light (LCPL) and right circularly polarized light (RCPL) directed on the sample denoted by A and LCPL A respectively. The difference in absorbance RCPL Acan be calculated from A= A LCPL A illustrated in the plot of Figure 4. Lipid tubules show RCPL significantly higher absorption of left circularly polarized light than that of right circularly polarized light for the laser of wavelength 532 nm and 633 nm and no difference is found for an 810 nm laser. From the plot in Figure 4, absorbance difference is high in the high cooling rate which normally would refer to higher chirality for chiral molecule detector. However, this result contradicts with the physical appearance of helical winding of lipid tubules because in the higher cooling rate, smaller β with shorter helical pitch is observed which means the helical winding is weak. To gain a complete parameter of circular dichroism other than A for historical reasons since most measurements are reported in degrees of ellipticity (Rodger and Nordén 1997), molar ellipticity [ θ ] can be computed from A [ θ ] = , where c is the sample concentration (molar) and l is the path length of light c l passing through the sample (cm). Computed molar ellipticities are plotted in Figure 5. Thus molecular 0

6 organization of lipid molecules into lipid tubules is found such that looser helical winding causes high absorbance difference, thus high molar ellipticity of the structure. Figure 4 Plot of the difference in absorbance different laser sources. A= A LCPL A versus inverted cooling rate (1/R) for RCPL Figure 5 Plot of molar ellipticity versus inverted cooling rate (1/R) for different laser sources.

7 Conclusion Investigation of physical structure of lipid tubules by AFM imaging obtained the helical angle (with respect to tubule length normal) dependence on cooling rate. With lower cooling rate, higher helical angle is observed. We further examined the chirality of lipid tubules by laser circular dichroism study. The result shows that lipid tubules formed from R-enantiomer molecules absorbing left circularly polarized light more than right circularly polarized light in the visible range region (532 nm and 633 nm wavelengths in our study). Absorption is found depending on helical angle of lipid tubules, thus depending on cooling rate during the formation process. With higher cooling rate, lower helical angle, higher chirality of lipid tubules is found based on the observation of higher molar ellipticity from circular dichroism. Acknowledgement This work is supported by Kasetsart University Research and Development Institute, National Research Council of Thailand and National Nanotechnology Center, Thailand. References Clark, Noel A., and Sven T. Langerwall Submicrosecond bistable electro-optic switching in liquid crystals. Appl. Phys. Lett. 36 (11): Georger, Jacque H., Alok Singh, Ronald R. Price, Joel M. Schnur, Paul Yager, and Paul E. Schoen Helical and tubular microstructures formed by polymerizable phosphatidylcholines. J.Am.Chem.Soc 109 (20): Rodger, Alison, and Bengt Nordén Circular Dichroism and Linear Dichroism. Oxford, UK: Oxford University Press. Schnur, Joel M Lipid Tubules: A Paradigm for Molecularly Engineered Structures. Science 262: Selinger, Jonathan V., and Joel M. Schnur Theory of Chiral Lipid Tubules. Phys. Rev. Lett. 71 (24): Spector, Mark S., Ronald R. Price, and Joel M. Schnur Chiral Lipid Tubules. Adv. Mater 11 (4): Thomas, Britt N., Cyrus R. Safinya, Robert J. Plano, and Noel A. Clark Lipid Tubule Self- Assembly: Length Dependence on Cooling Rate Through a First-Order Phase Transition. Science 267: Yager, Paul, and Paul E Schoen Formation of tubules by a polymerizable surfactant. Mol. Cryst. Liq. Cryst. 106: