Toward the Rational Control of Nanoscale Structures Using Chiral Self

1888

Langmuir 2003, 19, 1888-1898

Toward the Rational Control of Nanoscale Structures Using Chiral Self-Assembly: Diacetylenic Phosphocholines† Alok Singh,* Eva M. Wong, and Joel M. Schnur Center for Bio/Molecular Science & Engineering, Code 6900, Naval Research Laboratory, 4555 Overlook Avenue SW, Washington, D.C. 20375 Received August 1, 2002. In Final Form: November 12, 2002 Results reported in this study provide, for the first time, a simple approach to tune the diameter of nanoscale diacetylenic phospholipid-based tubules. We have found a clear correlation between the n segments of the acyl chains in diacetylenic phosphocholines and the diameter of nanotubules formed from lipid mixtures containing diacetylenic phosphocholine and a short-chain “spacer” lipid. Microscopic structures in the aqueous dispersions of equimolar mixtures of diacetylenic phospholipid, DCm,nPCs, and saturated spacer lipids (DCm′PCs) were studied to understand and formulate the basis for developing the “design” rules for the formation of stable nanoscale structures. An increase in the number of n resulted in an increase in the diameter of the tubules produced by the mixtures. Circular dichroism (CD) spectra of these mixtures were analyzed and the very complex CD spectra clearly confirmed the chiral nature of the nanotubule architecture. Tubules from most of the mixtures demonstrated long-term storage stability.

Introduction It is clear that ultrasmall objects of different shapes can have significant application value.1-3 This paper describes methods of making diacetylenic lipid based nanoscale hollow cylinders and design rules for controlling their diameter. Phospholipids by virtue of their unique self-assembling properties are among the most effective building blocks for making structures at the scale ranging from micrometer to nanometer.4-11 An understanding of the self-organization process of small molecules into identifiable morphologies is the key to developing versatile approaches for building nanoscale materials.1-3 Current research activities signifying the technological importance of tubule morphology1,4-7,12-15 motivated us to focus our research efforts on the exploration of the phospholipid self-assembling process to create structures of desired dimensions predictably and reproducibly. The formation † Part of the Langmuir special issue entitled The Biomolecular Interface.

(1) Schnur, J. M. Science 1993, 262, 1669. (2) Shimizu, T. Macromol. Rapid Commun. 2002, 23, 311. (3) Lehn, J. M. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 4763. (4) Singh, A.; Schnur, J. M. Polymerizable Phospholipids. In Phospholipids Handbook; Cevc, G., Ed.; Marcel Dekker: New York, 1993; pp 233-291. (5) Bong, D. T.; Clark, T. D.; Granja, J. R.; Ghediri, M. R. Angew. Chem., Int. Ed. 2001, 40, 988. (6) Ariga, K.; Kikuchi, J.; Naito, M.; Koyama, E.; Yamada, N. Langmuir 2000, 16, 4929. (7) Fuhrhop, J. H.; Helfrich, W. Chem. Rev. 1993, 93, 1565. (8) Zastavker, Y. V.; Asherie, N.; Lomakin, A.; Pande, J.; Donovan, J. M.; Schnur, J. M.; Benedek, G. B. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 7883. (9) Markowitz, M. A.; Chang, E. L.; Singh, A. Biochem. Biophys. Res. Commun. 1994, 203, 296. (10) Svenson, S.; Messersmith, P. B. Langmuir 1999, 15, 4464. (11) Spector, M. S.; Singh, A.; Messersmith, P. B.; Schnur, J. M. Nano Lett. 2001, 7, 375. (12) Lasic, D. D. Liposomes: from physics to applications; Elsevier: New York, 1993. (13) Wilson-Kubalek, E. M.; Brown, R. E.; Celia, H.; Milligan, R. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 8040. (14) Yu, X.; Dillon, G. P.; Bellamkonda, R. V. Tissue Eng. 1999, 5, 291. (15) Lvov, Y. M.; Price, R. R.; Selinger, J. V.; Singh, A.; Spector, M. S.; Schnur, J. M. Langmuir 2000, 16, 5932.

10.1021/la026337r

of tubular structures from diacetylenic phospholipids demonstrates that modest chemical alteration in natural phospholipids exerts a profound influence on the morphology of resultant structures.1,4,16,17 Subsequent studies following Yager and Schoen’s initial report on the formation of micron-sized tubules with a fixed internal diameter of 500 nm16 have revealed the formation of other forms of self-assembling structures including helices, ribbons, and tubules17 and the means to produce tubules with variable diameters.18,19 The goal of the current study is to understand and formulate the basis for developing the “design” rules for the formation of stable nanoscale structures. Initial studies on binary lipid mixtures comprised of a short-chain saturated phospholipid, 1,2-dinonanoyl-snglycero-3-phosphocholine (DC7PC, previously named as DNPC), and a diacetylenic phospholipid, 1,2-bis(tricosadiyn-10,12-oyl)-sn-glycer-3-phosphocholine (DC8,9PC), demonstrated the formation of nanotubules and helices.9,20,21 In this system, the acyl chain length of the saturated lipid was equal to the number of methylene groups present between the diacetylenic moiety and the ester group on the glycerol backbone. This system produced 28 nm wide ribbons in water and salt solution depending on the lipid concentration and nanotubules upon careful thermal manipulation of aqueous dispersions of an equimolar mixture of lipids.10,11 The observation of chiraloptical signatures of nanotubule, twisted ribbon, and microtubule morphologies in circular dichroic spectra has been reported, and their use for monitoring the temporal and thermal stability of structures has been suggested.10,22 Recent observations on the influence of individual lipid (16) Yager, P.; Schoen, P. E. Mol. Cryst. Liq. Cryst. 1984, 106, 371. (17) Georger, J. H.; Singh, A.; Price, R. R.; Schnur, J. M.; Yager, P.; Schoen, P. E. J. Am. Chem. Soc. 1987, 109, 6169. (18) Markowitz, M. A.; Singh, A.; Schnur, J. M. Chem. Phys. Lipids 1992, 62, 193. (19) Singh, A.; Markowitz, M. A.; Tsao, L. Chem. Phys. Lipids 1992, 63, 191. (20) Singh, A.; Gaber, B. P. Applied Bioactive Polymeric Materials; Gebelein, C. G., Carraher, C. E., Forster, V. R., Eds.; Plenum Press: New York, 1988; p 239. (21) Rhodes, D. G.; Singh, A. Chem. Phys. Lipids 1991, 59, 215.

This article not subject to U.S. Copyright. Published 2003 by the American Chemical Society Published on Web 12/19/2002

Control of Nanoscale Structures

Langmuir, Vol. 19, No. 5, 2003 1889

Figure 1. Schematic representation of groups of binary lipid mixtures of diacetylenic DCm,nPC with saturated DCm′PC.

content in lipid mixtures on microscopic morphologies and the effect of methylene units in the distal segment (methylterminated segment) of diacetylenic phospholipid acyl chains on the diameter of nanotubules led to the initiation of the current studies.22 The focus of this study is to determine a means of rational control of these small structures, particularly tubules from binary lipid systems. We selected three groups of lipid mixtures derived from binary lipid systems involving diacetylenic DCm,nPCs and saturated spacer lipids DCm′PCs as shown in Figure 1. The first group consists of DC8,9PC mixtures with 1,2-disaturated phosphocholines containing variable m′ (6, 7, 8, 9, and 10) segments (group A) and explores the influence of the acyl chain length of spacer lipids on tubule structures; the second set consists of DC7PC mixtures with diacetylenic lipids containing variable distal chain length while keeping proximal chain segment fixed (m,n ) 8,11 and 8,13; group B); and the third set contains 1,2-diundecanoyl phosphocholine (DC9PC) mixtures with diacetylenic lipids consisting of fixed proximal chain length and variable distal chain length, DC10,nPC (n ) 9,11,13; group C). The latter two groups probe the influence of the n segment on the tubule formation. The mixtures examined in this study consisted of equimolar lipid ratios while keeping the total lipid concentrations fixed at 2 mM. The morphologies from each mixture were characterized using transmission electron microscopy and circular dichroic spectroscopy at different temperatures. Materials and Methods All diacetylenic phospholipids used in this study were synthesized in our laboratory following the previously reported procedures.23,24 Saturated phosphocholines 1,2-bis-nonanoyl (DC7PC), 1,2-bis-octanoyl (DC6PC), 1,2-bis-decanoyl (DC8PC), 1,2-bis-undecanoyl (DC9PC), and 1,2-bis-dodecanoyl (DC10PC) were purchased from Avanti Polar Lipids. The following protocol was used for making nanotubules from lipid dispersions: equimolar amounts of a diacetylenic phospholipid, DCm,nPC, with an appropriate saturated phosphocholine, DCm′PC, were mixed in chloroform, and the solvent was evaporated in a gentle stream of nitrogen, leaving a thin lipid film on the walls of the container. This thin lipid film was vacuumdried and hydrated in ultrapure water (18.2 MΩ cm) at 70 °C for 3 h. Lipids were dispersed by intermittent vortex mixing while maintaining the temperature at 70 °C until a homogeneous dispersion was formed. All the lipids dispersed easily to produce a translucent solution. Lipid dispersions were then placed in a Dewar flask filled with hot water (70 °C) and left at room temperature for 2 h. The flask was then placed in a chamber maintained at 4 °C for least 10 h before characterization. All (22) Singh, A.; Wong, E. M.; Spector, M. S.; Schnur, J. M. Mater. Res. Soc. Symp. Proc. 2002, 711, HH3.36.1. (23) Singh, A.; Schnur, J. M. Synth. Commun. 1986, 16, 847. (24) Singh, A. J. Lipid Res. 1990, 31, 1522.

Table 1. Diameter of Nanotubules Formed from Equimolar Binary Lipid Mixtures phospholipid composition

average tubule diameter (nm)

DCmPC DCm,nPC fresh sample 105 days m′ m n (4 °C) (4 °C)

120 h (RT)

6 8 9 10

8 8 8 8

9 9 9 9

Group A 57 ( 4 58 ( 2 52 ( 4 52 ( 5 124 ( 7 63 ( 3 93 ( 6 55 ( 3

7 7

8 8

11 13

Group B 63 ( 4 70 ( 5 108 ( 4 88 ( 6

95 ( 6 94 ( 6

9 9 9

10 10 10

9 11 13

Group C 60 ( 4 69 ( 2 81 ( 7 75 ( 3 100 ( 6 117 ( 6

79 ( 8 83 ( 8 127 ( 12

30 (filament width) 89 ( 11, shards 75 ( 12, shards 167 (vesicle diam), 25 (ribbon width)

preparations were very sensitive to photopolymerization and therefore were protected from direct laboratory light throughout the experiment. The following is the list of phospholipid amounts in each lipid mixture used in the preparations of 2 mL dispersions in water. Each mixture prepared has a total lipid concentration of 2 mM. For group A, 1.82 mg of DC8,9PC was mixed with DC6PC (1.08 mg), DC8PC (1.13 mg), DC9PC (1.18 mg), and DC10PC (1.24 mg); for group B, DC7PC (1.06 mg) was mixed with DC8,11PC (1.93 mg) and DC8,13PC (2.05 mg); and for group C, 1.18 mg of DC9PC was mixed with DC10,9PC (1.93 mg), DC10,11PC (2.05 mg), and DC10,13PC (2.16 mg). The microscopic structures in dispersions of each binary lipid system were characterized by transmission electron microscopy (TEM) and circular dichroism (CD) spectroscopy. These characterizations were carried out on fresh samples just after their preparation was complete, after annealing for 105 days at 4 °C, and after incubating at room temperature for 120 h. Samples were maintained at 4 °C during the characterization process involving CD spectroscopy. Circular dichroism studies were carried out on a Jasco J-720 spectropolarimeter. Samples were removed from the cold chamber and immediately placed in a quartz cell (1.00 or 0.5 mm path length) that had been precooled to 4 °C by immersing it in a water bath. Transmission electron microscopic images of samples maintained at 4 °C were taken on a Zeiss EM-10C electron microscope operating at 60 kV. The room-temperature samples were done on the Hitachi H8100 at 200 kV. Stained samples were prepared by placing a few microliters of aqueous 1% (w/v) uranyl acetate onto a drop of tubule dispersion placed on a carbon-coated grid. The excess solvent was removed by wicking.

Results All the positional isomers of diacetylenic phosphocholines (DCm,nPC) used in this study produced microtubules of 500 nm diameter when dispersed in water as single components.4 Table 1 summarizes results from electron microscopy studies of the structures produced by various

1890

Langmuir, Vol. 19, No. 5, 2003

Singh et al.

Figure 2. TEM images and CD spectra of equimolar dispersions of a DC8,9PC/DC6PC mixture (2 mM) in water at 4 °C: (a,c) fresh sample; (b,d) annealed for 105 days. Table 2. Circular Dichroic Properties of Dispersions from Equimolar Binary Lipid Mixtures at 4 °C lipid composition DCm,nPC DCmPC, m′

m

n

peak wavelength (λnm) and molecular ellipticity (deg cm2/dmol) fresh samples

annealed samples (105 days) Group A

192 (-6.0 × 103); 194 (+4.9 × 103); 196 (-1.2 × 104); 198 (-1.0 × 104) 194 (+4.5 × 104); 204 (-5.0 × 102) 196 (+6.1 × 104); 194 (+4.1 × 104); 203 (-4.8 × 104) 194 (+2.3 × 105)

6

8

9

8 9 10

8 8 8

9 9 9

7 7

8 8

11 13

200 (+1.2 × 105); 207 (-6.9 × 104) 201 (+1.7 × 105); 197 (+3.8 × 104)

9 9 9

10 10 10

9 11 13

198 (+5.4 × 105); 208 (-3.8 × 104) 199 (+3.5 × 105) 201 (+9.8 × 105); 200 (+9.9 × 105)

Group B

Group C

combinations of binary lipid mixtures at three time intervals: immediately after the formation was complete, after equilibrating by annealing at 4 °C for 105 days, and after storing at room temperature for 120 h. Table 2 compiles the results from CD studies on the molecular ellipticities and the absorption peaks of the mixed lipid dispersions soon after the formation of nanotubules and after equilibration at 4 °C for 105 days. TEM images from all the freshly prepared mixtures typically show the formation of nanotubules in abundance along with a few vesicles. In mixed lipid dispersions of any specific lipid composition, only small variations in diameter were observed. The diameter of nanotubules produced by each lipid mixture was obtained by following a single standard protocol, and no special efforts were made toward optimization of nanotubule formation. The diameters presented in Table 1 represent those observed from an overwhelming majority of the tubules. Figures 2-10 are compiled to provide information on the morphological features of nanotubules and CD spectral characteristics from each mixed lipid system. Figures 11 and 12 show

194 (+8.3 × 104); 198 (-2.9 × 104) 192 (+1.2 × 104); 194 (+8.0 × 103); 199 (-2.9 × 104) 192 (+7.0 × 103); 198 (-1.6 × 105) 195 (+3.9 × 105) 199 (+3.9 × 104); 207 (-9.8 × 104) 201 (+1.0 × 105); 194 (+4.9 × 103) 199 (+3.9 × 105) 199 (+4.4 × 105) 201 (+1.6 × 105); 194 (-1.4 × 105)

changes in the morphologies from each mixed lipid system after incubating at room temperature (25 °C) for 120 h. Since the morphological features observed for DC9PC mixtures with DC10,9PC and DC10,11PC were identical, only the nanotubule image from the DC10,11PC/DC9PC mixture is included. All the mixtures examined in this study produced nanotubules showing their outer boundary as a parallel track and being devoid of any striation-like features that are typical of microtubules produced from single-component diacetylenic lipid systems.1 The freshly prepared nanotubules produced from equimolar mixtures of diacetylenic and saturated phosphocholines produced nanotubule structures with three main morphological features represented by fully formed tubules of 57-108 nm diameter (DC7PC with DC8,11PC and DC8,13PC, and a DC6PC mixture with DC8,9PC), not fully formed tubules with curled-up edges (DC8,9PC/DC10PC, DC8,9PC/DC8PC, and DC10,9PC/DC9PC mixtures), and angular twisted ribbons that attain tubule shape (DC8,9PC/C9PC). The majority of the mixtures upon annealing at 4 °C for 105 days produced


Langmuir, Vol. 19, No. 5, 2003 1891

Figure 3. TEM images and CD spectra of equimolar dispersions of a DC8,9PC/DC8PC mixture (2 mM) in water at 4 °C: (a,c) fresh sample; (b,d) annealed for 105 days.


tubules with larger diameter. In only one case were helical spirals observed (Figure 14). Room-temperature incubation produced three morphologies: large vesicles, nanotubules with further increased diameter, and nanometerwide ribbons. No microtubules were observed within the reported time period. Self-Assembling Properties of Diacetylenic Lipids from Group A Lipid Mixtures. Figures 2 and 3 show the morphologies of nanotubules prepared by mixing DC8,9-

PC with saturated phosphocholines consisting of 8 carbon to 12 carbon long acyl chains. The nine carbon atom long acyl chain completely masks the proximal segment of DC8,9PC, leaving the diacetylenic moiety free to interact with diacetylenes of neighboring lipid molecules. This perfect match leads to the formation of nanotubules with an average diameter of 50 nm. Saturated PC with m′ ) 6 produced 57 nm diameter tubules. When the chain length was increased by 2 carbon units (m′ ) 8) to mask the first

1892


Singh et al.



carbon of the diacetylenic moiety, 52 nm diameter tubules were produced. Annealing caused these structures to equilibrate and produced well-formed nanotubules. The differences in the diameters were small and obtained by independently measuring the tubules from each mixture. The small changes reported here, though real, can be considered similar in a broader sense. CD spectra of these two samples showed completely opposite behavior (Figures

2 and 3). One carbon unit short spacer produced two peaks of opposite polarity, the negative peak being larger. Annealing showed a moderate change in diameter, but the CD spectra showed the exclusive appearance of a positive peak at 194 nm. One carbon long spacer, DC8PC, produced nanotubules that showed only two positive peaks. In this case, annealing produced a predominantly negative CD peak at 198 nm. Similarly, room-temperature


Langmuir, Vol. 19, No. 5, 2003 1893



storage produced 30 nm wide ribbons for DC6PC and the latter produced 78-100 nm diameter tubules (Figure 11a,b). A further increase in spacer acyl chain length to 11 and 12 carbon atoms (m′ ) 9 and 10) to completely mask one acetylene segment of diacetylene and both acetylenes, respectively, produced 117 nm diameter tubules made from twisted ribbons (DC9PC, Figure 4) and 93 ( 6 nm diameter tubules (Figure 5). In both cases, annealing caused the nanotubule diameter to shrink to 63 ( 3 and 55 ( 3 nm, respectively. In the case of DC9PC, the CD spectrum showed one large negative peak at 198 nm upon

annealing and the absence of two large positive peaks previously observed in fresh preparation. The DC10PC spacer produced a positive peak that remained positive upon annealing. Room-temperature incubation produced a stable nanotubule morphology for the mixture with the DC9PC spacer (Figure 11c), whereas for the DC10PC spacer only vesicles were observed. Self-Assembling Properties of Diacetylenic Lipids from Group B Lipid Mixtures. The lipid mixtures of this group were examined to evaluate the influence of the n segment on the diacetylenic lipid assembly using the saturated spacer lipid that perfectly matched the m

1894


Singh et al.



segment. In this case, m is 8 and n is 9, 11, or 13. Results on n ) 9 have been previously reported in the literature.10,11 Results from the current study are compiled in Figures 6, 7, and 12a,b. The inclusion of the previously reported diameter of tubules (55 nm) from n ) 9 with the results reported here shows the trend that nanotubule diameter increases with an increase in the n segment. Thus, 55, 63, and 108 nm diameter nanotubules were observed for n ) 9, 11, and 13, respectively. CD spectra of a freshly prepared

sample of the DC8,11PC/DC7PC mixture showed both positive (200 nm) and negative (207 nm) peaks, which retained the peak position upon annealing for 105 days; however, the intensity of the negative peak increased. The mixed lipid system involving the DC8,13PC/DC7PC mixture showed only a single positive CD peak with large molecular ellipticity. Following annealing, the peak narrowed and the molecular ellipticity value decreased, but the nanotubules remained almost unchanged (Figure 7).


Langmuir, Vol. 19, No. 5, 2003 1895

Figure 11. TEM images of equimolar dispersions of binary mixtures incubated for 120 h at 25 °C after annealing at 4 °C: (a) DC8,9PC/DC6PC; (b) DC8,9PC/DC8PC; (c) DC8,9PC/DC9PC; (d) DC8,9PC/DC10PC.

Figure 12. TEM images of equimolar dispersions of binary mixtures incubated for 120 h at 25 °C after annealing at 4 °C: (a) DC8,11PC/DC7PC; (b) DC8,13PC/DC7PC; (c) DC10,11PC/DC9PC; (d) DC10,13PC/DC9PC.

Upon incubating at room temperature, both lipids produced nanotubules, which have a larger diameter than those observed in annealed samples. A remarkable stability of the nanotubules was observed in these mixtures, and unlike the DC8,9PC/DC7PC mixture, helical ribbons and microtubules were not observed. Self-Assembling Properties of Diacetylenic Lipids from Group C Lipid Mixtures. This mixed lipid system is similar to the group B system except the m and m′ segments of both the diacetylenic and spacer lipid are two carbon atoms longer than those of the group B system. The goal of this study was to examine the influence of overall acyl chain length on the tubule diameter. The results are compiled in Figures 8-10 and 12c,d. Overall, mixtures in these groups demonstrated nanotubule sta-

bility and an increase in tubule diameter with respect to an increase in n. The lipid mixtures from DC10,9PC/DC9PC and DC10,11PC/DC9PC showed marked similarities in their CD and structural behavior. Both of these mixtures showed nanotubules of 60 ( 4 and 81 ( 7 nm diameters and single positive peaks in their CD spectra at 198 and 199 nm with large molar ellipticities similar to those reported for nanotubules.21 The CD spectra remained unchanged in both cases even after 105 days of annealing at 4 °C (Figures 8 and 9). The DC10,13PC/DC9PC on the other hand showed a positive peak at 201 nm and a negative peak at 194 nm. This is the reverse of the trend in CD described previously, since the peaks at shorter wavelength were always positive and the peaks at longer wavelength changed their polarity with no obvious

1896


predictable trend. Figure 12c,d shows the TEM images of the nanotubules examined after incubating the annealed tubules for 120 h at room temperature. The close similarity in nanostructures from each mixture indicates the stability of the tubules. In this case, the trend in increasing nanotubule diameter with an increase in the n segment is also clear. For n ) 9, 11, and 13, lipid mixture tubule diameters of 69 ( 3, 75 ( 3, and 117 ( 6 nm were observed (Table 1). Discussion Hollow cylindrical structures with high aspect ratios have important technological applications.1,5 A reduction in tubule diameter leads to higher aspect ratio structures at constant tubule lengths. Therefore, control over the tubule diameter will have a significant impact on those technologies that utilize nanotubules for their aspect ratios. This study demonstrates such control. Numerous published reports are emerging that focus on the formation of nanotubules from precursors of biological origin, for example, lipids, peptides, and carbohydrate-based surfactants.1,5-8,25 The production of strong circular dichroic signatures by the helical aggregates of amphiphiles provides a simple means to characterize surfactant dispersions for the presence of helical structures.25-27 We have previously reported that the bilayer assemblies prepared by the addition of a short-chain phosphocholine (DC7PC) to a diacetylenic phosphocholine, DC8,9PC, enhanced polymerization of diacetylenes in bilayers20 without interrupting the formation of regular bilayer structures.21 However, the formation of a gel consisting of 28 nm wide ribbons at room temperature and the formation of nanoribbons and nanotubules in water at 4 °C9,10 led us to examine the mixed lipid system by CD spectropolarimetry.11 The circular dichroic spectral behavior of microtubules (500 nm diameter) prepared from diacetylenic phosphocholines and their positional isomers have been previously studied. All diacetylenic lipids studied so far have produced 500 nm diameter tubules irrespective of their acyl chain length and the placement of diacetylenes within. Their CD spectra were reported to produce several peaks of large molar ellipticities in the range of 0.05-2.5 × 106 deg cm2/ dmol between 192 and 195 nm due to the diacetylenic chromophore and between 202 and 206 nm due to the carbonyl chromophore,26-28 and the peak intensity observed due to carbonyl (∼205 nm) was lower than those observed at 195 nm.27 The peak polarity (positive or negative) was dependent on the optical isomer used in the study. Previous studies reported that nanotubules or nanohelices produce a strong negative CD peak at ∼202 nm along with a positive CD peak at the shorter wavelength as opposed to microtubules, which produced peaks with a single polarity.11,22 In the current study, CD was used as a tool for screening the lipid dispersions for the presence of structures at the sub-500-nm scale. Figures 2-10 present a side-by-side visual comparison of the nanotubule morphologies and the CD spectral trends of mixed lipid groups examined in this study. The following sections discuss the observed trends in a simple but systematic fashion to understand the following: (1) the (25) Nakashima, N.; Ando, R.; Muramatsu, T.; Kunitake, T. Langmuir 1994, 10, 232. (26) Schnur, J. M.; Ratna, B. R.; Selinger, J. V.; Singh, A.; Jyothi, G.; Easwaran, K. R. K. Science 1994, 264, 945. (27) Spector, M. S.; Easwaran, K. R. K.; Jyothi, G.; Selinger, J. V.; Singh, A.; Schnur, J. M. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 12943. (28) Spector, M. S.; Selinger, J. V.; Singh, A.; Rodriguez, J. M. Langmuir 1998, 14, 3493.

Singh et al.

effect of saturated phosphocholine chain length on the structure-forming capabilities of DC8,9PC, (2) the influence of diacetylenic acyl chain length on the structure-forming capabilities of diacetylenic phosphocholines, and (3) the effect of annealing on nanotubules at 4 °C and room temperature. An analysis of the circular dichroic properties of both freshly prepared and annealed nanotubules from binary lipid systems is discussed separately. Effect of the Saturated Acyl Chain Length of Spacer Lipids on Nanotubule Formation. One of the roles of the diacetylenic moiety of phospholipids in bilayer membranes is as a morphology modulator. This is achieved through electronic and geometrical interaction with neighboring molecules. In the DC8,9PC/DC7PC mixture, DC7PC makes a perfect spacer for DC8,9PC without masking the interaction between neighboring diacetylenic moieties. In DC8,9PC/DC6PC and DC8,9PC/DC8PC mixtures, both of the saturated lipids, DC6PC and DC8PC, fall short in masking the diacetylene functionality from interacting with neighboring diacetylenes in bilayers and produce nanotubules of 43 and 47 nm diameter. Equilibrium structures obtained after annealing at 4 °C have a diameter in the range of 55 nm, similar to that reported for the DC8,9PC/DC7PC system.10,11 On the other hand, acyl chains of saturated lipids, DC9PC and DC10PC, are capable of masking diacetylenes in bilayers. Interesting behavior was observed for the DC8,9PC/DC9PC and DC8,9PC/DC10PC mixtures. The first such behavior was the formation of large-diameter tubules from the DC8,9PC/ DC9PC mixture in the freshly prepared samples held at 4 °C (Figure 4). A closer look at the structures revealed that the large diameters (140-240 nm) are caused by two types of crack patterns presumably produced by ruptures in the walls of nanotubules (Figure 13). These ruptures appear to have occurred at the chiral domain boundaries producing high-pitch (Figure 13a) and low-pitch (Figure 13b) patterns. These observations support the model proposed by Selinger et al.31 The second unusual behavior of the DC9PC/DC8,9PC mixtures is the healing of the breaks in domain boundaries upon annealing the tubule dispersions at 4 °C for 105 days (Figure 4). The formation of 63 ( 3 nm diameter tubules follows the trend set for DC8,9PC-based systems. The CD spectrum shows an exclusive negative peak at 198 nm with large molar ellipticity. Structures produced by equimolar mixtures of DC8,9PC/DC6PC constitute the only system apart from the DC8,9PC/DC7PC system that produced low-pitch helical ribbons upon annealing at 4 °C for 105 days. Figure 14 shows the formation of 100 nm diameter helices involving 36 nm wide ribbons along with tubules of varying diameter ranging from 45 to 80 nm. Effect of Diacetylenic Acyl Chain Length on Nanotubule Formation. From a technology point of view, this is an important variable. Diacetylenic acyl chains of phosphocholines pose a unique opportunity in interpreting their self-assembling properties based on more than one variable, for example, the effect of chain length in the m and n segments and the overall length of the acyl chains. Since this study involves saturated phospholipid spacers with saturated acyl chains equal to m in length, only two variables, n and the overall length of acyl chains, are discussed regarding their influence on nanotubule diameter. Table 1 shows the effect of the n segment on the tubule diameter formed from DCm,nPC and DCm′PC mixtures (groups A and B). An incremental increase in the diameter of nanotubules in binary lipid mixtures is observed as the value of n is increased. This change in diameter is a


Langmuir, Vol. 19, No. 5, 2003 1897

Figure 13. TEM images showing high-pitch (a) and low-pitch (b) breaks in chiral domains during the formation of nanotubules from an equimolar mixture of DC8,9PC/DC9PC at 4 °C.

Figure 14. TEM images showing production of low-pitch helical ribbons after equilibrating nanotubules from a DC8,9PC/DC6PC mixture at 4 °C for 105 days.

property that is uniquely associated with the mixed lipid systems. Previous studies have reported the formation of only 500 nm diameter tubules from more than 20 phospholipids from the DCm,nPC series.4 Values of annealed and equilibrated samples were used for comparison. Samples from DC8,nPC (n ) 9, 11, 13) mixtures with DC7PC produced nanotubules of 55 ( 5 nm (n ) 9),27 70 ( 5 nm (n ) 11), and 88 ( 6 nm (n ) 13) diameter. A similar trend was observed for DC10,nPC (n ) 9, 11, 13) mixtures with DC9PC, which produced 69 ( 2 nm (n ) 9), 75 ( 3 nm (n ) 11), and 117 ( 6 nm (n ) 13) diameter tubules. The noteworthy trend is that the diameter is not dependent upon the overall length of diacetylenic acyl chain. DC8,11PC and DC10,9PC have 25 carbons in their acyl chains, but the diameters of their tubules are 70 ( 5 and 69 ( 3 nm, respectively. Similarly, DC8,13PC and DC10,11PC with 27 carbons in their acyl chains produced 88 ( 6 and 75 ( 3 nm diameter tubules. Both of these examples reveal that overall chain length has no large effect upon tubule diameters. If we compare the diameter of tubules produced from the lipids containing identical n segments ignoring the value of m, we find the following values for tubule diameter: DC8,9PC and DC10,9PC produced 55 ( 5 and 69 ( 2 nm diameter tubules; DC8,11PC and DC10,11PC produced 70 ( 5 and 75 ( 3 nm diameter tubules; and DC8,13PC and DC10,13PC produced 88 ( 6 and 117 ( 6 nm diameter tubules.

Influence of Annealing on Nanotubule Morphologies. Tubules produced by the mixed lipid systems are stable at 4 °C, and the diameter changes are minimal even after annealing for 105 days. In the cases of DC8,9PC/DC9 PC and DC8,9PC/DC10PC mixtures, annealing helped the formation of tubules leading to smaller diameter tubules. This property makes these tubules technologically attractive. Noticeable changes are observed when tubules are incubated at room temperature (25 °C) for 5 days. Most of the samples retained their nanotubule morphologies except in two cases studies here (Figure 11a,d). While TEM images did not show any noticeable changes in tubule morphologies examined in this study, CD spectra showed complex patterns (Figures 2-10). CD Properties of Self-Assembled Structures from Mixed Lipid Systems. The CD signals in diacetylenic systems arise from the association of diacetylenic and carbonyl functionalities present in the phospholipid molecules. In all of the structures reported here, the diacetylenic moiety has two roles: as a chromophore to produce a specific absorption maximum (∼192-196 nm) and as a morphology modulator to produce chiral assemblies. Lower molecular ellipticity values observed for binary systems as opposed to single diacetylenic lipids may be due to a decrease in diacetylenic content in the mixture caused by addition of spacer lipids (1,2-disaturated phosphocholines). A decrease in signal is also associated with a decrease in π-π* overlap influenced by the insertion of spacer lipids between the diacetylenic acyl chains of neighboring molecules. The carbonyl chromophore, on the other hand, is responsible for an absorption maximum around 198-205 nm, and the molar content in the binary mixture remains unchanged when compared with that in pure diacetylene based systems. Figures 2-10 and the values in Table 2 show that in all the mixed lipid systems major changes are taking place around 200 nm, indicating that in mixed lipid systems carbonyls are experiencing some perturbations presumably from the dispersion medium. The location of carbonyl groups in phospholipids is at the interface of the hydrophilic and hydrophobic segments, which permits easy interaction of water molecules with carbonyls. This interaction may be dependent on the type, size, and morphological features of the assemblies. Polar solvents including water are known to interact with carbonyl groups to produce a hypsochromic shift in their

1898


CD spectrum caused by an nfπ* transition.29 The molecules from water arrange themselves around the carbonyl group in such a way that the ground state is favored. In a brief interval during which the carbonyl group is in the excited state, water molecules do not have sufficient time to reorient themselves in accordance with the new charge distribution. This interaction results in decreasing the energy of the ground state more than that of the excited state leading to a shift of the absorption band toward a shorter wavelength.30 Water is capable of hydrogen bonding with carbonyl oxygen and hence blocking the lone pair of electrons on the oxygen atom. This makes the nfπ* transition more difficult and consequently produces a hypsochromic shift. As stated earlier, in the binary lipid systems variations in the CD absorptions mainly due to carbonyls are observed. These may be due to a variable degree of interaction of water molecules with the carbonyls depending on the subtle changes in molecular assembling patterns in the microscopic structures. (29) Sidman, J. W. Chem. Rev. 1958, 58, 689. (30) Velluz, L.; Legrand, M.; Grosjean, M. In Optical Circular Dichroism: Principles, Measurements and Applications; Verlag Chemie: Weinheim, 1965; p 79. (31) Selinger, J. V.; Spector, M. S.; Schnur, J. M. J. Phys. Chem. B 2001, 105, 7157.

Singh et al.

This hypothesis needs to be validated by additional experiments. Conclusions Results from this study show that mixed lipid systems consisting of diacetylenic phospholipids and short-chain saturated phospholipids provide a key for the development of novel, simple, and efficient strategies for controlling the diameter of “nanotubules”. The n segment of diacetylenic lipids in the presence of a saturated spacer lipid of matching m plays a pivotal role in determining the nanotubule diameter. Our results indicate that the overall length of the acyl chain has less influence on tubule diameter than the chain length of the n segment of diacetylenic phosphocholine. The long-term storage stability of nanotubules over 105 days at 4 °C and the shortterm stability of nanotubules at room temperature make them suitable candidates for developing practical applications. Acknowledgment. This work was supported by the Office of Naval Research. E.M.W. is an NRC Research Associate. Discussions with Drs. Jonathan Selinger and Mark Spector are greatly appreciated. LA026337R

Toward the Rational Control of Nanoscale Structures Using Chiral Self

Recommend Documents