Nanotubes Self-Assembled from Amphiphilic Molecules via Helical

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Nanotubes Self-Assembled from Amphiphilic Molecules via Helical Intermediates Thomas G. Barclay,*,† Kristina Constantopoulos,§ and Janis Matisons‡ Flinders Centre for Nanoscale Science & Technology, School of Chemical and Physical Sciences, Flinders University, Adelaide, South Australia 5042, Australia 3.1. Self-Assembled Helically Based Tubes Found in Nature 3.1.1. Cholesterol from Bile Mixtures 3.1.2. Galactosylceramides 3.1.3. (S)-Nonacosan-10-ol 3.2. Self-Assembled Helically Based Tubes from Isolated Natural Compounds 3.2.1. 12-Hydroxy Stearic Acid and Derivatives 3.2.2. Derivatives of Steroidal Acids 3.2.3. Oleic Acid 4. Helically Based Nanotubes Self-Assembled from Synthetic Phospholipids 4.1. Diacetylenic Phosphatidylcholine 4.1.1. Nanotube Stabilization via Polymerization 4.1.2. How Changes to Molecular Structure Affect Supramolecular Organization 4.1.3. Modification of Supramolecular Structure by External Additives and Forces 4.1.4. Alignment of Nanotubes 4.1.5. Template Directed Synthesis Using DC8,9PC 4.2. Other Phospholipids 5. Helically Based Nanotubes Self-Assembled from Glycolipids 5.1. Aldonamide Head Groups 5.1.1. N-Alkylaldonamides 5.1.2. Diacetylenic Aldonamides 5.1.3. DAP Linked Aldonamides 5.1.4. Mesogenic Glycolipids 5.2. Cardanol-Based Glycolipid Nanotubes 5.3. Vaccenic Acid-Based Glycolipid Nanotubes 5.3.1. Encapsulation of Hydrophobic Molecules into the Tube Walls 5.3.2. Template Directed Synthesis 5.3.3. Control of Nanotube Dimensions 5.3.4. Manipulation and Alignment of Nanotubes 5.4. Oleic Acid-Based Glycolipid Nanotubes 5.5. Glycoamphiphiles with Fluorocarbon Hydrophobic/Oleophobic Tails 5.6. Other Glycolipids 5.6.1. D-Amygdalin Head Group 5.6.2. Diacetylenic Glycopeptidolipids 5.6.3. Disaccharide Head Groups

CONTENTS 1. Introduction 1.1. Molecular Self-Assembly as a Method for the Construction of Nanostructures 1.2. Previous Reviews Discussing Self-Assembly of Helically Based Nanotubes 1.3. Benefits of Helical Construction 1.4. Descriptive Terms 2. Theory of the Self-Assembly of Amphiphiles into Helically Based Nanotubes 2.1. Introduction to the Self-Assembly of Amphiphiles 2.2. Lyotropic and Thermotropic Phases of Amphiphiles in Bilayers 2.2.1. Liquid Crystalline or Crystalline Packing? The Argument 2.3. Theoretical Models for the Self-Assembly of Helically Based Nanotubes 2.3.1. Use of Macro-Scale Morphology To Assign Constants for Micro-Scale Interactions: Continuum Theory 2.3.2. Use of Micro-Scale Pair Potentials between Individual Chiral Molecules 2.3.3. Meso-Scale: Effect of Tilt Direction on Elastic Properties 2.4. Chiral Symmetry Breaking 2.5. Relating the Hand of Helices to the Molecular Structure of the Amphiphile 2.6. Stability of Ribbon Edges 2.7. Supramolecular Chiral Ordering by Low Quantities of Chiral Species 2.8. Specific Molecular Factors Affecting Helical Self-Assembly of Amphiphiles 3. Nanotubes Self-Assembled via Helical Intermediates from Biological Sources © 2014 American Chemical Society

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10228 Received: February 9, 2013 Published: October 7, 2014

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Chemical Reviews 5.6.4. Phenyl Glucosides 5.6.5. Glyco-bolaamphiphiles 6. Amphiphiles Constructed with Amino Acid Head Groups and Backbones 6.1. Amino Acid Backbones Linking Ammonium/ Amine Head Groups and Hydrophobic Tails 6.2. Amino Acid Backbones Linking Amino Acid Head Groups and Dual Hydrophobic Tails 6.3. Amino Acid Head Groups with Single Hydrophobic Tails 6.3.1. Amino Acid Head Groups with Diacetylenic Hydrophobic Tails 6.4. Amino Acid Backbones Linking Tris-Based Head Groups to Hydrophobic Tails 6.5. Tube Forming Amphiphiles Comprised Entirely of Amino Acids 7. Helically Based Nanotubes Self-Assembled from Synthetic Amphiphiles with Ammonium and Amino Head Groups 7.1. Ammonium Head Groups 7.2. Amino Head Groups 8. Helically Based Tubes Self-Assembled from Synthetic Gemini Surfactants 8.1. Gemini Surfactants with Quaternary Ammonium Head Groups 8.2. Other Gemini Surfactants 9. Helically Based Tubes Self-Assembled from Synthetic Amphiphiles Based on Extended Aromatic Stacking 9.1. Graphitic Nanotubes (Hexa-peri-hexabenzocoronene Derived Amphiphiles) 9.2. Ester-diamide Amphiphiles 9.3. Macrocyclic Amphiphiles 10. Helically Based Tubes Self-Assembled from Synthetic Multipiece Amphiphiles (Requiring Supramolecular Organization before Helical Assembly) 11. Applications for Self-Assembled Helices and Tubes 11.1. Drug Delivery and Controlled Release 11.2. Helical Crystallization of Proteins 11.3. Helical and Tubular Organic Templates for the Production of Inorganic Replicas 11.3.1. Vacuum Field Emission Cathodes 12. The Rational Approach to Controlling the SelfAssembly of Amphiphiles into Helices and Nanotubes 12.1. Tuning the Dimensions of Self-Assembled Helices and Tubes 12.1.1. Amphiphile Molecular Structure 12.1.2. Annealing Conditions 12.1.3. Addition of Other Substances 12.1.4. External Forces 12.2. Rational Design of New Amphiphiles for Self-Assembly into Helices and Tubes Author Information Corresponding Author Present Addresses Notes Biographies Acknowledgments References

Review

1. INTRODUCTION

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1.1. Molecular Self-Assembly as a Method for the Construction of Nanostructures

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In the miniaturization of manufactured goods, the practical limits of what can be achieved when engineering from the “top down” are being approached.1−9 Despite the continuing development of lithography, vapor deposition, and other microfabrication methods, the cost and complexity in achieving submicron resolution, together with the limited variety of what can be produced with each of these techniques, prevent them from being realistic options for many nanoscale constructions.3,4,9−14 An alternative is engineering from the “bottom-up”, or molecular self-assembly. The molecular self-assembly approach to generating nanoscale artifacts is ubiquitous in nature as it provides a powerful, efficient, and convenient means of fabrication.6−11,15−20 In the last 50 years, our understanding of the supramolecular associations that generate these natural selfassembled structures has progressed significantly, and synthetic techniques to produce functional, artificial nanostructures have been developed.9,17,21−27 The structures that can be created artificially follow one of two major methodologies: (i) using nature as a guide in the creation of biomimetic systems;1,8,10,14,15,17,18,20,28−30 or (ii) creating entirely synthetic and distinctively unnatural assemblies.1,8 While both methods enjoy the benefits of the “bottom-up” approach,1,3−5,18,31 only the biomimetic route benefits from enhancements made during millennia of natural selection, while still retaining the flexibility to take advantage of new ideas.7,10,17,32,33 The self-assembly of amphiphilic molecules in aqueous environments is an area of research where the “bottom-up” approach observed in nature is readily translated into workable synthetic nanostructures.4,15−18,27,28,34,35 In nature, selforganized, amphiphilic lipid assemblies have a variety of purposes including their roles as integral components of the cell wall, the cytoskeleton, and cellular organelles.7,16,18,36−38 These natural lipid assemblies exist in a variety of macrostructures, the morphology of which is largely controlled by the molecular structure of the lipid.14,35,39 In turn, the overall morphology of the lipid aggregation is crucial in determining its biological function.15,16,18,28,36,40−44 It is therefore possible to synthetically tune the shape of lipid assemblies, and consequently their biochemical function, by making simple changes to the chemical structure of the lipid precursors.35 It is this feature, along with the ability of lipid membranes to support useful biological species such as receptors and enzymes,32,45 that provides so much potential for useful self-assembled nanostructures based on amphiphilic molecules. Self-assembling amphiphile systems have already been utilized in a large variety of useful nanostructures. A planar membrane assembly is the simplest of lipid structures and has been most often exploited in research. An example of this occurs in biosensors where both self-assembled monolayers and supported bilayer membranes have been used to isolate an electrode from a solution containing ions.46 Changes in membrane permeability resulting from some recognition event are registered through changes in the current measured at the electrode, and so the concentration of key analytes may be determined.22,47 Flat membrane structures can additionally be used to stabilize biological species, such as enzymes, and consequently the enzyme function can then be utilized in synthetic systems.45 Vesicular lipid structures can also stabilize biological species. For instance, parts of cellular molecular machinery have

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been used to assist solar energy transduction in vesicles.32 More usually though, lipid vesicles are employed as encapsulation vessels.15,31 Examples of this type of application have included their use as microreaction chambers48 or as chemical delivery vehicles, where their adjustable permeabilities made them ideal as controlled release agents.49 This is of particular relevance in medicinal applications5,15,28,31,34,50 as lipids are generally biocompatible and have the potential to support biological molecular recognition systems, allowing a drug to be targeted to specific locations within the body.50,51 Lipid nanotubes (Figure 1) are suitable for a diverse range of additional applications as a result of their high aspect ratio52

substances;57,59,88,89 the use of lipid nanotubes as templates for inorganic deposition;57,60,63,81 nanotubes self-assembled from biobased amphiphiles56 and phospholipids;37 the alignment of nanotubes and consequent applications;53 the construction and application of one-dimensional supramolecular assemblies;90 and the expression of chirality in self-assembled fibrillar systems.91 Clearly, this area is of broad interest, and this Review brings a current perspective incorporating all of these foci. 1.3. Benefits of Helical Construction

Helices are ubiquitous in biological self-assembled structures, examples being the double helices of DNA, the secondary, tertiary, and quaternary structures of proteins, the preferred organization of several polysaccharides, and the capsids of viruses.43,92−100 This prevalence of the helical motif has less to do with a requirement for helices per se and more to do with simplicity and economy of production.92,95 That is, selfassembly through helices is a simple and economic method for construction of organized structures from a number of identical objects that have some mechanism to pack at a fixed angle about a vertical axis, like molecular chirality.92,93,95 Such assembly simplifies the continuing production of organized structures from a single type of building block with high fidelity.95 Therefore, utilizing a helical basis to design tubular structures is a rational way to proceed for simple construction from a single amphiphilic precursor. The coiled ribbon structure of helically based tubes also provides the structural rigidity to form long, straight tubes,58,59,101−105 which are useful for templating. In addition, the inherent helicity can be used in diverse, novel applications such as imaging bound proteins75 and possibly as a molecular solenoid.106 1.4. Descriptive Terms

Figure 1. TEM images of nanotubes self-assembled from N-(11-cisoctadecenoyl)-β-D-glucopyranosylamine. Reprinted with permission from ref 85. Copyright 2011 Materials Research Society.

In describing the self-assembled structures in this Review, the terms “lipid” and “amphiphile” will be used interchangeably as will “tube” and “tubule”. The prefixes of “nano-” and “micro-” will also be used to describe different sized tubes. In general, the language used by the authors of the original literature will be cited.

and their hollow cylindrical morphology.53 These characteristics complement their suitability for many of the applications already mentioned, such as use in biosensors and in drug delivery.4,29,53−57 As distinct from other nanostructures with similar aspect ratios, such as carbon nanotubes, lipid nanotubes can be synthesized with a range of internal diameters, from 10 to 1000 nm,6,31,58−60 and have hydrophilic surfaces with a range of available surface functionalities.6,13,20,53,61−64 Further, chemical modification postassembly can provide external nanotube chemistries that are not themselves able to support self-assembly into nanotubes.65 This flexibility in size and chemistry provides further applications specific to lipid nanotubes including use in micro fluidic networks,66,67 in molecular recognition devices,68 and as liquid-crystalline biomaterials.69,70 Furthermore, lipid nanotubes have been shown to act as ideal templates for synthesizing useful structures from materials that otherwise do not self-assemble.53,56,71−81 The tubes can also be used in combination with other materials as nanocomposites.29,53,78,79,82−84

2. THEORY OF THE SELF-ASSEMBLY OF AMPHIPHILES INTO HELICALLY BASED NANOTUBES 2.1. Introduction to the Self-Assembly of Amphiphiles

In 1925, Gorter and Grendel107 demonstrated experimentally that lipid membranes were composed of a bilayer (two molecules thick), and so postulated a structure in which the polar head groups faced out from a nonpolar center (later verified by electron microscopy and XRD108). Driving the self-assembly of lipids in water is both the amphiphilic nature of lipid molecules and the character of the intermolecular forces of the water itself.44,109 The strong, directional forces between water molecules lead to an isotropic arrangement that minimizes free energy.109 As such, it is thermodynamically unfavorable for the nonpolar hydrophobic regions of lipids to be dispersed in an aqueous phase because these hydrophobic regions cannot provide alternate interactions to compensate for the loss of the intermolecular associations between the water molecules. Consequently, they aggregate in a fashion that creates the least disruption and distortion to the organized arrangement of the aqueous phase.7,40,44,87,109 At the same time, the attached hydrophilic head groups help to further minimize any disruption to the organization of water by facing out from the

1.2. Previous Reviews Discussing Self-Assembly of Helically Based Nanotubes

This Review focuses on nanotube self-assembly from amphiphilic molecules via a helical intermediate species. Detailed reviews on this area were previously undertaken by Shimizu et al.31 in 2005 and Spector et al.55 in 2003. A few short general reviews have been written,4,28 as well as many review articles that focus more narrowly on topic areas such as self-assembly theory;86,87 the use of self-assembled nanotubes to encapsulate 10219

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head groups facing out into the aqueous environment, sandwiching the hydrophobic interior (Figure 2b). When on a large enough scale, planar membranes will curve back onto themselves forming a vesicle in which the hydrophobic edge regions are completely sealed off from water.40,55,86 The size of the vesicles is determined by balancing the free energy minima associated with the packing requirements of the lipid and the smaller sizes that are favored entropically.40,86 For helices and helically based tubes, there are many forces driving the formation of the aggregated structures. One of these forces is the amount of curvature that the lipid will tolerate in the assembly based upon the relative volumes of the hydrophobic and hydrophilic parts of the molecule. Generally, the molecule must have an appropriate geometry to be part of a bilayer assembly. Many nanotube forming molecules are found to assemble into bilayer vesicles at temperatures higher than their liquid-crystalline transition temperature. Below this temperature, a liquid-crystalline phase of increased order forms, thus generating the organized structure required to form nanotubes.6 These nanotubes usually have diameters much less than those of vesicles for the same species. Therefore, molecules that afford the low curvature organization in vesicles must also be able to accommodate the high curvature of nanotubes. Relieving some of this potential for molecular packing problems in obtaining highly curved nanotubes is the ordered nature of the packing itself, with molecular orientation constant in the ribbon-like structure. That is, in one direction of orientation, along the length of the tube, the lipid can pack with minimal curvature, but this same molecule must then be able to accept highly curved packing in the orthogonal direction. This means that the shape of the head group and its orientation relative to the tail are vitally important for the formation of helically based nanotubes.

aggregation where the polar interactions between water and the head groups can be maximized. The lipid head groups can also interact with each other, creating a director for the shape of the assembled aggregate110 (described in more detail in sections 2.3, 2.5, and 2.8). The combined interactions, whether between the head groups and water or the van der Waals attractive forces occurring between the closely packed hydrophobic chains, reduce the overall free energy of the system36,40,44,109 to such an extent that the entropic advantage that favors a completely dispersed system can be overcome.36,40,111 The macrostructure of an aggregated lipid system is further influenced by the molecular structure of the lipid.27,40,44,55,87,110,112,113 The hydrophobic incentive for the hydrocarbon chains to associate is balanced against the hydrophilic, ionic, and/or steric repulsion of the head groups.44,110,114 This balance then defines the interfacial area of the lipid molecule exposed to water, which in combination with the maximum effective length and volume of the hydrocarbon chain establishes the geometric packing constraints of the lipid.6,40,110,114 Short, saturated, single-chain lipids with bulky or charged head groups, having a large interfacial area and a small length and volume of the hydrocarbon chain, have been described as having a coneshaped geometry. Cone-shaped molecules can be packed such that the outer surface of the aggregated structure is highly curved. Such high curvature, along with smaller structures being favored entropically, supports the formation of small, micellar morphologies with an internal hydrocarbon center (Figure 2a).40,44 In contrast, lipids containing hydrophobic bulk

2.2. Lyotropic and Thermotropic Phases of Amphiphiles in Bilayers

Bilayer aggregated amphiphiles in aqueous systems exist in several lyotropic and thermotropic mesomorphic phases.115,116 At physiological temperatures, most natural lipids are in a lamellar fluid Lα-phase, analogous in symmetry to the liquidcrystalline smectic A phase (Figure 3a).117,118 This phase has a highly disordered short-range structure due to the rapid transgauche isomerization of carbon−carbon bonds in the hydrocarbon chains,44,119,120 and in an ideal system the lipids move freely within the plane of the bilayer.44,109,121−123 The longrange organization of this phase, however, is ordered in the one-dimensional lattice of the bilayer structure, and the overall average of the lipid’s long axis is oriented parallel to the bilayer normal.115,124,125 The macrostructure of such lipid assemblies remains set by the thermodynamics of the system, which favor spherical structures such as vesicles in the absence of other forces.40,126,127 Reduction in temperature or water content can induce a transition in which the lipids cooperatively organize, and the great majority of hydrocarbon chains are fully extended in the trans conformation.128 The orientation of these chains relative to the normal now depends on head group packing.129,130 If the head group is relatively small, the chains orient parallel to the bilayer normal, and this is described as an Lβ phase (see Figure 3b). However, if the head group is relatively bulky, the lipid may tilt with respect to the bilayer normal, such that the head group can be better accommodated within the plane of the bilayer surface.129,130 This organization is known as the Lβ′ phase (Figure 3c) and usually has long-range hexatic

Figure 2. Effect of lipid molecular geometry on aggregate morphology. (a) When the lipid molecular geometry is cone shaped, aggregates will tend toward micellar character. (b) In contrast, cylinder shaped lipids will tend to form bilayer aggregates.

in the form of a second hydrophobic chain and/or the inclusion of unsaturated bonds generate molecular geometries described as cylinders or truncated cones. The packing constraints of the bulky hydrophobic portion in comparison to the head group mean that highly curved structures cannot exist.40,44,86 Instead, locally planar bilayer membranes form with the hydrophilic 10220

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Figure 3. Liquid-crystalline phases of membrane bilayers. Some of the different liquid-crystalline phases of lipids include: (a) Lα or smectic A phase that has a one-dimensional layered structure. The mean orientation of the molecules is perpendicular to the bilayer, but there is no short-range positional order. (b) The Lβ or hexatic smectic B phase brings short-range positional order, in which the molecules are evenly spaced in a twodimensional lattice. The Lβ phase also has long-range bond orientational order, which is expressed as a hexagonal packing of the molecules in which orientation of the lines joining adjacent molecules, or bonds, possesses 6-fold symmetry. (c) The Lβ′ phase is often a hexatic phase observed when a force drives these molecules to tilt relative to the bilayer normal. This is hexatic F (e) when the molecular tilt is perpendicular to the sides of the hexagon, and hexatic I (f) when the molecular tilt is toward the corner of the hexagon.118,135 Sometimes the hexagonal arrangement of molecules in the Lβ′ phase is distorted and there is orthorhombic perpendicular symmetry (g). Finally, (d) the rippled membrane phase, Pβ′, can occur just before the transition from the Lβ′ phase to Lα phase.

crystal phases already mentioned. It seems that crystallinity has often been used to describe the phase of tubules, and crystallization as the tubule forming process, simply for want of better terms to characterize the prevailing high degree of order. Nevertheless, some researchers argue that the tubule phase is crystalline.126,142−150 Evidence to support this argument includes X-ray and electron diffraction studies on diacetylenic lipid tubules, which have shown chain packing with crystalline character over the length scales probed by these techniques.143−145 Such experiments have so far produced insufficient resolution to examine the extent of the correlation length, and the large number of higher harmonics is only really indicative of a highly defined lamellar order.37,117 Part of the argument for the crystalline nature of tubes is based on the X-ray diffraction (XRD) analysis of dried tube samples,144 where it has been suggested that the hydration of the bilayer does not significantly affect lipid packing,144 although this is a dangerous assumption to make.91 Indeed, Thomas et al.151 found that dry tubes showed 3-D crystallinity, while a suspension in a water/ethanol solution had no evident interlayer correlations. From this, Shashidar and Schnur37 concluded that the hydrated tubule exists either as a two-dimensional crystal with a finite correlation length or as a hexatic liquid crystal, as originally postulated by Nelson and Peliti.123 Polymerization occurs for some types of diacetylenic tubes via 1−4 addition between diacetylenic moieties. Such a polymerization requires specific alignment and spacing of diacetylenic groups, and this, it is argued, is indicative of the crystallinity within the self-assembled tubes.152,153 Similarly, Fuhrhop126 postulates that the regular nature of the helices

bond order similar to that of hexatic smectic liquid-crystalline materials (Figure 3e and f, top views of lipid orientation relative to hexatic organization).87,117,118,125,128 In some instances, the organization amphiphiles in the Lβ′ phase have been defined as a distorted, quasihexagonal lattice in which the hexagonal arrangement is compressed and there is orthorhombic perpendicular symmetry (Figure 3g).39,128,131 In either arrangement of the Lβ′ phase, its tilt is an example of a force that can alter the shape of the aggregate.87 In this case, molecular chirality of the amphiphile can be reflected in the membrane morphology leading to the formation of helices and related tubules.44,55,132−134 Another significant mesomorphic lipid phase, Pβ′ (Figure 3d), occurs when the water content is sufficiently high136,137 and just below the temperature of the transition between the Lα phase and the Lβ′ phase.124 At this “pretransition”, the tilt angle of the lipid molecules becomes reduced as compared to the Lβ′ phase,138 and as the bulky head groups strive to maintain an optimum exposure to water, a periodic ripple is incorporated into the lamellae (Figure 3d). This ripple produces two-dimensional order in a plane normal to the membrane.136,137 Alternatively, the rippled structure is attributed to coupling between membrane curvature and molecular tilt.136 The Pβ′ phase has been seen in helically based tubules, although it is not a prerequisite for tube formation or stabilization.139,140 2.2.1. Liquid Crystalline or Crystalline Packing? The Argument. While there is no doubt that the transition to tubules is accompanied by increased organization of the amphiphile packing,141 debate exists as to whether the tubes are actually in the crystalline phase, Lc, rather than the ordered liquid 10221

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that form tubes (long ribbons with constant width) provides sufficient evidence for crystallinity. Likewise, researchers working on helically based tubes of cholesterol142,149,150 suggest that the organization of cholesterol into tubules is crystalline, as these tubules are the precursor for cholesterol monohydrate crystals. These arguments that are based on apparent amphiphile organization, however, are equally well sustained by a tubule existing in highly correlated hexatic phases;154 a hypothesis that is supported by the majority of theoretical argument and the weight of experimental evidence.54 Theoretical models fall on both sides of the crystalline or noncrystalline divide. Oda and Huc155 have developed a model to explain their experimental results for twisted and helical ribbons observed for Gemini surfactants. They argue that for a fluid system the twisted ribbon will be the minima of free energy, and that helical ribbons cannot be the equilibrium structure unless the membrane solidifies where cylindrical curvature becomes favored over the Gaussian curvature of twisted ribbons. Such a postulation is at odds with research that shows that the transition from twisted ribbons to helical ribbons is not first-order.156,157 The most important theoretical data from which it is inferred that the amphiphiles in self-assembled tubules exist in highly ordered liquid-crystalline phases arise from observations that a membrane that can buckle in three dimensions to form a helix cannot have equilibrium crystalline order.158 Supporting this perspective is experimental evidence from a variety of techniques. For example, NMR and electron spin resonance experiments indicated that the lipids were ordered, yet highly mobile, molecular components within diacetylenic phospholipid tubules.159 Similarly, electron diffraction studies of lithocholic acid steroidal nanotubes showed no evidence of crystallinity.58 Lipid nanotubes cut by repeated scanning of an AFM tip in constant force mode underwent a slow self-annealing process. This was believed to be due to viscous flow of lipids from the bilayer around the cut, suggesting that the lipids within the tubes were not crystalline.160 Finally, investigations into the transition between vesicles and tubes using CD, DSC, and XRD have all concluded that the formation of tubules is via an Lα to Lβ′ transition, rather than by crystallization.161,162

Figure 4. Electron microscopy image of self-assembled helices of lithium 12-hydroxystearate. Reprinted with permission from ref 168. Copyright 1970 The Chemical Society of Japan.

deoxycholate in the presence of trace amounts of magnesium chloride.167 Following various observations of helical ribbons selfassembled from naturally derived amphiphilic molecules, a relationship between chirality and the organization of lipid packing was quickly established. However, the earlier studies provided no in-depth understanding of the self-assembly mechanisms that created such structures, or the ways in which rational design could be used to synthesize new organized structures. More information began to emerge with the work of Kunitake et al.,169 who were the first to create a totally synthetic bilayer membrane in 1977. An extension of this work was published in 1981,16 when 62 different synthetic lipids were presented and their aggregated morphologies from aqueous dispersions investigated. Hence, it was established that three structural elements are required for stable self-assembly: (i) a flexible tail; (ii) a rigid segment; and (iii) a hydrophilic head group. Furthermore, two other elements are found to affect the morphology of the aggregate: (iv) a spacer group and (v) an additional interacting group.16 In particular, Kunitake et al.16 found that rigid segments that are bent, with respect to the rest of the chain (due to angles within the rigid section, or in the connections to the other sections), will tend to form curved elongated structures such as micellar rods and bilayer tubes (Figure 5). Later work has suggested that these bent rigid sections cause the molecules to pack at some specific angle with respect to each other, which can break the symmetry of the membrane in a chiral fashion.55,91,132,170 In the work of Kunitake et al.,16 four out of the 62 synthesized lipids produced tubular structures based on assembly from bilayers. These results, combined with the observed self-assembly of chiral amphiphiles,163−166,171 provided the first real understanding of the types of molecular architectures that promote the aggregation of amphiphiles into organized structures. 2.3.1. Use of Macro-Scale Morphology To Assign Constants for Micro-Scale Interactions: Continuum Theory. In 1984, three groups independently released work on the self-assembly of helical ribbons from synthetic chiral amphiphiles.172−174 Furthermore, two of these groups observed tube formation. By 1986, the theoretical work on how selfassembled tubes might form was starting to be released. Models included those that described a competition between the spontaneous torsion of ribbon edges and the bending rigidity of the membrane.175,176 Another theory argued that a tilted, chiral,

2.3. Theoretical Models for the Self-Assembly of Helically Based Nanotubes

Some of the more complex and interesting macroscopic lipid morphologies are helices and helically assembled tubes. Among the first reports of helices self-assembled from amphiphilic molecules was that for single enantiomers of various chain length carboxylic acids, terminated by an s-butyl group. When dispersed in mineral oil, these species formed tubes, arising from counter clockwise, helical ribbons.163 The self-assembly of helical ribbons from aqueous dispersions of amphiphilic molecules was first noticed for metallic salts of 12-hydroxystearate. In 1952, Hotten and Birdsall164 observed single-handed, helical ribbons in aqueous dispersions of single stereoisomers of lithium 12-hydroxystearate (Figure 4). This relationship between chirality and helix formation was confirmed by Tachibana and Kambara165,166 who found that the D-form of metallic salts of 12-hydroxystearate produced right-handed helices and the L-form produced left-handed helices, while no helices were produced from a racemic mixture. Another early example of helical ribbons arising from chiral amphiphilic molecules was the formation of helical ribbons from aqueous solutions of the steroid sodium 10222

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Figure 5. Self-assembly of chiral amphiphilic molecules with a bent rigid segment in the hydrophobic tail. Reprinted in part with permission from ref 85. Copyright 2011 Materials Research Society.

and kinked shape.39,44,55 Such twist propagation in a membrane in which the molecules are tilted with respect to the bilayer normal creates an intrinsic bending force that helically twists the membrane ribbon into a tube (Figure 5).44,54,58,186 Developments on the continuum theory of Helfrich and Prost have followed a variety of different paths. Chapell and Yager187 suggested a model where packing interactions are essentially one-dimensional (along the length of the ribbon) as the interactions between molecules in this direction are substantially stronger here than those in a transverse direction. It was proposed that this relationship, rather than the direction of the molecular tilt, supports the bilayer curvature.187 Pursuing an alternate direction, Nelson and Powers188,189 used renormalization-group calculations to show that bulk chiral coupling suffers logarithmic renormalization. They suggested that temperaturedependent fluctuations in the tilt and curvature changed the way the tubule radius scaled with chiral coupling. In expanding the application and scope of continuum theory, Ou-Yang and Liu190,191 used a cholesteric, or helical liquid crystal model, instead of the original chiral smectic model. They assumed that the chiral free energy effect was much stronger than all other effects, even though the elasticity free energy was also important.191 This theory, moreover, stated that helical ribbons should be metastable with respect to a tube (i.e., the tubular structure is necessarily of lower free energy than the helix), but it has been shown that this is not always the case.54,164,173,192,193 In addition, CD evidence suggests that lipids in tubules were not in a cholesteric phase.194 Finally, this model, along with other early theoretical models on the formation of nanotubes via helical intermediates, specified that the pitch of the helix was 45°.175,176,190 Although this matched all previous experimental evidence for these types of tubes,152,195−198 more recent results show that this is not always true.199,200 In 1992 it was reported that the crystallization of cholesterol from supersaturated solutions of bile produced two different metastable helical ribbon intermediates that had pitch angles of 11° and 54°.199 Later work also revealed a third minor pitch angle between 30° and 47° for cholesterol helices, with the ultimate angle dependent on the other chiral components of the bile mixture, even though such components were not included in the helical structure.142 Lvov et al.197 also found multiple pitch angles upon the self-assembly of a diacetylenic phospholipid (DC8,11PC) when mixed with 2% loading of a charged lipid (DC8,9PEOH), and Shimizu et al.12 found three distinct pitch angles for a glycolipid.

bilayer ribbon would undergo spontaneous ferroelectric polarization with a positive charge forming along one long edge and a negative charge along the other. Electrostatic attraction between these edges was thought to buckle the ribbon into a tube.177 However, this did not explain the helical nature of the tubes. Further, experimental evidence disproved the electrostatic theory by showing that the tubule radius was unaffected upon tube formation in electrolyte solutions, showing the electrolytes did not screen any putative electrostatic attraction.178−180 Indeed, conversely, it has been shown for lithocholate helical ribbons that electrolytes screen the electrostatic repulsion of ribbon edges with the same charge, and allow closure of the helices into tubes.181 Experimental evidence also rejected another theory that suggested shapes other than spherical vesicles, such as tubes, formed to prevent the formation of energetic disclinations during the freezing of a spherical vesicle.182 This theory predicted a relationship between the tubule radius (r) and its length (L), such that r ∝ L1/2.152 However, no such relationship between r and L has ever been found.151 Another theory proposed that the tubule radius was independent of chirality and molecular tilt, as well as tube length, but was equal to one-half the radius of a precursor vesicle.170,183 This theory asserted that the redistribution of lipids in spherical vesicles between the outer and inner layers of the membrane breaks the symmetry of the bilayer, and, upon transition to the Lβ′ phase, tubes are formed by anisotropy of the bending rigidity of the membrane caused by tilting of the lipids.170 Again, experimental evidence does not support this hypothesis as the tubule radii are often much smaller than one-half the radius of any precursor vesicles and do not follow the vesicle radii polydispersity. Furthermore, tubules are not exclusively formed from vesicles; they are also formed from lipids dissolved in saturated solution.86 Fournier et al.184 showed that such a theory can only hold for symmetrical bilayers and not for bilayers in which the monolayer tilt directions are different.132 In 1988, Helfrich and Prost176 developed a theoretical framework based on continuum theory describing the self-assembly of amphiphiles into tubes via helical intermediates. The essence of this theoretical framework is that long, chiral molecules do not pack parallel to each other, but rather pack with some nonzero twist relative to nearest neighbors (Figure 5).44,92,185 For a single enantiomer this twist has a preferred direction, allowing the propagation of the twist throughout a membrane. Moreover, this twist can be amplified by intramolecular rigidity 10223

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Figure 6. Kinetic evolution of tubes from bilayer membranes. Domain walls form where tilt modulation changes abruptly and ribbons separate. These ribbons twist into a helix as molecules pack with an offset relative to neighbors. The ribbons then widen via the deposition of lipids from saturated solution and close the helices to form tubes.54

Chung et al.200 were the first to attempt to explain the variation in pitch angle theoretically, whereby they used elastic anisotropy to relate the pitch angle to the ratio of two elastic constants. These constants characterize bending of the bilayer in two directions, that is, along the length of the ribbon in the case of one constant and perpendicular to the length of the ribbon for the other. Such analysis included kinetic arguments that explained why helices were more stable for shorter ribbons, and why tubules would form as ribbons grew longer. Moreover, theoretical support for the helix to tubule transition was now provided, explaining the presence of the precursor species.200 A further significant expansion on Helfrich and Prost’s continuum theory of an intrinsic bending force in tilted chiral bilayers was made by Selinger and Schnur.201 Their modeling, utilizing theories of three-dimensional liquid-crystal free energy, confirmed theoretically that chirality, be it molecular chirality or chiral symmetry breaking within the bilayer, can cause a membrane to helically twist into a cylinder.201 Additionally, they predicted that this chirality modulated the direction of molecular tilt in the bilayer, which led to domains of modulation in the curvature of the membrane. The theory predicted sharp domain walls forming where the tilt modulation changes abruptly. It is along these domains that the membrane splits into ribbons that subsequently twist into helices, which can then mature into tubes. The domain walls continued to be evident in the tubule, explaining why tubules formed from helices often displayed a helical pattern (Figure 6).11,82,157,197,198,202−205 Further development of this theory occurred in 1996, when Selinger et al.54 combined their earlier theory of tilt modulation with Chung’s model200 using elastic anisotropy. This modified model helped to explain an anomalously low ratio of elastic constants found by Chung et al.200 by separating the tilt

direction from the direction of the ribbon.54 Selinger et al.54 also showed that helical ribbons, for certain parameters, can be stable equilibrium structures, and not just an intermediate species in tube formation,54 and these theoretical studies have been supported by a body of experimental work.157,164,173,192,193 Finally, Selinger et al.54 described the tube surface as a rippled phase resulting from the modulation in tilt and the domains of curvature this modulation predicted. Overall, this conjecture was supported by CD analyses of tubules and vesicles of DC8,9PC.194,206 The concept of tilt modulation produced by Selinger et al.54 was developed further by Komura and Ou-Yang,207 who introduced a boundary condition that the molecular tilt direction must align with the helical direction at the ribbon edges, a condition supported by earlier XRD studies.144 Their work proposed that there are two different organizations of molecular tilt that represent a decreasing succession in the free energy of metastable, intermediate states in the formation of tubes. This provided an excellent explanation for the two major observed pitch angles for cholesterol helical ribbons and tubes prepared from bile.207 For the high pitch angle of 54°, the model predicted that molecules at either edge of the bilayer tilt parallel to each other with no tilt modulation. In contrast, for the low pitch angle of 11°, the edge molecules tilt in an antiparallel fashion, with a modulation between the two tilts across the ribbon.207 Zastavker et al.,142 who have experimented with diverse helix forming systems based on the crystallization of cholesterol in bile, found it unlikely that all of these systems, involving diverse molecular species, would have precisely the same two angles of molecular packing and consequently sought other explanations. Indeed, researchers in investigating the crystallization of cholesterol tend to believe that the helical 10224

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the enantiomers of 12-hydroxystearic acid, calculated correctly, was the opposite to the handedness of its lithium salts. Reasons for the failure of this model included that it was an extreme approximation with molecules represented by a single chiral carbon bonded to four groups. Each of these groups was approximated as spheres whose size depended solely on the space-filling volume of the attached chemical group. This meant that calculations were carried out on a distorted tetrahedral shape and not the elongated structure that actually represented most lipids. Thermal fluctuations, which were important in organized structures,55,86 were also ignored. Nonetheless, this work represented an important theoretical advance assisting researchers in the rational design of self-assembled, helically based tubes. 2.3.3. Meso-Scale: Effect of Tilt Direction on Elastic Properties. Further work by Selinger et al.214,215 was conducted on the meso-scale, using Monte Carlo simulations of the growth of chiral aggregates to evaluate the values of the elastic parameters required for both a helical ribbon and a tubule to form. The simulations showed short-range chiral interactions leading to twisted ribbon- or helical ribbon-based shapes. These shapes were able to switch between one another as changes were made to the elastic parameters.214,215 At this point, it is important to note the difference between a helical ribbon and a twisted ribbon; the former has a nonzero mean curvature, while the latter exhibits saddle geometry with a negative Gaussian curvature (Figure 7).91,111,155,157,216−218 The apparent continuous transition between twisted and helical morphologies suggested that these were not first-order transitions,111,156,157,218,219 and both CD spectroscopy and DSC indicated greater chiral order within the helical species.192,216,220 Ghafouri and Bruinsma218 theorized that the transition from twisted to helical ribbons was related to ribbon width, with thinner ribbons supporting twisted structures and wider ribbons supporting helices. Ziserman et al.111,217 later demonstrated experimentally that twisted ribbons were a precursor to helical ribbons, the transition occurring upon ribbon widening. Further complicating this issue is that twisted ribbons have also been identified as an intermediate structure between micellar fibers and vesicles.221 The meso-scale research of Selinger et al.215 also showed that the handedness of macroscopic shapes depended not only on the microscopic chiral parameters, but also on the orientation of the molecular tilt. For example, hexatic smectic I has a ribbon edge molecular tilt director shifted 90° from hexatic smectic F (Figure 3e and f), and given the same chiral parameters, such structures are modeled to have helical macrostructures of opposite hand.214 This factor helps to explain why assigning the hand of the aggregated morphology of an amphiphile may be inaccurate when the assignment is purely based on its chiral parameters,213,222 and why enantiomerically pure material can form aggregates of either hand, as noticed experimentally.222 Finally, the research of Selinger et al.215 also proposed that changes in tilt direction relative to nearest neighbors would alter the elastic properties and consequently the diameter and pitch of the final aggregate. This explains how a structure composed of the same material can still vary in these geometric parameters, such as the two major pitches noticed for ribbons formed from supersaturated bile solutions.199 This seemingly complex phenomenon of the formation of helically based tubes has systematically yielded its secrets to rational theoretical developments.

assemblies that they work on are distinct from the others discussed in this Review and provide good evidence to this effect.149,150 Despite Zastavker’s reservations, further developments on the theory of modulated tilt angle arose from Hu and Lo208 who varied the molecular tilt as a function of the cylindrical coordinates, and found an inverse relationship between the tilt angle and the cylinder, or tube, radius. Experimental confirmation that tubules can indeed display both modulated and uniform tilt states then came from Zhao et al.209 who imaged the molecular tilt order of self-assembled tubes by using liquid crystals as an optical amplification probe. The partial penetration of the tube’s surface lipid bilayer by the liquid crystal molecules allowed the liquid crystals to adopt the lipid orientation. After exposure to the liquid crystals, lipid tubules, which were featureless under standard optical microscopy, were observed under a polarizing optical microscope. Two distinct types of image were discovered, reflecting the two distinct tilt orders. Most prevalent were tubes that displayed a directional dependence on the intensity of transmitted light, reflecting a uniform tilt state. However, approximately 10% of the tubules exhibited a birefringent modulated helical motif. Analysis of the variation in transmitted light intensity and subsequent modeling of the predicted orientation of the liquid crystal molecules showed that the lipids exhibited modulated tilt order.209 This analysis was disputed by Tu and Seifert,210 and they suggested that the result could be explained by helical ripples in the tube surface with amplitudes below the resolution of the methods used. The reasoning for this was that the modulated tilting of lipids is not an equilibrium structure (based on the authors’ concise theory of chiral lipid membranes). This theory introduced a concise free energy density based on contributions from bending and surface tension, chirality, and molecular tilt. The relative simplicity of this free energy expression, as compared to those that deal with modulated tilt states,54,207 allowed the preparation of Euler−Lagrange equations for tubes and ribbons in an effort to determine the ultimate equilibrium structures.210 These theoretical results were used to explain various specific experimental data in the assembly of diacetylenic phospholipids and Gemini surfactants.210 2.3.2. Use of Micro-Scale Pair Potentials between Individual Chiral Molecules. Up to this point, the previously described theoretical work is based on the longer-length scale degrees of freedom such as continuum elastic theory, which uses assumptions made from macro-scale morphology to assign constants for microscale interactions.91,180,211 That is, the system is considered continuous instead of a series of discrete molecular interactions. Indeed, there is theoretical evidence that nanostructures, such as ribbons, may have helical morphology induced by anisotropic surface forces in a purely mechanical model, entirely ignoring chemical and physical factors.212 While these results are both important and valid, they do not establish the specific connections between molecular structure and aggregate morphology that are required for the rational design of macrostructures.91,180,211 Nandi and Bagchi211,213 attempted to correct this gap in understanding by developing a model in which the pair potential between two chiral molecules was calculated. In doing so they predicted that a racemate has no twist between molecules, but that single enantiomers do pack with an angle. They also predicted helical direction, or hand, that matched the experimentally observed hand for four different systems. Unfortunately, the model was unable to explain why the handedness of 10225

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Figure 7. Helical ribbons have mean curvature, and twisted ribbons have Gaussian curvature.155

2.4. Chiral Symmetry Breaking

suggesting that each side of the bilayer has a different tilt. Such work establishes a theoretical framework for explaining the formation of tubules from racemic mixtures,103,196,206 from achiral molecules,99,166,225,234−239 and for the formation of mixed-handed precipitates from an enantiomerically pure amphiphilic starting material.142,224,240

The previous section highlighted the importance of chiral molecular packing in the expression of the molecular chirality in the self-assembled morphology of helically based aggregates. Nonetheless, racemic mixtures can also assemble into helical ribbons and tubes, despite the fact that previous theories suggest that the sum of chiral interactions is zero, meaning chiral aggregates should not form. Early discussions suggested that ribbon and tube formation in racemates was due to partial or complete phase separation of the enantiomers,162,223 which has subsequently been shown not to be the case.222,224,225 Instead, spontaneous chiral symmetry breaking within the bilayer best supports the experimental evidence.222,225 Spontaneous symmetry breaking occurs when a symmetrical system is reduced in energy to a state where a particular symmetry is lost. When mirror symmetry is prevalent, symmetry breaking (loss of mirror symmetry) leads to chirality in the system.226,227 For aggregated systems of lipids, the exothermic transition from the Lα phase to the Lβ′ phase involves loss of symmetry, as molecular tilt is introduced. If Lβ′ phase molecules are packed into a skewed hexatic phase, where the tilt direction is locked at some angle relative to the one of the local bond directions, then mirror symmetry can be broken upon the transition from the Lα phase. This induces chirality into the membrane morphology.94,226,228 An example of a molecular structure that induces this effect is the diacetylenic group used in some types of tube forming phospholipids.146,206,222,223 The diacetylenic group provides a rigid, bent, tilt-inducing segment229,230 in the hydrophobic chain so that upon transition to hexatically correlated and tilted phases, the chains cannot rotate relative to local bond directions and have limited packing possibilities with superimposable mirror images.231,232 Consequently, mirror symmetry can be broken and chirality may be expressed in the aggregate.16,206,222,223 Chiral symmetry breaking specifically relating to helically based tubes was first theoretically investigated by Seifert et al.132 who introduced the concept that each side of a membrane bilayer, below the Lα to Lβ′ phase transition, may have different tilt angles relative to the bilayer normal. This model favors cylindrical and saddle curvature rather than flat or spherical shapes, implying that the origin of membrane curvature is not necessarily a chiral interaction between molecules. Instead, it is proposed that such curvature is due to the collective tilt of the molecules with respect to the bilayer normal.132 Support for this is provided by Spector et al.233 who discovered that head groups are involved in chiral interbilayer interactions,

2.5. Relating the Hand of Helices to the Molecular Structure of the Amphiphile

The self-assembly of lipids into tubes can occur via helical intermediates. How the sense of the helix then relates to the molecular structure of the precursor lipid has received intense scrutiny. From the earliest work on forming helices from natural and synthetic products, the hand of a helix was related to the chirality of the precursor molecule; specifically, each enantiomer formed helices of a different hand, and most racemic mixtures formed achiral aggregates.12,55,91,163−166,171,172,211,213,223,237,241−247 Of particular interest was that helices formed from 12-hydroxystearic acid were of the opposite hand to the lithium salts of this acid “for the same enantiomer”.165,166 This contributed to some confusion among researchers until a model was developed to relate the hand of the helix to the orientation of molecular tilt, rather than just the chiral packing parameters (see section 2.3).214,215 Confusion in relating the hand of helices to the molecular structure of the lipid was also created because a large proportion of the early work on self-assembled tubules was conducted on a single diacetylenic phospholipid, 1,2-bis(10,12-tricosadiynoyl)sn-glycero-3-phosphocholine (DC8,9PC). As will be discussed, it is now known that DC8,9PC is atypical as two forces, packing of chiral molecules and chiral symmetry breaking, are apparent in driving helix formation. However, before this was known, substantial difficulties were encountered in modeling the helix behavior of DC8,9PC, and the phenomenon became the topic of significant investigation. DC8,9PC helices are left-handed for the D-enantiomer and right-handed for the L-enantiomer.194,248 Spector et al.206 found that both L- and D-enantiomers had opposite signs for molar ellipticity in CD spectra, as per other helical systems. However, racemates produced helices of both hands and tubular characteristics identical to those seen in single enantiomer preparations, except for having a CD signal of zero.206,223 This was in contrast to the achiral aggregates created from racemates noted for most other systems.163−166,171,172,223,241,242 Examination of the CD spectra also revealed that enantiomeric mixtures have a direct linear relationship between the sign and magnitude of molar ellipticity and enantiomeric excess.206 Such 10226

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small amounts of left-handed helices among the majority of righthanded helices in the aggregation of cholesterol from a bile mixture, despite this mixture coming from enantiomerically pure starting materials. Lending further support to these theories was the self-assembly of an achiral analogue of DC8,9PC, where the phosphatidylcholine head group was exchanged with the adjacent ester-linked diacetylenic tail, placing the head group centrally on the glycerol backbone and removing the chirality.225 The resultant achiral phospholipid produced helically based tubules with equal numbers of both the left and the right-hand. This provides confirmation that molecular chirality is not essential to the formation of diacetylenic phospholipid tubules, and that the bent diacetylenic group can induce supramolecular chirality via the symmetry breaking model.225,235,238

behavior is unusual in self-assembled helices, where a small enantiomeric excess can often control the hand of the aggregate in mixtures according to the “majority rules” principle.249 Thomas et al.,222 in arguing against the enantiomeric phase separation models proposed by others,162,223 suggested that kinetically induced symmetry breaking in the membrane provides a good explanation for the atypical behavior of DC8,9PC. For example, CD results showing zero net chirality for the racemic mixture is accounted for by the random nature of the chiral symmetry breaking mechanism. Also, tubule diameters do not increase in nearly racemic mixtures, as would be predicted by the dilution of molecular chirality. Rather, the diameters remain constant, as expected for a chiral symmetry breaking model.189,224,225 Thomas et al.222 used optical microscopy to follow the formation of tubules from vesicles of enantiomerically pure DC8,9PC in water. In this process, helically based tubes were spontaneously extruded from the vesicles at a rate of 1.0 μm s−1 when the temperature was reduced to below the transition temperature. Both left- and right-handed helices formed during this process, with approximately 60% being right-handed. Tens of minutes after the initial formation of the tubes, electron microscopy showed all tubes had right-handed exteriors (in agreement with previous results of Spector et al.206). This was not due to the conversion of the core of left-handed helices, but rather the wall thickness increased via lipid deposition from the saturated solution directly onto the tube at a slower rate of 0.1 μm s−1.222 The slow accretion of lipid from solution was similar to that seen for tubes formed directly from mixed solvent solutions without vesicular intermediates. It was thought that in the initial fast tube extrusion, rapid kinetics allowed nucleation of the metastable left-handed state, most likely directed by spontaneous chiral symmetry breaking.222,250 The molecular chirality in this instance simply provided a slight weight toward the right-handed twist.55,206,222 Slower aggregation of monomers from solution, however, allowed thermodynamic equilibrium to be reached, and, in this instance, the molecular chirality was fully expressed in the overall morphology. This idea was supported by a theoretical model provided by Selinger et al.86 in which there are two free energy minima for the packing of diacetylenic lipids, one for each hand of packing. One of these minima is slightly lower than the other, as directed by the chirality of the molecule, explaining why there can be metastable packing to both hands, but only one hand provides ultimate thermodynamic equilibrium. As such, in this case the molecular chirality determines the handedness of the final aggregate.55,206 Ultimately, tubule formation in DC8,9PC and its analogues is best described by an integration of the tilt-based chiral symmetry breaking model with chiral molecular packing theories.55,240 Support for the suggestion that DC8,9PC and its analogues self-assemble into helically based tubes via a combination of tiltbased chiral symmetry breaking and molecular chiral packing was provided by research where mixed-handed helix and tube populations resulted from enantiomerically pure diacetylenic phosphonate molecules.224,240 These tubules had twice the diameter of those observed for DC8,9PC, from which the head group varies only slightly. This suggested that molecular changes near the chiral center resulted in less intense chiral molecular packing. Consequently, tilt-driven spontaneous chiral symmetry breaking was responsible for tubule formation, thereby explaining the lack of a single hand.224 The combination of tiltbased chiral symmetry-breaking and molecular chiral packing may also explain the results of Zastavker et al.142 who found

2.6. Stability of Ribbon Edges

The previous sections have discussed how helically based tubes are able to form from bilayer ribbons, but what then drives the formation of seemingly unstable long, thin ribbon structures (Figure 8a)? At the most basic level it is clear that the point at

Figure 8. Ribbon edge models with (a) straight edges, (b) hemispherical edges, and (c) deflated tube ribbon.

which a helical ribbon becomes a tube physically restricts the ribbon’s width,91 but this does not help to explain stable helical ribbons. A more comprehensive explanation comes from polymer science in which the theory of strain-induced crystallization suggests that crystallization perpendicular to the tilt direction is not favored by molecular tension, and so supports the formation of narrow ribbons of lipid.248 The theory that tilt modulation in the surface of the vesicular membranes creates sharp domain walls that split the membrane into ribbons also supports ribbon formation.201 Moreover, specifics of the hydrophobic organization and strong noncovalent interactions can direct fast uniaxial elongation.217 Slower aggregation along the width is driven by the hydrophobic interactions of the high-energy edges, but this force becomes relatively less as the ribbon widens, slowing this process generating ribbon structures.111,217 10227

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described as “sergeants and soldiers” is attributed to the phenomena where a small percentage of chiral units can direct the chiral packing of a large number of cooperative achiral units.43,94,254,255 In a similar way, slight enantiomeric excess within a mixture of chiral units can lead to a strong preference for the chiral order conferred by the enantiomer in excess. This is referred to as the principle of “majority rule”.249,254,256 An early example of “sergeants and soldiers” was found for poly(alkylisocyanate) copolymers where a small proportion of optically active monomers were able to direct the sense of the polymer helix.255,257 Similarly, “sergeants and soldiers” has also been noticed in helical bilayer assemblies with just 20% of a chiral cerebroside able to direct the morphology of a phospholipid membrane into a tube.258 This valuable property has been used to incorporate specifically functionalized, yet nontube forming lipids, into the cerebroside membrane as sites for inorganic mineralization.29,259 Also, up to 80% of a nontube forming, peptide-binding lipid has been incorporated into a mixture with cerebrosides, producing helical tubes used in imaging proteins.75 “Majority rule” exists when the energy required to pack an enantiomer by the wrong hand is less than that of reversing the hand of the helix and the strength of chiral amplification depends on the difference in these energies.99,256,260−263 This force was first observed for isotactic vinyl polymers synthesized from chiral monomers,264,265 and then the idea was pursued in polyisocyanates where steric constraints enforced the helical packing of the side chains.254 For side-chains of mixed chirality, enantiomeric excesses as small as 10% had optical activities measured by CD indistinguishable in terms of intensity and sign from the homopolymer.256,260,262 The 2,6-dimethylheptyl aliphatic chains also caused a “majority-rule” effect in the selfassembly of nonpolymeric C3-symmetrical compounds into helical aggregates.249 The consequences of “majority rule” for the organization of amphiphiles were first seen experimentally in 1981 by Arnett and Thompson266 who found that a racemic stearamide in a Langmuir−Blodgett film could be made to express the lower surface pressure of the single enantiomer by seeding the film surface with crystals of one specific enantiomer. “Majority rule” is also effective in directing the self-assembly of helical structures from lipids. The assembly of nonacosan-10-ol into helical ribbons occurred from an enantiomeric excess because single enantiomers could not be synthesized or isolated in a practical manner.242 Graphitic chiral amphiphiles also followed “majority rule”, with CD spectra of enantiomeric mixtures showing enantiomeric excesses from 20% to 99% having essentially the same CD profile as the pure enantiomer.99

So, theoretically, ribbons can form, but it would seem that this process still involves the unfavorable exposure of hydrophobic portions of the molecules to the aqueous environment.156 Again, it could be said that this instability is removed when these ribbons mature into tubes. However, some molecules are stable for extended periods as ribbons,164,172,173,192,193,241 while for others the helical ribbon is actually the stable equilibrium structure.54,219 Consequently, theories to explain the relatively stable structures of helical ribbons have been developed. Georger et al.152 suggested that the bilayer edges could be initially stabilized by organic solvents bound to the edge. Whether the edges retain the solvent or simply become trapped in the exposed edge conformation is not clear. Also, this suggestion does not work as a general explanation for ribbon edge stability as many assembly techniques do no involve organic solvents.152 Hydration of partially charged head groups and the repulsion of charged surfaces has also been suggested as a stabilizing factor.247 A more universal explanation relates the structure of the bilayer ribbon edges to a highly curved hemispherical arrangement of lipids, such as that found at the ends of rod-like micelles and at the edge of disc-like micelles (Figure 8b). In this model, the alkyl chains of lipids at the edge of the ribbon are less exposed to the solution than those in a straight edge model (Figure 8a), and so are more stable, although still of relatively high surface energy. This model, with high surface energy edges in which the head groups are relatively more solvated and accessible due to lower packing density,251 still allows for the preferential absorption of various species at the ribbon edges and the resultant helical patterning of tubules noted experimentally.75,82,197,203−205,251 Experimental evidence has also suggested that the ribbons are actually themselves flattened tubes and that the edges are highly curved bilayer assemblies (Figure 8c).156,248 When lysozymes were added to DC8,9PC, crack discontinuities resulted in the surface of the air-dried samples prepared for SEM, allowing images of the morphologies of the lipids in bulk solution.156 A previously unseen morphology observed at the crack discontinuities was a “tubulet” that had the appearance of an uninflated fire hose with a width of 1 μm. The observation of helically wound versions of this tubulet promoted the idea that they were the precursors to the tubules, previously described as helical ribbons.156 Arguing against such a model is the existence of tubules having odd numbers of bilayers in the tubule walls, such as those assembled from methanol−water mixtures, while tubes formed from tubulets can only have walls with multiples of two bilayers.133,195 Furthermore, the transition from helical ribbon to tubule is found to mostly occur by ribbon widening rather than by changing pitch.172,241,252,253 In this case, it would mean the unlikely growth of the tubulet to larger diameters. Perhaps these tubulets formed the initial helical ribbons upon extrusion from vesicles,222 and the gaps were filled in from lipids dissolved in solution?140 Nounesis et al.133 perhaps provided the answer when they found that while direct formation of lipid tubules from isotropic phases led to single bilayer construction, tubules formed from the vesicular phase always had walls in even multiples of bilayers.

2.8. Specific Molecular Factors Affecting Helical Self-Assembly of Amphiphiles

The shape of amphiphile aggregates is determined by the molecular structure of the precursor.39,112 In general, the molecular factors that contribute to helical self-assembly include the molecular volume, the volume ratio of hydrophobic and hydrophilic parts of the molecule, the shape and rigidity of the molecule, the chirality, the hydration, and the molecular potential for hydrogen bonding.16,20,40,55,91,112,134,252,267−271 Predominantly, the specific components of the molecular structure that intrinsically effect helical self-assembly lie within the head group, or the interfacial region linking the hydrophobic tail to the hydrophilic head group. Small changes to lipid structure in these regions can have profound effects on the morphology of lipid

2.7. Supramolecular Chiral Ordering by Low Quantities of Chiral Species

There are two different labels applied to situations in which the chiral ordering of a supramolecular structure can be directed by a small percentage of a specifically chiral species. The principle 10228

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Table 1. Characteristics of Nanotubes Self-Assembled from Naturally Sourced Amphiphiles amphiphile

solvent

1 C16:0-NFA-Cer C16:0-NFA-Cer 5 7 8 + ethanolamine 8 + ethanolamine 8 + ethanolamine 11 Na+ salt 11 NH4+ salt 11 11 11 + ethylenediamine 11 + NAENMEDc 12 14 (1.18 mM) 14 (3.99 mM) 15 16 17 + 18 oleic acid + diamminesilver(I)

b

aq bile aqueous ethylene glycol aqueous chloroform aqueous aqueous aqueous aq pH 12.3 aq pH 12.3 aq pH 7.4 aq pH 9.5 aqueous aqueous aq pH 10 aqueous aqueous aqueous aqueous aqueous aqueous

length (μm)

external diameter (nm)

10

60 >1 >7

20−40 >50

3000−15000 100 50−100 25−30 100−500 600a 2000a 4000a 49 49 600 170−250 30 100 450 241 546 92 99 2000−3000 155−200

internal diameter (nm)

trans temp (°C)a

90.1 59.3 400

200 50

25−70

37a 46a 48a 57.5 52.5

ref 142,200,324 180 180 274 336 103 103 103 58,348,349 102 101 64 345 345 353 347 347 355 355 356 52

For 8 the “trans temp” are temps identified for different sized aggregates. 1 assembled from native and synthetic bile mixtures. cNAENMED = N-(2-aminoethyl)-N′-methylethylenediamine. a

b

aggregates.15,28,31,50,126,127,172,180,244,258,259,268,272−275 Tubule morphology is less sensitive to alterations made exclusively to the hydrophobic tail.152,172,206,233,245,276,277 Perhaps an efficient analogy describing the effects of the head group and hydrophobic tail on the formation of helically based tubes is that the head group can be seen as an on/off switch, while the hydrophobic tail is a tuning dial. The head group either works or it does not, but the tail has more utility in making subtle changes to the aggregate structure. Nevertheless, the overall molecular geometry plays an important role in directing the resultant shape of self-assembled morphologies,268,270 and the geometry of the hydrophobic tail is a primary influence on the selfassembly of nanotubes driven both by chiral molecular packing16 and by chiral symmetry breaking.222,225,229 This Review has already stated that for the self-assembly of tubes from lipids via helical intermediates, a structural feature that causes asymmetric, nonparallel packing between nearest neighbor molecules in the membrane, such as chirality, is required.16,91,176,271 The intensity of the packing around these helix-inducing centers is important in the expression of the asymmetric packing that leads to helical aggregates.224,240 Static van der Waals forces between molecules are unable to provide the requisite intensity of packing and are theoretically insufficient to support a helical structure on their own.278 In contrast, strong binding interactions between head groups have been isolated as an example of a force created by the molecular functionality of a lipid that supports elongated macrostructures in aggregated lipid systems.15,28,31,39,50,126,127,180,244,258,259,271,274,275 Such interactions between head groups ensure tight packing between neighbors and will therefore express any tendency toward asymmetry.36,87,121,126,259 To achieve optimum chiral expression in an aggregate, the strong binding interactions between molecules should be directional, happen close to the helix inducing center, and occur in the interfacial region.279 Group interactions that provide noncovalent directional binding include the hydrogen bonding between amide groups

(refs 12, 39, 50, 71−73, 83, 91, 172, 173, 180, 193, 198, 234, 241, 243−246, 258, 259, 267, 269, 271−274, 280−299) and cyclic sugar moieties (refs 12, 38, 39, 50, 61, 69, 71−73, 81, 83, 161, 180, 192, 198, 220, 258, 259, 272−274, 289−291, 297, 298, 300−303), as well as aromatic π−π stacking.12,61,69,91,99,106,192,220,302−313 All group interaction types have been used extensively in synthetic lipids designed to self-assemble into tubes.

3. NANOTUBES SELF-ASSEMBLED VIA HELICAL INTERMEDIATES FROM BIOLOGICAL SOURCES 3.1. Self-Assembled Helically Based Tubes Found in Nature

Amphiphile-based tubular structures are common in nature.199,258,314−318 Some are made up of proteins314 or are created and stabilized by the action of proteins (e.g., the tubules that are part of the Golgi apparatus, endoplasmic reticulum, and the inner mitochondrial membrane) (Table 1).317,319−322 The incorporation of proteins into the tubular structure has both advantages and disadvantages. Protein incorporation is advantageous for directed drug delivery and sensing applications, where other functions of a protein may be of use.318 In general, however, proteins are best excluded whenever possible from the self-assembled tubes due to their associated chemical and environmental sensitivity.322,323 So far, two types of lipid nanotubes that form from helical intermediates without structural direction from proteins have been discovered within the human body, the intermediate helices and tubes in the formation of gallstones324−326 and nanotubes that are the result of lipid storage diseases.327 To date, the processes that are involved in forming these helical and tubular deposits is not well understood, and it is possible that improvements in this knowledge could be used to develop treatments for these afflictions. 3.1.1. Cholesterol from Bile Mixtures. Natural bile is, for the most part, a mixture of enantiomerically pure cholesterol (1), bile acids such as taurocholic acid (2) (and their salts), 10229

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Chart 1. Major Organic Components of Native Bile Mixtures

Figure 9. Pathway of the formation of cholesterol monohydrate crystals from supersaturated bile solutions. Reprinted with permission from ref 200. Copyright 1993 National Academy of Sciences.

tures had been optimized. The specific pitch angle of the intermediate was dependent on the noncholesterol (yet chiral) bile components. The tubes based on helices with pitch angles of 11° have been found to directly transition from fractures in the tube into plate-like crystals of cholesterol monohydrate,199,200 and were a major part of the aggregations that made cholesterol-based gallstones.199 While it seems there was a direct transformation from the helices with a low pitch angle to cholesterol monohydrate crystals, there was no evidence of a similar transition between high and low pitch angle helices. The belief was that these were separate intermediate states for anhydrous and hydrated bilayers, respectively (Figure 9).142,199,200 There has been interest in these helical structures, rather than the tubes, that has focused on the elastic character associated with their helical morphologies. For example, stretching of a low pitch angle helix attached to an AFM cantilever enabled the calculation of the spring constant, the determination that the spring or elastic behavior was completely reversible, and the observation of a linear relationship between force and helix extension was discovered.329 This, along with calculations based on other physical properties of the tubes, has made it possible to calculate the spring constant for any given helix, and so enabled the selection of helical springs appropriate to a particular application, such as measuring the forces between nanoscopic objects.150 There is a strong belief among researchers working on these particular helical and tubular structures that such structures have crystalline character142,149,330 and are distinct from the majority of the liquid-crystalline helically assembled tubes discussed in this Review.149,150 While this is likely to be true,149,150 at the very least these cholesterol structures have a close relationship to the other types of helices and tubes discussed here, and both types have similar forces driving their assembly. 3.1.2. Galactosylceramides. Galactocerebrosides are composed of a single galactose head group attached to a ceramide tail comprising a fatty acid attached to a sphingosine through an amide linkage (Chart 2 shows some examples).50,274 The amide moiety and the hydroxyl groups of the galactose provide strong and directional hydrogen-bonding interactions between the lipids,258 both raising the transition temperature and providing anisotropic ordering to aggregates.274 Similar monoglycosyl lipids have been shown to form ordered hexagonal and highly

phosphatidylcholine (3), bile pigment, and inorganic salts (Chart 1). The crystallization of the cholesterol from bile occurs via several pathways from aqueous solutions of micellar bile in a complex process.324−326 Helically based intermediates in the crystallization of cholesterol from supersaturated bile were first observed by Konikoff et al.,199 using phase contrast and video enhanced light microscopy for both model and fresh human bile’s. Analysis of native bile taken from patients suffering from gallstones showed that not all crystallization progressed through the helical intermediate.199,328 The crystallization pathway followed depended on the ratios of chiral bile components, and only bile mixtures relatively rich in bile salts resulted in helical intermediates.324 Zastavker et al.142 experimented with sterols other than cholesterol and found that no helices formed when the sterol nucleus was nonplanar and contained a 3α-hydroxy group. Kinetic pathways that resulted in helical structures initially formed long filamentous microstructures surrounded by a lipid monolayer,199 and these slowly developed an arc-like morphology324 that converted, almost invariably, into right-handed helical ribbons.142,200,324 The small proportion of left-handed helical species noted by Zastavker et al.142 was caused by the chirality of components other than cholesterol because when cholesterol was the only chiral component, the helices were exclusively right-handed.142 Contribution to this effect may also have come from chiral symmetry breaking, as discussed in section 2.4. The width of the helical ribbons increased with time, eventually closing the gaps to create helically marked tubules.199,324 Initially, helices with high pitch angles of ∼54° and diameters between 3 and 15 μm were found before dissolution.142,200,324 Forming later, but coexisting for a period with high-pitch angle helical structures, were helices having larger diameters and smaller pitch angles of ∼11° (Figure 9).142,200,324 Also, Zastavker et al.142 have noted small amounts (less than 10%) of helices with an intermediate pitch angle of between 30° and 47° in experiments where the yields of helical struc10230

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mixture (bCer) formed helically based multilamellar nanotubes from aqueous dispersions. These cerebrosides were also added to model phosphatidylcholine membranes. At high concentrations (59 mol %), bCer was able to induce a change from liposomes to multilamellar tubule morphology in the partly unsaturated lipid membrane. A greater effect, however, occurred with the synthetic C16:0-NFA-Cer that was able to produce the tubule morphology with a diameter of 100 nm at 20 mol %. Because of the difficulty in dispersing cerebrosides in water,298 research has also been conducted into the self-assembly of cerebrosides in other solvent systems.50,180,259 Variances in the assembly of assembly of HFA-Gal-Cer (cochleate cylinders259,333) and NFA-Gal-Cer (helically based tubes, diameters 50−100 nm180) in the presence of glycols were attributed to the additional hydroxyl group situated at the hydrophobic interface of HFA-Gal-Cer (Chart 2).259,274 This indicates that head group interactions in the interfacial region played a critical role in the formation of helically based nanostructures.180,258,259 In contrast, when partial modification of the exterior head group functionalities was effected29 or variations to the hydrophobic chain length were made, the morphology was not significantly altered. Such results indicated that these changes separated from the interfacial region were less crucial in directing the morphology.274 The ability to make chemical modifications to the precursor amphiphiles without significantly affecting aggregate morphology was exploited for increased aggregate functionality. For example, anionic sulfated galactocerebrosides (Chart 2, 6) were used as sites for inorganic mineralization.29,259 Despite 6 showing no evidence that it could form helically based structures on its own, a mixture of NFA-Gal-Cer and 6 (15% w/w) formed unilamellar helical structures.259 Also, as part of an effort to create a new drug delivery system, a species of C24:1-NFA-Cer, modified at the primary hydroxyl group at C1 with napthonic acid, assembled into tubes when precipitated from a 1 mM dimethylformamide solution by the addition of water (35% by volume).334 Another aspect of amphiphile molecular structure decisive in chiral assembly is unsaturation in the hydrophobic chain. For instance, unilamellar nanotubes with diameters of 25−30 nm were produced from aqueous dispersions of isolated 24:1-NFAGal-Cer, while isolated 24:0-NFA-Gal-Cer produced ribbons, some with helical morphology.274 Thus, this research on helical structures constructed from galactosylceramides showed that optimal bilayer nanotube formation via anisotropic chiral interactions could occur when the chains of Gal-Cer were long and included a single cis unsaturated group located in a position that limited disruption to chain−chain packing.335 As such, this work generally supported the theoretical framework for the formation of helically based nanostructures in the amphiphilic aggregation of lipids based on chiral elasticity.274,335 3.1.3. (S)-Nonacosan-10-ol. Waxy tubules of 0.1−0.5 μm diameter made up of (S)-nonacosan-10-ol (Chart 3, 7) have been found to occur in a dense mesh covering the stomata on the needles of pine trees. The function of the tubules is to filter particulates from the air during CO2/O2 gaseous exchange.316 In the laboratory, the natural product reforms into tubes through self-assembly from chloroform solution.336 In contrast, a synthetic racemic mixture was not able to form tubules, but only flat platelets.242 Purification of the racemic mixture concentrated the (S)-form to create an enantiomeric ratio of 8:1 (S:R enantiomers), and this mixture was able to self-assemble into tubes, similar to the natural material. That such a simple structure formed tubes reinforces the intrinsic role that chirality

Chart 2. Examples of 2-Hydroxy Fatty Acid (HFA, 4), Non-Hydroxy Fatty Acid (NFA, 5), and Sulfonated Galactosylceramides (6)

curved membrane phases in bacteria and plants.331,332 Providing order seems to be the primary physiological function of these lipids in mammalian systems as well, as the monogalactose heads do not protrude far enough into the extracellular matrix to be utilized as a cell surface receptor, as is the case with other sphingolipids.258 Galactocerebrosides are enriched in myelin and brush border aggregates, where they act in conjunction with proteins to form and maintain tubular structures.258,274 When such lipids accumulate at high concentrations within the body, they provide sufficient anisotropic order to form helical nanotube structures independently of peptide-based morphology directors. This is particularly so when nonhydroxy cerebrosides (NFA-Gal-Cer) have a significant presence (Chart 2).180,258,274 The resulting undesired intracellular deposits are the consequence of lipid storage disorders such as Gaucher’s and Krabbe’s diseases.50,180,258,274 Cylindrical nanostructures in membranes containing cerebrosides were first noted by researchers investigating Gaucher’s and Krabbe’s diseases,297 and later examined by those investigating lipid aggregate morphologies involving cerebrosides.258,298 The early work by Curatolo and Neuringer258 investigated how cerebrosides were able to direct the shape of membrane bilayers. Here, natural bovine brain cerebrosides (bCer) were fractionated into HFA-Gal-Cer (4) and NFA-Gal-Cer (5, Chart 2), and then the NFA-Gal-Cer was further separated into fractions, primarily composed of C24:0-NFA-Gal-Cer and C24:1-NFA-Gal-Cer (5). A shorter chain C16:0-NFA-Gal-Cer was also synthesized (in this naming convention, the number following the C denotes the length of the fatty acid chain and the number after the colon indicates the number of double bonds in the fatty acid chain). In isolation, both the synthetic C16:0-NFA-Gal-Cer and the natural 10231

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sense as assembly from organic solvent.171,340 It was also possible to influence the sense of the helices through assembly in long-chain alcohols, which interfered with molecular packing of the hydrophobic tails and resulted in mixed hand aggregates.168 These important early studies showed that both the head groups and the hydrophobic tails had influence on the chirality of the aggregate structures and illustrated the complexity involved in the self-assembly mechanisms that result in helically based tubules. In more recent research, Xie et al.339 found that the sodium salt of D-12-hydroxystearate self-assembled into left-handed twisted ribbons when cooled from solutions in pure water, rather than the mixed handed helices observed previously,168 and exclusively right-handed twisted ribbons when cooled from ethanol:water mixtures (1:4, v/v). These ribbons had a width of 250 nm, thickness of 40 nm, a pitch length of 2.0 μm, and were used as a template for silica deposition by adding N-trimethoxysilylpropyl-N,N,N-trimethylammonium chloride and tetraethoxysilane to the ribbons. During the sol−gel processing, the left-handed twisted ribbons assembled from water converted into helical ribbons resulting in helical silica replicas with a ribbon width of 450 nm.339 In contrast, the right-handed twisted ribbons assembled from ethanol:water mixtures did not undergo the helical conversion during sol−gel processing, and instead twisted silica replicas were found. Other recent research investigated the self-assembled structures of the ethanolamine salt of racemic 12-hydroxystearic acid in water.196 Equimolar aqueous solutions of racemic 12-hydroxystearic acid and ethanolamine were heated until isotropic (75 °C). Upon cooling, helically marked nanotubes formed (outer diameter 600 nm, inner diameter 400 nm, wall thickness 4 bilayers) in yields greater than 98%.196 The fact that these tubes formed from a racemic mixture provided strong evidence that the formation mechanism for helices constructed from 12-hydroxystrearic acid and its salts was influenced by chiral symmetry breaking.103,196 Once the 12-hydroxy stearic acid−ethanolamine tubules were formed, the diameter of these tubes had atypical temperature dependence. Upon heating a 1% solution above 37 °C, the diameter increased to 2 μm at 46 °C, and then to 4 μm at 48 °C. At 50 °C, substantially below the melting point, neither tubules, nor any other structure, were found. At 52 °C, 4 μm tubules were again observed, while still further heating caused the diameter to decrease, until the tubes melted at 70 °C.103 The 12-hydroxy stearic acid−ethanolamine mixture was exposed to a range of different assembling conditions that varied in ionic strength, pH, concentration of 12-hydroxy stearic acid, and relative concentrations of 12-hydroxy stearic acid and ethanolamine, as well as adding additional components such as ethanol and doping lipids.341 Generally the formation of tubes was not found to be very sensitive to assembly conditions.341 Experiments with sonication showed that the tubes formed using from a potassium salt of 12-hydroxy stearic acid were broken apart, but those formed with ethanolamine became flexible and were able to be bent or transformed into vesicles.342 It seems the energy of sonication was able to cause the same transition from Lβ′ to Lα noticed upon heating for the ethanolamine-based tubes, and that a similar ordered phase was not accessible to the potassium salt-based tubes.342 Further investigations of the unusual behavior of the selfassembly of 12-hydroxy stearic acid established that tubes also formed when ethanolamine was exchanged for 1-amino-2propanol, or when the original acid was substituted by ricinelaidic

Chart 3. (S)-Nonacosan-10-ol

takes, whether of the molecule or induced into the macrostructure by symmetry breaking, in the formation of helically based tubes. 3.2. Self-Assembled Helically Based Tubes from Isolated Natural Compounds

The isolation of natural lipid compounds is advantageous because it provides a source of relatively inexpensive molecules with inherent chiral selectivity, difficult to achieve synthetically,274 and provides access to a large pool of precursors.76,196 Natural lipid compounds that do not self-assemble into helices and tubes from native mixtures, but which do form such structures when isolated, are discussed below. 3.2.1. 12-Hydroxy Stearic Acid and Derivatives. The aggregation of amphiphilic precursors into helical ribbons was first noticed in 1945, when Weitkamp163 reported that natural carboxylic acid species isolated from lanolin generated helically based tubes when dispersed in mineral oil. While this work seems not to have been revisited, there has been significant research into the self-assembly of other carboxylic acids, such as 12-hydroxystearic acid and its salts, into helical ribbons.164−166,168,171,196,337−339 It was found that the lithium salts of 12-hydroxystearic acid (Chart 4, 8) self-assembled into helical ribbons of consistent Chart 4. L-12-Hydroxystearic Acid

hand, and this was linked to the chirality of the initial single enantiomer;164 that is, lithium D-12-hydroxystearate produced right-handed helices, and lithium L-12-hydroxystearate produced left-handed helices, while the racemic mixture produced no helical aggregates, only flat platelets.165 Each enantiomer maintained the sense of the helix despite changes to formation conditions that altered the gross morphology, such as the width of the ribbon.166,168 Interestingly, exchanging the metal ion associated with 12-hydroxystearate had an effect on the sense of the helix that was dependent on ion size. That is, for L-12hydroxystearate, the sodium and potassium salts formed helices of mixed hands, while the rubidium and cesium salts formed left-handed helices, the opposite to that observed for the lithium salt.168 There are other influences to the hand of assembled helices of 12-hydroxystearic acid and its salts. These include the assembly of the acid from organic solvents, which resulted in helices opposite in hand to those formed by aqueous dispersions of lithium salts.166 12-Hydroxystearic acid has also been collapsed into helical structures by compressing the monolayers that formed on water, resulting in the same helical 10232

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acid and combined with either ethanolamine or L-alaninol.103 These new tubes displayed temperature-dependent diameters and characteristics similar to those of the original tubes, but with lower transition temperatures.103 The self-assembly of 12-hydroxy stearic acid was also attempted with other alcoholic amines varying in the number of carbon units in the chain and the position and number of alcohol and amine groups.338 All variants formed tubes initially that had temperature tunable diameters. Generally it was found that as the chain length for linear hydroxylalkylamine species increased, the melting temperature of the associated aggregates decreased. 12-Hydroxystearic acid-ethanolamine nanotubes formed by cooling through the main transition in the presence of iron oxide nanoparticles led to the incorporation of the particles within the tubes, and their association with the tube walls.342 These tubes had essentially the same characteristics as those formed without the particles except that there were no observed helical striations.342 The hybrid nanotubes were able to be aligned in a magnetic field and were also found to stretch under these conditions. Upon removal of the magnetic field, the orientation was lost due to Brownian motion, and the stretched nanotubes relaxed to their previous dimensions.342 The stretching motion was used to calculate the Young’s modulus for these hybrid tubes at 33 MPa, which is roughly an order of magnitude less than that observed for other helically based tubes,61,344 and similar to natural nonhelical microtubules.342 3.2.2. Derivatives of Steroidal Acids. Steroidal salts based on bile acids such as cholic acid (9), deoxycholic acid (10), and lithocholic acid (Chart 5, 11) can self-assemble to form

portions of the structure had a large effect on the overall aggregate morphology.275 3.2.2.2. Metal Ion Cholates. Research on bile acid salts has been reprised more recently with the increasing interest in selfassembled nanostructures. Similarly to the originally observed deoxycholate helices, cholate has been found to self-assemble into helical ribbons in the presence of a divalent metal cation.346 Calcium, nickel, zinc, cobalt, and copper can all combine with cholate anions to form supramolecular helices. In the case of calcium cholate, these structures were exclusively right-handed helical ribbons that were several nanometers thick, 10−40 nm wide, and had a pitch of ∼70 nm.346 Small-angle X-ray scattering (SAXS) results showed a periodicity of 2.78 nm, which was roughly twice the length of the cholate backbone, and another periodicity of 1.06 nm, which was roughly twice the width of the cholate backbone. High-resolution TEM was able to resolve stripes along the helical ribbon of ∼2.8 nm, a close match to the first SAXS periodicity, and was attributed to a bilayer structure that formed across the width of the ribbon. The authors reasonably predicted this to be the lengthwise bilayer association of cholate molecules, the two carboxylate anions binding a calcium ion from each side, and van der Waals interactions then occurring between the hydrophobic faces of the cholate molecules (Figure 10). Cholate has both a hydrophobic and a hydrophilic face, due to having all of the hydrophilic groups on one side of the molecule,347 which allowed another bilayer type assembly to form in a direction orthogonal to the first. This face-to-face bilayer assembly involved hydrogen bonding between hydroxyl groups of the hydrophilic faces, and was directed through the ribbon, this being the direction more classically envisaged for the assembly of surfactants in aqueous systems.346 This represents the discovery of a unique mode of assembly. That is, metal ion coordination occurring orthogonally to hydrogen-bond associations and the bidirectional association caused by the hydrophobic effect created a bilayer ribbon constructed of narrow strips. The helical structures constructed from calcium cholate were used as templates in the construction of inorganic replicates. Silica helices were created by a sol−gel process after tetraethyl orthosilicate (TEOS) and an ammonium catalyst were added to aqueous dispersions of the organic helices.346 Similarly, inorganic zinc sulfide nanotubes were created upon the addition of sodium sulfide to an aqueous dispersion of the zinc cholate helices. The semiconductor nanotubes had 30−70 nm external diameters, 10−50 nm internal diameters, and distinct helical markings with a pitch of 50 nm.346 These dimensions were in close agreement with those of the zinc cholate helical ribbons,346 but whether the formation of inorganic tubes was due to some alteration of the organic structure, or just due to inorganic material filling the gaps in the helices, was not discussed. 3.2.2.3. Lithocholic Acid and Lithocholate Salts. Terech et al.58,181,348,349 have conducted work on lithocholate salts finding that helical nanotubes, among other morphologies, can be made in alkaline aqueous conditions. When made from lithocholic acid with a large excess of sodium hydroxide, these tubes displayed unilamellar morphology, had highly monodisperse internal diameters of 49 nm, high aspect ratios, a melting transition of 57.5 °C, and exhibited stiffness, despite having no crystallinity (as determined by electron diffraction studies).58,348,349 For sodium lithocholate, the dominant tubular aggregates coexisted with helical ribbons, twisted ribbons, and composite structures, confirming that the tubes had a helical basis.58,181,349 Further, helical ribbons became the dominant

Chart 5. Steroidal Acids Found in Bile That Self-Assemble into Tubes

helically based nanostructures. These nanostructures, which start their assembly process as dimers, were created through the formation of hydrogen bonds between hydroxyl groups of adjacent molecules, balancing the loss of hydration of the alcohol groups contained within the hydrophobic portion of the assembly.343,345 3.2.2.1. Sodium Deoxycholate. In 1960, while investigating the assemblies of vaccina virus, McCrea and Angerer167 found that sodium deoxycholate formed helical fibers in the presence of trace amounts of magnesium chloride. Following this initial work, Ramanathan et al.275 investigated the formation of these helices and their subsequent conversion to “rod-shaped” aggregates or tubes. A pH dependence of the formation of helical structures was discovered, with tubes unable to form above pH 7.4.275 In agreement with current theories, the exclusive righthanded twist of the helices was ascribed to the characteristic asymmetry of the molecule, and minor variations in the polar 10233

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Figure 10. Schematic model of the calcium cholate helical nanoribbon. Reprinted with permission from ref 346. Copyright 2009 American Chemical Society. (a) Molecular structure and backbone of cholate illustrating the facial amphiphilicity and molecular size; (b) demonstration of the intermolecular and intramolecular associations between dimers (the blue dotted lines denote H-bonds); (c) scheme of twisted nanoribbon composed of parallel, longitudinal stripes; and (d) top view and (e) cross-sectional view of the molecular model.346

split from one end into thinner tubes, which then coiled into spirals. The observation of vesicles and spirals by Fang et al.101 contrasts to the results of Terech et al.58,348,349 described previously despite there being only minor differences in assembly procedures; that is, Fang et al.101 used a marginally lower pH (12.0 versus 12.3 for Terech et al.) and did not utilize buffers. Furthermore, Fang et al.101 observed varied tube diameters, whereas Terech et al.58,348,349 found monodisperse diameters. This variation in observed morphologies was surprisingly not discussed by the authors. Interestingly, the spiral structures, which were stable at pH 12, reversibly straightened upon the introduction of hydrochloric acid and the subsequent reduction of pH to 7.4. TEM images of these straight tubes showed they were 600 nm in diameter, with tube walls 200 nm thick.101 Over several months at pH 7.5 the straight tubes assembled from lithocholic acid aggregated into bundles 4−5 μm in diameter. Subsequent aging led to the splaying of the tube bundles equally from either end, thus creating sheaf-like assemblies that eventually matured into spherulites made up of tubes (Figure 12).350 Gentle sonication of the sheaves and spherulites led to the break up of these structures into well-dispersed tubes.350 Finally, upon returning the solution to pH 12, tubes with larger diameters reformed into spiral shape tubes, and tubes with smaller diameters curled into a helical structures.350 Lithocholate tubes have been used as a template for the deposition of cadmium sulfide nanoparticles.64 This was achieved by dissolving lithocholic acid in aqueous solution (pH 9.5) and then adding cadmium perchlorate hydrate and thioacetamide. The basic conditions deprotonated the acid and provided a coordination site for the cadmium cations.64 The coordinated cadmium then reacted in situ with the sulfide anion, forming nanoparticles of 4−6 nm diameter that deposited within the walls of the tubes and also onto the tubes surfaces when cadmium concentration was high enough.64 In these mildly basic conditions, the hybrid nanotubes were straight, with internal diameters of ∼50 nm, external diameters of between 170 and 250 nm, and lengths of up to 60 μm. Further work on templating from the self-assembled structures of lithocholic acid in basic conditions was accomplished by Fang et al.351 They used the tunable properties of the

morphology when using the minimum stoichiometric amount of sodium hydroxide to deprotonate the lithocholic acid. In this instance, there was insufficient electrolyte to screen the charged ribbon edges, and complete closure of the helical ribbons into tubes did not occur.181 A subsequent increase in ionic strength through the addition of sodium chloride was enough to screen the repulsion of the ribbon edges and allow formation of tubes from solution containing the minimum quantity of sodium hydroxide.181 Further work on the self-assembly of lithcholate showed that ammonium salts of lithocholic acid self-assembled into unilamellar tubes that were more rigid, shorter, and had a lower melting than their sodium analogues, but had the same internal diameter of 49 nm. The ammonium salts of lithocholic acid also assembled into nanotubes at pH 11.3, lower than that possible for sodium lithocholate for which a pH > 12 was required.76 Cationic poly(ethylene-imine) absorbed onto the anionic surface of the tube provided a suitably charged surface for the metallization of the tubes with copper.76 Optical microscopy by Fang et al.101 showed the self-assembly of lithocholic acid in alkaline aqueous suspensions at pH 12 gave vesicles as the initial assembled structure, which then aggregated into long lines that eventually made a transition into tubes. These tubes continued to grow until all of the vesicles had been consumed, and with increasing length the tubes coiled into spirals that were exclusively left-handed (Figure 11). The widest of the tubes found were unable to form spirals, but did eventually

Figure 11. Spiral tube formed from sodium lithocholate. Reprinted with permission from ref 101. Copyright 2010 John Wiley & Sons. 10234

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Chart 6. p-tert-Butylphenylamide Derivatives of Cholic and Deoxycholic Acids

Figure 12. Bright-field and polarizing optical microscopy images of sheafs (a and b) and spherulites (c and d) formed from aggregates of sodium lithocholate tubes. Reproduced with permission from ref 350. Copyright 2010 The Royal Society of Chemistry.

self-assemble structures to create varying templates64,101,350 for the synthesis of silica replicas of straight, coiled, and helical tubes as well as the sheaf like structures. This was achieved by combining preassembled structures with TEOS prehydrolyzed using ammonium hydroxide. Replication took place at room temperature for a week before calcination under nitrogen removed the organic template. 3.2.2.4. Bile Acid Salts Modified by Alkyl Ammonium Counterions and Chemical Alteration. The research into the formation of helically based tubes from steroidal salts inspired further work in which the naturally produced single enantiomeric species of steroidal salts were modified. This was done to increase the variety of morphologies possible, while maintaining the selected chirality and minimizing cost. A simple example of this compromise was the addition of alkyl ammonium chloride to aqueous sodium cholate, creating an alkyl ammonium cholate salt that self-assembled into tubes via helical intermediates.343 The tubule structure was supported by the strong ionic and hydrogen-bonding interactions between the oppositely charged head groups of the cholate and alkyl ammonium counterion.343 Lithocholic acid also self-assembled in the presence of di-/oligomeric amines when aqueous solutions were cooled from ≥ 60 °C.345 The morphology of the gels formed varied with the nature of the amine, with nanotubes found when the amine was either ethylenediamine, forming tubes of ∼30 nm diameter and lengths of more than 1 μm, or N-methyl diethylenetriamine, forming tubes of ∼100 nm diameter.345 Recent work focused on the synthetic modification of deoxycholic acid and cholic acid by creation of 3β-amino derivatives of the acids, which were subsequently reacted with p-tert-butyl-benzoyl chloride to attach p-tert-butylphenyl groups through an amide bond (Chart 6).347,352,353 The modified sodium cholate derivative (12), when incubated at 40 °C and at pH 10, was found to self-assemble into tubes with diameters of ∼450 nm and lengths of up to 7 μm. This occurred in a fashion similar to that observed by Fang et al.64,101 in which vesicles

initially assembled, then aligned and aggregated, undergoing a transition into tubes.353 In this case, during the incubation process, low diameter tubes formed first from the vesicles, and then the tubes themselves laterally associated and transformed into larger diameter tubes. In a subsequent paper, the initial small diameter tubes were described as fibers, without any explanation as to why the description of the structure changed.352 It was found in this paper that the morphology could reversibly change with temperature. Fibers were observed at room temperature and tubes formed from ∼35 °C, which were then subsequently converted to micelles above 55 °C.352 It was predicted that changes in the balance between the relative strengths of the hydrophobic interactions and hydrophilic interactions were responsible for this temperature responsive surfactant behavior.352 The large diameter modified sodium cholate (12) tubes were initially constructed with walls of a single bilayer, as seen earlier for the much smaller tubes observed for lithocholate selfassembly by Terech et al.58,76,102,348,349 Whether these modified cholate tubes had the same helical basis that Terech et al. had established was not discussed.353 In one related example, deprotonated 12 combined with a positively charged analogue, 13, self-assembled into tubes that were believed to form from rolled up sheets,354 similar to cochleate cylinders. In contrast, for the assembly of pure modified sodium cholate derivative, 12, CD showed there was a chiral aspect to the intermolecular interactions, suggestive of helical assembly, and the intensity of the chirality increased with incubation time.352 Further evidence that modified sodium cholate nanotubes may be helically based was provided by an identically modified deoxycholate, 14, that self-assembled into tubes. In this case, helices were observed as an intermediate structure in the incubation of 14 (1.18 mM) at 37 °C.347 Tubes formed though pitch reduction of the helices after 3−6 h, with an average external diameter of 241 ± 28 nm.347 These tubes retained helical markings with a final pitch of around 45°, and the chirality of 10235

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the molecular packing increased as the aggregate transformed into tubes.347 Related p-tert-butylphenyl derivatives of the sodium salts of chenodeoxycholic (15) and ursodeoxycholic acids (16) (with the hydroxyl group from the 12 position shifted to the 7 position as compared to deoxycholic acid) also self-assembled from aqueous solutions into bilayer tubes with average diameters of 92 and 99 nm, respectively. The differing diameters between these and other bile acid derivatives were attributed to varying hydrogen-bonding organizations.355 While cholesterol is not a bile acid, it is a precursor to those species, and so the work of Pescador et al.356 in which they chemically modified cholesterol with adenosine (17) is described here (Chart 7). The chemically modified cholesterol

Figure 13. Mixed organization of oleic acid/diammine silver(I) complexes that lead to nanotubes.52

Chart 7. Nucleoside Modified Cholesterol, 2′-N-(2(Cholesteryl)-succinyl)-2′-deoxy-2′-aminouridine, and DOPC

and the silver complex were a mixture of chelating bidentate and bridging bidentate (Figure 13).52

4. HELICALLY BASED NANOTUBES SELF-ASSEMBLED FROM SYNTHETIC PHOSPHOLIPIDS 4.1. Diacetylenic Phosphatidylcholine

By far the most research on amphiphiles that self-assemble via helical intermediates into nanotubes has been conducted on phospholipids, in particular 1,2-bis(10,12-tricosadiynoyl)sn-glycero-3-phosphocholine (DC8,9PC, 19), shown in Chart 8, and related diacetylenic lipids. A large proportion of the early work was conducted through the Bio/Molecular Engineering Branch at the Naval Research Laboratory in Washington, DC (NRL). The NRL was among the first to publish work on nanotubes self-assembled from chiral, synthetic lipids in 1984.174 From their research on the polymerization of lipid membranes came the serendipitous finding of the tubular morphology, which formed after their inclusion of the polymerizable diacetylenic moiety into phosphatidylcholine.174,233,357 Phospholipid tubule research was subsequently pursued with tenacity by the NRL throughout the 1980s and 1990s, resulting in many significant publications.84,145,152,162,174,179,195,206,358,359 A general convention has been established in the naming of the diacetylenic lipids where DCm,nPC represents a diacetylenic phosphocholine with m methylene units in the alkyl chain between the ester group and the diacetylenic moiety (the proximal chain), and n methylene units between the diacetylenic group and the terminal methyl group (the distal chain, Chart 8). DCmPC represents spacer lipids used in this research with no diacetylenic group and with m methylene units between the ester and the terminal methyl group (Chart 8). The initial work by the NRL found that tubules self-assembled from aqueous dispersions of DC8,9PC vesicles when gently cooled from above 50 °C.146,174 Such tubes had diameters of 0.4−1 μm, lengths of tens to hundreds of μm,174,360 and the width of tube walls varied from 2 to 10 bilayers (Figure 14).152,248 Visually, the tubes were straight, appeared rigid,141,361 and had open ends, and thin walled tubes displayed an exclusively right-handed helical striation on their surface.139,152,174 Despite AFM studies that confirmed the tubes were rigid,362 the bilayers retained some fluid character (illustrated by NMR and electron spin resonance spectroscopy), which suggested they were not crystalline aggregates.159 In water, diacetylenic phospholipid tubes only formed from vesicles that were larger in diameter than the tubes, and if the cooling rate was sufficiently slow.139,146,174 However, even under optimized conditions, a substantial portion of lipid

was driven to assemble into helically based tubes in binary mixtures with dioleoylphosphatidylcholine (DOPC, 18). The resultant tubes had outer diameters of 2−3 μm and lengths of 20−40 μm and coassembled with vesicles and small populations of thinner tubes with diameters of less than 1 μm. Tubes were found for mixtures containing from 20 to 50 mol % of 17 and tube yield was maximized at 30 mol %, with the population of the thinner tubes increasing with increasing concentration of 17.356 3.2.3. Oleic Acid. When oleic acid was combined with diamminesilver(I) (Figure 13) in a ratio of 50:1, it self-assembled into tubes with walls comprised of approximately 20 interdigitated bilayers.52 The outer diameter was between 155 and 200 nm, with wall thicknesses of 60−70 nm and tube aspect ratios of around 250.52 The nanotube suspensions were treated with formaldehyde, creating nanocables with a metallic core, having a diameter of 30−45 nm.52 Adjusting reducing conditions by the fast addition of formaldehyde, or using sodium borohydride, led to nanoparticle decorated tubes, with silver nanoparticles forming both inside and outside of the tube, but not in solution.52 FTIR analysis of the assembled tubes suggested the interactions between the oleic acid head group 10236

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Chart 8. 1,2-Bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine (DC8,9PC, 19) and 1,2-Dinonanoyl-sn-glycero-3phosphatidylcholine (DC7PC, 20)

Figure 15. Height mode AFM image of DC8,9PC helical ribbon to tube transition illustrating the helical basis of the tubes. Reprinted with permission from ref 140. Copyright 2006 American Chemical Society. Figure 14. Light microscopy image of DC8,9PC tubules. Reprinted with permission from ref 363. Copyright 2001 Elsevier BC.

assembly. For example, decreasing the cooling rate for ethanol/ water solutions through the transition temperature significantly increased average tube length, and decreased the wall thickness.151 For ethanol/water mixtures, it was also found that 70% ethanol maximized tube formation, resulting in tubes averaging ∼500 nm exterior diameters141,152,364,366,367 with tube walls consisting of 5−10 bilayers.152 In contrast, tubes formed from methanol/water mixtures had predominantly single bilayer walls at low lipid concentration, and still only 2−4 bilayers in the walls at high lipid concentration.133,162,195,206,233 Analysis of the main thermotropic transition illustrated that, although the packing of the lipids was not crystalline in tubules, it nonetheless did have a highly ordered liquid-crystalline character.162 This was supported by FTIR, Raman,37,146,147 and fluorescent361 spectroscopy, which showed both the hydrophobic chains and the hydrophilic head groups were organized and tightly packed. Further, XRD studies suggested that the lipid bilayer was highly organized in the Lβ′ liquid crystal phase with noninterdigitated chains tilted at 32° and packed into a distorted hexagonal lattice having no interlayer correlations.37,144,151 To further resolve the details of the formation of tubules, high-resolution video was taken through an optical microscope of the formation of DC8,9PC tubules from vesicles in an ethanol/water solvent mixture cooled at 0.25 °C per hour (Figure 16).222 The video showed that at the transition

material still failed to form tubes, and this lipid was difficult to remove from the tubes.152 For this reason, the formation of tubules from mixed solvents was investigated whereby the lipid was dissolved in an alcohol and heated before the addition of water and subsequent cooling to room temperature, precipitating the tubes.195 Overall, the precipitation of tubules from aqueous alcohol was a simpler, more efficient method for the assembly of tubules, resulting in higher yields, longer and less fragile tubes,206 and narrower distributions of exterior diameters and tube lengths than those found from assembly in water alone.195,364 At low lipid concentration in alcohol/water mixtures, the diacetylenic lipids remained dissolved in an isotropic solution up to the point of tubule formation, and therefore no vesicles were observed.195 This type of assembly generated a large proportion of helices (much rarer in liposome derived precipitates), with the helical pitch of 42 ± 6°152,195,197 (Figure 15).365 At higher concentrations of lipid, an intermediate vesicular liquidcrystalline Lα phase was formed, like those observed in the selfassembly from water alone, before the transition to tubules.195 This showed that there were at least two pathways to the formation of these tubes from lipids.147,195 The properties of the tubes assembled from water and alcohol mixtures were adjusted by altering the conditions for 10237

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Figure 17. Contact mode AFM image of DC8,9PC tube showing helical structure and reduction in diameter toward the end of the tube.367 Reprinted with permission from ref 367. Copyright 2009 American Physical Society.

modulus was determined using this method to be 0.703 GPa.344 This work also indicated that the stiffness of the tubes reduced progressively toward the tube ends (Figure 17).344,367 This decrease in tube stiffness was correlated to a stepped decrease in tube thickness occurring toward the ends of the tubes,367 something also noticed for glycolipid nanotubes.368 Fluorescence microscopy techniques, fluorescence recovery after photobleaching, and fluorescence correlation spectroscopy were used to track the diffusion of fluorophores through the lumen of the tubes and the association of fluorophores with, or diffusion through, the tube walls.369 Rhodamine 6G had two distinct diffusion profiles, and the diffusion of the dye in the bulk water of the lumen of the tube was 2−4 orders of magnitude faster than that of dye molecules interacting with the membrane wall.369 Furthermore, the diffusion of the fluorophore was slower in the middle of the tubes than at the ends of the tubes, and this was attributed to variation in lipid packing densities in these locations.369 4.1.1. Nanotube Stabilization via Polymerization. Diacetylenic lipid-based tubules have been stabilized by polymerization of the diacetylenic groups when UV light, or other more energetic irradiation, excited the diacetylenic groups. The irradiated samples displayed a characteristic red color of 1−4 addition between the diacetylenic moieties. In contrast, for irradiation occurring between 0 and 4 °C, the polymerized tubes developed a deep blue color. Upon warming the blue tubes to room temperature, a thermochromic effect occurred and the color changed to red.139,152,153 The blue to red color change was also induced by solvation stresses, exposure to an X-ray beam, and by being mechanically scratched or sheared.370 Despite this evidence of polymerization, the continued presence of the 42 °C melting endotherm153,174 and the detection of unreacted triple bonds by Raman spectroscopy371 indicated that overall conversion was low. The polymerization of the tubes nonetheless provided enhanced stability toward physical and chemical perturbations, and a reduced propensity to leak entrapped substances.146,160 The topotactic polymerization of diacetylenic groups requires that the groups are properly aligned and spaced. As such, the polymerization that occurred in tubules was indicative that the aggregated structure was highly ordered.139,152,154,174,248,372−374

Figure 16. Optical microscopy of helices extruding from a diacetylenic phospholipid vesicle.222 Reprinted with permission from ref 222. Copyright 1999 American Physical Society.

temperature, tubules based on helices of mixed hand grew directly from the vesicle at a rate of 1 μm s−1 along the tubule axis in a reversible process.222 Tubule growth from saturated DC8,9PC solutions without vesicles also occurred, but at a much reduced rate of 0.1 μm s−1 and based on helices of a single hand. Analysis of these observations showed that the tubule forming drive of DC8,9PC and its close analogues was best described by an integration of the kinetically induced fast extrusion of the tubes from vesicles, based on purely tilt-based chiral symmetry-breaking, and the thermodynamically directed chiral molecular packing associated with the agglomeration of lipids from solution (see section 2.5).240 The ripple (Pβ′) phase of self-assembled diacetylenic tubes has been observed by several authors. The first to record such a ripple phase was Yager et al.139 Using freeze-fracture electron microscopy, they observed a ripple on the surface of DC8,9PC tubules, having a period of 100 nm and an angle of 60° relative to the tube equator. More recently, the rippled phase in DC8,9PC tubules has been investigated by the research group of Fang et al.140 using amplitude mode AFM. In this case, 7% of the tubules had a ripple phase with a period of 200 ± 30 nm, and a bimodal distribution of angles (relative to the equator of the tube) being 28° and 5°. The fact that the 28° ripple phase runs at the same angle as the helical bilayer ribbons140,209 and similar amounts of tubules are shown to exhibit a tilt modulation (10%) as those that have a ripple phase (7%)140 corroborates current theories on the packing of lipids during the formation of helically based tubes that predict there is a modulated tilt order that can induce the observed rippled phase in tubules.86 The group of Fang has studied the mechanical properties of the tubes assembled from DC8,9PC.105,367 In one experiment, the buckling of polymerized DC8,9PC multiwall nanotubes trapped inside shrinking liquid droplets was studied by optical microscopy, and the Young’s modulus for the bending rigidity of the tubes was estimated to be 1.074 GPa.105 Tubes were also buckled under the force of an AFM tip, and the radial Young’s 10238

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At the same time, some flexibility in the self-assembled structure was also indicated, as movement of the monomers within the structure was induced by the polymerization, causing both strain and deformation to the tube structure, ultimately preventing the formation of long polymer chains.139,153 This was improved to an extent by the addition of short-chain spacer lipids, such as DC8PC or DC7PC, to the lipid matrix for self-assembly to provide enhanced flexibility in packing and allowing more extensive polymerization of diacetylenic groups.153 Polymerization of tubes assembled from DC8,9PC and DC7PC stabilized the macrostructure against detergent lysis and also increased the permeability of the membrane.375 4.1.2. How Changes to Molecular Structure Affect Supramolecular Organization. 4.1.2.1. Addition of a ShortChain Spacer Lipid. The inclusion of short spacer lipids like DC7PC in the self-assembly of DC8,9PC, used as discussed in the previous section to help stabilize polymerized tubes, also alters the aggregate morphology. Using lipid ratios of DC8,9PC to DC7PC of close to 1:1 in aqueous dispersions formed networks of interconnected right-handed twisted ribbons216 that gelated the solvent at just 0.4% (v/v) of lipid.375 Svenson and Messersmith376 found the gel progressed through two metastable morphologies. Vesicles with an average diameter of 827 nm initially formed on cooling of the lipid solution from 65 °C to room temperature. Narrow tubules of low rigidity relative to those constructed purely of DC8,9PC then formed, before the final gelation occurred.376 The rate of transformation to the final gelated structure increased with increasing lipid concentration.216,376 The narrow mixed lipid tubules were stabilized against the transformation to gels by cooling to 4 °C.216,376 These tubes had a narrow distribution of diameters between 45 and 55 nm, and had lengths of up to tens of micrometers.216 No helical markings were evident on the tubes, but CD spectroscopy showed a large signal arising from chirally organized molecular packing,216 and the split into positive and negative peaks for the diacetylenic group indicated there was exciton coupling between neighboring groups in the mixed lipid aggregate.216 Such exciton coupling suggested not only that the packing around the diacetylenic group was highly organized in the mixed lipid tubes, but also that chirality, whether of the molecule or the macrostructure, played an important role in the tube formation.86,216 Further work has since varied the lengths of saturated chains in the lipid spacers. DC6PC and DC8PC work similarly to DC7PC when mixtures with DC8,9PC were self-assembled as none of these chains were long enough to block diacetylenic interactions between neighboring DC8,9PC lipids.276 Selfassembly of DC8,9PC with a DC9PC spacer, however, produced larger diameter tubes of 140−240 nm, apparently caused by rupture of the tubule walls along the helical seams at the edges of the chiral domains. Annealing at 4 °C for 105 days allowed the repair of such ruptures and resulted in tubes with 63 ± 3 nm diameters, almost as low as the DC7PC system.276 Further investigations showed that increasing the distal chain length in mixed lipid systems in which the spacer lipid has one less methylene unit than the proximal chain of the diacetylenic lipid led to an increase in tube diameter.276 This was in contrast to pure diacetylenic systems for which the distal chain length had little influence on the self-assembling properties of the lipid.223 4.1.2.2. Alterations to Alkyl Chain Length. A range of diacetylenic phospholipids have been synthesized, varying in distal and proximal chain lengths, and many of these self-assembled

into tubes.152 Moderate increases in the length of the distal chain showed little effect on the properties of the aggregate; for instance, DC8,11PC and DC8,13PC self-assembled in an almost identical manner to DC8,9PC.276,277 Similarly, there were few obvious changes to the self-assembly upon moderate change to the proximal chain, as long as the proximal chain length was kept as an even number of methylene units. However, odd numbers of methylene units in the proximal chain created variations in assembly, such as the number of bilayers in the tube walls.206 This difference was attributed to changes in molecular packing, supported by FTIR, CD, DSC, and electron density analysis, all of which indicated an increased level of order in the intramolecular organization for self-assembled lipids with even numbers of methylene units in the proximal chain.206,277,377 This was because the kink in the chain arising from the diacetylenic group had different orientations relative to the head group for odd and even proximal chains, significantly altering the packing of the lipid.206,377 Not only did even numbers of methylene units in the proximal chain provide superior packing for the alkyl chains, but also provided an orientation of the head groups that allowed for improved chiral interactions between the head groups in adjacent bilayers.233 4.1.2.3. Alterations to the Head Group and Backbone. 4.1.2.3.1. Exchanging Choline for Glycol Groups. The choline on the phosphocholine head group of DC8,9PC was exchanged for a short alkyl chain attached to a hydroxyl group (Chart 9, 21).378 These lipids only formed tubules in significant Chart 9. Diacetylenic Phospholipid Similar to DC8,9PC, but with the Choline Head Group Exchanged for a Hydroxyl Group Attached through (x) Methylene Units

populations for aqueous solutions at pH 5.6 in the presence of either a relatively high concentration of monovalent metal ions (0.1 M) or a lower concentration of divalent metal ions (1−1.5 mM).358 The metal ions neutralized the charge on the head groups, and so allowed the lipids to aggregate.378 The anionic counterions to the metal ions also had an effect on lipid self-assembly. Chloride and perchlorate counterions created bimodal distributions of tube diameters for lipids containing two or three methylene units in the short alkyl chain in the head group.358 In contrast, fluoride, sulfate, and nitrate counterions led to the production of single large diameter tubes for the same lipids.358 Some specific applications for lipid-based tubules are assisted, or only possible, if the tubule is coated in metal (Figure 18).379 Tubules were formed from 21 and palladium tetraammine chloride at pH 5.6, and then metallization occurred from a plating solution through electroless deposition. This dual use of the palladium ions to assist amphiphile assembly and also act as a metallization catalyst represents an improvement in metallization of tubes as compared to the physisorption of a Sn−Pd catalyst required for DC8,9PC tubes (section 4.1.5.3), which results in the retention of tin oxides in the metal coating.379 10239

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Figure 18. TEM image of metallized phospholipid lipid tubules.380

4.1.2.3.2. Phosphonate Head Groups. Rational tubule design looks to achieve new internal diameters, as this is important in terms of both the storage capacity and the diffusion rate associated with any encapsulated materials. One way of doing this was to make small changes in lipid backbones such that the tubular morphology could be retained, but the dimensions of the tubes altered.240 Phosphonate lipids with a C−P link between the tail and the head group (instead of C−O−P seen for DC8,9PC, Chart 8), still formed tubules readily with the same helical basis.240 The tubule formation was very similar between DC8,9PC and its “C4” and “C3” phosphonate analogues (Chart 10, 22 and 23, respectively), precipitation of

Figure 19. TEM images of phosphonate lipid helically based tube. Reprinted with permission from ref 240. Copyright 1998 American Chemical Society.

Chart 11. Ether Linked Diacetylenic Phospholipid

Chart 10. Diacetylenic Phosphonate Lipids Chart 12. Achiral Diacetylenic Phospholipid

(Chart 12, 25). This placed the head group centrally on the glycerol backbone, and removed the chirality.225 The achiral phospholipid self-assembled into helically based tubules, with left- and right-handed versions of equal numbers, and no CD measurable signal was observed. Consequently, molecular chirality is apparently not essential in the formation of diacetylenic phospholipid tubules, and chiral symmetry breaking must be a factor in its the assembly.225 When 25 was doped with 6.1% DC8,9PC, the chiral dopant was able to chirally direct the assembly in the manner of the “sergeants and soldiers” model (see section 2.7), producing a CD signal 5 times larger than would be predicted on the basis of concentrations alone. This type of direction by molecular chirality is not unexpected for chiral symmetry breaking models of self-assembly.206,225 4.1.3. Modification of Supramolecular Structure by External Additives and Forces. Alteration of tubule parameters is most important for rational design, and the simplest way this can be achieved is by altering aspects of assembly other than the molecular design of the lipid. Various external forces have been shown to affect the aggregate morphology of the tubes self-assembled from DC8,9PC. For example, extremes of pH reduced tubule diameter, but also caused hydrolysis of the ester groups.152 Tubule length has been increased by decreasing the cooling rate,151 while self-assembly in acetonitrile led to transitions having 3−5 kcal mol−1 higher enthalpy values as

tubules occurring with essentially the same length, pitch angle, yield, and interlamellar spacing for the same assembly conditions. However, the phosphonate lipids had a 2-fold increase in diameter (22, 1.182 ± 0.135 μm; 23, 1.076 ± 0.090 μm) and a reduction in the wall thickness by one-half as compared to DC8,9PC.156,224,240 Another variation in the assembly of 22 and 23 as compared to DC8,9PC was that the helical precursor assemblies (Figure 19) of the phosphonate tubes were of mixed hand despite 99% enantiomeric purity in the starting material.156,224,240 This suggested that self-assembly occurred via the chiral symmetry breaking model for tubule formation.240 4.1.2.3.3. Ether Links to the Alkyl Chains. Exchanging ester linkages for ether linkages between the head group and chains (Chart 11, 24) still allowed tube formation. These tubules had the ability to polymerize, indicating a highly ordered structure.381 4.1.2.3.4. Achiral Phospholipid. An achiral analogue of DC8,9PC was created by exchanging the phosphatidylcholine head group with the adjacent ester linked diacetylenic tail 10240

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to the weak anisotropic diamagnetism of the hydrocarbon chain.148 Diacetylenic phospholipid tubes orient parallel to magnetic field lines, which is indicative of chain tilt with a direction away from the tubule axis, as orientation of the tube is driven by the polar angle between the hydrocarbon chain and the tubule axis.148 DC8,9PC tubules mixed with aqueous dispersions of ferric oxide aligned themselves along the field lines of a much weaker magnetic field (25−800 G) than that for pure aqueous dispersions of tubes.141 Cationic magnetic nanoparticles coated the DC8,9PC tubules and filled the internal tubule cavity. In contrast, anionic nanoparticles did not enter the lumen of the tube, and did not appear to bind to the outer surface.141 These factors suggested a model in which the tubules behaved as negatively charged objects. This was ascribed to the conformation of the head group exposing the negatively charged phosphate group to the aqueous environment more than the positively charged choline group.141 This model was disputed by Lvov et al.197 who explained the phenomena by a flexoelectric effect in which the curvature of a membrane induces a net electrostatic polarization normal to the membrane.384 The dipole moment of this polarization should point outward for the phospholipids, providing sufficient electrostatic energy to attract positively charged particles inside the lumen, and keeping negatively charged particles out.197 Lipid tubules have also been oriented magnetically after being coated with ferromagnetic permalloy (Ni80Fe20) using electroless deposition. Because of the magnetic properties, these tubes were aligned in a small magnetic field of less than 100 G.385 The modulated tilt of direction of the amphiphiles in diacetylenic nanotubes provides birefringent properties. As such, tubes constructed from a single enantiomer were manipulated using 532 nm, linearly polarized laser tweezers. The tubes spontaneously rotated due to the torque applied by the radiation. Light passing through the tubes was right circularly polarized by the tube structure, and in maintaining angular momentum the rotation of the tubes was in a single direction, rotation continuing until their long axis was aligned with direction of polarization.386 Optical tweezers using circularly polarized incident light led to continuous rotation of the tubes, with the direction of rotation decided by the direction of the circular polarization of the light.386 Phospholipid tubules were also aligned mechanically by drawing a droplet containing the tubules into microcapillaries, formed between a glass substrate and a PDMS stamp etched with parallel channels.67 These aligned tubules on a glass substrate were immersed in colloidal silica solution at pH 8.4, and the deposited colloidal particles formed rough silica films of 420−470 nm thickness surrounding the portion of the nanotubes exposed to the solution.67 Similar structures were created with the sol−gel reaction of TEOS and the tube surfaces, but in this case the silica surface was smooth.53 In related experiments, the PDMS stamp used to align the DC8,9PC tubes has also been used as a microcontact printing device.53 This was achieved dipping the stamp into the tubule solution, and surface tension retained the aligned tubes in the channels of the stamp (Figure 21a). The aligned tubes were then printed onto appropriate surfaces, including textured and otherwise nonflat surfaces. One example was the orthogonal printing of nanotubes onto 250 nm thick gold electrodes fabricated on a silica substrate (Figure 21b). The tubes were able to span the electrodes without breaking, suggesting applications in nanoelectronics. Another example was the deposition of

compared to those formed in water, but retaining similar characteristic dimensions.382 Self-assembly of DC8,9PC from various salt solutions at molar concentrations has resulted in both increased (di- and trivalent cations) and reduced (sodium iodide) transition temperatures, reduced tube length (sodium and calcium chloride as well as trivalent cations), and increased tube wall thickness (divalent cations).179 From this general lack of a significant, consistent electrolyte effect, it was inferred that in this system polar and ionic interactions between head groups are not as important to the ordered packing of the molecules as the interactions between hydrophobic parts.179 As mentioned previously (section 4.1), the precipitation of tubules from aqueous mixtures with various alcohols has resulted in differing physical characteristics,195 one example being that tube wall thickness increases with alcohol chain length.195 This has enabled the optimization of the tubule wall thickness for metal plating by adding both methanol and ethanol to the solvent mixture (64:16:20 methanol/ethanol/ water), resulting in a consistent two bilayer tube wall thickness for metallization.206 Lysozymes were added to DC8,9PC, and the resulting aggregate morphology consisted of gently tapered hollow cones (Figure 20).383 The protein caused crack discontinuities in the

Figure 20. DC8,9PC cone-shaped aggregate morphology in the presence of lysozymes. Reprinted with permission from ref 156. Copyright 2005 American Chemical Society. Upper electron microscopy image illustrates cone morphology and crack discontinuity from which helical (A) and twisted (B) tubulets appear, in close-up in the lower image.156

surface of air-dried samples prepared for SEM. This allowed unprecedented microscopic access to the morphologies existing in bulk solution, such as the tubulets described in section 2.6.156 4.1.4. Alignment of Nanotubes. Specific orientation of nanotubes is important for applications in optical and mechanical devices. Biological membranes can be oriented by magnetic fields of 1 T or more, and this effect has been attributed 10241

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Figure 21. Microcontact printing of tubes aligned in a PDMS stamp. Reproduced and adapted from ref 53. Copyright 2007 The Royal Society of Chemistry. Optical microscopy images of (a) tubes aligned inside PDMS stamp, (b) aligned tubes printed across gold electrodes on a silicon surface, and (c) a diagram of the alignment of tubes on a glass tube.53

allel channels 0.8 μm high and 1.0 μm wide.389 These tubes, when imaged by AFM, displayed buckling at the sites of the bends of the zigzag. The extent of the buckling increased with increasing bend angle from straight up to a maximum at 30°.389 Using contact mode in AFM, the researchers were able to determine the stiffness of the tubes at 1.2 N/m for the straight sections and one-half that for the bent sections.389 4.1.5. Template Directed Synthesis Using DC8,9PC. 4.1.5.1. Inorganic Deposition. Silica can chemically bind to nanotubes in several ways. In one method, inorganic silica particles were deposited onto the phospholipid nanotube surface to make a strongly adherent amorphous film 50 nm thick.390 Subsequent heating to 600 °C decomposed the organic phospholipids, but retained a silica tubular morphology.390 Although the average length of tubules was reduced during this process, an improvement in thermal and mechanical stability was realized.390 The resultant silica tubes were more easily functionalized than their precursor lipid tubes, with metallization by nickel electroless deposition more efficiently and effectively achieved.390 A second method of chemically binding silica to diacetylenic nanotubes involved the addition of aqueous hydrobromic acid to a solution of DC8,9PC and TEOS in ethanol. The TEOS hydrolyzed, instantly forming a white precipitate.370 The precipitate contained silicified helical ribbons and tubes with diameters of 0.5−1.5 μm and lengths of greater than 5 μm.370 The lack of amorphous colloidal silica together with the presence of continuous lattice fringes in high-resolution TEM images suggested that the sol−gel reaction occurred in association with the lipid molecules as they self-assembled. Calcination of the microstructures at 410 °C removed the organic template, leaving a silica replica.370 Interestingly, an increase in interbilayer spacing of 3 nm indicated that the silica is intercalated between the bilayers. In a related method, DC8,9PC was combined with 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphoethanolamine (DC8,9PE) at a loading of 10% of DC8,9PE (DC8,9PE differs from DC8,9PC only in that the terminal ammonium moiety has no methylation).80 The lipids were then mixed with a combination of TEOS and 3-aminopropyl-triethoxysilane (APTES, 11:1 v/v) in ethanol. On the addition of water, the sol−gel reaction started despite the absence of acid or base catalysts. It was proposed that this deposition matched that which occurs in biomineralization with inorganics being deposited on phosphatidylcholine head groups.80,391 The inclusion of APTES in the self-assembly provided external amine groups that were subsequently used in standard bioconjugation reactions

Figure 22. Optical microscopy of tubules aligned on gold. Reprinted with permission from ref 387. Copyright 2006 American Chemical Society.

the aligned nanotubes onto the curved surface of a glass tube (Figure 21c).53 Microcontact printing was also used to pattern gold substrates with hydrophobic dodecanethiol monolayer stripes (Figure 22).387 Such patterned gold substrates were immersed in aqueous dispersions of lipid nanotubes, and then withdrawn in the direction of the dodecanethiol stripes.387 The lipid tubules selectively absorbed onto the naked gold substrate between the monolayer stripes, and were aligned with the stripes, as the hydrophilic lipid exterior was repelled from the hydrophobic stripes.387 Alterations to the long, straight tubule shape were achieved by drying a droplet of suspended polymerized lipid tubules on a gold substrate coated in a dodecanethiol monolayer. The shrinking contact line of the droplet then bent the tubules uniformly into looped shapes. These loops had a radius of 14− 25 μm and an estimated persistence of 41 μm, larger than that found for carbon nanotubes, indicating that the lipid nanotubes were stiffer.362 Zigzag shapes were also created with 42° bends by moving contact lines,388 but in contrast to the uniform bending of the loops, these zigzag shapes displayed some buckling in the bending regions.388 The tubules bent into loops and zigzags were able to be filled with a nematic liquid crystal through capillary action, thereby proving that the hollow core remained open and accessible.388 DC8,9PC nanotubes have also been bent into zigzag shapes by a moving contact line of water held loosely between a glass substrate and polydimethylsiloxane stamp patterned with par10242

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Figure 23. TEM image of silica nanoparticles helically deposited on DC8,11PC tubes under 5000× magnification. Reprinted with permission from ref 197. Copyright 2000 American Chemical Society.

to functionalize the hybrid tubes.80 Section 11.1 describes how such tubes were used as drug delivery vehicles to treat cancer.80 Germania is closely related chemically to silica, with germanium having the same organization of valence electrons, but is varied enough to provide extra potential for applications due to its optical and electronic properties as well as catalytic activity. Diacetylenic phospholipid tubules have been used in the synthesis of germania nanoparticles and germania coated tubes.391 In this process, which closely mimicked the uncatalyzed silica deposition technique discussed above, germanium tetraethoxide (TEOG) was crystallized in the presence of DC8,9PC.391 As with silica deposition, the self-assembly of DC8,9PC in the presence of TEOG led to precipitation of germania exclusively on the surface of the self-assembled structure. Lvov et al.197 used the multiple adsorptions of oppositely charged polymers and silica spheres to amplify the small underlying charge patterns caused by defects in the packing of diacetylenic phospholipids in tubules. Initially, DC8,11PC tubes adsorbed an anionic polymer, followed by absorption of a cationic polymer and finally absorption of the negatively charged silica nanoparticles. In this case, the silica spheres were exclusively absorbed onto the ends of the tubes. Inclusion of 2% of the negatively charged lipid 1,2-bis(10,12- tricosadiynoyl)sn-glycero-3-phosphohydroxyethanol (DC8,9PEOH, Chart 9, 21, x = 2) with the zwitterionic DC8,11PC created tubules with a mean diameter of 800 nm.197 Cationic polymers were absorbed to these tubes followed by anionic polymers and then cationic polymers again before absorbing the silica nanoparticles. The silica spheres bound to the ends of the tubes again, but in this case about 5% of tubes also had silica deposited along the helical seams, exclusively on the inside of the tubes (Figure 23). This highlighted that the lipid mixture had three pitch angles (as discussed in section 2.3) instead of the single pitch angle previously noted for DC8,9PC tubes.197 The patterning indicated that the negatively charged lipids were enriched in the helical defects in the tubule structure. When 3-(2-aminoethyl-3-aminopropyl)trimethoxysilane was added to an ethanolic aqueous dispersion of preformed DC8,9PC

tubule templates, the addition of aqueous magnesium chloride caused the construction of magnesium phyllo(organo)silicate clay micropipes.392 The resulting helical ribbons and tubes had outer diameters between 0.55 and 0.80 μm. The outer walls of the lipid tubes were coated with a smooth layer of clay 25−50 nm thick, with no evidence of exogenous clay particles or of intercalation between bilayers. The lipid was removed from these structures by dissolution in ethanol, leaving behind intact organoclay tubes.392 Ferromagnetic nanotubes have also been created by absorption of ammonium iron(II) sulfate from solution onto the internal and external surfaces of tubes self-assembled from DC8,9PC (19). The tubes were then treated with hydrogen peroxide, oxidizing the iron and initiating polymerization of the DC8,9PC diacetylenic groups. Subsequent calcination of the tubes resulted in the formation of a carbon tube coated with iron oxide on the internal and external surfaces, with iron oxide phases exhibiting extraordinary magnetization.393 By altering the solvent composition of DC8,9PC assembly (discussed in section 4.1), 6, 10, and 50 nm carbon wall thicknesses were generated for the calcined tubes. The thicknesses of the magnetite layers were also controlled by altering the ratios of reagents for the redox reactions, resulting in iron oxide layers from 12−45 nm thick. The increase in thickness resulted in an almost 2-fold increase in saturation magnetization.394 4.1.5.2. Deposition of Conducting Polymers. In an extension of application of tubes to template directed synthesis of nanostructures, polypyrrole was polymerized in the presence of phospholipid nanotubes, deposition occurring exclusively at the edges of the helical striations and at the ends of the tubes with minimal polymerization occurring in solution.203 It was thought that the partially exposed diacetylenic groups at the high energy edges of the helical ribbons provided an electron-rich and hydrophobic environment to preferentially absorb the pyrrole, allowing their polymerization into long polymer strands.203 4.1.5.3. Deposition of Metals. The polar head groups of the phospholipids can provide a suitable substrate for the absorption of colloidal palladium and tin ions that can subsequently 10243

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act as a catalyst in the electroless deposition of nickel, copper, iron, and cobalt.33,360 The size of the metal particles formed and their density of deposition can be controlled by adjusting the parameters of metallization.33 Unfortunately, the metallization process subjects the fragile lipids to environmental stresses that result in significant tubule breakage, which reduces the average tube length by 50%.357 Lipid tubules have been coated with permalloy (Ni80Fe20) using electroless deposition, with interior and exterior coating thicknesses between 20 and 50 nm.395 The alloy coating exhibited a saturation magnetization moderately reduced from that expected for the bulk metal, which was attributed to oxidation during the coating process.395 Magnetic alignment of such tubules within a polymer matrix then allowed the production of materials with extremely anisotropic electromagnetic properties.84,385 This could allow the reduction in size of microwave electronics, and has already been applied in the development of variable phase shifters.33 Metallization of DC8,9PC tubes has also taken advantage of the higher chemical reactivity of lipids located in the helical striations to create helical templates.204 An example of this was the chemical reduction of hydrogen tetrachloroaurate hydrate from aqueous ethanolic solution by lipids at the edge of preformed tubes.204 Here, it was presumed the exposed diacetylenic groups took part in the redox reaction,204 resulting in the patterning of discrete gold nanoparticles that followed the helical ribbon structure of the tube, as well as being deposited at the ends of the tube.204 Further work on the helical deposition of gold nanoparticles onto phospholipid tubules used electroless plating in which tin and silver intermediates facilitated the subsequent deposition of gold.205 Through the careful control of the plating time, almost exclusive deposition of gold nanoparticles along the helical seams was achieved, resulting in continuous wires. Upon extraction of the phospholipid template with ethanol, the gold nanowires held their helical structure.205 Price et al.11 postulated that the specificity of metal deposition along the seams of diacetylenic phospholipid could be improved by following Lvov et al.’s197 lead in using a mixture of DC8,9PC with 2% DC8,9PEOH to make tubes, followed by the subsequent sequential adsorption of polyelectrolyte to amplify the charge along the helical seams. Price et al.11 found three polyelectrolyte adsorption steps were required to bind sufficient negatively charged palladium nanoparticles to the mixed lipid tubules to subsequently catalyze the deposition of copper. This resulted in well-formed copper spiral structures in 30% yield.11 Colloidal palladium particles also preferentially decorated the helical edges on the interior and exterior of phospholipid tubules. The palladium then catalyzed the deposition of nickel metal from solution, and this nickel then retained the original helical patterning of the palladium.82 Adding electrically conductive particles, such as copper and nickel coated phospholipid nanotubes, to an insulating polymer increases the permittivity and conductivity of the resulting composite.396 Metallized nanotubes display percolation conduction (or macroscopic conduction) at significantly lower loading densities than those measured for spherical conducting particles.396 Combined with the ability to align the metallized tubes in magnetic fields,385 this may then lead to applications in coatings with controlled anisotropic dielectric properties.357,360 4.1.5.4. Deposition of Proteins. DC8,9PC was coassembled with 1,2-dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (Chart 13, 26) and egg phosphatidylcholine (27) in a ratio of 3:1:1 from a

Chart 13. 1,2-Dioleoyl-sn-glycero-3-phospho-(1′-racglycerol), 26, and Egg Phosphatidylcholine, 27

dispersion in methanol:water (80:20) forming a mixture of helices and tubes, the tubes being 570 nm wide and 10−100 μm long.397 The incorporation of the charged lipid into the tubes was used to support the assembly of FtsZ protein rings from the single-stranded filaments usually found in vitro. The filaments encircled the tubes, closely packed into ribbon like rings variably spaced along the tubes length (Figure 24). FtsZ is

Figure 24. FtsZ protein rings encircling phospholipid tubes. Reprinted with permission from ref 397. Copyright 2012 Biophysical Society.

a major cytoskeleton protein of prokaryotes; however, the rings are difficult to image from bacterial samples and do not usually assemble in vitro. This experiment has provided new evidence for the structural organization of these rings.397 Horseradish peroxidase was encapsulated in DC8,9PC nanotubes at a mass of 0.16 μg per mg of tubes. The internalized proteins were more stable than external proteins under conditions of time, heat, and exposure to a denatureant.398 4.2. Other Phospholipids

1,2-Dimyristoyl-sn-glycero-3-phosphochiline (DMPC, Chart 14, 28) with saturated hydrocarbon chains forms helical superstructures under certain conditions (Table 2).399,400 One method is to expose hydrated DMPC to elevated temperatures (30− 50 °C) in the presence of sodium azide for extended periods.399 This leads to some degradation of the lipid, with the complementary byproducts of myristic acid and lyso-phosphatidylcholine produced and mixed together with the lipid. After a few weeks incubation, a small population of right-handed 10244

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Chart 14. 1,2-Dimyristoyl-sn-glycero-3-phosphochiline (DMPC)

Chart 15. Dilauroyl Phosphotidyl Adenosine

Chart 16. Amide Linked Phospholipids Developed by Sommerdijk et al.244

helically wound ribbons was observed, having a wall thickness of 3−5 bilayers.399 DMPC was also found to self-assemble into chiral ribbons when combined with cetyltrimethylammonium bromide (CTAB) protected gold nanorods (16.0 ± 2.4 nm × 43.5 ± 7.4 nm) and a small amount of fluorescent lipid and then heated above the lipid phase transition temperature. Upon cooling, the self-assembled structures proved to be helical ribbons with the nanorods helically aligned in the manner of mesogenic molecules in a cholesteric liquid-crystalline mesophase.401 A dilauroyl-phosphatidyl derivative with an adenosine head group (Chart 15, 29) self-assembled into helical ribbons via micellar intermediates in aqueous solutions with pH 7.5 at 25 °C.113 The helical twist of these aggregates was of both hands, even though CD analysis suggested the existence of chirality in the supramolecular packing. It was proposed that this anomalous behavior was due to the helical assembly being driven by base stacking of the nucleoside head groups rather than chiral interactions, but a chiral symmetry breaking mechanism could also explain such behavior.113 In an effort to further understand the forces driving supramolecular chirality, a series of chiral, amide-containing surfactants was synthesized, and their aggregate morphologies examined.244 The molecules created are shown in Chart 16 with molecules 31 and 33 being positional isomers of 30 and 32. Molecule 31 formed left-handed, 22 nm diameter helices upon dispersal in water.244 The enantiomer of 31 formed identical right-handed helices, but the positional isomer, 30, only formed planar structures. This variation was due to the organization of the butyrate group that for 30 points toward the

hydrophobic layer, making the head group too large to allow organized packing of the hydrocarbon chain.244 Molecule 32 forms bilayer ribbons in aqueous dispersions at pH 6.5. As pH is reduced to pH 2.5, protonation of the head groups induced a twist in the ribbons resulting in left-handed helices that matured into tubes.244 These helical aggregates had a phase transition of 26 °C, which is 5 °C higher than that for the achiral ribbons at pH 6.5, indicating that the helical aggregates had a higher degree of order in the molecular packing.244 The positional isomer of 32, 33, only forms fibrous micellar aggregates, with a low degree of packing order.244

Table 2. Characteristics of Nanotubes Self-Assembled from Phospholipids under Varying Conditions amphiphile

solvent

19 19 19 19 + 20 1:1 DC8,11PCa + 21 (2%) DCm,nPC (odd)a DCm,nPC (even)a DC10,7PCa DC10,7PC (5 mg/mL)a 21 22 23 24

aqueous EtOH:water 70:30 MeOH:water 85:15 aqueous aqueous MeOH:water 80:20 MeOH:water 80:20 MeOH:water 80:20 MeOH:water 80:20 aqueous EtOH:water 70:30 EtOH:water 70:30 EtOHc

length (μm)

external diameter (μm)

10−1000 50−170 60 10

0.4−1 0.5 0.5 0.05 0.8

0.08−0.96b 1.2 1.1

23.5 16.6

no. of bilayers

trans temp (°C)

ref

2−10 5−10 1

43 36 33 25.4

multiple 1 1 6−10

48 52

152,366 152,366 133,162 216 197 206 206 206 206 358 224,240 224 381

3.2 3.2 30.0

a

DC8,11PC, DCm,nPC, and DC10,7PC are as described in Chart 8 and section 4.1.2.2 and assembled at a concentration of 1 mg/mL unless otherwise specified. Phospholipids DCm,nPC have either odd or even numbers of methylene units in the proximal chain. bSpecific diameter dependent on pH and the identity and concentration of salts in self-assembly mixture. c24 dissolved in ethanol precipitated with water. 10245

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Chart 17. N-Alkyl Aldonamides: 34−38, Chiral Micellar Aggregate Forming Species; 39−42, Helical Ribbon and Tube Forming Species; 43, Cochleate Cylinder Forming Amphiphile

5. HELICALLY BASED NANOTUBES SELF-ASSEMBLED FROM GLYCOLIPIDS 5.1. Aldonamide Head Groups

5.1.1. N-Alkylaldonamides. In 1985, Pfannemüller and Welte280 were the first to observe the formation of anisotropic chiral aggregates from aldonamide molecules in water. These aqueous gels were comprised of highly ordered, high-axial ratio structures with a right-handed twist and formed from N-alkyl-Dgluconamides at concentrations of just 1−2%.280 Extensive work has been conducted to analyze the ultimate aggregate structure of two N-alkyl-D-gluconamides (Chart 17, 34 and 35) using high-resolution electron microscopy,243,402,403 NMR,404,405 XRD,406 and AFM.407 The final conclusion is that they are quadrupole helices made from micellar cylinders with only the micellar diameter varying between the two aldonamide species due to the different chain lengths (Figure 25).243,402,407 Following on from the research into the N-alkyl-Dgluconamides, Fuhrhop et al.293 synthesized eight diasteromeric N-octyl-D-aldonamides and three enantiomeric analogues, all of which varied only in the stereochemical organization of the head group. It was found through NMR investigations that the head group conformation and the consequent intermolecular or hydration opportunities for the various hydrogen-bonding groups drove the aggregation behavior.404,405 The aldonamides (Chart 17) with multiple gauche bonds in the head group (36−38) were water-soluble,293 and the molecules with a single gauche bond in the head group close to the amide bond, such as 34, were less soluble and precipitated with a twisted micellar packing that forms the anisotropic micellar helices mentioned above.293,294,404,405 N-Octyl-D-galactonamide (39) and N-octylL-galactonamide (40), with a fully extended all-trans head group conformation, formed relatively insoluble, high-axial-ratio bilayer assemblies such as tubes and helical ribbons (described as “twisted” ribbons in this work) with the chirality of the aggregates related to the molecular chirality of the precursors.293,294 The all-trans head group conformation allowed all of

Figure 25. Computer graph model of the N-octyl-D-gluconamide quadrupole micellar helix. Reprinted with permission from ref 402. Copyright 1993 American Chemical Society.

the hydroxyl groups to be involved in intermolecular hydrogen bonding, reducing hydration and supporting extended chains of hydrogen bonding between amide moieties. This prevented 10246

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molar amounts of enantiomers were mixed together, flat planar platelets resulted.245 When the galactose head group was exchanged for L-mannose (45), similar tubes were created, but with a larger external diameter of 370 nm.246 Frankel et al.245 also investigated changing the length of the head group for diacetylenic aldonamides, creating both a pentose (46) and a heptose (47) based on the stereochemistry of 45. For 46 and 47, tubular structures resulted, with outer diameters from 0.27 to 0.4 μm. Polymerization of the diacetylenic groups in the N-alkylaldonamides by UV-irradiation stabilized the tubular structure, with treated tubes able to withstand exposure to organic solvents that dissolved unpolymerised tubes.246 UV characterization of polymerized tubule structures showed that those lipids with heptose and hexose head groups had the highest level of polymerization, and therefore generated the most well-ordered structures prior to polymerization.245 Indeed, these diacetylenic aldonamides seemed to provide an improved arrangement of the diacetylenic moieties for the polymerization as compared to DC8,9PC, which polymerizes to relatively low yields.245 Interestingly, diacetylenic gluconamides with 1, 3, and 5 methylene groups between the amide moiety and the nonadiynyl chain (48, 49, and 50) did not self-assemble into helical bilayer-based structures.292 While nanotubes did form from 48 and 50, the researchers believed these much smaller tubes (external diameters from 50 to 70 nm) were constructed of bundles of chiral micellar fibers similar to those found in the quadrupole helices based on N-alkyl-gluconamides discussed in section 5.1.292 5.1.3. DAP Linked Aldonamides. A different type of alkylaldonamide was created with the incorporation of a 2,6diaminopyridine (DAP) linker between an acyclic glucose head group and either oleoyl or stearoyl single hydrophobic tails (Chart 19). When the oleoyl-based lipid (51) was dispersed in water at 100 °C, and then allowed to cool slowly, it formed helical ribbons that matured into tubes upon aging. These tubes had an external diameter of 60−80 nm, an internal diameter of 20 nm, and a main thermotropic transition at 70 °C.295 The stearoyl-based saturated analogue (52) formed fibrous morphologies, and the lack of chiral aggregated structures for 52 demonstrated the importance of the bent molecular shape of the cis double bond in 51 in generating chiral supramolecular assemblies.295 SAXS studies of aggregates of 51 showed the bilayer was constructed with only the distal chains interdigitated295 an effect previously proposed for similar glycolipids by the Shimizu group.12,220,291 The DAP group also provided molecular recognition functionality, exploiting that the native fluorescence of DAP can be quantitatively quenched by specific molecules.295 Furthermore, the DAP linker is able to complex with divalent cationic metal ions such as copper(II), zinc(II), and cobalt(II). 296 This afforded the opportunity for template directed deposition of these metals in a highly organized fashion, valuable for both catalysis and nanoelectronics.296 5.1.4. Mesogenic Glycolipids. Incorporating an aromatic segment as a spacer between the glucose derived head group and saturated alkyl chains of varying lengths produced a series of 4-alkyloxybenzylidene aminophenyl-α-D-glucopyranosides (Chart 20, 53). Here, the imine linker between aromatic rings provided rigidity to the spacer, generating thermotropic liquid-crystalline behavior in all products.270 Precipitation occurred upon the addition water (9:1 v/v) to the glycolipids

highly curved packing into micellar aggregates,293 encouraging anisotropic structures293 and enhanced aggregate stability.294 Indeed, unlike the relatively unstable anisotropic structures formed from 34, which precipitated into crystals within a few days, the tubes and helices of 39 and 40 were stable for several months.294 Consistent with the behavior of the N-alkyl-D-gluconamides, the aggregate structures for the tube-forming galactonamide species were not significantly altered with an increase in chain length; N-dodecyl-D-galactonamide (41) assembled into helically based tubes similarly to its shorter chain analogue (39).245 Alterations to the head group conformation, however, were significant. N-Dodecyl-L-mannonamide (42) forms aggregates similar to 39, 40, and 41,245 but N-octyl-D-mannonamide (43), despite having an all-trans conformation in the head group, did not form ribbons. Instead, bilayer sheets formed, which rolled up like a cigar into cochleate cylinders.293 5.1.2. Diacetylenic Aldonamides. Diacetylenic groups were incorporated into the alkyl chains of N-alkylaldonamides to stabilize the aggregate structures through polymerization.139,146,152,153,160,174 As described previously for DC8,9PC phospholipids (section 4),146,206,222,223 these diacetylenic groups can also help to drive assembly into helices. Helically based tubules were formed from N-dodeca-5,7diyne-D-galactonamide (Chart 18, 44).246 These tubes had Chart 18. Diacetylenic Aldonamides

average external diameters of 300 nm, wall thicknesses containing a single bilayer, and tube lengths of several micrometers (Figure 26).246 The D- and L-enantiomers formed morphologies that differed only in the hand of the helices, and when equi10247

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Figure 26. TEM of assemblies of N-dodeca-5,7-diyne-D-galactonamide. Reprinted with permission from ref 246. Copyright 1991 American Chemical Society.

pyranoside head groups with 4-(1,2,3)-triazolephenyl 4-alkoxybenzoate mesogenic hydrophobic tails (Chart 20, 54). The aggregates of these molecules were found to be similarly dependent on hydrophobic chain length for the molecular chirality to be expressed in the supramolecular aggregates.408 Here, chain lengths of less than 10 carbons formed platelets, chain lengths of 11 and 12 carbons formed helical ribbons, and intermediate chain lengths of 10 carbons produced mixed aggregates.408

Chart 19. DAP Linked Aldonamides

5.2. Cardanol-Based Glycolipid Nanotubes

Cardanol is a mixture of N-alkylphenols with varying degrees of unsaturation in the alkyl chain that are isolated from cashew nut shells. Shimizu et al.61,68,69,192,220,302,303 have used this biologically based, renewable resource to replace petroleum based chemicals in the synthesis of self-assembling molecules. The meta-substituted phenols were modified by the addition of a cyclic sugar unit to the hydroxyl group, creating a mixture of amphiphiles suitable for self-assembly in water (Chart 21, 55−58). When added to boiling water and then gradually cooled to room temperature, the mixture formed helical ribbons over 24 h, which then transformed into nanotubes after several days.302 These nanotubes had internal diameters of 10−15 nm, external diameters of 50−60 nm, and lengths of 10−100 μm.302 The tube walls consisted of two to four interdigitated bilayers with a tube to vesicle phase transition occurring at 46 °C.302 Cardanol analogues synthesized without the aromatic ring were unable to form high-axial-ratio nanostructures, which illustrated the effect of the phenyl ring on polymorphism.69 To gain further understanding of the supramolecular assemblies of the cardanol-based glycolipids, the components were isolated by chromatography and their individual supramolecular assemblies investigated. The diene (55) and triene (56)

Chart 20. Mesogenic Glycolipids

dissolved in tetrahydrofuran. Glycolipids with 7 and 8 carbons in the alkyl chains self-assembled into platelet morphologies, and those with 9 and 10 carbons in the alkyl chains formed elongated platelet morphologies having a left-handed twist. Glycolipids with 11−13 carbons in the alkyl chains formed left-handed helices, and the helical pitch length decreased with increasing chain length.270 Liquid-crystalline glycolipids similar to the mesogens described above were synthesized by combining β-D-galacto10248

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cardanyl glucosides was investigated by time-resolved fluorescence and attenuated-total-reflectance FTIR (ATR FTIR) spectroscopies.409 The peak wavelength of ANS confined within the nanotube, when compared to ANS dissolved in bulk water, was not only significantly blue-shifted, indicative of less polar environments, but also broadened. From this the polarity of the water within the tubes was estimated to be 20% less than that in the bulk,409 and was of character similar to that of intracellular water.88 Comparison of ATR FTIR spectra of nanotube samples dried under atmospheric conditions and under vacuum was used to show that the peak for water within the cavity of the tube differed from that for bulk water by having reduced absorbance at higher wavelengths.409 This was consistent with the fluorescence analysis and indicative of highly organized hydrogenbonding networks for water within the lumen of the tube. Such insight into the internal aqueous environment of the tube helps to predict the behavior of aqueous solutions involved in applications such as controlled release.409 The Young’s modulus of the nanotubes synthesized from natural cardanol mixtures was measured using optical tweezers.61 Significantly, it was shown that there was a temperature dependence of the flexural rigidity, which was reduced by one-half between 23 and 27 °C, despite the lack of macroscopic transformation in the tubules.62 Concomitant FTIR and DSC analyses in this temperature range confirmed that the softening of the tube walls was indeed a thermodynamic phenomenon.62 A microscopic change in the hexatic ordering between lipid molecules, like that between smectic I and smectic F (Figure 3) predicted by Chen et al.,410 may be responsible for such microscopic transformations.214 To investigate the influence of the position and number of double bonds in the hydrophobic chain on the aggregate morphology, various synthetic analogues have been made of the cardanol-based amphiphiles. These analogues varied in having a para-substituted phenol attached to the longer hydrocarbon chains through an amide link to increase the temperature of the Lα-Lβ′ transitions for ease of analysis (Chart 22).12,220 Three of the unsaturated analogues formed high-axial-ratio nanostructures in aqueous solutions. The monoene (59) formed a twisted fiber structure; the diene (60) formed a 5% population of left-handed helically based tubes with internal diameters of 150−200 nm and a wall thickness of 20 nm; and the triene (61) formed helically based tubes with 70 nm internal diameter and wall thicknesses of 20−30 nm, together with some left-handed helical ribbons.220 CD spectroscopy again showed supramolecular chirality of much greater intensity for the helically based nanostructures than for the twisted ones, indicative of the greater chiral order in the helical species.220 XRD and FTIR analyses revealed 60 and 61 as compared to 59 had weaker hydrophobic interactions and stronger head group interactions, and consequently were better able to express the molecular chirality in the helical aggregate morphology.220 Further structural variation in the long-chain phenyl glucoside lipids was provided by the inclusion of 4 (62) and 5 (63) cis double bonds in the alkyl chain, the use of trans double bonds in a diene (64) analogue.12 62 and 63 formed morphologies similar to that of 61, having left-handed helical ribbons and tubules possessing an inner diameter of 80−100 nm and wall thickness of 20−30 nm.12 In contrast, 64 formed a plate-like morphology having CD signal intensity much lower than its cisdouble bond analogue. This again highlighted the role of the kink generated by the cis-double bonds in driving the

Chart 21. Cardanol-Based Synthetic Long-Chain Phenyl Glucosides

components remained liquid at room temperature, and did not form high-axial-ratio nanostructures on their own.68,192,220 The assembly of 57 produced nanotubes and helical ribbons similar to those seen in the natural mixture, with a phase transition of 38.5 °C. The isolated saturated component (58) gelated many organic solvents, as well as aqueous/organic solvent mixtures, the gels comprising three-dimensional networks of fibers with both twisted and left-handed helical motifs.68,302 Binary mixtures of 57 and 58 containing 10−40% of 57 formed twisted ribbons. However, for monoene concentrations of 50% and higher, stable helically wound ribbons were created, with their helical pitch decreasing as the content of 57 increased, eventually transitioning to helically marked tubules at 90% 57.192 CD spectroscopy showed supramolecular chirality of much greater intensity for the helically based nanostructures than for the twisted ribbons, indicative of greater chiral order in the helical species. This chiral order was essentially lost upon the phase transition to vesicular structures, again showing that the intensity of the signal was due to the supramolecular chirality of the aggregate.192 As such, it was suggested that 57 drives supramolecular chirality and helicity, due to the nonparallel packing of the bent hydrocarbon tails (Figure 27).192

Figure 27. Nonparallel packing between cardanol monoene fraction driven by bends in the hydrocarbon chains.

The local environment of water confined within the internal cavity of lipid nanotubes assembled from the natural mixture of 10249

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Chart 22. Long-Chain Phenyl Glycoside Lipids

nonparallel packing of the molecules, and leading to the subsequent supramolecular chirality. The glucose head group of long-chain phenyl glucoside lipids was exchanged for galactose in both cis diene (65) and triene (66) molecules (Chart 22). For the compounds having galactose head groups, 65 was only able to form very loosely coiled helical ribbons, while 66 formed a double-helical structure consisting of two intertwining 5 nm micellar fibers. The chiral interaction between molecules was lessened by the axial hydroxyl moiety of the galactose head group as illustrated by both a significant reduction in the CD intensity for these molecules as compared to their glucose analogues and XRD results that showed an increased hydrophobic interaction.12 To capitalize on the aqueous gelation properties of the saturated component of the cardanol mixture, an analogue was synthesized in which a glucose head group was linked through an aromatic ring and an amide group to an 11 carbon hydrocarbon chain (Chart 23, 67).303 This glycolipid was able to form stable gels at room temperature with a range of organic solvents, as well as with water when trace amounts of methanol or ethanol were present. The gels contained a three-

Chart 23. Phenyl Glucoside with an 11 Methylene Unit Saturated Chain (67) and Aminophenyl Glucopyranoside (68)

dimensional network of fibers, having an interdigitated lamellar organization that showed both twisted and left-handed helical motifs.303 When aminophenyl glucopyranoside (68, 1:1 w/w) was included with 67 in the assembly process, the resultant gels comprised double-helices with diameters from 3 to 25 nm and lengths of several micrometers.411 The aminophenyl glucopyranoside provided a hydrogen-bonding group to bind the anionic sol−gel precursor of hydrolyzed TEOS. Subsequent 10250

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condensation and calcination successfully transcribed the organic template into an inorganic silica double helix.411 5.3. Vaccenic Acid-Based Glycolipid Nanotubes

The systematic research on self-assembling glycolipids from the group of Shimizu71−73,83,198,289−291 progressed from cardanol derived lipids to vaccenic acid derived lipids as the higher gel− liquid transition temperature created more practical tubes.412 The basic glycolipid molecular structure investigated now featured a β-D-glucopyranosylamine head group linked through an amide bond to a C-18 hydrocarbon chain. Initial experiments focused on six synthesized glycolipids (Chart 24), in Chart 24. Glycolipids Used To Optimize Self-Assembly into Helically Based Tubes

Figure 28. Electron microscopy images of tubes self-assembled from N-(11-cis-octadecenoyl)-β-D-glucopyranosylamine (a,b) before and (c,d) after lyophilization. Reprinted with permission from ref 73. Copyright 2004 American Chemical Society.

layer of 1.3 nm291 contained in tube walls made up of 18−21 interdigitated bilayers.289,291 Shifting the double bond either toward the head group (69) or toward the terminal methyl group (71) as compared to 70 reduced the nanotube yield to 90%, with the remaining aggregate material being amorphous.289 Shifting the double bond in this manner did not significantly affect the magnitude of the diameter measurements; however, the size distribution was notably larger, particularly for the exterior diameter.289,413 Further movement of the monoene toward the head group to the C6 (72) position produced only fibrous and amorphous aggregates, while including two double bonds at the C9 and C12 positions (73) formed deformed nanotubes in only 30% yield together with amorphous aggregates. Finally, the completely saturated analogue (74) produced exclusively amorphous material from aqueous dispersion.289 Contrastingly, in recent experiments, 74 and its analogue (with two less carbons in the hydrophobic chain) were able to self-assemble into bilayer nanotubes with an internal diameter of 70 nm, and a wall thickness of 70 nm from a water/methanol mixture (1:1, v/v).414 It is of interest that the group of Shimizu et al., who have conducted almost all of the work on lipid nanotubes selfassembled from 70, have consistently favored that the selfassembly is via helical intermediates, except in one paper.368 In this instance, they describe the assembly occurring from the rolling of a sheet in a cigar-like fashion similar to that described for cochleate cylinders126,333 despite the fact that the method of self-assembly appears to be the same as other papers supporting assembly via helical ribbons.198,289,415 Subsequently, Zernike phase-contrast TEM was conducted on the initial structures formed upon self-assembly of 70 and showed the tubes were indeed helically based.39 Here, it was observed that aggregates isolated at 60 °C, just below the gel−liquid-crystalline phase transition, were “core tubes” with helical striations at the tips

which the number and position of double bonds in the alkyl chain were carefully and systematically varied. The lipid selfassembly took place when aqueous dispersions were heated to 95 °C for 30 min and then incubated for 10 days at room temperature.289 Glycolipids with a single cis-double bond at C9 (69), C11 (70), and C13 (71) produced nanotubes as the major product, with 70 (the vaccenic acid derivative) particularly efficient, producing them in greater than 98% yield. These helically based tubes generally had smooth surfaces,85,198,291 with average external diameters of 200 nm, average internal diameters of 61 nm, tube lengths varying from 0.5 μm to hundreds of micrometers, and a hydrated phase transition that occurred at 71 °C (Figure 28).71,73,289 Thermal diffuse reflectance infrared Fourier transform (DRIFT) spectroscopy, DSC,85 and optical microscopy289 on lyophilized tubes showed that there was a dry melting transition at ∼148 °C. XRD and electron density analysis of the nanotubes formed from 70 showed the lipids were in the lamellar phase, with bilayer thickness of ∼4.5 nm39,79,368,415,416 and an interlamellar water 10251

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and of much more uniform dimensions than generally found for mature tubes. After the rapid formation of the core tube at the transition temperature, the walls of the tube grew thicker with the addition of more helical bilayers as the solution cooled, both internally and externally. These new layers appear to still be helically based,198 growing along the length of the tube with the growing end visible as a diagonal step across the tube. Perhaps several such steps of growing internal bilayers, each not quite reaching the end of the tube, is what gave the stepped increase in internal diameter that led to the conclusion that these tubes are cochleate-types cylinders,368 as discussed previously. 5.3.1. Encapsulation of Hydrophobic Molecules into the Tube Walls. A new method to self-assemble 70 into nanotubes used a mixed solvent technique.418 The amphiphile dissolved in methanol−ethyl acetate (1:1, v/v) was precipitated by dilution with water. The resultant nanotubes had characteristics essentially the same as those self-assembled through heating dispersions in water by TEM and XRD measurements.418 This technique has been used to coassemble hydrophobic molecules with 70 into nanotubes. The nanotubes coassembled with the fluorescent probe (ANS) and with Zn-phthalocyanine had similar bilayer measurements and the same internal diameter, but the tube wall thickness was reduced from 130 to 25 nm as compared to the nanotube self-assembled without the hydrophobic molecules. Fluorescence microscopy showed that the ANS was well dispersed in the hydrophobic walls of the nanotubes, rather than encapsulated within the lumen. Consequently, the coassembled molecules were not released from the nanotubes into aqueous solution until heated above the thermal phase transition of the tubes.418 5.3.2. Template Directed Synthesis. There are many examples of the use of 70 in template directed synthesis as described over the following pages. The shape of the selfassembled nanotubes provides a template that can be used in various ways, but there is also value of the structure in protecting the synthesized inorganic nanostructures from exposure to destructive and contaminating elements from the environment.13,415 5.3.2.1. Template Directed Synthesis of Metals and Semiconductors. Ethanolic aqueous solutions of hydrogen tetrachloroaurate (III) were internalized into empty lipid tubes self-assembled from 70 using capillary action, and then the gold was photochemically reduced to metal nanoparticles within the tubes.72 Electron microscopy and energy dispersive X-ray analysis revealed gold nanoparticles having 3−10 nm diameters were densely packed within the nanotubes.72 Similar work drew aqueous ethanolic silver nitrate solutions into lyophilized tubes and then reduced the silver ions to nanoparticles by UV irradiation (254 nm).85,419 Preparations of tubes washed of excess silver nitrate led to the formation of silver nanoparticles within the tubes upon UV irradiation. When UV irradiation was conducted on tubes still dispersed in silver nitrate solution, then silver nanoparticles were found decorating the outer surface of the nanotubes.85,419 The disadvantages to the method of using the nanotubes as nanosized reaction flasks to create nanoparticle hybrids included that only photoreducible metals could be used, and the size of the nanoparticles in the hybrids was not very controllable. Consequently, the lipid nanotubes self-assembled from 70 have also been used to encapsulate preformed metallic nanoparticles directly. Gold nanoparticle dispersions with 1−3 nm diameters were internalized by this method with close packed structural arrays (Figure 29). Silver nanoparticles with

Figure 29. TEM images of lipid nanotubes (a) filled with gold nanoparticles at low and (b) high magnification. Reprinted with permission from ref 73. Copyright 2004 American Chemical Society.

diameters of 2−4 nm were also effectively encapsulated within such nanotubes by this method.73 The tubules encapsulating both gold and silver nanoparticles were heated in air to decompose the organic nanotube template and fuse the encapsulated nanoparticles into nanowires. For the gold nanoparticles, heating to 750 °C produced the optimal nanowires. Silver nanoparticles, however, were oxidized by the heating process, so only a one-dimensional alignment of discrete particles could be achieved.73 The iron storage protein, ferritin, has also been encapsulated (at a volume ratio of 20%) into vaccenic acid derived glycolipid nanotubes using capillary action from an aqueous dispersion.71 The ferrihydrate core of ferritin can be exchanged for other inorganic materials, such as semiconductors and metal complexes, protecting labile species and preventing particulate agglomeration.71 In this way, a greater variety of useful hybrid structures can be generated. Equimolar quantities of an aminophenyl glucopyranoside (68) additive and 70 were mixed to make tubes, resulting in a 16% loading of 68198 that caused minor alteration to the nanotube aggregate morphology, with outer diameters ranging from 150 to 400 nm and wall thicknesses from 30 to 40 nm.198 By adding cadmium nitrate and thioacetamide to a dispersion of these nanotubes, CdS nanoparticles formed exclusively in one-dimensional helical arrays of a single hand and a pitch of 45° on the surface of the nanotubes.198 Over time, the initially thin lines of CdS nanoparticles filled out to cover the entire surface of the tube. Thermal decomposition of the template then led to the creation of CdS nanotubes.198 There was no organized deposition of CdS on pure lipid nanotubes, and, therefore, it was 68 that directed the helical organization of the CdS nanoparticles on the mixed lipid nanotube surface. The fact that the CdS formed in helical arrays provided experimental evidence to support the theory of Selinger et al.201 that abrupt changes in tilt modulation are required for tubes in which a modulated tilt state provides the drive for tube formation via helical intermediates. Whether 68 was preferentially attracted to assemble at the domain walls due to its altered functionality or packing requirements,197 or if these domain walls simply provided zones where the hydrophobic groups were more exposed to the aqueous environment,203 was not determined. CdS nanodots were confined within the lumen of nanotubes assembled from 70, both ready made as well as synthesized within the tube from a solution of cadmium(II) ions and thioacetamide.13 Subsequent calcination converted the CdS nanodots into nanowires13 and resulted in single crystalline nanowires of 15−40 nm diameter.72,73 10252

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N-(11-cis-Octadecenoyl)-β-D-glucopyranosylamine (70) lipids have been self-assembled around copper(II) oxide nanowires as a sheath to prevent exposure to the deleterious effects of moisture and air.415 The aligned, separated copper(II) oxide nanowires were characterized by diameters of 30−100 nm and lengths of up to 15 μm. The sheathing of the nanowires occurred by the addition of a boiling solution of 70, forming vesicles that attach to the hydrophilic surfaces of the nanowires. Subsequent incubation converted the vesicles to the desired helically based nanotube sheath that coated over 95% of the nanowires in a layer 70−120 nm thick.415 5.3.2.2. Template Directed Synthesis with Silica and Other Inorganics. Hybrid structures based on vaccenic acid derived glycolipid (70) nanotubes were made with silica by utilizing multiple variations on the templating technique described above for metals.83 One such method used preformed silica nanotube templates with an internal diameter of 200 nm, large enough that an aqueous glycolipid solution can be internalized through capillary action. Electron microscopy imaging showed that the lipid was deposited in a layer 15 nm thick along inside surface of the silica nanotubes.83 XRD confirmed that the deposited lipid layer is well ordered, with a lamellar organization similar to that found in their self-assembly from bulk water.83 Glycolipid nanotubes were also used for templating silica nanotubes through the sol−gel deposition of TEOS to create hybrid structures.83 The most effective way to prepare such hybrids was to introduce 10% by weight of aminophenyl glucopyranoside (68) to the standard glycolipid, which imparted a positive charge on the tubes and catalyzed the sol-gel process.83 The TEOS was added to an aqueous dispersion of mixed lipid nanotubes, and then left at room temperature over 7 days.83 The resulting hybrid structure consisted of silica nanorods internalized within the tubes, as well as silica deposited on the external wall of the nanotube (Figure 30).83 The positively charged lipid mixture was also used in the templated self-assembly of lipid nanotubes by a silica nanotube.83 In this case, the silica nanotube provided the suitably sized confined space for the self-assembly of the lipids, as well as the negatively charged surface silanol groups to promote the adsorption of the positively charged aminophenyl glucopyranoside.83 The resultant hybrid structure was a tube and had an internal diameter of 200 nm and a wall thickness of 100 nm, which scanning TEM showed it to consist of two lipid layers deposited on the internal and external surfaces of the original silica nanotube.83 The positively charged lipid layers were then further used to subsequently template the deposition of silica using the sol−gel process discussed above. Hybrid nanorods were produced in this way with a silica core encased in alternating organic and silica layers.83 Sol−gel reactions were similarly used to template titania onto the surfaces of nanotubes.417 In this case, aqueous dispersions of lipid tubes assembled from 70 only were frozen. An ethanolic solution of titanium isopropoxide was added to the chilled tubes at −20 °C, and the ethanolic solution diffused through a layer of “nonfreezing” water generated by the strong association with the surface sugar moieties of the tube (Figure 31). This layer of water confined the sol−gel reaction to the tube surface, with the deposition of the titania as particles absorbed to the surface, and the amount of coverage increased with time.417 After lyophilization had removed the surrounding ice, thermal decomposition of the nanotube template revealed that titania

Figure 30. STEM images of self-assembled, hybrid, and templated structures. Reproduced with permission from ref 83. Copyright 2005 The Royal Society of Chemistry.

Figure 31. Schematic for sol−gel deposition inside glycolipid nanotubes. Reprinted with permission from ref 417. Copyright 2006 The Chemical Society of Japan.

was deposited on both internal and external surfaces of the tube and the heat treatment combined them into a nanorod.417 This showed that electrostatic attraction between the template and a precursor was not essential to the templating of sol−gel reaction products.417 5.3.3. Control of Nanotube Dimensions. For nanotubes to be effective as controlled release agents, the diameter and length of the self-assembled structure should be consistent to allow optimal control of incipient release. To narrow the size distribution of both the internal and the external diameters of the lipid nanotubes, without resorting to time-consuming incubation methods, extrusion and in-pore self-assembly techniques were applied.420 Extrusion of lipid vesicles at temperatures above the phase transition temperature through a series of polycarbonate filters, whose pore sizes decreased from 5 μm to 100 nm, produced vesicles of consistently small size. When these vesicles were assembled within an anodic alumina membrane filter, having a pore size of 200 nm,420 it resulted in nanotubes with a small average external diameter of 148 nm, and narrow distributions in wall thicknesses and in internal 10253

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and external diameters.420 However, there still remained a wide length distribution.420 This can be controlled in a separate process using mechanical stirring that effectively cut these types of tubes to relatively consistent lengths between 1 and 10 μm long, the ultimate length being dependent on stirring speed.290 5.3.4. Manipulation and Alignment of Nanotubes. Nanotubes made from cardanol extracts with average external diameters of 50 nm were aligned on a glass substrate, via extrusion through a 500 nm femtotip.61 This technique was repeated with larger tubes assembled from 70 using a microcapillary and a two-step microextrusion technique.421 A similar method was used to make a nanopipette with a lipid nanotube sealed into the end of a 2 μm diameter glass micropipette using a photocross-linked resin.104 Once the nanopipette was made, liquid was transported through the pipette end by applying a direct current between the solution in the pipette and the destination solution, the flow rate through the pipette controlled by the applied voltage.104 An off-chip potential valley created by a pair of electrode needles in a drop containing lipid nanotubes was able to align the tubes through colloidal dielectrophoresis and create a nanotube film of parallel nanocapillaries.422 When dried, these films of parallel tubes were able to encapsulate gold nanoparticles of 5 nm diameter through capillary action, illustrating a potential application as a high throughput nanofluidic device.422

bled from this mixture of oleic acid and 69, then lyophilized and used to encapsulate magnetite spherical nanoparticles with a 10 nm diameter. This encapsulation was pH dependent, occurring at pH’s between 5.4 and 6.8, which was close to the isoelectric point of the nanoparticles and where the nanotube had a negative zeta potential.423 The magnetite nanoparticles remained encapsulated within the tubes upon drying or dilution, and the hybrid tubes could also be aligned oriented parallel to an external magnetic field. Magnetic testing showed the hybrid structure to be a “semihard” magnetic material suitable for magnetic recording.423 5.5. Glycoamphiphiles with Fluorocarbon Hydrophobic/Oleophobic Tails

A key driving force in the formation of high-axial-ratio nanostructures from amphiphiles in water is the hydrophobic aggregation of the nonpolar parts of the molecules. Strengthening the hydrophobicity through the substitution of hydrocarbon hydrophobic moieties for fluorocarbon analogues has proven successful in the formation of highly ordered nanostructures, such as tubes.427 Fluorocarbons have very strong intramolecular interactions and weak intermolecular interactions (as compared to hydrocarbons),428 and not only are they extremely hydrophobic, but also oleophobic. This means that fluorocarbons can impart increased stability and order to supramolecular systems by introducing new organization with their ability to segregate from both aqueous and lipidic phases.429,430 Fluorocarbons also have greater bulk and stiffness than hydrocarbons due to the fact that fluorocarbon chains in the all-trans conformation have a helical twist caused by the packing requirements of the larger fluorine atoms (as compared to hydrogen).428,431,432 This increased stiffness of the fluorocarbon moiety may provide a source for a spontaneous break in the symmetry of the molecular packing, leading to the formation of a chiral macrostructure from achiral constituents.132 Some interesting ways of incorporating fluorocarbon groups into self-assembling nanostructures have emerged. For instance, amino acids were used as the backbones between lactose derived head groups and dual hydrophobic tails, where one tail consisted of a hydrocarbon chain and the other consisted of a fluorocarbon chain, to produce a series of glycolipid surfactants varying in the amino acid backbone (Chart 25 shows an

5.4. Oleic Acid-Based Glycolipid Nanotubes

Glycolipid nanotubes assembled from an oleic acid derivative (69) have already been shown to self-assemble into tubes with appearance similar to that of the vaccenic acid derived lipid (70).289 Using the oleic acid derived lipids solves the problem of the prohibitive cost of the vaccenic acid component, while making little change to the physical properties of the tubes. Further, Shimizu et al.423,424 have recently moved onto alternate self-assembly procedures (section 6.3), which have been reported to make the self-assembly of the oleic acid species almost quantitative. This technique also generates much larger yields, with 100 g produced within the lab and 10 kg within a factory setting.423,425 This makes a more commercially practical product.412 Safety tests were conducted on the oleic acid-based nanotubes, and biodegradation tests showed these organic nanotubes had almost no effect on humans, animals, and plants and were essentially completely degraded by environmental microbes within 4 weeks. Toxicity tests on rats showed no fatalities after oral administration of 5000 mg/kg after 2 weeks. Similarly, no issues were discovered with toxicity, mobility, or growth for aquatic organisms grown in 100 mg/L dispersion of nanotubes, and a mutagenicity test was negative.412 Oleic acid-based amphiphiles (69) self-assembled from methanol into nanotubes having an internal diameter of 80 nm, an external diameter of 200 nm, and lengths of 0.2−60 μm.423 It is important in the dynamics of the release of encapsulated substances that the dispersity of the tube lengths is minimal. For this reason, nanotubes self-assembled from 69 were physically cut using a mortar and pestle and a mechanical mill, and then measured using fluorescence optical microscopy.413 Tubes ground with the mortar and pestle showed a positively skewed length distribution with a single maximum at 5.52 μm, and after mechanical milling the maximum was shifted to 2.66 μm.413 Together with oleic acid, 69 was used as a surfactant coating to stabilize magnetite nanoparticles.426 Tubes were also assem-

Chart 25. Lactose Derived Glycolipid with Dual Hydrophilic Tails, One Fluorocarbon and One Hydrocarbon

example structure, 75). Glycine backbones produced multilayer vesicles, while glycylglycine links produced single-walled vesicles. Interestingly, lysine backbones produced helically twisted structures and tubules, illustrating the influence of the backbone in the interfacial region on the final morphology of the aggregate.272,273 Anionic glycophospholipids were synthesized by grafting a phosphate group onto the O-6 position of D-glucose, D-galactose, and D-mannose. Attached to the phosphate group was a saturated hydrophobic double chain, that was either fully hydrogenated or contained one fluorinated chain (Chart 26, 76, 10254

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structures were stabilized by photopolymerization of the diacetylenic moieties, thereby illustrating the high level of order in the aggregate.42 Derivatives omitting the galactose head group were also synthesized. These new lipids self-assembled mostly into vesicles, demonstrating the importance of the sugar group in creating intermolecular hydrogen bonds that can generate ordered assemblies.42 5.6.3. Disaccharide Head Groups. Another alkylaldonamide that formed organized macrostructures is N-hexadecyl-Dmaltosylamine. This lipid was cosolubilized in a methanol/ water (1:5) mixture and heated to 70 °C, before cooling to room temperature, and then annealed for 6 h. The aged suspension formed nanotubes with diameters of ∼25 nm and lengths of 600 nm.300 5.6.4. Phenyl Glucosides. A low molecular weight sugarbased gelator (Chart 28, 80) was found to self-assemble into

Chart 26. Anionic Glucophospholipid

example with a fluorinated chain).38 Only the glucose-based lipids self-assembled from pure water into tubes, which occurred after cooling below the main transition in a reversible process. The lipids with fully hydrogenated alkyl chains formed multiple bilayer tubes that were 1 μm in diameter and 100 μm in length at 4 °C. The glycolipids with fluorinated chains also formed nanotubes. For these species, the fluorinated mannose derivative could form a low population of tubules at pH 11 and 4 °C, as could the glucose derivatives. The fluorinated glucolipid, however, also formed nanotubes at neutral pH and at room temperature. These latter tubes were of comparable length to those created using lipids with dual hydrocarbon chains, but had much smaller diameters, with an external diameter of 14.2 nm and an internal diameter of 3.6 nm.38,161

Chart 28. Terphenyl Glucoside

5.6. Other Glycolipids

5.6.1. D-Amygdalin Head Group. Sometimes gels are formed as a result of molecular packing of lipids that is very similar to that observed in the self-assembly of helically based nanotubes. These gels consist of twisted or helical ribbons that fuse into three-dimensional networks, which have many biomedical applications, including the controlled delivery of hydrophobic drugs (Table 3).301 Hydrogel forming lipids have been synthesized from D-amygdalin linked through an ester group to 3 (77), 13 (78), and 17 (79) carbon saturated hydrophobic tails (Chart 27).301 These molecules readily selfassembled into organized tilted bilayers when dispersed in water.301 The lipids having the longer hydrocarbon tails formed helical ribbons of 50 nm width and lengths of several micrometers.56,301 These ribbons fused into dense networks that immobilized the solvent. No morphological difference occurred to the gels formed in the presence of the hydrophobic drug curcumin, which was solubilized in small hydrophobic pockets in the hydrogel.56 Subsequent release of the drug occurred on the enzymatic degradation of the lipid, breaking apart the supramolecular structure.56,301

helical ribbons upon cooling from above the transition temperature in toluene and 1,4-dioxane and mixtures of the later solvent with water (60/40−100/0 v/v).134 The selfassembly was driven by aromatic stacking interactions in the hydrophobic chain and hydrogen bonding between sugar groups producing helices of 20−150 nm diameter in the 1,4dioxane/water solvent.134 In this solvent, it was found that the hand of the helix was dependent on the state of the nuclei formed when cooling through a small window of temperature between 52.5 and 50 °C. Fast cooled solutions led to the kinetically controlled formation of right-handed helices with a pitch of ∼45°, while slowly cooled solutions were controlled thermodynamically forming left-handed helices with a pitch of ∼60°.134 This behavior is suspected to be related to that of diacetylenic phospholipid nanotubes in which chiral symmetry breaking driven by the hydrophobic tail was able to create a kinetically allowed metastable state and subsequent thermodynamically controlled aggregation to the stable state was much more influenced by chiral packing within the head group.222 In work related to that on the terphenyl glucoside, a family of diphenyl glucosides self-assembled into helical ribbon-based gels from water/dioxane (60/40 v/v). The length of the alkyl chain was important to the self-assembly of the diphenyl glucoside, and alkyl chains of 3−6 carbons (left-handed) had opposite helical sense to those with 11−12 carbons (righthanded).433 5.6.5. Glyco-bolaamphiphiles. Bolaamphiphiles have two hydrophilic head groups placed at each end of a linear hydrophobic chain and self-assemble into monolayer structures from polar solvent. These structures can include nanotubes,434−436 and some structures assembled from glycolipid-based bolamphiphiles have a chiral basis.89,437,438 Bolaamphiphiles with dual glucosamide-based head groups connected to a central 10, 12, or 14 methylene unit alkyl chain assembled chirally into highaxial-ratio twisted ribbons with widths from 8 to 25 nm.438 In another example, asymmetrical bolaamphiphiles having glucose and amino-based head groups and varying alkyl chain length have self-assembled under both basic and acidic conditions

Chart 27. D-Amygdalin-Based Glycolipids

5.6.2. Diacetylenic Glycopeptidolipids. A glycopeptidolipid that used a peptidic backbone to link a galactose head group to palmitoyl and 10,12-pentacosadiynoyl hydrophobic chains has been synthesized for its supramolecular properties. This glycopeptidolipid formed high-axial-ratio nanostructures, believed to be rods or tubes when dispersed in water.42 These 10255

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Table 3. Characteristics of Nanotubes Self-Assembled from Glycolipids Under Varying Conditions amphiphile

solvent

41 44 45 46 47 N-hexadecyl-D-maltosylamine 51 55−58 mixture 57 60 61 62 63 69 69 70 70 + 68 70 70 70 70 70 71 74 76 76 analoguee 80 81

aqueous aqueous aqueous aqueous aqueous MeOH:water 1:5 aqueous aqueous aqueous aqueous aqueous aqueous aqueous aqueous MeOH aqueous aqueous acetonitrile:MeOH 1:1 + water acetonitrile:MeOH 1:1 + watera acetonitrile:MeOH 1:1 + waterb aqueousc aqueous (60 °C) aqueous MeOH:water 1:1 aq pH 11 aqueous dioxane:water 6:4 aqueous

length (μm) several

0.6 10−100 10−100

0.2−60

100

external diameter (nm) 1000 300 370 270−400 270−400 25 60−80 50−60 50−60 190−240 110−130 120−160 120−160 220 200 200 150−400 320 110 140 148 180 214 210 14.2 1000 20−150 32

internal diameter (nm)

20 10−15 10−15 150−200 70 80−100 80−100 73 80 61 90−340 60 60 60 52 130 76 70 3.6

trans temp (°C)

70 46 38.5 65.2 90.0

58 71

59 71 88 106−108d 11−13

20

ref 245 246 246 245 245 300 295 302 302 220 220 12 12 289 423 289 198 418 418 418 420 39 289 414 38 38 134 437

70 self-assembled in the presence of ANS fluorescent probe. b70 self-assembled in the presence of Zn phthalocyanine fluorescent probe. cIn-pore assembly of extruded vesicles. dGlass transition temperature. e76 is an anionic glycophospholipid with one fluorocarbon chain in the hydrophobic tail, while its analogue has fully hydrogenated alkyl chains in the hydrophobic tail. a

less irritant and less toxic than petrochemical derived surfactants.442,443 The use of amino acids as part of a strategy to design lipids for tubule formation was among the earliest examples of helical bilayer assemblies based on synthetic lipids,172,173 and research is ongoing in this area.

Chart 29. Asymmetrical Bolaamphiphile

6.1. Amino Acid Backbones Linking Ammonium/Amine Head Groups and Hydrophobic Tails

into nanotubes.437,439−441 Specifically, the bolaamphiphile with a 20 carbon linking chain (Chart 29, 81) assembled into helical coils from acidified aqueous dispersion that then matured into nanotubes with inner diameter of 20 nm and wall thickness of 6 nm. The glucose groups were on the external surface of the tubes, while the amino groups were presented on the internal surface. The internal amino groups were exclusively chemically modified with a fluorescent donor dye for use in optical recognition of the encapsulation of guest molecules using fluorescence resonance energy transfer (FRET).437

CD measurements on some chiral ammonium surfactants suggested that the bilayer aggregates had asymmetric molecular packing, and further that this chirality could be expressed in the morphology of the aggregate.172,241,285 This early work attached a trimethylammonium head group through an alkyl linker to a chiral glutamic acid backbone that supported a dual hydrocarbon chain tail (Chart 30, 82).172,241,285 Optical and electron microscopy revealed that helical ribbons were formed by dispersing a single enantiomer of the lipid in water above its phase transition temperature, and then cooling to 10 °C followed by annealing over 2 days. The helical sense of the ribbons was determined by the chirality of the amino acid backbone, while racemic mixtures formed only fibers without any helical morphology.172,241,285,444 In addition, when the annealing process was continued for a week, the helices matured into tubes via widening of the ribbon.241 The resultant tubes had a length of 10 μm, an external diameter of 150 nm, and a 28 nm wall thickness (4−5 bilayers thick).172,444 The effect of the lipids’ molecular structure on the aggregate morphology was investigated in several ways. While it was found that alterations to the hydrophobic tails had no effect, changes to the amino acid

6. AMPHIPHILES CONSTRUCTED WITH AMINO ACID HEAD GROUPS AND BACKBONES Amino acid molecules, particularly those with carboxylic acid side-chains, have frequently been used in the synthesis of tube forming lipids, for example, as head groups, as backbones between head groups and hydrophobic tails, and as other linking groups. This is due to amino acids providing both multiple functionality and high chiral fidelity in a cost-effective manner. Amino acids are also a renewable resource, and the derived surfactants are biofriendly materials, generally being 10256

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Chart 30. Aspartic Acid and Glutamic Acid Derived Dialkylammonium Surfactants

Chart 32. Glutamic Acid Derived Lipids

backbone and the alkyl linker were significant.172 Exchanging the glutamic acid backbone for an aspartic acid backbone (Chart 30, 83) created a lipid that formed tubes more readily, with complete conversion within 1 day.172 For lipids with either amino acid backbone, shortening the spacer to the ammonium head group prevented the formation of chiral aggregates.172 Other lipids with trimethyammonium head groups linked though a spacer to amino acid backbones also formed helical aggregates. In this case, the lipids had single hydrophobic tails and aromatic groups were incorporated into the spacer, and two examples, 84236 and 85444 (Chart 31), formed helical ribbons upon cooling of heated aqueous solutions.236 Chart 31. Biphenyl and Azo Derived Ammonium Surfactants

the low temperature helices, and to twisted fibers for the high temperature species.286 Various nonphoto-reactive analogues of the previously described lipid based on glutamic acid backbones were synthesized, as shown in Chart 31 (87−90). These amphiphiles also self-assembled into helical bilayer ribbons when dispersed in water, 90 forming helically based tubes with an external diameter of 18.8 ± 1.9 nm, single bilayer walls 6.0 ± 0.7 nm thick, and lengths of 50−400 nm.445 CD demonstrated the supramolecular interactions in the structures were chiral. However, analogues of molecules 87−90 with the amide bonds between the alkyl tails and the glutamic acid backbone exchanged for ester bonds had no CD signal and did not assemble into helical aggregates.446 This highlights the need for strong, directional hydrogen-bonding interactions, such as those provided between amide moieties, to afford the order required for the self-assembly of supramolecular helices.446−448 Uncharged pyridine head groups have also been utilized to form helically based nanotubes.449 A glutamic acid backbone was attached through amide linkages to two octadecylamine groups, providing long hydrophobic tails, and to a pyridinebased head group with attachment ortho, meta, and para to the nitrogen of the pyridine (Chart 33, 91−93). Self-assembly was from dimethyl sulfoxide, and the ortho compound (91)

Ihara et al.286 synthesized a lipid with a positively charged pyridine head group linked through a dimethylene chain to a glutamic acid backbone having two long hydrocarbon chains that incorporated photoreactive 2,4-hexadienoyl groups at each terminal end (Chart 32, 86).286 This species self-assembled into a helical bilayer superstructures of 250−300 nm diameter, both above and below a gel−crystalline transition at 51 °C.286 These structures were differentiated by their UV−vis spectra. The lower temperature aggregate exhibited very strong exciton coupling attributed to the chiral stacking of the head groups that was absent in higher temperature aggregate. UV irradiation photopolymerized the 2,4-hexadienoyl groups at a rate that was 25 times greater for the low temperature species than it was for the high temperature species, indicating more highly ordered packing in the former. The photopolymerization induced a morphological transition to tubular aggregates for 10257

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-sarcosine, and -L-proline moieties, as well as a combination of these amino acids with L-lysine and L-(Z)-lysine.267 Aggregate morphologies were observed for lipids sonicated in water above the main transition and then annealed at room temperature. The lipid with the sarcosine trimer head group formed multiple bilayer twisted ribbons, while the glycine trimeric head group amphiphile formed amorphous crystals.267 Lipids with a L-proline trimer (Chart 35, 96) or mixed amino acid tetramer

Chart 33. Glutamic Acid Derived Lipids with Pyridine Head Groups

Chart 35. Tube-Forming Oligo-amino Acid Lipids Developed by Shimizu et al.267,283

self-assembled into achiral fibers, while the meta compound (92) formed right-handed twisted ribbons and the para compound (93) formed nanotubes. The nanotubes had an external diameter of 111.0 ± 1.5 nm, internal diameter of 77.0 ± 0.7 nm, and wall thicknesses of 17.0 ± 1.6 nm, and were found with right-handed helical ribbon precursors.449 NMR, FTIR, and CD showed that the difference in aggregation was caused by the variation in noncovalent interactions driven by the hydrogen-bonding opportunities of the aromatic amide proton. 6.2. Amino Acid Backbones Linking Amino Acid Head Groups and Dual Hydrophobic Tails

Another of the earliest examples of helical ribbons selfassembled from synthetic lipids was presented by Yamada et al.173 who used an L-glutamic acid oligopeptide as the lipid head group and the backbone, the oligomer attached at one end to two long hydrocarbon chains. Upon dispersal in water at pH 8−9, amphiphiles that contained 14 glutamic acid groups and 12 (94) or 16 (95) carbons in linear, saturated hydrophobic chains (Chart 34) formed unilamellar helical ribbons and low

(97) head groups assembled via a left-handed helical intermediate into stable nanotubes, having 80 nm external diameters and single bilayer walls.267,283,450,451 Self-assembly of 96 was also conducted in different solvent systems. In methanolic aqueous solution (volume fraction of methanol from 9%−50%), tubes were produced with diameters of 60 nm. In ethanolic aqueous solutions, the tubes decreased in diameter with increasing concentration of ethanol, with volume fractions of 2%, 9%, and 23.1% producing tubes with diameters of 80, 50, and 25 nm, respectively.451 The peptidic lipid nanotube containing the L-proline head group (96) has been used in the template directed synthesis of inorganic materials via a sol−gel reaction that does not require catalysts. One example similar to that described for glycolipids in section 5.3.2.2 used ethanol solutions of titanium isopropoxide, which were added to iced aqueous dispersions of nanotubes, and the temperature was maintained at 0 °C over 2 weeks (Figure 32).283 Titania nanotubes formed as the ethanolic solution diffused along organized, nonfreezing water closely surrounding both the interior and the exterior of the hydrophilic nanotube. Upon calcination at 500 °C, the intercalated organic template thermally decomposed, generating titania nanotubes with 80 nm external diameters and walls of 20−30 nm.283 Similarly, inorganic nanotubes of both tantalum and vanadium oxides could be made using the same iced lipid templates. A slightly different method was used in the template directed synthesis of silica using the same tri-L-Pro (96) lipid nanotubes. In this case, freezing of the nanotubes was not required, and TEOS was added to an aqueous dispersion of the tubes. The mixture was left to stand for 7 days before lyophilization,

Chart 34. Oligo-L-glutamic Acid Headed Lipid

populations of tubes.173,282 Analogues using an L-glutamic acid backbone, but having a head group comprised of oligo-Laspartic acid, with 13 aspartic acid residues, formed very similar helical aggregates. However, those amphiphiles containing only four aspartic acid residues, or a simple alkyl ammonium head group, were unable to form such organized helical aggregates. Consequently, it was suggested that intermolecular hydrogen bonding between head groups was the key factor that stabilized the organized structure.282 In similar work conducted by Shimizu et al.,267,283,450−452 linear oligo peptides were connected through a glutamic acid backbone to a dialkylamide hydrophobic moiety. The trimer and tetramer peptide head groups consisted of oligo-glycine, 10258

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Figure 32. Sol−gel transcription mechanism for the iced lipid nanotube. Reproduced with permission from ref 283. Copyright 2005 The Royal Society of Chemistry.

followed by calcination.450−452 Mole ratios of TEOS to lipid of 20, 40, 60, and 80 produced abundant silica nanotubes that increased in wall thickness from 7 to 15 nm with increasing TEOS concentration.452 These are single walled tubes and were believed to be a combination of the silica deposited on the inner and outer surfaces combining upon calcination of the thin organic tube (4.5 nm).450 The lack of a solution catalyst meant that the sol−gel reaction only took place at the tube surface, the positively charged lipid head groups being mildly acidic and performing the catalytic task.450,452 Following the work of Shimizu et al.,83,267,283,450−452 Lee et al.193 synthesized several glutamic acid dialkyl amides with varying alkyl chain length and peptide-based head groups to evaluate the utility of the aggregated structures as drug delivery vehicles.193 For lipid 96 heated above the main transition temperature and then cooled slowly in aqueous buffers mimicking physiological conditions, the aggregate morphologies were similar to those found by Shimizu.267 The differences included larger average diameters of 140 ± 50 nm, and the formation of stable helical structures and tubes that did not grow with long-term incubation.193 Analogues of 96 with longer hydrophobic tails incorporating 14 and 16 carbons decreased the efficiency of tube formation, with significant fractions of amorphous material observed.193 The tube formation efficiency, however, could be improved to almost 100% by using 30% methanol in the self-assembling medium.193 Tube size was also found to be related to chain length with an external diameter of 220 ± 20 nm for a 14 carbon chain, which increased to 280 ± 30 nm for a 16 carbon chain.193 Heating the nanotubes at 40 °C in fluids that mimic physiological conditions for almost 2 days made no significant difference to the self-assembled structure, further enhancing their potential use as drug delivery vehicles.193 The previously discussed work that used a glutamic acid backbone between a hydrophilic head group and dual hydrophobic tails had both tails connected through the acid groups and the head group attached through the amine. In a variation to this work, Cescato et al.453 attached the hydrophobic alkyl tails through the amino and carboxylic acid groups of the main chain of the glutamic acid, leaving the side chain carboxylic acid free to act as the head group (Chart 36, 98). In its deprotonated form, this type of molecule self-assembled into helices and tubes when cooled below the transition temperature in buffered aqueous solution with pH’s from 9 to 10. The diameters of the helices and tubes were several micrometers and the lengths up to dozens of micrometers. An analogue synthesized with the glutamic acid group exchanged for arginine (99) also assembled into helical ribbons and tubes, this time

Chart 36. Glutamic Acid and Arginine Derived Amphiphiles Utilizing the Side Chain as the Head Group

at pH 8. The diameters of these aggregates were smaller by an order of magnitude than those using the glutamic acid group.453 N,N′-Bis(octadecyl)-L-glutamic diamide (100) and its enantiomer (101) are amphiphiles in which the central glutamic acid derived linking group also acts as the hydrophilic head group in polar solvent (Chart 37).454 Lipid 100 precipitates from ethanol Chart 37. N,N′-Bis(octadecyl)-glutamic Diamide

as a xerogel comprised of nanotubes when solutions are cooled to room temperature. The nanotubes are generated from righthanded helical ribbons and have dimensions that are highly consistent, having wall thicknesses of 45.6 ± 1.0 nm, an outer diameter of 116.7 ± 1.3 nm, and lengths of several hundred nanometers.454 The D-enantiomer showed similar behavior, but in this case self-assembling into nanotubes based on left-handed helical ribbons. Upon mixing the enantiomers using a decreasing molar percentage of 101, the morphologies of the self-assembled structures showed a continuous change from left-handed helical ribbon-based tubes (100% 101), to nanotubes with a left-handed helical seam (95% 101), then to left-handed twisted ribbons (75% 101). The twisted ribbons became less tightly wound as the concentration of 101 reduced until achiral sheets resulted 10259

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exposed to hydrogen sulfide vapor released the cadmium cations, which then reacted with sulfur anions to form cadmium sulfide (CdS) nanodots. These 4−5 nm diameter nanodots were homogeneously dispersed all over the internal and external lipid bilayer surfaces and confined within the bilayer structures.79 The nanotube assemblies resulting from the complexation of copper ion with 107 were able to catalyze oxidation of a number of organic compounds by hydrogen peroxide and tert-butyl hydrogen peroxide in a reusable fashion, with the chemistry and structure of the tubes unaffected by the process.458 Unfortunately, the multiwall bilayer structure of the tubes occluded copper ions within the tube walls supporting the formation of peroxo bridges, which were suggested to decompose the hydrogen peroxide reagent.458,459 Different methods of self-assembly were investigated for the oligoglycine lipids (102−109), and one method enabled assembly by dissolution in water through the addition of 1 equiv of sodium hydroxide followed by precipitation by the neutralization of the solution using vapor diffusion of dilute acetic acid.424 FESEM and STEM showed that the precipitates were sheets and nanotubes, with populations of each determined by the head group and the length of the hydrophobic tail with longer and even numbers of carbons in the hydrophobic tails tending to form nanotubes. Selected examples of the glycylglycine amphiphiles (105 and 107) were also found to self-assemble into nanotubes from alcoholic solutions (methanol, ethanol, and for one example from isopropanol and n-butanol).424 The amphiphiles were dissolved at 40 °C using sonication, and the solution was left to evaporate at 40 °C, leaving a white powder of tubes. This temperature of evaporation was key as the nanotubes are only kinetically stable in alcohol, and higher and lower temperatures of evaporation resulted in flat morphologies. In contrast to the aqueous assembly of these amphiphiles, assembly from alcohol resulted in the alkyl chains being presented externally.424 The alcohol self-assembly process was efficient, using 5 times less solvent, and production time was decreased to 1/10 as compared to aqueous methods.424 Further, while lyophilization is required to remove water from tubes internal cavities, evaporation of alcoholic solutions gives dry solids that can be washed with volatile organic solvents. This method lends itself to the bulk production of useable nanotubes in short periods of time and so is valuable commercially.424 Another variation on the self-assembly procedure investigated the self-assembly of lipids 105 and 107 from alcoholic aqueous dispersion in the presence of metal ions. In one example, an aqueous solution of zinc acetate was added to a dispersion of 107 in methanol.456 The initial solid, plate-like morphologies of the lipid dispersed in methanol converted to nanotubes (outer diameter 85 nm; inner diameter 35 nm) over the course of 3 h upon complexation of zinc ions to the lipid head groups.456 In a similar method, nickel ion coordinated nanotubes (outer diameter 30 nm; inner diameter 20 nm) were self-assembled from 105 from an ethanolic aqueous dispersion containing nickel chloride and 1 equiv of triethylamine.456,459 The tubes had an average wall thickness of 5 nm, consistent with a single bilayer structure. The tubes were able to catalyze oxidation of a wide range of organic compounds by hydrogen peroxide, without organic solvent and with at least five cycles of reusability.459 This system showed improved catalytic activity and stabilized the oxidation agent as compared to the related system using copper ions complexed into multibilayer tubes.458

from the racemic mixture. With further reductions to the concentration of 101, the aggregates transitioned to right-handed twisted ribbons, and then to right-handed helical ribbon-based tubes as the concentration of 100 approached 100%.454 6.3. Amino Acid Head Groups with Single Hydrophobic Tails

In a variation to their previous work on amino acid-based lipids with dual hydrophobic tails, Shimizu et al.79,239,416,424,425,455 have also conducted research on amino acid-based lipids with single hydrophobic tails. One section of this work investigated the self-assembly and the resultant applications of a series of amphiphiles with oligoglycine head groups attached to longchain fatty acids.79,239,416,424,425,456 Two triglycyl lipids were created with 13 (102) and 14 (103) carbon hydrophobic chains (Chart 38), and six diglycyl lipids were made with 11−16 Chart 38. Amphiphiles with Oligoglycyl Head Groups Connected to Linear Alkyl Chains by an Amide Bond

carbons in the hydrophobic tail (104−109).239 Sodium salts of these lipids dissolved in water formed precipitates upon the addition of a range of di- and trivalent metal ions, the precipitates of the diglycyl lipids being split between nanotubes and sheets in the presence of copper(II), cobalt(II), iron(III), and manganese(II) ions. For nickel(II) ions, fibrous hydrogels formed, and for zinc(II), silver(II), and with lanthanum(III) ions, only sheets formed.239 From this, it was concluded that octahedral coordination of the metal ions favored the formation of tubes for the diglycyl species.239 The triglycyl lipids formed nanotubes in complexes with manganese(II), iron(III), nickel(II), copper(II), and zinc(II) ions. This suggested that assembly for the triglycyl species was driven by head group effects and was not as reliant as the diglycyl species on the metal ion coordination structure. For the tube forming diglycyl lipid−metal ion complexes, a general trend was seen that even numbers of carbons in the alkyl chain supported tubes better than odd numbers of carbons, attributed to the way the terminal methyl group was accommodated in the aggregate structure.457 This led to relatively thinner bilayers for the even numbered hydrophobic chain lipids,239 which was ascribed to increased bilayer tilt, a characteristic of membranes important for tubular assembly.239 The diglycyl lipid with 14 carbons in the hydrophobic chain (107) complexed with copper and manganese was used as a template to create metal oxide nanotubes upon calcination of the organic component. Cadmium ions complexed to 107 also self-assembled into tubes (outer diameter 100−200 nm; inner diameter 40−80 nm; and wall thickness 40−80 nm) that when 10260

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propyl triethoxysilane was grafted to the silica tubes, and this was found to be an efficient catalyst for the Knoevenagel condensation reaction.461 Pashuck and Stupp219 investigated the self-assembly of an amphiphile containing a phenylalanine trimer linking a 15 carbon, saturated alkyl chain and a triglutamic acid head group (Chart 40, 119). This amphiphile was found to assemble from a

The utility of these types of structures as biomolecule delivery vehicles was investigated with an emphasis on providing it with antimicrobial properties.416 In this instance, 107 self-assembled into nanotubes by complexation with silver(II) ions from ethanol solution. Subsequent UV irradiation reduced the silver ions to the metal, forming nanoparticles within the bilayers of the nanotube.416 These nanotubes were shown to have a bacterial inhibition ratio of over 90%.416 Even though the lipid itself was shown to have no antimicrobial effect, it appeared that the effect of the nanotubes alone, without silver nanoparticles, was not investigated. Therefore, antimicrobial action being due to the aspect ratio of the tubes cannot be entirely discounted. Alternatives to the oligoglycine headed nanotubes included those in which the head group was turned around and became an amine terminated head group for hydrophobic tails containing 11−16 carbons (Chart 39, 110−115). The amphiphiles

Chart 40. Amphiphile with Tri-phenylalanine Linker and Tri-glutamic Acid Head Group

Chart 39. Other Amphiphiles with Glycine Derived Head Groups 10 mM aqueous solution into short twisted ribbons after 30 s, which then grew into long twisted ribbons after 10 min. The right-handed, long twisted ribbons further matured into right-handed helical ribbons over the course of 4 weeks when annealed at 25 °C. This showed that the twisted ribbon was a metastable state in the formation of the thermodynamic minima of the helical ribbons (Figure 33).

coordinated with hydrochloric acid were soluble in water and were precipitated by the neutralization using vapor diffusion of dilute triethylamine. White precipitates appeared within a few days, tubes found for those amphiphiles with the longest hydrophobic chains.424 These nanotubes were surfaced with amine moieties and were able to absorb anionic nanoparticles into their lumens.424 A diglycylvaline head group was also developed (117, Chart 39). 117 self-assembled by dissolving the sodium salt in aqueous solution followed by precipitation through the diffusion of dilute acetic acid formed nanotubes with an external diameter of 44 ± 6 nm. In contrast, 116, with one less methylene unit in the hydrophobic tail, self-assembled under the same conditions formed sheets.424 For Shimizu’s group,424 it was found that the glycylglycyl unit was required for nanotube assembly as carboxy terminated glycyl, glycyl−valine, and valine−valine were all unable to form tubes. However, Park et al.460,461 found that using amine terminated single glycine head group did allow self-assembly of glycyldodecylamide (Chart 39, 118) into single wall nanotubes (outer diameter 45 nm), which was followed by templating silica.460 The amphiphile template was removed, the porosity of the silica nanotubes was measured, and the surface area was found to be 677 m2 g−1.461 Subsequently, amino

Figure 33. CryoTEM images monitoring the self-assembly of 119 over time. Reprinted with permission from ref 219. Copyright 2010 American Chemical Society.

In work related to that of Pashuck and Stupp,219 an amphiphile with a 16 carbon hydrophobic tail connected to a head group based on the core motif of the amyloid beta peptide selfassembled into helically wound nanotubes from aqueous solution (1 wt %) at room temperature.462 The nanotubes with a diameter 10261

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of 276 nm and a wall thickness of 4.4 nm underwent a reversible transition to twisted ribbons on heating to 55 °C, suggesting possible application in drug delivery.462 A further example of amino acid-based amphiphiles investigated by Shimizu et al.284 featured a single aspartic acid head group connected via an amide bond to a single linear hydrocarbon chain (Chart 41, 120). These amphiphiles self-assembled into Chart 41. Aspartic Acid Headed Lipids

Figure 34. CryoTEM images of platinum-shadowed ribbons showing (a) left-handed helix for D-122 and (b) right-handed helix for L-122. Reprinted with permission from ref 247. Copyright 2001 American Chemical Society.

helical nanostructures, but in narrow pH ranges that increased in magnitude as the hydrophobic chain length increased. As this matched the trend in titration end point for these amphiphiles, it illustrated that the degree of ionization was an important factor in the organization of the resultant aggregate structure.284 Ragunathan et al.463 synthesized an amphiphile with a hydrophobic tail having a terminal alkyne group and linked via an amide bond to a serine head group (Chart 42, 121). This mole-

in methanol:water (1:3 v/v) were cooled to room temperature. Co-assembled with these tubes were clusters of smaller diameter tubes and square crystal-like structures.85 In another method of self-assembly, 122 self-assembled into nanotubes (outer diameter 300−800 nm; length 10−40 μm) from toluene when cooled at 2 °C min−1 in the presence of 6 nm diameter Fe3O4 nanoparticles coated with the surfactant.464 Using different cooling rates of 10 and 0.5 °C min−1, no tubular aggregates were found. The nanoparticles assembled with the tubes were bound to the inner and outer surfaces of the nanotubes and highly organized, having a close-packed hexagonal symmetry. Upon calcination of the hybrid structures at 400 °C, in some instances the nanoparticles rearranged to form helically coiled wires, and occasionally double helices of wires generated from the inner and outer surfaces were observed.464 The aqueous self-assembly of 122 created by Boettcher et al.247 was dependent on the pH, with the molecule soluble above pH 7 and precipitating into amorphous and crystalline aggregates below pH 4.0. Between these extremes, helical ribbons and planar sheets were found at pH 6.4, a mixture of closed tubes and helical ribbons at pH 5.6, and tubes were the dominant species at pH 4.9. This apparent trend in increasing order in molecular packing with the decrease in pH was supported by DSC that showed aggregates increased in melting temperature with decreased pH of assembly.247 Clearly the formation of helically based tubes was best supported by a particular degree of ionization of the head group, and the authors suggested that the aggregates relied on some degree of carboxylic acid to carboxylate anion hydrogen bonding.247 The pH of assembly was also very important in the selfassembly of an amphiphile constructed from a valine head group.465 This amphiphile was without the benefit of an amide link and used a secondary amine attachment to a 12 carbon hydrophobic chain that had a hydroxyl grafted onto the second carbon (Chart 44, 123). At pH 13, 123 self-assembled into

Chart 42. Amphiphile with Serine Head Group and Acetylene Terminated Hydrophobic Tail

cule was found to self-assemble into large multilamellar spherical structures, right-handed helical ribbons 15−30 μm wide and long tubules up to 3000 μm long and of 5−20 μm diameter. The population of spherical structures was found to decease over time, being replaced by helical ribbons.463 A serine head group was also used by Boettcher et al.247 who combined it with a hydrophobic tail based on dodecanoic acid linked to the amino acid via an amide bond (Chart 43, 122), Chart 43. Amphiphile with Serine Head Group

and self-assembly was investigated from both aqueous and organic solvents. From toluene, tubules of 1 μm length, 80 nm external diameter, and 25 nm internal diameter formed with low populations of helically striated tubes and helical ribbons. From aqueous solution larger tubes with the same internal diameters, similar, but more varied, external diameters (80− 130 nm) and lengths of up to 10 μm formed along with much larger populations of helical ribbons (Figure 34).247 The hand of the helical ribbons was found to vary with molecular chirality and racemic mixtures in both solvents assembled only into flat platelets.247 An analogue amphiphile to 122 utilizing a vaccenic acid-based hydrophobic tail also self-assembled into nanotubes with an external diameter of 292 nm when heated solutions

Chart 44. Amphiphile with Valine Head Group and a Hydroxyl Group at the β-Position to the Head Group in the Hydrophobic Tail

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tubes, and it was believed that hydrogen bonding between hydroxyl and amino groups of adjacent molecules created the ordered directional packing, more usually driven by amide hydrogen bonding for these types of amphiphiles.465 An amphiphile with an L-lysine derived head group and containing naphthalene diimide within the hydrophobic tail self-assembled into helically based tubes.466 The zwitterionic amphiphile (Chart 45, 124) was insoluble in pH neutral water, Chart 45. Amphiphiles with L-Lysine Head Group and Naphthalene Diimide Derived Hydrophobic Tail Figure 35. Progression of structures self-assembled from 127 over time. Reprinted with permission from ref 217. Copyright 2011 American Chemical Society.

struction with ∼10 bilayers making up the walls of the tubes,111 most likely with a folded orientation of the amphiphile217 that provides a mixture of bolaamphiphile and regular amphiphile configuration. An amine terminated glycine unit was used as a head group linked through an amide bond to an ethylenediamine linker, itself linked through an amide bond to an azobenzene group (Chart 47, 128).467 The amphiphile was self-assembled by dis-

but self-assembled in aqueous solutions of pH 1.2 and 12 into identical nanotubes with diameters of 14 ± 1 nm and wall thicknesses of 4.2 nm. XRD results suggest that the walls consist of fully extended molecules in a bilayer with no overlap of the hydrophobic tails. Contrasting with the self-assembly behavior of 124, the methyl ester 125 self-assembled from neutral pH water into nanotubes having an external diameter of 18 ± 1 nm and a wall thickness of 7 nm. The XRD results indicate that in this case the walls were comprised of two bilayers containing tilted or interdigitated arrangements of amphiphiles. The amino derivative of the naphthalene diimide-based amphiphile (126) self-assembled from aqueous solution in a concentration-dependent manner.466 At 15 mM, the nanotubes formed were 100−250 nm wide and several micrometers long. At 5 mM, a variety of structures self-assembled from water including helical ribbons, twisted ribbons, and nanotubes, while at 250 μM the predominant self-assembled structures were twisted ribbons. Given this progression, the authors suggest that the tubes self-assembled at 15 mM proceed through unseen intermediate twisted and helical ribbons. N-Lauryl-lysyl-aminolauryl-lysine-amide (Chart 46, 127) was found to self-assemble into nanotubes via twisted and helical

Chart 47. Amine Terminated Glycine Head Group Linked through Ethylenediamine to an Azobenzene Hydrophobic Tail

persal in refluxing aqueous solution, and then gradually cooled to room temperature. At pH 6.1 the protonated amphiphile formed fibers, and at pH 9.2 the uncharged amphiphile formed sheets. Between pH 7.6−8.2, the amphiphile was partially positively charged and assembled into nanotubes (inner diameter 20 nm; wall thickness 8 nm).467 Upon UV irradiation (365 nm, 4 min), the nanotubes were converted to cylindrical nanofibers as the trans-azobenzene moiety was photoisomerised to the cis form. Subsequent visible light irradiation (436 nm, 15 min) induced complete photoisomerisation back to the trans form, creating helical nanotapes that varied in the bilayer organization as compared to the original nanotubes.467 Carboxyfluorescein was encapsulated within the nanotubes assembled from 128 by adding lyophilized nanotubes to a solution of carboxyfluorescein at pH 9. The carboxyfluorescein was rapidly released into solution upon UV irradiation of the tubes, with 40% released within 4 min. In contrast, from the nonirradiated nanotubes, there was constant slow release from the nanotubes, 40% of the probe being released after 12 h.467 Four amphiphiles were synthesized with aspartic acid head groups and an azo group contained within the hydrophobic tails that varied in proximal and distal chain lengths (Chart 48, 129−132). These amphiphiles were self-assembled from aqueous methanolic solution forming chiral H-aggregates.468 Those compounds with 10 carbon proximal chains and 4 (129) or 7 (130) carbon distal chains self-assembled into nanotubes when annealed from mixtures of methanol and water (1:3, v/v)

Chart 46. N-Lauryl-lysyl-aminolauryl-lysine-amide

ribbon intermediates by cryo-TEM.111,217 Early structures were fibers that transformed into thin twisted ribbons with widths of