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Remembrances of Self-Assemblies Past Fredric M. Menger Department of Chemistry, Emory University, 1515 Dickey Drive, Atlanta, Georgia 30322, United States ABSTRACT: Research on four types of self-assemblies (micelles, coacervates, gels, and vesicles) is discussed via a particular investigative methodology (in order of appearance): kinetics, dynamic NMR, PGSE-NMR, double-13C labeling, molecular dynamics computations, phase diagrams, cryo-HRSEM, rheology, light/electron microscopy, electrophoretic mobility, electroformation, confocal microscopy, and calorimetry. The emphasis here is on how a given method, each in its own special way, illuminates a complex system.
W
hen Langmuir asked me to write a feature article reviewing our self-assembly work, I pointed out that I had just published two related reviews entitled “Self-Assembling Systems: Mining a Rich Vein”1 and “Amphiphiles I Have Known”.2 Thus, my instinct was to offer Langmuir something different (e.g., a survey of past papers by other scientists who have most influenced my understanding of self-assemblies and to whom I obviously owe a great debt). Unfortunately, as gravity tugs at my briefcase with ever-increasing malice, I felt I lacked the wherewithal to do all this work justice in a reasonable amount of time. As an alternative, I once again sought refuge in our own work, egocentric though this might seem. But I did not want to repeat coverage of papers already cited in the two reviews just mentioned, so I devised the following plan using additional papers: I would organize this review in terms of self-assembly type (micelles, coacervates, gels, and vesicles.). Within each of these categories, I would include subheadings encompassing papers that exploit a particular methodology. For example, the vesicle topic consists of papers dealing with electrophoretic mobility, electroformation, confocal microscopy, and calorimetry. Because progress with any complex system, including selfassemblies, depends on a diversity of experimental methods, selfassembly is a delightful platform from which to become broadly educated and, in turn, to train others. (Indeed, a course in analytical chemistry could be taught using only self-assemblies as examples.) So this feature article is not only for the specialist but also for the student/novice. The latter are more impressionable, and, let us admit it, any instilled interest has a potentially longer time span with which to express itself. Each subheading describes a single relevant observation arising from a particular methodology. Additional observations and details, if so desired, can be obtained by consulting the original work. Two outside references per methodology are also given. Thus, this is a meandering journey but one that is unified by the allure of diverse molecules, all of which spontaneously gather themselves into assemblies. If politicians can claim that “in unity there is strength”, then chemists interested in self-assemblies can claim that “in unity there is possibility”. r 2010 American Chemical Society
As with all reviews, what follows is a recapitulation of the past. In this connection I can quote from a recent paper:1 “Papers from past years of research become like a chain of pearls stored in the drawer of an old attic closet. On occasion one removes the chain from the drawer and blows the dust off a particular pearl in hopes that the pearl will shine as, once upon a time, it seemed to do.” In the text below, we blow the dust off certain papers that, unaccountably, are among our favorites. Before beginning this missive, I should point out that there exists one particular discipline that we have found generally useful in self-assembly research: organic synthesis. Organic synthesis expands possibilities, although often with sweat and tears. However, only a synthesis effort allows one to explore the properties of self-assemblies as a function of molecular structure. Consider the example of 1, a compound that required almost 2 years to prepare.3 Thus, the synthesis was complicated by four functional groups residing in close quarters, by a long hydrocarbon chain producing oils, by intermolecular hydrogen bonding of the diketopiperazine ring reducing solubility, by two identical oxygens requiring unsymmetrical derivatization, and by two chiral centers tending to racemize and give diastereomers. However, we cheerily accepted the notion that there is no highway for effortless travel, and in any case, we badly wanted to get hold of 1 in order to learn how attractive intermolecular hydrogen-bonding forces, acting in concert with hydrophobic forces, would perturb self-assembly. The reward came when we discovered that compression/relaxation in a monolayer displays a remarkable hydrogen-bond-induced hysteresis involving horizontal-to-vertical reorientations.4 In the text about to follow, I will not delve further into synthesis issues, but behind the scenes, they were an ever-present concern.5,6
Received: August 16, 2010 Revised: September 17, 2010 Published: October 14, 2010 5176
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1. MICELLES (a). Kinetics (Menger, F. M.; Portnoy, C. E. On the chemistry of reactions proceeding inside molecular aggregates. J. Am.Chem. Soc. 1967, 89, 4698).
A Saul Winstein 1961 publication, in which solvolysis kinetics were used to detect three intermediates (an intimate ion pair, a solvent-separated ion pair, and a free carbocation),7 left me with a deep admiration of rate constants’ power. This high regard for kinetics led us to quantify rates of reactions occurring in micellar solutions (Scheme 1). In this scheme, Sn is the micelle composed of n surfactant molecules S; k1 is the rate constant for the substrate in the absence of micelles; k2 is the rate constant of the substrate within the micelles; and K is the binding constant between the substrate and micelle. Linear double-reciprocal plots allow k2 and K to be determined. Widespread application of the construct (which has evolved into the so-called pseudophase ion exchange model) was pleasant enough, but there was also a disappointment. With micellar rate constants now in hand, it was evident that in no way did they measure up to the efficiency of enzymatic catalysis. Information on micelle environments (e.g., their polarity) and on their sensitivity to additives is perhaps the most useful benefit derived from micellar kinetics.8,9 (b). Dynamic NMR (Menger, F. M.; Lynn, J. L. Fast proton transfer at a micelle surface. J. Am. Chem. Soc. 1975, 97, 948). Rates of NH-proton exchange for N,N-dimethyldodecylamine (C12NHMe2þ) in aqueous acidic media were deduced from the slow-passage 1H NMR signal of the N-methyl protons. When the pH is lowered sufficiently, the methyl signal transforms from a sharp singlet (where fast Hþ exchange washes out spin-spin splitting) into a doublet (where Hþ exchange is slow on the NMR timescale). Computer-assisted NMR line-shape analysis at each pH gave the corresponding rate constant. Rate data were analyzed in terms of the Grunwald mechanism (Scheme 2).10 Amine protonation (k-a), a reverse step, shows why the observed rate decreases with decreasing pH (increasing H3Oþ). One key point was immediately obvious from our study: At concentrations above the cmc of C12NHMe2þ, the rate of proton transfer is much faster than that with a monomeric amine, C6NHMe2þ. Thus, the micellar system requires a pH range of 0.0 to 0.45 to bring the rates into the NMR timescale (compared with a pH range of 3.25 to 3.66 for the monomer). It was shown that the proton-delivery step (ka) is about 30 times faster at the micelle surface than from an amine monomer. The pKa of the micellized amine is lowered to 8.7 (1.4 units less than the pKa of C6NHMe2þ). Both the ka and pKa effects can be ascribed to electrostatic repulsion among the cationic headgroups at the surface of the C12NHMe2þ micelles. The NMR-determined pKa is useful because previous attempts to determine micellar pKa values had used probes (e.g., ionizable dyes) whose self-inflicted environmental perturbations always present an uncertainty.11,12 (c). Pulse Gradient Spin-Echo NMR (PGSE-NMR) (Menger, F. M.; Lu, H.; Lundberg, D. A-B-A-B-A and B-A-B-A-B block amphiphiles. Balance between hydrophilic and hydrophobic segmentation. J. Am. Chem. Soc. 2007, 129, 272).
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Penta-segmented molecules with the general structure E-C-EC-E or C-E-C-E-C (where E is a hydrophilic polyethylene oxide and C is a hydrophobic carbon chain) were synthesized and examined by PGSE-NMR for possible self-assembly. PGSENMR is a technique that provides diffusion coefficients (D) in solution. They were obtained with a 600 MHz spectrometer equipped with a pulse field gradient generator using a Hahn echo sequence (the details of which are well described in the literature).13 Assuming that solute particles are spherical, then D values provide effective sizes using the Stokes-Einstein equation. Plots of D versus concentration for E3C6E3C6E3 and E6C10E6C10E6 are given in Figure 1. The molecular volume of E3C6E3C6E3 at 0.5 mM is about 1.4 nm3, corresponding to a monomeric species. At 25 mM, the volume increases by 50%, which is indicative of a dimer. Thus, despite its 12 methylenes, E3C6E3C6E3 fails to self-assemble, presumably owing to hydrocarbon segmentation. In contrast, the D versus concentration plot of E6C10E6C10E6 does indicate self-assembly. Approximate aggregation numbers at 5 and 25 mM were found to be 20 and 60, respectively. Apparently, E6C10E6C10E6 forms assemblies that, lacking a high degree of cooperativity, grow continuously with increasing concentration (as opposed to the precipitous micelle formation at a cmc, with discrete aggregation numbers, characteristic of classical surfactants). PGSE-NMR is clearly a source of valuable information that is difficult to obtain by other means.13,14 (d). Double-13C Labeling (Menger, F. M.; Carnahan, D. W. A new method for studying chain conformation. Proof of nonradial binding to micelles; chain-bending at an enzyme surface. J. Am. Chem. Soc. 1986, 108, 1297). The chain conformation is fundamental to the overall structure of micelles, bilayers, membranes, films, and most other selfassemblies. We have developed a method for studying chain conformation that has a unique capability: it experimentally provides the dihedral angle at a specific carbon/carbon bond within a long hydrocarbon chain. The method is based on longrange coupling between two 13C atoms spaced four carbons apart (-13C-C-C-13C-). In a type of Karplus relationship, 3J decreases from about 4.0 Hz to about 1.5 Hz when an anti conformation (linear) rotates around the central C-C bond into a gauche conformation (bent). When double-labeled 2 was adsorbed onto a cationic micelle, the 13C spectra gave a 3J of 3.5 Hz, showing that bound guest molecules are substantially linear about their central linkage. But when 3 was adsorbed onto trypsin (in what is probably a nonspecific complex), 3J was reduced to only 0.8 Hz corresponding to a bent chain with a dihedral angle of about 70°. Linear and kinked conformations associated with micellar and enzyme binding, respectively, are depicted in Figure 2.
Double-13C labeling was also valuable in proving (as no other current method is able to do) that surfactant chains become highly bent at three known sites when they enter the micellar state.15 (e). Molecular Dynamics Computations (Menger, F. M.; Zhang, H.; de Joannis, J.; Kindt, J. T. Solubilization of paclitaxel (Taxol) by peptoad self-assemblies. Langmuir 2007, 23, 2308). 5177
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Scheme 2
Figure 1. Observed self-diffusion coefficients of E3C6E3C6E3 (b) and E6C10E6C10E6 (O) at different concentrations (25 °C).
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of 4 have a useful property: they water-solubilize paclitaxel (Taxol), a very insoluble anticancer drug (5). The question arose as to why 4 is such an effective excipient (rivaling the commercial Cremphor EL now used with the drug). The best way to get a fix on this question was through the aid of a 10 ns molecular dynamics (MD) simulation whose results are now briefly summarized: Compound 4 forms transient hydrogen bonds at two to six sites on the drug, thereby partially encasing the drug in a hydrophobic shell. This hydrophobicized species then resides in the water-free center of the surfactant clump. In a sense, 4 makes the drug more water-soluble by first rendering it less water-soluble so that it can then enter the hydrophobic center of the watersoluble clump. Structure 5 shows a drug/assembly complex (4.2 ns simulation) with four hydrogen-bonding sites of the drug, each being occupied by a molecule of 4 (as indicated by dotted lines and hydrogen bond distances). Because the molecules of 4 in the complex are mobile, extending the simulation to 4.5 ns leads to a different hydrogen-bonding pattern. It would be difficult to obtain such information from any current experimental method; there are times when a computation is the only option.16,17
2. COACERVATES (a). Phase Diagram (Menger, F. M.; Peresypkin, A. V. A combinatorially-derived structural phase diagram for 42 zwitterionic geminis. J. Am. Chem. Soc. 2001, 123, 5614).
Figure 2. Schematics of 2 in a linear conformation binding to a cationic micelle and 3 in a bent conformation binding to an enzyme.
Compound 4, with its n-heptyl chain plus three amide groups, was obtained in the course of our designing, synthesizing, and studying new amphiphilic systems. We found that a series of amide groups could serve as a suitable hydrophilic headgroup for short-chain surfactants that form “clumps” in water.
Self-assemblies of 4 are termed clumps rather than micelles because the aggregates appear to grow continuously with concentration and thus do not have a cmc. In any case, self-assemblies
Ternary phase diagrams, those triangular graphs in which the concentrations of three components are systematically varied, can be extraordinarily complex. For example, a water, oil, and surfactant phase diagram possesses over 30 discrete regions.18 Our approach to phase diagrams was quite different and, fortunately, simpler. For one thing, structure, not concentration, was the key parameter. The parent amphiphile in question was compound 6, a zwitterionic gemini in which A and B were varied such that the chains were short-short, short-long, long-short, and longlong, respectively. All told, 42 compounds were synthesized and examined for phase behavior in water. A structural phase diagram, given in Figure 3, plots A versus B for the 42 compounds. Four phases were encountered: micelles, gels, vesicles, and coacervates (each represented by a different symbol as shown).
To summarize the data briefly, short-short (e.g., A6B4) forms micelles; long-long (e.g., A18B18) forms bilayer structures; long-short and short-long (e.g., A22B9) form gels; and (most 5178
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Figure 3. Structural phase diagram of 42 zwitterionic geminis. Numbers in the right top corner of the circles represent gel-transition temperatures of the vesicles (Tm,°C).
interesting) intermediate lengths (e.g., A10B10) form coacervates. A coacervate is created when an amphiphile exposed to water separates into two immiscible aqueous phases: a coacervate layer that is rich in colloidal material and a so-called “equilibrium liquid” that is poor in colloidal material. Thus, one is confronted with a remarkable situation in which two aqueous layers, one with up to 95% water and the other with 99-100% water, do not freely mix. Self-assemblies are often in delicate thermodynamic wells. In the case of our compound 6, small structural changes dictate whether a coacervate forms, once again emphasizing the value of surveying a large variety of structural analogs with the aid of synthetic chemistry. In the absence of systematic screening, discovery of the coacervate phase, with its rather narrow range of A and B values, would have been fortuitous.19,20 (b). Cryo-High-Resolution Scanning Electron Microscopy (cryo-HRSEM) (Menger, F. M.; Peresypkin, A. V.; Caran, K. L.; Apkarian, R. P. A sponge morphology in an elementary coacervate. Langmuir 2000, 16, 9113). How can two aqueous phases be immiscible as described above? One explanation is that the coacervate phase adopts a “sponge morphology”. As with a kitchen sponge, the coacervate can absorb considerable water without actually dissolving. This model was supported by electron microscopy. Thus, a suspended coacervate droplet of A8B10 was rapidly frozen (ca. 10 000°/s),
Figure 4. Fractured coacervate droplet as detected by cryo-HRSEM (bar = 667 nm).
fractured, coated with a 1 nm layer of Cr, and directly observed in the upper stage of a field-emission scanning electron microscope (Figure 4). The image in Figure 4 shows a frozen droplet where fracturing has exposed the seldom-observed honeycomb or spongelike inner morphology of a coacervate. Electron microscopy is an invaluable friend of the self-assembly investigator. How else could one have uncovered such a honeycombed structure?21,22 5179
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Figure 5. (A) Single molecule of 6. (B) Self-assembly into large gel fibers. (C) Bundling of small 15 nm fibers in the xerogel. (D) Side-by-side packing of small fibers into sheets. (E) Interconnected sheets form a porous solid.
3. GELS (a). Rheology (Menger, F. M.; Caran, K. L. Anatomy of a gel. Amino acid derivatives that rigidify water at submillimolar concentrations. J. Am. Chem Soc. 2000, 122, 11679).
Fourteen L-cystine derivatives, 7, were synthesized and examined for their ability to gelate water. Several members of this amino acid family are remarkably effective aqueous gelators, with the best one (R = NH2, R0 = H, and R00 = 2-naphthoyl) being able to rigidify aqueous solutions at 0.25 mM in less than 30 s. SEM showed that all of the good gelators self-assemble into fibrous structures that entrain the solvent in the capillary spaces between them. As usual, multiple techniques were applied to the systems, but for the purposes of this review, I will focus briefly only on one not yet discussed: rheology.
Although textbooks warn that “rheology is not an easy branch of science”, the method is critical to answering important questions such as the following: What happens to the gel fibers when they are exposed to mechanical stress? By what mechanism does gelator concentration affect gel rigidity? How do temperature and cosolvents affect fiber dynamics? In our rheometer experiments, a thin layer of gel was placed between a round flat plate at the bottom and a round conical plate (40 mm diameter), fixed to a rotatable shaft, at the top. The cone rotated back and forth at 0.1-10 Hz and at a constant specified torque. The torque was converted directly into “stress” (expressed in pascal units). A position sensor on the oscillating shaft measured the amplitude of the gel’s deformation to give a unitless “strain”. Although details can be obtained in the above reference and textbooks, I need to mention here that stress and strain automatically generate two useful parameters, G0 and G00 . Elastic modulus G0 represents the ability of the deformed gel to “snap back” to its original geometry. Viscous modulus G00 represents the tendency of the gel to flow under stress. One can easily appreciate the values of G0 and G00 in characterizing a gel. For example, rheology revealed that our fastest and lowest-concentration gelator (the one mentioned above) has a small elastic modulus indicative of a “weak” gel. Apparently, fast gel formation yields only short fibers that fail to produce a robust gel. Certain of our gels were stable to a 1 Hz, 3 Pa oscillating shear stress at 90 °C whereas other gels experienced a catastrophic break at lower temperatures. Any time
that self-assembly enhances the viscosity of a system, rheology comes to mind as a source of unique insight.23,24 (b). Light and Electron Microscopy (Kobel, M.; Menger, F. M. Hierarchical structure of a self-assembled xerogel. Chem. Commun. 2001, 275). Cooling a warm solution of glycouril 8 in benzyl alcohol (20 mg/mL) creates a stable, thermoreversible, self-supporting gel. Light microscopy (400) revealed flexible fibers several hundred micrometers long and about 2 μm in diameter. Removing the solvent from the gel produced a brittle xerogel of resinlike appearance.
Macroscopic fibers, as originally observed by light microscopy, were no longer visible. Instead, curved, wrinkled, and interconnected sheets were evident under the light microscope. Examination by SEM (60 000) showed a fibrous fine structure, with fibers 15 nm in diameter and several micrometers in length packed into craggy, planar structures. Owing to the high disorder and curvature of the sheets and to their multiple junctions with one another, cavities were formed to give a macroporous solid. The hierarchical levels of the gel and xerogel are depicted in Figure 5.25,26
4. VESICLES (LIPOSOMES) (a). Electrophoretic Mobility (EPM) (Yaroslavov, A. A.; Melik-Nuberov, N. S.; Menger, F. M. Polymer-induced flipflop in biomembranes. Acc. Chem. Res. 2006, 39, 702).
This vesicle work was a funded Russian/U.S. collaboration with all of the experimental work being carried out in Russia. Doublechained amphiphiles, such as phospholipids, self-assemble into bilayers that in turn form hollow spheres called vesicles. The cited experiments utilized vesicles constructed from dipalmitoylphosphatidylcholine (DPPC) containing 10 mol % cardiolipin (CL-2), a lipid bearing two negative charges and four long tails. Calorimetry showed a single symmetrical peak, indicating a nearly uniform distribution of CL-2 throughout the two leaflets of the vesicle bilayer. 5180
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Figure 6. (A to B) Cationic polymer-induced flip-flop of the anionic lipid from the inner leaflet (left side) to the outer leaflet (right side) of the bilayer. (B to C) Removal from the bilayer of the cationic polymer by the anionic polymer. (C to D) Drifting back of anionic lipid to the equilibrium distribution.
The electrophoretic mobility (EPM) was used to examine bilayer properties when a cationic polymer (quaternized poly(4vinylpyridine), P2) was bound externally to the anionic vesicles. Laser-detected migration of the polymer/vesicle complex in an electric field ceases only when the anionic charge of the vesicle’s outer leaflet is precisely charge neutralized by the cationic polymer. In other words, a plot of EPM versus [P2] (using a polymer of known charge content) reveals, at zero mobility, the anionic charge of the vesicle. It is as if the charge on the outer leaflet has been titrated. This is a very useful piece of information as seen by the results given in the next paragraph. Below the transition temperature Tm = 41.5 °C of DPPC, the membrane is “solid” (a rigid gel with little chain mobility). Above Tm, the membrane is “liquid” (a liquid crystal with considerable chain disorder). When our vesicle was solid, it was charge neutralized by externally bound polymer whose charge corresponded to 50% of the total CL-2 content in the bilayer, but neutralizing a liquid vesicle required polymer equivalent to 100% of the CL-2. Clearly, the polymer had induced the CL-2 to flipflop from the inner to outer leaflets of the bilayer (Figure 6A,B). Calorimetric experiments demonstrated that the CL-2, now exclusively in the outer leaflet, was not randomly distributed in the outer leaflet but instead resided in domains where a maximum number of ionic contacts with the polymer could be achieved (Figure 6C). When the cationic polymer was removed from the vesicle (with poly(acrylic acid)), about half of the CL-2 drifted back to the inner leaflet (Figure 6D), allowing the outer-to-inner flip-flop (an important process in biomembranes) to be measured quantitatively.27,28 (b). Electroformation (Menger, F. M.; Angelova, M. I. Giant vesicles: imitating the cytological processes of cell membranes. Acc. Chem. Res. 1998, 31, 789. Seredyuk, V. A.; Menger, F. M. Membrane-bound protein in giant vesicles: induced contraction and growth. J. Am. Chem. Soc. 2004, 126, 12256). Electroformation is an excellent method for preparing giant vesicles (GVs) visible under the light microscope (10-200 μm). An electroformation cell is composed of two parallel 0.5-mmdiameter Pt wires spaced 0.5 cm apart. After a lipid film is coated onto the wires, the cell is filled with water at >Tm and a 0.2 V voltage is applied at an ac frequency of 10 Hz. Elevating the voltage to 1-4 V produces giant vesicles lined up like pigeons on a telephone line. Gentle pulling by a micropipet under suction allows a single giant vesicle to be completely removed from a wire
Figure 7. Schematic of GV growth as promoted by a protein, zein, embedded in the bilayer. The diagram is not to scale with a GV being in fact 500 larger than an SUV. Zein, shaded ovals; GV lipids, open dumbbells; SUV lipid, solid dumbbells. Zein provides hot spots for membrane fusion. After fusion, rapid lateral diffusion would randomize the SUV-derived lipids (not shown).
and manipulated. Direct observation of bilayer behavior via the GV has great advantages. For example, one can physically or chemically injure a single GV and then monitor its recovery, a feat not possible with the more common small (ca. 50 nm) unilamellar vesicles (SUVs). Anionic GVs were constructed of POPC (a zwitterionic phospholipid), POPG (an anionic phospholipid), and zein (a hydrophobic globular protein) in a ratio of 93:5:2, respectively. These giant vesicles were observed to grow when supplied with microscopically invisible “food” consisting of cationic SUVs (plus a small amount of SDS, a surfactant growth initiator). The growth can be reasonably attributed to GV/SUV fusion. For growth to occur, the following conditions must be met: (a) Zein had to be present and (b) the GVs and SUVs had to have opposite charges. We reasoned that GV/SUV fusion probably arises from membrane defects at the periphery of the protein molecules (fusion hotspots) as depicted in Figure 7. One is reminded of virus fusion proteins that reconfigure host membranes prior to viral entry. Giant vesicle systems were also used to 5181
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Scheme 3
Figure 8. Confocal microscopy picture where organic synthesis and self-assembly combine to form blistered hollow spheres (bar = 50 μm).
observe the fusion, fission, birthing, healing, and layering of bilayers. Electroformation is a superior method for making giant vesicles free from lipid chunks and other nonvesicular “trash”.29,30 (c). Confocal Microscopy (Menger, F. M.; Bian, J.; Seredyuk, V. A. Vesicular latex. Angew. Chem. Int. Ed. 2004, 43, 1265). One end of compound 9 consists of a cholesterol unit that readily embeds in a vesicle bilayer. On the other end of 9 was placed a maleimide group whose double bond reacts with thiols in a sort of “click” Michael addition. When 200 nm phospholipid vesicles bearing about 5 mol % 9 were rapidly mixed with excess dithiothreitol, the vesicles were cross linked (Scheme 3), producing a white material resembling shaving cream.
Figure 9. Transition enthalpy, ΔHc, in kcal/mol resulting from the disordering (melting) of phospholipids as a function of the position of methyl branching at carbon 4, 6, 8, 10, 12, or 16 of chain no. 2.
dependence of ΔHc on the methyl location with position 10, in the middle of the chain, being by far the most disruptive to the lipid assembly.
An examination of the material by confocal microscopy (Figure 8, Zeiss laser scanning microscope, λ = 488 nm) showed that blisters (nonremovable by suction with a micropipet) are integrated into the surfaces of spherical particles ranging from 5 to 150 μm in diameter. Particles are believed to be hollow, waterfilled structures, with shells of
