Langmuir 2002, 18, 4847-4853
4847
Surface Characterization of Cholesterol Monohydrate Single Crystals by Chemical Force Microscopy Richard S. Abendan and Jennifer A. Swift* Department of Chemistry, Georgetown University, 37th and O Streets NW, Washington, District of Columbia 20057-1227 Received February 21, 2002. In Final Form: March 22, 2002 Atomic force microscopy (AFM) and chemical force microscopy (CFM) techniques have been used to characterize the chemical functionality of cholesterol monohydrate single-crystal surfaces in different solution environments. Both synthetic and natural crystals adopt a platelike habit in which the largest face is (001). Under aqueous and organic solution conditions, in situ contact mode topographical images reveal that the plate face is primarily terminated by bilayer molecular structures and therefore largely homogeneous. Adhesion force measurements obtained with chemically modified tips demonstrate that the functionality of the crystal surface can be altered by changes in the solution composition. The 3-hydroxyl end of cholesterol molecules is presented on the plate face in aqueous media, while alkyl tail groups terminate the surface in organic solutions. Contact angle measurements on (001) surfaces exposed to solvents of different hydrophilicity also show similar trends, providing additional support to these AFM assignments. These studies link molecular-level and macroscopic adhesion properties and demonstrate that solution environment can exert a strong influence on the surface properties of this important biomaterial.
Introduction Cholesterol is ubiquitous in all living systems: it is essential for cell membrane structure, the insulation of nerves, the production of certain hormones, and the synthesis of bile acids. Though highly insoluble in water,1,2 cholesterol is solubilized in vivo in lipid bilayer or micelle systems, such as cell membranes and bile salts, or with lipoproteins in blood serum. When cholesterol becomes insoluble in biological systems, it separates as an oily phase and/or crystallizes. The causes for cholesterol precipitation are not fully understood but may be due either to a marked increase in cholesterol concentration or to a decrease in solubilizing lipid concentrations.3 The unregulated deposition of cholesterol solids in humans has been implicated in diseases such as gallstone attacks and atherosclerosis, which afflict over 10 million people annually in the United States alone. Studies on human gallstones and arterial plaques by light microscopy4,5 and X-ray diffraction6 have confirmed the presence of cholesterol monohydrate crystals having a characteristic platelike morphology. Cholesterol monohydrate crystal deposition can occur under a variety of hydrophilic and hydrophobic chemical environments in vivo. Gallstone formation occurs in an arguably aqueous environment by the formation and aggregation of microcrystals of cholesterol monohydrate. These crystals constitute up to 80% of the total stone mass.5 Cholesterol monohydrate crystals are also typically found in lipidrich core regions of advanced fibrous plaques, in patients as young as age 10.7 The removal of these arterial crys* To whom correspondence should be addressed: e-mail
[email protected]. (1) The Merck Index, 12th ed.; Merck and Co.: White House Station, NJ, 1996. (2) Saad, H. Y.; Higuchi, W. I. J. Pharm. Sci. 1965, 54, 1205-1206. (3) Admirand, W. H.; Small, D. M. J. Clin. Invest. 1968, 47, 10431052. (4) Katz, S. S.; Shipley, G. G.; Small, D. M. J. Clin. Invest. 1976, 58, 200-211. (5) Sedaghat, A.; Grundy, S. M. New Engl. J. Med. 1980, 302, 12741277. (6) Bogren, H.; Larsson, K. Biochim. Biophys. Acta 1963, 75, 65-69.
tals through natural metabolic processes would be difficult, because it would require crystal dissolution in an already supersaturated lipid environment.8 To elucidate the crystal nucleation and growth properties of this important biomaterial, we thought it would be beneficial to gain an intimate understanding of the molecular nature of the crystal surfaces under a range of organic and aqueous solution conditions. By elucidating the crystal surface structure, one is in a better position to rationally devise agents that could modify the surfaces such that crystal growth processes in vivo might be suppressed.
Craven9 previously determined the crystal structure of cholesterol monohydrate in the 1970s. Like most 3-hydroxy steroids, cholesterol monohydrate adopts a bilayer structure in the crystalline state (space group P1; a ) 12.39, b ) 12.41, c ) 34.36, R ) 91.9°, β ) 98.1°, γ ) 100.8°). Polar 3-hydroxyl ends form hydrogen bonds with each other and with water molecules, giving a slightly puckered two-dimensional hydrogen-bonded network (Figure 1). The long axes of the molecules are nearly parallel and are tilted by ∼17° from the normal to the (001) plane. Bilayers measuring 33.9 Å in height are stacked through van der Waals forces along the crystallographic c axis. Synthetic crystals grown from aqueous organic solutions and/or model bile solutions adopt a characteristic platelike morphology identical to those formed in vivo (Figure 2). (7) Stary, H. C. Eur. Heart J. 1990, 11 (Suppl. E), 3-19. (8) Small, D. M.; Shipley, G. G. Science 1974, 185, 222-229. (9) Craven, B. M. Nature 1976, 260, 727-729.
10.1021/la025649r CCC: $22.00 © 2002 American Chemical Society Published on Web 05/04/2002
4848
Langmuir, Vol. 18, No. 12, 2002
Figure 1. Bilayer structure of cholesterol monohydrate constructed from fractional coordinates provided in ref 9. Dark spheres represent oxygen atoms from C-3 hydroxyl and water; hydrogen atoms are omitted for clarity.
Figure 2. Optical micrograph of a typical synthetic cholesterol monohydrate crystal. (scale bar ) 5 mm) The largest plate face bisects the crystallographic c axis.
The largest plate face is the c face, which is consistent with faster growth within the bilayer plane (stronger intermolecular interactions) than growth between bilayers. The angle between the smaller side faces is typically ∼101° (γ ) 100.8°), indicating that these faces are likely a combination of low-index (100), (010), (101), and (011) surfaces as suggested by previous reports.10 The anhydrous crystal phase of cholesterol is metastable and has also (10) Perl-Treves, D.; Kessler, N.; Izhaky, D.; Addadi, L. Chem. Biol. 1996, 3, 567-577.
Abendan and Swift
been characterized crystallographically.11 Anhydrous crystals deposit as needles, making them easily distinguishable from the thermodynamically favored monohydrate form. Each crystal face exposes a slightly different functionality due to the different orientations of the cholesterol and water molecules at the surfaces. All of the smaller side faces expose primarily the cholesterol backbone and are expected to be largely hydrophobic in character. The chemical character of the terminal groups on the plate face was somewhat less obvious. We rationalized a priori that the molecular surface structure of the plate face might be chemically homogeneous, terminating in either alkyl tails or hydroxyl groups, or heterogeneous, displaying regions of both functionalities. Though (001) and (002) planes of cholesterol monohydrate are crystallographically equivalent and defined solely by the location of the unit cell origin, we use the conventions (001) and (002) to denote hydrophobic (CH3-terminated) and hydrophilic (OHterminated) surface planes, respectively. This study aims to characterize both the surface structure and the chemical composition under very different but well-defined solution conditions. In particular, we were interested to test whether the terminal surface undergoes any chemical or morphological changes as a function of solution composition. Central to this study is the use of the atomic force microscope12 (AFM), which has sufficient vertical resolution ( (hydrophobic-hydrophobic) > (hydrophilic-hydrophobic). Others17 have suggested that adhesion forces are greater between matched hydrophobic surfaces than hydrophilic surfaces in some solvents, due to the increased work of solvent exclusion in hydrophobic interactions. However, invoking hydrophobic effects in our case would not change our functional group assignments. Differences in the adhesion force between hydrophobic and hydrophilic tips and the cholesterol monohydrate plate face in solution should be sufficient to unambiguously assign the functional group character of the cholesterol monohydrate plate face. AFM tips used in this study were chemically modified with either carboxyl (COOH) or methyl (CH3) functionalities, by self-assembling alkanethiols on gold-coated conventional silicon nitride tips. The cholesterol monohydrate crystal surface is terminated with either hydroxyl (OH) or methyl (CH3) groups. Though the hydrophilic groups on the tip (COOH) and surface (OH) are not identical in this study, we assume that a stronger adhesive interaction would still exist between these two hydrophilic surfaces than between a hydrophilic tip and a hydrophobic surface. Due to more favorable hydrogen-bonding capabilities, COOH-OH adhesive forces are stronger than identical OH-OH interfacial interactions. We expect hydrophobic tips to have a greater adhesion to (001) than to (002) surfaces. A variety of CFM methods were surveyed in this study, including friction force and phase imaging studies, as well as direct adhesion measurements by means of force-distance curves. The latter proved to be the most useful for surface functional group identification. Friction Force Imaging. Friction and contact-mode topographical images can be collected simultaneously. Various studies that map lateral friction or adhesion forces measured at the scanning AFM tip have shown regional contrast at chemically heterogeneous surfaces.38-40 Much effort was made to locate stable monolayer regions on cholesterol monohydrate crystal surfaces, because comparison between the friction on these areas and on the (37) Johnson, K. L.; Kendall, K.; Roberts, A. D. Proc. R. Soc. London A 1971, 324, 301-313. (38) Overney, R. M.; Meyer, E.; Frommer, J.; Guntherodt, H.-J.; Fujihara, M.; Takano, H.; Gotoh, Y. Langmuir 1994, 10, 1281-1286. (39) Green, J.-B. D.; McDermott, M. T.; Porter, M. D.; Siperko, L. M. J. Phys. Chem. 1995, 99, 10960-10965. (40) Eaton, P. J.; Graham, P.; Smith, J. R.; Smart, J. D.; Nevell, T. G.; Tsibouklis, J. Langmuir 2000, 16, 7887-7890.
CFM of Cholesterol Monohydrate Single Crystals
predominant flat planes might provide a means to assign chemical functionality. However, cholesterol monohydrate plate faces are terminated almost exclusively with homogeneous bilayer steps, and the general rarity of finding stable monolayers made such friction comparisons difficult. The small monolayer regions near step edges do show some frictional contrast, but this could easily be ascribed to higher lateral stresses and/or tip-induced elastic deformation at step edges, rather than to actual differences in surface functional groups. Tapping-Mode/Phase Imaging. Simultaneous phase41 and tapping-mode topographical imaging can minimize sample damage because an oscillating tip intermittently in contact with the surface greatly reduces the lateral stresses imposed. In fact, we saw no indication of tipinduced surface damage under our in situ tapping-mode imaging conditions. Phase images are essentially distribution maps of the measured changes in phase lag of the oscillating cantilever. A variety of factors have been found to influence phase contrasts, including material hardness,42 scanning parameters,43 and more importantly surface chemical composition44,45 which could provide some indication as to the chemical functionality of both monolayers and bilayers. A number of optimal scanning parameters46,47 proposed to make phase images more sensitive to chemical interactions between tip and surface were incorporated in this study. Tapping-mode images of cholesterol monohydrate plates collected under aqueous solution occasionally show areas adjacent to bilayer edges that have heights indicative of monolayers. However, we note that these stable monolayer regions are generally only detectable in AFM images smaller than ∼0.5 µm wide. Typical monolayer regions such as those in Figure 4 are 12-22 Å deep (predicted monolayer height ) 17 Å) and approximately ∼35 nm wide, or about 100 molecules. The corresponding phase images show contrast that suggests a different functionality than that on the predominant bilayer surface, though we cannot exclude the possibility that the contrast is due to differences in the mechanical properties of bilayers and monolayers. While this method remains promising, the relatively small and poorly defined dimensions of the monolayers have so far limited a more detailed structural analysis of these image features. Direct Adhesion Force Measurements. The most successful means to assess surface functionality proved to be direct measurement of tip-sample adhesion forces by force curve analysis. Since there is predominantly a single functionality present on the surface, the types of tip-sample interactions present would be either “matched” (hydrophilic-hydrophilic or hydrophobic-hydrophobic) or “mismatched” (hydrophilic-hydrophobic). The chemical character of the homogeneous surface layer can be then deduced by comparing adhesion forces measured with both hydrophilic and hydrophobic tips. If, under a given set of solution conditions, the crystal surface is hydroxylterminated (002), then greater adhesion forces should be (41) Babcock, K. L.; Prater, C. B. Digital Instruments Application Notes; Digital Instruments (www.di.com). (42) Magonov, S. N.; Elings, V.; Whangbo, M.-H. Surf. Sci. 1997, 375, L385-L391. (43) Bar, G.; Thomann, Y.; Brandsch, R.; Cantow, H.-J.; Whangbo, M.-H. Langmuir 1997, 13, 3807-3812. (44) Viswanathan, R.; Tian, J.; Marr, D. W. M. Langmuir 1997, 13, 1840-1843. (45) Noy, A.; Sanders, C. H.; Vezenov, D. V.; Wong, S. S.; Lieber, C. M. Langmuir 1998, 14, 1508-1511. (46) Brandsch, R.; Bar, G.; Whangbo, M.-H. Langmuir 1997, 13, 6349-6353. (47) Chen, X.; Davies, M. C.; Roberts, C. J.; Tendler, S. J. B.; Williams, P. M.; Burnham, N. A. Surf. Sci. 2000, 460, 292-300.
Langmuir, Vol. 18, No. 12, 2002 4851
Figure 4. Tapping-mode topographical image (top) (z-scale ) 40 nm) and corresponding phase image (bottom) (z-scale ) 10°) indicating exposed monolayers.
obtained with a hydrophilic tip. Similarly, methylterminated (001) crystal surfaces should show a greater adhesion force with hydrophobic tips. Adhesion force values typically exhibit significant scatter due to variations in the area between contacting surfaces, so a large sampling of ∼140 unique force curves was collected for each tip-solvent combination. The calculated adhesion forces are tabulated as histograms with the mean and standard deviation determining the normal distribution curve. Most adhesion values fall underneath the distribution curve, with a few outlying values present, particularly in measurements performed in aqueous conditions. A small number (∼5%) of these force curves were omitted due to extremely high adhesion values (>12 nN). Such force curves display the effects of strong layer forces,30,48 probably due to a significant cholesterol contamination of the tip. After the tip was rinsed with ethanol, this behavior promptly disappeared. Histograms of the measured adhesion forces obtained with HOOC- and CH3- terminated tips under aqueous solution conditions are shown in Figure 5. The average adhesion force obtained with hydrophilic tips (4.4 ( 1.9 nN) is about double that observed with hydrophobic tips (2.0 ( 0.9 nN). This is strong evidence that the hydrophilic (002) surfaces of cholesterol monohydrate are favored in (48) Burnham, N. A.; Colton, R. J.; Pollock, H. M. Nanotechnology 1993, 4, 64-80.
4852
Langmuir, Vol. 18, No. 12, 2002
Figure 5. Frequency distribution of adhesion forces measured in aqueous solution. Measurements were made with COOHterminated tips (top) and CH3-terminated tips (bottom).
Figure 6. Frequency distribution of adhesion forces measured in “organic” ethylene glycol solution. Measurements were made with COOH-terminated tips (top) and CH3-terminated tips (bottom).
an aqueous environment. Adhesion forces were also measured with hydroxyl-coated tips. While adhesion forces show the same general trend, the difference in mean adhesion forces between hydroxyl and hydrophobic tips was much smaller. Adhesion force measurements obtained under an ethylene glycol environment showed the opposite trend (Figure 6). Larger adhesion forces were obtained with hydrophobic tips (2.1 ( 1.1 nN) compared to hydrophilic tips (0.41 ( 0.42 nN). The higher adhesion force with the
Abendan and Swift
hydrophobic tips indicates the crystal surface is also hydrophobic (001). Measured step heights on the surface still correspond well with predicted bilayers, an observation that argues against a gross surface reconstruction. The most likely mechanism by which a (002) surface converts to a (001) surface is presumably by dissolution of (2n + 1) monolayers. With hydrophilic tips, about 30% of the force curves obtained in ethylene glycol showed adhesion values less than the detection limit of ∼0.08 nN. For the purpose of calculations and curve fitting, such curves were assigned conservative adhesion force values of 0.08 nN, so the actual difference in adhesion forces measured in ethylene glycol may be slightly larger than our reported value. The general shift to lower adhesion forces for all tips in organic solvents as compared to water is consistent with previous adhesion reports.17,28 Lower adhesion in organic media is typically attributed to the fact that organic solvents tend to reflect “bare” chemical interactions with minimal solvation effects, while stronger hydrophobic and potential electrostatic forces can be present in water. Contact Angle Measurements. Contact angles have been used extensively as a means to characterize the hydrophobicity of a surface. Though measurements are typically done in air, we rationalized that contact angles might still provide supplementary evidence for the chemical functionality presented at different crystalsolution interfaces. Freshly grown crystals were thoroughly rinsed with distilled water to expose surfaces that are in equilibrium with “aqueous” solution conditions and/ or immersed in and removed from methanol solution for surfaces in equilibrium with a more “hydrophobic” environment. Rapid immersion in other organic solvents (e.g., ethers, hexanes, and acetates) was attempted but the cholesterol monohydrate crystals dissolved, cracked, and/ or dehydrated too quickly, thereby challenging the integrity of the bulk material. Crystals dipped in methanol did not show any obvious signs of damage. All crystals remained translucent throughout the duration of the measurement. Surface assignments made from CFM adhesion force experiments are supported by contact angle measurements. The plate faces of crystals taken from aqueous solutions display a significantly smaller equilibrium contact angle (θ ) 87° ( 3°) than those dipped in methanol (θ ) 99° ( 3°). Though samples must be extremely flat for meaningful data and all crystal surfaces are imperfect, higher contact angles generally indicate an increase in surface hydrophobicity. The full 12° difference observed on cholesterol monohydrate crystals that differ only in their solvent exposure is a good indication that solution environment can readily alter terminal surface groups. Alternatively, we considered the possibility that the hydrophobic character might be due to an increase in the step density, since the step “rises” expose the cholesterol backbone and are largely hydrophobic. In ethylene glycol solution, AFM topographical images revealed that the surface area of the cholesterol monohydrate plate face covered by step rises is only a small fraction of the total area, similar to those images collected in aqueous solution. There is currently no standard method for measuring contact angles or predicting absolute values as a function of surface composition, but comparisons to contact angles obtained on surfaces with similar functionality can be drawn. The contact angle of the methanol-exposed cholesterol monohydrate surface (θ ) 99° ( 3°) falls nicely between the reported advancing (θ ) 107°) and receding (θ ) 93°) contact angles obtained on self-assembled monolayers of thiocholesterol.49 This is arguably the most
CFM of Cholesterol Monohydrate Single Crystals
similar comparison available. The contact angle of the aqueous-exposed crystal is substantially lower than the one exposed to methanol, but the distribution of contact angles reported for hydroxyl-terminated surfaces in the literature is very broad. Very hydrophilic surfaces such as those of carboxylic-terminated self-assembled monolayers can have typical contact angles as low as 60-65°.50 Conclusion Atomic force microscopy has demonstrated that the molecular surface structure of the cholesterol monohydrate crystal plate face is predominantly terminated with bilayer steps under aqueous and ethylene glycol solutions. Monolayer regions under these solution conditions are rare and are therefore assumed to be less stable. Such features are typically only observed under in situ tapping-mode imaging conditions. Adhesion force experiments with chemically modified AFM tips indicate that the cholesterol monohydrate plate face is terminated with 3-hydroxyl groups (002) in aqueous environments, while the surface becomes predominantly terminated with alkyl groups (49) Yang, Z. P.; Engquist, I.; Kauffmann, J.-M.; Liedberg, B. Langmuir 1996, 12, 1704-1707. (50) Drelich, J.; Wilbur, J. L.; Miller, J. D.; Whitesides, G. M. Langmuir 1996, 12, 1913-1922.
Langmuir, Vol. 18, No. 12, 2002 4853
(001) under anhydrous ethylene glycol solution. This study demonstrates that stable bilayer crystal surfaces, which present either hydrophobic or hydrophilic functionalities, can be achieved in response to solution changes at the crystal-solution interface. Contact angle measurements taken on the plate face of crystals exposed to different solution environments indirectly support the conclusions drawn from chemical force microscopy experiments. Surfaces exposed to methanol have contact angles 12° higher than analogous surfaces from aqueous solution. This macroscopic adhesion supports our molecular-level AFM data. Knowledge of the structural and chemical characteristics of cholesterol monohydrate surfaces in different solution environments marks an important step for studies into the pathogenic crystallization events of cholesterol. Acknowledgment. We are grateful for the financial support provided by the National Science Foundation (DMR-0093069), the ACS Petroleum Research Fund (36457-G5), and the Henry Luce Foundation. We additionally thank Michael Ward (University of Minnesota), Mak Paranjape (Georgetown), and Paul Goldey (Georgetown) for their kind assistance with gold-coating AFM tips. LA025649R
