Dissolution on Cholesterol Monohydrate Single-Crystal Surfaces Monitored by in Situ Atomic Force Microscopy† Richard S. Abendan and Jennifer A. Swift* Department of Chemistry, Georgetown University, 37th and O Streets NW, Washington, D.C. 20057-1227 Received May 27, 2005;
CRYSTAL GROWTH & DESIGN 2005 VOL. 5, NO. 6 2146-2153
Revised Manuscript Received June 27, 2005
ABSTRACT: The mechanism(s) and dissolution rates on the (001) surface of cholesterol monohydrate (ChM) in aqueous solutions containing 10-50% ethanol were monitored using in situ atomic force microscopy (AFM). Dissolution was found to proceed mechanistically via the layer-by-layer retreat of 34 Å bilayer or multilayer steps and/or the creation and expansion of bilayer and multilayer etch pits. In general, the dissolution rate is very strongly dependent on the local surface topography, which is highly variable on (001) ChM. Since the overall dissolution on the surface is by necessity a function of the relative density of different types of surface features, dissolution on both typical “smooth” and “rough” micron-sized areas was examined. For areas exhibiting low topographical relief, the rate of solute loss from isolated surface features was monitored by tracing the position of particular step fronts in sequential images over time. Although the absolute local dissolution rates are variable, the loss of solute molecules from isolated bilayer islands and/or pits occurred on the order of ∼105 molecules/min. The critical interstep distance for nondissolving features (observed in multiple experiments) appeared to decrease with increasing ethanol concentration, suggesting that surface diffusion effects likely influence this process. Dissolution in regions exhibiting much greater topographical relief tended to occur most readily by the rapid expansion of multilayer pits rather than by bilayer step retreat. In such areas, the alternative method of roughness analysis provided a more reliable means to track surface changes over time. The relatively high frequency with which such pit features were observed presumably speaks to the significantly large defect density in conventionally grown ChM crystals. Introduction The formation of cholesterol monohydrate (ChM) crystals is an important step in the pathogenesis of human gallstones, since this material can compose up to 80% of a gallstone by mass.1 The dissolution of such crystals within the gallbladder is very limited due to cholesterol’s low solubility in water2,3 and the supersaturated lipid environment maintained in bile solutions.4 While cholesterol transport in vivo is typically mediated through micelles and vesicles formed by bile components, gallstone dissolution therapies have found limited success, and surgery is still the most effective and common approach to alleviate gallstone attacks. Although metastable and/or anhydrous cholesterol forms have been implicated in the early nucleation and growth stages in bile,5,6 ChM is the predominant crystalline form observed in gallstones and late-stage atherosclerotic plaques.1,7,8 It adopts a characteristic platelike morphology and has a bilayer crystal structure (P1; a ) 12.39, b ) 12.41, c ) 34.36, R ) 91.9°, β ) 98.1°, γ ) 100.8°)9 with the polar 3-hydroxyl ends forming hydrogen bonds with each other and with water molecules, resulting in a slightly puckered two-dimensional hydrogen-bonded network. Bilayers measuring 33.9 Å in height are stacked along the crystallographic c axis (Figure 1). The largest plate face of the macroscopic crystal is the c face or (001), with smaller side faces a combination of low-index (100), (010), (101), and (011) surfaces.10-12 * To whom correspondence should be addressed. E-mail: jas2@ georgetown.edu. † Dedicated to Professor J. Michael McBride on the occasion of his 65th birthday.
In previous atomic force microscopy (AFM) work,13 we demonstrated that the ChM plate face is predominantly terminated with bilayer steps under both static aqueous and ethylene glycol solution environments. The bilayer surface termination implies that a single chemical functionality is presented on the surface under a particular set of solution conditions. Chemical force microscopy experiments revealed that the hydroxylterminated (002) face predominated in aqueous environments; however, alkyl tail groups (001) terminated the crystal surface in ethylene glycol solutions. The topography of crystal surfaces undergoing dissolution must obviously change over time, yet little is know about this process mechanistically on the molecular level. Bulk dissolution studies on ChM have traditionally been pursued using methods that measure total sample solubility in various organic solvents.14-16 Solubility values are obtained through analyzing cholesterol concentrations in supernatant solutions in which excess ChM crystals have reached equilibrium under constant agitation. Using such a methodology, solubility is the average of the intrinsic solubilities of all the crystal faces, with dissolution kinetics affected by factors such as crystal size and morphology. The orientation of cholesterol molecules on each crystal surface is unique, such that the molecular-level dissolution activity of each type of crystal face can vary significantly. The detachment of molecules from different crystal surfaces may therefore be expected to proceed with different rates and/or mechanisms. In situ AFM studies on molecular-level surface dissolution processes have been previously reported for a number of different minerals17-21 and protein crystals.22-25 A comparatively smaller number of dis-
10.1021/cg050236k CCC: $30.25 © 2005 American Chemical Society Published on Web 09/07/2005
Dissolution on ChM Single-Crystal Surfaces
Crystal Growth & Design, Vol. 5, No. 6, 2005 2147
Figure 2. Schematic of flow system used with the AFM fluid cell. Solution is flowed from a custom-built fluid reservoir (A) through a ∼30 µL glass AFM fluid cell (B) mounted above a temperature-controlled stage (C). A syringe pump (D) regulates solution flow.
Figure 1. Crystal structure of cholesterol monohydrate (ChM) constructed from fractional coordinates provided in ref 9. Dark spheres represent oxygen atoms from C-3 hydroxyl and water (top). Schematic of the ChM crystal morphology (bottom).
solution studies have been pursued on organic molecular crystal surfaces, including those of amino acids,26,27 aspirin,28 and 1,2-diphenyl ethanes.29 To our knowledge, no previous molecular-level dissolution studies have been reported for cholesterol or any of its structural analogues. The focus of the present study is to determine the real-time molecular-level dissolution mechanism of ChM (001) in binary ethanol-water solutions. The bulk solubility of cholesterol is higher in ethanol (∼30 mg/ mL)15 than in water (0.002 mg/mL)2, so increasing ethanol concentrations can be used as a means to systematically drive dissolution. An understanding of the dissolution units, rates, and the roles of defect sites on ChM should help contribute in developing insight into the molecular-level crystal surface properties and the mechanisms by which ChM dissolution might occur in vivo. Experimental Section Sample Preparation. Platelike ChM crystals with large (001) faces were grown from slow evaporation of saturated cholesterol (Aldrich, 99+%) solutions prepared in 95% ethanol
(Aldrich, 99.5%) and 5% water by volume. Crystals formed after ∼14 days exhibited dimensions ranging from a few hundred microns up to 4 mm long. The solvent content in crystalline samples was determined by thermal gravimetric analysis using a Q600 TGA (TA Instruments, New Castle, DE) and NMR spectroscopy (300 MHz Varian, Palo Alto, CA). In Situ Atomic Force Microscopy. Single crystals with well defined and visually flat (001) plate faces were removed from the mother liquor and immediately mounted with epoxy on an AFM sample puck with the (001) face up. Crystals were rinsed with distilled water to remove any particulates and imaged as soon as possible, usually within 3 h. All images were obtained with a Nanoscope IIIa Atomic Force microscope (Digital Instruments, Santa Barbara, CA) equipped with a 30 µL glass fluid cell. The flow of solutions was facilitated with use of a custom-built fluid reservoir from which solution is drawn by a syringe pump (Kd Scientific, Holliston, MA) through the fluid cell (Figure 2). Temperature was maintained at 35-37 °C at both the reservoir and the temperature stage beneath the AFM sample puck. Narrow, 115-µm-long triangular-shaped silicon nitride cantilevers (Digital Instruments, Santa Barbara, CA) with ∼9 kHz resonant frequencies in solution were used. To minimize any potential surface damage caused by the scanning AFM tip, tapping mode imaging was used in all experiments. Larger area images were routinely collected at the conclusion of each experiment to check for tip-induced artifacts. Under the minimal tip-sample forces applied, no obvious surface deformation was observed. Observable dissolution was brought about by exchanging water in the fluid cell with solutions of greater solubilizing power. The etching solutions used consisted of binary mixtures of ethanol and water. Solutions with a higher fraction of ethanol bring about greater dissolution activity on ChM surfaces. The fastest measurable surface change within the scanning capabilities of the AFM was determined to be 30:70 v/v ethanol/water solutions. Other etching solutions evaluated included mixtures of water with methanol (Fisher, 99.9%), 1-propanol (Fisher, standard grade), 2-propanol (Fisher, HPLC grade), and acetone (EM Science, 99.5%). For particular surface features to be effectively tracked in sequential images during dissolution, optimal scan rates of 3-4 Hz were used. Higher scan rates resulted in a deterioration of image quality, while slower scan rates prohibited tracking of individual surface features in sequential images. All in situ AFM experiments first began by imaging in pure aqueous solution. After a period of about 5 min, the etching solution is flowed quickly (10 mL/h) into the fluid cell so as to fully replace the water in the crystal environment. Imaging continues throughout the solution exchange, although it was
2148
Crystal Growth & Design, Vol. 5, No. 6, 2005
Abendan and Swift
smallest surface steps measure ∼34 (( 5) Å in height, corresponding to bilayers.13 Virtually no monolayer steps (∼17 Å) are observed. Hillocks of multiple cascading bilayer steps or multilayer steps with height values corresponding to multiple bilayers can also be found. Some large (∼10 µm2) flat regions exist in which only a few different bilayer steps are exposed (see Figure 3, top). Such areas also typically have a minimal number of stacked bilayers, with typical lateral distances between individual steps greater than 2 µm. In contrast, some areas are marked with numerous hillocks and multilayer steps with height values typically greater than that of 10 bilayers (see Figure 3, bottom). This local surface type is accentuated with extreme height variation over a small (
