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Biomimetic Gallstone Formation: Crystallization of Calcium Carbonate by the Evolving Taurocholate-Lecithin-Cholesterol Complex Lipid System Tu Lee*,†,‡ and Jheng Guo Chen†

CRYSTAL GROWTH & DESIGN 2009 VOL. 9, NO. 8 3737–3748

Department of Chemical & Materials Engineering, Institute of Materials Science & Engineering, National Central UniVersity, 300 Jhong-Da Road, Jhong-Li City 320, Taiwan, R.O.C. ReceiVed April 20, 2009

ABSTRACT: The aim of our research reported in this paper is to mimic and understand gallstone formation. The precipitation of calcium carbonate by reacting CaCl2 with NaHCO3 in model bile containing 68.6 mol % of taurocholate, 22.9 mol % of lecithin, and 8.5 mol % of cholesterol with a total lipid (taurocholate + lecithin + cholesterol) concentration of 29.2 g/dL was followed by a calcium ion meter. The 36-h old biomimetic stones collected were analyzed for cholesterol content (CC) by thermal gravimetric analysis, for vaterite-to-calcite ratio (Rv-c) by powder X-ray diffraction, and for morphology by scanning electron microscopy. The stability of model bile was also disturbed with seeds of anhydrous cholesterol, cholesterol monohydrate, lysozyme, sodium taurocholate, and CaCO3 crystals. We found that lecithin itself was capable of inducing the formation of vaterite or slowing down the vateriteto-calcite transformation probably through its positively charged choline moiety to produce high Ca2+ ion concentrations near the vesicular surface and to stabilize the negatively charged [001j] plane of vaterite nanocrystals. The mixture of vaterite and calcite nanocrystals was then aggregated and aligned under the direction of the crystal surface adsorbed lecithin molecules to form mesocrystals. Disturbing the stability of the microscopic structure of the complex lipid system could in principle release the vesicular adsorbed Ca2+ ions and the solubilized cholesterol so that calcium carbonate and cholesterol microcrystals could be precipitated out even when the cholesterol saturation index (CSI) < 1. We proposed that this was an autocatalytic cycle because the newly precipitated calcium carbonate and cholesterol microcrystals could then serve as seeds to further worsen the stability of the complex lipid system. Besides the microcrystals of calcium carbonate and cholesterol, other biliary components such as Ca2+ and HCO3- ions, bile salts, and proteins were also capable of destabilizing the complex lipid system. Introduction The formation of calcium carbonate crystals is prevalent in a wide range of biological systems1-4 such as planktons, gastropods, shells, otoconias, corals, and eggshells. Not only have these carbonate biominerals contributed significantly to the definition of continental morphology and the sequestration of CO2 throughout geological time,1 they have also given superb lessons in crystal engineering through the mediation of organic matrices.3,5-14 But surprisingly, to the best of our knowledge to date, those underlying molecular mechanisms of biological control of calcium carbonate crystallization15 have not been realized in gallstone formation despite the pathological importance of calcium carbonate growth. Gallstone is a composite material of organic and inorganic components. Much of the attention to gallstone formation has still been paid to the prerequisites of the other organic components and complex fluid structures in gallbladder bile, including cholesterol and its solubilization,16-32 bile salt micellar aggregation,33-36 phospholipid vesiculation,37-41 cholesterol-phospholipid vesiculation,42-45 bile salt-phospholipid mixed micelle formation,46-52 and bile salt-cholesterol-phospholipid mixed vesiculation.53,54 Yet, there are a few papers addressing the contributions23,55 of calcium ions and calcium carbonate phase on cholesterol nucleation,25,56 calcium carbonate-cholesterol interactions,57,58 calcium-phospholipid bindings,59 and calcium-bile salts interactions.60-62 Although calcium carbonate is known to have three crystalline polymorphs,63 vaterite, aragonite, and calcite, the common type * Corresponding author. Phone: +886-3-422-7151ext. 34204. Fax: +886-3425-2296. E-mail: [email protected]. † Department of Chemical & Materials Engineering. ‡ Institute of Materials Science & Engineering.

of cholesterol-rich, nonpigmented gallstones55,64-67 contain two polymorphs of calcium carbonate: spheroliths of the thermodynamically unstable vaterite and stable calcite, mixed with about 70 wt % of broken sheets of cholesterol monohydrate68 microcrystals. These findings raise a number of interesting questions. For instance, what causes the stabilization of vaterite in the gallstone during lithogenesis?63 What controls the crystallization of calcium carbonate? How is the nucleation event of cholesterol linked to the nucleation, polymorphic phase selection, and morphology of calcium carbonate microcrystals in gallbladder bile? What is the role of biliary proteins in the crystallization of calcium carbonate?66 The aim of our research reported in this paper is to answer these questions by mimicking the gallstone formation. Calcium carbonate was crystallized under the mediation of the wellcharacterized model bile,54 which was a complex system containing 68.6 mol % of taurocholate (Figure 1a), 22.9 mol % of lecithin (Figure 1b), and 8.5 mol % of cholesterol (Figure 1c) with the total lipid (taurocholate + lecithin + cholesterol) concentration of 29.2 g/dL at 22 °C. This particular combination pinpointed by the blue arrow along with the other reported physiological and simulated biliary combinations20,24-26,44,53,54,64,66,69 summarized as red dots is shown in Figure 2. The chosen model bile underwent a microscopic structural transformation via Pathway C25 during cholesterol precipitation: small unilamellar vesicles f multilamellar vesicles f lamellar liquid crystals (aggregated and fused) f cholesterol monohydrate crystals (plate-like) f cholesterol anhydrous crystals (arc, helical, and tubular) in 10 days.25,30,54 The cholesterol precipitation normally began when the degree of supersaturation of cholesterol (cholesterol saturation index (CSI))20 rose above 1.66 In this particular model bile system,

10.1021/cg900440p CCC: $40.75  2009 American Chemical Society Published on Web 06/11/2009

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Figure 2. Equilibrium phase diagram of the taurocholatelecithin-cholesterol system. The composition of the model bile (blue arrow) was 68.6 mol % of taurocholate, 22.9 mol % of lecithin, and 8.5 mol % of cholesterol with a total lipid concentration of 7.3 g/dL in 0.15 M NaCl at pH 7.0 and 22 °C. The system was placed in Region C of the central three-phase zone. The three phases at equilibrium were saturated micelles, liquid crystals, and cholesterol monohydrate crystals. Other reported physiological and simulated biliary combinations were also labeled (red dots).

bile was closely followed by polarizing optical microscopy (POM) and dynamic light scattering (DLS) at different time points, whereas the crystallization kinetics of calcium carbonate was continuously monitored by a calcium ion meter.70 The polymorphism of calcium carbonate, the interfacial interactions between lipids and growing calcium carbonate crystals, the composition, and the morphology of the biomimetic stones were analyzed by powder X-ray diffraction (PXRD), thermal gravimetric analysis (TGA), and scanning electron microscopy (SEM), respectively. Materials and Methods

Figure 1. Molecular structures of (a) sodium taurocholate hydrate, (b) lecithin (L-R-phosphatidylcholine), and (c) cholesterol.

the CSI was increased from 0.97 to 1.21 upon a 4-fold dilution to 7.3 g/dL.54 The microscopic structural evolution of the model

Chemicals. Lecithin (L-R-phosphatidylcholine, C42H82NO8P, from dried egg yolk, type X-E g 60% (TLC), mp > 200 °C, MW ) 768, lot 047K0736 and 128H8003), cholesterol (C27H46O, g 99%, mp ) 148 °C, MW ) 386.66, lot 045K5311), sodium azide (NaN3, g 99.5%, mp ) 275 °C, FW ) 65.01, lot 057K0153), lysozyme (from chicken egg white, g 99%, mp ) 60 °C, MW ) 14.7 kDa, lot 015K6144), and Tris buffered saline 10× (0.22 µm filtered, working solution contains 20 mM Tris, pH ∼7.4, 0.9% NaCl, bp ) 103° to 107 °C, MW ) 121.41, lot 107K6077) were used as received from Sigma-Aldrich (St. Louis, MO). Sodium taurocholate monohydrate (as determined by thermal gravimetric analysis identification test) (taurocholate or bile salt, C26H44NaO7S · H2O, g 97% (TLC), mp ) 230 °C, MW ) 555.7, lot 1384482) was obtained from Fluka Analytical (Milan, Italy). It was dehydrated in a vacuum oven at 100 °C for one day before use (MW ) 537.7). Sodium chloride (NaCl, g 99.5%, mp ) 801 °C, FW ) 58.44, lot SP-2642 V) and sodium hydrogen carbonate (NaHCO3, g 99%, decompose ∼60 °C, FW ) 110.98, lot SR-3330P) were purchased from Showa Chemical, Co. Ltd. (Tokyo, Japan). Calcium chloride (CaCl2, g 99%, mp >1600 °C, FW ) 110.98, lot E51638) was obtained from Mallinckrodt Baker, Inc. (Tokyo, Japan). Solvents. Chloroform (CHCl3, g 99.99%, bp ) 61 °C, MW ) 119.38, lot E554180) and methanol (CH3OH, g 99.9%, bp ) 64 °C, MW ) 32.04, lot 411070) were received from Echo Chemical Co. Ltd. (Taipei, Taiwan) and TEDIA Company (Fairfield, USA), respectively. Reversible osmosis (RO) water was clarified by a water purification system (model: Milli-RO Plus) bought from Millipore (Billerica, MA) and was boiled afterward to remove dissolved CO2. Instrumentations. Calcium Ion Meter. The calcium ion concentration in the model bile solution was monitored by a hand-held calcium

Biomimetic Gallstone Formation ion/pH meter IM-22P (DKK-TOA Corporation, Tokyo, Japan). It was calibrated by 10 standard CaCl2 solutions of 0.001, 0.01, 0.4, 1, 2, 4, 6, 8, 10, and 12 mg/mL prepared in 0.15 M NaCl and 3 mM NaN3 at pH 7.0 at 22 °C. An aqueous KCl solution (Code No. 143A333, DKKTOA corporation, Tokyo, Japan) used as an ionic strength adjuster for calcium ions was added to all calibrations and sample measurements with a volume ratio of 1-10. A calibration curve of conductivity (mV) ) 11.589 × ln(Ca2+ concentration (M)) + 44.13 with R2 ) 0.9971 was determined and employed to covert the measured conductivity into the meaningful calcium ion concentration. The conductivity was measured for every 30 s for the first hour and every 5 min for the rest of the 35-h course. All measurements were repeated twice to verify reproducibility. All calcium concentration vs time plots were based on the average values, and some of the average calcium concentration values and their standard deviations of specific time points were tabulated for discussion use. Dynamic Light Scattering (DLS). The micellar and vesicular size distribution of lipids in model bile at the vesicular stage was measured by Malvern Zetasizer Nano (Malvern, Worcestershire, UK) with a He-Ne laser source of 4 mW and 633 nm. The refractive index of 1.33 and viscosity of 0.8872 cP for water at 25 °C were used. About 2.5 mL of sample was introduced in a low volume glass cuvette. Polarizing Optical Microscopy (POM). Crystal habit and solution morphology were examined and imaged by a polarized optical microscope (BX51; Olympus, Tokyo, Japan) equipped with a digital camera (Moticam 2000 2.0 M Pixel USB2.0; Motic, Inc., Xiamen, China) to take images of samples. Data were visualized using Motic Images Plus 2.0 ML (Motic, Inc., Xiamen, China) at the time points of 6, 12, 24, and 36 h. Thermal Gravimetric Analysis (TGA). TGA analysis was carried out by TGA 7 (Perkin-Elmer, Norwalk, CT, USA) to monitor sample weight loss as a function of temperature. The heating rate was 10 °C/ min ranging from 50 to 700 °C. Weight loss was usually associated with solvent evaporation close to the boiling point of a solvent as in the case of solvates or sample decomposition. The open platinum pan and stirrup were washed by ethanol and burned by a spirit lamp to remove all impurities. All samples were heated under nitrogen atmosphere to avoid oxidization. About 3 mg of a solid sample were placed on an open platinum pan suspending in a heating furnace. Powder X-ray Diffraction (PXRD). PXRD patterns were obtained from samples using a wide-angle powder X-ray diffractometer (model D/Max-IIB, Rigaku Co., Tokyo, Japan). X-ray radiation CuK R1 (λ ) 1.5405 Å) was set at 30 kV and 20 mA passing through a nickel filter with divergence slit (0.5°), scattering slit (0.5°), and receiving slit (1 mm). Samples were subjected to X-ray powder diffraction analysis with a sampling width of 0.01° in a continuous mode with a scanning rate of 1°/min over an angular range of 20° to 60° 2θ. Transmission Fourier Transform Infrared (FTIR) Spectroscopy. Transmission FTIR spectroscopy was utilized to measure purity, detect bond formation, and verify chemical identity. Transmission FTIR spectra were recorded on a Perkin-Elmer Spectrum One spectrometer (Perkin-Elmer Instruments LLC, Shelton, CT, USA). The KBr sample disk was scanned with a scan number of 8 from 450 to 4000 cm-1 having a resolution of 2 cm-1. Scanning Electron Microscopy (SEM). A scanning electron microscope (SEM) (Hitachi S-3500N, Tokyo, Japan) was used to observe the morphology of the crystals. Both secondary electron imaging (SEI) and backscattered electron imaging (BEI) were used for the SEM detector and the magnification was 15 to 300 000-fold. The operating pressure was 10-5 Pa vacuum and the voltage was 15.0 kV. All samples were mounted on a carbon conductive tape (Prod. No. 16073, TED Pella Inc., California, USA) and then sputter-coated with gold (Hitachi E-1010 Ion Spotter, Tokyo, Japan) with a thickness of about 6 nm. The discharge current used was about 0 to 30 mA and the vacuum was around 10 Pa. Experiments. Procedure A: Preparation of Model Bile.54 Model micellar bile was prepared by coprecipitation in a 100 mL glass bottle of 1865.2 mg of taurocholate, 889.3 mg of lecithin, and 166.2 mg of cholesterol from 50 mL of CHCl3-MeOH (2:1 v/v) solution in a vacuum oven at 40 °C to achieve constant weight. Dried lipid film was then dissolved in a 10 mL aqueous solution of 0.15 M NaCl and 3 mM NaN3 adjusted with a small amount of 1 M NaOH to pH 7.0 to yield a complex lipid system containing 68.6 mol % of taurocholate, 22.9 mol % of lecithin, and 8.5 mol % of cholesterol with the total lipid (taurocholate + lecithin + cholesterol) concentration of 29.2 g/dL.

Crystal Growth & Design, Vol. 9, No. 8, 2009 3739 The model bile was sonicated at a full sonic power for 1 h at 50 °C by a PowerSonic UB410 water bath (Thermoline Scientific, Smithfield, Australia) to ensure complete solubilization of coprecipitated cholesterol in micelles. The model bile solution was then microfiltered through a 250 mL fritted glass funnel made by NDS, Technologies, Inc. (New Jersey, USA). Procedure B: Precipitation of Cholesterol.54 To induce supersaturation of cholesterol, the model bile was diluted 4-fold with the aqueous solution of 0.15 M NaCl and 3 mM NaN3 at pH 7.0 in the 100 mL glass bottle at 22 °C. Procedure C: Preparation of Calcium Carbonate Crystals.70 The 40 mL diluted model bile solution was divided into two halves in volume. 443.92 mg of CaCl2 were added into the first 20 mL and 336 mg of NaHCO3 were added into the second 20 mL of model bile solution to yield 20 mL of 0.2 M CaCl2 and 20 mL of 0.2 M NaHCO3 solutions at 22 °C. A small volume of Tris buffered saline was added to both 20 mL portions of solution to achieve a pH ∼7.5.23,61,71 CaCO3 crystals began to form when the two bottles were mixed and stirred at 750 rpm. The final concentrations of taurocholate, lecithin, and cholesterol in a 40 mL aqueous solution were 0.0839, 0.0289, and 0.0107 M, respectively. A small amount of Tris buffered saline was added to achieve a pH ∼7.5 and 4 mL of KCl ionic strength adjuster for Ca2+ ions were also introduced to all the conditions below: Condition A: Model Bile without Calcium Carbonate Precipitation. Details of Procedures A and B were followed. Condition B: Model Bile with Calcium Chloride Only. Details of Procedures A, B, and C were followed without any addition of NaHCO3 in Procedure C. There was no waiting period between Procedures B and C. Condition C: Calcium Carbonate Precipitation in Saline Solution (Control). A 40 mL aqueous solution of 0.15 M NaCl and 3 mM NaN3 at pH 7.0 in the 100 mL glass bottle at 22 °C was divided in halves. 443.92 mg of CaCl2 were added into the first 20 mL and 336 mg of NaHCO3 were added into the second 20 mL of saline solution to yield 20 mL of 0.2 M CaCl2 and 20 mL of 0.2 M NaHCO3 solutions at 22 °C. CaCO3 crystals began to form when the solutions from the two bottles were mixed and stirred at 750 rpm. Condition D: Calcium Carbonate Precipitation in the Presence of Lecithin. Details of Procedures A, B, and C were followed. However, Procedure A was modified by the addition of 889.3 mg of lecithin only. There was a waiting period of 12 h (vesiculation time) between Procedures B and C. Condition E: Calcium Carbonate Precipitation in Model Bile at the Micellar Stage. Details of Procedures A, B, and C were followed. There was a waiting period of 10 min (micellation time) between Procedures B and C. Condition F: Calcium Carbonate Precipitation in Model Bile at the Vesicular Stage. Details of Procedures A, B, and C were followed. There was a waiting period of 12 h (vesiculation time) between Procedures B and C. Condition G: Calcium Carbonate Precipitation in Model Bile at the Cholesterol Monohydrate Stage. Details of Procedures A, B, and C were followed. There was a waiting period of 10 days (cholesterol monohydrate formation time) between Procedures B and C. Condition H: Calcium Carbonate Precipitation in Sodium Taurocholate Solution. Details of Procedures A, B, and C were followed. However, Procedure A was modified by the addition of 1865.2 mg of sodium taurocholate only. There was no waiting period between Procedures B and C. Condition I: Calcium Carbonate Precipitation in Model Bile at the Vesicular Stage Seeded with Calcium Carbonate Crystals Produced in Condition F. Details of Procedures A, B, and C were followed. There was a waiting period of 12 h (vesiculation time) between Procedures B and C. Four milligrams of calcium carbonate crystals was added into the 20 mL CaCl2 solution in Procedure C as seeds. Condition J: Calcium Carbonate Precipitation in Model Bile at the Vesicular Stage Seeded with Anhydrous Cholesterol Crystals. Details of Procedures A, B, and C were followed. There was a waiting period of 12 h (vesiculation time) between Procedures B and C. Four milligrams of anhydrous cholesterol crystals were added into the 20 mL CaCl2 solution in Procedure C as seeds.

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Condition K: Calcium Carbonate Precipitation in Model Bile at the Vesicular Stage Seeded with Sodium Taurocholate Crystals. Details of Procedures A, B, and C were followed. There was a waiting period of 12 h (vesiculation time) between Procedures B and C. 36 mg of sodium taurocholate crystals were added into the 20 mL CaCl2 solution in Procedure C as seeds. Condition L: Calcium Carbonate Precipitation in Model Bile at the Vesicular Stage Seeded with Cholesterol Monohydrate Crystals. Details of Procedures A, B, and C were followed. There was a waiting period of 12 h (vesiculation time) between Procedures B and C. Four milligrams of cholesterol monohydrate crystals was added into the 20 mL CaCl2 solution in Procedure C as seeds. Condition M: Calcium Carbonate Precipitation in Model Bile at the Vesicular Stage Seeded with Lysozyme. Details of Procedures A, B, and C were followed. There was a waiting period of 12 h (vesiculation time) between Procedures B and C. Four milligrams of lysozyme was added into the 20 mL CaCl2 solution in Procedure C as seeds.

Results and Discussion Upon a 4-fold dilution, the CSI rose from 0.97 to 1.21, and the model bile became supersaturated (CSI > 1) with cholesterol. This would cause a disturbance to the equilibrium of our chosen taurocholate-lecithin-cholesterol complex system pinpointed by the blue arrow in Figure 2.54 According to Portincasa et al.30 and Gantz et al.,54 cholesterol originally carried in 40-80 Å mixed micelles containing taurocholate and lecithin became thermodynamically unstable and started to solubilize into 40-200 nm aggregated unilameller vesicles composed of lecithin and a small concentration of taurocholate. Further fusion of unilamellar vesicles resulted in 200-500 nm multilamellar vesicles (liquid crystals). Later, more cholesterol (cmc ) 30.08 × 10-9 M)42 started to precipitate out in hydrate and anhydrous forms giving solid crystals of different shapes, such as platelets, arcs, needles, filaments, spirals, helices, and tubules.30,54 Cholesterol monohydrate appeared more rapidly than anhydrous cholesterol because the taurocholate to lecithin ratio was 2.9 e 7.0.56 The whole microscopic structural evolution of the complex system mentioned above followed a so-called Pathway C.28,41 As cholesterol was being crystallized, taurocholate and lecithin in the sludge might begin to form taurocholate micellar aggregates, lecithin multilamellar vesicles, and taurocholatelecithin mixed micelles simultaneously as summarized by us schematically based on refs 30 and 54 (Figure 3). To ensure Pathway C was under our control, dynamic light scattering and optical microscopy were used to characterize model bile at the vesicular stage (12 h into Condition B) before and after the addition of CaCl2. The aggregated vesicles were observed in model bile after 12 h (Figure 4a). But upon the addition of CaCl2, platelets (possibly cholesterol monohydrate), arc-like and right-handed helical crystals (possibly anhydrous cholesterol) began to appear after 12 h (Figure 4b). Normally, cholesterol monohydrate did not form until 3-4 days. Dynamic light scattering data also demonstrated that the second size distribution peak shifted from 400 nm (Figure 4a) to 2000 nm due to vesicular fusion and crystallization of anhydrous cholesterol and cholesterol monohydrate after the addition of CaCl2 (Figure 5) (the addition of NaHCO3 also showed a similar result). The precipitation of calcium carbonate was achieved here by reacting calcium chloride with sodium bicarbonate in an aqueous phase with a supersaturated Ca2+ concentration of 0.1 M (Condition C). Although the bicarbonate ions, HCO3-, from sodium bicarbonate could either dissociate into carbonate ions, CO32-, or become carbonic acid, H2CO3, HCO3- ions were the dominant species (∼95 mol %)72 at a constant buffered pH of

Figure 3. The microscopic structural evolution of the taurocholatelecithin-cholesterol complex system. In unsaturated bile (i.e., CSI < 1) cholesterol was carried in (a) 40-80 Å mixed micelles containing taurocholate and lecithin. As the CSI rises above 1, cholesterol was solubilized in (b) 40-200 nm aggregated unilameller vesicles composed of lecithin and small concentration of taurocholate. Further fusion of unilamellar vesicles resulted in (c) 200-500 nm multilamellar vesicles (liquid crystals) with a “maltese cross” birefringent aspect under polarizing light microscopy. Micron-sized (d) plate-like (possibly cholesterol monohydrate) crystals and (possibly anhydrous cholesterol) crystals with the shapes of (e) arcs, needles, filaments, (f) spirals, helices, and (g) tubules were formed. As cholesterol was being crystallized out, taurocholate and lecithin in the model bile might begin to form (h) taurocholate micellar aggregates, (i) lecithin multilamellar vesicles, and (j) taurocholate-lecithin mixed micelles.

7.5. Therefore, the crystallization of calcium carbonate could be described by the reaction:70 + Ca2+(aq) + HCO3 (aq) f CaCO3(s) + H (aq)

(1)

The rate of precipitation (Figure 6a) declined steeply from ∼0.17 M due to the modes of dilution (from 0.2 to 0.1 M theoretically) and reaction upon mixing of Ca2+ and HCO3solutions. The precipitation then slowed down significantly toward the end of the precipitation as the concentration of Ca2+ ions in the mother liquor decreased. However, the 36-h concentration of Ca2+ ions, [Ca2+]36h, of ∼63 mM was still far away from the equilibrium concentration of 0.06 to 0.1 mM (Ksp25°C of vaterite and calcite ) 1.23 × 10-8 M2 and 3.31 × 10-9 M2, respectively11) due to the extremely small precipitation rate constants73 of 10-5 to 10-7 mM L-1 s-1 and the atmospheric uptake of CO2. On hydration of CO2, HCO3-, and H+ ions were generated and reacted with CO32- ions generated from the solubilization of CaCO3 to form additional HCO3- ions. This action caused a shift of the equilibrium of CaCO3 (s) ) Ca2+ (aq) + CO32- (aq) to the right.74 The 36 h old calcium carbonate crystals produced in the control experiment (Figure 6a) were calcite. This agreed with the usual fact that the polymorphs of crystalline calcium carbonate are normally 50 mol % vaterite and 50 mol % calcite at the instant of precipitation and all vaterite crystals would be transformed into calcite crystals irreversibly by Ostwald ripening after 200 to 370 min at 25 to 30 °C in an aqueous solution.75 But in the absence of water, the solid-solid transition of vaterite to calcite phase will never occur due to the high activation energy barrier11 of 252.8 ( 48.7 kJ mol-1. Interestingly, the concentration profiles of Ca2+ ions of calcium carbonate precipitation in the presence of lecithin (L)

Biomimetic Gallstone Formation

(Condition D) (Figure 6b), in sodium taurocholate solution (STC) (Condition H) (Figure 6c), in model bile at the micellar stage (Mi) (Condition E) (Figure 6d), in model bile at cholesterol monohydrate stage (ChM) (Condition G) (Figure 6e), and in model bile at the vesicular stage (Ve) (Condition F) (Figure 6f) exhibited different degrees of decrease in the concentration of Ca2+ ions, g, from 0.5 to 4.5 min and values of [Ca2+]36h. The parameters of, g and [Ca2+]36h, instead of the kinetics rate coefficient, k, or the rate expression, would be used to depict the concentration profiles because of the difficulty in decoupling the complicated total precipitation rate of calcium carbonate73 involving the surface area of the CaCO3 crystals, the modes of homogeneous nucleation (without seeds), crystal growth, heterogeneous nucleation on CaCO3, and anhydrous and monohydrate cholesterol microcrystals in sludge, adsorption, catalytic nucleation, and heterogeneous nucleation in the evolving complex lipid system. However, to quantify the synergistic events of adsorption, catalytic nucleation, or any heterogeneous nucleation occurring in the evolving complex lipid system, we defined a total interfacial activity ratio, R:

Crystal Growth & Design, Vol. 9, No. 8, 2009 3741

R)

[Ca2+]control - [Ca2+]systems 36h 36h systems [lipids]total

(2)

where [Ca2+]control36h and [Ca2+]systems36h were the 36 h concentrations of Ca2+ ions in the control and other systems, respectively, and [lipids]systemstotal was the total lipids concentration. [lipids]systemsTotal of the model bile was 7.3 g/dL ÷ [(0.686)(537.7) + (0.229)(768) + (0.085)(386.66)]g/mol × 1 dL/0.1 L × 1000 mmol/mol ) 126 mM. [lipids]systemstotal of lecithin solution was 0.8893 g ÷ 768 g/mol ÷ 0.04 L × 1000 mmol/mol ) 28.9 mM. [lipids]systemstotal of anhydrous sodium taurocholate solution was 1.8652 g ÷ 537.7 g/mol ÷ 0.04 L × 1000 mmol/mol ) 86.7 mM. Additionally, the 36 h old biomimetic stones produced from those systems were also characterized for their CC, in wt % by TGA (decomposition temperature around 250 °C) and for their relative vaterite-to-calcite ratio, Rv-c, by the PXRD peak intensities, I, at 25° 2θ and 29.5° 2θ, respectively.76 Typical PXRD patterns of calcium carbonate stones obtained from a model bile in a vesicular stage with the addition of lysozyme seeds, a model bile in a vesicular stage with the addition of

Figure 4. (a) Dynamic light scattering of unilamellar vesicles and multilamellar vesicular aggregates, and (b) optical micrographs of platelet crystals (possibly cholesterol monohydrate), arc-like crystals (possibly anhydrous cholesterol) and regular right-handed helical crystals, in a 12-h model bile before and after the addition of CaCl2, respectively (scale bars ) 20 µm).

Figure 5. Dynamic light scattering data of a 12-h model bile after the addition of CaCl2.

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Figure 6. Concentration profiles of Ca2+ ions as a function of time: (a) calcium carbonate precipitation in saline solution (control), (b) calcium carbonate precipitation in the presence of lecithin, (c) calcium carbonate precipitation in sodium taurocholate solution, (d) calcium carbonate precipitation in model bile at the micellar stage, (e) calcium carbonate precipitation in model bile at cholesterol monohydrate stage, and (f) calcium carbonate precipitation in model bile at the vesicular stage.

Figure 7. PXRD diffractograms of 36-h old CaCO3 crystals harvested from calcium carbonate precipitation (a) in a model bile in a vesicular stage with the addition of lysozyme seeds, (b) in a model bile in a vesicular stage with the addition of cholesterol monohydrate seeds, (c) in a system in the presence of lecithin, and (d) in a saline solution (control) (c: calcite, and v: vaterite).

cholesterol monohydrate seeds, a system with the presence of lecithin and a saline solution (control) were illustrated in Figure 7. Values of g, R, CC, and Rv-c for the systems of control, L,

Lee and Chen

STC, Mi, ChM, and Ve were summarized in Table 1 for comparisons. Six new important lessons were learned: Lesson 1. Lecithin micelles or vesicles in aqueous solution alone could induce the formation of vaterite or hinder the transformation of vaterite to calcite in an aqueous phase by slowing down the dissolution rate of preformed vaterite.75 This was reflected by a high Rv-c value of 0.20. A high charge density at the lecithin micellar or vesicular surface was believed to kinetically favor vaterite nuclei through stabilization of disordered clusters of excess charge. Interestingly, the very same phenomenon was also observed and verified in the egg yolk spherocrystal.4 At the same time, the high charge density also promoted a relatively high HCO3-:Ca2+ ratio in aqueous solution favorable for the formation of vaterite in the bulk.5 This pointed to the fact that the inclusion of proteins in CaCO3 crystal structure63 does not have to be a prerequisite for the formation of vaterite as many have suggested. Yabusaki et al. had already demonstrated that in methanolic solution, the Ca2+ ion and free molecular lecithin formed a 1:1 complex (i.e., R ) 1.0) as determined by proton magnetic resonance59 and the relative distance from the center of the Ca2+ ion to the various centers of the carbon atoms bearing the protons under consideration were C3 > N(CH3)3 > C2 > C1 as shown in Figure 1b.59 However, in our lecithin vesicular system, the total interfacial activity ratio, R, was 0.51 and not 1.0 (Table 1). The discrepancy implied that either the total interfacial activity was not purely due to adsorption mechanism or the binding of the Ca2+ ions was dependent on the density variation of the choline head groups of lecithin in micelles or vesicles. In contrast to the micrometersized calcium carbonate cubic crystals grown in a saline solution (Figure 8a), the obtained 36 h old biomimetic stones in the lecithin system composed of hundreds of primary nanosized small plates of calcite and vaterite (Figure 8b-d), and exhibited broader peak width (Figure 9c) than the micrometer-sized calcite crystals in the control at half-maximum at 29.5° 2θ (Figure 9d) which resembled the rugged and faceted look of vaterite mesocrystals grown in the presence of N-trimethylammonium (-N+(CH3)3) derivative of hydroxyethyl cellulose.8 This led us to believe that the positively charged, structurally similar choline moiety of lecithin could likewise stabilize the negatively charged [001j] plane of vaterite nanocrystals.8 Lecithin lowered the interfacial energy of vaterite nuclei from the supersaturation solution through binding the Ca2+ ions and preferentially adsorbed on vaterite nanocrystals relative to calcite nanocrystals to balance the relative loss in the crystallization energy between vaterite and calcite which stabilized the vaterite phase at early stages.77 At later crystallization stages, lecithin might act as cooperative agents to promote an oriented attachment of the many nanocrystals to form mesocrystals.78 No obvious shift of calcite ν3 IR absorption peak at 877 cm-1 was observed (Figure

Table 1. Degree of Decrease in Ca2+ Ions (between 0.5 to 4.5 min), Total Interfacial Activity Ratio (at 36th hour), Cholesterol Content (at 36th hour), and Vaterite/Calcite Intensity Ratio for CaCO3 Precipitation Systems (at 36th hour) of Control, in the Presence of Lecithin (L), in Sodium Taurocholate Solution (STC), in Model Bile at the Micellar Stage (Mi), in Model Bile at Cholesterol Monohydrate Stage (ChM), and in Model Bile at the Vesicular Stage (Ve) CaCO3 precipitation systems without seeds

degree of decrease in Ca2+ ions, g (mM/min)

total interfacial activity ratio, R

cholesterol content, CC (wt %)

vaterite/calcite intensity ratio, Rv-c ) I25°2θ/I29.5°2θ

control in the presence of lecithin (L) in sodium taurocholate solution (STC) in model bile at the micellar stage (Mi) in model bile at cholesterol monohydrate stage (ChM) in model bile at the vesicular stage (Ve)

8.09 ( 4.45 5.49 ( 1.14 3.81 ( 0.79 5.62 ( 1.94 3.90 ( 3.57

0 0.51 ( 0.13 0.47 ( 0.07 0.35 ( 0.05 0.39 ( 0.04

0 0 0 30.10 ( 0.21 29.81 ( 2.54

0.06 ( 0.003 0.20 ( 0.001 0.11 ( 0.01 0.03 ( 0.01 0.20 ( 0.04

5.93 ( 0.08

0.40 ( 0.03

31.33 ( 0.28

0.24 ( 0.01

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Figure 9. PXRD 29.5° 2θ calcite peak of 36 h old CaCO3 crystals harvested from calcium carbonate precipitation (a) in a model bile in a vesicular stage with the addition of lysozyme seeds, (b) in a model bile in a vesicular stage with the addition of cholesterol monohydrate seeds, (c) in a system with the presence of lecithin, and (d) in a saline solution (control).

Figure 10. FTIR spectra of 36 h old CaCO3 crystals harvested from calcium carbonate precipitation (a) in a saline solution (control), (b) in a model bile in a vesicular stage with the addition of cholesterol monohydrate seeds, (c) in a model bile in a vesicular stage with the addition of lysozyme seeds, and (d) in a system in the presence of lecithin.

Figure 8. Scanning electron micrograph of 36-h old CaCO3 crystals obtained from calcium carbonate precipitation (a) in a saline solution (control), (b-d) in a system in the presence of lecithin. Nanosized crystals are indicated by black arrows.

10).12 This implied that interactions between lecithin and growing calcite were not strong. Lesson 2. A model bile in a vesicular stage gave the highest Rv-c value of 0.24, whereas a model bile in a micellar stage with the same chemical compositions had a very low Rv-c value of only 0.03. This clearly indicated the key role of the microscopic structure of the complex lipid system. Different

patches of different local combinations of taurocholate, lecithin, and cholesterol made up the substructures of the micellar or vesicular entity. The surfaces of these intermixed colloids looked like a fluctuating mosaic; the balance and time scale of fluctuation were controlled by factors such as counterions and temperature.23 This kind of dynamic surface activity could influence the local surface charge distribution and chemical microenvironments which determined the degree of supersaturation of CaCO3 in the interfacial region and the magnitude and long-range periodicity of the nonspecific electrostatic interactions and stereochemical recognitions between the headgroup and the nucleating crystal.11 To verify the interplay between Ca2+ ions and the dynamic surface activity and to decouple that from the CaCO3 precipitation at the same time, we monitored the concentration of Ca2+ ions completely dissociated from CaCl2 in a model bile without any addition of NaHCO3 (Condition B). Figure 11 illustrated that the Ca2+ ion concentration plummeted from about 200 mM (0.2 M) to 30 mM after a 2-fold

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Lee and Chen

Figure 12. Scanning electron micrograph of CaCO3 crystals obtained from calcium carbonate precipitation in sodium taurocholate solution. Figure 11. Concentration profiles of Ca2+ ions in a model bile as function of time without any addition of NaHCO3 (Condition B). The original Ca2+ ion concentration was 200 mM (blue line) and the theoretical Ca2+ ion concentration of 100 mM (red line) after a 2-fold dilution.

dilution by mixing with 20 mL model bile. Since 30 mM was less than 100 mM, the discrepancy implied that 70 mM Ca2+ ions were adsorbed to the interface of micelles or vesicles in a model bile at the early stage of after-mixing. But as time went by, more and more Ca2+ ions were released from the evolving complex lipid system, most likely, due to the decrease in the adsorption area of micelles or vesicles per unit volume because of the fusion of vesicles upon the presence of Ca2+ ions and the precipitation of cholesterol monohydrate.23,54,56 Our speculation agreed with the optical micrographs in Figure 4b and dynamic light scattering data in Figure 5. Figure 11 showed that the Ca2+ ion concentration began to rise and went beyond 100 mM toward the later stage of after-mixing (Condition B). Concentrations higher than 100 mM suggested that some of the Ca2+ ions might be excluded from some newly formed complex lipid volumes such as the interior volume of the fused vesicles. Many times, the microenvironment near the vesicle surface was supersaturated with Ca2+ ions which could have become the primary factor in determining CaCO3 polymorphs.79 Lesson 3. Sodium taurocholate was not as good as lecithin in promoting the formation of vaterite with a moderate Rv-c value of only 0.11 although it had been suggested that bile salt micelles could act as a chelating system for Ca2+ ions80 and the negatively charged sulfonate (-SO3-) headgroup in general11,77 could stabilize the vaterite phase by lowering the interfacial energy of vaterite nuclei. Probably the inefficiency of sodium taurocholate was due to the dimerization of two facing antiparallel sodium taurocholate linked by head-to-tail hydrogen bonds together with Na+ · · · SO3- Coulombic interactions33,34 with a major size distribution of only 2 nm as shown by dynamic light scattering (not shown). This competing process would drastically reduce the number of available binding sites for Ca2+ ions. Therefore, CaCO3 grew through a fast vaterite-to-calcite transformation and crystallization into large rhombohedral crystal plates with calcite as the dominant phase (Figure 12). Lesson 4. Uniform round 36-h old biomimetic stones with a size distribution of 5-10 µm and a Rv-c value of 0.20 (Table 1) exhibiting well-defined terraced facets on the outer surfaces of the spheres were obtained (Figure 13) in a model bile in a cholesterol monohydrate stage. Vesicles for both systems in Lessons 1 and 4 became larger in size and smaller in number probably by the fusion, aggregation, or clustering of small

Figure 13. Scanning electron micrograph of CaCO3 crystals obtained from calcium carbonate precipitation in model bile at cholesterol monohydrate stage.

unilamellar vesicles upon the addition of Ca2+ ions23 at the very early stage of CaCO3 precipitation and monolayers of cholesterol were known to give calcite crystals.5 As cholesterol was gradually crystallized out at the later stage of CaCO3 precipitation, the complex lipid system would be filled with slowly evolved mixed micelles and vesicles of taurocholate and lecithin. From Lessons 1 and 3, we know that lecithin played a dominant role in controlling the morphology and polymorphism of CaCO3 crystals. In addition, the epitaxial growth of cholesterol monohydrate on calcite57 might have also hindered the growth rate of newly formed calcite which was the rate-determining step of the transformation of vaterite to calcite.75 Therefore, the slowly evolved complex lipid system provided a better control in CaCO3 nucleation, particle size distribution, and nanoaggregation than the pure lecithin system in Lesson 1. Lesson 5. The degree of decrease in Ca2+ ions for model bile at the vesicular stage of g ) 6.01 was higher than the one for model bile at the cholesterol monohydrate stage of g ) 3.76. This indicated that the precipitation of solubilized cholesterol in model bile could slow down the total precipitation rate of CaCO3 by affecting the microenvironments of the micellar or vesicular surfaces. But the similar respective cholesterol contents (CC) of 29.81 and 31.33 wt % implied that the fast precipitation of the CaCO3 due to the local high Ca2+ ion concentration near the micellar or vesicular surfaces could also speed up the precipitation rate of cholesterol in return due to the fusion and

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Crystal Growth & Design, Vol. 9, No. 8, 2009 3745

Figure 14. Concentration profiles of Ca2+ ions as a function of time with seeds: (a) calcium carbonate precipitation in model bile at the vesicular stage seeded with anhydrous cholesterol crystals, (b) calcium carbonate precipitation in model bile at the vesicular stage seeded with lysozyme, (c) calcium carbonate precipitation in model bile at the vesicular stage seeded with cholesterol monohydrate crystals, (d) calcium carbonate precipitation in model bile at the vesicular stage seeded with sodium taurocholate crystals, and (e) calcium carbonate precipitation in model bile at the vesicular stage seeded with calcium carbonate crystals produced in Figure 6f.

aggregation of vesicles.23,54,56 In other words, the crystallization of cholesterol could be induced by the instability of the complex lipid system. This might explain why patients without cholesterol crystals could have supersaturated bile (CSI ) 1.51 and 1.25) and patients with cholesterol crystals could have unsaturated biles (CSI < 1.0).43 Therefore, the old concept of a two-step formation of cholesterol gallstones64 first through exceeding of the capacity of bile to solubilize cholesterol and then the aggregation of microcrystals of cholesterol does not tell a complete story. Lesson 6. To further prove that the instability of the complex system could be interrupted by factors other than the precipitation of CaCO3 and the instability of the complex system was indeed related to the crystallization of cholesterol, we added seeds of CaCO3 (obtained from Condition F), anhydrous cholesterol, cholesterol monohydrate, sodium taurocholate, and lysozyme to the precipitation of CaCO3 in model bile at the vesicular stage. This could mimic cyclic growth of gallstone with periods of rapid growth interspersed with intervals of quiescence.63 The concentration profiles of Ca2+ ions were illustrated in Figure 14. The concentration of Ca2+ ions in model bile seeded with anhydrous cholesterol21 and lysozyme13,51 rose from 100 to 400 min and then dropped from 400 to 1200 min (Figure 14a,b). Obviously, the ready incorporation of anhydrous cholesterol and lysozyme destabilized the complex system and facilitated the process of micellar or vesicular fusion. This caused a sudden release of surface-adsorbed Ca2+ ions to the

Figure 15. Scanning electron micrograph of biomimetic CaCO3 stones (round in shape) and cholesterol monohydrate (plate-like crystals) obtained in model bile seeded with anhydrous cholesterol.

Figure 16. Schematic diagram for the proposed pathogenesis of gallstone formation.

solution phase in which the Ca2+ ions were consumed by the reaction with NaHCO3. A delayed rise in the Ca2+ ions happening at around 900 min (Figure 14c) was due to the relative difficulty of breaking three hydrogen bonds of cholesterol monohydrate to release one water molecule of hydration into the surrounding water before it could be inserted into the bilamellar leaflets of phospholipids to destabilize the complex system.21 However, abrupt rises in the concentration profiles of Ca2+ ions of model bile at the vesicular stage seed with sodium taurocholate and CaCO3 crystals were not observed (Figure 14d,e). The negatively charged patches on the globular lysozyme surface and the sulfonate headgroup of sodium taurocholate might have interacted more strongly with Ca2+ ions than anhydrous cholesterol, cholesterol monohydrate, and CaCO3 crystals. Therefore, the stronger adsorptions caused the respective degrees of decrease in Ca2+ ions, g ) 10.81 and 14.00

Table 2. The Degree of Decrease in Ca2+ Ions (between 0.5 to 4.5 min), Total Interfacial Activity Ratio (at 36th hour), Cholesterol Content (at 36th hour), and Vaterite/Calcite Intensity Ratio for CaCO3 Precipitation Systems (at 36th hour) Seeded with Anhydrous Cholesterol, Lysozyme, Cholesterol Monohydrate, Sodium Taurocholate and CaCO3 Crystals from Condition F CaCO3 precipitation systems in model bile at the vesicular stage

degree of decrease in Ca2+ ions, g (mM/min)

total interfacial activity ratio, R

cholesterol content, CC (wt %)

vaterite/calcite intensity ratio, Rv-c ) I25°2θ/I29.5°2θ

with anhydrous cholesterol seeds with lysozyme seeds with cholesterol monohydrate seeds with sodium taurocholate with CaCO3 (Rv-c ) 0.24) seeds

4.34 ( 0.17 10.81 ( 1.06 7.09 ( 6.05 14.00 ( 1.23 5.87 ( 0.53

-0.01 ( 0.13 -0.01 ( 0.18 -0.05 ( 0.58 0.02 ( 0.19 0.40 ( 0.04

31.04 ( 0.28 34.05 ( 1.37 34.97 ( 1.52 31.04 ( 1.37 22.36 ( 1.47

0.39 ( 0.02 1.12 ( 0.05 1.36 ( 0.1 0.39 ( 0.09 0.38 ( 0.04

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higher than the other g values (Table 2). The total interfacial activity ratios, R, of most cases were very close to zero (Table 2) because the released Ca2+ ions from adsorption were reacted with NaHCO3 to produce more CaCO3 so that the 36-h concentration of Ca2+ ions, [Ca2+]36h, of ∼63 mM of the control experiment was approached. The destabilization of the complex lipid system caused by seeds gave rise to the formation of more cholesterol monohydrate microcrystals as shown by the relatively high cholesterol content of 31 to 34 wt % (Table 2). The high vaterite/calcite intensity ratios, Rv-c, such as 1.12 and 1.36 were due to the occurrence of a second surge of high Ca2+ ion concentration originated from the irreversible destabilization of the complex system upon seeding (Table 2). Interestingly, the CC of 22.36 wt % for the model bile seeded with CaCO3 crystals was quite low. This pointed to the fact that the introduction of CaCO3 seeds would preferentially induce the nucleation of CaCO3 and not that of cholesterol. Therefore, CaCO3 crystals might not serve as a nidus for stone formation as many have suggested.51,63 All biomimetic CaCO3 stones obtained looked like the ones seeded with anhydrous cholesterol (Figure 15). They were round in shape having a size distribution of 1-2.5 µm mixed with cholesterol monohydrate plate-like crystals. Although lysozyme itself favored calcite formation over aragonite and vaterite,13 in the light of the lysozyme seeding experiment, any imbalance of proteins in human bile, such as66 immunoglobulin A and M, concanavalin (a lectin)-reactive proteins, a 42-kDa acidic glycoprotein, fibronectin, apolipoproteins A-I and A-II, concanavalin A-reactive proteins, and Helix pomatia (a lectin)-reactive proteins, occurred prior to the formation of biliary sludge or stone could have caused a destabilization of the microscopic structure of bile. The destabilization of the complex lipid system would then release the adsorbed Ca2+ ions and the solubilized cholesterol to the surrounding bile fluid with high bicarbonate concentration. If that happened, precipitation of CaCO3 and crystallization of cholesterol could be initiated at the same time. Conclusions Lecithin itself was capable of inducing the formation of vaterite or slowing down the vaterite-to-calcite transformation probably through its positively charged choline moiety to produce high Ca2+ ion concentrations near the vesicular surface and to stabilize the negatively charged [001j] plane of vaterite nanocrystals. However, the interactions between lecithin and growing calcium carbonate were not strong. The mixture of vaterite and calcite nanocrystals were then aggregated and might be aligned under the direction of the crystal surface adsorbed lecithin molecules to form mesocrystals. Disturbing the stability of the microscopic structure of the complex lipid system could in principle release the vesicular adsorbed Ca2+ ions and the solubilized cholesterol to induce the crystallization of calcium carbonate and cholesterol microcrystals even for CSI < 1. This turned out to be an autocatalytic cycle because the newly precipitated calcium carbonate and cholesterol microcrystals could serve as seeds to further worsen the stability of the complex lipid system. Besides the microcrystals of calcium carbonate and cholesterol, other biliary components such as Ca2+ ions, bile salts, and proteins were also capable of destabilizing the complex lipid system. The proposed pathogenesis of gallstone formation was illustrated in Figure 16. Acknowledgment. This work was supported by a grant from the National Science Council of Taiwan, Republic of China (NSC 97-2113-M-008-006). Assistance from Ms. Jui-Mei Huang

Lee and Chen

in DSC, Ms. Shew-Jen Weng in PXRD, and Ms. Ching-Tien Lin in SEM and DLS, and all with the Precision Instrument Center at National Central University are gratefully acknowledged.

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