Deposition and Aggregation of Aspirin Molecules on a Phospholipid

Dec 22, 2004 - The bilayer stripe pattern exposes the cross section of the lipid bilayer lamellae and enables the direct visualization of the molecula...
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Langmuir 2005, 21, 578-585

Deposition and Aggregation of Aspirin Molecules on a Phospholipid Bilayer Pattern Guangzhao Mao,* Dongzhong Chen,† and Hitesh Handa Department of Chemical Engineering and Materials Science, Wayne State University, 5050 Anthony Wayne Drive, Detroit, Michigan 48202

Wenfei Dong, Dirk G. Kurth,‡ and Helmuth Mo¨hwald Max Planck Institute of Colloids and Interfaces, Research Campus Golm, 14424 Potsdam, Germany Received September 2, 2004. In Final Form: November 2, 2004 Aspirin and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE) are deposited from their alcoholic mixed solution onto highly oriented pyrolytic graphite (HOPG) by spin coating. The film structure and morphology are characterized by atomic force microscopy (AFM). The barely soluble DMPE forms a highly oriented stripe phase as a result of its one-dimensional epitaxy with the HOPG lattice. The bilayer stripe pattern exposes the cross section of the lipid bilayer lamellae and enables the direct visualization of the molecular interactions of drug or biological molecules with either the hydrophobic or the hydrophilic part of the phospholipid bilayer. The bilayer pattern affects the aspirin molecular deposition and aggregation. AFM shows that the aspirin molecules prefer to deposit and aggregate along the aliphatic interior part of the bilayer pattern, giving rise to parallel dimer rods in registry with the underlying pattern. The nonpolar interactions between aspirin and the phospholipid bilayer are consistent with the lipophilic nature of aspirin. The bilayer pattern not only stabilizes the rodlike aggregate structure of aspirin at low aspirin concentration but also inhibits crystallization of aspirin at high aspirin concentration. Molecular models show that the width of the DMPE aliphatic chain interior can accommodate no more than two aspirin dimers. The bilayer confinement may prevent aspirin from reaching its critical nucleus size. This study illustrates a general method to induce a metastable or amorphous form of an active pharmaceutical ingredient (API) by chemical confinement under high undercooling conditions. Metastable and amorphous solids often display better solubility and bioavailability than the stable crystalline form of the API.

Introduction The ability to manipulate and characterize materials at the nanoscale has led to explosive research activities in the molecular thin films, crystals, and devices. In supramolecular pharmaceutics, the same active pharmaceutical ingredient (API) molecules are manipulated by various noncovalent interactions (hydrogen bonding, van der Waals, π-π stacking, and electrostatic interactions) into different solid-state forms ranging from amorphous to crystalline states.1 The solid-state form of the API affects its compressibility, solubility, dissolution rate, chemical stability, and bioavailability. Some new drug discovery methods involve screening different crystallization conditions in small volume2 and on engineered surfaces.3 Micropatterns of self-assembled monolayers have been used to control crystallization by relying on guest molecules to recognize different surface functional groups and by microconfinement.4 The integration of bottom-up to top-down approaches in the developing technologies, such as the high-throughput screening of * Corresponding author. E-mail: [email protected]. † Current address: Key Laboratory of Mesoscopic Chemistry of MOE and Department of Polymer Science & Engineering, College of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, People’s Republic of China. ‡ National Institute for Materials Science (NIMS), 1-1 Namiki, Tsukuba, Ibaraki 305-0044, Japan. (1) Rodrı´guez-Spong, B.; Price, C. P.; Jayasankar, A.; Matzger, A. J.; Rodrı´guez-Hornedo, N. Adv. Drug Delivery Rev. 2004, 56, 241. (2) Perepezko, J. H. Mater. Sci. Eng., A 1994, 178, 105. (3) Ward, M. D. Chem. Rev. 2001, 101, 1697. (4) Aizenberg, J.; Black, A. J.; Whitesides, G. M. Nature 1999, 398, 495.

drugs, requires the knowledge of placing molecules on ever diminishing patterns. Alkanes and alkane derivatives are known to self-assemble into a long-range ordered stripe phase on highly oriented pyrolytic graphite (HOPG) as a result of the onedimensional (1-D) epitaxial match between the 1,3-methylene group distance ()0.251 nm) and the distance of the next nearest neighbor of the HOPG lattice ()0.246 nm).5,6 Recently, this molecular pattern has been used to align small organic molecules7,8 as well as macromolecules.9-12 The amphiphilic pattern at the solid and liquid interface has been reproduced in thin solid films by spin coating and has been used to synthesize sulfide molecular rod arrays on the copper arachidate template.13 Both the arachidate pattern and the subsequent inorganic rod arrays have been characterized by atomic force microscopy (AFM). AFM is capable of resolving the location and shape of guest molecular aggregates in relation to a specific type (5) McGonigal, G. C.; Bernhardt, R. H.; Thomson, D. J. Appl. Phys. Lett. 1990, 57, 28. (6) Rabe, J. P.; Buchholz, F. Science 1991, 253, 424. (7) Wei, Y.; Kannappan, K.; Flynn, G. W.; Zimmt, M. B. J. Am. Chem. Soc. 2004, 126, 5318. (8) Hoeppener, S.; Chi, L.; Fuchs, H. ChemPhysChem 2003, 4, 494. (9) Mena-Osteritz, E.; Meyer, A.; Langeveld-Voss, B. M. W.; Janssen, R. A. J.; Meijer, E. W.; Ba¨uerle, P. Angew. Chem., Int. Ed. 2000, 39, 2679. (10) Kurth, D. G.; Severin, N.; Rabe, J. P. Angew. Chem., Int. Ed. 2002, 114, 3681. (11) Loi, S.; Butt, H.-J.; Hampel, C.; Bauer, R.; Wiesler, U.-M.; Mu¨llen, K. Langmuir 2002, 18, 2398. (12) Severin, N.; Rabe, J. P.; Kurth, D. G. J. Am. Chem. Soc. 2004, 126, 3696. (13) Mao, G.; Dong, W.; Kurth, D. G.; Mo¨hwald, H. Nano Lett. 2004, 4, 249.

10.1021/la047802i CCC: $30.25 © 2005 American Chemical Society Published on Web 12/22/2004

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of functional group on the host pattern. The amphiphilic pattern of phospholipids serves as a model for the study of the molecular interactions of drug or biological compounds with biomembranes and cells. The phospholipid stripe phase is structurally similar to the plane perpendicular to the bilayer lamellae. The bilayer pattern exposes the hydrophobic and hydrophilic parts simultaneously for guest molecular recognition and also for AFM imaging. This enables detailed structural analysis of drug or biological molecular interactions with specific functional groups of the phospholipid bilayer. This paper reports the effect of the phospholipid bilayer pattern on the deposition and aggregation behavior of aspirin. Aspirin, also called acetylsalicylic acid or 2-(acetyloxy)-benzoic acid, was first synthesized by Bayer in 1897. Aspirin is a nonsteroidal anti-inflammatory drug (NSAID), widely used to treat human inflammatory disorders, such as blood coagulation, thrombosis, and atherosclerosis, by inhibiting platelet aggregation.14 Recently, aspirin has also been found to reduce the risk of heart attack and to be effective against colorectal cancer. The primary mechanism of aspirin drug action is its interference with the biosynthesis of inflammatory prostaglandins.15 Another known effect of NSAIDs is their capability to perturb the phospholipid ordering in the biomembranes, thus, affecting the normal functions of the membrane proteins.16 Aspirin is a weak acid and lipophilic.17 Aspirin is found to increase the fluidity of liposomes by inserting itself in between the hydrocarbon chains of the phospholipid molecules. Aspirin has only one crystal form,18 which makes its structural analysis less complicated. The aspirin crystal consists of hydrogen-bonded dimers. The hydrogenbonded dimers are the most common supramolecular synthon for monocarboxylic acid crystals.19 The supramolecular synthon is the smallest molecular building block, whose symmetry and connectivity predispose the symmetry and packing in the final crystal structure. This paper presents a first study on the adsorption of supramolecular aggregates in a predefined way on the graphite-based templates. The study of the aggregation behavior of the aspirin dimers may shed light on nanoscale confinement means to engineer crystals with self-replicating building blocks. Experimental Section Materials. Aspirin (Aldrich, +99.5%), 1,2-dimyristoyl-snglycero-3-phosphoethanolamine (DMPE; Sigma, 99%), ethanol (Pharmco, 100%), and methanol (Mallinckrodt Chemicals, 100%) are used as received. ZYB grade HOPG (Mikromasch) is handcleaved with an adhesive tape just before film preparation until a smooth surface is obtained. Film Preparation. Aspirin and DMPE are dissolved in methanol or ethanol in different molar ratios. The solubility of aspirin in alcohol is 1.11 M,17 while DMPE dissolves sparingly and slowly in alcohol. Approximately 10-5 M DMPE is dissolved after shaking the solution for 10 min. The DMPE concentration is maintained at 10-5 M, while the molar ratio of aspirin to DMPE is varied from 1 to 5000. A total of 100 µL of a freshly prepared solution, filtered with a 0.22-µm poly(tetrafluoroethylene) filter (Millipore), is placed on a HOPG substrate rotating at 3000 rpm (PM101DT-R485 photo resist spinner, Headway Research) at room temperature for 1 min. (14) Lasslo, A. Blood Platelet Function and Medicinal Chemistry; Elsevier: New York, 1984. (15) Vane, J. R. Nat. New Biol. 1971, 231, 232. (16) Das, M. M., Dutta, S. K., Eds. Modern Concepts on Pharmacology and Therapeutics, 24th ed.; Hilton & Co.: Calcutta, 1989; p 26. (17) Budavari, S., O’Neil, M. J., Smith, A., Heckelman, P. E., Kinneary, J. F., Eds. The Merck Index: an Encyclopedia of Chemicals, Drugs, and Biologicals; Merck & Co., Inc.: Rathway, NJ, 1996; p 144. (18) Wheatley, P. J. J. Chem. Soc. 1964 (Suppl.), 6039. (19) Frankenbach, G. M.; Etter, M. C. Chem. Mater. 1992, 4, 272.

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Figure 1. Optical micrograph of aspirin recrystallized from ethanol by slow solvent evaporation. Characterization. The optical images are captured by an Olympus BX60 microscope with a SONY DXC-970MD camera in transmitted light. AFM images are obtained with either an E scanner (Nanoscope III, VEECO) or a Dimension Scan Head (Dimension 3100, VEECO). Height, amplitude, and phase images are obtained in tapping mode in ambient air with silicon tips (TESP, VEECO). Only height images are shown here unless specified. The scan rate is 1 Hz. Integral and proportional gains are approximately 0.4 and 0.7, respectively. The crystal structures and structural analysis are made with the Materials Studio software programs from Accelrys.

Results and Discussion Aspirin Crystals Recrystallized in Alcohol. Aspirin recrystallized from methanol and ethanol by slow solvent evaporation forms rectangular plates as shown in Figure 1. The largest aspirin crystal face from alcohol is the (001) face, followed by two other low-energy faces (100) and (011).20 Aspirin recrystallized from hexane forms needles. Aspirin crystals with the needle habit dissolve faster in water than the crystals with the plate habit.21,22 Therefore, commercial aspirin tablets contain a significant amount of needle crystals. Spin-Coated DMPE Films. Spin coating of the 10-5 M DMPE methanol solution produces the stripe phase on HOPG with close to a monolayer coverage. Figure 2a-c displays the spin-coated film structures as captured by AFM. Similar film structures are obtained also from ethanol. The stripe phase domain is rectangular in shape with an average domain size of 200 nm. The domain size is consistent with the single crystalline domain size of ZYB grade HOPG. Two sides of the rectangle are terminated clearly at the stripe edges, while the other sides are less obvious because of higher boundary energy. The domain orientation displays the threefold symmetry of the HOPG lattice, as expected from the 1-D epitaxy (Figure 2a). The domain height is 0.7 nm by the sectional height analysis (Figure 2b). At full monolayer coverage, the film appears to be identical to HOPG at the micrometer scale, showing the typical folded layer ledges of HOPG. The AFM tip is capable of sweeping the DMPE molecules away when scanning at a force ) 100 nN repeatedly in contact mode. The AFM tip digs a square trench with the same size of the previous scans as a result. Sectional height (20) Masaki, N.; Machida, K.; Kado, H.; Yokoyama, K.; Tohda, T. Ultramicroscopy 1992, 42, 1148. (21) Kim, Y.; Machida, K.; Taga, T.; Osaki, K. Chem. Pharm. Bull. 1985, 33, 2641. (22) Yesook, K.; Matsumoto, M.; Machida, K. Chem. Pharm. Bull. 1985, 33, 4125.

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Figure 2. AFM images of DMPE films on HOPG spin coated from 10-5 M methanol solution. (a) A 600-nm scan showing submonolayer domains. The angle between domains as shown is 59.48°. (b) The cross-sectional height profile along the dotted line in part a. The domain height is 0.7 nm. (c) A 200-nm scan of the DMPE bilayer stripes. The periodicity is 5.2 nm as determined from the 2-D Fourier transform pattern in the inset. The arrow points to the direction of the interlinear spacing R.

analysis shows that the unperturbed film portion is 0.7 nm above the square trench. It is found that the tip does not damage the HOPG surface at the same force level. The measured thickness is much smaller than the DMPE chain length, which excludes the normal monolayer structure with vertically oriented hydrocarbon chains. The interchain spacing in triclinic alkane crystals is 0.42 and 0.48 nm in the direction perpendicular and parallel to the zigzag carbon chain plane, respectively.23 The film thickness of 0.7 nm is consistent with the DMPE double chains lying parallel to the HOPG basal plane but in a tilted configuration to maximize the chain packing. The centerto-center distance between neighboring stripes is measured to be 5.2 nm by the two-dimensional (2-D) Fourier (23) Kitaigorodskij, A. I.; Mnjukh, J. B. Bull. Acad. Sci. U.S.S.R., Div. Chem. Sci. 1959, 1992.

transform analysis (Figure 2c inset). This periodicity, of which 3.3 nm belongs to the hydrophobic tails,24 agrees well with the DMPE bilayer thickness as determined by X-ray scattering.25 Energetically the lipid bilayer should be continuous across its hydrophobic interior in a tailto-tail configuration with a small separation existing between the opposing headgroups from neighboring bilayers. The same energy consideration also requires the stripe phase terminating at the hydrophilic headgroups. Figure 2c shows that the domain edge matches the edge and not the center of a bright stripe. It is evident that the center of the bright stripe should correspond to the (24) The hydrophobic layer thickness, LH, in the DMPE bilayer can be calculated as follows: LH ) 2 × [12 + (9/8)] × 0.1265 nm ) 3.32 nm. (25) Helm, C. A.; Tippmann-Krayer, P.; Mo¨hwald, H.; Als-Nielsen, J.; Kjaer, K. Biophys. J. 1991, 60, 1457.

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Figure 3. AFM images of aspirin films on HOPG. (a) A 500-nm scan showing disordered molecular aggregates spin coated from 10-3 M methanol solution. (b) The cross-sectional height profile along the line in part a. The aggregate height is 0.5 nm. (c) A 5-µm scan showing crystalline layers spin coated from 10-2 M ethanol solution. The arrow points to a top crystalline layer with a step edge angle close to 90°. Some edges display a 60° angle as marked. (d) The cross-sectional height profile along the dotted line in part c. The minimum step height of the aspirin crystal is 1.1 nm.

hydrophobic center of the lipid bilayer, while the dark gap should correspond to the hydrophilic headgroup region. Spin-Coated Aspirin Films. No aspirin molecules are observed on HOPG when the aspirin concentration is below 10-3 M. At 10-3 M, the HOPG surface is sparsely covered by the molecular aggregates presumably belonging to aspirin. The aggregates are irregular in shape and do not bind strongly to HOPG (Figure 3a). A minimum force in tapping mode can still sweep the aggregates away. The average height of the aggregates is 0.5 nm (Figure 3b). Above 2 × 10-3 M, localized crystalline layers of aspirin of at least 10 nm in thickness appear on HOPG. Figure 3c shows the aspirin film spin coated at 10-2 M in ethanol. The layers have orthogonal boundaries with a minimum differential thickness of 1.1 nm (Figure 3d). This is consistent with the texture orientation of the aspirin crystal (001) face. Some 60° angles also exist, which indicates likely azimuthal correlation between the aspirin (001) face and the HOPG basal plane. Spin-Coated Aspirin and DMPE Films. The molar ratio of aspirin to DMPE is varied from 1 to 5000 with the DMPE concentration fixed at 10-5 M in methanol or ethanol. When the aspirin-to-DMPE molar ratio is less

than 5, only the DMPE stripe phase is observed on HOPG. On the other hand, when the aspirin-to-DMPE molar ratio is greater than 50, the film morphology is largely that of the aspirin crystalline layers. When the molar ratio is between 5 and 50, the film shows both the molecular aggregates of aspirin on top of the DMPE stripe domains and the platelike aspirin crystals on bare HOPG areas between the DMPE domains, as illustrated by the scheme in Figure 4a. The spin-coated films from binary solutions generally show phase separation. If the two components are similar to each other in solubility and surface affinity, lateral phase separation occurs. A significant difference in solubility or surface affinity between the two components results in vertical phase separation and the formation of a wetting layer.26-28 This AFM study reveals a largely vertical phase separation between aspirin and DMPE, with DMPE forming the wetting layer. This is due to the large difference in alcohol solubility between (26) Jones, R. A. L.; Norton, L. J.; Kramer, E. J.; Bates, F. S.; Wiltzius, P. Phys. Rev. Lett. 1991, 66, 1326. (27) Steiner, U.; Klein, J.; Eiser, E.; Budkowski, A.; Fetters, L. J. Science 1992, 258, 1126. (28) Walheim, S.; Bo¨ltau, M.; Mlynek, J.; Krausch, G.; Steiner, U. Macromolecules 1997, 30, 4995.

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Figure 4. AFM images of aspirin and DMPE mixed films on HOPG with an aspirin-to-DMPE molar ratio ) 10. (a) Schematic graph of topographical features observed in the mixed film of aspirin and DMPE. (b) An 800-nm scan. (c) A 300-nm scan. (d) A 150-nm scan. The arrows point out that the aspirin molecular aggregates sit directly on top of the DMPE stripes, not in between. (e) The cross-sectional height profile along the line in part c. The height difference between the top and the bottom layer is 0.7 nm.

aspirin and DMPE. For example, when the DMPE concentration is 10-5 M and the aspirin-to-DMPE molar ratio is 50, the solution is saturated with DMPE but highly undersaturated with aspirin (aspirin concentration/ solubility ) 0.000 45). DMPE precipitates upon the slight solvent evaporation. Its epitaxial interaction with HOPG warrants the formation of the wetting monolayer as well as its self-organization into the stripe pattern. Aspirin precipitates only when its solubility limit is reached with further solvent evaporation, either on the DMPE layer or, if the surface is not fully covered, on HOPG. AFM images show that aspirin molecules recognize the amphiphilic pattern inherent in the DMPE stripe phase. Aspirin

molecules adsorb only on the hydrophobic interior of the DMPE bilayer stripe while avoiding the hydrophilic boundaries. This molecular recognition should be due to the strong lipophilicity of aspirin. The bilayer pattern should promote the 1-D aggregation of aspirin dimers, while limiting the 2-D and three-dimensional crystallization. Inhibition of the growth of the (001) face is desirable because this face is less water-soluble than the (100) face. Here the nonpolar interactions are sufficient to stabilize an otherwise highly unstable molecular rod structure. Meta-stable and amorphous forms of APIs are often more desirable than the stable crystalline form because they tend to show higher bioavailability.

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Figure 4b-d shows the film made from a mixed solution with the aspirin-to-DMPE molar ratio of 10, as imaged at 800-nm, 300-nm, and 150-nm scan sizes, respectively. The individual domains display the threefold azimuthal orientation as defined by the stripe edges. The domain has a rough appearance, which is different from the parallel stripe pattern of the DMPE layer. A closer look (Figure 4c,d) shows an additional layer partially covering the DMPE stripes. The second layer is 0.7 nm above the first stripe layer (the stripe layer thickness is still 0.7 nm) as measured in Figure 4e. Pure DMPE spin coated at 10-5 M, on the other hand, forms a submonolayer structure. Presumably the bottom layer belongs to the DMPE wetting layer and the second layer is made of aspirin aggregates. Figure 4d shows that the height undulation of the rodlike aspirin aggregates is in phase with that of the underlying DMPE stripes. This means that the aspirin aggregates are situated directly on top of the hydrophobic center of the DMPE bilayer stripe. This is consistent with the lipophilic nature of aspirin, which interacts through nonpolar interactions with the hydrophobic interior of biomembranes and the hydrophobic pockets of the protein receptors. When the aspirin-to-DMPE molar ratio is increased to 20, elongated particles with ill-defined shapes are distributed randomly but evenly on the surface, as shown in Figure 5a. The orientation of these particles is analyzed by measuring the orientation angles of those particles whose long axes are clearly defined. The orientation histogram is presented in Figure 5b. The mutual orientation angle of the majority of the particles maintains the threefold symmetry of the HOPG basal plane. Figure 5c,e shows two characteristic features at higher magnifications. Figure 5c shows that aspirin forms a continuous layer on the DMPE stripe phase. The layer is 0.7 nm above the DMPE stripes (Figure 5d). The long sides of the aspirin layer are parallel to the DMPE stripe and, thus, better defined. The other sides display no clear boundaries against the DMPE background. Figure 5e shows rectangular aspirin crystals located between the DMPE stripe domains. The height of the crystals, 1.1 nm, is one unit length along the [001] axis. It is clear that the DMPE bilayer pattern inhibits the crystallization of aspirin by stabilizing the rodlike molecular aggregates. Structural Analysis and Modeling. It is possible to understand the AFM images further by studying the molecular packing structures of aspirin and DMPE in the solid state. The structural analysis provides a likely model for the aspirin molecular aggregation on the DMPE bilayer pattern. The DMPE crystal structure is unavailable, and, therefore, the crystal structure of 1,2-dilauroyl-sn-glycero3-phosphoethanolamine acetic acid (DLPE) is used instead. DLPE has the same molecular structure as DMPE with the exception that it is shorter by two carbons in each aliphatic chain. DLPE forms a monoclinic crystal structure P21/c with the following unit cell parameters: a ) 47.700 Å, b ) 7.770 Å, c ) 9.950 Å, and β ) 92.000°.29,30 One reaches a hydrophobic layer thickness of 32.920 Å from the crystal structure similar to the value by X-ray scattering25 by adding an ethylene unit ()4.992 Å) to each of the DLPE chains. The hydrophilic headgroup thickness of the DMPE bilayer is 14 Å according to the molecular model. The periodicity in the AFM images is slightly larger than the expected bilayer thickness of DMPE, which is (29) Elder, M.; Hitchcock, P.; Mason, R.; Shipley, G. G. Proc. R. Soc. London, Ser. A 1977, 354, 157. (30) CCDC LAPETM10 contains the supplementary crystallographic data for DLPE. These data can be obtained free of charge via www.ccdc. cam.ac.uk/data_request/cif, by e-mailing [email protected], or by contacting The Cambridge Crystallographic Data Centre, 12, Union Road, Cambridge CB2 1EZ, U.K.; fax, +44 1223 336033.

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also its crystal unit cell length along the [100] direction. The hydrocarbon chains in the DLPE crystal lie along the [100] axis perpendicular to the bilayer plane. The height of the DMPE layer measured by AFM matches the unit cell length along the [010] direction, about one and a half the hydrocarbon chain diameter. The plane containing the two hydrocarbon chains of a DMPE molecule should be tilted against the HOPG basal plane for maximum packing. The zwitterionic phosphoethanolammonium headgroup dipoles are parallel to the bilayer surface and along the [010] axis in the DLPE crystal. The ammonium groups form short bonds (bond length ) 2.7 Å) with neighboring phosphate groups with partial hydrogen, and the rest have ionic bonding character.31 This rigid bonding structure among headgroups may enhance the epitaxial ordering of DMPE on HOPG. It can be concluded that DMPE exhibits an azimuthal orientation on the HOPG basal plane with the hydrocarbon chains along the interhexagonal direction of the HOPG lattice. Aspirin recrystallizes from ethanol into a monoclinic crystal structure P21/c with the following unit cell parameters: a ) 11.430 Å, b ) 6.591 Å, c ) 11.395 Å, and β ) 95.68°.18,32 Aspirin forms the inversion-symmetric, hydrogen-bonded carboxylic acid dimer as illustrated in Figure 6a. The (001) and (100) faces are displayed in Figure 6b as viewed along the [010] direction. The (001) face contains the methyl and phenyl groups, while the (100) face contains the ester groups. Aspirin dimers most likely prefer to face the DMPE bilayer with the (001) face to maximize the nonpolar-nonpolar interactions. A previous AFM study by Danesh et al. shows that the methylterminated AFM tip interacts more favorably with the more hydrophobic (001) face, while the carboxyl-terminated AFM tip is attracted more to the (100) face.33 Aspirin dimers are free to aggregate through π-π stacking along the [010] axis along the DMPE stripe direction; however, the aggregation in the direction perpendicular to the stripe is inhibited by the hydrophilic bilayer boundaries. The DMPE hydrocarbon region is 3.3 nm wide, which can fit up to two unit cells along the aspirin [100] axis. Aspirin aggregates may not be able to reach the required critical nucleus size for crystallization to occur on the DMPE pattern. Therefore, aspirin forms only molecular aggregates on DMPE at the same concentration where crystals are observed on the bare HOPG substrate. The AFM-determined aggregate height, 0.7 nm, less than the unit cell length along [001], also indicates that crystallization has not occurred. The height is close to the size of aspirin, 7.433 Å, as measured lengthwise from the methyl to the phenyl group from its crystal structure. Figure 6c displays the possible model of the aspirin aggregation on the DLPE bilayer, which is consistent with the AFM study. Conclusions This paper describes the molecular aggregation behavior of aspirin molecules on the bilayer stripe pattern of DMPE. The pattern is formed spontaneously by spin coating the DMPE alcoholic solution on HOPG. When aspirin is added to the DMPE solution, it deposits either on top of the DMPE stripe pattern or on the uncovered HOPG surface during spin coating. Aspirin molecules do not deposit randomly but prefer to aggregate along the hydrophobic center of (31) Pascher, I.; Lundmark, M.; Nyholm, P.-G.; Sundell, S. Biochim. Biophys. Acta 1992, 1113, 339. (32) CCDC ACSALA01 contains the supplementary crystallographic data for aspirin. (33) Danesh, A.; Davies, M. C.; Hinder, S. J.; Roberts, C. J.; Tendler, S. J. B.; Williams, P. M.; Wilkins, M. J. Anal. Chem. 2000, 72, 3419.

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Figure 5. AFM images of aspirin and DMPE mixed films on HOPG with an aspirin-to-DMPE molar ratio ) 20. (a) A 2-µm scan showing elongated but otherwise ill-defined aspirin aggregates. (b) Particle orientation analysis of the previous image. (c) A 300-nm scan showing an aspirin layer on top of the DMPE stripes. (d) The cross-sectional height analysis along the line in part c. The aspirin layer height is 0.7 nm. (e) A 250-nm scan showing rectangular aspirin crystals deposited on HOPG between the DMPE stripe domains. The phase image is shown here because the corresponding height image does not show the aspirin crystal shape as clearly.

the DMPE bilayer. It shows that the aspirin molecules can bind to a specific region of the lipid bilayer through

the nonpolar van der Waals interactions. While the bilayer pattern promotes the aspirin dimer aggregation via π-π

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Figure 6. Aspirin structural analysis by the Materials Studio software program. (a) The basic building block of the aspirin crystal, the aspirin dimer. The hydrogen bonds are marked by the dotted lines. (b) The functional groups on the (100) face and (001) face as viewed along the [010] direction. (c) A structural model of aspirin rodlike molecular aggregates on the DLPE bilayer pattern on HOPG. The HOPG surface is not drawn to avoid crowding. Only one dimer row is drawn for every bilayer stripe even though the stripe width can accommodate up to two dimer rows. The DLPE layer is created by cleaving the crystal along its (010) face. A vacuum slab is created above the DLPE supercell. Nine aspirin dimers along the [010] axis are selected from the aspirin crystal structure and are docked above the DLPE supercell. A vacuum slab with the DLPE (010) face at the bottom enables the docking of aspirin dimers within the vacuum above so that the dimer does not see a periodic image of the surface above it. The dimer row is arranged to parallel the [001] direction of the DLPE layer.

stacking along the length of the stripe, the narrow width of the stripe inhibits the crystallization of aspirin, probably by limiting the association among aspirin dimers in the width direction of the bilayer stripe. The experimental approach described here may offer a simple strategy to study the effect of surfactant mesophases and liposomes on the crystal engineering and encapsulation of the APIs. It is especially relevant to the solid-state preparation methods that require a small volume and high undercooling. One may utilize the various noncovalent interac-

tions between the API molecule and the surfactant or polymer additive molecule to control the degree of crystallinity and even the solid form to achieve different aggregate structures from the same API molecule. Acknowledgment. This work is partially supported by the National Science Foundation (CTS-0221586 and CTS-0216109). G.M. acknowledges the financial support from the German-American Fulbright Commission. LA047802I