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Crown Ether Functionalized Lipid Membranes: Lead Ion Recognition and Molecular Reorganization Darryl Y. Sasaki,*,† Tina A. Waggoner,† Julie A. Last,† and Todd M. Alam‡ Biomolecular Materials and Interface Science Department and Organic Materials Department, Sandia National Laboratories, Albuquerque, New Mexico 87185 Received November 6, 2001. In Final Form: January 14, 2002 A fluorescent lipid bilayer, functionalized with 18-crown-6, was developed to examine the mechanism of chemical recognition-induced molecular reorganization events in a membrane system. The synthetic receptor-lipid, PS18C6, was prepared with the crown ether at the headgroup position and a pyrene fluorescent tag on the hydrophobic tail. When incorporated into bilayers of distearylphosphatidylcholine, the receptorlipid aggregated into domains, evidenced by the relatively large pyrene excimer emission from the bilayer. Langmuir pressure-area (π-A) isotherm measurements and atomic force microscopy (AFM) further aided in characterizing the receptor aggregation at the macro- and nanoscale, respectively. The functionalized bilayer exhibited selective affinity for mercuric and lead ions in aqueous buffered solution (pH 7.4), with a fluorescence response that was linear over the concentration range 10-7 to 10-4 M metal ions. 1H NMR studies established that the binding stoichiometry of PS18C6 with lead was 1:1, with Ka ) 105 M-1 in methanol. Recognition and binding of lead ions at the membrane surface resulted in a rapid and prominent reorganization of the receptor-lipids in the membrane that was measurable at both the macro- and nanoscales. Removal of the lead ions, through the addition of EDTA, resulted in recovery of the original fluorescence and the reaggregation of structures in the membrane.
Introduction Chemical recognition events on lipid membranes are the initiating steps toward cellular signaling, which activate numerous processes including cell adhesion, endocytosis, division, and immune response.1 Particularly interesting are the recognition phenomena that occur with the immune system’s cells, where pathogenic agents must be rapidly and precisely distinguished from innocuous and “self” agents. T cells, for example, use a combination of receptor subunits (proteins and lipids) that organize into specific superstructures upon binding with targeted ligands, subsequently activating a signaling pathway, which is then amplified into a cellular response.2 The chemical recognition processes that occur on these membrane surfaces form the basis of the most versatile and specific sensor systems known. By harnessing even a fraction of the capability of cellular membrane recognition systems, it may be possible to build unique sensor systems that not only are rapid and specific in response but also are innately biocompatible. Furthermore, the idea of using chemical recognition to form specific structures in the membrane may be a potent tool to aid in the development of controllable nanoscale architecture. Recent efforts have focused on the development of artificial lipid membranes for sensor systems and nanostructured materials. Examples of sensors include ve* To whom correspondence should be addressed. Fax: (505) 8445470. E-mail:
[email protected]. † Biomolecular Materials and Interface Science Department. ‡ Organic Materials Department. (1) Alberts, B.; Bray, D.; Lewis, J.; Raff, M.; Roberts, K.; Watson, J. D. Molecular Biology of The Cell, 3rd ed.; Garland Publishing: New York, 1994. (2) For example, see: (a) Grakoui, A.; Bromley, S. K.; Sumen, C.; Davis, M. M.; Shaw, A. S.; Allen, P. M.; Dustin, M. L. Science 1999, 285, 221-227. (b) Boyington, J. C.; Motyka, S. A.; Schuck, P.; Brooks, A. G.; Sun, P. D. Nature 2000, 405, 537-543. (c) Lang, P.; Stolpa, J. C.; Freiberg, B. A.; Crawford, F.; Kappler, J.; Kupfer, A.; Cambier, J. C. Science 2001, 291, 1537-1540. (d) Eichmann, K. Angew. Chem., Int. Ed. Engl. 1993, 32, 54-63. (e) Ravetch, J. V.; Lanier, L. L. Science 2000, 290, 84-89.
sicular lipid membranes (liposomes) that can detect virus,3 toxin,4 and sugars5 with a fluorescence or colorimetric response. Electrodes and optical platforms have also been coated with lipid monolayer films to create selective sensors for sugars,6 metal ions,7 and a variety of other substrates.8 As nanostructured materials, lipid bilayers can be complexed with proteins, DNA, or metal ions to generate hierarchical structures, such as concentric tubules,9 striated lamellar sheets,10 and pillared stacks of bilayers.11 Fluorescent optical sensor materials based on lipid bilayers have been a focus of our research for the selective detection of metal ions,12-14 polypeptides,15 and proteins.16 (3) Reichert, A.; Nagy, J. O.; Spevak, W.; Charych, D. J. Am. Chem. Soc. 1995, 117, 829-830. (4) (a) Pan, J. J.; Charych, D. Langmuir 1997, 13, 1365-1367. (b) Antes, P.; Schwarzmann, G.; Sandhoff, K. Chem. Phys. Lipids 1992, 62, 269-280. (c) Song, X.; Nolan, J.; Swanson, B. I. J. Am. Chem. Soc. 1998, 120, 4873-4874. (d) Song, X.; Nolan, J.; Swanson, B. I. J. Am. Chem. Soc. 1998, 120, 11514-11515. (5) Niwa, M.; Ishida, T.; Kato, T.; Higashi, N. J. Mater. Chem. 1998, 8, 1697-1702. (6) (a) Kanayama, N.; Kitano, H. Langmuir 2000, 16, 577-583. (b) Ludwig, R.; Harada, T.; Ueda, K.; James, T. D.; Shinkai, S. J. Chem. Soc., Perkin Trans. 2 1994, 697-702. (7) (a) Steinberg, S.; Rubinstein, I. Langmuir 1992, 8, 1183-1187. (b) Terrettaz, S.; Vogel, H.; Gra¨tzel, M. J. Electroanal. Chem. 1992, 326, 161-176. (c) Schaffar, B. P. H.; Wolfbeis, O. S.; Leitner, A. Analyst 1988, 113, 693-697. (d) Shimomura, M.; Kunitake, T. J. Am. Chem. Soc. 1982, 104, 1757-1759. (8) (a) Rojas, M. T.; Ko¨niger, R.; Stoddart, J. F.; Kaifer, A. E. J. Am. Chem. Soc. 1995, 117, 336-343. (b) Nyquist, R. M.; Eberhardt, A. S.; Silks, L. A., III; Li, Z.; Yang, X.; Swanson, B. I. Langmuir 2000, 16, 1793-1800. (c) Yamamura, K.; Hatakeyama, H.; Naka, K.; Tabushi, I.; Kurihara, K. J. Chem. Soc., Chem. Commun. 1988, 79-81. (d) Thomas, R. C.; Yang, H. C.; DiRubio, C. R.; Ricco, A. J.; Crooks, R. M. Langmuir 1996, 12, 2239-2246. (9) Wong, G. C. L.; Tang, J. X.; Lin, A.; Li, Y.; Janmey, P. A.; Safinya, C. R. Science 2000, 288, 2035-2039. (10) (a) Ra¨dler, J. O.; Koltover, I.; Salditt, T.; Safinya, C. R. Science 1997, 275, 810-814. (b) Koltover, I.; Salditt, T.; Ra¨dler, J. O.; Safinya, C. R. Science 1998, 281, 78-81. (11) Waggoner, T. A.; Last, J. A.; Kotula, P. G.; Sasaki, D. Y. J. Am. Chem. Soc. 2001, 123, 496-497. (12) Sasaki, D. Y.; Shnek, D. R.; Pack, D. W.; Arnold, F. H. Angew. Chem., Int. Ed. Engl. 1995, 34, 905-907.
10.1021/la011651r CCC: $22.00 © 2002 American Chemical Society Published on Web 03/16/2002
Crown Ether Functionalized Lipid Membranes
Synthetically prepared lipids, functionalized at the headgroup position with chemical receptors and at the tail position with a fluorescent probe, are incorporated into phospholipid bilayers to form the sensor materials. Binding of specific ligands from solution induces a change in the aggregational state of the receptors, resulting in a change in fluorescence of the bilayers. Depending on the ligand of interest, it is possible to induce the receptorlipids to either aggregate or disperse in response to the chemical recognition event. In the case of metal ions, the receptors initially are in the aggregated state, caused by the phase separation of the liquid-phase receptor-lipids from solid-phase matrix lipids (e.g., distearylphosphatidylcholine (DSPC)). Binding of metal ions activates the receptor-lipids and causes them to disperse into the DSPC matrix. For polyfunctional ligands, on the other hand, the receptors are initially in the dispersed state within a fluidphase matrix (e.g., stearyloleylphosphatidylcholine) and are then drawn toward aggregation through multiple recognition events between the ligand and the lipid membrane. In both systems, the fluorophore senses the change in aggregational state producing a readily monitored change in the intensity of emission wavelengths. Our previous studies focused on the sensing aspect of these bilayer materials but lacked a detailed understanding of the mechanism of molecular reorganization in the membrane and the macro- to nanostructural changes that take place. Herein, our intent was to design and prepare a lipid membrane that was functionalized with a receptor that would be minimally affected by solution conditions (e.g., pH, salt concentration), large enough to be detectable by atomic force microscopy (AFM) in small aggregates, simple in functionality to allow a straightforward analysis, and sufficiently polar to serve as a lipid headgroup. A functional group that accommodates all of these characteristics is the crown ether. The 18-crown-6 group, in particular, should not only offer excellent properties as the lipid headgroup but also may provide a material with selective affinity, and thus sensing capability, for toxic metal ions of interest such as Pb2+ or Hg2+.17,18 The resulting functionalized bilayer allowed us to gain some insight into the mechanism of the chemical recognitioninduced receptor aggregation and the macro- to nanoscale changes of receptor assemblies in the two-dimensional space of the lipid membrane. Several techniques used in this study allowed us to probe the molecular to supramolecular interactions within the bilayer. NMR studies were used to measure affinity and stoichiometry of binding, Langmuir monolayer investigations qualitatively determined the extent of lipid mixing at the macroscale while fluorescence spectra monitored the microscopic changes, and AFM imaging provided the means to observe nanoscale structures as they responded to ligand binding. Experimental Section General. All compounds were of reagent grade purity and used as supplied unless stated otherwise. Organic solvents were (13) Sasaki, D. Y.; Padilla, B. E. Chem. Commun. 1998, 1581-1582. (14) Sasaki, D. Y.; Waggoner, T. A. Proc. SPIEsInt. Soc. Opt. Eng. 1999, 3606, 46-54. (15) Maloney, K. M.; Shnek, D. R.; Sasaki, D. Y.; Arnold, F. H. Chem. Biol. 1996, 3, 185-192. (16) Ng, K.; Pack, D. W.; Sasaki, D. Y.; Arnold, F. H. Langmuir 1995, 11, 4048-4055. (17) Izatt, R. M.; Pawlak, K.; Bradshaw, J. S.; Bruening, R. L. Chem. Rev. 1991, 91, 1721-2085. (18) (a) Luca, C.; Azab, H. A.; Tanase, I. Anal. Lett. 1985, 18, 449465. (b) Buschmann, H.-J. Inorg. Chim. Acta 1985, 98, 43-46. (c) Chen, L.; Bos, M.; Grootenhuis, P. D. J.; Christenhusz, A.; Hoogendam, E.; Reinhoudt, D. N.; van der Linden, W. E. Anal. Chim. Acta 1987, 201, 117-125. (d) Hayashita, T.; Sawano, H.; Higuchi, T.; Indo, M.; Hiratani, K.; Zhang, Z.-Y.; Bartsch, R. A. Anal. Chem. 1999, 71, 791-795.
Langmuir, Vol. 18, No. 9, 2002 3715 of spectral grade from Fisher Scientific. Aqueous solutions were prepared from water purified through a Barnstead type D4700 NANOpure analytical deionization system with an ORGANICfree cartridge registering an 18.0 MΩ‚cm resistance. NMR analyses were performed on a Bruker DRX 400 (Billerica, MA) with a resonance frequency of 400.1 MHz for 1H and 100.6 MHz for 13C. Infrared absorption spectra were taken on a Perkin-Elmer 1750 FTIR spectrometer (Norwalk, CT). Elemental analyses were performed by Desert Analytics (Tucson, AZ). 8-[1-Octadecyl-2-(9-(1-pyrene)nonyl)-rac-glyceroyl]-1,4,7trioxanonyl-1-(18-crown-6) (PS18C6). To a solution of 8-[1octadecyl-2-(9-(1-pyrene)nonyl)-rac-glyceroyl]-3,6-dioxaoctan-1ol16 (0.52 g, 0.65 mmol) and triethylamine (0.27 mL, 0.20 g, 1.9 mmol) in CH2Cl2 (10 mL) cooled to 0 °C was added mesyl chloride (0.10 mL, 0.15 g, 1.3 mmol) in a dropwise manner. After 1 h, the reaction mixture was worked up in the following manner: The solution was diluted with CH2Cl2 (30 mL) and washed with H2O (50 mL). The aqueous layer was isolated and extracted with fresh CH2Cl2 (2 × 30 mL). The organics were combined and then washed with aqueous 1 N HCl (40 mL), followed by saturated aqueous NaHCO3 (40 mL) and finally brine (40 mL). After the solution was dried over anhydrous MgSO4, the organics were filtered and then evaporated, yielding a yellow-brown oil. The oil, dissolved in DMSO (5 mL), was added to a solution of 2-(hydroxymethyl)-18-crown-6 (0.40 g, 1.36 mmol) and KOH (0.10 g, 1.78 mmol) in DMSO (8 mL) and the mixture stirred overnight at 85 °C. The reaction was worked up in a similar manner as above. The residual yellow-brown oil was flash column chromatographed on silica gel (7% methanol/CH2Cl2, Rf ) 0.17). A light yellow-green oil was isolated in 25% (0.18 g) yield. 1H NMR (CDCl3, 298 K) δ 8.28 (d, 1 H, J ) 9.3 Hz, Py-H), 8.11 (m, 7 H, Py-H), 7.86 (d, 1 H, J ) 7.8 Hz, Py-H), 3.80 (t, 2 H, J ) 4.8 Hz, OCH2CH), 3.62 (m, 44 H, O-CH), 3.33 (t, 2 H, J ) 7.8 Hz, CH2-Py), 1.85 (m, 2 H, Py-CH2CH2), 1.54 (m, 6 H, OCH2CH2CH2 and Py-CH2CH2CH2), 1.24 (m, 38 H, CH2), 0.87 (t, 3 H, J ) 6.9 Hz, CH3). 13C NMR (CDCl3, 298 K) δ 137.6, 131.7, 131.2, 129.9, 128.8, 127.8, 127.5, 127.3, 126.7, 125.9, 125.3, 125.0, 124.8, 123.7, 78.6, 78.1, 71.9, 71.6, 71.5, 71.1, 71.0, 70.8, 33.8, 32.2, 32.1, 30.3, 30.1, 29.9, 29.8, 29.7, 29.6, 26.4, 26.3, 22.9, 14.3. IR (neat) 3034, 2924, 2854, 1466, 1351, 1297, 1247, 1118, 845 cm-1. Anal. Calcd for C65H106O12‚2H2O: C, 69.98; H, 9.94. Found: C, 70.29; H, 10.00. Liposome Preparation. Distearylphosphatidylcholine (DSPC) was purchased from Avanti Polar Lipids (Alabaster, AL). The liposomes were prepared by dissolving DSPC and PS18C6 in chloroform at a 5% mole ratio with the total lipid concentration ∼1.5 mM. The solution was evaporated to a thin film on the walls of a glass centrifuge tube using a rotary evaporator. Residual solvent was removed from the film by further drying overnight under high vacuum. The film was then hydrated in either 3.0 mL of MOPS buffer solution (0.02 M 4-morpholinopropanesulfonic acid, 0.1 M NaCl, in 18 MΩ water, pH 7.4) or 0.1 M NaCl aqueous solution at 65 °C with vortex stirring, producing a total lipid concentration of 3.5 mM. The completely suspended film was then degassed with N2 gas for several minutes, followed by sonication with a 3 mm probe tip at 25 W power under N2 gas. Sonication was performed in 4 min cycles with 1 min of rest between each cycle for a total of 20 min at room temperature. The translucent solution was centrifuged for 20 min at 15 000 g to remove large bilayer aggregates. The supernatant was then filtered through a 0.2 µm filter. NMR Binding Studies. All samples were dissolved in methanol-d4 and run on a Bruker DRX400 at 400.1 MHz for 1H. Spectra were obtained using direct single pulse excitation with an 8 s recycle delay, an 8 µs pulse, and 256 scan averages. Samples were referenced to residual protonated methanol at 3.3 ppm. For the experiments to determine binding constants, the concentration 0.1 mM lipid was used to allow reasonable acquisition times. Stock solutions of metal salts in methanol-d4 were used to prepare the lipid-metal solution samples. The Pb(II) binding stoichiometry was determined using the continuous variation method, with a Job’s plot being produced for the binding with PS18C6.19 The sum of the metal (guest) and the crown-ether lipid (host) concentrations was maintained at the constant value 1 mM. A (19) Job, A. Ann. Chim. (Paris) 1928, 9, 113-134.
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series of solutions were sampled where the host concentration was varied from 100 µM to 1.0 mM in increments of 100 µM, with additional samples, varying in 50 µM increments of the host concentration, near the maximum in the Job’s plot.20 Fluorescence Spectral Data. Fluorescence spectra of the PS18C6/DSPC liposomes were obtained on a SPEX Fluoromax II (Instruments S.A., Edison, NJ) spectrophotometer using the excitation wavelength 346 and 2 nm slit widths. All sample solutions were analyzed at 20 ( 0.1 °C using a water-jacketed cell. Stock solutions of metal ions, at the concentrations 17.5 µM, 175 µM, 1.75 mM, 17.5 mM, and 175 mM, were prepared in 0.1 mM NaCl aqueous solution. Aliquots (10 or 20 µL) of the metal ion solutions were added to the liposome solution in the fluorescence cuvette (3.5 mL) to yield the desired metal ion concentrations. All reported data are averages from at least three trials. TEM Images. The liposomes were stained using a standard TEM preparation protocol.21 TEM images were taken on a Philips CM-30 operated at 300 kV. Langmuir Isotherms. All π-A isotherms and Langmuir film preparations were performed on a KSV minitrough (Helsinki, FI). The trough was situated on a vibration isolation table inside a class-100 clean room. The subphase temperature was maintained at 20 ( 0.1 °C using a Neslab water circulator. Surfactants were spread on the water surface from a chloroform solution. A pure water subphase was used for studies examining the dependence of π-A isotherms on varying lipid mole fraction in the monolayer. For experiments that monitored the change in isotherm shape in response to heavy metal ions in solution, an aqueous subphase containing 0.1 M NaCl was used instead to maintain a high ionic strength. The pH of the Pb(NO3)2 containing subphases fluctuated between 5.5 and 6.5. MOPS buffer was not used in these studies because of the influence of MOPS on the isotherm shape. The compression rate for the π-A isotherms was 37.5 cm2/min (∼5 Å2/molecule‚min). AFM Images. AFM experiments were performed with a Nanoscope IIIa Multimode scanning probe microscope (Digital Instruments, Santa Barbara, CA). The images were acquired in tapping mode in solution using a commercially available liquid cell (Digital Instruments) with 120-µm oxide-sharpened silicon nitride V-shaped cantilevers. The nominal spring constant of the cantilever was 0.32 N/m. Images were collected with the E scanner, which has a maximum range of 12 µm × 12 µm, operating at a scan rate of 2 Hz. The cantilever drive frequency was ∼9 kHz, and the drive amplitude was between 150 and 250 mV. The images were collected with 512 data points per line. Supported lipid bilayers were prepared via vesicle fusion on freshly cleaved mica substrates. The clean mica was first imaged in aqueous 0.1 M NaCl solution to establish the baseline. The liposome solution, diluted to 1 mM total lipid concentration, was then injected into the AFM liquid cell, and the mica surface was imaged subsequently. The liposome solution was incubated with the mica at room temperature for approximately 1 h to allow optimal time for vesicle fusion, resulting in full bilayer coverage of the substrate. The saline solution was used instead of MOPS buffer solution to maximize the solubility of the lead salts.
Results The crown ether functionalized lipid, PS18C6, was prepared by coupling 8-[1-octadecyl-2-(9-(1-pyrene)nonyl)rac-glyceroyl]-3,6-dioxaoctan-1-ol (PSOH)16 with 2-(hydroxymethyl)-18-crown-6 (Scheme 1). Coupling of these two components was facilitated by mesylation of the PSOH lipid followed by reaction with 2-(hydroxymethyl)-18crown-6 in a DMSO solution with KOH, at elevated temperature. The product lipid, PS18C6, was isolated by flash chromatography as a light green, viscous oil. All spectral data corresponded with the proposed structure. Small unilamellar vesicles (SUVs) of 5 mol % PS18C6 in a DSPC membrane were prepared in a MOPS buffer (20) Hirose, K. J. Inclusion Phenom. Macrocylic Chem. 2001, 39, 193-209. (21) New, R. R. C. Liposomes, 1st ed.; Oxford University Press: New York, 1990.
Sasaki et al. Scheme 1a
a Conditions: (a) methanesulfonyl chloride, NEt3, CH2Cl2, 0 °C, 1 h; (b) 2-(hydroxymethyl)-18-crown-6, KOH, DMSO, 85 °C, 12 h.
Figure 1. Fluorescence spectra of 5% PS18C6/DSPC liposomes (λex ) 346 nm) in MOPS buffer solution (pH 7.4) in the absence (s) and presence of Pb(NO3)2 at 1 µM (- - -), 10 µM (‚ - ‚), 100 µM (- - -), and 1.0 mM (- - -).
solution via ultrasonication. The vesicle sizes ranged from 80 to 120 nm in diameter, as determined by transmission electron microscopy (TEM). A TEM image of an ammonium molybdate stained vesicle can be found in the Supporting Information (Figure S1). Vesicles of the 5% PS18C6/DSPC bilayers were irradiated at 346 nm, producing a fluorescence spectrum with emission from both pyrene monomer (λem max ) 376 nm) and excimer (λem max ) 464 nm) (Figure 1). The intensity ratio of the excimer (E) to monomer (M) was typical of a pyrenelabeled lipid with a hydrophilic headgroup (e.g., dithioamide, alcohol) in a DSPC bilayer.13,14 Bilayers prepared in buffered saline solution exhibited relatively constant E/M values over the pH range 4-9, indicating stability of the bilayers to pH (see Supporting Information, Figure S2). In contrast, similar studies performed with bilayers prepared with 5 mol % iminodiacetic acid-functionalized lipid PSIDA12 exhibited a large variance in normalized E/M value with pH, changing from 0.39 at pH 4 to 1.0 at pH 7.5.13 The 5% PS18C6/DSPC vesicles responded selectively to the presence of lead and mercuric ions with a change in excimer to monomer intensities from the pyrene fluorophore. An example of the optical response is shown in Figure 1, where Pb(NO3)2 was present at varying concentrations in the vesicle solution. As the lead concentration was increased, the excimer emission decreased with a concomitant increase in the monomer emission.
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Figure 2. Normalized (*) E/M fluorescence data for 5% PS18C6/ DPSC liposomes responding to a logarithmic increase in concentration of Pb(NO3)2 (b), HgCl2 (0), and the numerous metal salts listed in the text, which include CuCl2, NiCl2, CdCl2, CoCl2, CaCl2, CsCl2, and SrCl2 (2).
Numerous other metal ions were sampled, but no optical response was observed. Those sampled were NiCl2, SrCl2, CrCl3, CaCl2, CeCl3, CsCl2, CoCl2, and MnCl2 at concentrations up to 0.5 mM, AgCl and CdCl2 up to 1.0 mM, CuCl2 up to 0.1 mM, and KCl and RbCl up to 100 mM. Because of solubility issues, Pb(NO3)2 was used in preference to PbCl2 and Pb(Ac)2. The latter two salts had poor solubility in MOPS buffer solution at the higher concentrations (>100 µM). At lower concentrations, however, the responses of the different lead salts were virtually identical. Figure 2 shows a plot of the normalized E/M fluorescence of the 5% PS18C6/DSPC vesicles as they responded to increasing metal ion concentration in solution. The data show that the bilayer yielded a fairly linear response to Pb(II) from 10-7 to 10-4 M. At concentrations greater than 10-4 M, the response exceeded the linear response defined by the lower concentrations. The response for the mercuric ion was less pronounced but just as sensitive as the Pb(II) response. Sensitivities for Pb(II) and Hg(II) were at the sub-micromolar level (i.e., low-ppb sensitivity). The lipid bilayers could also be regenerated to their original optical state through the addition of stoichiometric amounts of EDTA to the solution. Previous studies with metal ion sensitive lipid bilayers12 found that the E/M changes observed upon metal ion addition were a reflection of the change in the aggregational state of the lipids upon chemical recognition. The pyrene-labeled lipid, which has a Tc well below room temperature, and the DSPC matrix with a Tc of 55 °C phase separate, resulting in the aggregation of PS18C6 lipids and a high local concentration of pyrene moieties in the two-dimensional space of the lipid bilayer. The high pyrene concentration thus promotes excimer formation. This lipid aggregation is, however, dependent upon the physical state of the lipid. Initially, a charge neutral receptor-lipid would have its distribution within the membrane dictated by the crystalline domain formation of the DSPC lipids. Changing the physical characteristics of the lipid, such as the addition of electrostatic charge from metal ion chelation, can introduce repulsive forces that override the phase separation of the lipid components. This repulsion will then result in the dispersion of the receptor-lipids (Figure 3). A high-resolution 1H NMR study was conducted to confirm that the metal ion is recognized and bound by the crown ether headgroup of PS18C6. Although the experiments would have preferably been done in water to obtain
Figure 3. Schematic of one leaflet of a PS18C6 (darkened headgroup)/DSPC mixed lipid membrane before (above) and after (below) addition of Pb2+ ion. Recognition of Pb2+ by the crown ether causes the lipid’s headgroup to become cationic, resulting in electrostatic repulsion between other Pb2+-bound lipids.
binding constants with conditions similar to those used in the fluorescence studies, the innate aggregation of lipids in aqueous solution prohibited analytical measurements by NMR. The experiments were instead performed in methanol- d4, which provided solubility for the lipid and metal salts and offered a protic and polar medium that mimicked, as closely as possible, aqueous conditions. It is plausible that methanol may in fact mimic the medium near the crown ether better than water because of the lipophilic environment near the lipid bilayer surface. Molecular recognition at the lipid film surface is wellknown to exceed the affinity of identical recognition sites that operate in bulk water, with affinities that more closely mimic those found in organic solution.22 NMR spectra of the methylene protons R to the ether linkages of PS18C6 (δ 3.90-3.40 ppm) in the presence of varying concentrations of Pb(NO3)2 are shown in Figure 4. With increasing concentration of the metal ion in solution, the resonances of the methylene protons associated with the crown ether experience a downfield shift from δ 3.59-3.50 to δ 3.79-3.69, due to the formation of a metal-crown ether complex. This complex is in fast equilibrium on the NMR time scale, such that an average chemical shift between those of the complexed and uncomplexed species is observed. A similar chemical shift has also been observed for 18-crown-6 binding with La(III).23 All other protons of the lipid, including those of the pyrene and triethylene glycol spacer, remained unchanged up to the highest concentrations examined (0.2 M) (data not shown), indicating minimal interaction between the Pb2+ and these portions of the lipid. (22) (a) Cha, X.; Ariga, K.; Kunitake, T. J. Am. Chem. Soc. 1996, 118, 9545-9551. (b) Sasaki, D. Y.; Kurihara, K.; Kunitake, T. J. Am. Chem. Soc. 1991, 113, 9685-9686. (c) Kawahara, T.; Kurihara, K.; Kunitake, T. Chem. Lett. 1992, 1839-1842. (d) Ikeura, Y.; Kurihara, K.; Kunitake, T. J. Am. Chem. Soc. 1991, 113, 7342-7350. (e) Ariga, K.; Kunitake, T. Acc. Chem. Res. 1998, 31, 371-378. (f) Ebara, Y.; Itakura, K.; Okahata, Y. Langmuir 1996, 12, 5165-5170. (g) Mertesdorf, C.; Plesnivy, T.; Ringsdorf, H.; Suci, P. A. Langmuir 1992, 8, 2531-2537. (23) Bu¨nzli, J.-C. G.; Wessner, D.; Klein, B. Complexes of the Heavier Lanthanoid Nitrates with Crown Ethers; Bu¨nzli, J.-C. G., Wessner, D., Klein, B., Eds.; Plenum Press: Fargo, ND, 1979; Vol. 2, pp 99-104.
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Figure 6. Langmuir monolayer isotherms of PS18C6/DSPC films at various mole fractions on a pure water subphase at 20 °C.
Figure 4. 1H NMR of PS18C6 (0.1 mM) with varying concentrations of Pb(NO3)2 in methanol-d4.
Figure 5. Binding isotherm of PS18C6 (0.1 mM) with Pb2+ from 1H NMR analysis. The chemical shift change of the methylene protons on the crown ether ring is plotted against increasing concentration of Pb(NO3)2 in methanol-d4.
The binding constant for PS18C6 with Pb(II) was obtained by plotting the chemical shift change of the crown ether protons with respect to the concentration of metal ion. Figure 5 shows the plot of the median chemical shift of the crown ether resonances versus the number of equivalents of Pb(NO3)2 in solution. The data were readily fit to a Langmuir isotherm equation to yield the association, or binding, constant (Ka) 1.0 × 105 M-1.20 The binding constant is comparable to values found in the literature for 18-crown-6 with Pb(II) in methanol.17 Binding experiments were also conducted with MnCl2 and CaCl2 under identical conditions to determine the relative binding constants of metal cations that did not yield a fluorescence response from the bilayers. For Mn(II), no changes in chemical shifts were observed up to 10 equiv (1.0 mM) of metal ion against PS18C6. Some line broadening
did appear, however, indicating cage effects. Binding of Ca(II) to the crown ether of PS18C6 was observed with a Ka (5.0 × 102 M-1) orders of magnitude smaller than that for Pb(II) (data not shown). We also performed a continuous variations experiment to establish the binding stoichiometry of the metal ion with the functionalized lipid. A plot of the change in chemical shift of PS18C6 against the ratio of Pb(II) to PS18C6 in solution was produced (see Supporting Information, Figure S3). The value of the x-coordinate at the maximum in the Job’s plot corresponds with the relative stoichiometry (a/a + b) of the complexation between the host (a) and the metal guest (b). A maximum at x ) 0.5 was observed, indicating a 1:1 binding stoichiometry of Pb(II) with PS18C6. The fluorescence data strongly suggest an initially phase-separated PS18C6/DSPC film that switches to a dispersed lipid film after recognition and binding of Pb(II) or Hg(II). To provide some measure of the degree of miscibility for the two lipids before and after introduction of metal ion, Langmuir monolayer studies with the mixed films were performed. By monitoring the changes in collapse pressure, expansion, and shape of the pressurearea (π-A) isotherms while varying the mole fraction of the lipids in the film, the degree of lipid miscibility can be qualitatively assessed.24 The π-A isotherms with varying mole fractions of the two component films are shown in Figure 6. The isotherm for DSPC alone exhibits a solid condensed phase with the collapse pressure 60 mN m-1 and the limiting molecular area, obtained by extrapolating the steepest part of the isotherm to zero pressure, 47 Å2/molecule. The isotherm for PS18C6, on the other hand, shows a highly expanded monolayer with the collapse pressure 37 mNm-1, the limiting molecular area ∼100 Å2/molecule, and the molecular area at the liftoff point (point at which surface pressure is first detected) 220 Å2/molecule. This highly expanded isotherm is similar to reports of other known crown ether functionalized amphiphiles.25 At the lower molar percentages of PS18C6 in DSPC, the two lipids appear to interact in a cooperative way as inferred by the increase in collapse pressure: the 5% PS18C6/DSPC monolayer collapses at 64 mN m-1, and the 20% PS18C6/DSPC monolayer (24) (a) Gaines, G. L., Jr. Insoluble Monolayers at Liquid-Gas Interfaces; Interscience Publishers: New York, 1966. (b) Ulman, A. An Introduction to Ultrathin Organic Films. From Langmuir-Blodgett to Self-Assembly; Academic Press: San Diego, CA, 1991. (25) Yoshida, S.; Okawa, Y.; Watanabe, T.; Inokuma, S.; Kuwamura, T. Chem. Lett. 1989, 243-246.
Crown Ether Functionalized Lipid Membranes
Figure 7. Langmuir isotherms of a PS18C6 monolayer on an aqueous 0.1 M NaCl subphase with varying concentrations of Pb(NO3)2 in the subphase (at 20 °C).
collapses at 68 mN m-1. With molar percentages above 50%, a prominent kink in the isotherms appears at the surface pressure 37 mN m-1, coincident with the collapse pressure of PS18C6. The data clearly indicate poor miscibility of the mixed lipid film, which is reasonable considering the differences in phase transition temperature, Tc, of the two lipids (i.e., Tc(DSPC) ) 55 °C, Tc(PS18C6) < 0 °C). The binding of Pb(II) to the crown ether headgroup of PS18C6 should generate significant physical changes in the lipid that would manifest themselves as observable alterations in the π-A isotherm of the lipid. A series of isotherms of PS18C6 monolayers spread on a 0.1 M NaCl subphase containing Pb(NO3)2 shows some small but distinct and consistent changes with increasing Pb(II) concentration in solution (Figure 7). In constrast, isotherms produced on subphases containing CuCl2, CaCl2, or AgCl exhibited no changes up to 1.0 mM concentration. Increasing the levels of Pb(II) in the subphase produced a discernible increase in orientation of the lipids, as evidenced by the increase in the slope at the liquid condensed region of the isotherm and the reduction in the area of the liftoff point. The slight expansion of the liquid condensed region in the presence of high concentrations of Pb(II) may be a result of the increase in headgroup size from metal ion chelation. Upon binding of Pb(II) to mixed monolayers of PS18C6 and DSPC, distinct changes in film structure were observed, suggesting heightened miscibility of the lipids in the film. Increasing the metal ion concentration in the subphase alters the isotherms of monolayers composed of 50, 70, and 90% PS18C6/DSPC, producing a marked decrease in the molecular area at the liftoff point, an increase in slope of the condensed region, an increase in collapse pressure, and a loss of severity of the kink at 37 mN m-1. Figure 8 shows a series of isotherms of 50% PS18C6/DSPC monolayers on various concentrations of Pb(NO3)2 in the 0.1 M NaCl aqueous subphase. The steeper slope and decrease in molecular area at liftoff can be attributed to the structural changes of PS18C6 upon binding with Pb(II), as described above. The increase in collapse pressure and reduction of the kink in the isotherm are good indicators of enhanced miscibility/cooperative effects of the PS18C6 lipid with the DSPC matrix. Monolayers with less than 50% PS18C6/DSPC, that is the 5% and 20% compositions, exhibited little or no response in their π-A isotherms to metal ion in the subphase. The 5% PS18C6/DSPC composition showed no difference in the isotherm in the absence or presence of
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Figure 8. Langmuir isotherms of a 50% PS18C6/DSPC monolayer on an aqueous 0.1 M NaCl subphase with varying concentrations of Pb(NO3)2 (at 20 °C).
up to millimolar concentrations of Pb(NO3)2, while the 20% composition showed only slight increases in collapse pressure and steepness of slope of the condensed region. Apparently, the molecular reorganization of PS18C6 lipids in the DSPC matrix induced by Pb(II) recognition, as revealed by the fluorescence experiments, does not result in gross structural changes in the monolayer that can be detected via Langmuir isotherm measurements. However, at the submicroscopic level the crown ether lipids must change their organizational state to an extent large enough to produce the fluorescence spectral changes observed. Visualization of submicroscopic changes in lipid aggregation was accomplished with AFM imaging of the lipid bilayers supported on a mica surface. A 20% PS18C6/ DSPC bilayer was formed on the mica surface via the vesicle fusion technique in an AFM solution cell. The 20% PS18C6/DSPC composition provided the most distinct AFM images, compared to lower mole ratios of 5 and 10%.26 The bilayer coverage was fairly homogeneous over the mica with holes of micron size or smaller sparsely distributed in the film. These holes provided a reference to measure film thickness, which was approximately 54 ( 3 Å, consistent with a DSPC bilayer (∼47 Å) supported upon an 8-10 Å water layer. Figure 9A shows a typical 1.8 µm × 1.8 µm AFM image of the bilayer. The area was free of holes but populated with dark and light regions. The height difference between the dark and light regions was 8 ( 1 Å,27 which is approximately the height difference between DSPC and PS18C6, as determined by a simple space-filling model. Furthermore, using a pixel-counting analysis, we found that the light regions in the membrane accounted for roughly 20% of the surface, coincident with the loading of the crown ether lipid in the DSPC matrix. From these pieces of data we could assign the dark regions to the DSPC matrix and the light regions as areas rich in PS18C6. The nanoscale features of Figure 9 changed in size and intensity as the membrane responded to the introduction or removal of Pb(II) to the solution. The images were taken from the same area over the course of the experiment (total experimental time < 20 min). The structures in the membrane are composed of submicron scale islands and (26) The fluorescence response of the 20% PS18C6/DSPC liposomes was generally the same as that of the 5% loaded bilayers, but with a larger initial E/M ratio. The selectivity for Pb2+ is identical between the bilayer materials, but the E/M response was slightly stronger for the 20% bilayer compared to the 5% bilayer. See Supporting Information, Figures S4 and S5. (27) See Figure S6 in the Supporting Information.
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Figure 9. AFM topographic images of a 20% PS18C6/DSPC bilayer supported on a mica surface demonstrating the actuation of the film from aggregated to dispersed states with Pb2+ ions. The membrane in the (A) initial state, then (B) after addition of 0.1 mM Pb(NO3)2, followed by (C) addition of 0.1 mM EDTA, and finally (D) after addition of 0.1 mM Pb(NO3)2 again. All solutions were aqueous saline (0.1 M NaCl).
10-30 nm wide filaments of PS18C6-rich regions. The widths of those filaments are roughly equivalent to one to two dozen PS18C6 molecules. Addition of Pb(NO3)2 (100 µM) causes those structures to diminish in size and intensity and causes significant restructuring of the membrane (Figure 9B). Removal of the Pb(II) ion from the membrane surface, by the addition of a solution of 100 µM EDTA, induces a rapid reaggregation of the PS18C6 lipids that is readily perceptible by the increase in size and intensity of the light regions (Figure 9C). And finally, reexposing the membrane to a 100 µM solution of Pb(NO3)2 reverts the nanoscale features again to reduced dimensions (Figure 9D). These chemical recognitioninduced membrane reorganization events occur on a time scale faster than that needed to inject the solutions and image the material (
