Langmuir 2000, 16, 9347-9351
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Photophysical Study of Pyrene-Labeled Phospholipids at the Gas/Water Interface M. I. Sa´ndez Macho,* A. Gil Gonza´lez, and A. Sua´rez Varela Departamento de Quı´mica Fı´sica, Facultad de Farmacia, Universidad de Santiago, 15706 Santiago de Compostela, Spain Received March 4, 2000. In Final Form: August 7, 2000 The surface monolayer technique was used to study the properties of biomembrane-like systems. The technique was used in combination with surface fluorescence measurements of fluorescent probes incorporated into a phospholipid matrix. Two types of fluorescent probe containing the chromophore pyrene bound to the hydrocarbon tail and the amino group of the phospholipid, respectively, were used. The fluorescent properties of the molecules were studied both in solution and in monolayers. On the basis of the results, fluorescence changes in the molecules upon compression of the monolayers studied arise from increased compactness in the phospholipid, which decreases permeation of atmospheric oxygen and hinders orientation changes in the chromophore at the interface.
1. Introduction One important property of biomembranes is their ability to act as physical barriers separating different chemical species. Thus, they can separate an aqueous medium from a lipid medium or the latter from a gas. The study of the factors that influence the kinetics and efficiency of the transfer of chemical species across membranes is crucial to a broad range of research topics from biosensors to the role of lung surfactants in gas transport. Oxygen is among the substances that can cross such membranes, which makes it of interest in determining the conditions under which it can permeate a phospholipid film. One way of establishing such conditions is by using fluorescent probes with a lipid structure.1 Especially prominent among the fluorescent chromophores that can bind to biomembrane phospholipids is pyrene, which possesses a relatively long lifetime in its excited state and also a high quantum yield. The fluorescence of this chromophore is sensitive to the presence of atmospheric oxygen and to the polarity and microviscosity of the medium. This has fostered the synthesis of molecules of variable complexity that incorporate this chromophore and mimic the structure of the components of the system concerned. The surface monolayer technique provides a straightforward, elegant method for reproducing the conditions under which membrane phospholipids occur at the air/ water interface. In this work we used the surface monolayer technique in combination with steady-state fluorescence measurements on mixed monolayers of phospholipids and phospholipids with a fluorescent probe.2-4 2. Experimental Section Dipalmitoylphosphatidylethanolamine (DPPE) (Figure 1) was purchased from Sigma. 1-Hexanedecanoyl-2-(1-pyrenedecanoyl)-sn-glycero-3-phosphoethanolamine (Py-DPPE) and N-(1* Corresponding author. (1) Haugland, R. P. Handbook of Fluorescent Probes and Research Chemicals; Molecular Probes Inc.: Eugene, OR, 1989. (2) Pethica, B. A. Structural and Functional Aspects of Lipoprotein in Living Systems; Academic Press: London, 1969; pp 37-72. (3) Cadenhead, D. A. Recent Progress in Surface Science; Academic Press: New York, 1970; Vol. 3, pp 169-192. (4) Phillips, M. C. Progress in surface and membrane science; Academic Press: New York, 1972; Vol. 5, pp 139-221.
pyrenesulfonyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine,triethylammoniumsalt(DPPE-Py),werebothpurchased from Molecular Probes. All solvents used were HPLC grade and purchased from Aldrich. Deionized water of 18 MΩ‚cm resistivity, obtained from a reverse osmosis Milli-RO, Milli-Q apparatus (Millipore) that provides “reagent grade” water, was also employed. Spreading solutions were prepared in 4:1 chloroform/ethanol. The number of phospholipid molecules deposited in each experiment was 1.6 × 1016, and that of probe molecules was 1.6 × 1015. π-A isotherms were recorded by using a Lauda TGW surface balance equipped with a hydrophobic (Teflon) trough resistant to most organic acids. The instrument includes a thermal circulator that delivers water at a preset temperature through a coil located in the bottom of the Teflon trough. The substrate temperature was automatically controlled with the aid of a thermistor immersed in it. The subphase temperature was kept constant at 20 °C throughout. The moving barrier used to compress the monolayer was driven by a motor that afforded speeds from 0.015 to 0.11 cm‚s-1; this latter was employed in all experiments. The spreading solution was deposited by means of a GilsonMicroman micropipet precise to within 0.2 µL. Once spread, the solution was allowed to stand for at least 10 min in order to ensure complete evaporation of the solvent. Emission and excitation spectra were recorded on a Fluoromax-2 Jobin-Ivon Spex instrument fitted with a fiber-optic probe, the free end of which was placed at an angle of 45° to the monolayer surface. Absorption spectra were recorded on a Uvicon 810 P spectrophotometer (Kontron Instruments) furnished with halogen and deuterium lamps. Wavelength reproducibility was better than (0.02 nm, and wavelength accuracy was (0.5 nm.
3. Results 3.1. Bulk Phase. Prior to studying the fluorescence behavior of the probes in monolayers, their photophysical properties in pure solvents (bulk phases) were examined. This was done using a 0.1 mg/L stock solution to prepare two 10-5 M working solutions by dilution in cyclohexane and ethanol, respectively. Neither solution was degassed, so the oxygen concentration at a partial pressure of 0.21 atm was virtually the same in both solvents.5 The absorption spectrum for Py-DPPE in cyclohexane (Figure 2) exhibits two sets of bands containing three peaks (5) Battino, R. Solubility Data Series: Oxygen and Ozone; Pergamon: Oxford, 1981; Vol. 7.
10.1021/la0003286 CCC: $19.00 © 2000 American Chemical Society Published on Web 10/11/2000
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Figure 3. Absorption (s) and fluorescence spectra for DPPEPy in cyclohexane (‚ - ‚) and ethanol (‚ ‚ ‚).
Figure 1. Chemical structures of the compounds under investigation.
Figure 2. Absorption (s) and fluorescence spectra for PyDPPE in cyclohexane (‚ - ‚) and ethanol (‚ ‚ ‚).
each: at 256, 266, and 277 nm (the strongest) in one and at 315, 329, and 344 nm (the strongest) in the other. The positions of the peaks are similar to those reported for the absorption bands of pyrene.6-8 The peaks in the first set of bands correspond to the 1Lb transition (with a dipole moment normal to the molecular axis); on the other hand, those in the second set correspond to the 1La transition (with a dipole moment parallel to the molecular axis).6,9 The absorption spectrum obtained in a polar solvent (ethanol) also exhibits two sets of bands containing three peaks each; however, these present great absorption and are bathochromically shifted by 1-2 nm. As can be seen from Figure 2, the emission spectrum in cyclohexane, obtained at λex ) 344 nm, exhibits a peak at 376 (I) and another at 397 nm (II); these can be ascribed (6) Birks, J. B. Photophysics of Aromatic Molecules; Wiley-Interscience: New York, 1970; Chapter 7. (7) Dabestani, R.; Ivanov, I. N. Photochem. Photobiol. 1999, 70, 10. (8) Perkampus, H.-H.; Sandeman, I.; Timmons, C. J. DMS UV Atlas of Organic Compounds; Verlag Chemie, Weinheim; Butterworth: London, 1966-1971; Vols. I-V. (9) Klessinger, M.; Michl, J. Excited States and Photochemistry of Organic Molecules; VCH Publishers: New York, 1995; Chapter 2.
to the fluorescence of a monomeric state that resembles that of pyrene in solution. In addition, the probe exhibits a very strong, nonstructured band at 472 nm (III) that can be ascribed to the excimer emission and is also similar to that for pyrene in cyclohexane.10 The spectrum in ethanol exhibits two peaks that are typical of the monomer. At the concentration used, however, it lacks the band for the excimer. One of the excitation spectra was recorded with the emission monochromator set at 376 nm (the emission wavelength for the monomer), and the other, with the emission monochromator at 474 nm (the emission wavelength for the excimer). Both spectra exhibit two series of bands with maxima similar to the absorption peaks. The peaks are at identical positions in both spectra and differ only in intensity, which is markedly greater in the spectrum recorded at the excimer wavelength. Only the excitation spectrum with the emission monochromator set at 376 nm was recorded in ethanol, as the excimer exhibited no band in this medium. The absorption spectrum for DPPE-Py in cyclohexane (Figure 3) exhibits two sets of bands less structured than those for Py-DPPE and consisting of two peaks each: at 269 and 280 nm for the former and at 335 and 350 nm for the latter. Similarly to the previous probe, the spectrum in ethanol only differs from that in cyclohexane in absorptivity, which is slightly higher in the latter. The fluorescence spectrum in cyclohexane exhibits two emission peaks at 376 (I) and 395 nm (II) for the monomer, in addition to the broad, strong band at 472 nm (III) corresponding to the excimer emission. The spectrum in ethanol exhibits the peaks for the monomer but not the band for the excimer. The excitation spectra exhibit the same bands as the absorption spectra in both solvents. 3.2. π-A Isotherms. The phospholipid/probe mixtures used as spreading solutions were prepared in a 9:1 ratio, which ensured that the DPPE/probe system would contain a large enough amount of chromophore to yield an adequate fluorescence signal.11 The incorporation of pyrene into the DPPE molecule, whether at the hydrocarbon chain (nonpolar residue) or the polar head, caused the monolayer to expand in relation to pure DPPE. At low surface pressures, the π-A isotherm for the DPPE/Py-DPPE monolayer (Figure 4) occurs in an expanded state that evolves to a condensed state at higher (10) Birks, J. B. Photophysics of Aromatic Molecules; Wiley-Interscience: New York, 1970; p 302. (11) Caminati, G.; Gabrielli, G.; Ahuja, R. C.; Mo¨bius, D. Prog. Colloid Polym. Sci. 1993, 93, 236.
Pyrene-Labeled Phospholipids at the Gas/Water Interface
Figure 4. π-A isotherms and compressibility modulus (inset) for pure DPPE (- - -), DPPE/Py-DPPE (‚ ‚ ‚), and DPPE/DPPEPy (‚ - ‚). πc denotes the collapse pressure of each substance.
Figure 5. Fluorescence spectra for a DPPE/Py-DPPE mixture, normalized at a constant probe surface density and a variable surface pressure: (9, 1; b, 10; 2, 20; 1, 30; [, 40; ×, 50 mN/m). Inset: Variation of the excimer fluorescence intensity (III) with the probe surface concentration. (a) In the air. (b) Under a nitrogen atmosphere.
pressures. The transition between the two phases was not clearly reflected in the isotherm, which was typical of a second-order transition, so we opted for plotting the compressibility modulus (Cs-1, the mean slope of the π-A curve) against π (see Figure 4, inset). As can be seen, the transition took place at a surface pressure of 15 mN/m. The monolayer collapse pressure was 55 mN/m, and the extrapolated area was 62.5 Å2/molecule. With the pyrene molecule at the polar head of the phospholipid, the monolayer also evolves from an expanded state to a liquid state, albeit at a lower surface pressure; the condensed zone reaches a slightly greater compressibility modulus; the film occupies a larger surface area; and the collapse pressure is 58 mN/m. 3.3. Fluorescence of DPPE/Py-DPPE Mixed Monolayers. Figure 5a shows the fluorescence spectra for a DPPE/Py-DPPE (9:1) mixture recorded at a surface
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Figure 6. Excitation spectra for a DPPE/DPPE-Py mixture obtained at the emission wavelength of the monomer (s) and that of the excimer in monolayer form at a surface pressure of 30 mN/m (‚ ‚ ‚). The spectra were normalized with respect to the maximum of peak II in order to better expose the shift. Those obtained at the other pressure values have been omitted for simplicity.
pressure of 1, 10, 20, 30, 40, and 50 mN/m. They exhibit two absorption bands at 376 (I) and 396 nm (II) for the monomer, as well as an emission band at 472 nm (III) for the excimer that is particularly strong at the surface pressure 50 mN/m. The excimer band increases slightly with increasing surface pressure up to 40 mN/m, beyond which the increase is more pronounced. Erratic behavior in the intensity of the excimer is observed at low surface pressures. This may be attributed to the experimental conditions under which the fluorescence measurement is carried out, given that greater compression of the monolayer can produce minor variation in the level of the surface upon which the film is deposited, thus producing small changes in the measurement of fluorescence. Whatever the cause, there is a significant increase in the intensity of fluorescence in the excimer band at pressures beyond 40 mN/m. In the fluorescence spectra recorded under a nitrogen atmosphere, the excimer emission is quite appreciable, even at low surface pressures; the emission increases steadily with increasing surface pressure up to 50 mN/m (Figure 5b). As can be seen from Figure 6, the excitation spectra recorded at the characteristic wavelengths of the excimer and monomer, at the different surface pressures studied, undergo a bathochromic shift the magnitude of which increases with increasing surface pressure. 3.4. Fluorescence of DPPE/DPPE-Py Mixed Monolayers. Figure 7a shows the fluorescence spectra obtained for the DPPE/DPPE-Py (9:1) system. As can be seen, whatever the surface pressure, the spectra exhibit the two bands for the monomer (at 379 and 398 nm) in addition to that for the excimer. Also, the bands undergo a hypsochromic shift of about 10 nm on increasing the surface pressure from 1 to 50 mN/m. The spectra recorded under a nitrogen atmosphere (Figure 7b) are similar to the previous ones. The excimer emission decreases steadily and a hypsochromic shift is observed as the surface pressure is raised. The excitation spectra obtained at the monomer and excimer wavelengths exhibit no significant shifts in their maxima and differed in intensity only.
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Figure 7. Fluorescence spectra for a DPPE/DPPE-Py mixture, normalized at a constant tested surface density and a variable surface pressure: (9, 1; b, 10; 2, 20; [, 30; [, 40; ×, 50 mN/m). Inset: Variation of the excimer fluorescence intensity (III) with the probe surface concentration. (a) In the air. (b) Under a nitrogen atmosphere.
4. Discussion On the basis of the above-described results, the probes studied behave differently in solution and in monolayer form; in the latter, they also exhibit a differential behavior depending on whether pyrene is bound to the polar or nonpolar residue of the phospholipid. Pyrene, at a 10-5 M concentration, forms no excimer in ethanol or cyclohexane; but at the same concentration, Py-DPPE and DPPE-Py form an excimer in cyclohexane but not in ethanol. These facts suggest that the excimer formation is not exclusively due to an intrinsic property of pyrene. In Py-DPPE and DPPE-Py, pyrene is part of a molecule with a relatively long hydrocarbon residue, so the probe behaves differently in a polar solvent and in a nonpolar one. The latter may favor the formation of clusters of a few molecules and hence the mutual approach of pyrene molecules to form excimers. This is consistent with experimental data not reported here that deserve some comment. Thus, although the intensity of the excimer emission in the emission spectra for both probes in cyclohexane decreases with increasing dilution, the emission is still appreciable at concentrations as low as 10-8 M. This suggests that excimers may be formed even at such low concentration levels. Also, the fact that the positions of the bands in the excitation spectra recorded at the monomer and excimer emission wavelengths in cyclohexane coincide suggests that the band at 474 nm corresponds to the typical emission of an excimer. Such an excimer referred to as a “dynamic excimer” by Birks must be formed as a result of two molecules (one in its ground state and the other in the excited state) approaching each other, the process being diffusion-controlled.12,13 The excitation and fluorescence spectra for DPPE-Py/ DPPE (9:1) and DPPE/DPPE-Py (9:1) mixed monolayers differ from those obtained in a spectrophotometric cuvette because the monolayer molecules adopt a specific orientation at the air/water interface that may change as the film is compressed. In this situation, some pyrene molecules may be close enough to one another to form clusters (D), which, once excited (D*), will be the species responsible (12) Birks, J. B. Acta Phys. Pol. 1968, 34, 603. (13) Winnik, F. M. Chem. Rev. 1993, 93, 587.
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for the emission. Because the emission is very similar to that of the dynamic excimer, the species is called a “static excimer”. As the monolayer is compressed, it adopts a condensed liquid state as a result of the molecular axis of pyrene lying increasingly normal to the interface. Under these conditions, the feasibility of the dimer being formed increases with an increase in surface pressure up to 30 mN/m, where molecules are fully packed (as revealed by the maximum value in the compressibility modulus). The previous assertion is supported by the fact that the excitation spectra for the monolayer at low surface pressures (where little dimer is formed) exhibit virtually no shift in their maxima. As the monolayer is compressed, however, the shift becomes more marked and peaks at 30 mN/m. Evidence of the orientation change in pyrene as the monolayer is compressed is provided by the change in intensity ratio between peaks I and II in the excitation spectrum: the ratio increases with increasing surface pressure up to 30 mN/m. This phenomenon had previously been reported14 to occur in the 1Lb and 1La bands of the reflection spectra for fluorescent probes in monolayers. The fact that the excimer emission in the fluorescence spectrum does not increase appreciably upon compression arises from the quenching effect of atmospheric oxygen on the fluorescence. Such an effect vanishes when the monolayer molecules are packed so tightly that atmospheric oxygen cannot reach the excimer, so the fluorescence intensity rises abruptly as a result (see Figure 5a, inset). This is confirmed by the fluorescence spectra recorded under a nitrogen atmosphere: under these conditions, quenching by atmospheric oxygen is hindered, so the excimer emission increases steadily with increasing compression (see Figure 5b, inset). A transfer of energy between the monomer and excimer might also occur. If so, it would increase the excimer emission at the expense of the monomer emission. An energy transfer of this type has been observed in LB films.15 However, for the transfer to take place, the excimer and monomer must be at a fairly short distance. In our systems, with probe mole fractions of 0.1, the transfer, if any, will be irrelevant, owing to the long distances between the excimer and monomer molecules, at least at low surface pressures. When the pyrene molecule is bound to the polar head of the phospholipid (DPPE-Py), the experimental π-A isotherms suggest that the pyrene group is located on the surface of the subphase, owing to its hydrophobicity. In this situation, the contribution of the group to the total area occupied by the monolayer is greater than that when it is bound to the nonpolar residue (see Figure 4). Moreover, because of its hydrophobic character, pyrene tends to decrease its hydrophilicity, and hence its attraction by water, which facilitates a change in its orientation as the monolayer is compressed. This situation leads to pyrene lying between hydrocarbon chains and parallel to them, which in turn decreases the collapse pressure relative to that of the pure DPPE monolayer. Using time-resolved fluorescence spectroscopy, Yamazaki et al.16 observed a shift in the excimer band for LB films of 16-(1-pyrenyl)hexadecanoic acid in stearic acid. The shift was caused by the presence of two types of excimer: a sandwichlike one (E2) emitting at 470 nm and a partially overlapped one (E1) emitting at 420 nm. At long times (14) Ahuja, R. C.; Mo¨bius, D. Langmuir 1992, 8, 1136. (15) Morita, T.; Kimura, S.; Imanishi, Y. Langmuir 1998, 14, 171. (16) Yamazaki, I.; Tamai, N.; Yamazaki, T. J. Phys. Chem. 1987, 91, 3572.
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(50-100 ns), which are closer to the experimental conditions of steady-state measurements, the fluorescence profile only exhibits the emission for the sandwichlike excimer. If the shift in the excimer band to shorter wavelengths as the film is compressed were due to increased emission from excimer E1, then the band at 420 nm would be quite substantial and distort the emission spectrum in that region. The steady-state spectra obtained do not allow the shift to be ascribed to E1. For this reason, changes in the chromophore orientation and environment must be the origin of the shift in the wavelength of the excimer maximum as the monolayer is compressed.6 The fact that the excitation spectra exhibit no shift suggests that the emitting species is an excimer formed in a diffusion-controlled process. The decrease in excimer emission with compression of the monolayer (Figure 7a) cannot be ascribed to quenching by atmospheric oxygen, as a similar effect was observed under a nitrogen atmosphere (Figure 7b). Consequently, the phenomenon must be ascribed to a decrease in the diffusion coefficient of the probe as the monolayer is compressed.
in the ground state. The excimer intensity for the probe can be used to monitor packing in a DPPE phospholipid matrix. An abrupt jump in intensity suggests that the degree of packing is high enough to prevent quenching of the excimer by atmospheric oxygen. Experiments conducted under a nitrogen atmosphere confirm this assertion. DPPE-Py forms dynamic excimers in DPPE/DPPE-Py mixed monolayers; unlike those of the previous probe, these excimers are formed in a diffusion-controlled process (they are the result of a molecule in its ground state and one in the excited state meeting). Because the chromophore in this probe is isolated from air, its emission cannot be quenched by atmospheric oxygen (as confirmed by the spectra recorded under a nitrogen atmosphere, which were not appreciably different from the previous ones), so the decrease in excimer emission upon compression must be due to the diffusion coefficient of the probe decreasing as the monolayer is compressed.
5. Conclusions In a DPPE matrix, the Py-DPPE probe exhibits excimer emission due to the formation of a cluster (static dimer)
Acknowledgment. This work was funded by a grant from the Conselleria de Educacion of the Xunta de Galicia (Spain) awarded for the realization of Proyect XUGA 20306A96. LA0003286
