Influence of Pyrene-Based Fluorescent Probes on the Characteristics

Langmuir 1996, 12, 4797-4802

4797

Influence of Pyrene-Based Fluorescent Probes on the Characteristics of DMPA/DMPC Langmuir-Blodgett Films M. Leonard-Latour, R. M. Morelis, and P. R. Coulet* Laboratoire de Ge´ nie Enzymatique UPRESA CNRS 5013, Universite´ Claude Bernard, Lyon 1 43, bd du 11 Novembre 1918, 69622 Villeurbanne Cedex, France Received January 22, 1996. In Final Form: May 21, 1996X Pyrene and pyrene-labeled phosphatidylcholine (PyC10PPC) were used as fluorescent probes embedded in behenic acid/behenate and in dimyristoyl-L-R-phosphatidic acid/dimyristoyl-L-R-phosphatidylcholine (DMPA/DMPC) monolayers and related Langmuir-Blodgett films (LB films). The influence of the probe on the integrity of the lipid monolayer was first controlled through the characteristics of surface pressuremolecular area isotherms. The characterization of LB films was based on the transfer ratio values and on the type of multilayers. Steady-state fluorescence studies performed on the corresponding multilayers enabled the advantages and drawbacks of each probe to be determined. Pyrene does not alter the molecular packing of the phospholipidic matrix but is not transferred in a sufficient amount to give a detectable signal. On the contrary, excellent results were obtained with PyC10PPC, which can be used at a molar fraction lower than 4.2%, avoiding any disturbance both in the integrity of the monolayer and in the stacking of the transferred layers in LB films.

Introduction One of the new and very promising ways for a better understanding of the behavior of biological membranes is to build up supramolecular systems having the ability of self-organization.1 Fluidity is one of the characteristics of these membranes, and the possibility of testing changes in rigidity in biomimetic membranes is of prime importance. Langmuir-Blodgett (LB) films associated with enzymes appear as a suitable approach for such a purpose. The goal of the present study is to select a fluorescent probe which enables us to give the most detailed information on supported planar layers without leading to major modification in the LB film’s stacking. LB films were prepared by transferring a compressed lipidic monolayer (docosanoic acid or mixed dimyristoylL-R-phosphatidic acid and dimyristoyl-L-R-phosphatidylcholine) spread on a water surface. The phospholipids were chosen among a large variety with regard to their transition temperature, so that further studies of fluidity do not damage the integrity of the proteolipidic membranes. Pyrene and several derivatives have been currently used as fluorescent probes in micelles, floating monolayers, lipidic vesicles, or phospholipid dispersions.2-6 The pyrene moiety can be covalently linked either to fatty acids7,8 or to phospholipids. Two different types of pyrene-labeled phospholipids were used: one bearing the pyrene moiety at the end of one or two aliphatic chains3,4,9 and the other with the fluorophore linked to the polar head group.10 To * Author to whom correspondence should be addressed. E-mail: [email protected]. X Abstract published in Advance ACS Abstracts, August 15, 1996. (1) Ringsdorf, H. Supramol. Sci. 1994, 1, 5. (2) Dorrance, R. C.; Hunter, T. F. J. Chem. Soc., Faraday Trans. 1 1977, 73, 1891. (3) Somerharju, P. J.; Virtanen, J. A.; Eklund, K. K.; Vainio, P.; Kinnunen, P. K. J. Biochemistry 1985, 24, 2773. (4) Tanaka, F.; Kaneda, N.; Mataga, N. J. Phys. Chem. 1986, 90, 3167. (5) Bohorquez, M.; Patterson, L. K. Langmuir 1990, 6, 1739. (6) Ahuja, R. C.; Mo¨bius, D. Langmuir 1992, 8, 1136. (7) Sluch, M. I.; Vituknovsky, A. G.; Petty, M. C. Thin Solid Films, in press. (8) Yamazaki, N.; Tamai, T.; Yamazaki, I. J. Phys. Chem. 1987, 91, 572. (9) Bohorquez, M.; Patterson, L. K. J. Phys. Chem. 1988, 92, 1835. (10) Caruso, F.; Grieser, F.; Thistlethwaite, P. J.; Almgren, M.; Wistus, E.; Mukhtar, E. J. Phys. Chem. 1993, 97, 7365.

S0743-7463(96)00072-8 CCC: $12.00

our knowledge, a few studies have been performed with fluorophores in LB films; some were conducted to determine lateral diffusion at the solid-liquid interface,11 and some others were performed to determine either the kinetics of pyrene fluorescence12,13 or the formation of intramolecular excimers.14 In our case, two fluorescent probes were chosen, pyrene and 1-palmitoyl-2-pyrenyldecanoylphosphatidylcholine (PyC10PPC). The influence of these fluorescent probes on the behavior of the lipidic monolayer was controlled through the shape of surface pressure-molecular area isotherms, which was directly related to the concentration of the probe. All the monolayers thus obtained could be transferred, but in different types. Steady-state fluorescence studies performed on multilayers then obtained led us to determine the advantages and drawbacks of each probe. Experimental Section Materials and Sample Preparation. The amphiphilic material, 1,2-ditetradecanoyl-sn-glycero-3-phosphate (dimyristoyl-L-R-phosphatidic acid, DMPA, purity 98%), 1,2-ditetradecanoyl-sn-glycero-3-phosphocholine (dimyristoyl-L-R-phosphatidylcholine, DMPC, purity 99%), 1,2-dihexadecanoyl-sn-glycero3-phosphocholine (dipalmitoyl-L-R-phosphatidylcholine, DPPC purity 99%), and docosanoic acid (behenic acid, purity 99%) were purchased from Sigma Chemical Co. (St. Louis, MO). Phospholipids were dissolved in chloroform/methanol (2/1 v/v) to prepare a 2 × 10-3 M DMPA solution, a 10-3 M DMPC solution, and a 10-3 M DPPC solution, which were stored at 4 °C. A 10-3 M solution for the cospreading of DMPA/DMPC (molar ratio 2/1) was prepared on use. Behenic acid was dissolved at a final concentration of 10-3 M in chloroform. Two fluorescent probes were used: pyrene (purity 99%, Aldrich-Chemie, Steinheim) or 1-hexadecanoyl-2-(1-pyrenyldecanoyl)-sn-glycero-3-phosphocholine (PyC10PPC) (fluorescent probes, Interchim). These probes were added to the amphiphilic solutions before spreading. The pyrene/lipid molar ratio was equal to 1, while the percent molar fractions of PyC10PPC in the phospholipidic monolayers were 1.7%, 4.2%, 8.3%, and 16.7%. (11) Caruso, F.; Thistlethwaite, P. J.; Grieser, F.; Furlong, D. N. Langmuir 1994, 10, 3373. (12) Lemmetyinen, H.; Ikonen, M.; Mikkola, J. Thin Solid Films 1991, 204, 417. (13) Kinnunen, K.; Virtanen, J. A.; Tulkki, A. P.; Ahuja, R. C.; Mo¨bius, D. Thin Solid Films 1985, 132, 193. (14) Sadovskii, N.; Shirov, P.; Kuzmin, M.; Lemmetyinen, H.; Ikonen, M. Thin Solid Films 1991, 204, 441.

© 1996 American Chemical Society

4798 Langmuir, Vol. 12, No. 20, 1996 CaCl2, NaCl, and NaHCO3 of the highest grade available from Sigma, Prolabo, and Chimie-Plus were added to pure water (resistivity ) 18.2 MΩ‚cm) obtained through a milli-Q fourcartridge purification system (Millipore) to prepare a cationic subphase. Prior to use, quartz substrates (35 mm × 9 mm × 1.25 mm) from Thuet-Biechelin (France) were cleaned as described elsewhere for CaF2 substrates15 and were silanized16 by treatment with dichlorodimethylsilane (1% in CHCl3). Surface Pressure Area Isotherms. Surface pressure area isotherms as well as transfer ratios were recorded by means of a computerized KSV 3000 Langmuir-Blodgett trough (Finland). The trough was made of one piece of Teflon with a well in the center and was mounted on a thermoregulated base plate. It was enclosed in a pure air flow cabinet to avoid dust and was mounted on an antivibrational table. A symmetric compression of the monolayer was achieved with two moving barriers in Delrin. The surface pressure was measured with a platinum Wilhelmy plate attached to a sensitive balance. After each experiment the plate was first soaked in ethanol and then rinsed in pure water. All the experiments were performed on a cationic subphase containing 10-4 M CaCl2 and 10-2 M NaCl thermostated at 18 or 23 °C with its pH adjusted at 7 by addition of NaHCO3. Before the experiments, the subphase surface was cleaned with a sucking system. The spreading solutions (10-3 M) were deposited very carefully on the surface using a 500 µL SGE microsyringe. A 10 min period was observed between spreading and the beginning of compression to favor solvent evaporation. Compression was carried out at speeds of 10 and 5 mm‚min-1 for behenic acid/ behenate and phospholipid monolayers, respectively. Langmuir-Blodgett Films Deposition. Before use, the silanized quartz substrates were rinsed with CHCl3. They were clamped parallel to the barriers. The surface pressure was maintained constant during the deposition. The dipping rates were 20 and 10 mm‚min-1, for behenic acid/behenate and phospholipid monolayers, respectively, both for the upstroke and the downstroke. Each substrate was covered with 10 layers on each face for the monolayers containing pyrene and with 4 layers for the monolayers containing PyC10PPC. The coated substrates were kept in glass tubes filled with nitrogen and sheltered from light. Microscopic Observations. To estimate the quality of the Langmuir-Blodgett films, micoscopic observations were performed. Defects in the last deposited monolayer were observed with a Nomarski differential interference contrast microscope (Carl Zeiss). The defects were characterized by structures more or less reflective. Steady-State LB Film Fluorescence Experiments. Steady-state fluorescence experiments were performed on LB films with a Jobin-Yvon spectrofluorimeter. Emission spectra were collected rigorously under the same conditions (temperature, time of exposition to atmospheric oxygen, and position of the substrate in the cuvette). The wavelengths of excitation (determined by absorption) were 337 nm for pyrene and 348 nm for PyC10PPC. The background emission was controlled using a bare quartz slide; the only peak observed was one of light diffusion around 360 nm due to the geometry of the system, whose intensity varied with the position of the substrate in the cuvette.

Results and Discussion 1. Influence of Pyrene on Both Behenic Acid/ Behenate Monolayers and Related LB Films. Behenic Acid/Behenate Monolayer. The ionic state of the monolayer depends on the nature of the subphase. When cations are added, some molecules of behenic acid are transformed into behenate. The ratio between the acid form and the salt form depends on both the nature of the cation involved and the pH of the subphase.17-19 Previous (15) More´lis, R. M.; Girard-Egrot, A. P.; Coulet, P. R. Langmuir 1993, 9, 3101. (16) Girard-Egrot, A. P.; More´lis, R. M.; Coulet, P. R. Langmuir 1993, 9, 3107. (17) Le´onard, M. A.; More´lis, R. M.; Coulet, P. R. Thin Solid Films 1995, 260, 227. (18) Chollet, P. A. Thin Solid Films 1978, 52, 343. (19) Daillant, J.; Bosio, L.; Benattar, J. J.; Blot, C. Langmuir 1991, 7, 611.

Leonard-Latour et al.

FTIR studies have shown that behenic acid exists in these two forms in the presence of Ca2+ in the subphase at pH 7 (results not shown). The interaction of Ca2+ with the carboxylate groups leads to a decreasing of head-group repulsion and, hence, an expansion of the solid-packed state, indicating a tighter packing of the molecules. Experiments were conducted with pyrene in a behenic acid/behenate monolayer spread onto a subphase containing 10-4 M CaCl2 and 10-2 M NaCl at pH 7. The presence of pyrene did not affect the molecular area of the amphiphilic molecules in the monolayer. However, pyrene leads to a slight decrease (24%) of the slope of the solidpacked state. This result is related to the surface compressional modulus (Cs-1) according to the definition proposed by Davies and Rideal.20 The value of this modulus is 1400 mN‚m-1 for the pure monolayer and is lower, 714 mN‚m-1, in the presence of pyrene, indicating that the pyrene monolayer is in a less condensed state. The modifications of both the slope and Cs-1 prove that pyrene leads to a slight modification of the molecular packing in the solid-packed state. From a 45 mN‚m-1 surface pressure, pyrene modifies strongly the integrity of the monolayer, since at this pressure a slow collapse starts, whereas in the absence of pyrene the collapse pressure of the monolayer is 68 mN‚m-1. Transfer of Pyrene/Behenic Acid/Behenate Monolayer. The transfer of such a monolayer was performed at a surface pressure of 32 mN‚m-1, where the monolayer is in the solid-packed state. The silanized substrate was covered with 10 pyrene/behenic acid/behenate layers on each face. The multilayers obtained were Y-type. All the transfer ratio (Tr) values are equal to 1 ( 0.1. Only the two last values are given in Table 1. The presence of pyrene in the monolayer did not modify the Tr value of the first transfer (1.07). This first transfer corresponds to the interaction of the tails with the hydrophobic quartz surface. This suggests that pyrene is not located on top of the tails. Fluorescence Study of Pyrene/Behenic Acid/Behenate Multilayer. Figure 1a depicts the emission fluorescence spectrum of these layers under excitation at 337 nm. This shows two components: a structured band due to excited pyrene monomers and another band at longer wavelengths, broad and structureless, attributed to excimers. This band originates either in “dynamic” excimers, i.e. formed upon collision of an excited pyrene with a ground-state pyrene as defined by Birks,21 or in “static” excimers22 formed from associative ground-state pyrene dimers. The monomer fluorescence band exhibits vibrational peaks with maxima at 372, 382, and 393 nm, while the excimer spectrum shows a band centered around 465 nm. The ratio of excimer to monomer fluorescence intensities (IE/IM, respectively, at 465 and 372 nm) was 1.6. It is proportional to the collision frequency of pyrene molecules, which in turn depends on their local concentration. Referring to Kalyanasundaram and Thomas,23 the IIII/II ratio (II and IIII being the intensities of the 372 and 382 nm vibrational peaks of the monomer emission band) gives information about the degree of polarity of the pyrene environment. This ratio varies from 0.63 for a polar solvent such as water to 1.65 for n-hexane.24 For pyrene in behenic acid/behenate multilayers, the ratio value was higher than 1 (1.21), which reflects the hydrophobic nature of the pyrene environment. (20) Davies, J. T.; Rideal, E. K. Interfacial Phenomena; Academic Press: New York, 1963; p 265. (21) Birks, J. B. Photophysics of Aromatic Molecules; WileyInterscience: New York, 1970; Chapter 7. (22) Winnik, F. M. Chem. Rev. 1993, 93, 587. (23) Kalyanasundaram, K.; Thomas, J. K. J. Am. Chem. Soc. 1977, 99, 2039. (24) Dong, D. C.; Winnik, M. A. Photochem. Photobiol. 1982, 35, 17.

Characteristics of DMPA/DMPC LB Films

Langmuir, Vol. 12, No. 20, 1996 4799

Table 1. Detection of Free Pyrene in LB Films through Both the Type of Transfer and the Emitted Fluorescent Signala transfer surface pressure (mN‚m-1)

temp (°C)

32

23

40

18 23

53

18 23

type of transfer (Y or YZ) and transfer ratio valuesb (Tr) without pyrene

with pyrene

Matrix: Behenic Acid/Behenate Y Y dw ) 0.97, up ) 0.90 dw ) 0.99, up ) 0.95 Matrix: DMPA/DMPC YZ dw ) 0.56, up ) 0.96 YZ dw ) 0.32, up ) 1.05 Y dw ) 0.98, up ) 1.06 Y dw ) 0.92, up ) 1.1

Y dw ) 0.89, up ) 1.03 YZ dw ) 0.54, up ) 0.95 Y dw ) 1.01, up ) 1.03 Y dw ) 0.98, up ) 0.99

fluorescent signal +++

+ ∼0 + ∼0

a The silanized quartz substrates were coated with 10 layers of a behenic acid/behenate or a DMPA/DMPC monolayer (with or without pyrene). b Tr values at the downstroke (dw) and at the upstroke (up) are given for the two last layers.

Figure 1. Fluorescence emission spectra of (a) 2 × 10 layers (pyrene/behenic acid/behenate) on a silanized quartz substrate; (b) the same coated substrate after a 20 min immersion in water; and (c) the water used for immersion: λexc ) 337 nm.

The pyrene/behenic acid/behenate-coated substrate was immersed for 20 min in water, and after withdrawal, its fluorescence emission spectrum was recorded (Figure 1b). The spectrum exhibits a pattern similar to that in Figure 1a but with lower fluorescence intensities. The fluorescence emission of the water used for soaking (Figure 1c) shows only the monomer emission, the ratio IIII/II being lower than 1, as expected in the case of a polar environment. Moreover, the fluorescence emission spectrum of the subphase used for the experiment exhibits a band characteristic of the monomer, which proves that some molecules of pyrene leave the surface and are found in the subphase. From these studies, it appears that pyrene is located in the hydrophobic part of the monolayer. According to Wistus et al.,25 the area occupied by a single pyrene molecule lying flat at the interface is about 35-50 Å2. As the molecular area of behenic molecules is not modified by the presence of pyrene, such a flat position in the tails cannot be considered. Pyrene is rather inserted between the tails, in a position which does not increase the molecular area of the behenic molecules, but its interaction with the tails is not tight enough to prevent its leakage into water. 2. Influence of Pyrene on Both Phospholipidic Monolayers and LB Films. Π-A Isotherms. In order (25) Wistus, E.; Mukhtar, E.; Almgren, M.; Lindquist, S.-E. Langmuir 1992, 8, 1366.

to assess the influence of pyrene on the molecular packing of a DMPA/DMPC monolayer, the surface pressure-area isotherms with and without pyrene were studied. DMPA/ DMPC molecules were spread onto a subphase thermostated at either 18 or 23 °C (23 °C corresponds to Tm, i.e. the temperature of the main transition of DMPC). Figure 2 shows that the behavior of a DMPA/DMPC monolayer (without pyrene) is rather independent of the temperature. It must be pointed out that the two components of the monolayer are perfectly miscible at these temperatures and that the collapse pressure is 66 mN‚m-1. At 18 °C (Figure 2a), it can be seen that pyrene modifies the isotherm shape in the liquid-expanded phase. In the presence of pyrene, the area per molecule is larger up to 53 mN‚m-1. Above this pressure, the two isotherms overlap and exhibit the same collapse pressure. At 23 °C (Figure 2b), pyrene slightly affects the packing characteristics of the DMPA/ DMPC monolayer, but nevertheless, the two isotherms are not superimposable. As the behavior of the pyrene/ DMPA/DMPC monolayer appears related to the temperature, further experiments were performed at both 18 and 23 °C. Transfer of Pyrene-Containing DMPA/DMPC Monolayer. Multilayers of phosphatidylcholine can be transferred only when this phospholipid is mixed with phosphatidic acid.26 DMPC was chosen because of its Tm value (23 °C). As the multilayers can be irregular with a DMPA/DMPC mixture poor in DMPA, a 2/1 ratio was

4800 Langmuir, Vol. 12, No. 20, 1996

Leonard-Latour et al.

Figure 2. Surface pressure-area isotherms of a DMPA/DMPC monolayer (molar ratio 2/1) with pyrene (dotted curve) and without pyrene (solid curve) at (a) 18 °C and (b) 23 °C: subphase 10-4 M CaCl2, 10-2 M NaCl, pH ) 7.

Figure 3. Fluorescence emission spectrum of 2 × 10 layers (pyrene/DMPA/DMPC) on a silanized quartz substrate at Π ) 53 mN‚m-1 and 18 °C: λexc ) 337 nm.

used. The cationic subphase adjusted at pH 7 was necessary to transfer more than 2 layers. The transfer of 10 layers of DMPA/DMPC with and without pyrene was performed at two surface pressures: 40 mN‚m-1 (beginning of the condensed phase on the isotherms in Figure 2a) and 53 mN‚m-1 (value for which the two isotherms overlapped) and at two temperatures. Results are given in Table 1 (Tr values are given for the two last layers). Without pyrene, at 40 mN‚m-1 it can be seen that the monolayer was transferred with a Tr clearly lower than 1 at the downstroke and with a Tr close to 1 at the upstoke. The multilayers obtained were called “YZ-type”, as defined in a previous work,27 by analogy with the XYtype described by Hasmonay et al.28 At 53 mN‚m-1, typical Y-type multilayers were obtained. With pyrene, Y-type multilayers were obtained at 40 mN‚m-1 and at 18 °C, while multilayers remained YZtype at 23 °C. At 53 mN‚m-1, Y-type multilayers were obtained in both cases. Fluorescence Study of Pyrene-Containing DMPA/ DMPC Multilayers. Figure 3 shows the emission fluorescence spectrum of 10 pyrene/DMPA/DMPC layers transferred at 53 mN‚m-1 on a silanized quartz substrate at 18 °C. The ratio IIII/II is slightly higher than 1. When pyrene is embedded in DMPA/DMPC multilayers, the (26) Hasmonay, H.; Caillaud, M.; Dupeyrat, M. Biochem. Biophys. Res. Commun. 1979, 89, 338. (27) Girard-Egrot, A. P.; More´lis, R. M.; Coulet, P. R. Langmuir 1996, 12, 778. (28) Hasmonay, H.; Vincent, M.; Dupeyrat, M. Thin Solid Films 1980, 68, 21.

intensity of the fluorescence of the bands was lower than the one of pyrene embedded in behenic acid/behenate multilayers. For transfers at 18 °C, results are similar for the two transfer pressures, 40 and 53 mN‚m-1 (Table 1), but for transfers at 23 °C, the fluorescence emission of pyrene is not significantly detectable. These results could be explained in different ways: (i) Pyrene is not in a propitious position to emit fluorescence. The value of the ratio IIII/II proves that pyrene embedded in a DMPA/ DMPC matrix is in an environment less hydrophobic than in behenic acid/behenate multilayers. (ii) Pyrene can be retained in multilayers but in a very low amount. To check the latter hypothesis, the substrate coated at 53 mN‚m-1 was immersed in water for 20 min. After withdrawal, the spectrum of water was collected and no characteristic band of pyrene was observable. This suggests that even if few molecules of pyrene leak out of the multilayers, they are in too low an amount to give a detectable signal. Considering the results of experiments performed at 23 °C, it is likely that pyrene is not retained in multilayers probably because it cannot interact with the hydrophobic tails of the phospholipids at this temperature. The fluorescence spectrum performed on the aliquot of the subphase exhibits only a band characteristic of the monomer, which proves that some molecules of pyrene sank into the subphase, as in the case previously seen with behenic acid/behenate. These results can be attributed to the interaction of pyrene with the hydrophobic tails, which depends both on the nature of the monolayer and on the temperature of the experiment. At 23 °C (temperature very low compared to the Tm of behenic acid), the isotherm of behenic acid spread onto a subphase containing calcium is characterized by a short liquid-condensed phase; hence, pyrene could be immediately trapped between the tails of behenic acid/behenate molecules. On the contrary, during compression, phospholipidic molecules do not exhibit the same behavior as the fatty acid molecules. In the liquid-condensed phase, even if calcium is present in the subphase, thus permitting the phosphatidic head groups to be close together, the tails are less close so the van der Waals forces are weaker. This phenomenon is enhanced at 23 °C, because the tails of DMPC are still in a fluid state. 3. Influence of Pyrene C10PPC on Both Phospholipidic Monolayers and LB Films. Π-A Isotherms. Another approach was then considered using pyrene linked to a phospholipid (PyC10PPC). In a first step the

Characteristics of DMPA/DMPC LB Films

Langmuir, Vol. 12, No. 20, 1996 4801

Table 2. Influence of the Pyrene Moiety in PyC10PPC on Both the Type of Transfer and the IE/IM Ratioa PyC10PPC molar fractionb (%)

DPPC type of transfer (Y or YZ) and transfer ratio valuesc

1.7 4.2

YZ f Y dw ) 0.75, dw ) 0.91 up ) 1.06, up ) 0.98

8.3 16.7

Y dw ) 0.99, up ) 0.98

type of transfer (Y or YZ) and transfer ratio valuesc

IE/IM

YZ dw ) 0.68, up ) 1.15 YZ dw ) 0.68, up ) 1.02

0.8

YZ dw ) 0.56, up ) 1.02 Y dw ) 0.9, up ) 1.01

7

2.5

6

a The silanized quartz substrates were coated with four layers of DMPA/DMPC (with or without pyrene) or with four layers of DPPC/ DMPA/DMPC. b Molar fraction of DPPC in DPPC/DMPA/DMPC monolayer or of PyC10PPC in PyC10PPC/DMPA/DMPC monolayer. c Tr values at the downstroke (dw) and at the upstroke (up) are given for the two last layers.

Figure 4. Surface pressure-area isotherms of PyC10PPC/ DMPA/DMPC monolayers. Percent molar fractions of PyC10PPC: (s) 0%; (- ‚ -) 1.7%; (- - -) 4.2%; (- - -) 8.3%; (‚‚‚) 16.7%. Subphase 10-4 M CaCl2, 10-2 M NaCl, pH ) 7, 18 °C.

molecular packing of a DMPA/DMPC monolayer was studied through the surface pressure-area isotherms for several concentrations of the fluorescent probe. The percent molar fraction of the fluorescent probe in the PyC10PPC/DMPA/DMPC matrix was varied from 1.7 to 16.7%, keeping the ratio DMPA/(DMPC + PyC10PPC) equal to 2. When the probe concentration was increased, the molecular areas shifted toward larger values (Figure 4). For PyC10PPC at 8.3% and 16.7%, isotherms show a discontinuity at a pressure of 44 mN‚m -1, which is the collapse pressure of PyC10PPC. These results indicate a nonideal mixing of PyC10PPC with the host DMPA/DMPC matrix. Isotherms obtained for 1.7% and 4.2% PyC10PPC overlapped the isotherm of DMPA/DMPC at pressures higher than 60 mN‚m-1. For these two lower concentrations of the probe, the collapse pressure of the DMPA/DMPC monolayer is not modified, which indicates that the presence of the probe does not lead to a major perturbation in the host DMPA/DMPC matrix. Transfer of PyreneC10PPC-Containing DMPA/ DMPC Monolayers. For transfer, the chosen pressure was 40 mN‚m-1, lower than the collapse pressure of PyC10PPC. Several sets of four layers were obtained by transfer of the DMPA/DMPC monolayer containing variable concentrations of PyC10PPC. Results are given in Table 2. (The Tr values were given for the two last layers.) YZtype multilayers were obtained for each molar fraction of the probe except for the highest (16.7%), for which multilayers are Y-type. When PyC10PPC is replaced by DPPC in the DMPA/DMPC matrix, the multilayers

obtained with 16.7% of DPPC are Y-type, whereas, with 4.2% DPPC, the YZ-type multilayers obtained tend to the Y-type as the number of layers increases. The presence of DPPC in the monolayer helps keep the value of the transfer ratio at the downstroke close to 1, whereas when the pyrene moiety is linked to the end of the sn-2 acyl chain of DPPC, the transfer ratio at the downstroke is lower than 1. As previously shown in Table 1, the transfer of the DMPA/DMPC monolayer, at 40 mN‚m-1 gives YZ-type multilayers; to obtain Y-type multilayers (Table 2), it is necessary either to add DPPC to the monolayer in a sufficient amount or to increase the transfer pressure (the latter possibility is in agreement with the results reported by Matuoka et al.29 with DPPA). However, when monolayers contain 4.2 or 8.3% PyC10PPC, the multilayers obtained are still YZ-type. This can be due to two concomitant effects, a structural effect of DPPC and a disturbing effect of the pyrene moiety which tends to maintain the multilayers in the YZ-type. An additional criterion of the disturbing effect of pyrene is the following: if the transfer of a monolayer with a probe molar fraction of 8.3% or 16.7% is performed at 48 mN‚m-1, higher than the collapse pressure of the probe but in the middle of the condensed phase, Y-type multilayers are obtained but with transfer ratios higher than 1. This proves that these monolayers are in the precollapse state. In addition, the quality of the LB films (and the related quality of the monolayers) was estimated through microscopic observations with a Nomarski differential interference contrast micoscope. Figure 5 shows photographs of DMPA/DMPC multilayers without PyC10PPC (Figure 5a) and with 8.3% and 4.2% PyC10PPC, respectively (Figure 5b and c). Figure 5a shows a few scattered defects, so the DMPA/DMPC multilayers appear relatively homogeneous. In Figure 5b, numerous pearl-like defects are observed all over the coated substrate surface. In Figure 5c the defects have the same structure but their density is lower. These observations led us to choose 4.2% PyC10PPC to perform the fluorescence studies. Fluorescence Study of PyreneC10PPC/DMPA/ DMPC Multilayers. Figure 6 shows the fluorescence spectrum (excitation 348 nm) of a substrate coated with four layers of PyC10PPC/DMPA/DMPC (4.2% in the probe). The spectrum displays a broad band around 470 nm corresponding to the excimer emission and only two peaks at 376 and 394 nm corresponding to the monomer emission. It must be stressed that the monomer emission differs noticeably from that of pyrene by the number of peaks and related wavelengths. The corresponding values are in agreement with those reported in the literature.12 For other concentrations of the probe, the fluorescence spectrum exhibits the same pattern and only the values (29) Matuoka, S.; Asami, H.; Hatta, I. Thin Sold Films 1989, 180, 123.

4802 Langmuir, Vol. 12, No. 20, 1996

Leonard-Latour et al.

Figure 5. Photographs of Nomarski microscope observations of 2 × 4 layers (DMPA/DMPC) on silanized quartz substrates without PyC10PPC (a) and with PyC10PPC at a percent molar fraction of 8.3% (b) and 4.2% (c). The arrows indicate defects.

Figure 6. Fluorescence emission spectrum of 2 × 4 layers (PyC10PPC/DMPA/DMPC) on a silanized quartz substrate at Π ) 40 mN‚m-1, 18 °C: λexc ) 348 nm.

of the ratio IE/IM differ. These values are given in Table 2 for each concentration of the probe. When the percent molar fraction of the probe is equal to 16.7%, the ratio value does not reflect the increase of the probe in the monolayer; in this case the monolayer is not homogeneous, as additionally exemplified by the discontinuities shown on the Π-A isotherms (Figure 4). Conclusion The aim of the present study was first to detect the influence of a fluorescent probe both on behenic acid/

behenate or DMPA/DMPC floating monolayers through isotherms and on the related LB films through the transfer ratio values in order to select a fluorescent probe with less disturbing effects. Fluorescence assays were conducted in the films with pyrene either free or in a bound form, i.e. pyrene-labeled phosphatidylcholine. In experiments conducted first on behenic acid/behenate monolayers, free pyrene at a high molar ratio gives a high intensity of fluorescence. In experiments performed with the DMPA/DMPC monolayers, even at a high molar ratio, the intensity of the fluorescent signal was very low. However in both cases, two major drawbacks occurred: (i) a too weak interaction of free pyrene with the hydrocarbon chains and (ii) its partial miscibility in water. Hence, free pyrene did not appear suitable for studies of rigidity in the case of the substrate-supported planar membranes. Considering now pyrene-labeled phosphatidylcholine (PyC10PPC), it exhibits a disturbing effect on the host lipidic matrix, related to its concentration. For values equal to or lower than 4.2% (probe molar fraction), it is noteworthy that the pyrene moiety does not affect the monolayer integrity, as shown by the isotherm in the condensed phases. In this case, the transfers are similar to those of a DMPA/DMPC monolayer. As the pyrene moiety is covalently linked to the end of one alkyl chain of the phospholipid, its interaction with water is not favored and its mobility is strongly decreased compared to that of free pyrene. One additional advantage is a sufficiently high signal when such a probe is involved in the building-up of LB films even at a low concentration. Therefore, pyrene-labeled phosphatidylcholine appears suitable for further studies in LB film molecular packing, notably if protein association is considered. Acknowledgment. This research was partly supported by CNRS-ULTIMATECH. We also thank the Service de Biochimie De´partement de Biologie Applique´e de l’IUTA of Universite´ Claude Bernard-Lyon 1 for the use of the Jobin-Yvon spectrofluorimeter. LA960072X