Organization of a Water-Soluble Porphyrin in Mixed Monolayers with

Langmuir 1998, 14, 4175-4179

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Organization of a Water-Soluble Porphyrin in Mixed Monolayers with Phospholipids Studied by Brewster Angle Microscopy I. Prieto, M. T. Martı´n Romero,† and L. Camacho Departamento de Quı´mica Fı´sica y Termodina´ mica Aplicada, Facultad de Ciencias, Universidad de Co´ rdoba, Avda. S. Alberto Magno s/n, E-14004 Co´ rdoba, Spain

D. Mo¨bius* Max-Planck-Institut fu¨ r biophysikalische Chemie, Am Fassberg 11, D-37077 Go¨ ttingen, Germany. E-mail: [email protected]. Received October 27, 1997. In Final Form: January 21, 1998 The organization of a cationic water-soluble porphyrin, TMPyP, in a complex monolayer containing an anionic phospholipid, DMPA, at the air-water interface has been directly inferred by Brewster angle microscopy, BAM. In a previous work (Martı´n, M. T.; Prieto, I.; Camacho, L.; Mo¨bius, D. Langmuir 1996, 12, 6554) the organization of this mixed monolayer TMPyP/DMPA, in the ratio 1:4, was explained on the basis of a model where the TMPyP molecules are in a monomer-dimer equilibrium depending on the surface pressure, that is, on the surface density of the porphyrin, by analysis of π-A isotherms and reflection spectra results. The morphology of the mixed monolayer was recorded at several surface pressures. The images are different from those obtained for the lipid matrix. At 5 mN/m a homogeneous and continuous film was observed. However, for surface pressures > 8 mN/m the coexistence of two phases is recorded, and small domains with higher brightness than that of the surrounding area are formed. The density of the bright domains increased until covering nearly the whole surface at 35 mN/m. The BAM results have been related with those obtained by reflection spectroscopy at the air-water interface. The appearance of the bright domains has been attributed to the formation of the dimer phase II where the DMPA molecules are dense-packed with an area ADMPA,II ) 0.40 nm2, and the homogeneous area has been attributed to phase I with porphyrin monomers. In phase I, the DMPA molecules are in a liquid-expanded state with the area ADMPA,I ) 0.84 nm2.

Introduction Mono- and multilayers are materials known as ultrathin films of thickness measured on the nanometer scale. The fundamental research on the designed molecular organization of ultrathin films has rapidly evolved from this type of assemblies involved in model systems.1 The Langmuir-Blodgett (LB) technique is well-known to fabricate molecular assemblies with well-controlled composition, structure, and thickness by sequential transfer of monolayers from the air-water interface to solid substrates. The methodology to fabricate those materials started to be developed in the 30s;2-4 however, only during the past decades has the assembly of monolayers in a desired organization with high order and for application of physicochemical system as well as models for biological systems become possible.5-7 The organization of the monolayer at the air-water interface is crucial and has to be characterized in order to establish the properties of LB films. * To whom correspondence should be addressed. † E-mail: [email protected]. (1) Kuhn, H. Pure Appl. Chem 1965, 11, 345. (2) Langmuir, I. J. Am. Chem. Soc. 1917, 39, 1848. (3) Blodgett, K. B. J. Am. Chem. Soc. 1935, 57, 1007. (4) Blodgett, K. B. Phys. Rev. 1937, 51, 964. (5) Swalen, J. D.; Allara, D. L.; Andrade, J. D.; Chandross, E. A.; Garoff, S.; Israelachvili, J.; McCarthy, T. J.; Pease, R. F.; Wynne, K. J.; Yu, H. Langmuir 1987, 3, 932. (6) Roberts, G. G. In Langmuir-Blodgett Films; Plenum Publishing Co.: New York, 1990. (7) Kuhn, H.; Mo¨bius, D. In Monolayer Assemblies, 2nd ed.; Rossiter, B. W., Baetzold, R. C., Eds.; John Wiley and Sons: New York, 1993; Vol. IXB, p 375.

Brewster angle microscopy (BAM) is one of the most attractive techniques recently developed for in situ studies of the organization of the amphiphilic molecules forming monolayers at the air-water interface. The morphology of floating monolayers can also be visualized with fluorescence microscopy.8 However, in contrast to fluorescence microscopy, BAM has the great advantage that it does not require probe molecules but rather uses the intrinsic optical properties of the monolayer. The Brewster angle technique is based on the fact that the presence of a monolayer at the air-water interface modifies the local reflectivity of the interface. By imaging a laser beam reflected off the interface at the Brewster angle of the pure gas-water interface, one can observe structures in the monolayer due to their different reflectivities. This makes BAM suitable for monolayer studies of a wide variety of monolayer classes, including fatty acids and alcohols, phospholipids, liquid crystals, and polymers. Also, BAM has been shown to be a powerful method for a qualitative characterization not only of monolayers9-21 (8) Qui, A. M.; Ruiz-Garcia, J.; Stin, K. J.; Knobler, C. M. Phys. Rev. Lett. 1991, 67, 703. (9) Ho¨nig, D.; Mo¨bius, D. J. Phys. Chem. 1991, 95, 4590. (10) He´non, S.; Meunier, J. Rev. Sci. Instrum. 1991, 62, 936. (11) Ho¨nig, D.; Mo¨bius, D. Chem. Phys. Lett. 1992, 195, 50. (12) Ho¨nig, D.; Mo¨bius, D. Thin Solid Films 1992, 210/211, 64. (13) Ho¨nig, D.; Overbeck, A.; Mo¨bius, D. Adv. Mater. 1992, 4, 419. (14) Overbeck, A.; Ho¨nig, D.; Mo¨bius, D. Langmuir 1993, 9, 555. (15) Kaercher, T.; Ho¨nig, D.; Mo¨bius, D. Int. Ophthalmol. 1993, 17, 341. (16) Rivie`re, S.; He´non, S.; Meunier, J.; Schwartz, D. K.; Tsao, M.W.; Knobler, C. M. J. Chem. Phys. 1994, 101, 10045.

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Scheme 1. Distribution of the Porphyrin Molecules in a Monolayer of TMPyP/DMPA, Molar Ratio 1:4, at the Air-Water Interface as a Monomer-Dimer Equilibrium Described in Ref 22a

a

The different intensities of the molecules indicate the top and lower planes of the dimer.

at the air-water interface but also of mono- and multilayers LB transferred on solid substrates. In this work, Brewster angle microscopy has been used to infer directly the organization of a cationic water-soluble porphyrin, TMPyP, in a complex monolayer containing an anionic phospholipid, DMPA, at the air-water interface. The organization of this mixed monolayer TMPyP/ DMPA, in the ratio 1:4, has been widely described previously,22 where the interactions between porphyrin and phospholipid have been analyzed by surface pressurearea isotherms (π-A) and reflection spectroscopy under normal incidence. The molecular organization was explained with a model where the TMPyP molecules are in a monomer-dimer equilibrium depending on the surface pressure, that is, on the surface density of the porphyrin. This model is illustrated in Scheme 1 as well as the vertical section for DMPA and TMPyP in a flat orientation. In this equilibrium, the molecules of porphyrin keep a flat orientation with respect to the interface. With increasing surface pressure a layer of dense-packed porphyrin molecules is formed by interaction of the charges of the porphyrin and the negative charge of the phospholipid. In this situation, the phospholipid molecules are not yet dense-packed. A further increase of the surface pressure by reduction of the area causes porphyrin molecules to assume a position underneath the first dense-packed porphyrin layer, forming a dimer with particular stacking, which allows the porphyrin molecules of the second layer to interact with the charged phospholipid molecules at the top the first porphyrin layer. The accessibility of the lipid head groups to the second porphyrin layer requires (17) Ahuja, R. C.; Caruso, P.-L.; Mo¨bius, D. Thin Solid Films 1994, 242, 195. (18) Wolthaus, L.; Schaper, A.; Mo¨bius, D. J. Phys. Chem. 1994, 98, 10809. (19) Overbeck, G. A.; Ho¨nig, D.; Wolthaus, L.; Gnade, M.; Mo¨bius, D. Thin Solid Films 1994, 242, 26. (20) Brezesinski, G.; Scalas, E.; Struth, B.; Mo¨hwald, H.; Bringezu, F.; Gehlert, U.; Weidemann, G.; Vollhardt, D. J. Phys. Chem. 1995, 99, 8759. (21) Friedenberg, M. C.; Fuller, G. G.; Frank, C. W.; Robertson, C. R. Langmuir 1996, 12, 1594. (22) Martı´n, M. T.; Prieto, I.; Camacho, L.; Mo¨bius, D. Langmuir 1996, 12, 6554.

a stacking of the porphyrin rings lying parallel with a twist of 45° with respect to each other under the phospholipid matrix.22 Experimental Section Materials. L-R-Dimyristoylphosphatidic acid, DMPA, was purchased from Sigma Chemical Co. and used as received. 5,10, 15,20-Tetrakis(1-methyl-4-pyridyl)-21H,23H-porphine, TMPyP, was obtained from Aldrich Chemical Co. and used without further purification. A mixture of trichloromethane and methanol, ratio 3:1 (v/v), was used as spreading solvent. The pure solvents were obtained from Baker Chemicals (Germany) and used without further purification. The water for the subphase was prepared with a Millipore Milli-Q Plus system. Monolayers Visualized by BAM. Monolayers of TMPyP/ DMPA and DMPA were prepared by spreading on a large rectangular Teflon trough equipped with a movable polyethylene oxide, Dynal, barrier to minimize film leakage. The barrier is moved continuously with an analog dc motor, and its position is detected with a potentiometer. The mixed monolayer was compressed or expanded by a movable barrier with a compression velocity of 0.05 nm2 min-1 molecule-1. The trough is mounted onto a solid aluminum plate that is thermostated with a Lauda RC6 thermostat. The temperature for all experiments was maintained at 21 ( 0.5 °C. To minimize evaporation, to ensure high humidity, and to avoid pollution, this system is enclosed in a plastic box. For controlling and measuring the surface pressure, a Wilhelmy balance provided with a filter paper plate was used.23 The Brewster angle microscope (NFT, BAM2) was designed by Ho¨nig et al.13 The morphology of the spread monolayer was continuously recorded by a highly sensitive CCD camera, which was connected to a video display monitor. The image was also recorded on videotape using a SVHS videocassette recorder followed by a frame grabber card that is plugged inside a personal computer. When p-polarized light is directed on an interface under the Brewster angle no light is reflected. This angle is R ) 53.1° for the air-water interface. Keeping the angle of incidence constant, the formation of a monolayer of amphiphilic molecules changes the optical situation and the reflected light may be observed with a microscope. The Brewster angle microscope is equipped with an argon-krypton laser (wavelength used, 514 nm; beam diameter, 0.5 mm) and an achromatic lens of focal length 25 mm, (23) Fromherz, P. Rev. Sci. Instrum. 1975, 46, 1380.

Water-Soluble Porphyrin in Mixed Monolayers

Figure 1. π-A compression isotherms of a DMPA monolayer and a mixed TMPyP/DMPA ) 1:4 monolayer at the air-water interface. These isotherms have been taken from Figure 1 of ref 22, and the narrows on the isotherms correspond to the surface pressure values of the recording of Brewster angle microscopy images. T ) 21 °C. imaging the illuminated spot on the water surface onto the active area of a small CCD camera with high sensitivity. The lateral resolution of the optical system in the plane of the water surface is limited to about 2 µm. The microscope is equipped with an analyzer in front of the CCD camera for observation of domain anisotropy in the monolayer. The scale bar corresponds to 50 µm for all images. In some images, it is possible to observe cross-vertical lines (e.g. see Figure 3b) due to mechanical problems with the scanner. Also, with the aim to improve the quality of images, the contrast has been enhanced.

Results and Discussion Figure 1 shows π-A compression isotherms of DMPA and TMPyP/DMPA ) 1:4 monolayers at the air-water interface taken from ref 22, and the narrows on the isotherms correspond to the surface pressures at the recording of Brewster angle microscopy images. First, a monolayer of DMPA, without porphyrin, was prepared and its BAM images were recorded (images not shown here) at low and high surface pressures. The morphology of the monolayer of DMPA at low surface pressure shows a discontinuous structure with dark holes and no optical anisotropy, while it is homogeneous and continuous without any defined structure at high surface pressure. The mixed monolayer TMPyP/DMPA ) 1:4 was formed by cospreading the components on the water subphase, and its behavior under compression-decompression was observed by BAM. The morphology of the cospread TMPyP/DMPA monolayer, in the ratio 1:4, at the airwater interface with increasing surface pressure is shown in Figure 2. Immediately after cospreading the mixed solution of TMPyP/DMPA, molar ratio 1:4, a surface pressure of π ≈ 0.2 mN/m was measured and no optical signal due to the presence of the monolayer was obtained from the airwater interface. This fact is related to a low density of the lipid monolayer in the gaseous phase. This morphology in comparison with that of the lipid matrix is totally different at low surface pressure as well as high π values. It could be concluded that the porphyrin molecules match somehow the distribution of the DMPA molecules in the matrix.

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When the surface pressure was increased to aproximately 5 mN/m, a continuous, homogeneous film with high reflectivity and no apparent optical anisotropy was observed (see Figure 2a). The increased reflectivity of the monolayer with respect to π ≈ 0.2 mN/m is related to the increased thickness and density of the film. However, it has not been possible to determine the thickness due to the limit of detection of the system. Under further compression, at a surface pressure of π ≈ 8 mN/m, small domains with higher brightness than that of the surrounding area are formed. Figure 2b shows those small domains for the mixed monolayer at 10 mN/ m. The surface density of TMPyP in the mixed monolayer increases with increasing surface pressure. Consequently, the density of the bright domains increases until they cover nearly the whole surface at 35 mN/m. During compression only the fractions of the homogeneous dark areas as well as of the bright domains change but not the reflectivities of these two phases. These BAM results have to be related with those obtained by reflection spectroscopy at the air-water interface as a basis of the model of monomer-dimer equilibrium analyzed in detail elsewhere (see Scheme 1).22 At 8 mN/m the mixed monolayer TMPyP/DMPA contains the porphyrin as monomer only. In this case, the porphyrin molecules occupy the whole area underneath the lipid monolayer in a flat orientation with respect to the air-water interface. At 8 mN/m the area per DMPA, ADMPA, in the pure monolayer is 0.46 nm2 and that in the mixed monolayer is 0.84 nm2 (see Figure 1). Therefore, in the mixed monolayer, the porphyrin determines the area, and according to the mixing ratio TMPyP/DMPA ) 1:4 (selected due to stoichiometry) an area of 3.36 nm2 per porphyrin spread is found at 8 mN/m. This value is in close agreement with the area of a flat-lying molecule of TMPyP, 3.2 nm2. At 8 mN/m, the porphyrin forms a densepacked layer underneath the DMPA matrix in a liquidexpanded state. Thus, a homogeneous structure as observed by BAM (see Figure 2a) is expected. Upon further compression, all porphyrin molecules cannot be accommodated in the monolayer plane. Therefore, a phase (phase II, see Scheme 1) with porphyrin dimers in equilibrium with the phase of the porphyrin monomer is formed (phase I in Scheme 1). As mentioned above, the formation of the dimer is attributed to the strong tendency of porphyrin to associate and to the accessibility of the negatively charged head groups of the lipid matrix.22 The beginning of the formation of the dimer phase seems to be in agreement with the appearance of domains for surface pressure values close to 10 mN/m (see Figure 2b). Therefore, we attribute the bright domains to the phase of porphyrin dimers underneath the matrix of densepacked DMPA molecules. The area fraction of the domains increases with increasing surface pressure, that is, surface density of the porphyrin molecules, as illustrated by the BAM images recorded at 15, 25, and 35 mN/m (see Figure 2c, d, and e, respectively). The reversibility of the observed molecular packing was tested by decompression of the cospread monolayer from π ≈ 35 to 11 mN/m (see Figure 2f). As can be seen, the area fraction of the bright, small domains is similar to that obtained for 15 mN/m during the compression process (see Figure 2c). This means a slight hysteresis. This fact was also observed in the π-A isotherms during the decompression cycles of the TMPyP/DMPA ) 1:4 monolayer (see Figure 2 in ref 22). When we attribute the appearance of the bright domains to the formation of the dimer phase II, the DMPA molecules

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Figure 2. Morphology of a mixed monolayer of TMPyP/DMPA ) 1:4 at the air-water interface under different surface pressures visualized by Brewster angle microscopy: (a) 5 mN/m; (b) 10 mN/m; (c) 15 mN/m; (d) 25 mN/m; (e) 35 mN/m; (f) ∼11 mN/m during the π-A expansion isotherm. T ) 21 °C.

are dense-packed with an area of ADMPA,II ) 0.40 nm2. The homogeneous darker area is attributed to the phase with porphyrin monomers. In phase I, the DMPA molecules are in a liquid-expanded state with the area ADMPA,I ) 0.84 nm2 according to stoichiometry. At surface pressure > 8 mN/m the average area per DMPA molecule is

ADMPA ) fIIADMPA,II + (1 - fII)ADMPA,I

(1)

where fII is the area fraction of phase II. Therefore

fII )

ADMPA,I - ADMPA ADMPA,I - ADMPA,II

(2)

The evaluation of the area fraction of the domains and of the darker homogeneous phase, respectively, at each surface pressure recorded was done to test this model. The fractions of the bright, dimer phase were calculated from the different areas of the images in a random manner. The result is shown in Figure 3, where the area fraction of the bright domains (full circles) evaluated by BAM is plotted versus average area per DMPA. The straight line

Figure 3. Plot of the area fraction of the bright domains (full circles) recorded by BAM versus the average area of DMPA ADMPA. For comparison, the area fraction of the dimer phase obtained by the analysis of reflection spectra described in ref 22 is introduced as empty circles. The straight line corresponds to the theoretical fraction of dimer22 described in eq 2.

represents eq 2 with the paremeters given above, assuming a dense-packed monolayer of phase I at ADMPA,I ) 0.84 nm2 and phase II at ADMPA,II ) 0.40 nm2. For comparison

Water-Soluble Porphyrin in Mixed Monolayers

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Scheme 2. Structure of the Phase of the Bright Domains, Dimer Phase Inferred from the BAM Imagesa

a

The different intensities of the molecules note the two planes of the dimer.

the values of the area fraction of the dimer phase II obtained by analysis of reflection spectra described in ref 22 are introduced as empty circles. An amazingly good agreement between the results obtained by both methods is found, strongly supporting the assignment of the dimer phase of porphyrin molecules in the mixed monolayer of TMPyP/DMPA, 1:4, to the bright domains observed directly at the air-water interface by BAM. In conclusion, the behavior of the mixed monolayer of TMPyP/DMPA ) 1:4 on water at surface pressures g 8 mN/m can be described by the coexistence of two phases. Phase I consists of dense-packed flat-lying porphyrin monomers underneath the DMPA matrix in a stoichiometric ratio, with lipid molecules in the liquid-expanded state. In phase II, the porphyrin molecules form flatlying dimers, and the lipid is dense-packed to a liquidcrystalline phase. Consequently, in the bright domains, eight molecules of DMPA are binding two flat molecules of porphyrin twisted 45° with respect to each other (Scheme 2),22 in contrast to the case of the darker homogeneous phase, where only four molecules of DMPA are bound to

one flat molecule of porphyrin. This morphology (see Schemes 1 and 2) consists of regions with high dipole density (domain areas) and a homogeneous phase of low dipole density. Therefore, the different electrostatic energy should give rise to some tension between both phases. This phenomenon has been observed elsewhere.24 To obtain more information about the effect of this energy difference on the morphology, further studies have to be done. As a contribution to this topic, the influence of the different ionic strengths in the subphase on the monolayer behavior has been studied, and the results have been published elsewhere.25 Acknowledgment. The authors wish to express their gratitude to the Spanish DGICyT for financial support (Project PB94-0446) and to fonds der Chemischen Industrie, Germany. LA971162K (24) Mo¨hwald, H. Annu. Rev. Phys. Chem. 1990, 41, 441. (25) Prieto, I.; Camacho, L.; Martin, M. T.; Mo¨bius, D. Langmuir 1998, 14, 1853.