Photochemistry and Photobiology Vol. 46, No. 6. pp. 953-957, 1987 Printed in Great Britain. All rights rcserved

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PORPHYRIN-LIPID INTERACTIONS AT THE AIR-WATER INTERFACE IN SPREAD MONOLAYERS. A FLUORESCENCE STUDY.

Maria Bohorquez l , Larry K. Patterson" and Daniel Brault" l p 2

Radiation Laboratory and Department of Chemistry,

Laboratoire de Biophysique, INSERM U.201, CNRS UA.481, University of Notre Dame, IN 46556, USA

Mus6um National d'Histoire Naturelle, 61 Rue Buffon, 75005 Paris, France

(Received 15 July 1987 : accepted 14 August 1987)

Abstract - In membrane systems, carboxylic porphyrins may interact with both the lipid pseudophase and the adjacent aqueous environment through their hydrophobic core and their polar acid chains, respectively. These interactions are monitored in model membrane systems, i.e. spread mono- layers of dioleoylphosphatidylcholine as functions of lipid organization and pH of the aqueous subphase using steady state and time resolved fluo- rescence techniques. In all cases contact between porphyrin and aqueous subphase, as indicated through quenching by I-, is observed at low surface pressure. This contact decreases and becomes almost insignificant as the monolayer approaches maximum organization through compression. On deproto- nation of the monocarboxylic porphyrin, methylpyrroporphyrin, increased contact with water is observed in liquid compressed monolayers. In liquid expanded layers, however, it appears that organization of lipid molecules surrounding this dissymmetric charged form affords some isolation from water. The effect of esterification of carboxylic chains is also examined.

INTRODUCTION

Damage to cellular membrane structures (plasmic membrane, mitochondria, lysosomes . . . I appears to be associated with key events in porphyrin sensitized pho- todynamic therapy of tumors as well as in porphyrie, a disease resulting from dis- turbance in porphyrin metabolism (Kessel, 1977 ; Salet and Moreno, 1981 ; Hilf et al.. 1984 : Santus et al., 1983 : Sandberg et al., 1982). As most porphyrins have very similar photosensitizing properties (Keene et al., 1986), the photobiological effects are mainly governed by the affinity of porphyrins for lipidic structures and by their localization within the membranes. Using egg phosphatidylcholine vesicles as membrane models, it has been shown that interactions of carboxylic porphyrins with the lipidic bilayer are dominated by both the hydrophobicity of the porphyrin core and the charge of the carboxylic side chains which depends on pH (Brault et al.. 1986). To elucidate the role that membrane organization can play in these pro- cesses, interactions of carboxylic porphyrins with lipids and water are now examined in spread monolayers of dioleoylphosphatidylcholine (D0L)I. This model system allows for continuous alteration of lipid organization by changing surface pressure (Caines, 1966). Additionally, the state of organization can be characterized via

* Correspondance to L.K.P. and D.B. to Notre Dame and Paris, respectively.

Abbreviations : DOL : dioleoylphosphatidylcholine : DP : deuteroporphyrin : DPDME : deuteroporphyrin dimethyl ester : PYR : methylpyrroporphyrin ; PYREE : methyl- pyrroporphyrin ethyl ester.

PAP 4b:b-A

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surface pressure-area measurements taken during compression of the layer. DOL was chosen as a lipid matrix because it exhibits liquid-like characteristics throughout the range of surface pressures used and complications due to phase transitions in the layer may be avoided. Porphyrin-lipid interactions are monitored at the molecu- lar level using steady state and time resolved fluorescence techniques. Quenching by iodide added to the aqueous subphase is used to amplify the changes in fluorescence lifetimes, and to probe porphyrin-aqueous phase interaction.

MATERIALS AND METHODS

The structures of the porphyrins used are shown in Fig. 1 . Methylpyrro- porphyrin ethyl ester (PYREE) was purchased from Aldrich. Methylpyrroporphyrin (PYR) was obtained from the ester form by hydrolysis in 25 $ HC1 for 48 hours. After neu- tralization of the solution with NaHC03, the porphyrin was extracted with CH2C12. PYREE and PYR were chromatographed on silica gel according to methods derived from Vever-Bizet et al. (1984). Mixtures of CH2C12 : CH3CH20H : CH3COOH and CH3COCH3 : CH2C12 : HC1 : H20, were used as eluent systems, respectively. As shown by high performance liquid chromatography, the final purity exceeded 98 $. Deuteroporphyrin (DP) and its dimethyl ester form (DPDME) were prepared and purified as described elsewhere (Brault et al., 1986).

Fig. 1. Structure of porphyrins. (a) R = H, methylpyrroporphyrin

(PYR) ; R = C2H5, methylpyrroporphyrin CH. ethyl ester (PYREE). (b) R = H, deuteropor-

phyrin IX (DP) : R = CH , deuteroporphyrin CH, cn,

I I CH, I CH, I IX dimethyl ester (DPDMEj.

b CO,R CO,R

Monolayers of dioleoylphosphatidylcholine DOL - (Avanti Polar Lipids, USA) were prepared in a rectangular teflon Langmuir trough enclosed in a temperature controlled chamber continuously flushed with nitrogen. The aqueous phase was buffe- red to pH 9 or 4.6 with 1 mM sodium borate o r sodium acetate, respectively. For the fluorescence lifetime measurements, 0.4 M sodium iodide was added. A chloroform solution of porphyrin and lipid was applied to the water surface and allowed to stand for 20 min. The porphyrin/lipid ratios were 1:lOO and 1:30 for the time resol- ved and steady state fluorescence mtasurements, respectively. The layer was then compressed at rate not exceeding 1 A2.molecule-l min-1. The surface pressure was measured using a Wilhemy plate attached to a Cahn 2000 electrobalance. The fluores- cence measurements on the mixed DOL-porphyrin monolayers were performed at various stages of compression. The background signal from the water phase was measured be- fore spreading the monolayer and substracted from subsequent measurements. Steady- state fluorescence spectra were recorded in ratio mode using a fiber optic system connected to a Spex-Fluorolog single photon counting fluorimeter as described else- where (Agrawal et al., 1985). The xenon lamp of the apparatus was used for excita- tion (400 nm). The fluorescence lifetime measurements were performed as described elsewhere (Subramanian and Patterson, 1985) except the exciting light (353 nm) was provided by the third harmonic of a Quantronix Neodynium-YAG laser giving 100 ps

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pulses with a repetition rate of 5 KHz.

RESULTS AND DISCUSSION

The emission spectrum of PYR in a compressed DOL monolayer is shown in Fig. 2a. It is characterized by major peaks at 625.7 and 692.7 nm and apparent fine structure. All these features are characteristic of monomeric porphyrins in a hydro- phobic environment (Brault et al., 1986). No significant shift in the peak position was observed when the pH of the aqueous subphase was changed from pH 9.0 to 4.6. This indicates that, in the compressed monolayer, PYR is essentially buried in the non polar part of the phospholipid layer whatever the pH of the subphase. The pK of the carboxylic side chains of porphyrins in water has been reported to be in the range 5-6 (Brault e t al., 1986). In vesicles of egg lecithin this pK was shown to move by = 1.5 units toward higher pH. A similar, though perhaps smaller, effect would be expected with DOL monolayers. In a study on phosphatidylcholine vesicles, it was shown that the deprotonated form of deuteroporphyrin obtained at high pH remains incorporated within the lipidic bilayer although it bears two negative charges (Brault et al., 1986). In the same way, the more hydrophobic PYR remains incorporated within the monolayer even when it is deprotonated. It is very likely that in both cases the porphyrin is oriented in the lipidic layer with its propionic side chain(s) pointed towards the polar water interface. In contrast with behaviour observed in vesicles, it was not possible to obtain mixed deuteroporphyrin DOL mono- layers, probably because this molecule, with two acidic groups is too soluble in water. Of course, this is not unexpected as the two model membrane systems differ in their lipid/water ratios by several orders of magnitude.

Fig.2. Emission profiles for porphyrins in DOL monolayers at the nitrogen-water inter-

face. Excitation wavelength was 400 nm. a) PYR, pH of the aqueous subphase = 9, surface pressure = 20 dyn.cm-l b) DPDME, pH of the aqueous subphase = 9, surface pressure = 0 (---I and 20 dyn.cm-l (-1.

The positions of the fluorescence maxima of PYR were found little depen- dent on surface pressure. A larger dependence was found with the esterified dicar- boxylic porphyrin, DPDME as depicted in Fig. 2b. On compression of the layer from 0 to 20 dynlcm, the main emission band of DPDME was shifted to the red by 3 nm and the fine structure became more apparent reflecting a decreased exposure of the porphyrin to the polar lipid water interface.

Fluorescence lifetimes of porphyrins are little dependent on their envi- ronment in homogeneous solutions (Andreoni et al., 1983). In the present study, a dynamic fluorescence quencher, sodium iodide, was added to the aqueous subphase. A decrease in fluorescence lifetime is thus expected if the chromophore interacts with the water interface containing iodide. Lifetime values as functions of surface pres- sure will thus give information concerning isolation of the chromophore from the aqueous phase. In all the systems investigated, each fluorescence decay consisted

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mainly of a single mono-exponential component. Continuous changes in fluorescence lifetime of porphyrins were observed on monolayer compression. These data are given in Fig. 3. The curves can be characterized by their contours as a function of sur- face pressure, the amplitudes of lifetime change and initial and final lifetime values. These characteristics are seen to be dependent on both the pH of the aqueous subphase and the esterification of the porphyrin carboxylic side chains. As expec- ted, in all cases, increasing isolation from water is observed on monolayer compres- s i o n which brings about a higher degree of lipid organization and, perhaps, extru- sion of the aqueous phase. However, in the compressed monolayer the extent of non- esterified porphyrin isolation appears to depend on the pH of the aqueous subphase (Fig. 3a). The carboxylic group of PYR will be ionized when the pH of the subphase is 9. The interaction of this more polar form with the aqueous interface is expected to be greater than that of the neutral form obtained in systems having a slightly acidic subphase. In the latter case, the neutral porphyrin is expected to be well buried into the non polar region of the monolayer. A longer life time is observed, accordingly.

_ _ _ _ _ _ _ _ - _ _ - - - - -

Fig.3. Fluorescence lifetime of porphy- Y rins in DOL monolayers as a func- - 0 tion of surface pressure. Y - - - - - - - - - - - - - - - - - - a) PYR, pH of the aqueous subphase = 4.6

1 -=/-MF

/:Ti m : . .

'"1 ST (-0-1 or 9 (-1. b) DPDME (+), PYREE (+), pH of the aqueous subphase = 9. The dashed line represents lifetime for unquenched fluorescence.

SURFACE m i s s u n i ldvn ern-'^

More surprisingly, in non-compressed monolayers, the deprotonated charged form of PYR appears more isolated than the neutral form. It is suggested that the polar and dissymmetrical structure of the ionized PYR lead to some organization of the lipids in its vicinity affording self isolation. In addition, the negative charge of the propionic group may repel iodide ions.

As exemplified in Fig. 3b, the curves of lifetimes versus surface pressure for the porphyrin ester DPDME and PYREE were characterized by S shapes. The esteri- fication of the carboxylic acids makes the molecule more hydrophobic but more impor- tantly strongly reduces the dissymmetry of the molecule with regard to polarity. In non-compressed monolayers, it is believed that the molecule may have little privi- leged orientation. The porphyrin core of DPDME may thus be little isolated. This situation will diminish with increased organization of the lipids which may force the porphyrin into non polar environment and/or extrude water from the lipid region. This scheme would also account for the larger dependence of steady state fluores- cence spectra on monolayer compression observed with DPDME. As expected, these phenomena are observed to a lower extent with the more hydrophobic PYREE . It is worth noting that for some cases, at the highest level of monolayer organization, interaction with the aqueous subphase, as reflected in I- quenching, is decreased by more than 90 $ when compared to the expanded layer.

In conclusion it has been shown that steady state and time resolved fluo- rescence spectroscopy in spread lipid monolayers provide an excellent approach to the characterization of porphyrin-lipid interactions. The interactions of porphyrins

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within membranes may be seen to depend in a subtle way on the dissymmetry of porphy- rin structure and charge. These parameters are believed to control to an important extent photodamage in biological membranes and must be considered in any mechanism which addresses phototherapeutic effects.

Acknowledgement. - The research described herein was supported by the Office of Basic Energy Sciences of the U.S. Department of Energy and ATP CBM 6931 from CNRS. This is Document No. NDRL 3006 from the Notre Dame Radiation Laboratory.

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Agrawal, M.L., J.P. Chauvet and L.K. Patterson (1985) Effects of molecular orga- nization on photophysical behavior : lifetime and steady-state fluorescence of chlorophyll a singlets in monolayers of dioleoylphosphatidylcholine at the ni- trogen-water interface. J. Phys. Chem. 2. 2979-2982.

Brault. D., C. Vever-Bizet and T. Le Doan (1986) Spectrofluorimetric study of por- phyrin incorporation into membrane models. Evidence for pH effects. Biochim. Biophys. Acta 857, 238-250.

Hilf, R., D.B. Smail, R.S. Murant, P.B. Leakey and S.L. Gibson (1984) Hematoporphy- rin derivative-induced vhotosensitivitv of mitochondria1 succinate dehvdroaenase - - and selected cytosolic -enzymes of R32jOAC mammary adenocarcinomas of rats. Can- cer Res. *, 1483-1488.

Keene, J.P., D. Kessel. E.J. Land, R.W. Redmond and T.C. Truscott (1986) Direct detection of singlet oxygen sensitized by hematoporphyrin and related compounds. Photochem. Photobiol. 3, 117-120.

Kessel, D. (1977) Effects of photoactivated porphyrins at the cell surface of leuke- mia L1210 cells. Biochemistry 16, 3443-3449.

Salet, C. and C. Moreno (1981) Photodynamic effects of hematoporphyrin on respira- tion and calcium uptake in isolated mitochondria. Int. J. Radiat. Biol. 2, 227-230.

Sandberg, S., I. Romslo, G. Hovding and T. Bjorndal (1982) Porphyrin-induced photo- damage as related to the subcellular localization of the porphyrins. Acta Derma- tovener (Stockholm) 100, 75-80.

Santus, R., C. Kohen, E. Kohen, J.P. Reyftmann, P. Morlikre, L. Dubertret and P.M. Tocci (1983) Permeation of lysosomal membranes in the course of photosensitiza- tion with methylene blue and hematoporphyrin : study by cellular microspectro- fluorimetry. Photochem. Photobiol. 3, 71-77.

Subramanian, R. and L.K. Patterson (1985) Effects of molecular organization on photophysical and photochemical behavior. Interactions involving l-pyrenedode- canoic acid and 4-dodecylaniline at the nitrogen-water interface. J. Phys. Chem. - 89, 1202-1205.

Vever-Bizet, C., 0. Delgado and D. Brault (1984) The purification of hematoporphyrin IX and its acetylated derivatives. J. Chromatogr. 2, 157-163.