2330
Langmuir 1991, 7, 2330-2335
Photochemically Controlled Ion Permeability of Liposomal Membranes Containing Amphiphilic Azobenzene Tomoo Sato,' Masato Kijima, Yoshihiro Shiga, and Yoshiro Yonezawa Department of Industrial Chemistry, Faculty of Engineering, Kyoto University, Yoshida, Sakyo-ku, Kyoto 606, J a p a n Received February 5, 1991. I n Final Form: May 21, 1991 We have succeeded in reversible control by the photochemical method of the K+ permeability across a membrane of liposomes consisting of L-a-dimyristoyl phosphatidylcholine (DMPC), dicetyl phosphate (DCP),and 4-octyl-4'-(5-carboxypentamethyleneoxy)azobenzene(8A5) (molar ratio, [DMPC]:[DCP]:[8A5] = 10:1:0-1). In the dark at 288 K, permeability coefficient, P, was 10-1"10-13 cm/s. When liposome dispersion was irradiated with UV light, trans-to-cis photoisomerization of 8A5 occurred and two regions distinguished by variation of P with time were observed, the 'main region" and 'following region". In the former region, Pincreased rapidly to saturated value immediately after the onset of UV light and remained constant for >30 min. In the latter region, P slowly increased again. In the "main region", 5000 times enhancement of P was observed, which resulted in P of lo-* cm/s. When the liposome dispersion was irradiated with visible light after UV irradiation, P decreased again to the original value. The exponential relation between P and molar ratio of [cis-8A5]to [DMPC] has been established. It has been found that permeability of the bulky ion having negative charges, [Fe(CN)#-, is also increased by UV irradiation. Steady increase of P in the "following region", which occurred after prolonged UV irradiation at 288 K, was attributed to deformation and mutual fusion of liposomes.
Introduction Photoinduced electron transfer and energy transfer processes in biomembranes and artificial membrane systems have received a considerable amount of attention.'* Photoinduced ion transfer, i.e., photoinduced change of ion permeability across the plasma membrane, is another important process in biomembranes in connection with visual systemsSeSimulation of visual systems by artificial membranes would be interesting in relation to exploration of novel optical sensors, information storage media, and imaging materials. From such viewpoints, the lipid membrane systems containing rhodopsin and bacteriorhodopsin have been prepared to investigate photoinduced ion transfer.'+ The lipid membranes containing synthetic dye molecules capable of photoisomerization, e.g., azobenzene and spiropyran, would be regarded as the simple model of the visual systems. Although a crucial role of photoinduced enzyme cascade in the visual system has been well-known,6 such a process seems to be far beyond the scope of biomimetic chemistry in the present status. Kano et al. observed that UV irradiation of the liposome including amphiphilic azobenzene caused the increase of water permeability from outer phase to inner phase.'O Okahata and co-workers prepared the microcapsules in which micropores were covered with lipid (1) Fendler, J. H. Chem. Reu. 1987,87,877; J. Phys. Chem. 1985,89, 2730; J. Phys. Chem. 1980,84,1486.
( 2 ) Kuhn, H.; Mtibius, D.; Bllcher, H. In Physical Methods of Chemistry; Weissberger, A., Roaaiter, B., Eds.; Wiley: New Yolk, 1972; Vol. 1, Part IIIB, Chapter VII, p 577. (3) Hurst, J. K.; Thompson, D. H. P. J. Membrane Sci. 1986, 28, 3. (4) Sato, T.;Yonezawa, Y.; Hada, H. J. Phys. Chem. 1989,93, 14. ( 5 ) Yonezawa, Y.; Hayashi, T.; Sato, T.; Hada, H. Thin Solid Films
1989,180, 167. (6) For example, Pugh, E. N., Jr., Miller, W. H., Eds. Annu. Reu.Physiol. 1987, 49, 711. (7) O'Brien, D. F. Photochem. Photobiol. 1979,29,679.
(8)Dmzon, A.; Montal, M.; Zarco, J. Biochem. Biophys. Res. Commun. 1977, 76, 820. (9) Racker, E. Biochem. Biophys. Res. Commun. 1973,55, 224. (10) (a) Kano, K.; Tanaka,Y.; Ogawa, T.;Shimomura, M.; Kunitake, T.Photochem.Photobiol. 1981,34,323. (b) Kano,K.;Tanaka,Y.; Ogawa, T.;Shimomura, M.; Okahata, Y.; Kunitake, T.Chem. Lett. 1980,421.
membranes containing amphiphilic az0benzene.l' Permeability of NaCl across the membrane of the microcapsules increased with UV light and gave back to the original value by visible light. Azobenzene-bridged crown ether12 and spiropyran with long alkyl chain13were used to control K+permeability of polymer membranes under irradiation. Although photoisomerization of azobenzene derivatives surely affects the ion permeability of the membranes, details of the change of the permeability, related with dye concentration and the ratio of isomerization, are still not clear. Since the liposomes are fairly defect-free, they are useful for the study of physicochemical properties of bilayer membranes. However, the structure of liposomes sometimes suffers considerable change by mutual fusion, which renders the reversible permeability control rather difficult. In this study, we have prepared the liposomes composed of L-a-dimyristoylphosphatidylcholine (DMPC) and a small amount of dicetyl phosphate (DCP) (DMPC: DCP liposome, molar ratio [DMPC]:[DCP] = 1O:l) and doped them with 2.5-10 mol 96 4-octyl-4'-(5-carboxypentamethy1eneoxy)azobenzene (8A5). The K+permeation and isomerization of 8A5 have been examined a t the same time to establish the relationship between photoisomerization and ion permeability change in the liposome dispersion. In addition, the first experimental observation of the reversible control of permeability by alternate irradiation with UV and visible light is reported here.
Experimental Methods Materials and Methods. DMPC (Sigma, 99% purity), BAS (DojindoLaboratories),and DCP, NaCl, KCl, KaFe(CN)e,CHCla, NaHzP04 (nacalai tesque, special grade), 1-propanol,KHpPO4, and Na2HP04(Wako Chemicals, special grade)were used without further purification. Singly distilled water was used throughout the work. (11) Okahata, Y.; Lim, H. J.; Hachiya, S . J.Chem. SOC., Perkin Tram. -2 -1984.989. - - -, - - -. (12) Kumano, A.; Niwa, 0.;Kajiyama, T.; Takayanagi, M.; Kano, K.; Shinkai, S. Chem. Lett. 1983, 1327. (13) Shimidzu, T.;Yoahikawa, M. Polym. J. 1983, 15, 631.
0743-7463/91/2407-2330$02.50/0 0 1991 American Chemical Society
Photochemically Controlled Ion Permeability The small unilamellar vesicles (SUV)which contained KC1in the inner aqueous phase were prepared in a conventional manner"-16 in the dark. The mixture of 72 pmol of DMPC, 7.2 pmol of DCP, and 1.8-7.2 pmol of 8A5 in CHCl3 was dried in a round-bottom flask in vacuoat 313K by using a rotary evaporator. DCP gave negative charges to the liposomal membrane. After CHCls was perfectly removed by the stream of N2 gas and thin lipid layers were formed at the bottom of the flask, a 20-mL aqueous buffer solution (SI buffer) was added into the flask and the flask was vigorously shaken at 313 K until all lipid films were stripped from the wall. The S1 buffer contained 0.5 M (1M = 1 mol/dmS) KCl, 0.1 M NaCl, 1.9 mM NaHzPO4, and 8.1 mM NazHPOl (pH = 7.4). The lipid solution was then sonicated with a 300-W probe-type sonicator, Model US-300(Nippon Seiki Co., diameter of the probe, 26 mm), for ca. 20 min at 313 K under Nzbubbling. The K+ in the bulk solution was removed by the gel-filtration method with a column filledup with SephadexG-50 (Pharmacia). The eluent was an aqueous buffer (G1 buffer) containing 0.6 M NaC1,l.g mM NaHzPOd, and 8.1mM NazHPOd (pH = 7.4) in order to reduce osmotic pressure difference across the liposomal membrane. The temperature of the column and the eluent was held below 283 K. The optical density of the liposome dispersion passing through the column was monitored at X = 400 nm with a UV 120-01spectrophotometer (Shimadzu). The concentration of DMPC in the liposome dispersion was determined by the choline-oxidase-phenol method" with a colorproducing kit, Phospholipid B-Testwako (Wako Chemicals). The reaction cell used for photoisomerization of 8A5 was a rectangular vycol vessel, 15x 38 X 48 mm in size with a light path length of 15 mm. The cell was set up in a dark box and ca. 20 mL of liposome dispersion was placed in it for irradiation. Irradiation of the dispersion was carried out at the temperature T = 288 K which was lower than the phase transition temperature of DMPC, T, = 296 K.18 The liposome dispersion was stirred with a magnetic stirrer during the reaction. Two types of the light source, a 500-W high pressure mercury lamp, USH500D, with a HB-501A power supply (Ushio Denki) (Hg lamp) and a 500-W xenon lamp, UXL-500D,with a DSBdOOSS power supply (Ushio Denki) (Xe lamp), were used. UV light from the Hg lamp, wavelength 350 nm d X d 400 nm, was selected by glass filters, UV-D2 and UV-35 (Toshiba), together with a 10 wt % CuSO, aqueous solution filter. The incident photon number, as measured by a ferrioxalate actinometer,'Qwas 7.9 X lOI6 cm-2s-l. Monochromatic light from the Xe lamp was selected with an aid of a monochromator (Shimadzu Bausch & Lomb 33-86-25, slit 20 nm). The incident photon number was (1.4-1.6) X 10" cm-2 8-1 at X = 365 nm (UV light) and (3.1-3.2) X 10" cm-28-1 at X = 450 nm (visible light), respectively. During irradiation of the liposome dispersion, the 0.2-mL dispersion was taken out periodically and was diluted 5 times with the G1 buffer. The extinction spectra were measured with a spectrophotometer UV-260 (Shimadzu) to monitor the progress of photoisomerization of 8A5 molecules. To estimate the molar ratio of cis-8A5 to DMPC, X ( t ) = [cis-8A5]/[DMPC], we paid attention to the change of the absorbance of trans-8A5 at X = 350 nm, A ( t ) . The following relation was adopted under an assumption that trans-to-cis isomerization has completed after prolonged UV irradiation X ( t ) = {A(O)- A(t)J/IA(O) - A ( m ) W (1) where R denotes the molar ratio of 8A5 to DMPC, A(0) the absorbance before UV irradiation, and A ( - ) the absorbance when all trans-8A5 molecules are converted to cis-8A5. (14)Papahadjopoulos,D.; Watkins, J.C.Biochim. Biophys. Acta 1967, 136,639. (15)Johnson, S. M.; Bangham, A. D. Biochim. Biophys. Acta 1969, 193,82. (16)Huang, C. Biochemistry 1969,8,344. (17)Takayama, M.; Itoh, S.;Nagasaki, T.; Tanimizu, I. Clin. Chim. Acta 1977,79,93. (18)Chapman, D. In Form and Function of Phospholipids; Aneell, G. B., Hawthorne, J. N.. Dawson. R. M. C... Ede.:. Elsevier Scientific: Am&dam, 1973;.p117; (19)(a) Parker, C. A. Roc. R. SOC. London, Ser. A 1963,220,104.(b) Hatchard, C. G.; Parker, C. A. Roc. R. Soc. London, Ser. A 1966,235, 518.
Langmuir, Vol. 7, No. 10, 1991 2331 Transport of the K+ from the inner phase to the outer phase through the liposomal membrane was monitored by measuring the increase of the K+ concentration in the outer phase, C. In situ measurement of C was carriedout with an EA-920ion analyzer equipped with a potassium ion selective electrode 93-19 (Orion Research). We gave heed to the drift of electrode potential immediately after inserting the electrode into the liposome dispersion. To estimate the final concentration of K+,C,, which corresponded to the situation where all possible K+ in the inner phase was leaked to the outer phase, the liposomes were destroyed by adding 0.5 mL of l-propanol to the 0.5-mL liposome dispersion and then diluted to 10 mL with water. C, was determined by atomic absorption spectrometry with a Jarrel-Ash AA-8200spectrophotometer. Calculation of Permeability Coefficient. The trapped volume of the liposomesper mole of lipid, VT (cm3/mol),is given approximately by VT = (C, - Co)/C: (l/LC) (2) where Co denotes K+ concentration in the outer phase of the liposome dispersionimmediately after gel filtration, Ci0 the initial K+ concentration in the inner phase, and LC (mol/cm3)the total lipid (DMPC + DCP) concentration. Assuming the spherical liposomewith the diameter d (cm) and thickness L (cm),another expression of VT is obtained VT = Vi OA NA/S OA
(d-!W3 d2+(d-2L)2
where Vi (cm3)denotes the total volume of the inner phase, S (cm2)the sum of the outer and inner surface area of the liposomalmembrane, OA (cm2)the occupiedarea of one lipid molecule in the membrane, and N AAvogadro's number. To estimate d from eq 3b, OA of 6 X 10-16 cm2 and L of 6 X lO-' cm were assumed.20 When K+permeation acrws the liposomalmembrane is caused by diffusion along the concentration gradient, the permeability coefficient, P, is introduced by the following equation16a1 (4) where V denotes the volume of outer phase and Ci the K+ concentration of inner phase. Mass balance of K+ leads to the following equation:
c,(v+vi)=civi+cv
(5)
After eliminationof Ci from eqs 4 and 5 and severalmanipulations, under consideration of eq 3a and the inequality, V >> Vi, P is finally given by
The dependence of C on time was measured and the differential value,d(C/C,)/dt, wasevaluated byusingapolynomialsmoothing method22 to estimate P.
Results Preparation of Liposomes. A gel filtration curve of the liposome dispersion had a single peak, indicating that most liposomes were in the form of SUV. The fraction corresponding to about 90 % of the peak area was collected and used in this study. The volume of the dispersion was ca. 30 mL. The liposome dispersion was transparent enough because of low light scattering in the visible region (20)A waa calculated from the surface area of DMPC or DCP monolayer spread on water at 40 mN/m. L was supposed to be twice of molecular length of DMPC. The molecular length waa estimated from the model in Gf IO. (21)Hamilton, R.T.; Kaler, E. W. J. Phys. Chem. 1990,94,2560. (22)(a) Savitzky, A.; Golay, M. J. E. Anal. Chem. 1964,36,1627.(b) Bromba, M. U.A,; Ziegler, H. AM^. Chem. 1979,51,1760.
Sato et al.
2332 Langmuir, Vol. 7, No. 10, 1991 4.0 I I
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Figure 2. Increase of X ( t ) ([~k-8A5][DMPC]) with UV irradiation (A = 365 nm, Hg lamp). [DI& C]:[DCP] = 101 liposome dispersion with [8A5]/[DMPC] = 0.10 (l),0.05 (21, and 0.025 (3). [DMPC]= (2.15-2.3) X lO+M. UVirradiationstarted at 10 min in the figure. T = 288 K.
Wavelength ( n m ) Figure 1. Extinction spectra of the liposome dispersion ([DMPC]:[DCP] = 101) with [8A5]/[DMPC] = 0.05 in aqueous buffer (see text) before (1) and after (2-10) UV irradiation (A = 365 nm, Hg lamp). [DMPC] = 2.3 X M. Irradiation time: 1,O min; 2, 1 min; 3, 2 min; 4 , 3 min; 5,4-30 min; 6,42 min; 7,54 min; 8, 65 min; 9, 84 min; 10, 120 min. T = 288 K. Light path length
1.01
I
= 1 cm.
(A = 400-700 nm). The DMPC concentration in the liposome dispersion was in the range of (1.89-2.29) X lo4 mol/cm3. CiO and Cowere 19 600 ppm (0.5M) and 1ppm (2.55 X M), respectively. Reduction of C" less than 1 ppm was difficult in this work, since a considerable amount of K+was contained as the impurity in the NaCl used for the eluent. C, in eq 2 was estimated to be 14-20 ppm. The ratio of the trapped volume in the liposomes to total dispersion volume, Ce/Cio, was 1/1OOO to 1/1500. It appeared that V T in eq 2 was 290-400 cm3/mol and d in eq 3b was 30-34 nm. This diameter is close to the usual value for phospholipid liposomes prepared by the strong sonicating m e t h ~ d . ' O J ~ $ ~ ~ P h o t o i s o m e r i z a t i o n of 8A5 i n L i p o s o m a l Membranes. It has been known that azobenzene derivatives in CHCla solution undergo trans-to-cis isomerization by UV irradiation and cis-to-trans isomerization by visible i r r a d i a t i ~ n .The ~ ~ liposome dispersion (molar ratio [8A5]/[DMPC] = 0.05) was irradiated with UV light (Hg lamp) a t T = 288 K and the change of the extinction spectrum is shown in Figure 1. trans-8A5 molecules in the liposomes converted to cis-8A5 as evident from the decrease of the strong absorption peak a t X = 350 nm and the concurrent increase of the weak peak a t X = 450 nm. The elevation of the extinction spectrum over X = 200700nm was observed after UV irradiation for 40 min, which will be discussed later. The rate of trans-to-cis isomerization by UV irradiation was slower in the liposomes than in the CHCl3 solution. Figure 2 shows the increase of X ( t ) with irradiation for the liposome dispersion with [8A5]/ [DMPC] = 0.025, 0.05, and 0.1. After irradiation for 2 min ([8A5l/[DMPC] = 0.025), 4 min ([8A5]/[DMPC] = 0.05), or 8 min ([8A5]/[DMPC] = 0.10), almost all trans8A5 molecules changed into cis-8A5. In the dark, the rate of cis-to-trans isomerization of 8A5 in the liposomes was very slow. Irradiation with visible light led to decrease of cis-8A5 and increase of trans-8A5 (cis-to-trans photoisomerization). (23) Hauser, H.; Phillips, M. C.; Stubbs, M.Nature 1972,239, 342. (24) For example, Kumar, C. S.; Neckers, D. C. Chem. Rev. 1989,89, 1915.
0.2
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Figure 3. Increase of (a) normalized K+ concentration (C/CJ in the outer phase of the liposome and (b) log (permeability coefficient, P)of the liposomal membrane with UV irradiation (A = 365 nm, Hg lamp). [DMPC]:[DCP] = 101 liposome dispersion with [8A5]/[DMPC] = 0.05. [DMPC] = 2.3 X 10-8 M. T = 288 K. UV irradiation started at 10 min. C denotes K+ concentration in the outer phase of the liposome. C. denotes final K+ concentration (see text). UV and dark indicates that liposome dispersion is under UV irradiation and in the dark, respectively. Permeation of K+ across Liposomal Membranes. The liposome dispersion ([8A5]/[DMPC] = 0.05)was irradiated with UV light (Hg lamp) a t T = 288 K. The increase of K+in the outer phase was represented as C/C,, where Ce was 19.5 ppm, and is shown in Figure 3a. After 100 min of irradiation, the K+trapped in the inner phase a t the beginning was almost completely released to the outer phase. When the liposome dispersion was placed in the dark, release of K+ was very little, even after 900 min. Permeability coefficient, P, was evaluated from C/Ce vs time curve and the change of log P is shown in Figure 3b. Before irradiation, log P was -12.6, whose value was coincident with the constant value of P when the lipo-
Langmuir, Vol. 7, No. 10, 1991 2333
Photochemically Controlled Ion Permeability I
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Figure 4. Increase of log (permeability coefficient, P) of the liposomal membrane with UV irradiation (A = 365 nm, Hg lamp). [DMPC]:[DCP]= 1 0 1 liposomedispersionwith [8A5]/[DMPC] = 0.10 (1),0.05 (2), q d 0.025 (3). [DMPC] = (2.15-2.3) X M. T = 288 K. UV irradiation started at 10 min.
V E
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i
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Time (min) Figure 6. Variations of (a) X ( t ) ([cis-8A5]/[DMPC]) and (b) log (permeabilitycoefficient,P)of the liposomal membrane with intermittent UV irradiation (A = 365 nm, Hg lamp). [DMPC]: [DCP] = 101 liposome dispersion with [8A5]/[DMPC] = 0.10. [DMPC] = 2.0 X M. T = 288 K. UV irradiation periods: 40-41,70-71,100-101, and 115-116 min. Arrows indicate onset of UV irradiation for 1 min. Liposome dispersion is in the dark between 0 and 40,41 and 70,71 and 100,101 and 115, and 116 and 150 min.
is noticeable that time course of the change of log P in Figure 4 is similar to that of X ( t ) in Figure 2. The liposome dispersions with [8A5]/ [DMPC] = 0.030.10 were irradiated with UV light (Xe lamp) at T = 288 K. Figure 5 shows variation of X ( t ) and concomitant increase of log P with time. Also in this case, time course of the change of log P i s similar to that of X ( t ) . A so-called time lag, in which P does not begin to increase in contrast to the beginning of trans-to-cis isomerization of 8A5, is seen in Figure 5. In the liposome dispersion with [8A5]/ [DMPC] = 0.10, permeation of K+was completed before trans-to-cis isomerization finished. Reversible Control of K+ Permeability. The liposome dispersion with [8A5]/ [DMPC] = 0.10 was irradiated intermittently with UV light (Hg lamp) a t T = 288 K. The irradiation period and dark period were 1 min and several ten minutes, respectively (ON/OFF experiment). Parts a and b of Figure 6 show variations of X ( t ) and log P with intermittent irradiation, respectively. Although log P increased in accordance with increasing X (t ) by irradiation, interruption of the UV light made the increase of log P stop, as X ( t ) became constant. The stepwise change of log P much resembles the change of X ( t ) . At the fourth irradiation, almost all 8A5 molecules were converted to cis-8A5 and P became 5000 times as large as that in the dark. log P can be fixed freely in the range of -12.5 to -8.5 by regulating the totalamount of incident UV light, which determines X ( t ) . The liposome dispersion with [8A5]/[DMPC] = 0.03 was alternately irradiated with UV and visible light (Xe lamp) at T = 288 K (UV-vis experiment). As shown in Figure 7a, X ( t ) was increased by UV irradiation until saturation value of 0.03. Irradiation with visible light made
-"F0 ....
**.I. *trCII"*..UC--*.-*
d
.-
-13 0" * * '
T i m e (min) Figure 5. Increase of (a) X ( t ) ([cis-dA5]/[DMPC]) and (b) log (permeability coefficient, P) of the liposomal membrane with UV irradiation (A = 365 nm, Xe lamp). [DMPC]:[DCP] = 101 liposome dispersion with [8A5]/[DMPC] = 0.10 (11, 0.05 (21, and0.03(3). [DMPC] =2.1 x 1 P M . T=288K. UVirradiation started at 10 min.
some dispersion was placed in the dark for 900 min. log
P rapidly increased in a few minutes after irradiation, reached -10.2 (P= 5 X W1cm/s), and remained constant for 30 min (designated as "main region"). After this region,
P gradually increased again (designated as "following region"). In this section, we examine the phenomena taking place in the "main region". T o focus on the details of the variation of P with time, we plot the dependence of log P on time shorter than 15 min after the onset of UV light (Figure 4). The similar plots for the liposome dispersions with [8A5]/ [DMPC] = 0.025 and 0.10 are also given in Figure 4. Before irradiation, log P was in the range of -12 to -12.6. On irradiation, log P was increased untilsaturationvaluesof-11.2after2min([8A5]/[DMPC] = 0.0251, -10.2 after 4 min ([8A5]/[DMPC] = 0.05), and -8.5 after 6 min ([8A5]/[DMPC] = 0.101,respectively. It
2334 Langmuir, Vol. 7,No. 10, 1991
Sat0 et al. -8
Vis -- _- ._ _ _ UV _ -Vis
