Langmuir 1991, 7, 918-922
918
Evaluation of Phase Transition Temperature of Liposomes by Using Tautomerism of Benzoylacetoanilide and Effects of Cholesterol M. Ueno,*ptJ S. Katoh,+ S. Kobayashi,t E. Tomoyama,f R. Obata,? H. Nakao,? S. Ohsawa,§ N. Koyama,§ and Y. Moritag Department of Applied Chemistry, Faculty of Science, Science University of Tokyo, Institute of Colloid and Surface Chemistry, Science University of Tokyo, 1-3 Kagurazaka, Shinjuku-ku, Tokyo 162,Japan, and Products Formulation Research Laboratory, Eisai Co., Ltd., 2-14 Minami, Honjo, Saitama 367,J a p a n Received April 20,1990.I n Final Form: October 29,1990 The properties of liposomes are known to be influenced by many factors. Especially, the stability of liposomes is effected by the change of the phase transition temperature (T,) of lipids. In this work, the keto-enol tautomerism of benzoylacetoanilide (BAA) was used to measure the T,. When BAA is added to the liposome suspensions, the ketonic form exists in bulk water and the enolic form in bilayers of the liposomes. Moreover, when the temperature for the system is changed, the change of the ratio of the enolic to ketonic absorances can be expected to show a sharp peak at a temperature corresponding to the T, of lipids. Therefore, the T,value for liposomes can be estimated from the change of the absorbance ratio caused by the change of the temperature for the system. Three kinds of phosphatidylcholines were used as the standard lipids, and these data were used for determination of the T,of hydrogenated egg lecithins. Further, additional effects of cholesterol in liposomes were investigated in the same way. These results indicated that the T, values of hydrogenated egg lecithins decreased linearly with the increase of iodine values, and the T,values for most liposomes dropped suddenly above the concentration of about 15 mol ?6 cholesterol. This suggests that above this concentration cholesterol molecules are incorporated homogeneously into the bilayers and then the T,values decrease due to formation of the mixed layers such as a palisade type between cholesterol and lipid molecules. Consequently, this method for T,determination can be applied even in the diluted aqueous solution with concentration below 0.15% of lipids and is very simple and useful in comparison with common methods.
Introduction Liposomes prepared from egg lecithins have been extensively studied as models for artificial biomembranes and drug carriers in a field of medicine and pharmacy.lv2 In previous work, we have studied the preparation of stable and small unilamellar liposomes from hydrogenated egg lecithins as drug carrier^.^ Moreover, the properties of these liposomes and the inner environments of their bilayer such as the particle sizes, polarity, encapsulation efficiency, and microviscosity have been investigated by using steady-state fluorescence spectroscopy with pyrene as a probe. From these results, liposomes prepared from egg lecithin with an iodine value of 28 had the smallest size and showed the highest stability for storage and the highest kinetic microviscosity of the bilayer. In general, the degree of unsaturation of fatty acids in the lipids is measured by the iodine value, which is defined as the number of grams of iodine that combine with 100 g of lipid. Further, the lecithin molecules in these liposomes were found to be closely packed and to have a moderate flexibility.3~~So, we tried to estimate the effect of temperature on the stability of liposomes from the measurement of the phase transition temperature (?',). In general, the molecular assemblies formed from lipids are known to transfer from the gel to the liquid crystalline states a t T, and have more flexibility and permeability5 + Department of Applied Chemistry.
Institute of Colloid and Surface Chemistry. Co., Ltd. (1) Kineky, S. C.; Nicolotti, R. A. Annu. Rev. Biochem. 1977, 46,49. (2) Hotta, T. Kagaku-no Ryouiki (Bio Material Science) 1977, 117. (3) Ueno,M.;Ohkuma,A.;Ohsawa, S.; Shibusawa,K.YakugakuZasshi 1986,105, 224. (4) Ueno, M.; Kikuchi, H.;Katoh, S.;Ohaawa, S.;Shibusawa, K. Yukagaku Zasshi 1987, 36, 272. f
fi Eisai
above T,. In the case of biomembranes consisting of many kinds of lipids, the phase transition occurs continuously and so it is very difficult to determine the definite point of transition by physicochemical measurements on biomembranes. The properties of liposomes are known to be influenced by many factors such as the degree of unsaturation of fatty acid in the lipid, its Tc, or the pH and temperature of the solution. Especially, the change of the Tcof lipids is the most important factor for affecting the stability of liposomes. Many studies for the T, have been reported."16 In this work, the keto-enol tautomerism of benzoylacetoanilide (BAA) was used to measure the T,. The UV spectra of BAA in various solvents show two peaks of absorbance near 250and 315 nm corresponding to the ketonic and enolic forms, respectively. As the percentage of enolic form is much higher in organic solvents than in water, this phenomenon has so far been applied to determine the critical micelle concentration in surfactant solution^.^^ When BAA is added to liposome suspensions, the ketonic (5) Nojima, S.;Sunamoto,J.; Inoue, K. The Liposomes;Nankohdou: Tokyo, 1988. (6) Mellier, A.; Diaf, A. Chem. Phys. Lipids 1988, 46, 51. (7) Allen, T. M. Biochim. Biophys. Acta 1981, 640,386. (8) Cevc, G.; Waata, A.; Marsh, D. Biochemistry 1981,20,4965. (9) Devam, P.; McConnel, H. M. J. Am. Chem. Soc. 1972,94,4477. (10) Maeda, T.; Ohnishi, 0. Biochem. Biophys. Res. Commun. 1974, 60, 1509. (11) Levin,Y.K.;Bixdd,N.J.M.;Lee,A.G.;Metcalfe,J.Eiochemistry 1972, 11, 1416. (12) Seelig, J. Biochim. Biophys. Acta 1978, 515, 105. (13) Shinitzky, M.;Barenholtz, Y. Biochim. Biophys. Acta 1978,515, 367. .
(14) Sunamoto, J.; Iwamoto, K.; Endo, T.; Nojima, S. Biochim. Biophys. Acta 1982,685,283. (15) Kawato, S.;Kinoshita, K.; Ikegami, A. Biochemistry 1977, 16,
---". 9.114
(16) Lichtenberg, D.; Menashe, M. Lipids 1984,19, 395.
0743-7463/91/2407-0918$02.50/0 0 1991 American Chemical Society
Phase Transition Temperature of Liposomes Table I. Contents of Egg Lecithin and Structure of Phoephatidylcholine & Lecithin contanta DhC4DhOEDide 98.3% phosphatidylcholine 69.0% phosphatidylethanolamine 17.4% lyeophosphatidylcholine 1.6% cholesterol 1.7% hydrocarbon chain length
Langmuir, Vol. 7, No. 5, 1991 919 , - -.... -.- - - - - -...- ...
33.0% 63.0% 3.4%
ClS CIS C20
Structure of Phosphatidylcholine CHIOCOR
I
R'OCOCH
i&O--P-
Sol-1 60 *C
0
II
d-
OCH&Hd
CIS, dipalmitoylphosphatidylcholine(DPPC) Cld, dimyristoylphoephatidylcholiie (DMPC)
form is in the bulk water and the enolic form is in the bilayer of the liposomes. Moreover, as the temperature for the system is changed, the change of the ratio of the enolic to ketonic absorbance can be expected to show a sharp peak a t a temperature corresponding to the Tc of the lipids. Therefore, the Tcvalues can be estimated from the change of the absorbance ratio caused by the change of various temperatures for the system.18 Three kinds of phosphatidylcholines were used as the standard lipid, and these datal9 were used for determination of the Tc of hydrogenated egg lecithins. Further, effects of cholesterol in liposomes were investigated in the same way.
Experimental Section Materials. Dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine(DPPC),and distearoylphosphatidylcholine (DSPC) were supplied from Nihon Yushi Co. and used without further purification. Nine kinds of hydrogenated egg lecithins with iodine values (IV) of 28,27,23,20,17,9,8,3, and 1were supplied from Asahi Kasei Kogyo Co. The contents of egg lecithins and the molecular structure of phosphatidylcholine are shown in Table I. These values have been confirmed by liquid chromatography. Before egg lecithins are used, the degree of oxidation on egg lecithins has been estimated by measuring UV spectra of the absorbance at 233 nm corresponding to oxidation of double bonds in the fatty acid chains. These egg lecithins were kept in the freezer at 4 "C under a nitrogen atmosphere. Benzoylacetoanilide (BAA) was purchased from Tokyo Kasei Kogyo Co. and used after recrystallization from pure hexane. Cholesterol was purchased from Kokusan Kagaku Co. and used after twice recrystallization from ethanol. Water used here was purified by passing through a Milli-R/Q water purifier system (Nippon Millipore Co.) and was used after confirming several properties such as pH (7.0) and specific conductivity (below lo" R m-9. Methods. Preparation of Liposomes. Liposomes were prepared by the ethanol injection method.20J!1 The apparatus is schematically illustrated in Figure 1. The ethanolic solution of lipids, abbreviated as Sol-1,was injected into pure water with the dispenser (Auto Dispenser FH-10s Hirasawa Seisakusho Co.). When liposomes are prepared by this (17)Shoji, N.;Ueno, M.;Meguro, K. J. Am. Oil. Chem. SOC.1978,55, 297. (18)Sila, M.; Au, M.;Weiner, N. Biochim. Biophys. Acta 1986,859, 165. (19)Umo, M.;Katoh, S.;Kobayaehi, S.;Tomoyama,E.; Oheawa, S.; Koyama, N.;Morita, Y. J. Colloid Interjace Sci. 1990,134, 589. (20)Haga,. M. Kagaku-no Ryouiki 1981,3630. (21)Batzn, S.;Korn, E. D. Biochim. Biophys. Acta 1973,298,1015.
+
Sol-1
(CHJ)J
R,R'= CIS,distearoylphosphati8dylcholine (DSPC)
Pure Water 50%
.1
Liposome suspension Lecithin Additive Marker in Ethanol
Figure 1. Apparatus for liposome preparation.
Table 11. Conditione of the Injection for Liposome Preparation (a) lecithin concentration of the ethanolic solution (Sol-11, 1.5 g/60 mL (b) temperature of Sol-1, 60 "C (c) internal diameter of needle for injection, 0.6 mm (d) injection rate of Sol-1, 6 mL/5 s (e) stirring speed of pure water, 8.9 rps (f) amount of pure water, 94 mL (9) temperature of pure water, 50 "C injection method, the sizes of liposomemay be affected by several conditions.% The preparationsof liposomewere carried out under the same conditionsas those described in the previously published papers894 as shown in Table 11. The oxidation of lecithins in the liposome suspensions prepared by this method was prevented by fillingnitrogen gas fully in the flask, and the flasks were stored in a darkened place at room temperature. The Particle Size of Liposome. In general, liposomes prepared by the injection method are of the SUV type. The size is about 20-100nm.ss23 The particle sizes of these samples were measured at 25 "C by using a submicrometer particle analyzer (Coulter Model N4MD). The Tautomerism of BAA. BAA was dissolved in 6 mL of ethanol together with lecithins and then this mixed solution was injected into 94 mL of pure water. The concentration of BAA in the liposome suspension was adjusted to 2 X lod mol/L. The UV spectra were measured at several temperatures by using a UV-visible recording spectrophotometer (UV-160, Shimadzu). The transfer of BAA molecules from the internal bilayer to the bulk water phase or the reversewas evaluated from the change of the ratio of the enolic to ketonic absorbances, Aenol/Aket,,. The ketonic peak of the absorbance appeared at 250 nm and the enolic peak at 315 nm. The sample was sealed in a quartz cell, and was heated to 20 "C for 10min. After equilibrium had been established,UVspectra were recorded in the range of 360-200 nm. The temperature of the sample was gradually increased at the intervals of 5 "C from 20 to 70 "C. However, near T,,the temperature was increased at intervals of 1"C to measure the precise effect of temperature. The heating rate of samples was about 1 "C/min. Effects of Cholesterol. Addition of cholesterol to the bilayers of liposome is known to decrease the permeabilityU and (22) Murakami, Y.;Sunamoto, J. Enzyme: The Chemistry of the Membrane Model in Vivo; Nankohdou: Tokyo 1981;p 88. (23)Yamauchi, H.; Kikuchi, H. Fragrance J. 1967,115,6. (24)Inoue, K.Biochim. Biophys. Acta 1974,339,390.
Ueno et al.
920 Langmuir, Vol. 7, No. 5, 1991
Ketonic Form Enolic Form Benzoylacetoanilide
BAA Z X I O ' ~moll1
(b) 0
5
0
3
Wavelength ( n m )
Figure 2. (a)Structuralformula of BAA. (b)Absorption spectra of BAA dissolved (A) in mixed solvent consisting of water and ethanol (94/6 (v/v)); (B) in liposome suspension prepared from lecithins.
the flexibility of membrane consisting of lecithins with unsaturated fatty acid groups and, on the other hand, to cause the phase transition to disappear and raise the flexibility in the case of lecithins with saturated fatty acid groups. In this way, the addition of cholesterol causes a dual effect on the liposomes.26 In this work, the added amounts of cholesterol were changed in the range from 5 to 30 mol 5%.
Results and Discussion The Particle Size of Liposome. The average of the particle size distribution for liposome suspensions was about 80 nm and the sizes were little affected by adding other materials such as cholesterol to the single lipid system. These results for the particle size of liposomes give strong support that these liposomes are SUV types. The Tautomerism of BAA. BAA shows a tautomerism of the ketonic form and the enolic form. The molecular structure and the UV spectra of BAA in water and liposome suspension are shown in Figure 2. In mixed solvent consisting of water and ethanol (94/6 (v/v)), only one peak appeared near 250 nm for the ketonic form. But in liposome suspensions, another peak appeared near 315 nm for the enolic form. This suggests that BAA molecules incorporated from bulk water phase into bilayer change to the enolic form. The change of BAA molecules from ketonic to enolic form was observed by measuring the ratio of the enolic to ketonic absorbance (Aenol/Akeh). At first, the ratios were obtained for the liposomes prepared from standard lipids such as DMPC, DPPC, and DSPC and were plotted against temperature for each liposome as shown in Figure 3. These ratios indicated the maximum points a t 23.0 "C for DMPC liposome in Figure 3a, at 40.7 "C for DPPC liposomes in Figure 3b, and a t 54.0 "C for DSPC liposomes in Figure 3c. Each of the temperatures corresponding to these sharp maximum peak was in good agreement with the published value of T, for each of these lipids.2e-29 Therefore, the temperatures corresponding to (25) Damel, R. A.; De Kruijff, B. Biochim. Biophys. Acta 1976, 457, 109. (26) Chapman, D.; Williame, R. M.; Ladbrooke, B. D. Chem. Phys. Lipids 1967, I , 445.
the peak of the ratio were concluded to be the T, of each of the lipids.19 We have confirmed that the position for each of these peaks does not shift even with changes in most conditions of the injection except for conditions b and g in Table I1 and is reproducible for several repeats of the increase and the decrease in the temperatures above and below T,. This method was applied for the determination of the Tc of hydrogenated egg lecithins with various IV from 1 to 28. The effects of temperature on the absorbance ratios are shown in Figure 4. Each curve indicated the maximum peak, but these peaks were not as sharp and comparedto the standard lipids. This broadness is attributed to the liposome being prepared from mixture consisting of several kinds of lipids as shown in Table I. The T, of hydrogenated egg lecithins decreased linearly with increase of IV as shown in Figure 5. These results are summarized in Table I11 together with the number of double bonds for each IV. Figure 6 illustrates the liposomes incorporated (releasing) BAA molecules into (from) the bilayer with increasing temperature. At first, the enolized BAA molecules located in the bilayer near the surface of liposome are released gradually to the outer or inner water phase from the bilayer with increasing temperature. This suggests that the ratio Aenol/fieb decreases with increasing temperature. At the higher temperatures, the bilayer of the liposomes becomes loosely packed and can easily incorporate the BAA molecules. When BAA molecules are incorporated into the bilayer, the ratioAenoi/ increases steeply. As the temperature of the system becomes much higher, after passing through the T,, the movement of lecithins becomes more active, which increases the flexibility of the bilayer. Then BAA molecules are released, and the ratio decreases again. Effects of Cholesterol. The addition of cholesterol causes the T, to decrease or disappearS3OThe change of the ratioA,,I/Akeb for the additional system of cholesterol in liposomes prepared from egg lecithin with IV = 8 is shown in Figure 7. The position of the maximum peak corresponding to T, remained almost constant until about 15-20 mol % cholesterol, and above this amount, T, shifted toward the low temperature region. The changes of T, for the additional amounts of cholesterol are shown in Figure 8. All systems showed the same tendency. The T, values remained almost constant up to near the concentration of 15 mol '% cholesterol, and above this Tcdropped suddenly. This constant value for T, suggests that molecules of cholesterol can not be dissolved homogeneously in the bilayer and a phase consisting of only phospholipids as well as a mixed phase of cholesterol and phospholipids still exists in the bilayer. However, above the addition of 20 mol '% cholesterol, as the cholesterol molecules dissolve homogeneously throughout the bilayer, the T, seems to decrease due to the formation of the mixed phase between cholesterol and lipid molecules. Ohnishi et al. have reported that the shape of the DPPC liposome changes by the addition above 15 mol 7% cholester01.3~This may be related to our results obtained here, concerning the decrease of T, above 15mol 5% cholesterol. Figure 9 shows the plots of T, values against IV. T, values decreased almost linearly with the increase of IV similar to the single system of lecithins. T,-IV curves shifted downward to (27) Chapman, D. Form and Function of Phospholipids; Ansell, G. B., Hawthorne, J. N., Eds.;Ekevier: Ameterdam, 1973; p 117. (28) Van Dijck, P. W. M.;De Kruijff, B.;Van Deenen, L. L. M.; De Gier, J.; Demel, R. A. Biochim. Biophys. Acta 1976, 455, 5'6. (29) Wilkinson, D. A,; Nagle, J. F. Liposomes; Knight, C. G.,Ed.; Elsevier/North-Holland: Amsterdam, 1981; p 273. (30) Mabrey, S.; Mateo, P. L.; Sturtevant, J. M. Biochemistry 1978, 17, 2464. (31) Houya, K.; Ohnishi,
S. Seibutsu-butsuri 1982,22, 39.
Phase Transition Temperature of Liposomes
Langmuir, Vol. 7, No.5, 1991 921
DM PC
80
*
s
u
70
0
0 Y
0
c
1 a4
2 60 u
a
50 b-
0
15
20
30
25
Temperature ('C) DPPC
30
0 20
-
0
0
--
70
0
a4
60
70
)
-
x
a
('C
Figure 4. Effect of temperature on absorbance ratio of enolic form and ketonic form in liposomes prepared from egg lecithin with various iodine values: ( 0 )IV 1; (A)IV = 8; (0)IV = 17; (m) IV = 20; ( 0 )IV = 27.
s
Y
e20 a
50
LO
Temperature
h
Y
30
i
10 -
e 30
0 20
30
40
50
60
70
Temperature ('C 1
-
20 .
10
-
DSPC
20
t
0
10
20
30
40
Iodine Value
s
Figure 5. Effect of hydrogenation for egg lecithins on phase transition temperature.
Y
0
0
Table 111. Phase Transition Temperature of Lecithins
X
0 a4
5- 10 2
a
0 20
30
40
50
60
70
Temperature ('C )
Figure 3. Effecta of temperature on absorbance ratio of enolic form and ketonic form in liposomes: (a) prepared from DMPC; (b) prepared from DPPC; (c) prepared from DSPC. lower Tcvalues parallel to other curves with increases in added cholesterol, and moreover these curves shifted remarkably downward above 20 mol ?6 cholesterol and showed two groups among the curves below 15 mol 7% and above 20 mol % This suggests that the effect of added cholesterol is very remarkable above 20 mol 7%.
.
iodine no. of double phase transition temperature (TJ, O C value bonds molecule uv published data 1 0.03 57.0 3 0.09 56.2 8 0.25 54.5 9 0.28 53.8 17 0.53 48.2 20 0.63 44.8 23 0.72 44.5 27 0.85 41.5 28 0.38 39.5
DSPC DPPC DMPC
0.00
0.00 0.00
54.0 40.7 23.0
54.1 41.0 23.0
Conclusions From the above results, the following conclusions can be derived: (1)Temperature dependence on the change of the absorbance ratio Aenol/Aketo showed the sharp maximum point corresponding to the T,for the system of phosphatidylcholine as standard lipids.
Uena et al.
922 Langmuir, Vol. 7, No.6,1991 0 0~
BAA Lecithin
0
0
0
0
60
1
V
e 0
below
c"
0
t -
Tc
Temperature
------+
50
above
('C
Figure 6. Schematic diagram of liposome suspension incorporating or releasing BAA in bilayer.
40
iv= a t
IO
0
h
5 10 15 20 25 30 Amount of Cholesterol ( m o l % )
Figure 8. Effect of added cholesterol on phase transition temperature of liposomes prepared from various iodine values: (0) I v = 1; ( 0 )I v = 3; (A)Iv = 8; (A)Iv = 9; (0)I v = 17; (W) Iv = 20; ( 0 )IV = 23.
s
v
0
0 X
0
c al
Y
-
: 5 0
6C
al
a
h
-kl I-
0
20
30 40 50 Temperature ('C
60
5c
U
70
Figure 7. Effect of temperature on absorbance ratio of enolic form and ketonic form in liposomes with various amounts of added cholesterol: (0) 5 mol % ; ( 0 )10 mol % ; (A)15 mol % ; (A)20 mol %; (0)25 mol 5%; (W) 30 mol %. (2) Even in the case of the mixtures of some kinds of phospholipids like egg lecithins, the maximum point appeared a t the temperature corresponding to the T,. (3) The Tc values decreased linearly with the increase of IV or the degree of unsaturation. (4) In the case of the additional system of cholesterol, the effects became remarkable a t the concentration of 20 mol %, a t which the T,values dropped suddenly. Consequently, this method for T,determination using tautomerism of BAA can be applied in the diluted aqueous solutions with concentrations below 0.15 % of lipids. In
4C
1 3
89 17 Iodine Value
20
23
27
Figure 9. Effect of added cholesterol on phase transition temperature of liposomes prepared from various iodine values: (0) 5 mol %; ( 0 )10 mol %; (A)15 mol % ; (A)20 mol % ; (0) 25 mol %; (m) 30 mol %. comparison with common methods for T,determination, the method proposed here is very simple and useful.
