Langmuir 1996,11, 1653-1657
1653
Partition Coefficients of Poly(ethy1ene glycol) n-Alkyl Ethers between Water and DPPC Liposome Membranes Hideo Kawamura,* Masahiro Manabe, and Kazunori Miyake Department of Industrial Chemistry, Niihama National College of Technology, Niihama, Ehime 792, Japan
Yasushi Sasaki Department of Research and Development, SAN-EIGEN FFI Co., Ltd., Toyonaka, Osaka 561, Japan Received March 8, 1994. I n Final Form: January 23, 1995@ The partition coefficients (Kx) for poly(ethy1ene glycol) n-alkyl ethers (C,(OE),OH, m = 2 , n. = 1; m = 4, n = 0-4; m = 6, n = 1) between water and dipalmitoylphosphatidylcholine liposome membrane were determined at the gel to liquid crystalline phase transition temperature of the liposome membrane. For poly(ethy1eneglycol)n-butyl ethers (C4(0E),OH),the plot of log Kx against the number ( n )of oxyethylene groups decreased linearly with increasing n in the range n = 1-4. The standard free energy change of transfer per oxyethylene group (AG"(0E) = 1.13 kJ mol-l) from water to the liposome membrane was determined from the slope of this straight line and compared with corresponding values for micelles and dodecane. The local polarity around the OE group located in these molecular assemblies (liposome and micelle) and dodecane was estimated from ESR spectra by using a spin probe. We found that AG"(0E) values for these systems decreased linearly from a positive value (indicating the hydrophilic nature of the OE group) to a negative value (hydrophobicnature) as the local polarity around the OE group increased.
Introduction It is well known that the critical micelle concentration (cmc)of polyoxyethylenated nonionic surfactants increases with a n increase in the oxyethylene (OE) chain length.lI2 Inoue et al. determined the partition coefficient (Kx) of poly(ethy1ene glycol) n-dodecyl ethers with various OE chain length between water and dipalmitoylphosphatidylcholine (DPPC) liposome membrane and found that the Kxvalues decreased with increasing OE chain length.3 For short chain ethers (C,(OE),OH; m = 4, 5, n = 0-41, Kx values between water and dodecane as determined by Manabe et al. were found to decrease when the number of OE groups increased for a given alkyl chain length.4 These facts can be easily understood from the view point that the OE group is hydrophilic. On the contrary, it was found by Schwuger et al. that the cmc of sodium dodecyl ether sulfate (C12(0E),S04Na; n = 0-4) decreased when the number of OE group increased.j Furthermore, Manabe et al. reported an interesting result for micellar solution.6 The addition of short chain ethers described above to sodium dodecyl sulfate (SDS) solution led to the decrease of the cmc of SDS, and the decreasing tendency (dcmcJdC,, where C, is the concentration of the ether) was emphasized as the number of OE groups increased for a series of ethers with a given alkyl chain. The results suggest that in this case the OE group acts as a hydrophobic group. Recently, Marangoni and Kwak determined the KXof such short chain ethers between water and micelles of the cationic surfactant dodecyltrimethylammonium bromide (DTAB), Abstract published in Advance ACS Abstracts, April 1, 1995. (1)Schick, M. J.J. Colloid Sei. 1962,17,801. (2)Rosen, M. J.;Cohen, A. W.; Dahanayake, M.; Hua, X. Y. J . Phys. Chem. 1982,86,541. (3)Inoue, T.;Fukushima, K.; Shimozawa, R. Bull. Chem. SOC.Jpn. 1988,61,1565. (4)Manabe, M.; Koda, M.; Shirahama, K. Bull. Chem.SOC.Jpn. 1976, 48,3553. (5)Lange, H.; Schwuger, M. J. Colloid Polym. Sci. 1980,258,1264. (6)Manabe, M.; Shirahama, K.; Koda, M. Bull. Chem. SOC.Jpn. 1978, 51,1599. @
as well as the anionic surfactant SDS, and pointed out that the OE group had a negligible contribution to the solubilization of the ethers into DTAB micelles but had a significant contribution when the ethers were solubilized in SDS micelles.' These two apparently oppositebehaviors (hydrophilic and hydrophobic) of the OE group may be related to its environment. However, until now, no quantitative relation has been discussed. In the present study, KXfor the short chain ethers mentioned above has been determined in the DPPC liposome system in order to compare the results to the reported data in the systems of micelles and dodecane. The standard free energy change of transfer per OE group (AG"(0E)) of the short chain ethers from water to the oil phase (liposome,micelle, or dodecane) is calculated from the KXvalue obtained in order to compare quantitatively the contribution of the OE group in the solubilization. Furthermore, we attempt to demonstrate how the contribution of the OE group is influenced by the environment around the OE group, on the basis ofthe correlation between AG"(0E) and the local polarity of the environment where the group locates.
Experimental Section Materials. Dipalmitoylphosphatidylcholinewas purchased from Sigma and was used without further purification. Poly(ethylene glycol) (PEG)n-alkyl ethers, C,(OE),OH (where OE, m, and n represent oxyethylene group, the carbon number in the alkyl chain, and the number of oxyethylene group, respectively; m = 2,n = 1;m = 4,n = 0-3; m = 6 , n = l),were commercially available and were used as received. Cd(OE)40Hwas synthesized ~ dodecyl sulfate was and purified in our l a b ~ r a t o r y .Sodium synthesized and purified by the same procedure as described elsewhere.8 Dodecyltrimethylammonium bromide was purchased from Tokyo Kasei and was recrystallized twice from acetone. Dodecane was also purchased from Tokyo Kasei. The spin probe 4-octanoyl-2,2,6,6-tetramethyl-l-piperidinyloxy (C8TEMPO),was synthesized from 4-hydroxy-2,2,6,6-tetramethyl1-piperidinyloxy (TEMPOL) and 1-octanoyl chloride using the (7) Marangoni, D. G.; Kwak, J. C.T. Langmuir 1991,7,2083. (8)Manabe, M.; Koda, M. Bull. Chem. SOC.Jpn. 1976,49, 2904.
0743-7463/95/2411-1653$09.00/00 1995 American Chemical Society
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1654 Langmuir, Vol. 11, No. 5, 1995
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0
20
40
60
80
100
120
140
C, / mmol L" Figure 2. Plot of -ATm against C, for the DPPC-C4(OE)nOH system. (0)n = 0, (0) n = 1, (A) n = 2, ( 0 )n = 3, and (A)n = 4.
38
40 42 TEMPERATURE / 'C
44
Figure 1. Typical traces of changes in light intensity at 400 nm associated with a temperature increase for the DPPCC~(OE)BOH system. Concentration of C4(0E)30H (C,/mmol L-I): (a) 0, (b) 23.35, (c) 46.70, and (d) 70.05. method reported by Waggoner et aL9 Water was deionized and distilled before use. Preparation of DPPC Liposome. A 1mL sample of a stock solution of DPPC dissolved in chloroform was placed in a test tube. When the solvent was evaporated by a rotary evaporator, a thin film was formed at the bottom of the test tube. The solvent was then further evaporated by a vacuum pump. After 1mL of solvent (water or aqueous solution of PEG n-alkyl ether at a given concentration) was added into the tube and the content was agitated by a vortex mixer, an additional 9 mL of the solvent was added into the tube, and then the suspension was sonicated for about 1 min by a sonicator (Tomy-Seiko,UR-200 P) to form liposomes. Agitation and sonication were carried out around 45 "C, which is above the phase transition temperature of the liposome membrane. The formed liposome was considered to be multilamellar judging from the phase transition temperature and the phase transition pattern (see Figure 1). The concentration of the resultant DPPC liposome solution was about 1mmol L-1. Determination of Gel to Liquid Crystalline Phase Transition Temperature. The phase transition temperature of the DPPC liposome membrane from gel to liquid crystalline form in the absence and the presence of the PEG n-alkyl ethers was determined by monitoring a transmitted light intensity at 400 nm as a function of temperature on ax-Y recorder. Details concerning the procedure were described previously.1° ESR Measurements. A 1mL sample of the stock solution of DPPC mentioned above was mixed with a small amount (a few microliters) of a stock solution of Ca-TEMPO dissolved in chloroform in a test tube. Liposomes including the probe were prepared in the same manner as described above and packed in a capillary tube. For SDS and DTAB micellar solutions (70mmol L-I) and dodecane, the labeling was performed as follows. A (9) Waggoner,A.S.;Keith, A. D.; Griffith, 0.H. J . Phys. Chem. 1968, 72,4129. (10)Kawamura, H.;Manabe, M.; Yamashita, R.; Kagimoto, H. J . Solution Chem. 1994,23,85.
small amount of the stock solution of Ca-TEMPO was placed in a test tube. When the solvent was evaporated by avacuumpump, a thin film of the probe was formed at the bottom of the test tube. Next, 1mL of the micellar solution or dodecane was added into the tube. The solution including the probe was left to stand for 2 h at room temperature to allow the probe to be solubilized, and then the probe was packed in a capillary tube. The concentration of the spin probe was about 0.01 mmol L-l. ESR measurements were performed on a JEOL RE-1X ESR spectrometer equipped with a temperature controller. Experimental conditions were as follows: power, 24 mW; modulation, 0.1 mT; scan range, 328.4 & 5 mT.
Results and Discussion Typical traces of changes i n the transmitted light intensity associated with temperature increase a r e illustrated in Figure l. The curves a r e sigmoid, and the temperature at t h e inflection point is taken as t h e phase transition temperature from the gel to the liquid crystalline phase of t h e liposome membrane. The phase transition temperature of DPPC lipsome solution in the absence of PEG n-alkyl ethers is 42.0 "C, denoted by Tm,0,which is in good agreement with t h e temperature (41.4f0.5"C) obtained by other methods." The phase transition temperature (T,) in t h e presence of PEG n-alkyl ethers was determined in t h e same manner. The measurements were repeated four times for a given sample. The reproducibility of T , was typically &O. 1 "C. T , decreases with an increase in t h e concentration (C,) of PEG n-alkyl ethers, as seen for triethylene glycol n-butyl ethers in Figure 1. The relation between t h e depression of T , (AT, = T, - T,,o) and C, is shown for poly(ethy1ene glycol) n-butyl ethers (CI(OE),OH; n = 0-4) i n Figure 2. It is apparent that the relation is linear through the origin in the low C, region. This linear relation is also obtained for all other ethers studied. Considering that T , is the melting point of t h e gel state of the DPPC liposome membrane to t h e liquid crystalline state, t h e depression of T , induced by additives can be treated with a van't Hoff model for the freezing point d e p r e s s i ~ n . ~ JThere~-~~ (11)Marsh, D. CRC Handbook oflipid Bilayers; CRC Press: Boca Raton, FL, 1990; Section 11.7. (12) Kamaya, H.; Kaneshina, S.; Ueda, I. Biochim. Biophys. Acta 1981,646,135. (13)Inoue, T.;Miyakawa, K.; Shimozawa, R. Chem. Phys. Lipids 1986,42,261. (14)Inoue, T.;Iwanaga, T.; Fukushima, K.; Shimozawa, R. Chem. Phys. Lipids 1988,46,25.
Partition Coefficients of PEG n-Alkyl Ethers
Langmuir, Vol. 11, No. 5, 1995 1655
Table 1. Values of K,, AGYOE), and dcmcldC,,
DPPC m
n
Kx
2 4 4 4 4 4 6
1 0 1 2 3 4 1
4.75 77.1 102 40.3 29.1 1550
SDS
AG"(OE)/ kJ mol-l
AG"(OE)/ Kxb
kJ mol-'
143
1.13a
ii: }
487
" Calculated from eq 2. Taken from ref 7.
DOD
DTAB
AG"(OE)/ kJ mol-I Kxb
-(dcmc/dCa)c 0.028 0.072
128
-0.905"
-0.74d
0.14 0.18
131
AG"(0E)I kJmol-l
}
Kxe
AG"(OE)/ kJ mol-'
1.70 0.275"
0.856 0.284 0.0940
2.67a
Taken from ref 6 for SDS. Calculated from eq 3. e Taken from ref 4.
fore, the linear relation between AT,,, and C, in Figure 2 can be expressed from the van't Hoff equation a s follows, assuming that added PEG n-alkyl ether is solubilized in DPPC liposome membrane in the liquid crystalline state but not in the gel state:13
energy change of transfer per OE group (AG"(0E))from water to the oil phase (liposome, micelle, or dodecane) as
d In K, AG'(0E) = -RT dn
and as long a s C, is very low. AHo and CL are the enthalpy change associated with the phase transition and the concentration of DPPC, respectively, C, is the total concentration of PEG n-alkyl ether and R is the gas constant. KXis the partition coefficient defined in mole fraction units as Xal/Xa", where Xal and X," are the mole fractions of PEG in the liposome membrane in the liquid crystalline state and in the bulk water, respectively. Applying eq 1 to the linear relation in Figure 2, KX is determined from the slope of the straight line, using the followingvalues: Tm,0=315.0KandAH0= 36.4kJmol-l.ll The KXvalues obtained are summarized in Table 1. The semilogarithmic plot of KX vs the number of oxyethylene groups is illustrated in Figure 3 for poly(ethylene glycol) n-butyl ethers. The linear decreasing tendency ofKx ( n 2 1)indicates the hydrophilic character of the OE group. This property of the group is in agreement with the well-known fact that the cmc of a series ofnonionic surfactants such as palyoxyethylene alkyl ethers increases with an increase in n.'n2 It is interesting to compare the present dependence of KXwith that in some partitioning systems for the same ethers: KXin micellar systems of SDS and DTAB' and in a dodecane (DODI-water system4 and dcmc/dCa in a SDS micellar system.6 In all systems in Figure 3, the relation can be regarded to be linear except for that of the ether with n = 0. For the DOD-water system, the slope is the steepest and negative, but for the SDS system, the slope is positive. The slope gives us the standard free
AG"(0E) = -RT
d In(-dcmc/dC,) dn
(3)
The relation of eq 3 was derived by Manabe et al.15 These values are also listed in Table 1. The differences in the AG"(0E) values for each system may be due to the influence of each environment of the OE group. It is noticed that in each system shown in Figure 3, KX a t n = 0 (butanol) deviates downward from the linear relation. The deviation indicates that the strong hydrophilic OH group weakens the hydrophobicity of methylene groups in the alkyl chain connected to the OH group. In order to estimate a reasonable hydrophilicity (or hydrophobicity) of the OE group, it is necessary to check the dependence of KX on the alkyl chain length ( m )in the ethers, since the result in Figure 3 is for the ethers with a butyl group only. The dependence of KX on m for 1-alkanols(C,OH) and monoethylene glycol n-alkyl ethers (C,(OE)OH) in the DPPC system is shown in Figure 4. It is apparent that the relation gives a straight line with a positive slope and that the lines are parallel to each other. The slope provides the standard free energy change of transfer per methylene group (AG'(CH2)) in the alkyl chain from water to the DPPC liposome membrane as AG"(CH2)= -RTdInKx/dm = -3.79 (DPPC-C,(OE)OH (15) Manabe, M.; Koda, M.; Shirahama, K. J.Colloid Interface Sei. 1980, 77, 189.
Kawamura et al.
1656 Langmuir, Vol. 11, No. 5, 1995 10000
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I
1
I
I
3
4
5
6
m Figure 4. Dependence of log KXon the carbon number ( m )in
the alkyl chain for C,(OE)OH and C,OH. ( 0 )DPPC-C,(OE)OH system, ( 0 )DPPC-C,OH system (taken from ref 161, and (A) DPPC-C,OH system (taken from ref 12).
system; m = 2 , 4 , 6 ) and -3.72,16 -3.4612 (DPPC-C,OH system; m = 3-6) k J mol-l. The values for these systems are in good agreement with each other. The result shows that the strong hydrophilicity of the terminal OH group in C,(OE)OH is screened by the OE group located between the OH group and the alkyl group. It should be noted in Figure 4 that the linear relation of C,(OE)OH holds even for m = 2, but the KX value of C2OH is lower than the extrapolated value of the straight line. The result indicates that the hydrophobicity of the methylene group adjacent to the OH group in C,OH is effectivelyweakened by the OH group with strong hydrophilicity but that of the methylene group attached to the OE group is little influenced by the OE group in C,(OE)OH. This may be ascribed to weaker hydrophilicity of the OE group compared with that of the OH group. Inoue et al. determinedKx values of poly(ethy1eneglycol) n-dodecyl ethers (C12(0E),OH) in the range n = 4-8 between water and DPPC liposome membrane in the liquid crystalline state and reported AG"(0E) = 1.20 k J m01-l.~ Although the OE chain length of PEG n-alkyl ethers used in the present study is shorter than that used by Inoue et al., the estimated AG"(OE),1.13 kJmol-l (see Table l), in the present study is in good agreement with that determined by Inoue et al. This result suggests that the OE chain of the PEG n-alkyl ether, regardless of its chain length, does not penetrate into the DPPC liposome membrane and locates around the interfacial region of the liposome membrane when the PEG n-alkyl ether is solubilized, which is consistent with the observation that poly(ethy1ene oxide) does not interact with phospholipid vesicles and is located in the aqueous phase.17 Therefore, the discrepancy of AG"(0E) values for the four systems shown in Table 1 may be ascribed to an environmental factor of the interfacial region of these molecular assemblies (liposome and micelles) and dodecane. Here, the local polarity of the interfacial region is considered to be a preferential factor controlling AG"(0E). To support this hypothesis, the local polarity of the regions was estimated by the spin label method. The method has been widely used for estimating the local (16) Kawamura, H.; Manabe, M.; Ihara, H.;Tokunaga, S. Proceedings of the 23rd IUPAC Conference on Solution Chemistry, Leicester, U.K., August 16-19, 1993. (17) Labonte, R.; Gao,Z.; Kwak, J. C. T. ColloidsSurf. 1993,78,271.
Figure 5. ESR spectra for Cs-TEMPO in (a) SDS micellar solution (25 "C), (b) DTAB micellar solution (25 "C),(c) DPPC liposome solution (45 "C), and (d) dodecane (25 "C). Concentration of SDS and DTAB solutions was 70 mmol L-I. ESR
measurements were made at the temperatures given in parentheses. 3 3
'
I
2
1
1S O
1.55
1.60
1.65
aN / mT Figure 6. Dependence of AG"(0E)on aN. AG"(0E)for SDS micellar system ( 0 )taken from ref 7 and (0)taken from ref 6.
polarity and viscosity of these molecular assemblies.18J9 Cs-TEMPO is used a s a spin probe which has a N - 0 radical near the hydrophilic group, as shown in Figure 5. We expect the radical of the probe must locate around the polar interfacial region of these molecular assemblies when it is solubilized. ESR spectra of C8-TEMPO in SDS and DTAB micellar solutions (25 "C), DPPC liposome solution (45 "C),and (18)Baglioni, P.; Minten, E. R.; Dei, L.; Ferroni, E. J . Phys. Chem. 1990,94, 8218. (19) Ristori, S.; Martini, G. Langmuir 1992,8, 1937.
Partition Coefficients of PEG n-Alkyl Ethers
Langmuir, Vol. 11, No. 5, 1995 1657
dodecane (25 "C) are shown in Figure 5. The hyperfine coupling constant (aN),which is a parameter representing the degree of the local polarity around the radical of the spin probe, increases as the local polarity around the radical increases.18J9 aNis determined from the magnetic field difference between low-field and high-field peaks of the spectrum (see Figure 5). In order to show how AG"(0E) depends on the local polarity around the OE group, the relation between AG"(0E) a n d a Nis plotted in Figure 6. It can be seen that AG"(0E) decreases almost linearly with increasing u N . The relation determined by the least mean squares method is
AG"(0E) = 44.9 - 2 7 . 8 ~ ~
(4)
This result suggests that the local polarity around the OE group determines whether the OE group can behave as a hydrophilic group (i.e., AG"(0E) > 0) or as a hydrophobic one(i.e.,AG"(0E) < 0)in thesolubilizationofPEGn-alkyl
ethers. Since the hydrophilicity ofthe OE group is weaker than that of the OH group, as seen in Figures 3 and 4, it seems that the contribution of the OE group in the solubilization ofPEG n-alkyl ethers depends on the degree of the local polarity around the group. As seen in Figure 6, the OE group can act as a hydrophobic group when the PEG n-alkyl ether is solubilized into the polar and hydrophilic environment with aN > 1.61 mT, e.g., the interfacial region in SDS micelles, and when the environment is less polar, such as dodecane and DPPC liposome, the OE group can act a s a hydrophilic group. The local polarity of the interfacial region of cationic DTAB micelles is intermediate: AG"(0E) 0. Therefore, in DTAB micelles, the OE group does not contribute to the solubilization either as a hydrophobic group or as a hydrophilic one.
Acknowledgment. The authors thank a reviewer for many helpful suggestions. LA9402074
