5184
Langmuir 1998, 14, 5184-5187
Electron Magnetic Resonance Study on the Mobility of Nitroxide Spin Probes in the Dipalmitoylphosphatidylcholine Lipid Bilayers: Effect of Poly(ethylene glycol) Don Keun Lee, Jin Soo Kim, Young Min Lee,† and Young Soo Kang* Department of Chemistry, Pukyong National University Dae-yeon-3-dong, Nam-gu, Pusan 608-737, Korea
Byung Kyu Kim Department of Polymer Science and Engineering, Pusan National University, Pusan 609-735, Korea Received November 20, 1997. In Final Form: May 26, 1998 The degree of interaction of the lipid bilayer of a dipalmitoylphosphatidylcholine (DPPC) vesicle with poly(ethylene glycol) (PEG) was studied by determining electron spin resonance (ESR) line widths of nitroxide spin probes and measuring interaction distances between nitroxide groups in the hydrophobic core region and interface water (D2O) with electron spin-echo modulation (ESEM). The increasing ESR line width with increasing concentration of PEG up to 10 mM indicates that the PEG intercalates between the headgroups of the DPPC vesicle due to hydrophobic interaction with surfactant alkyl chains. This action of PEG is also reflected in increased deuterium modulation depths of the ESEM spectra. Thereafter, some interface water (D2O) is replaced by PEG molecules with increasing concentration of PEG at DPPC interface. This interpretation is supported by the decreased deuterium modulation depth up to 10 mM of PEG and slightly decreased deuterium modulation depth thereafter.
Introduction The interaction of polymers with surfactant molecular assemblies such as micelles, vesicles, and reverse micelles has been studied in the mixed surfactant systems.1,2 The polymer-surfactant interaction results from a relatively weak dipolar interaction. In a polymer-micelle complex the properties of both micelles and polymer can be modified.3,4 The important aspects for industrial applications are the solubilization power and viscosity of an aqueous solution of polymer-bound micelles that have applications in paints, coatings, cosmetics, and detergents.3 Polymer-micelle interaction also plays a role in tertiary oil recovery.3 Recently, much attention has been focused on the utilization of surfactants for catalysis and materials for environmental problems such as oil spills because of their excellent ability to disperse an exposed oil in the ocean and their easy biodegradability and photodegradability.4-12 † HS Chemical Company, Ltd, R&D Center, Kyong Nam 626800, Korea.
(1) Goddard, E. D. Colloid Surf. 1986, 19, 2225. (2) Goddard, E. D.; Hanna, K. B. In Micellization, Solubilization and Microemulsion; Mittal, K. L.; Ed.; Plenum: New York, 1977; Vol. 2. (3) Brackman, J. C.; Engberts, J. B. F. N. Chem. Soc. Rev. 1993, 85. (4) Hunter, M. L.; da Motta, M.; Lester, J. N. Environ. Technol. Lett. 1998, 9, 1. (5) Faust, S. D. Advances in Environment and Technology, Part 2, Vol. 8; Suffet, I. H., Ed.; Wiley: New York. (6) Swisher, R. D. J. Am. Oil Chem. Soc. 1963, 40, 648. (7) Swisher, R. D. J. Water Pollut. Control Fed. 1963, 35, 877. (8) Mittal, K. L.; Fendler, E. J. Solution Behavior of Surfactants, Vol. 1; Plenum: New York; p 149. (9) Larson, R. J.; Payne, A. G. Environ. Microbiol. 1981, 41, 621. (10) Yoneyama, H.; Yamashita, Y.; Tamura, H. Nature 1979, 282, 817. (11) Hidaka, H.; Kubota, H.; Gratzel, M.; Serpone, N.; Pelizzetti, E. Nouveau J. de Chemie 1985, 9, 67. (12) Jones, C. E.; Mackay, R. A. J. Phys. Chem. 1978, 82, 63.
Such a dispersing ability of surfactants with oil slicks exposed in seawater is based on the considerable hydrophobic interaction between them in water.8 As a model study of surfactant interaction with hydrophobic alkyl chains, some kinds of polymers were used to investigate the interaction with surfactants.8 Winnick et al.13 studied the interaction of sodium dodecyl sulfate (SDS) micelle with nonionic poly(ethylene oxide) (PEO) with pyrene at both ends of the PEO chain by measuring the fluorescent properties of bound pyrene. Despite the numbers of applications of polymer-micelle complexes, little is known about the details of the interaction mechanism. Nuclear magnetic resonance (NMR) has been used to study the micellization process of sodium ω-phenyldecanoate and the interaction between surfactant and PEO.14,15 Electron spin resonance (ESR) has been used to investigate the microenvironment of nitroxide spin probes in micelles and vesicles by measuring the nitrogen coupling constant and ESR spectra line widths.16 The coupling constant is affected by the local polarity of the nitroxide. A more polar environment gives larger values of coupling constant because of greater electron density in nitrogen. The line widths are controlled by rotational and lateral diffusion of the spin probes, which in turn is affected by viscosity, orderedness, and temperature at the local (13) Hidaka, H.; Kubota, H.; Gratzel, M.; Pellizzetti, E.; Serpone, N. J. Photochem. 1986, 35, 219. (14) Gerischer, H.; Weller, A. J. Electrochem. Soc. 1992, 139, 113. (15) Gao, Z.; Wasylishen, R. E.; Kwak, J. C. T. J. Phys. Chem. 1991, 95, 462. (16) (a) Bratt, J. P.; Kevan, L. J. Phys. Chem. 1993, 97, 7371. (b) Bratt, J. P.; Kevan, L. J. Phys. Chem. 1992, 96, 6849. (c) Ristori, S.; Martin, G. Langmuir 1992, 8, 1937. (d) Bonosi, F.; Gabrielli, G.; Margheri, E.; Martini, G. Langmuir 1990, 6, 1769. (d) Ristori, S.; Ottaviani, F.; Lenti, D.; Martini, G. Langmuir 1991, 7, 1958.
S0743-7463(97)01278-X CCC: $15.00 © 1998 American Chemical Society Published on Web 08/05/1998
Effect of Poly(ethylene glycol)
environment in the micelles and vesicles of the spin probe.17,18 The larger ordering parameters of x-doxylstearic acids (x-DSAs) at the interface regions of micelles or vesicles compared with the hydrocarbon region indicate oriented x-DSAs with its polar headgroup at the interface. This results in a broader line width due to the slower tumbling rate of the spin probe with a longer relaxation time. This can also be controlled by changing local viscosity in the nitroxide of x-DSAs by adding poly(ethylene glycol) (PEG) to the vesicle interface. The relative interaction distance of the nitroxide group of a spin probe with respect to the interface water (D2O) can be measured by deuterium modulation of electron spin-echo modulation (ESEM).19,20 ESEM can be measured only in the frozen state because the weak dipolar interactions are averaged out in liquids. However, numerous studies have demonstrated that micellar and vesicular structure is retained in rapidly frozen aqueous solutions and that meaningful ESEM studies are possible.19 In the present study, the interaction of PEG with dipalmitoylphosphatidylcholine (DPPC) vesicle surfactants was studied with electron spin resonance (ESR) by determining line widths on the spectra of nitroxide spin probes of x-DSAs. The interaction distance between the nitroxide group in the hydrophobic core region and the interface water (D2O) was determined with ESEM. The results are interpreted with respect to the miscibility of PEG with the surfactant headgroup at the vesicle interface by intercalation of PEG molecules between DPPC headgroups. Experimental Section PEG (average molecular weight of 200 and average numberto-average weight molecular weight Mn/Mw ) 1.2) was purchased from Aldrich and used without further purification. DPPC and x-DSA were obatined from Sigma Chemical Company and used without further purification. Deuterium oxide (D2O) was purchased from Aldrich Chemical Company (99.8 atom %) and was deoxygenated by purging with nitrogen gas for 20 min before use. A stock 30 mM solution of the vesicle monomers (DPPC) was prepared in chloroform. Each 13 mM stock solution of x-DSAs, of which structures are shown in Figure 1 was prepared in chloroform. A 17-µL quantity of each spin probe stock solution and 0.6 mL of DPPC stock solution were transferred into a 1.6cm (o.d.) by 1.25-cm (i.d.) test tube, and then the chloroform was evaporated by bubbling nitrogen gas into the surface of the solution. This procedure resulted in the formation of a thin film on the test tube wall. After the film had formed, 1 mL of D2O was added to the resulting films. The resulting solutions were sonicated for 30 min at 55 ( 3 °C under nitrogen gas to form completely solubilized clear solutions. Then, each quantity of PEG was added to the x-DSA/DPPC vesicle solutions to result in 0, 0.5, 2, 10, 20, and 40 mM concentrations of PEG in vesicle solutions. The spin probe concentration was 2.2 × 10-4 M. The 100 µL of sample solutions was introduced into 1.0-mm (i.d.) by 1.1-mm (o.d.) Pyrex diposable micropipets (CMS Company) for ESR experiments and 2-mm (i.d.) x 3-mm (o.d.) Suprasil quartz tube that was flame sealed at one end for ESEM experiments. The sample tubes were shaken to equilibrate the solution, and then the samples for ESEM were frozen by rapidly plunging into liquid nitrogen. (17) Baglioni, P.; Rivera-Minten, E.; Dei, L.; Ferroni, E. J. Phys. Chem. 1990, 94, 8218. (18) Meirovitch, E.; Nayeem, A.; Freed, J. H. J. Phys. Chem. 1984, 88, 3454. (19) Schreier, S.; Polnaszek, C. F.; Smith, I. C. P. Biochim. Biophys. Acta 1978, 515, 375. (20) Ichikawa, T.; Kevan, L.; Narayana, P. A. J. Phys. Chem. 1979, 83, 3378.
Langmuir, Vol. 14, No. 18, 1998 5185
Figure 1. The structures of nitroxide spin probes and DPPC vesicle molecule. The ESR spectra were recorded at room temperature at X-band using a Bruker ESP 300 spectrometer with 100-kHz field modulation. The sample tubes for ESR were directly placed in a TE102 rectangular cavity. The loaded Q factor of this cavity was measured as ∼1700. The microwave power was 1.97 mW. The microwave frequency was measured with a Hewlett-Packard 5350B frequency counter, and the magnetic field was monitored with a Bruker ER032M Hall effect field controller. Two-pulse ESEM signals were recorded at 4.2 K on a homebuilt spectrometer using 40- and 80-ns exciting pulses.20,21 The deuterium modulation appeared with ∼0.5-µs periodicity. The modulation depths were normalized by dividing the depth at the first deuterium minimum by the depth to the baseline at the same interpulse time. The ESR and deuterium modulation experiments were performed in triplicate. The simulation of the ESE signal was carried out with ESFT software by fitting the isotropic coupling constant (Aiso ) 0.1 MHz), the dipolar interaction distance (R), and the number of nuclei interacting with the unpaired electron (N). The isotropic coupling constant and the number of deuterium nuclei were fixed as 0.1 MHz and 6, respectively. This assignment does not result in a serious error during ESEM signal sumulation and allows the relative interaction distance data to be calculated.
Results and Discussion The building unit of a biological membrane is a surfactant bilayer consisting of two layers of surfactant molecules with their polar ends pointing out and contacting solvent water. The surfactants that form bilayers and vesicles typically have two alkyl chains attached to a polar headgroup. Added nonpolar solutes dissolve to a greater or less degree into the hydrocarbon region. The degree of order and compactness at the vesicle interface affect the solubilization of solutes in the hydrocarbon region. DPPC is neutral and its structure is shown in Figure 1. The size and flexibility of surfactant headgroups influence the solubilization of solutes into vesicles and create different degrees of order at the vesicle interface. The location and degree of the interaction of PEG polymer chains with surfactants like micelles, vesicles, and reverse micelles are only partially understood. The orientation and relative location of the nitroxide moiety at the micellar interface has been investigated with ESEM by determining deuterium modulation depth in a previous study.17 The nitroxide moiety of 5-DSA is located relatively close to the micellar interface and goes deeper into the (21) Narayana, P. A.; Kevan, L. Magn. Reson. Rev. 1983, 87, 239.
5186 Langmuir, Vol. 14, No. 18, 1998
Lee et al.
Figure 3. Normalized deuterium modulation depths at 4.2 K of 5-DSA (O), 7-DSA (4), 12-DSA (0), and 16-DSA (b) in DPPC/ D2O vesicles versus the concentration of PEG.
Figure 2. The first-derivative ESR spectra at room temperature of 5-DSA in DPPC/D2O vesicles with 0, 0.5, 2, 10, 20, and 40 mM concentration of PEG. Table 1. First-Derivative ESR Line Widths to ( 0.02 G for the Low and High Magnetic Field Lines of x-Doxylstearic Acids in 0.1 M SDS/D2O Micelles with Different Concentrations of PEG at Room Temperature [PEG], mM 0 0.5 2 10 20 40
ESR line low high low high low high low high low high low high
5-DSA
7-DSA
12-DSA
16-DSA
2.43 3.35 2.80 4.64 3.09 5.85 4.52 6.39 4.53 6.37 4.48 6.38
2.42 3.31 2.60 3.89 3.21 6.17 4.27 6.48 4.26 6.44 3.98 6.31
1.69 2.23 1.69 2.29 1.75 2.29 1.94 2.45 1.80 2.40 1.88 2.44
1.80 3.30 2.00 3.42 2.36 3.95 2.74 3.76 2.71 3.78 2.74 3.77
hydrophobic region of the micelle of 7-DSA and 10-DSA. The nitroxide moiety of 12-DSA and 16-DSA is located close to the micellar interface by alkyl chain bending of x-DSAs and becomes even closer for 12-DSA. For 16DSA, nitroxide moiety is closer than 7-DSA, but slightly deeper than 5-DSA into the hydrophobic region. In the present study, the interaction of the DPPC headgroup with the solubilized PEG was studied by ESR by measuring the line widths of nitroxide of x-DSAs solubilized in the DPPC vesicle. The representative firstderivative ESR spectra of 5-DSA in DPPC/D2O vesicles with different concentrations of PEG are shown in Figure 2. The first derivative ESR line widths at low and high magnetic field lines of the spin probes are given in Table 1. The line widths of 5- and 16-DSAs spin probes increased with increasing concentration of added PEG up to 10 mM, and thereafter was constant. The line widths of 7- and 12-DSA spin probes were not affected significantly by the concentration of PEG compared with 5- and 16-DSA. The
smallest line width was obtained from 12-DSA. The largest line width was obtained for 5-DSA, which decreased for 7- and 12-DSA and then increased slightly for 16DSA. As PEG was added to the DPPC solution, the local viscosity of the nitroxide environment increased, as shown by the increased ESR line widths in Table 1. This result can be explained by the increased local viscosity around nitroxide spin probes due to the intercalated PEG through the vesicle interface. The correlation time of the nitroxide group of spin probes in a micelle is related to the order parameter of the interface region and the rotational diffusion tensor.17 Previous studies have shown that a detailed line-shape simulation can give the order parameter and rotational diffusion tensor of the nitroxide in the vesicles, but this time-consuming analysis is not necessary to establish the trends with PEG concentration investigated here. Deuterium modulation depths of 5-DSA increased with increasing concentration of PEG added to DPPC/D2O vesicles. The normalized deuterium modulation depths of 5-, 7-, 12-, and 16-DSAs in DPPC/D2O vesicles versus the concentration of PEG are shown in Figure 3. The relative interaction distance change between nitroxide moiety of x-DSAs and vesicular interface water (D2O) was determined by deuterium modulation depths. The normalized deuterium modulation depths rapidly increased with increasing concentration of PEG up to 10 mM and thereafter slightly decreased up to 40 mM. The rapidly increasing deuterium modulation depths up to 10 mM of PEG indicates that PEG interacts with the vesicular interface probably by intercalation between the headgroups of DPPC vesicle surfactants. This interaction allows deeper water (D2O) penetration into the interface, which in turn decreases the interaction distance of the nitroxide moiety from the x-DSAs with interface D2O. But the slightly decreasing deuterium modulation depth after 10 mM of PEG can possibly be interpreted as the result of the replacement of some interface water (D2O) with PEG. This result is in contrast to the results obtained in the previous studies of micelles with PEG and cyclodextrins.22,23 In micelles, the higher concentration of added surface active agents destroyed the micelle structures by intercalation between surfactant headgroups. In the vesicle, as in the present study, the interface structure (22) Kang, Y. S.; Kevan, L. J. Phys. Chem. 1994, 98, 7624. (23) Lee, D. K.; Kang, Y. S.; Kevan, L. J. Phys. Chem. 1997, 101, 519.
Effect of Poly(ethylene glycol)
Langmuir, Vol. 14, No. 18, 1998 5187
Figure 4. Schematic drawing of the PEG intercalated interface structure of DPPC/D2O vesicles.
was stable enough not to be destroyed by the intercalation of surfactant headgroups but to replace the interface water (D2O) with PEG molecules at high concentrations of PEG. This result is indicated by the normalized deuterium modulation depth trend of slightly decreased deuterium modulation depths at PEG concentrations >10 mM. Added PEG molecules interact with the DPPC vesicle interface and penetrate into the nitroxide position of 5and 16-carbon of x-DSA, but not close enough to 7- and 12-carbon of x-DSAs, as shown in Figure 4. This result is indicated by the increasing ESR line widths of 5- and 16-DSA with increasing concentrations of PEG. The almost constant ESR line widths of 7- and 12-DSA show that the added PEG molecules do not penetrate into the hydrophobic region of DPPC vesicles close to 7- or 12carbon of x-DSA. The interaction distance between the vesicle interface and interface water (D2O) was studied with two-pulse ESEM signal simulations with fitting parameters of the isotropic coupling constant (Aiso), interacting nuclei numbers (N), and the interacting distance between the nitroxide moiety and interface water (D2O). The representative ESEM signals with computer-simulated signals are shown in Figure 5. The interaction distance between deuterium nuclei and nitroxide spin probe was changed between 3.2 and 4.4 Å. These results show that the interaction distance between interface water (D2O) and nitroxide moiety was changed by the intercalation of PEG and some interface water (D2O) replacement with PEG. Conclusions ESR line widths of nitroxide for 5- and 16-DSAs solubilized in DPPC/D2O vesicle increased with increasing concentrations of PEG in DPPC/D2O vesicle solution. On the other hand, ESR line widths of nitroxide for 7- and 12-DSAs were not changed significantly by the concentration of added PEG. This difference is interpreted as
Figure 5. Two-pulse X-band ESEM signals of 5-DSA in DPPC/ D2O vesicle with (top) 40 mM and (bottom) 2 mM PEG. Experimental (__) and computer-simulated (....) signals are shown with fitting parameters of (top) Aiso ) 0.1 MHz, N ) 6, and R ) 3.75 Å and (bottom) Aiso ) 0.1 MHz, N ) 6, and R ) 4.37 Å.
the result of the action of the added PEG in the interface of the DPPC/D2O vesicle of increasing the local viscosity of nitroxide of 5- and 16-DSAs by intercalation between DPPC surfactant molecule headgroups up to 10 mM of PEG and thereafter the replacement of some interface water (D2O) by PEG molecules. The increased local viscosity around nitroxide results in the slow tumbling of the nitroxide and thus an increase in ESR line widths. This interpretation is supported by normalized deuterium modulation depth trends. Normalized deuterium modulation depths of x-DSAs solubilized in DPPC/D2O vesicle solutions rapidly increased with increasing concentrations up to 10 mM of PEG and thereafter decreased up to 40 mM. This result indicates that PEG intercalates between DPPC surfactant hedagroups up to 10 mM and thereafter replaces some interface water (D2O) with PEG molecules. Acknowledgment. The authors thank the Regional Research Center (RRC) the Institute for Environmental Technology and Industry (IETI) for its finacial support to the project number 96-01-02. We give special thanks to Prof. Larry Kevan at University of Houston, Chemistry Department, for his kind assistance for ESEM measurement and sincere discussions. LA971278G
