VOLUME 104, NUMBER 24, JUNE 22, 2000
© Copyright 2000 by the American Chemical Society
LETTERS High-Magnetic-Field Effects on Liposomes and Black Membranes of Dipalmitoylphosphatidylcholin: Magnetoresponses in Membrane Potential and Magnetofusion Sumio Ozeki,*,† Hutoshi Kurashima,† and Haruo Abe‡ Department of Chemistry, Faculty of Science, Shinshu UniVersity, 3-1-1 Asahi, Matsumoto, Nagano 390-8621, Japan, and National Research Institute for Metals, 3-13 Sakura, Tsukuba, Ibaraki, 305-8565, Japan ReceiVed: September 23, 1999; In Final Form: March 1, 2000
Deformation in membranes of dipalmitoylphosphatidylcholin (DPPC) led to the fusion of its liposome and large changes in the membrane potential of its black membrane under high magnetic fields of up to 28 T. The magnetofusion of DPPC liposomes significantly depended on the particle size and aromatic compounds doped. Although a theory for the magnetic deformation of liposomes predicts magnetofusion and magnetodivision, only magnetofusion was experimentally observed. There seem to be discrete liposome sizes stabilized at 10 T. The changes in liposome size due to magnetofusion give an estimation of the local curvature of the membrane. The membrane potential of black DPPC membranes markedly increased with high magnetic fields and doped molecules, corresponding to magnetofusion. Undulation of a membrane due to high magnetic fields may relax any orientational defects in a black lipid membrane, leading to a ripple-like structure, which may cause the magnetoresponse in the membrane potential.
It is well established that diamagnetic assemblies having magnetic anisotropy will become oriented and rotate in a magnetic field so as to obtain the minimum-energy state.1-15 The magnetic orientational energy (Er) of a diamagnetic lipid domain containing N molecules (volume NV), whose long molecular axis is at an angle φ to H and the diamagnetic anisotropy ∆χ () χ| - χ⊥, where χ| and χ⊥ are the magnetic susceptibility parallel and perpendicular to H), is given by the following equation:7
Er ) -(H2/2)(χ⊥ + ∆χcos2φ)NV
(1)
When H and/or N is large enough, the long molecular axis of * Address correspondence to: Sumio Ozeki, Department of Chemistry, Faculty of Science, Shinshu University, 1-33 Asahi, Matsumoto, Nagano 390-8621, Japan. Telephone: 81-263-37-2567. Fax: 81-263-37-2559. E-mail
[email protected]. † Shinshu University. ‡ National Research Institute for Metals.
lipid molecules in a domain can be cooperatively aligned in the direction of averaged φ. Orientational effects are expected when N is on the order of 105 for ∆χ∼ -1.1 × 10-6 cm3 mol-1 under 10 T and 318 K or when H ) 4.0 T for a 200 nm diameter liposome with a 5 nm membrane thickness,9 because under these conditions the magnetic orientational energy is comparable with the thermal energy. Another magnetic-field effect on a bilayer membrane comes from the elastic properties of the membrane. Helfrich predicted theoretically the magnetic deformation of phospholipid liposomes from a sphere to an ellipsoid, which was shown experimentally by Tenforde and Liburdy9 as well as Maret and Dransfeld.10 In a previous paper, it was reported that the membrane potential and resistance of black lipid membranes (BLM), comprising didodecyl phosphite or dipalmitoylphosphatidylcholin, changed remarkably under low, steady magnetic fields of less than 0.5 T.14 The magnetic field effects on the electrical properties seem to occur not via the Lorentz force on the ion
10.1021/jp9934073 CCC: $19.00 © 2000 American Chemical Society Published on Web 05/26/2000
5658 J. Phys. Chem. B, Vol. 104, No. 24, 2000
Letters
Figure 1. Variations of the DPPC liposome radius (b) and membrane potential of a black DPPC membrane (O) at 318 K with a steady magnetic field intensity. After DPPC liposomes were exposed to magnetic fields at 318 K for 3 h, their radii were measured by dynamic light scattering outside of the magnetic field at 298 K. The liposome size in all systems was unchanged as long as liposomes were under no magnetic field. The magnetic response in the membrane potential ∆Ψ (≡ ΨH - Ψ0, where the subscripts 0 and H mean H)0 and H, respectively) was measured with the application of magnetic fields perpendicular to the membrane.
flux but via a cooperative orientation of lipid molecules. The addition of molecules having different magnetic anisotropy to a BLM modified the magnetoresponses of the membrane. We have examined whether deformation in the diamagnetic molecular assemblies of DPPC due to high magnetic fields can induce fusion among liposomes and response in the membrane potential of a BLM of DPPC. DPPC liposomes, prepared from chloroform solutions of DPPC with the vortex method, grew when they were exposed to high magnetic fields of more than 12 T at 318 K for 3 h (Figure 1); e.g., from 160 to 275 nm in radius by applying a magnetic field of 20 T, which may be referred to as the magnetofusion. The average liposome size was determined as a sphere from the Einstein-Stokes equation by dynamic light scattering (using an Otsuka ELS800 spectrophotometer) at 298 K under no magnetic fields. Figure 1 also includes the magnetic response in the membrane potential (Ψ) of a BLM of DPPC by applying magnetic fields (H) perpendicular to the membrane at 318K (∆Ψ ≡ ΨH - Ψ0, where subscripts 0 and H mean H ) 0 and H, respectively) as a function of H. The BLM was formed at room temperature in a 0.8 mm-diameter hole of a 0.5 mm-thick Teflon sheet which divided the Teflon cell into two compartments filled with 1 × 10-3 and 1 × 10-2 mol/dm3 aqueous NaCl solutions.14 BLMforming solutions were prepared by mixing 20 ppm of DPPC with (3+2) octane-dodecane. The membrane potential arising across a BLM was measured with a couple of Ag-AgCl electrodes and a Keithley multimeter 2001. The change in the membrane potential (Ψ0 ≈ -25mV) was appreciable even at 0.02 T and maximum (+6mV) at 0.15 T. Over 0.15 T, ∆Ψ decreased across 0 at 0.3 T, along with an increase in H, and reached a plateau (-8mV) in the range 10 to 12 T, followed by a steep decrease in the range 12 to 15 T, and then approached to -18mV at 28 T. The steep ∆Ψ change at around 12 T seems to correspond to liposome growth, suggesting that the large change in ∆Ψ should arise from certain changes of the membrane itself. The changes in ∆Ψ below 12 T, where the liposome size (r) was nearly unchanged, should arise from a
Figure 2. Irreversible changes of H NMR (500 MHz, 11.7 T) signals of DPPC liposomes with a 11.7 T magnetic field. The charts were measured in the order of A (30 °C), B (38 °C), C (45 °C), D (30 °C, 11.7 T) and of A, B, C, E (30 °C). D was quenched from 45 to 30 °C at 11.7 T, while E experienced no magnetic field. Chart D is very different from chart E and rather similar to chart B (the ripple phase), suggesting an irreversible magnetic orientation of lipid molecules.
reversible deformation of a membrane, because the liposome size represents the memory of the state under a magnetic field. The ∆Ψ behavior in the region of low magnetic field ( 1) and magnetodivision (n < 1) for F ) 25 nm. When n ) 1, a liposome of size r0 is stable against or insensitive to a certain magnetic field. Figures attached to each line are the F values.
(5)
When H is large enough (∼10 T) (case 1) or the shape of a liposome is exactly an ellipsoid of revolution (case 2), the following relation is obtained if c0 is unchanged during the size change:
6(1 - n) - c0r0 (n1/2 - n) g 0
(6)
This relation was derived from eqs 2, 3, and 4 using the two equations (Ec and EH) in ref 13a for case 1 or the following two equations13b for case 2:
Ec ) (8π/5)kc(6 - c0r0)(s2/r0)2
(7)
EH ) -(16π/15)∆χbH 2r0s2
(8)
Solving eq 6 and adding the condition ∆χ(6 - c0r0) > 0, possible association in liposome having radius r0 may be estimated for a given radius of curvature F () 1/c0), as shown in Figure 3. The shadow in the figure illustrates the regions of magnetofusion (n > 1) and magnetodivision (n < 1) for F ) 25 nm. When n ) 1, a liposome of size r0 is stable against or insensitive to a certain magnetic field; that is, the apparent ∆χ of a whole liposome should be zero. Figure 4 shows the square root of the experimental association number (n1/2) observed after liposomes were exposed to a 10 T magnetic field at 318 K for 3 h as a function of r0. Pure DPPC liposomes remained unchanged in size at 10 T, except for magnetofusion only at r0 ) 190 nm. The addition of benzene and anthracene promoted magnetofusion but not magnetodivision. Fusion seems to occur easily when anthracene instead of benzene was added at higher concentrations. Then, a comparison of Figure 3 to Figure 4 gives a F value for each liposome. However, from another viewpoint, the liposome size seems to approach a definite value (e.g., 160 nm in radius) with an increase in the initial size or F under a 10 T magnetic field, irrespective of the additive. In other words, no liposomes having
Figure 4. Square root of the experimental association number (n1/2) observed after liposomes were exposed to a 10 T magnetic field at 318 K for 3 h as a function of r0. The liposome size in all systems was unchanged as long as liposomes were stood under no magnetic field. Symbols: 0, pure DPPC; b, benzene (10 mol %); O, anthracene (10 mol %); 2, benzene (20 mol %); 4, anthracene (20 mol %).
the radii of 160 nm and probably 430 nm will change their size under a 10 T magnetic field. The addition (20 mol %) of benzene and anthracene to a BLM of DPPC also promoted membrane potential development, as in the liposome size. Figure 5 demonstrates that anthracene made the BLM very susceptible to a magnetic field; a steep drop in ∆Ψ occurred at around 1 T, which, when compared, was much smaller than that of 8 T for benzene. This difference seems to be anomalously large beyond expectation from their difference in ∆χ (about 3 times), suggesting cooperative deformation. The undulation structure for a BLM under a high magnetic field is
5660 J. Phys. Chem. B, Vol. 104, No. 24, 2000
Letters It was found that the fusion of DPPC liposomes and the great responses in the membrane potential of black DPPC membranes was brought about by high magnetic fields, and controlled by the addition of diamagnetic aromatic molecules, such as benzene and anthracene. Although temporary models for the magnetofusion of liposomes and magnetic responses in a black lipid membrane are proposed, the relationship between the magneticfield-induced undulation of the membrane and the changes in the membrane potential and liposome size is still unclear. Thus, we conclude at this stage only that magnetic regulation of the functions and structure of artificial organized membranes may be possible. References and Notes
Figure 5. Variations of the membrane potential of a black DPPC membrane containing additives at 318 K with a steady magnetic field intensity. The magnetic response in the membrane potential, ∆Ψ (≡ ΨH - Ψ0, where the subscripts 0 and H mean H ) 0 and H, respectively) was measured with the application of magnetic fields perpendicular to the membrane. Symbols: 0, pure DPPC; 2, benzene (20 mol %); 4, anthracene (20 mol %).
quite unknown, but it seems that the structure should not be very different from that for liposome, except for a contribution to the deformation of the compression energy of the aqueous phase in a liposome. Thus, intuitively the plateau in ∆Ψ would relate to a stable liposome size which may come from stable undulation modes.
(1) Biophysical Effects of Steady Magnetic Fields; Maret, G., Boccara, N., Keipenheuer, J., Eds.; Springer-Verlag: Berlin, 1986; pp 2-51. (2) Ueno, S.; Iwasaka, M.; Tsuda, H. IEEE Trans. Magn. 1993, 29, 3252. (3) Maret, G.; Dransfeld, K. Physica 1977, 86-88B, 1077. (4) Gaffney, B. J.; McConnell, H. M. Chem. Phys. Lett. 1974, 24, 310. (5) Hong, F. T. J. Colloid Interface Sci. 1977, 58, 471. (6) Seeling, J.; Borle, F.; Cross, T. A. Biochim. Biophys. Acta 1985, 814, 195. (7) Bragonza, L. F.; Blott, B. H.; Coe, T. J.; Melville, D. Biochim. Biophys. Acta 1984, 801, 66. (8) Liburdy, R. P.; Tenforde, T. S.; Magin, R. L. Radat. Res. 1986, 108, 102. (9) Tenforde, T. S.; Liburdy, R. P. J. Theor. Biol. 1988, 133, 385. (10) Maret, G.; Dransfeld, K. Top. Appl. Phys. 1985, 57, 143. (11) Boroske, E.; Helfrich, W. Biophys. J. 1978, 24, 863. (12) Higashi, T.; Yamagishi, A.; Takeuchi, T.; Date, M. Bioelectrochem. Bioenerg. 1995, 36, 101-108. (13) (a) Helfrich, W. Phys. Lett. 1973, 43A, 409. (b) Helfrich, W. Z. Naturforsch. 1973, 28C, 693. (14) Khan, H. R.; Ozeki, S. J. Colloid Interface Sci. 1996, 177, 628. (15) Ozeki, S.; Kurashima, H.; Miyanaga, M.; Nozawa, C. Langmuir 2000, 16, 1478.
