5584
Langmuir 2007, 23, 5584-5590
Individual Electrophoretic Mobilities of Liposomes and Acidic Organelles Displaying pH Gradients Across Their Membranes Yun Chen and Edgar A. Arriaga* Department of Chemistry, UniVersity of Minnesota, 207 Pleasant Street Southeast, Minneapolis, Minnesota 55455 ReceiVed NoVember 14, 2006. In Final Form: February 7, 2007 This report focuses on measuring the individual electrophoretic mobilities of liposomes with different pH gradients across their membrane using capillary electrophoresis with laser-induced fluorescence detection (CE-LIF). The results from the individual analysis of liposomes show that, using surface electrostatic theories and the electrokinetic theory as the first approximation, ζ potential contributes more significantly to the electrophoretic mobility of liposomes than liposomal size. For liposomes with an outer pH 7.4 (pHo 7.4) and a net negative outer surface charge, the most negative electrophoretic mobilities occur when the inner pH (pHi) is 6.8; at higher or lower pHi, the electrophoretic mobilities are less negative. The theories mentioned above cannot explain these pH-induced electrophoretic mobility shifts. The capacity theory, predicting an induced electrical charge on the surface of liposomes, can only explain the results at pHi > 6.8. In this report, we hypothesize that there is a flip-flop process of phospholipids, which refers to the exchange of phospholipids between the outer and inner layers of the membrane. This flip-flop is caused by the pH gradient and membrane instability and results in the observed electrophoretic mobility changes when pHi is 10, the O’Brien model suggests that the dependence of ζ on R could be neglected.4 The electrophoretic mobility, µ, and reduced mobility, µr, of individual liposomes with different pH gradients were calculated using eqs 1 and 2, respectively. The µr and κR for each liposome were then compared with the predictions of the O’Brien model. As an example, Figure 3 shows the values for liposomes with pHi 7.4 and pHo 7.4 (small dots) overlaid on the theoretical plots
5588 Langmuir, Vol. 23, No. 10, 2007
Figure 4. Electrophoretic mobilities at the 25%, 50%, and 75% quartiles. The number by each trace indicates the pHi. The pHo was 7.4.
of µr vs κR (eq 4) for different ζr potentials (indicated with numbers next to each trace). As seen in this figure, the measured liposomal data fall very close to the isopotential ζr ) 1, clearly indicating that electrophoretic mobility is nearly independent of κR. This representation confirms that the electrophoretic mobilities in this system are mainly a function of ζ potential. Since ζ potential is related to the surface charge (see Supporting Information, Part B), the variations in ζ potentials reflect the heterogeneity of surface charge density among the liposomes. Therefore, the individual electrophoretic mobility measurements appear adequate for investigating the effect of pH gradients on the surface charge characteristics of the liposomes. As earlier suggested by the Hayes group, the effect of the pH gradient across the liposomal membrane on the electrophoretic mobility requires comparison of the distributions of this parameter at various pHi’s while pHo is held constant.20 Since comparison of the electrophoretic mobility distributions is visually difficult, we chose to compare the 25%, 50%, and 75% quartiles of these distributions (Figure 4). As indicated near each trace, liposomes with pHi 4.0, 5.0, 5.8, 6.8, 7.4, and 8.0 have different quartiles. It is clear that electrophoretic mobilities shift to more positive values when the pHi decreases from 6.8 to 4 and when the pHi increases from 6.8 to 8. Acidic organelles (e.g., endosomes and lysosomes) are known to have a pHi lower than their cellular environment (i.e., pHo ∼7.4); thus, they have a pH gradient across their membrane. These organelles have also been analyzed individually by CELIF.36 Here, we investigated the electrophoretic mobility properties of acidic organelles isolated from CEM/C2 cells that have endocytosed and accumulated FRD in their acidic organelles. Using eqs 5 and 6, the acidic organelles radii range between 52 ( 11 and 167 ( 24 nm, which are slightly smaller than the liposomes used here (cf. Table 1). The acidic organelle’s interior pH was calculated on the basis of a calibration curve of the fluorescence intensity ratio (pH-dependent fluorescence/pHindependent fluorescence) versus pH. Figure 5 plots the mobility for acidic organelles in the pH ranges 4, the capacitively induced charge will reduce the net charge, bringing the liposomal mobility closer to zero. Consistent with this prediction, Figure 4 shows that the electrophoretic mobility becomes less negative when the pHi increases from 6.8 to 8.0. On the other hand, the capacitive model does not predict the trend in electrophoretic mobility when pHi becomes more acidic. Figure 4 clearly shows that the electrophoretic mobility becomes less negative when pHi decreases from 6.8 to 4. When pHi is lower than pHo (i.e., 7.4), the interior surface charge density decreases in relation to the exterior one (see Supporting Information, Part C, Table 2S). Capacitively induced positive charge will be mostly generated on the inner membrane of the liposome and this charge cannot directly affect its electrophoretic mobility. The amount of induced charge densities in the outer layer of liposomal membrane are estimated to be 2.99 (pH 8), 2.32 (pH 7.4), 2.22 (pH 6.8), 1.52 (pH 5.8), 0.51 (pH 5), and -0.30 µC/cm2 (pH 4), respectively (see Supporting Information, Part D). Other factors are needed to explain the electrophoretic mobility changes induced by low pHi. Flip-flop of phospholipids in liposomal membranes have been reported.13,21,23 We hypothesize that this process may contribute to the observed electrophoretic mobility changes at low pHi. This bidirectional transbilayer movement of phospholipids in biological membranes can be either by passive diffusion (flip-flop) or a more complex proteinmediated process.24 Typically, the former process is very slow due to the unfavorable passage of a hydrophilic headgroup across the hydrophobic membrane core. However, it has been found that the flip-flop rate varies for the membranes with different components and temperature.24 For the DMPC liposomes labeled on the outer membrane with 7-nitrobenz-2-oxa-1,3-diazol-4-yl
Langmuir, Vol. 23, No. 10, 2007 5589
(NBD) fluorescent analogue, the rate constants of the outward (flop) and inward (flip) movement and the half-time of transbilayer movement are 0.15 min-1, 0.30 min-1, and 1.6 min, respectively. After ∼10 min, a steady-state distribution was reached with ∼65% of the analogue in the outer membrane and the other 35% in the inner membrane.24 Cholesterol may reduce flip-flop of phospholipids;20 while there is obvious suppression of flip-flop of DPPC when cholesterol is 25% (mole), others have likely observed similar effects at 20% (mole) cholesterol.20 Due to the low percentage of cholesterol used in these studies (i.e., 7.5% (mole)), it is less likely that flip-flop of phospholipids is decreased. It has been proposed that the flip-flop process can be induced by the different surface charges in the inner and outer layers of the phospholipids membrane as a result of a pH gradient.39 This process is also needed to maintain the mechanical stability of the liposomes.39 Additionally, the change in the inner surface charge tends to destabilize the membrane,25 which results in membrane bending instability which then causes the rapid redistribution of charges in the bilayer membrane.25 The liposome behavior could be better interpreted if the effect of flip-flop process was incorporated into the theoretical model. Even in the absense of equilibrium, the fraction of a given phospholipid at both sides of membrane influenced by pH gradient is hard to predict. Hope et al. have predicted the equilibrium transmembrane distribution of lipids.13 Their results show that full equilibrium is not reached, as it is highly dependent on the membrane composition. However, fluorescent analogues may be used in future studies to estimate the fraction of some phospholipids in the outer membrane under certain circumstances.24 It should be noted that positive charged phospholipids on the outer surface of a liposome may interact with the residual negatively charged sinanol groups found on the surface of covalently modified capillary walls; this would cause delay in the liposome movement during capillary electrophoresis biasing the electrophoretic mobility determination. It is unlikely that phospholipid-silanol interactions are highly relevant in this study because we use AAP-coated capillaries, which present a more inert surface and would sterically prevent access of liposomes to the residual silanol groups in the capillary.40 In addition, at pHi < 6.8, the liposomes do not have external positive charges, which rules out the possibility that electrostatic interactions between the liposomes and the silanol groups are slowing down the movement of liposomes with low pHi. Finally, it is worth to mentioning that the theories presented in this report (i.e., capacity effect and flip-flop transport) can be further refined by taking into account the deformation, polarization, and multipole effects.12 The effect of deformation causes the elongation of liposomes in the direction of the applied field due to shear forces, as well as field-induced polarization. A more asymmetrically shaped particle (e.g., a more elongated ellipsoid) would have a larger electrophoretic mobility. An uneven surface charge distribution results from the negatively charged surface components migrating to one end of the liposomes and positive components to the other end. This asymmetric distribution of charges could increase the migration of particles. The multipole moment effect suggests that the electrophoretic mobility of a particle is dependent on all three moments (i.e. monopole, dipole, and quadrupole) rather than just the monopole moment. A large quadrupole determined by both the magnitude and distribution (39) Pohl, E. E.; Peterson, U.; Sun, J.; Pohl, P. Biochemistry 2000, 39, 18341839. (40) Meagher, R. J.; Seong, J.; Laibinis, P. E.; Barron, A. E. Electrophoresis 2004, 25, 405-414.
5590 Langmuir, Vol. 23, No. 10, 2007
of surface can significantly increase the mobility of particles. As an example of effects that these three factors may have on the electrophoretic mobility of liposomes with pH gradient across the membrane, qualitatively, the more negative end of a polarized liposome will be more adequate for induction of a positive charge when pHi is higher than pHo. In contrast, the positive or neutral end of the liposome will not experience this inductive charge. Therefore, the induced charge at the negative end will slow down less the liposomes which have a smaller pH gradient. Does pHi Affect the Electrophoretic Mobilities of Acidic Organelles? The pH of acidic organelles is known to decrease progressively during their maturation process from 6 to 6.5 in early endosomes, to 5.5-6 in late endosomes, and to 4.5-5 in lysosomes, while the pH of the cytosol remains at ∼7.4.41 The size of acidic organelles is ∼70 nm;42 therefore, the κR value suggests that their electrophoretic mobility is mainly dependent on their surface charge density and independent of R. Although organelles, as functional biological vesicles, are more complex than liposomes, both vesicle types share several properties. For example, the transbilayer distribution of lipids across biological membranes is asymmetric. It has been reported that the cholinecontaining lipids, phosphatidylcholine (PC) and sphingomyelin (SM), are enriched primarily in the internal phospholipid layer of internal organelles, while the amine-containing glycerophospholipids, phosphatidylethanolamine (PE) and phosphatidylserine (PS), are located preferentially in the cytoplasmic phospholipid layer.43 Even when the redistribution of phospholipids in organelle membranes appears mainly mediated by proteins (e.g., flipase enzymes), this asymmetry appears to be related to the stability of the membrane and vesicular transport as it is for liposomes experiencing a pH gradient.23 Until now, no report ever compared the phospholipids distribution between acidic organelles and liposomes. Our findings (Figure 5) indicate that the liposomes (41) Zen, K.; Biwersi, J.; Periasamy, N.; Verkman, A. S. J. Cell Biol. 1992, 119, 99-110. (42) Macdonald, P. E.; Eliasson, L.; Rorsman, P. J. Cell Sci. 2005, 118, 59115920. (43) Daleke, D. L. J. Lipid Res. 2003, 44, 233-242.
Chen and Arriaga
can be considered a good model for predicting the migration behavior of acidic organelles.
Conclusions Using CE-LIF, the electrophoretic mobilities of individual liposomes with the same pHo but different pHi were determined. The finding reveals that several mechanisms may be responsible for the electrophoretic mobility changes that are associated with a pH gradient. The effect of deformation, uneven surface charge distribution, and the multipole moments, as well as the surface electrostatic theories and the electrokinetic theory, cannot explain the behavior of liposomes with pH across their membrane. The capacity effect applies to liposomes with alkaline pHi, when pHo < pHi. The flip-flop of phospholipids in liposomes and acidic organelles with pHo > pHi nicely describes the trend in the electrophoretic mobility changes. Further evidence of the flipflop process at the surface of the electrophoresed liposomes is still lacking. This may require the use of fluorescently labeled charged phospholipids in making liposomes, bleaching the exposed fluorophores,44 and then observing both spectroscopic and electrophoretic properties of such liposomes. Indeed, prediction of the observed trend in the electrophoretic mobility of acidic organelles using liposome models with pH gradients across their membrane is a significant step in our efforts to predict and understand the electrophoretic behavior of biological particles. Acknowledgment. This work was supported through the NIH (AG20866) and a Grant-in-Aid from the University of Minnesota. E.A. is supported through NIH Grant No. K02-AG21453. Supporting Information Available: Part A: Determination of fluorescence emission response of FRD as a function of pH.; Part B: Theoretical derivation of the ζ potential; Part C: Charge density as a function of pH; Part D: Calculation of induced charge density in the outer layer of liposome membrane as a function of pH. Part E: Calculation of ionic strength, I, and osmolarity for buffers used in this study. This material is available free of charge via the Internet at http://pubs.acs.org. LA0633233 (44) McIntyre, J. C.; Sleight, R. G. Biochemistry 1991, 30, 11819-11827.
