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Comments Comment on “Kinetics of the Adhesion of DMPC Liposomes on a Mercury Electrode. Effect of Lamellarity, Phase Composition, Size and Curvature of Liposomes, and Presence of the Pore Forming Peptide Mastoparan X”
In a recent paper, H. A. Herna´ndez and F. Scholz1 (henceforth also referred to as H&S) reported on experimental evidence for a kinetic model of liposome adhesion on mercury electrodes. Rather remarkably, H&S claimed equivalence between molecular processes involved in the fusion of two vesicles2 (cf. schematic diagrams for sequential reaction steps leading to phospholipid vesicle fusion, Figure 1 in ref 2) and a liposome adhesion at a metallic surface (Figure 1 in H&S1). In this comment, we question the validity of such an equivalence and the H&S interpretation of the amperometric adhesion signals on a molecular level. The technique H&S used is based on measuring the displacement of the surface charge of a mercury electrode by the adhesion of deformable particles and living cells, (i.e., the amperometric adhesion signals), introduced by Zˇ utic´ and co-workers.3-6 In a note published in Langmuir4 and highlighted in Analytical Chemistry,7 we presented the amperometric response of singlecell adhesion in real time, from the initial attachment to a finite state of spread cell at the growing mercury drop electrode. We stated that “phospholipid Vesicles, the classical model in studying physical mechanisms of cell adhesion, would be well suited for future electrochemical studies”. The key ingredient in such a measurement is the versatile potentiostatic control of adhesion forces by changing the surface charge and tension at the electrode/ aqueous suspension interface.8 The adhesion force can be finetuned to study the interplay of complex processes involved in deformable particle-electrode double-layer interactions.3 In particular, the signature of a single adhesion event at the mercury electrode is the spike-shaped current-time transient,3,4-6 consistent with the classical model of the electrical double layer at the electrode/solution interface.9,10 The flow of such a current reflects the dynamics of adhesive contact formation of the deformable particle with the electrode and the subsequent rupture and spreading of particle constituents to a film of a finite surface area4-6 (scheme A in Figure 1). Yet, there is another process revealed by the Compton group11-14 (cited as refs 23-26 in * Corresponding author. E-mail:
[email protected]. (1) Herna´ndez, A. V.; Scholz, F. Langmuir 2006, 22, 10723. (2) Lee, J.; Lentz, B. R. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 9274. (3) Zˇ utic´, V.; Kovacˇ, S.; Tomaic´, J.; Svetlicˇic´, V. J. Electroanal. Chem. 1993, 349, 173. (4) Svetlicˇic´, V.; Ivosˇevic´, N.; Kovacˇ, S.; Zˇ utic´, V. Langmuir 2000, 16, 8217. (5) Svetlicˇic´, V.; Ivosˇevic´, N.; Kovacˇ, S.; Zˇ utic´, V. Bioelectrochemistry 2000, 53, 79. (6) Svetlicˇic´, V.; Hozic´, A. Electrophoresis 2002, 23, 2080. (7) News-Analytical Currents, Anal. Chem. 2000, 72 (23), 720A. (8) Ivosˇevic´, N.; Zˇ utic´, V. Langmuir 1998, 14, 231. (9) Bockris, J. O’M.; Reddy, A. K. N. Modern Electrochemistry; Plenum/ Rosetta Edition: London, 1973; Vol. 2, pp 718-801. (10) Israelachvili, J. Intermolecular and Surface Forces; Academic Press, Ltd: London, 1992; Chapter 12. (11) Maisonhaute, E.; White, P. C.; Compton, R. G. J. Phys. Chem. B 2001, 105, 12087. (12) Maisonhaute, E.; Brookes, B. A.; Compton, R. G. J. Phys. Chem. B 2002, 106, 3166. (13) Banks, C. E.; Rees, N. V.; Compton, R. G. J. Phys. Chem. B 2002, 106, 5810. (14) Rees, N. V.; Banks, C. E.; Compton, R. G. J. Phys. Chem. B 2004, 108, 18391.
H&S1) where electroinactive particles produce spike-shaped current transients termed impact eVents under potentiostatic conditions at solid microelectrodes (scheme B in Figure 1). Here we point out the major differences between the two processes: (1) transient particle-electrode contact, the impact eVent, is driven by acoustic agitation at the solid microelectrode,11-14 as opposed to the liposome-mercury electrode interaction where the permanent contact is established through adhesion forces under quiescent conditions; (2) the impact signals occur at a much shorter time-scale (microseconds vs milliseconds), and the current polarity is opposite that of adhesion signals while the spike amplitude is not sensitive to the size of the impacting particle.14 Common to both mechanisms is the phenomenon that an electroinactive particle produces a spike-shaped current transient, and the polarity of spikes inverts at the potential of zero charge of the electrode (Epzc). This apparent similarity seems to have caused the ambiguity (cf. Figure 1 in H&S1) in formulating the reaction steps for the liposome-mercury electrode interaction,1,15 as will be discussed below. Herna´ndez and Scholz1 examined the effects of liposome lamellarity, phase composition, size, curvature, and the presence of Mastoparan X (MPX) on the kinetics of liposome adhesion by analyzing the spike-shaped adhesion signals at the mercury electrode (as we discussed above) for various temperatures. This study was confined to only one potential of the mercury electrode (-0.9 V vs Ag/AgCl, 3 M) with a corresponding electrode charge density (σel) of approximately -10 µC cm-2, rather than taking the advantage of changing the adhesion force by varying the electrode potential. As we show in Figures 2 and 3 (our results discussed below), the change in the electrode potential reveals important information about the nature of the adhesion process, which was completely omitted by H&S.1 The treatment of the charge of the adhesion signals, recorded at a single electrode potential and temperature variation,1 leads us to question the relevance of the extracted parameters used in the validation of the kinetic model of the liposome adhesion. Specifically, the kinetic model of H&S1 for liposome adhesion on a mercury electrode is constructed by analogy to the mechanistic events in fusion between curved lipid bilayers of two vesicles2 (ref 6 in H&S1) where three sequential steps could be distinguished: (1) reversible first intermediate, (2) conversion to a semistable second intermediate, and (3) irreversible fusion pore formation. The empirical equation
Q(t) ) Q0 + Q1[1 - exp(-t/τ1)] + Q2[1 - exp(-t/τ2)] (1) used by H&S1 contains five parameterssQ0, Q1, Q2, τ1, and τ2sfor fitting the experimental current-time curves after an integration procedure. Q(t) is the total displaced charge by liposome interaction with the mercury electrode. The first term on the right side in eq 1 is associated with the docking of the liposome on the mercury surface (“only a few lecithin molecules with the hydrophobic tails pointing outward of the bilayer are (15) Hellberg, D.; Scholz, F.; Schubert, F.; Lovric´, M.; Omanovic´, D.; Herna´ndez, V. A.; Thede, R. J. Phys. Chem. B 2005, 109, 14715.
10.1021/la063712x CCC: $37.00 © 2007 American Chemical Society Published on Web 06/27/2007
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Figure 1. Comparison of processes postulated to occur at the electrode/aqueous electrolyte interface (schematic presentations) during the adhesion and impact event of a particle. (A) The attractive interaction between a hydrocarbon microdroplet and a positively charged mercury electrode; the double-layer charge displacement current (I ) dA/dt) has the polarity of a reduction current (reprinted with permission from ref 3, Copyright 1993, Elsevier). (B) Particle impact: the electrode is positive (a) and negative (b) relative to the Epzc (reprinted with permission from ref 14, Copyright 2004, American Chemical Society).
Figure 2. Adhesion signals of individual liposomes at the static mercury drop electrode (experimental details are given in ref 16) for surface charge densities of +9.5 µC cm-2 and -10.0 µC cm-2, which is equivalent to σel in the H&S experiment.1
Figure 3. Bidirectional adhesion signals in a liposome suspension recorded at potentials negative to the Epzc, within the range of the electrode charge densities 0 > σel > -4 µC cm-2. Experimental conditions are the same as those in Figure 1.
anchoring the liposome to the mercury surface”). The second term accounts for the opening of the liposome (“irreVersible restructuring of the liposome/mercury interface resulting in a liposome that is bound Via a few lecithin molecules to the mercury surface”) without formation of an open pore to the outer solution. The third term accounts for the spreading of the lecithin monolayer film on the mercury surface; the pore opening in the bilayer is claimed to be the rate-determining step. Equation 1 for the time evolution of the total displaced charge completely neglects the initial adhesion step of the liposome at the electrode surface and flattening of an intact liposome. More
specifically, H&S have assumed (a) that the adhesion of intact liposomes is a fast and weak reversible reaction with no measurable displacement of surface charge on the electrode; (b) that the polar heads of the liposome outer surface would not alter the electrode charge at the contact interface; and (c) that the polar-head-to-mercury orientation of the liposome outer membrane is strongly disfavored since the mercury surface is hydrophobic. H&S have not addressed why these limitations are valid for their model. These limitations are in direct contradiction with the theory and the experimental evidence on liposome deformability and the orientation of polar groups of bilayer membranes in the process of liposome adhesion on hydrophilic and hydrophobic substrates.18-23 The sequence of events involved in the transition from attached liposomes to bilayer patches on hydrophilic and hydrophobic surfaces were visualized in situ by tapping-mode atomic force microscopy (AFM) in liquid.24 The AFM images indicated that the attached liposomes start to flatten from the outer boundaries. Favorable adhesion forces may thus be the reason for the flattening of the liposomes and, at a certain point when the bending energies become too high, the liposomes rupture. Moreover, the direct experimental evidence of liposome polar head interaction with the gold electrode over a broad range of negative surface charge densities was presented in the electrochemical and neutron reflectivity study of Burgess et al.25 We argue that, analogously to fluid membranes of living cells,4-6,26 liposomes will first establish close contact with the charged surface of the mercury electrode with their intact outer (16) Liposomes were prepared by a thin-film hydration method from phosphatidylcholine, cholesterol, and dihexadecyl phosphate in the molar ratio 7:5:1.17 The electrochemical experiment was performed in phosphate-buffered saline (PBS), pH 7.47, at 20 °C under a nitrogen atmosphere, using a PAR 174A interfaced to a PC, and a 303A EG&G PARC electrode. Analogous data acquisition was performed by a DAQ card-AI-16-XE-50 input device, and data were analyzed using the application developed in LabView 6.1 software. (17) Tomasˇic´, J.; Hrsˇak, I. Biochim. Biophys. Acta 1982, 716, 217. (18) Seifert, U.; Lipowsky, R. Phys. ReV. A. 1990, 42, 4768. (19) Lipowsky, R.; Seifert, U. Langmuir 1991, 7, 1867. (20) Ra¨dler, J.; Strey, H.; Sackmann, E. Langmuir 1995, 11, 4539. (21) Keller, C. A.; Kasemo, B. Biophys. J. 1998, 75, 1397. (22) Reviakine, I.; Brisson, A. Langmuir 2000, 16, 1806. (23) Richter, R. P.; Be´rat, R.; Brisson, A. R. Langmuir 2006, 22, 3497. (24) Jass, J.; Tja¨rnhage, T.; Puu, G. Biophys. J. 2000, 79, 3153. (25) Burgess, I.; Li, M.; Horswell, S. L.; Szymanski, G.; Lipkowski, J.; Majewski, J.; Satija, S. Biophys. J. 2004, 86, 1763. (26) Institut Rudjer Boskovic; Svetlicic, V.; Zutic, Hozic Zimmermann, A. International Application No. PCT/HR2006/000008, 2006.
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membrane, without the “turning around of one or few lecithin molecules”1 or pore opening in the lipid bilayer.1 It is difficult to imagine the pulling out of only one or a few lecithin molecules from the supramolecular assembly of a phospholipid bilayer. Energetically, it is more favorable for a liposome to deform and flatten on the substrate and then rupture at the position of highest curvature.18-19 Deformability in contact with substrates and the variability of stable shapes are the intrinsic properties of giant unilamellar vesicles.27-29 Therefore, in contrast to H&S assumptions,1 the initial contact of a giant unilamellar liposome with the charged mercury surface results in a measurable flow of the displacement current. The effects of the electrostatic interaction of liposome polar groups with the electrode double layer could be selectively resolved by recording the liposome adhesion response near the point of zero charge of the electrode. For example, the surface charge density of living algal cells was determined by scanning electrical potential to a point of no net current flow, where the charge density of the cell compensates the electrode charge density.6 We previously proved4 that the distance of the closest approach, for cell adhesion at a positively and negatively charged mercury electrode, is equal to or less than the distance of the plane going through the charge centers of the hydrated counterions nearest to the electrode.30 We recorded the same type of adhesion signals in liposome suspensions as described by H&S (exemplified by Figure 2 in H&S1). However, on the basis of the studies of single adhesion events of oil microdroplets3,31-33 and living cells, unicellular algae,4-6 and blood cells,26 our interpretation of the liposome adhesion signals (as exemplified in Figures 2 and 3) is different. Figure 2 illustrates the shape of adhesion signals and the direction of flow of the compensating current at high surface charge densities of the mercury electrode (σel), positive (σel ) +9.5 µC cm-2) and negative (σel ) -10.0 µC cm-2). The flow of the compensating current reflects the dynamics of adhesive contact formation with a highly charged electrode and subsequent spreading to the supported film of a finite area. The adhesion signals we presented in Figures 2 and 3 were of a larger class of liposomes and differ by a factor of 50 in the current intensity compared to the corresponding signal in H&S1 Figure 2. We performed closer examination of liposome suspension by scanning the electrode potential around Epzc and revealed characteristic bidirectional adhesion signals. Figure 3 illustrates such adhesion signals occurring over the surface charge density range 0 > σel > -4 µC cm-2. Rather significantly, the ratios of negative to positive current maxima are constant for a given σel. In our view, the bidirectional adhesion signals originate from specific electrostatic interaction of positively charged choline groups of liposome polar heads when in immediate contact with the mercury electrode. Such an interaction is excluded by the H&S model1 on the basis of their initial assumption that the “polar-head-to-mercury orientation of the liposome outer membrane is strongly disfavored since the mercury surface is hydrophobic”. An intimate contact of phospholipid polar groups with the electrode surface over a broad range of charge densities is depicted explicitly in the model of a phospholipid bilayer at (27) Bernard, A.-L.; Guedeau-Boudeville, M.-A.; Jullien, L.; di Meglio, J.-M. Langmuir 2000, 16, 6809. (28) Tanaka, T.; Sano, R.; Yamashita, Y.; Yamazaki, M. Langmuir 2004, 20, 9526. (29) Sapper, A.; Janshoff, A. Langmuir 2006, 22, 10869. (30) Marcˇelja, S. Nature 1997, 385, 689. (31) Ivosˇevic´, N.; Zˇ utic´, V. In Contact Angle, Wettability and Adhesion; Mittal, K. L., Ed.; VSP: Utrecht, The Netherlands, 2002; Vol. 2, pp 549-561. (32) Ivosˇevic´, N.; Tomaic´, J.; Zˇ utic´, V. Langmuir 1994, 10, 2415. (33) Ivosˇevic´, N.; Zˇ utic´, V.; Tomaic´, J. Langmuir 1999, 15, 7063.
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the gold electrode25 and is supported by molecular-level information on the conformational changes in the polar head region of a lecithin molecule located in the plane of the metal surface.34 The overall consistency of the findings presented by H&S1 is also affected by their treatment of MPX.1 H&S have not provided conclusive evidence that the pore-forming peptide molecules have been incorporated into the liposome membranes by exposure to the 10-7 M MPX aqueous solution, as used in their experiments. Schwarz and Abruzova35 (cited as ref 42 in H&S1) also added MPX to the aqueous suspensions of liposomes, but at higher MPX concentrations. They concluded that the pore formation involved only a small fraction of lipid-associated peptide.35 On the other hand, Whiles et al.36 (cited as ref 43 in H&S1), applied MPX (with molar ratios to total long-chain lipid of 1:10 and 1:40) in the very initial step of the phospholipid bicelles preparation. Furthermore, MPX was found36,37 (cited as refs 43 and 44 in H&S1) to be more efficient in phospholipid bilayers doped with anionic lipids than in the undoped ones used by H&S.1 Therefore, the MPX effect on the adhesion events at the mercury electrode (Figures 13 and 14 in H&S1) originates from the peptide molecules disrupting the lipid order by residing on the membrane surface,35-38 rather than from the peptide molecules forming pores in the bilayer membrane, as claimed by H&S.1 In conclusion, the significance of single adhesion events at the mercury electrode has been apparent for more than two decades, since the discovery of stochastic adhesion signals of vesicles in seawater samples.39-41 An impressive subsequent progress made in the time-resolved data acquisition of stochastic amperometric signals has been illustrated by the H&S experiments on liposome adhesion at the static mercury drop electrode.1 However, this achievement is only a prerequisite for a consistent analysis and interpretation of the adhesion signals of liposomes in order to extract the kinetic data that are not accessible by other methods. Acknowledgment. This work was supported by Grant No. 098-0982934-2744 and Grant No. 021-0212432-2431 of the Ministry of Science, Education and Sports of Croatia. We thank Igor Zˇ utic´ at SUNY, Buffalo, for discussion and his comments. Vera Z ˇ utic´ ,* Vesna Svetlicˇ ic´ , Amela Hozic´ Zimmermann, and Nadica Ivosˇevic´ DeNardis
Group for Bioelectrochemistry and Surface Imaging, DiVision for Marine and EnVironmental Research, Rudjer BosˇkoVic´ Institute, P.O. Box 180, 10000 Zagreb, Croatia Ruzˇ a Frkanec
Institute of Immunology, Inc., P.O. Box 266, 10000 Zagreb, Croatia ReceiVed December 22, 2006 In Final Form: March 9, 2007 LA063712X (34) Bin, X.; Zawisza, I.; Goddard, J. D.; Lipkowski, J. Langmuir 2005, 21, 330. (35) Schwarz, G.; Arbuzova, A. Biochim. Biophys. Acta 1995, 1239, 51. (36) Whiles, J. A.; Brasseur, R.; Glover, K. J.; Melacini, G.; Komives, E. A.; Vold, R. R. Biophys. J. 2001, 80, 280. (37) Yu, K.; Kang, S.; Kim, S. D.; Ryu, P. D.; Kim, Y. J. Biomol. Struct. Dyn. 2001, 18, 595. (38) Sackmann, E. FEBS Lett. 1994, 346, 3. (39) Zˇ utic´, V.; Plesˇe, T.; Tomaic´, J.; Legovic´, T. Mol. Cryst. Liq. Cryst. 1984, 113, 131. (40) Zˇ utic´, V.; Legovic´, T. Nature 1987, 328, 612. (41) Zˇ utic´, V.; Svetlicˇic´, V. In The Handbook of EnVironmental Chemistry: Marine Chemistry; Wangersky, P., Ed.; Springer-Verlag: Berlin-Heidelberg, Germany, 2000; Vol. 5, Part D, pp 150-164.
