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Membrane Dynamics of a Myelin-like Giant Multilamellar Vesicle Applicable to a Self-Reproducing System Katsuto Takakura and Tadashi Sugawara* Department of Basic Science, Graduate School of Arts and Sciences, The University of Tokyo, 3-8-1 Komaba, Meguro, Tokyo 153-8902, Japan Received January 30, 2004 A real time observation of a myelin-like giant multilamellar vesicle (mGMV) revealed that it divided into relatively small mGMVs when an aqueous solution of an electrolyte was added. Furthermore, the mGMV showed a division process accompanied by the growth of the dividing mGMVs when a bolaamphiphile which was composed of an electrolyte unit and a vesicular amphiphile unit was added. This vesicular system can be regarded as a self-reproduction of mGMV, where the added amphiphile acts as a supplier of both the vesicular amphiphile and the division initiator.
Real-time observation of morphological changes in a giant unilamellar vesicle (GUV) or a giant multilamellar vesicle (GMV), induced by the variation in an osmotic pressure or temperature, by additives, or by chemical transformation occurring in giant vesicles, has drawn much attention as a pronounced example of membrane dynamics.1 On the other hand, the morphological change in a myelin-like giant multilamellar vesicle (mGMV), in which bilayer membranes are tightly packed, has scarcely been studied, although it is expected that mGMVs exhibit unique morphological changes as a result of the cooperative interlamellar reorientation of amphiphiles. Herein we describe the novel dynamics of mGMVs triggered by the addition of electrolytes, exhibiting the division of mGMVs. It is also found that mGMVs divide into mGMVs, accompanying the growth of the dividing mGMVs, especially when a bolaamphiphile2 is hydrolyzed to an electrolyte and the same amphiphile as the constituent of the original mGMV within the membrane of the original mGMV. The observed dynamics in this system can be regarded as a self-reproduction of mGMV. The vesicular amphiphile V with two hydrophobic chains, bearing a benzaldehyde moiety at one end, was prepared, and amphiphile V was found to form mGMVs.1k After mixing a dispersion of mGMV (5 mM) with a 10 mM solution of tetramethylammonium bromide (TMAB), sodium chloride, or sodium acetate, the morphological change in mGMVs was monitored by differential interfer* To whom correspondence should be addressed. E-mail:
[email protected]. Phone: +81-3-5454+6765. Fax: +81-3-5454-6997. (1) (a) Sackmann, E.; Duwe, H.-P.; Engelhardt, H. Faraday Discuss. Chem. Soc. 1986, 81, 281. (b) Ka¨s, J.; Sackmann, E. Biophys. J. 1991, 60, 825. (c) Menger, F. M.; Balachander, N. J. J. Am. Chem. Soc. 1992, 114, 5862. (d) Menger, F. M.; Gabrielson, K. J. Am. Chem. Soc. 1994, 116, 1567. (e) Wick, R.; Walde, P.; Luisi, P. L. J. Am. Chem. Soc. 1995, 117, 1435. (f) Menger, F. M.; Lee, S. J.; Keiper, J. S. Chem. Commun. 1998, 957. (g) Menger, F. M.; Angelova, M. I. Acc. Chem. Res. 1998, 31, 789. (h) Jaeger, D. A.; Schilling, C. L., III; Zelenin, A. K.; Li, B.; KubiczLoring, E. Langmuir 1999, 15, 7180. (i) Nomura, F.; Nagata, M.; Inaba, T.; Hiramatsu, H.; Hotani, H.; Takiguchi, K. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 2340. (j) Kahya, N.; Pe´cheur, E.-I.; de Boeji, W. P.; Wiersma, D. A.; Hoekstra, D. Biophys. J. 2001, 81, 1464. (k) Takakura, K.; Toyota, T.; Yamada, K.; Ishimaru, M.; Yasuda, K.; Sugawara, T. Chem. Lett. 2002, 404. (l) Jaeger, D. A.; Clark, T., Jr. Langmuir 2002, 18, 3495. (m) Takakura, K.; Toyota, T.; Sugawara, T. J. Am. Chem. Soc. 2003, 125, 8134. (2) Fuhrpop, J.-H.; Ko¨nig, J. Membranes and Molecular Assemblies: The Synkinetic Approach; Royal Society of Chemistry: Cambridge, U.K., 1994.
Figure 1. Differential interference contrast optical micrographs of morphological changes in a mGMV composed of V: (a-d) images obtained 0, 0.5, 1.5, and 2 min, respectively, after mixing a dispersion of GMV with a solution of TMAB. The white bars correspond to 10 µm.
ence contrast optical microscopy at 23 °C.3 We found that mGMVs showed the division to form several small mGMVs (Figure 1).4 One of the plausible driving forces to promote morphological changes in mGMVs is as follows. In the case of budding of a GUV,1c the vesicular membrane was locally disturbed by the change in the osmotic pressure or by the effect of an added electrolyte on the electric double layer of the membrane surface. If this phenomenon occurs in the mGMV, the destabilization in the outer membrane induces the interlamellar reorientation of the tightly packed bilayer membranes. The deformation, then, may be transmitted across membranes, resulting in the division of mGMV. Incidentally, it has been reported that loosely packed GMVs exhibit a separation process or birthing process;1d,f,i however, they never exhibit such division dynamics. Therefore, the division of mGMV can be regarded as characteristic dynamics in the closely packed aggregates. Because vesicular amphiphile V bears a formyl group in the hydrophobic terminal, it can be converted to the bolaamphiphilic form V* through the dehydrocondensation with an amine bearing a hydrophilic head. The structural feature of amphiphile V* prompted us to add V* to the mGMV containing a small amount of catalyst C for the chemical reaction V* f V + E, where bolaamphiphile V* is expected to supply both the electrolyte as a trigger of the division of mGMV and the vesicular (3) An Olympus Power BX51 (objective lens ×40) microscope was used. (4) The microscopic images of the division process of mGMV after the addition of the aqueous solution of NaCl or NaOAc are shown in Supporting Information, part A.
10.1021/la049738a CCC: $27.50 © 2004 American Chemical Society Published on Web 04/06/2004
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Figure 3. Differential interference contrast optical micrographs of morphological changes in a mGMV composed of V and 10 mol % C: (a-g) images obtained 0, 1.5, 3.5, 4, 5, 10, and 50 min, respectively, after mixing of a dispersion of mGMV and a solution of precursor V*. The white bars correspond to 10 µm. Figure 2. Formation of V through the hydrolysis of V* in an aqueous solution at room temperature. Initial conditions: (a) aqueous 1 mM solution of V* (2), and (b) aqueous 1 mM solution of V* in the presence of mGMVs (1 mM) containing 10 mol % of C ([). The inset shows the UV spectral change under the condition (b) at 5 min intervals. The absorption at 500 nm is assigned to the BODIPY moiety of catalyst C. Scheme 1
amphiphile V to the mGMV (Scheme 1). Bolaamphiphile V* was synthesized through the dehydrocondensation of V and the electrolyte E according to the similar method reported in the literature.5 A dynamic light-scattering measurement revealed that V* does not form giant vesicles but forms small vesicles with diameters of 40-100 nm which could not be detected under an optical microscope.6 The formation of vesicular amphiphile V and electrolyte E through hydrolysis of V* in 1 mM aqueous solution was monitored as a function of time by the decrease in the UV absorbance at 330 nm which was assigned to the diphenylazomethine moiety (Figure 2).7 Although the hydrolysis of V* proceeded slowly without C (plot a), it was greatly accelerated in the presence of 1 mM mGMVs, containing 10 mol % C. The conversion reached 90% after 35 min (plot b), indicating that incorporated catalyst C effectively catalyzed the hydrolysis of the imine. Be(5) Okahata, Y.; Kunitake, T. J. Am. Chem. Soc. 1979, 101, 5231. (6) The size of the aggregates was measured by a dynamic lightscattering method with a NIKKISO MicrotracUPA150 at room temperature. The solution of V* was sonicated for 5 min before analysis. (7) UV spectra were recorded on a SHIMADZU UV-3100 by using a quartz crystal cell with an optical path length of 0.1 cm.
cause electrolyte E diffused into the water layer, the equilibrium of the imine hydrolysis shifted toward the products side (V and E). After mixing a solution of precursor V* (10 mM) with a dispersion of mGMV (5 mM) containing 10 mol % of the amphiphilic acid catalyst C, the morphological change in mGMVs was monitored by differential interference contrast optical microscopy at 23 °C. Several minutes after the addition of the solution of V*, mGMVs exhibited a dramatic morphological change, as shown in Figure 3. A bud was derived from the spherical GMV of the first generation (P) after 1.5 min (Figure 3b). Then, the size of the bud grew and it divided into two mGMVs of the “second generation” (Q, Figures 3c-e).8 Note that the volume of each mGMV of the second generation was almost the same as that of the original mGMV: the sum of the volumes of the second-generation mGMVs was estimated to be 1480 µm3 from their radii, whereas the volume of the original one was only 620 µm3. The result supports the view that the morphological change in the GMVs occurs in conjunction with the increase in the size of the dividing mGMVs. This dynamics can be explained by the increase in the number of vesicular amphiphiles V caused by the hydrolysis of V* in the mGMV. Because the produced mGMV is composed of the same amphiphile as that of the original mGMV, these dynamics can be regarded as a selfreproduction1e,m of the mGMV. Although not all of the original GMV divided into two mGMVs almost equally as shown in Figure 3e, the increase in the number of mGMVs was a reproducible phenomenon. Intriguingly, it was observed that several mGMVs of the second generation again divided to form mGMVs of the “third generation” (R), as shown in Figure 3f,g. Because all the precursors V* were converted to V 1 day after the first addition, judging from plot b in Figure 2, we added a second portion of the solution of V* to the dispersion of mGMVs. It was found that the division of mGMVs was restarted by this treatment. Furthermore, real-time observation with a fluorescence microscope showed that the mGMVs after the second addition of V* also contained catalyst C (see Supporting Information, part C). To examine the effect of the liberated electrolyte E on the morphological change in mGMV, the dynamics of an aqueous dispersion of mGMV (5 mM) after mixing with a solution of E (10 mM) was monitored by differential interference contrast optical microscopy at 23 °C (see Supporting Information, part D). The mGMV also exhibited the self-division similar to the case of the addition of (8) Some mGMVs, in particular, those with diameters larger than 20 µm, did not divide completely but stopped their morphological change at the stage of squeezing (see Supporting Information, part B).
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V*, but the mGMV did not grow. Judging from the previously mentioned data, the results strongly suggest that the current system is the self-reproducing system of the mGMV with the following cycle: incursion of the precursor into mGMVs; growth of mGMVs, accompanied by the production of vesicular amphiphile V; and division of the mGMV triggered by the electrolyte E. In conclusion, we found that mGMVs exihibited selfdivision when several electrolytes were added. The mGMV composed of V and a catalytic amount of C, in particular, showed the self-reproducing dynamics by the addition of the bolaamphiphile V*, accompanied by the hydrolysis of V* into V and E. The reproducing dynamics was confirmed to restart by the second addition of V*. Such a system is attractive from the point of view of construction of an artificial cell.
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Acknowledgment. This work was supported by a grant-in-aid from the Center of Excellence (Study of Life Science as Complex Systems) of the Ministry of Education, Culture, Sports, Science and Technology, Japan. Supporting Information Available: (A) Microscopic images of morphological changes in the mGMV after the addition of an aqueous solution of sodium chloride or sodium acetate (PDF). (B) Representative images of incomplete separation of the mGMV (PDF). (C) Fluorescence microscopic observation of self-reproducing dynamics after the second addition of V* (PDF). (D) Microscopic images of morphological changes in the mGMV after the addition of E (PDF). These materials are available free of charge via the Internet at http://pubs.acs.org. LA049738A
