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Articles Light-Induced Shape Transitions of Unilamellar Vesicles E. Bru¨ckner, P. Sonntag, and H. Rehage* Institute of Physical Chemistry, University of Essen, D-45141 Essen, Germany Received February 23, 2000. In Final Form: January 29, 2001 In the present study fluorescence microscopy is used in order to investigate the light-induced shape transitions of giant unilamellar vesicles. Therefore pyrene was solubilized in the lipophilic core of the bilayer which allows the transformation of the shape of unilamellar vesicles upon UV irradiation. It turns out that tubular vesicles tend to collapse after illumination forming a string of nearly monodisperse spherical pearls. Giant unilamellar spherical vesicles with small excess areas can be changed reversibly into elliptical aggregates after irradiation with UV light.
Introduction Among the many structures that can be formed by surfactant molecules, vesicles made of phospholipids have attracted particular attention. The unique properties of vesicles result from their closed bilayer structure encapsulating an aqueous volume. The bilayer in the fluid state as a thin and soft membrane allows a large variety of different shapes so that vesicles can be considered as a good model for understanding the properties of cell and organelle membranes.1 Many applications as drug delivery systems2 or microreactors3 have used the extraordinary properties of these objects. One of the most interesting and potentially useful properties of vesicles is their ability to entrap hydrophilic or hydrophobic compounds, which can also be released in a defined manner. These phenomena are important in many pharmaceutical and cosmetic applications. Vesicles may, hence, serve as drug carriers or, in more advanced techniques, as targeting systems. In the latter case, the vesicles must release their content at defined conditions at a desired site. This has the advantage of lowering the drug concentration and protecting the surrounding tissue from any unwanted side effects of the drugs. To evaluate possible delivery and release mechanisms, a further insight into the nature of the interaction of solubilized molecules and the phospholipid membrane is necessary. The incorporation of foreign molecules into the bilayer of giant unilamellar vesicles has been studied to some extent using various techniques.4,5 One characteristic parameter of the bilayer membrane is its bending energy, which controls the fluctuations of a vesicle from one shape to another. Therefore the knowledge of kc is important for the understanding of the dynamic features of vesicles. To evaluate the elastic properties such as the effective tension σ j and the bending rigidity kc, video microscopy investigations of the fluctuations of giant quasi-spherical vesicles (1) Lipowsky, R.; Sackmann, E. Structure and dynamic of membranes; Elsevier: Amsterdam, 1995; Vol. 1. (2) Gregoriadis, G. Liposome Technology; CRC Press: Boca Raton, FL, 1983. (3) Barenholz, Y.; Lasic, D. D. Handbook of Nonmedical Applications of Liposomes; CRC Press: Boca Raton, FL, 1996. (4) Ha¨ckel, W.; Seifert, U.; Sackmann, E. J. Phys. II 1997, 7, 1141. (5) Vanderkooi, J. M.; Fischkoff, S.; Andrich, M.; Podo, F.; Owen, C. S. J. Phys. Chem. 1975, 63, 3661.
originating from Brownian motion can be used.6,7 Membrane properties as bending rigidity were found to be sensitive to the composition.8 The influence on the shape of vesicles controlled by the total membrane area and the enclosed volume is not yet well understood.9 In this work we therefore investigate the influence of solubilized pyrene on the shape of unilamellar vesicles using video microscopy. It turns out that it is possible to affect the shape of the vesicles via UV irradiation. Materials and Methods Chemicals. Synthetic 1,2-dimyristoyl-sn-phosphatidylcholine (DMPC) with a purity of 99% and greater was obtained from Fluka (Biochemika). Pyrene was purchased from Aldrich and purified by repeated crystallization from ethanol. The water employed in this study was bidistilled, deionized, degassed, and purged with argon. Vesicle Preparation. Giant vesicles were prepared according to the swelling method of Reeves and Dowben.10 A mixture of DMPC and pyrene (10 mol %) was placed in an airtight glass bottle in the presence of argon. After addition of oxygen-free water up to a DMPC concentration of 1 mM, the bottle was sealed using a Teflon cap, gently shaken, and stored at 30 °C for a period of at least 2 days. For the microscopic observations, a small amount of the dispersion consisting of uni- and multilamellar vesicles was placed in an argon-saturated observation microchamber as previously reported.8 The chamber was sealed with a quartz cover slip. All samples were kept at constant temperature for about 3 h prior to investigation. Measurements of the bending rigidity were performed in the fluid phase of quasi-spherical DMPC vesicles. Observations were performed with a phase-contrast microscope BX-50 (Olympus Optical Company) equipped with a 40× objective (Ph 2, NA ) 0.75) and a black and white CCD camera (Sensi Cam, PCO). Fluorescence measurements have been carried out using a mercury lamp and an excitation filter of 350 ( 25 nm, monitoring the emission at wavelengths larger than 420 nm. Evaluation of the Bending Elastic Constant. The bending elastic constant kc of single giant unilamellar vesicles was (6) Bivas, I.; Hanusse, P.; Bothorel, P.; Lalanne, J.; Aguerre-Chariol, O. J. Phys. (Paris) 1987, 48, 855. (7) Engelhardt, H.; Duwe, H. P.; Sackmann, E. J. Phys. Lett. 1985, 46, L-395. (8) Bru¨ckner, E.; Sonntag, P.; Rehage, H. J. Phys. Chem. 2000, 104 (10), 2311. (9) Do¨bereiner, H.-G.; Ka¨s, J.; Noppl, D.; Sprenger, I.; Sackmann, E. Biophys. J. 1993, 65, 1396. (10) Reeves, J. P.; Dowben, R. M. J. Cell. Physiol. 1969, 73, 49.
10.1021/la000256i CCC: $20.00 © 2001 American Chemical Society Published on Web 03/15/2001
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Figure 1. Transformation of the shape of a tubular DMPC vesicle (r ) 1.5 µm, 10 mol % pyrene) after the onset of UV irradiation (λexc ) 330-385 nm). Images were taken with a time delay of 0.5 s (a-c) and 2 s (d-e), respectively.
Figure 2. Pearling instability of a tubular DMPC vesicle with 10 mol % pyrene (rc ) 0.7 µm) after a short time span of UV irradiation. The images show the development of a peristaltic mode and the formation of a string of pearls (rP ) 1.1 µm). investigated noninvasively by analyzing the thermal induced fluctuations using phase-contrast microscopy. The evaluation of the bending elastic constant kc was performed as described in detail previously.8
Results and Discussion The shape of fluid DMPC vesicles is governed by the area of the vesicle and the enclosed water volume. It is possible to change the shape of a vesicle by variation of the spontaneous curvature11,12 or simply by modifying the area/volume ratio. An example for the latter process is the temperature-induced change of the vesicle shape. Due
to the larger area expansion coefficient of the membrane compared to volume increase of the enclosed water reservoir, a spherical vesicle deforms elliptically into a prolate one.13 On the basis of this perception, it should be possible to transform the vesicle shape by varying the occupied space of a lipophilic substance solubilized in the bilayer. To induce shape transitions, we used pyrene, which shows a diffusion-controlled quenching process attributed to the formation of excimers (excited dimers).14,15 Therefore pyrene was solubilized in the bilayer of giant DMPC vesicles and the influence of the irradiation with
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Figure 3. Spherical vesicle with a small excess of surface area and membrane-incorporated pyrene (10 mol %) before (a) and after 30 s of UV irradiation (b and c). The vesicle exhibits an excess surface and starts to fluctuate (b).
Figure 4. Relaxation of the elliptically deformed vesicle shown in Figure 3. Relaxation starts with a domain formation and growth until a sphere is finally formed again.
UV light was investigated on single unilamellar vesicles using video microscopy. Tubular DMPC vesicles containing the fluorophore pyrene within the nonpolar bilayer regime showed variations of the membrane curvature on irradiation at wavelengths of about 350 nm (Figure 1). After a time interval of a few seconds the tube collapsed, a process that could not be observed with pure DMPC vesicles or vesicles with solubilized benzene and toluene. With tubular vesicles with smaller diameters, after a short irradiation time the propagation of a peristaltic mode could be provoked. The tubular vesicle structure was transformed into a string of equal-sized pearls (Figure 2). Such a pearling instability is known to be induced using optical tweezers16 due to the dielectric effect of the laser which creates a local lateral tension within the membrane.17 One possible explanation for the influence of the UV irradiation might be a temperature increase due to the absorption of the UV light by the fluorophore. To clarify this problem in detail, we investigated spherical vesicles containing pyrene. It was found that these vesicles can also be influenced. Spherical vesicles with negligible amounts of excess area show an interesting effect: the morphology can be reversibly switched from a sphere to
an ellipse. In Figure 3 a typical example of this kind of shape transformation of an unilamellar vesicle upon UV irradiation is shown. After irradiation, the vesicle is elliptically deformed and thermally induced fluctuations became visible. After termination of the irradiation a relaxation of these fluctuations occurs. This process starts with a domain formation that could be visualized using video microscopy (Figure 4). The membrane exhibits different curvatures. Only the “elliptical part” of the vesicle undulates while in the developing domain region of the membrane no further fluctuations can be observed. With increasing time the domain grows in diameter until finally a thoroughly spherical vesicle results. This shape transformation was completely reversible and could be repeated several times. The influence on the total vesicle area and the time scale of this process is shown in Figure 5. Upon irradiation the area of the vesicle membrane increases significantly and decreases slowly to the initial value after illumination with UV light was terminated. To evaluate whether the shape transition is dominated by a local temperature increase within the bilayer, measurements of the bending rigidity of pyrene containing DMPC vesicles were performed in the presence and in the
Shape Transitions of Unilamellar Vesicles
Figure 5. Variation of the total vesicle area of a pyrenecontaining DMPC vesicle as a function of the observation time.
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Furthermore the reversibility of the observed phenomenon indicates that no chemical reaction such as dimerization of the pyrene has taken place. An examination of the excimer fluorescence was performed to clarify whether there is a molecular mechanism of the shape transformation. Therefore fluorescence measurements were carried out monitoring only the excimer emission of pyrene at wavelengths larger than 420 nm. A plot of the relative intensity of the excimer fluorescence of a spherical vesicle which is deformed elliptically upon UV irradiation is shown in Figure 6. The excimer fluorescence decreases upon irradiation until the vesicle achieves excess areas and starts to fluctuate. Hence an explanation for the decrease can be the diffusion of the slightly more polar excited monomers into the headgroup region of the bilayer18 while the excimers are retained in the lipophilic part.19 Hence the increase of the lateral membrane area seems to be the consequence of the arrangement of the “bulky” excited pyrene and the enhanced free volume needed for the solubilization within the bilayer. After cessation of UV irradiation the system relaxes relatively slowly on grounds of diffusion-controlled reorganization processes of the components. Conclusions The shape of fluid vesicles is governed by the constant inner volume and the area of the vesicle. Upon UV irradiating, the membrane area of vesicles with solubilized pyrene laterally increases because of the enhanced space requirement of the excited fluorophore within the bilayer. The inner vesicle volume remains nearly constant. Consequently the vesicle morphology changes. Spherical vesicles were elliptically deformed while tubular vesicles containing pyrene tended to collapse upon irradiation forming a string of nearly equal-sized spherical particles. This method points out a noninvasive way of influencing the vesicle shape.
Figure 6. Relative intensity of the excimer fluorescence (λem g 420 nm) of a unilamellar vesicle as a function of the excitation time. The arrow marks the appearance of undulations.
absence of UV irradiation. Consequently a temperature increase in the bilayer on irradiation should lead to a reduced value of the bending elastic constant kc compared to the nonilluminated pyrene-containing vesicles. The bending elastic constant kc shows a reduced value of (0.8 ( 0.1) × 10-19 J compared to kc of pure DMPC vesicles of (1.4 ( 0.2) × 10-19 J.8 In the presence of UV light the bending elastic constant kc increases and reaches a value of (1.1 ( 0.2) × 10-19 J. Thus an increase in the temperature can be neglected and seems not to be the major driving force of the observed shape transition.
Acknowledgment. This work is a research project of the Sonderforschungsbereich SFB 1690. We thank R. Lipowsky, U. Seifert, and H.-G. Do¨bereiner for fruitful discussions. LA000256I (11) Do¨bereiner, H.-G.; Selchow, O.; Lipowsky, R. Eur. Biophys. J. 1999, 28, 174. (12) Petrov, P. G.; Lee, J. B.; Do¨bereiner, H.-G. Europhys. Lett. 1999, 48, 435. (13) Ka¨s, J.; Sackmann, E. Biophys. J. 1991, 60, 825. (14) Galla, H.-J.; Sackmann, E. Biochim. Biophys. Acta 1974, 339, 103. (15) Dembo, M.; Glushko, V.; Aberlin, M. E.; Sonenberg, M. Biochim. Biophys. Acta 1979, 522, 201. (16) Bar-Ziv, R.; Moses, E. Phys. Rev. Lett. 1994, 73, 1392. (17) Seifert, U. Adv. Phys. 1997, 46, 13. (18) L’Heureux, G. P.; Fragata, M. Biophys. Chem. 1988, 30, 293. (19) Vekshin, N. L. J. Biochem. Biophys. Methods 1987, 15, 97.
