Morphology Transformations of DODAB Vesicles Reminiscent of

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Morphology Transformations of DODAB Vesicles Reminiscent of Endocytosis and Vesicular Traffic† Dominicus H. W. Hubert,*,‡ Martin Jung,‡ Peter M. Frederik,§ Paul H. H. Bomans,§ Jan Meuldijk,| and Anton L. German‡ Laboratory of Polymer Chemistry and Coatings Technology, Eindhoven University of Technology, P.O. Box 513, 5600 MB Eindhoven, The Netherlands, Departments of Pathology and Electron Microscopy, University of Maastricht, P.O. Box 616, 6200 MD Maastricht, The Netherlands, and Process Development Group, Eindhoven University of Technology, P.O. Box 513, 5600 MB Eindhoven, The Netherlands Received February 15, 2000. In Final Form: June 1, 2000 Mimicking the complex morphological transformations of living cells is an area of ongoing interest to membrane scientists. To this end, liposomes and surfactant vesicles are often regarded as adequate membrane models. Here, we report the morphological transformations reminiscent of endocytosis and vesicular transport for a very basic model system. It is demonstrated that structural conversions of vesicles composed of dioctadecyldimethylammonium salts are triggered by the presence of the cationic initiator V50. On the basis of experimental data (cryo transmission electron microscopy, dynamic light scattering, and turbidity measurements) and theoretical considerations, we propose and discuss a phenomenological model that accounts for these observations. It is shown that even simple triggers can give rise to interesting and complex morphological transformations. Finally, polymerizations of styrene in these model vesicle systems are performed, and it appears that the vesicle transformations have a pronounced effect on the final vesicle-polymer morphology.

Introduction Background. Liposomes and surfactant vesicles are considered as simple, yet adequate, models of biological membranes. Their chemistry and physics have been studied in great detail.1,2 An area of ongoing interest to the membrane scientist is the mimicking of the complex morphological transformations of living cells. These simple model membranes are useful for obtaining an improved understanding of the fundamental aspects of these complex cell activities.3 Their gross oversimplification when compared to a living cell is generally appreciated. However, due to the possibilities of structural and chemical variations and their controllability, these systems are frequently encountered in studies of membrane aggregation, fusion, and budding.4-6 Membrane fusion is an ever-present event in the functioning of living organisms.7 Among its other func* To whom correspondence should be addressed at FEI Co./Philips Electron Optics, Building AAE, P.O. Box 218, 5600 MD Eindhoven, The Netherlands. E-mail: [email protected]. † Part of the Special Issue “Colloid Science Matured, Four Colloid Scientists Turn 60 at the Millennium”. ‡ Laboratory of Polymer Chemistry and Coatings Technology, Eindhoven University of Technology. § University of Maastricht. | Process Development Group, Eindhoven University of Technology. (1) (a) Lipowski, R.; Sackmann, E. Structure and Dynamics of Membranes: From Cells to Vesicles; Elsevier: Amsterdam, 1995. (b) Lasic, D. D. Liposomes: From Physics to Applications; Elsevier: Amsterdam, 1993. (2) Lasic, D. D.; Barenholz, Y. Handbook of Nonmedical Applications of Liposomes: Theory and Basic Sciences; CRC Press: Boca Raton, FL, 1995. (3) Engberts, J. B. F. N.; Hoekstra, D. Biochim. Biophys. Acta 1995, 1241, 323-340 and references cited therein. (4) (a) Schullery, S. E.; Schmidt, C. F.; Felgner, P.; Tillack, T. W.; Thompson, T. E. Biochemistry 1980, 19, 3919-3923. (b) Fraley, R.; Wiltschut, J.; Du¨zgu¨nes, N.; Smith, C.; Papahadjopoulos, D. Biochemistry 1980, 19, 6021-6029. (c) Ellens, H.; Bentz, J.; Szoka, F. C. Biochemistry 1984, 23, 1532-1538. (d) Ellens, H.; Bentz, J.; Szoka, F. C. Biochemistry 1985, 24, 3099-3106.

tions, it is required during endocytosis and intracellular membrane traffic (see Figure 1). During membrane fusion, two membranes completely coalesce, leading to the formation of one membrane-enclosed compartment out of two originally separated compartments (vesicle docking in vesicular transport) or to two compartments out of one (as in cell division or endocytosis). Extensive model membrane studies have shown that divalent cations can induce membrane fusion of acidic phospholipid membranes.4 Supposedly, the fusion mechanism involves a combination of charge neutralization, “cross-linking” of the juxtaposed membranes, and local dehydration. A disordering of the lipid molecules in the contact area would eventually result in membrane fusion.4,7 The exact molecular basis of the mechanism of fusion is not yet understood. However, it is generally accepted that fusion is a local-point event that involves an infinitesimally small area of interacting membranes. The mechanism of molecular rearrangements is referred to as the “stalk-pore” model.7,8 The local-point-fusion concept implies that fusion only requires a local loss of the bilayer configuration of the interacting membranes. Excellent reviews and discussions of this topic can be found elsewhere.6-8 Apart from bringing bilayers together, effective fusogenic agents must induce local perturbations on the structures of the (5) (a) Menger, F. M.; Balachander, N. J. Am. Chem. Soc. 1992, 114, 5862-5863. (b) Menger, F. M.; Gabrielson, K. J. Am. Chem. Soc. 1994, 116, 1567-1568. (c) Wick, R.; Walde, P.; Luisi, P. L. J. Am. Chem. Soc. 1995, 117, 1435-1436. (d) Menger, F. M.; Keiper, J. S. Adv. Mater. 1998, 10, 888-890. See also references cited therein. (6) Tartakoff, A. M. The Secretory and Endocytic Paths: Mechanism and Specificity of Vesicular Traffic in the Cell Cytoplasm; John Wiley & Sons: New York, 1987. (7) (a) Burger, K. N. J. In Structural and Biological Roles of Lipids Forming Nonlamellar Structures; Epand, R. M., Ed.; JAI Press: Greenwich, CT, 1995. (b) Burger, K. N. J., In The Encyclopaedia of Molecular Biology; Meyers, R. A., Ed.; VCH Publishers: New York, 1995. (8) Ravoo, B. J. Membrane Fusion of Vesicles of Oligomerisable Lipids. Thesis, University of Groningen, The Netherlands, 1998.

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Figure 1. Schematic representation of cellular endocytosis uptake of extracellular material (adapted from ref 8).

Figure 2. The structural formula of the cationic initiator 2,2′azobis(2-amidinopropane hydrochloride) (V50).

opposed bilayers to permit the formation of nonbilayer intermediate structures leading to fusion. Overview of This Study. In this paper, we investigate morphological transformations reminiscent of cellular endocytotic uptake and vesicular trafficking. Initially, we observe that the addition of the azo-derived cationic initiator 2,2′-azobis(2-amidinopropane hydrochloride) (abbreviated V50) to large unilamellar dioctadecyldimethylammonium bromide (DODAB) vesicles results in the production of peculiar twinned vesicular structures. A set of experimental techniques is employed to asses the conditions for this vesicle duplication (dynamic light scattering (DLS), turbidity measurements), to explore the effect of vesicle duplication on vesicle morphology (cryo transmission electron microscopy, DLS), and, importantly, to determine the rate constant of duplication (stoppedflow measurements). Dioctadecyldimethylammonium (DODA) salts are used as vesicle-forming amphiphiles, and the role of the counterion (bromide, chloride, or methacrylate) in the duplication process is studied. An explanation of our observations is given in terms of basic physicochemical phenomena and intrinsic vesicle properties. The observed morphological transformations are accordingly interpreted as basic physicochemical events, whereas in a living cell an intricate machinery of proteins and specific molecules is responsible for the induction of fusion to strictly control and regulate these events.6,7 Additionally, we present structural evidence for the local contact sites involved in the fusion process. Finally, we briefly investigate the influence of the gemination process on the polymerization of styrene in these vesicles with respect to the final vesicle-polymer morphology, which is indicative of processes similar to vesicular trafficking. Experimental Section Materials. Dioctadecyldimethylammonium bromide (DODAB; >99%, Arcos), dioctadecyldimethylammonium chloride (DODAC; Fluka), and 2,2′-azobis(2-amidinopropane hydrochloride) (V50; Polysciences; for structural formula, see Figure 2) were used as received. Dioctadecyldimethylammonium methacrylate (DODAM; Jung) was obtained after passage of methanolic DODAB solution through an anionic ion-exchange resin that was converted into the methacrylate form.9 Styrene was purchased from Merck (>99%), distilled under reduced pressure, and stored at -16 °C. Prior to use, the monomer was passed over an inhibitor removal column (DeHibit 200, Polysciences). Aqueous solutions were prepared from water purified by a Millipore Milli-Q system (reverse osmosis). Vesicle Preparation. DODAB, DODAC, and DODAM vesicles were produced according to the standard preparation procedures presented previously. Briefly, extrusion vesicle solutions were prepared from 4-fold extrusions of DODAX/water solutions (1.0 (9) Fukuda, H.; Diem, T.; Stefely, J.; Kezdy, F. J.; Regen, S. L. J. Am. Chem. Soc. 1986, 108, 2321-2327.

Hubert et al. × 10-2 kmol/m3) through a stack of three polycarbonate filters (200 nm pore size; Millipore Co.) at 65 °C. For a more detailed description, see ref 10. Mixed Solutions. Stock solutions of V50 were used to study the influence of its concentration on the behavior of the vesicles (DODAB, DODAC, or DODAM) in terms of their responses according to turbidity, dynamic light scattering (DLS), and cryo transmission electron microscopy (cryo-TEM) measurements. Solutions of different V50:surfactant molar ratios were obtained by simple mixing of stock solutions at elevated temperatures. Typically, a 3.0 mL vesicle solution was mixed with 0.10 mL of the V50 stock solution at 65 °C. The actual molar ratio was set by changing the stock concentration. Prior to sample analysis, an equilibration period of at least 10 min at this elevated temperature was allowed. Stopped-Flow Measurements. A stopped-flow apparatus (Hi-Tech) was used to achieve rapid and complete mixing. Mixing times were better than 0.3 s. Incident light of 350 nm wavelength was used to monitor the turbidity changes, and the sample pathway was 20 mm. The recorded light transmission data were converted to absorption data to allow interpretation in terms of turbidity. Degassing of the samples and water was found to be crucial. Solutions were deaerated by vacuum and argon-rinsing cycles. Typically, a vesicle solution (1.0 × 10-2 kmol/m3 DODAX) was mixed with an equivolumetric amount of the V50 stock solution. The temperature of the system was controlled by a circulating heating bath (Lauda). DLS Measurements. Dynamic light scattering experiments were performed using a Malvern 4700 multiangle DLS instrument (λ ) 488 nm). Samples were diluted by a factor of more than 50 before measuring the intensity autocorrelation functions. The method of cumulant analysis11 was employed to calculate the intensity-averaged particle diameter and the so-called polydispersity index. The latter is a measure for the broadness of distribution, as it represents the square of the ratio of the variance and the mean of a presumed Gaussian decay rate distribution. DLS measurements were performed at 25 °C. Typically, five independent measurements were taken to obtain a mean hydrodynamic radius and a mean variance. See also ref 10. Cryo-TEM. Cryo transmission electron microscopy is a cryogenic technique that allows direct morphological characterization at a detailed level and with the least possible perturbation. Essentially, the preparation of a sample for cryoTEM involves the physical fixation of a sample contained in a thin film. This physical fixation comprises rapid vitrification of a sample by rapid cooling accomplished by plunging it into liquid coolant. Micrographs of the vitrified sample can be taken under low-dose conditions after transfer to a cryoholder and placement of the holder in the electron microscope (Philips CM12). A detailed description of this technique and corresponding protocols can be found elsewhere.12 In our experiments, ethane was used as the liquid coolant and vitrification was performed with a controlledenvironment vitrification system. Polymerization in DODAB Vesicles. Under stirring, styrene was injected into the vesicle dispersion with a microliter syringe to obtain a typical overall monomer concentration of 20 mM. Stirring for 2 days at room temperature ensured a completed takeup of monomer in the amphiphile aggregates.13 Thermally induced polymerizations were carried out in a 50 mL doublewalled glass reactor at 60 °C. The water-soluble azo initiator V50 was dissolved in 0.5 mL of water, and the solution was then injected into the preheated dispersion to reach a concentration (10) Hubert, D. H. W.; Cirkel, P.; Jung, M.; Koper, G. J. M.; Meuldijk, J.; German, A. L. Langmuir 1999, 15, 8849-8855. (11) (a) Ruf, H.; Georgalis, Y.; Grell, E. Methods Enzymol. 1989, 172, 364-390. (b) Hiemenz, P. C.; Rajagopalan, R. Principles of Colloid and Surface Chemistry; Marcel Dekker: New York, 1997. (12) (a) Frederik, P. M.; Stuart, M. C. A.; Bomans, P. H. H.; Busing, W. M.; Burger, K. N. J. Microsc. 1991, 161, 253-262. (b) Frederik, P. M.; Stuart, M. C. A.; Bomans, P. H. H.; Busing, W. M. J. Microsc. 1989, 153, 81-92. (c) Frederik, P. M.; Stuart, M. C. A.; Bomans, P. H. H.; Lasic, D. D. In Handbook of Nonmedical Applications of Liposomes; Lasic, D. D., Barenholz, Y., Eds.; CRC Press: Boca Raton, Fl, 1996; Chapter 15. (13) Hubert, D. H. W. Surfactant Vesicles in Templating Approaches. Ph.D. Thesis, Eindhoven University of Technology, 1999.

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Langmuir, Vol. 16, No. 23, 2000 8975 Table 1. Average Diameters Obtained by Dynamic Light Scatteringa dz, nm vesicles

before

after

size ratio

DODAB DODAC DODAM

150 (0.15) 115 (0.45) 136 (0.19)

131 (0.08) 109 (0.32) 119 (0.09)

0.87 0.95 0.88

a “After” corresponds to 10 min equilibration at 65 °C. The concentrations of surfactants and V50 were 1.0 × 10-2 and 5.5 × 10-3 Kmol/m3, respectively. The numbers in parentheses represent the polydispersity indices according to the cumulant analysis

Figure 3. Cryo-TEM micrograph (bar ) 100 nm) of a DODAB (1.0 × 10-2 kmol/m3) vesicle solution equilibrated for 10 min at 65 °C in the presence of V50 (1.9 × 10-3 kmol/m3). Vitrified from room temperature. of 2 mM. Finally, samples for conversion analysis were withdrawn through a septum (for details see ref 14).

Results In this work, we propose a mechanistic model for the observed phenomena of endocytotic and vesicular transport mimicry. As stated previously, the gemination or doubling of the initially unilamellar vesicles is anticipated to be triggered by the addition of the cationic initiator V50. To address this hypothesis, experiments using bare unilamellar vesicles were conducted. Next, polymerizations of styrene in these vesicles were carried out. Cryo-TEM. In a first set of experiments, we investigated the morphological influence of V50 directly by means of cryo-TEM visualization. To this end, unilamellar DODAB extrusion vesicles were equilibrated for 10 min at 65 °C, i.e., above the main phase transition temperature of DODAB (Tm ) 45 °C)14 in the presence of V50. In an equivalent experiment, DODAC extrusion vesicles were used. Remember that, with increasing temperature, vesicle bilayers exhibit a transition from a gel-like to a liquid-crystalline phase state at Tm. Cryo-TEM micrographs of these samples unambiguously demonstrate the presence of twinned vesicles (see Figure 3). Note that the original vesicle population was strictly unilamellar (see Figure 1 of ref 10). A representative series of micrographs indicates that twinning of the unilamellar DODAB vesicles takes place in a quantitative manner. A closer look at the micrographs reveals local perturbations of the bilayers. Remarkably, these irregularities of the inner and outer vesicles are strictly aligned. Also, the shapes of the inner and outer vesicles strongly resemble each other. The equilibrated DODAC extrusion vesicles display a comparable behavior in that, again, twinned vesicles are found. However, in contrast to the DODAB case, unilamellar lens-shaped vesicles were also visible (photographs not shown). Clearly, this system lacks quantitative transformation, though the same concentration of initiator was applied. Careful examination of the cryo micrographs leads to the conclusion that large unilamellar DODAC vesicles underwent transformation, whereas small vesicles did not. In this context, “small” qualifies lens-shaped vesicles with long axes on the order of 60 nm and short axes of about 20 nm, whereas “large” denotes spherical structures with diameters larger than 80 nm. Importantly, analysis of the micrographs of the untreated DODAC (14) Jung, M.; Hubert, D. H. W.; van Veldhoven, E.; Frederik, P. M.; Blandamer, M. J.; Briggs, B.; Visser, A. J. W. G.; van Herk, A. M.; German, A. L. Langmuir 2000, 16, 968-979.

Figure 4. Measured diameters of architectures in solution as obtained by DLS. DODAB vesicles were equilibrated at 65 °C in the presence of different V50 concentrations.

vesicle solution show a comparable bimodal distribution of shapes and sizes. Dynamic Light Scattering. Next, the influence of different levels of V50 during equilibration was assessed by using dynamic light scattering. The DLS technique is an effective tool for obtaining an averaged measure of the size of colloidal structures in solution.10 Vesicles prepared by extrusion of DODAB, DODAC and DODAM were investigated. As stated earlier, these molecules differ only in their counterions: bromide, chloride, and methacrylate, respectively. First, the effects of a fixed V50:surfactant ratio (0.55) on the final sizes of the structures in solution were determined for the three designated vesicles (see Table 1). The results show that the observed reductions for vesicles composed of DODAB and DODAM virtually coincide, while the size reduction in the case of the DODAC vesicle is less pronounced. The presence of V50 also causes significant reductions in the polydispersity indices. Again, these effects are comparable for DODAB and DODAM and are more pronounced than the effect for DODAC. Second, the effect of different levels of V50 on the final size of equilibrated DODAB vesicles was determined experimentally (see Figure 4). The degree of size reduction is seen to be a function of the molar ratio of the initiator to DODAB. At a certain critical value, i.e., a molar ratio of 0.70, the effect levels off. The corresponding size reduction amounts to 80%. Apparently, this situation represents a boundary. Turbidity. The turbidity of a suspension of colloidal scatterers is strongly dependent on the refractive index of the scatterer and on its shape and size.15 Hence, changes in these properties will effect changes in turbidity. The structural alteration of the unilamellar vesicles upon (15) Kerker, M. The Scattering of Light and Other Electromagnetic Radiation; Academic Press: New York, 1969.

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Figure 5. Turbidity as a function of temperature for DODAB extrusion vesicles in the presence of V50: (a) first series and (b) second series of temperature scans. Molar ratio [V50]/ [DODAB] ) 0.72.

equilibration in the presence of V50 suggests that changes in turbidity are inevitable. Indeed, an increase in turbidity is perceptible, even to the eye. This finding makes it possible to use another technique for further characterization of the structural transformation process without being restricted to electron microscopy or dynamic light scattering. Apart from being less laborious, this technique represents a way to assess the kinetics of the transformation process, whereas the former techniques do not. The turbidity of DODAB extrusion vesicles in the presence of V50 was measured as a function of temperature (see Figure 5). In the first heating scan, a significant increase in solution turbidity is observed at 43-45 °C. Also, at 48 °C a transition is observed. The cooling scan displays just one transition at T ) 40 °C. Interestingly, pronounced hysteresis in turbidity is apparent. Clearly, the structural transformations induced by heating are not reversed upon cooling, at least not on the time scale of the experiment. The second heating and cooling scans display transitions at 43 and 40 °C, respectively, and repeated runs result in virtually identical turbidity traces. These turbidity scans suggest that irreversible structural changes emerge in the first run at a temperature in the range of 43-45 °C. Turbidity evolution measurements were conducted at temperatures >45 °C using a stopped-flow apparatus. The results of these experiments are summarized in Figure 6. Typically, the course of turbidity displayed a minimum. The reported turbidity traces were normalized with respect to this minimum value and the final turbidity. From these results, it is apparent that an increase in temperature results in a faster transformation. This is obvious from

Hubert et al.

Figure 6. (a) Normalized turbidity responses of DODAB vesicles upon mixing with a V50 stock solution at different temperatures. [V50]/[DODAB] ) 0.97 (λ ) 350 nm). (b) Rate constants of the relaxation process at long times (see text).

the increasingly steeper “valleys” and from a shift toward shorter times. Structural transformations are complete in times on the order of seconds. The nontrivial response of the turbidity traces complicates a simple interpretation of the underlying kinetic events. Clearly, the mechanistic pathway involves intermediate structures with decreased intrinsic turbidities. In an attempt to quantify the kinetics of these transformation processes, the rate constants (s-1) of the first-order relaxation processes that were apparent at long times, i.e. at times where an increase of turbidity was observed, were determined. The results are presented in Figure 6. The slope of the fitted straight line is -1.53 × 104, and the activation energy, therefore, amounts to 127 kJ mol-1. Polymerization of Styrene in DODAB Vesicles: Cryo-TEM Observations. We previously reported that the polymerization of styrene in DODAB vesicles leads to “parachute-like” phase-separated vesicle-polymer architectures.10,14,16 In particular, the thermal polymerization of monomer-laden vesicles using an azo-derived cationic initiator, 2,2′-azobis(2-amidinopropane hydrochloride), V50, results in the production of peculiar twinned vesicular structures carrying one polymer particle (see Figure 7). The leading structures were classified as double parachutes. Remarkably, most structures appear to confine only one polymer bead in the outer vesicle of the twin. A closer look at some of these structures reveals two interesting details (see Figure 8): First, there is a small (16) Jung, M.; Hubert, D. H. W.; Bomans, P. H. H.; Frederik, P. M.; Meuldijk, J.; van Herk, A. M.; Fischer, H.; German, A. L. Langmuir 1997, 13, 6877-6880.

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Figure 9. Proposed pathway of morphological transformations. Supposedly, the gemination occurs via an intermediate of a stomatocyte morphology.

Figure 7. Cryo-TEM micrograph (bar ) 100 nm) of twinnedvesicular structures obtained after thermal polymerization using V50 as initiator.

Figure 8. Translocation of a polymer bead from the inner to the outer vesicle by vesicle transport mimicry (bar ) 100 nm).

third vesicle of about 40 nm diameter being entrapped in the water compartment between the two bilayers. Second, the polymer bead that is confined to the outer vesicle exhibits a strong asymmetry. Discussion The results presented above allow us to sketch a simple model that can account for the observed phenomena. The findings present unambiguous evidence that the gemination is triggered by the presence of the cationic initiator. Moreover, the accompanying reduction in the size of the vesicles demonstrates that this gemination is a univesicular process; i.e., a single vesicle is transformed into a twinned-vesicular architecture. As such, this gemination is reminiscent of endocytosis. The latter pathway involves subsequent invagination and self-fusion of a single membrane vesicle (vide supra). Accordingly, the initiator must induce or facilitate these structural transformations, in one way or the other. We ascribe a 2-fold action of the initiator to its ionic nature, in the following way (see Figure 9): The addition of this ionic solute induces an osmotic pressure difference across the semipermeable vesicle bilayer. As a result, water flows from the interior of the

vesicle to the bulk environment. This displacement of volume gives rise to vesicle deflation. Simultaneously, the curvature energy of the deformed vesicle increases. Deflation proceeds until the osmotic pressure and the curvature stress balance. The collapsed vesicle assumes a cuplike shape, in which the poles of the vesicle approach each other. This close approach results in a local-point contact of the poles, and fusion is triggered. This contact is facilitated by the initiator, likely by reducing the electrostatic bilayer repulsion and by bilayer surface dehydration. Local-point fusion necessarily leads to twinned vesicular structures. Interestingly, the presence of the initiator does not affect the “intervesicular” stability, as no signs of coagulation, aggregation, or unbridled fusion were perceptible, probably due to a synergy of reduced repulsion and forced close approach of poles. In essence, the action of the initiator comprises the induction of osmotically driven deflation and screening of the headgroup repulsion to finally force the poles of the vesicle to approach each other and fuse. Before turning to the experimental results, we analyze the proposed pathway in more detail. First, the hypothesis of osmosis-driven deflation requires that the bilayer be a semipermeable barrier. Indeed, reports on the osmotic response of DODAB vesicles can be found.17 Time constants for the transmembrane transport of bromide and chloride counterions are on the order of 103 s.18 The transport of a cationic species is expected to be even more strongly retarded. Therefore, permeation of the initiator is virtually nonexistent, certainly on the time scale of the gemination process (i.e., seconds; vide supra). Second, gemination is proposed to proceed via cuplike intermediates whose poles are forced to come into close contact. Interestingly, theoretical reflections concerning the understanding of vesicle shape transformations have been reported in the literature.19 Systematic analysis of the underlying curvature models revealed a large variety of shapes which minimize the energy for certain physical parameters such as the enclosed volume and the area of a vesicle. These theoretical results lead to the prediction of the trajectory of shape transformation as the physical parameters change. Shapes of lowest bending energy for vesicles with zero spontaneous curvature have been calculated and reported for several values of reduced entrapped volume.20 Indeed, deflation proceeds via shape transition from spheres to prolates, to oblates, and finally to stomatocytes (“cups”). Cryo-TEM. Micrographs of equilibrated vesicles clearly demonstrate the twinned structures that are expected from the process of gemination. The local and aligned (17) (a) Carmona Ribeiro, A. M.; Chaimovich, H. Biochim. Biophys. Acta 1983, 733, 172-179. (b) J. Colloid Interface Sci. 1984, 100, 433443. (18) (a) Lissi, E. A.; Abuin, E. B.; Zanocco, A.; Backer, C. A.; Whitten, D. G. J. Phys. Chem. 1989, 93, 4886-4890. (b) Cuccovia, I. M.; Chaimovich, H. Langmuir 1990, 6, 1601-1604. (19) (a) Svetina, S.; Zeks, B. In Handbook of Nonmedical Applications of Liposomes: Theory and Basic Sciences; Lasic, D. D.; Barenholz, Y., Eds.; CRC Press: Boca Raton, FL, 1995. (b) Seifert, U.; Lipowski, R. In Handbook of Nonmedical Applications of Liposomes: Theory and Basic Sciences; Lasic, D. D., Barenholz, Y., Eds.; CRC Press: Boca Raton, FL, 1995. (20) Seifert, U.; Berndl, K.; Lipowski, R. Phys. Rev. A 1991, 44, 11821202.

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Figure 10. Cryo-TEM snapshot of the transformation process. The micrograph was taken directly after mixing (