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Langmuir 2006, 22, 1976-1981
Fluorescence Microscopic Investigation on Morphological Changes of Giant Multilamellar Vesicles Induced by Amphiphilic Additives Taro Toyota,† Hirotatsu Tsuha,† Koji Yamada,† Katsuto Takakura,†,‡ Kenji Yasuda,§ and Tadashi Sugawara*,† Department of Basic Science and Department of Life Sciences, Graduate School of Arts and Sciences, The UniVersity of Tokyo, 3-8-1 Komaba, Meguro, Tokyo 153-8902, Japan, and Department of Chemistry and Biochemistry, Suzuka National College of Technology, Shiroko-cho, Suzuka, Mie 510-0294, Japan ReceiVed October 31, 2005. In Final Form: December 27, 2005 Adding an artificial bolaamphiphile to a dispersion of giant multilamellar vesicles (GMVs) made of 1-palmitoyl2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) induced a cup-shaped deformation in GMVs accompanied by partial extrusion of the inner vesicle; thereafter, the deformed vesicles returned to their original shape. On the other hand, when the artificial bolaamphiphile together with a surfactant was added to the vesicular dispersion, these deformation and reformation dynamics were transmitted from the outer membranes in GMVs to the inner membranes until an intact inner vesicle was extruded out of the outer membrane. The microscopic aspects of these processes were investigated using amphiphiles tagged with individual fluorophores.
1. Introduction Giant vesicles (GVs) consisting of phospholipids, for example, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC; 1), have drawn much attention as models with which to study the membrane dynamics of living cells.1 When a dispersion of surfactants or other additives is added to a suspension of GVs, the morphologies of the GVs are gradually changed.2 It has recently been found that chemical transformations of membraneforming amphiphiles induce changes in the shapes of GVs.3 Although physicochemical studies have pointed out several factors behind these dynamics,4 the precise mechanism of the morphological changes in GVs is still veiled. If the addition of amphiphilic additives can induce these morphological changes in giant multilamellar vesicles (GMVs), it would be intriguing from the aspect of constructing a minimal cell model that has a nested structure.5 We have already reported * To whom correspondence should be addressed. Phone: +81-3-54546742. Fax: +81-3-5454-6997. E-mail:
[email protected]. † Department of Basic Science, The University of Tokyo. § Department of Life Sciences, The University of Tokyo. ‡ Suzuka National College of Technology. (1) (a) Menger, F. M.; Angelova, M. I. Acc. Chem. Res. 1998, 31, 789. (b) Luisi, P. L.; Walde, P. Giant Vesicles (PerspectiVes in Supramolecular Chemistry); John Wiley & Sons, Ltd.: New York, 2000. (2) (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.; Gabrieison, K. J. Am. Chem. Soc. 1994, 116, 1567. (e) Menger, F. M.; Lee, S. J.; Keiper, J. S. Chem. Commun. 1998, 957. (f) Nomura, F.; Nagata, M.; Inaba, T.; Hiramitsu, H.; Hotani, H.; Takiguchi, K. Proc. Natl. Acad. Sci. 2001, 98, 2340. (g) Wick, R.; Walde, P.; Luisi, P. L. J. Am. Chem. Soc. 1995, 117, 1435. (h) Jeager, D. A.; Schilling, C. L., III; Zelenin, A. K.; Li, B.; Kubicz-Loring, E. Langmuir 1999, 15, 7180. (i) Kahya, N.; Pe´cheur, E.-I.; de Boeji, W. P.; Wiersma, D. A.; Hoekstra, D. Biophys. J. 2001, 81, 1464. (3) (a) Holopainen, J. M.; Angelova, M. I.; Kinnunen, P. K. J. Biophys. J. 2000, 78, 830. (b) Jeager, D. A.; Clark, T., Jr. Langmuir 2002, 18, 3495. (c) Holopainen, J. M.; Angelova, M. I.; So¨derlund, T.; Kinnunen, P. K. J. Biophys. J. 2002, 83, 932. (d) Takakura, K.; Toyota, T.; Yamada, K.; Ishimaru, M.; Yasuda, K.; Sugawara, T. Chem. Lett. 2002, 404. (e) Takakura, K.; Toyota, T.; Sugawara, T. J. Am. Chem. Soc. 2003, 125, 8134. (f) Takakura, K.; Sugawara, T. Langmuir 2004, 20, 3832. (4) (a) Zhelev, D. V.; Needham, D. Biochim. Biophys. Acta 1993, 1147, 89. (b) Moroz, J. D.; Nelson, P.; Bar-Ziv, R.; Moses, E. Phys. ReV. Lett. 1997, 78, 386. (c) Karatekin, E.; Sandre, O.; Guitouni, H.; Borghi, N.; Puech, P.-H.; BrochardWyart, F. Biophys. J. 2003, 84, 1734. (5) (a) Luisi, P. L. Anat. Rec. 2002, 268, 208. (b) Hanczyc, M. M.; Szostak, J. W. Curr. Opin. Chem. Biol. 2004, 8, 660.
that a GMV composed of reactive amphiphiles exhibited direct extrusion of the inner vesicle through a pore in the outer layers when a solution of the amphiphilic reactive partner, which can produce a bolaamphiphile,6 was added to the dispersion of the above vesicles.3d Hotani’s, Menger’s, and Brochard-Wyart’s groups, independently, have also reported these dynamics, which were induced by physical stimuli or addition of additives, and these dynamics are termed “birthing” or “separation”.2f,2e,4c Here, to clarify the role of bolaamphiphiles during such morphological changes in GMVs, a dispersion of GMVs of a nonreactive lipid, 1, labeled with fluorescent probe 2a, the fluorophore of which is a derivative of 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY), was added to a dispersion of bolaamphiphile 3 under a fluorescence microscope. Furthermore, we successfully applied a real-time two-color fluorescence imaging technique to the analysis of the birthing process, which was induced by the addition of a dispersion of a bolaamphiphile (3, 4) and a surfactant (5, 6) to GMVs. 2. Experimental Section 2.1. General. POPC (1) was purchased from Avanti Polar Lipids (AL), and cethyltrimethylammonium bromide (CTAB; 5) was purchased from Wako Pure Chemical Industries (Osaka, Japan). All other commercially available reagents were purchased from Tokyo Kasei Co. (Tokyo, Japan) or Sigma Aldrich Japan Co. (Tokyo, Japan), and were used without further purification. Reaction solvents were distilled. 1H NMR spectra were recorded on a JEOL GSX-270 spectrometer. Fast atom bombardment mass spectra (MS-FAB) were recorded on a JEOL JMS-600H spectrometer with 3-nitrobenzylalcohol as the matrix. Time-of-flight mass spectra (TOF-MS) were recorded on an Applied Biosystems Voyager-DE spectrometer with 2,5-dihydroxybenzoic acid as the matrix. Bolaamphiphile 3 was synthesized according to the previously reported method.3d 2.2. Synthesis of Red-Fluorescent Probe (2a). 11-[3′′,5′′-bis(4′′′-methoxyphenyl)-4′′,4′′-difluoro-4′′-bora-3a,4a-diaza-s-indacenyl]-3′,5′-dimethylphenoxy undecanoic acid7 (159.3 mg, 0.22 mmol), 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (107.2 mg, 0.22 mmol), 1,3-dicyclohexylcarbodiimide (89.7 mg, 0.43 mmol), and (6) Fuhrhop, J.-H.; Wang, T. Chem. ReV. 2004, 104, 2901. (7) Yamada, K.; Toyota, T.; Takakura, K.; Ishimaru, M.; Sugawara, T. New J. Chem. 2001, 25, 667.
10.1021/la0529198 CCC: $33.50 © 2006 American Chemical Society Published on Web 01/26/2006
AdditiVe-Induced Morphological Changes in GMVs 4-(dimethylamino)pyridine (6.9 mg, 0.06 mmol) were mixed in anhydrous CH2Cl2 (3 mL), and then the mixture was stirred for a week at room temperature under a nitrogen atmosphere. After filtration and evaporation of the solvent, the residue was purified on a silica gel chromatography column eluted with a mixture of CHCl3/CH3OH/28% NH3 (aq) (65:35:10) to afford a red-fluorescent phosphatidylcholine (2a) (85.9 mg, 33%). 1H NMR (270 MHz, CDCl3): δ 7.91 (4H, d, J ) 8.9 Hz), 6.94 (4H, d, J ) 8.9 Hz), 6.69 (2H, s), 6.60 (2H, d, J ) 4.3 Hz), 6.54 (2H, d, J ) 4.3 Hz), 5.23 (1H, m), 4.37 (2H, m), 4.36 (1H,m), 4.13 (1H,m), 3.85 (6H, s), 3.81 (2H,m), 3.78 (2H, br), 3.31 (9H, s), 2.31-1.08 (50H, m), 2.20 (6H, s), 0.87 (3H, t, J ) 6.2 Hz). MS-FAB (m/z): 1186 (M+); calcd, 1185.68 (M+). 2.3. Synthesis of Green-Fluorescent Probe (2b). 11-[1′′,3′′,5′′7′′tetramethyl-4′′,4′′-difluoro-4′′-bora-3a,4a-diaza-s-indacenyl]-3′,5′dimethylphenoxy undecanoic acid7 (80.7 mg, 0.15 mmol), 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (85.7 mg, 0.16 mmol), 1,3-dicyclohexylcarbodiimide (62.6 mg, 0.30 mmol), and 4-(dimethylamino)pyridine (5.3 mg, 0.05 mmol) were mixed in anhydrous CH2Cl2 (2 mL), and then the mixture was stirred for a week at room temperature under a nitrogen atmosphere. After filtration and evaporation of the solvent, the residue was purified on a silica gel chromatography column eluted with a mixture of CHCl3/CH3OH/ 28% NH3 (aq) (65:35:10) to afford a green-fluorescent phosphatidylcholine (2b) (37.0 mg, 24%). 1H NMR (270 MHz, CDCl3): δ 7.13 (2H, d, J ) 7.6 Hz), 6.96 (2H, d, J ) 8.6 Hz), 5.92 (2H, d, J ) 5.7 Hz), 5.18 (1H, br), 4.29 (2H, br), 4.11 (2H, m), 3.97 (2H, t, J ) 6.5 Hz), 3.83 (2H, br), 3.37 (9H, s), 2.55 (6H, s), 2.28 (2H, m), 1.81 (2H, t, J ) 7.8 Hz), 1.48 (2H, br), 1.40 (6H, s), 1.30-0.98 (42H, m), 0.88 (3H, t, J ) 3.9 Hz). 2.4. Synthesis of Red-Fluorescent Bolaamphiphile (4). 2,5bis-dihydroxybenzaldehyde (0.29 g, 2.0 mmol), 1,12-bromododecane (2.65 g, 8.0 mmol), 18-crown-6-ether (0.02 g, 0.08 mmol), and potassium carbonate (1.96 g, 35 mmol) were dissolved in 30 mL of dry acetone, and the mixture was refluxed overnight. After cooling and evaporation of the solvent, the residue was purified on a silica gel column eluted with a mixture of CHCl3/hexane (3: 2). Removal of the solvent afforded a transparent liquid that was 2,5-bis-(12bromododecyloxy)benzaldehyde (0.39 g, 31%). 2-(4-methoxyphenyl)pyrrole (0.07 g, 0.4 mmol) and 2,5-bis-(12-bromododecyloxy)benzaldehyde (0.13 g, 0.2 mmol) were dissolved in CH2Cl2 (20 mL), and the mixture was bubbled with N2 gas for 30 min. After one droplet of CF3COOH was added, the solution was stirred under nitrogen gas overnight. Then 2,3-dichloro-5,6-dicyano-1,4-benzoquinon (0.045 g, 0.2 mmol) was added, and the mixture was stirred again for 2 h. The reaction mixture was washed 4 times with saturated NaHCO3 (aq) and with saturated NaCl (aq), then dried over excess Na2SO4. The residue was purified on an alumina chromatography column eluted with a mixture of CHCl3/hexane (3:2), dried in a vacuum, and the product dissolved in toluene (5 mL) with triethylamine (0.25 mL, 3.4 mmol) and BF3‚Et2O (0.36 mL, 1.9 mmol). The solution was refluxed for 1 h and then washed twice with saturated NaHCO3 (aq) and saturated NaCl (aq) and then dried over excess Na2SO4. Purifying the reaction mixture on a silica gel chromatography column eluted with CHCl3 and further purifying on gel permeation chromatography eluted with CHCl3 afforded a purple oily product. 1H NMR (270 MHz, CDCl3): δ 7.87 (4H, d, J ) 8.6 Hz), 6.96 (7H, m), 6.75 (2H, d, J ) 4.3 Hz), 6.53 (2H, d, J ) 4.3 Hz), 3.90 (4H, m), 3.84 (6H, s), 3.39 (4H, m), 1.78 (8H, m), 1.561.14 (32H, m). MS-TOF (m/z): 1005.51 (M+); calcd, 1004.37 (M+). The product was dissolved in ethanol (10 mL), together with 10 mL of an aqueous solution of trimethylamine (30%), and the reaction mixture was stirred at 85 °C overnight. The resulting solution was cooled and dried in a vacuum to afford red-fluorescent bolaamphiphile (4) (20 mg, quant.). 1NMR (270 MHz, d6-DMSO): δ 7.84 (4H, d, J ) 8.6 Hz), 7.13 (1H, s), 7.10 (1H, d, J ) 2.9 Hz), 7.25 (4H, d, J ) 8.6 Hz), 6.96 (1H, d, J ) 2.9 Hz), 6.77 (4H, m), 3.92 (4H, m), 3.82 (6H, s), 3.07 (18H, s), 1.64 (4H, m), 1.42-1.04 (40H, m). 2.5. Synthesis of Green-Fluorescent Surfactant (6). 4-(10Bromo-n-decyloxy)benzaldehyde (342 mg, 1.0 mmol) and 2,4-
Langmuir, Vol. 22, No. 5, 2006 1977 dimethylpyrrole (190 mg, 2.0 mmol) were dissolved in CH2Cl2 (25 mL), and the solution was degassed by bubbling nitrogen (30 min). One drop of CF3COOH was added, and the solution was stirred overnight at room temperature under nitrogen atmosphere. Then, 2,3-dichloro-5,6-dicyano-1,4-benzoquinon (0.25 g, 1.1 mmol) was added, and the mixture was stirred for 4 h. The reaction mixture was washed with saturated NaHCO3 (aq) and saturated NaCl (aq), and then dried over Na2SO4, filtered, and concentrated. The crude product was purified on an alumina chromatography column using CHCl3 to afford a dipyrromethane derivative as a colored oil. The product and BF3‚Et2O (0.72 mL, 3.8 mmol) were dissolved with triethylamine (0.50 mL, 6.8 mmol) in toluene (10 mL), and the solution was refluxed for 1 h. The reaction mixture was washed with saturated NaHCO3 (aq) and saturated NaCl (aq), dried over Na2SO4, filtered, and concentrated. The fluorescent oil was purified on a silica gel chromatography column using hexane/ethyl acetate to afford a fluorescent alkyl bromide (73 mg, 13%) as a vermilion powder. The obtained bromide (50 mg, 0.09 mmol) was added to a 30% aqueous solution of trimethylamine (10 mL), and the suspension was heated at 80 °C for 30 h. After cooling, water and excess trimethylamine were removed under a reduced pressure, and the resulting solid was washed with acetone to afford a green-fluorescent amphiphile (6) (33 mg, 60%). 1NMR (270 MHz, CDCl3): δ 7.15 (2H, d, J ) 8.2 Hz), 6.99 (2H, d, J ) 8.2 Hz), 5.97 (2H, s), 4.00 (2H, t, J ) 6.6 Hz), 3.55 (2H, m), 3.41 (9H, s), 2.54 (6H, s), 1.90-1.52 (4H, m), 1.50-1.25 (18H, m). MS-FAB (m/z): 537 (M+ - Br); calcd, 538.38 (M+ - Br). 2.6. Preparation of GMVs and Observation of Their Dynamical Behavior. Labeled GVs of 1 were prepared according to the filmswelling method1 in the presence of 5 mol % of BODIPY derivative 2a or 2b as a red- or green-fluorescent probe, respectively. The red fluorophores (2a and 4) and the green fluorophores (2b and 6) were excited simultaneously with a band-pass filter (460-490 nm) and were detected with a detecting filter (>515 nm) in a fluorescence microscope system (IX-70; Olympus, Japan).7 Real-time simultaneous detection of the red- and green-fluorescence images was accomplished with a recording video system (WV-DR9; Sony, Japan), equipped with a color CCD (charge-coupling device) camera (DC330; Dage-MTI, USA). The resulting dispersion of POPC vesicles (final concentration of POPC ) 1 mM) contained myelin-like GMVs with diameters larger than 5 µm observed as solid red- or green-fluorescent spheres, although smaller vesicles with diameters less than 2 µm were also detected.8 We used these GMVs as a target to observe the morphological changes caused by the addition of surfactants or bolaamphiphiles or both. For tracing the morphological transformations of the GMVs in real time, we used a previously reported channeltype chamber3d,3e to add dispersions of additives to the dispersions of GMVs under the fluorescence microscope. On the other hand, to enable us to observe the morphological changes in the GMVs induced by adding nonfluorescent bolaamphiphile 3 or nonfluorescent surfactant 5 or both, we used a differential interference contrast microscope (BX51; Olympus, Japan) equipped with the same recording system and CCD camera as mentioned above.
3. Results 3.1. Cup-Shaped Transformation and Reformation of GMVs by the Addition of Bolaamphiphile. A dispersion of bolaamphiphile 3 in Figure 1 (10 mM) was added to a dispersion of GMVs of 1 labeled with red-fluorescent probe 2a. We observed that the outer layers of the GMVs became cup-shaped within a few seconds (Figure 2). After a while, the cup-shaped outer layer gradually reformed into a spherical shell, encapsulating the inner vesicle again. Such morphological changes in the vesicular membranes occurred repeatedly from the outer layer to the inner. Moreover, Figure 2b indicates the temporary generation of fluorescent aggregates in the inner water region between the (8) The fact that the red- or green-fluorescent color of the GMV of 1 labeled with 2a or 2b lasted for at least 2 days indicates that the synthesized fluorescent probe stained well in the hydrophobic layers of 1 in both cases.
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Figure 3. Fluorescence micrograph of a cup-shaped GMV (site a) with an incompletely exposed inner core, the surface membrane of which is also transformed into a cup shape (site b). This image was obtained at t ) 11 min. For comparison, a schematic illustration of the GMV is also shown.
Figure 1. Schematic illustrations of amphiphilic molecules.
Figure 2. Sequential fluorescence micrographs of the transmission of deformation to a cup shape and subsequent reformation to a spherical shape through the layers of a GMV of 1 labeled with red-fluorescent probe 2a following the addition of bolaamphiphile 3. For comparison, schematic illustrations of the GMV are also shown. The red concentric disks correspond to the vesicular membranes in the GMV of 1 labeled with red-fluorescent phospholipid 2a, and the red solid circles correspond to red-fluorescent aggregates observed in the inner water region between the outermost layer and the second outer layer.
cup-shaped outer layer and the inner core. We confirmed under a differential interference contrast microscope that nonfluorescent GMVs composed of 1 also exhibited the same cup-shaped transformation and reformation when bolaamphiphile 3 was added.
After the addition of bolaamphiphile 3 to GMVs of 1 labeled with 2a, some GMVs with the cup-shaped outer layers did not reform completely. In these cases, we observed that part of the inner core was exposed outside of the GMV (Figure 3; t ) 11 min) through the pore of the cup-shaped outer layer (indicated by a white arrow at site a) and that bolaamphiphile 3 also intruded into the exposed part of these inner vesicles, also generating cup-shaped layers (indicated by a white arrow at site b). 3.2. Birthing of GMVs by Addition of Both Bolaamphiphile and Surfactant. As described above, the birthing phenomenon of GMVs did not take place when the dispersion of bolaamphiphile 3 alone was added to the dispersion of GMVs of 1 labeled with 2a. This is presumably because bolaamphiphile 3 cannot substantially disturb the bilayer structure of the phospholipid because of its membrane-spanning conformation of the bolaamphiphile 3.6,9 Therefore, to clarify the factors of GMV birthing, a mixed dispersion of two types of amphiphiles was added to GMVs of 1; one of the additives was a bolaamphiphile and the other was a surfactant. Moreover, we carried out two independent real-time observations using two sets of fluorescence-labeled additives and GMVs: (i) green-fluorophore-labeled additives and red-fluorophore-lipid-containing GMVs, (ii) red-fluorophorelabeled additives and green-fluorophore-lipid-containing GMVs. A surfactant operating together with a bolaamphiphile is expected to change the membrane-surface tension and the line tension of the rim of the pore more efficiently than the addition of a bolaamphiphile alone. Here, the membrane-surface tension is defined as the energy of the surface of vesicular membrane per unit area, and the line tension is defined as the energy along the rim of the pore per unit length.4 A mixture (20 mM) of both bolaamphiphile 3 and greenfluorescent surfactant 6 was added to a dispersion of GMVs (1 mM).10 Immediately after the mixed micellar solution of 3 and 6 (molar ratio of 3/6 ) 20:1) was introduced gently to the dispersion of the red-fluorescent GMVs of 1 labeled with probe 2a (Figure 4a), the outer layers of the GMV first became yellow and then turned yellowish green (Figure 4b). This color change indicated that 3 and fluorescent 6 had started to intrude into the outer layers of the GMV of 1. The intact inner core then moved vigorously toward the inner surface of the outer layers of the GMV (Figure 4c). Eventually the intact red core of 1 was extruded from the GMV, exhibiting the so-called birthing phenomenon (Figure 4d). After the inner core was extruded, the spherical shape of the outer layers remained. Such a morphological change (9) Kunitake, T. Angew. Chem., Int. Ed. Engl. 1992, 31, 709. (10) Dynamic light-scattering experiments (NIKKISO Microtrac UPA150 at room temperature (23 ( 1 °C)) revealed that, in a micellar solution (20 mM) of surfactant 6 and bolaamphiphile 3 (3/6 ) 20:1, molar ratio), micelles with average diameters of 6 nm (standard deviation ) 1 nm) formed in water.
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Figure 4. Sequential fluorescence micrographs of the extrusion of an encapsulated vesicle of 1 labeled with red-fluorescent probe 2a. For comparison, schematic illustrations of the birthing phenomenon of the GMV are also shown. The red concentric circles correspond to the vesicular membranes in the GMV of 1 labeled with redfluorescent phospholipid 2a, and the green circles correspond to the layers of 1 intruded into by 3 and 6.
Figure 5. Change in the diameters of the intact red-fluorescent core in GMVs of 1 when accompanied by the sole addition of surfactant 6 (plot a) or by the addition of a mixture of surfactant 6 and bolaamphiphile 3 (plot b). The relative diameter of the core of 1 was determined by comparison with the diameter of the original GMV.
was frequently observed in the GMVs with diameters in the range of 5-20 µm. On the other hand, the addition of a micellar solution (2 mM) of green-fluorescent surfactant 6 alone induced the complete dissolution of GMVs of 1 labeled with 2a (See Supporting Information).11 The process of dissolution of the GMV layers, into which 6 had intruded, occurred repeatedly from the outer layers to the inner, and, within 1 min, all of the GMV layers disappeared. The typical change in diameter of the red-fluorescent intact core was monitored with imaging software (Figure 5, plot a). In contrast to the dissolution of GMVs by the addition of surfactant 6 alone, in the presence of both 3 and 6, the relative diameter of the intact core decreased to 40% of the original within the initial 40 s, but then stayed almost constant until the intact core was extruded at around 110 s (Figure 5, plot b). By selecting a site where the intrusion of the amphiphiles into the GMVs occurred slowly, we were able to closely monitor the intrusion process of bolaamphiphile 3 and green-fluorescent surfactant 6 into GMVs of 1 labeled with red-fluorescent probe 2a until the intact core was extruded through the invaded outer layers (Figure 6). The fluorescence color changed from red to intense green, while, at the same time, the outer layer deformed rapidly into a cup shape (within ∼0.2-0.5 s). The cup-shaped outer layer was observed for up to a minute, and then the intact inner core was pushed outward (Figure 6A). The deformed outer (11) Dynamic light-scattering experiments (NIKKISO Microtrac UPA150 at room temperature (23 ( 1 °C)) revealed that the 2 mM suspension of greenfluorescent surfactant 6 itself formed micelles with average diameters of 5 nm (standard deviation ) 1 nm) in water.
Figure 6. (A) Sequential fluorescence micrographs showing the transmission of pore formation and reformation through the invaded layers of a GMV of 1 labeled with red-fluorescent probe 2a. The white arrows in c indicate temporarily formed self-aggregates between the layers in the GMV. For comparison, schematic illustrations of the stepwise cup-shaped transformation and reformation resulting from the intermembrane diffusion of bolaamphiphile 3 and greenfluorescent surfactant 6 are also shown. (B) Time-course diagram of the change in the distance d between the center of the intact red-fluorescent core of 1 (labeled with 2a) and the edge of the cupshaped outer layer invaded by 3 and 6, which is determined from the observed images of typical dynamics within a GMV (indicated by the white arrow in b of panel A). Squares: outermost layer; circles: secondary outer layer; triangles: ternary outer layer.
layer then gradually reformed itself into a spherical shape within ∼5-10 s. Note that amphiphiles 3 and 6 (and maybe also including 1) were released, forming green-fluorescent aggregates (indicated by white arrows in Figure 6A). These aggregates diffused across the aqueous region into the inner layers, inducing the same morphological changes as that in the outer layers. These sequential dynamics were transmitted successively inward (Figure 6B), until the extrusion of the intact core of the GMV took place. On the other hand, a micellar solution (10 mM) of redfluorescent bolaamphiphile 4 and nonfluorescent surfactant 5 (4/5 ) 10:1, molar ratio) was added to a dispersion (1 mM) of GMVs of 1 labeled with 5 mol % of green-fluorescent probe 2b. The green-fluorescent outer layers of the GMV became orange within 1 min of adding the mixed dispersion of 4 and 5, and then the movement of the intact inner green core of the GMV of 1 occurred, which is associated with the cup-shaped transformation of the intruded outer layers. The birthing of the intact core was observed at 79 s (Figure 7). Furthermore, to exclude the possibility that the observed birthing phenomenon was caused by the large fluorophores attached to the bolaamphiphile or the surfactant, we also confirmed that the mixing of a micellar solution of nonfluorescent bolaamphiphile 3 and nonfluorescent surfactant 5 (20 mM, 5/3 ) 1:1, molar ratio) with the GMVs also caused the birthing phenomenon (Table 1).
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Figure 7. Sequence of fluorescence micrographs of the extrusion of an encapsulated vesicle of 1 labeled with green-fluorescent probe 2b (b, c, and d). A typical fluorescence micrograph of a greenfluorescent GMV is shown in panel a. For comparison, schematic illustrations of the GMV birthing phenomenonare also shown. The green concentric circles correspond to the vesicular membranes in the GMV of 1 labeled with green-fluorescent phospholipid 2b, and the red circles correspond to the layers of 1 intruded into by redfluorescent bolaamphiphile 4 and nonfluorescent surfactant 5. Table 1. Morphological Changes in GMVs of 1 in the Presence of Bolaamphiphile 3 and/or Surfactant 5a observed morphological changesb
additives 3 (mM)
5 (mM)
cup-shaped deformation
birthing
stepwise dissolution
20 10 0
0 10 20
++ + -
++ +
+ ++
a Each microscopic observation of the morphological changes in the GMVs within the mixing chamber was repeated 10 times. The concentration of the dispersion of GMVs of 1 was 1 mM in each manipulation. b Symbols: - ) not detected; + ) sometimes detected (frequency of detection was about 30%); ++ ) detected reproducibly.
4. Discussion Before we discuss the mechanism of the cup-shaped transformation and reformation of GMVs, we will introduce several interpretations of the birthing phenomenon (i.e., the direct extrusion of the inner core vesicle through the outer layer) that have been considered elsewhere. Hotani’s and Menger’s groups reported, independently, that dually nested GVs exhibited direct extrusion of the encapsulated inner vesicle accompanying shrinkage of the outer vesicle (the so-called birthing phenomenon) when a solution of surfactants was added to the dispersion of GVs. They discussed the mechanism of birthing on the basis of osmotic pressure induced by the diffusion of amphiphiles from the outer layer into the inner water region.2d,2e,2f On the other hand, the birthing of dually nested GVs has been found to be induced by changes in the temperature or osmotic pressure of the dispersion of GVs, or by electroporation or light irradiation.4 Brochard-Wyart’s group recently discussed the physicochemical features of the birthing phenomenon in fluorescent GVs induced by light irradiation.4c According to their discussion, the birthing phenomenon consists of 3 steps: the formation of a pore in the outer layer of the GV due to a change of membrane-surface tension and a decrease in the line tension; the extrusion of the inner vesicle through the pore, driven by the hydrostatic pressure difference; and the closing of the pore in the outer membrane due to the recovery of the line tension along the rim of the pore. Here, the membrane-surface tension is defined as the energy of the surface of the vesicular membrane per unit area, and the line tension is defined as the energy along the rim of the pore per unit length. In our current study, the birthing dynamics is also thought to be induced by the formation of a pore in the outer layer. Although
Figure 8. Schematic illustration of the pore formation of an outer layer, the membrane-surface tension of which decreases because of the invasion of surfactant and bolaamphiphile. Bolaamphiphile is capable to be inserted in the phospholipid bilayer as both the membrane-spanning conformation and the U-shaped conformation.
bolaamphiphile 3 alone forms a monolayer structure in its aqueous dispersion,9 there is experimental evidence suggesting that bolaamphiphiles dissolve in a vesicular membrane, not only in a membrane-spanning conformation, but also in an U-shaped conformation (Figure 8).6 Therefore, intrusion of bolaamphiphile 3 into the outer layer of a vesicle of POPC causes a stress on the outer layer to form a pore with a temporal increase in membrane-surface tension and a decrease in the line tension of the pore. The oversaturation of bolaamphiphiles in the membrane induces the dissolution of amphiphiles into the water layer between the membranes and disturbs the pressure balance between the inside and outside of the membrane. As a result, water flowing in pushes the inner vesicle out through the pore. However, by releasing bolaamphiphiles into the water layer, the membranesurface tension and line tension of the outer layer recovers to some extent, and the pore closes by itself. Even though the pore closed, such molecular diffusion of additives and water across the invaded layer induces a relaxed membrane-surface tension of the outer layer. In comparison with previous reports,4 our analysis of the birthing phenomenon based on our real-time fluorescent microscopic observations can be interpreted plausibly at a microscopic level: (i) the amphiphilic additives (i.e., the bolaamphiphiles and surfactants) are transmitted from the intruded outer layer to the intact inner layer, and (ii) the intruded outer layer alone can exhibit the transformation and reformation dynamics. These results are the first experimental evidence supporting the notion that the stepwise inward diffusion of amphiphilic additives and the temporary formation of a pore within the intruded layer induces the birthing phenomenon in nested GMVs.
5. Conclusion In summary, we found that addition of an artificial bolaamphiphile to a dispersion of GMVs made of POPC induces a sequential-dynamics, cup-shaped deformation, causing the partial extrusion of the inner vesicle and subsequent reformation of the outer vesicle. When both the artificial bolaamphiphile and a surfactant were added to the vesicular dispersion, the deformation and reformation dynamics were transmitted from the outer to the inner membranes until the intact inner vesicle was extruded out of the outer membrane. The microscopic aspects of these processes were investigated using amphiphiles tagged with individual fluorophores. Our investigation showed that the birthing process of GMVs induced by amphiphilic additives can be interpreted as a disturbance of the outer layer alone, with the inner vesicle remaining intact, and the flowing-in dynamics of water occurring through the disturbed outer layer.
AdditiVe-Induced Morphological Changes in GMVs
These findings concerning the dynamics of GMVs might be utilized for developing a novel multistep drug delivery system,12 which is different from the current system using unilamellar vesicles, and for constructing artificial cells with a multilamellar inner structure that can be regarded as a model of living cells in hierarchical structures. Acknowledgment. We thank Katsuhiko Sato (The University of Tokyo) for his critical reading of the manuscript and valuable (12) McPhail, D.; Tetley, L.; Dufes, C.; Uchegbu, I. F. Int. J. Pharm. 2000, 200, 73.
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discussion. This work was supported by a grant-in-aid from the Center of Excellence (Study of Life Science as Complex Systems) from the Ministry of Education, Culture, Sports, Science and Technology. Supporting Information Available: Sequential fluorescence micrographs of stepwise dissolution of POPC GMVs labeled with redfluorescent probe 2a induced by the addition of green-fluorescent surfactant 6 only. This material is available free of charge via the Internet at http://pubs.acs.org. LA0529198
