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Temperature dependent membrane phase reorganization in giant vesicles
Chemistry and Physics of Lipids 94 (1998) 53 – 61
Temperature dependent membrane phase reorganization in giant vesicles Alok Singh a,*, Paul E. Schoen a, Marie-Alice Guedeau-Boudeville b b
a Center for Bio/Molecular Science and Engineering, Na6al Research Laboratory, Washington DC 20375, USA Physique de la Matie`re Condense´e, Colle`ge de France, 11 Place Marcelin-Berthelot, 75231 Paris Ce´dex 05, France
Received 13 February 1998; received in revised form 6 April 1998; accepted 6 April 1998
Abstract Giant unilamellar vesicles (GUV) from diacetylenic phospholipid, 1,2 bis (heptacosa-8,10-diynoyl)-sn-glycero-3phosphocholine (DC6,15PC), have been prepared by applying electric field to the aqueous dispersion maintained above its chain melting transition temperature (Tm) of 58.9°C. Previous studies have shown that large vesicles ( 1 mm or greater diameter) of the diacetylenic lipid transform into the tubule morphology when the temperature of the dispersion medium is dropped below its Tm but these giant vesicles (200 mm diameter) are stable far below Tm. Then within a 2° temperature range at about 32°C (26° below Tm) the giant vesicles suddenly collapse, leaving oddly branched structures. Giant vesicles formed from the saturated lipid 1,2 distearoyl phosphatidylcholine, by contrast, remain intact as the temperature is lowered, eventually adhering to one another to form a gel. © 1998 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Giant vesicles; Diacetylene; Phospholipid; Tubule; Optical microscopy
1. Introduction A considerable amount of effort has been devoted to studying the different morphologies that can form with diacetylenic lipid membranes and the circumstances under which they can be transformed from one to another (Schnur, 1993; Singh and Schnur, 1993). Generally such lipids * Corresponding author. Tel.: + 1 202 4046060; fax: +1 202 7679594; e-mail: [email protected]
have a diacetylenic moiety inserted symmetrically into both acyl chains and behave much like nondiacetylenic lipids, in that they disperse into water, forming bilayer membrane vesicles in their high temperature phase. They display a phase transition, transforming to an ordered, low temperature phase below their main chain transition temperature (Tm). This phase transformation can be accompanied by remarkable morphological changes, including formation of helical and tubular shapes (Yager and Schoen, 1984). Lipid
0009-3084/98/$19.00 © 1998 Elsevier Science Ireland Ltd. All rights reserved. PII S0009-3084(98)00023-1
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A. Singh et al. / Chemistry and Physics of Lipids 94 (1998) 53–61
tubules are hollow, open ended cylinders formed from ribbons or sheets of bilayer membrane, about 0.5 mm in diameter and 10s to 100s of micrometers in length. They are of considerable technological interest because of their potential as templates for submicron high aspect ratio structures and as an encapsulating medium (Price et al., 1998). The presence of the diacetylenes in the lipids also allows polymerization to take place upon irradiation with UV light if the acyl chains are in their low temperature, ordered phase (Tieke et al., 1979; Hub et al., 1980; Lever et al., 1983). Polymerization in bilayer membranes was expected to provide a means for stabilization of membrane structures and to make them less permeable (Regen et al., 1982), but in fact polymerized diacetylenic vesicles turned leakier, i.e. their contents can exchange with the external medium (Lever et al., 1983). In addition, when partially polymerized diacetylenic lipid is used in membrane formation, a new, toroidal morphology is observed to form (Mutz and Bensimon, 1991; Mutz et al., 1991). In previous studies of temperature dependence, diacetylenic vesicles have demonstrated a variety of behaviors depending on their size. Large vesicles (\1 mm) usually, but not always, transform into tubules while smaller vesicles do not (Burke et al., 1988). To further probe the role of vesicle size on diacetylenic membrane transformation we have studied the giant unilamellar vesicle (GUV) morphology. The technique of forming GUVs, vesicles of 50 mm and greater diameter, was developed in the early 1980’s and has been used to study issues of vesicle stability (Menger and Gabrielson, 1994, 1995; Menger and Lee, 1995). GUVs could be observed individually and their behavior over time recorded by video microscopy. In this manner, for instance, GUVs were studied as reaction vials, as was the effect of injection of material into GUVs by micropipette (Menger and Lee, 1995) or by electroporation (Zimmerman, 1983; Bradley et al., 1997). In the present case, GUVs provided a system in which the stability and transformation of morphology of diacetylenic lipids could be studied when the
membrane was almost planar. In order to have a large range of observable temperatures, a diacetylenic lipid, 1,2 bis (heptacosa-8, 10diynoyl)-sn-glycero-3-phosphocholine (DC6,15PC), with 27 carbons in its acyl chains, was selected. Its main chain melting transition temperature of 58.9°C fell in the desired temperature range. The notation 6,15 refers to the number of methylenes above (proximal to the head group) and below the diacetylene group in the acyl chains, as shown in Fig. 1.
2. Experimental
2.1. Materials The lipid 1,2 bis (heptacosa-8, 10-diynoyl)-snglycero-3-phosphocholine (DC6,15PC) was prepared in 69% yield by following the literature procedure by reacting heptacosa- 8,10-diynoic anhydride (melting point 75°C) with glycerophosphoryl choline-CdCl2 (Singh and Schnur, 1986; Singh, 1990). Lipid gave satisfactory elemental analysis and its mass was determined FAB-MS (M + 1 = 1026). The purity of compound was maintained by intermittently checking chain melting transition temperature by DSC (58.9°C, DH 7.2 Kcal/mol). Distearoyl phosphatidylcholine (DSPC), sucrose and glucose were obtained from Sigma Chemical Company. Double distilled water was used in all preparations.
Fig. 1. Chemical structure of phospholipid DC6,15PC.
A. Singh et al. / Chemistry and Physics of Lipids 94 (1998) 53–61
3. Preparation of giant vesicles Unilamellar, spherical giant vesicles were obtained from DC6,15PC by modifying the technique described in the literature (Angelova and Dimitrov, 1987). A 10 ml chloroform/methanol (9:1) solution (10 mg/ml) of the lipid was spread on two conductive In— SnO2 — coated glass plates (3.5 × 6 cm) and then dried under high vacuum for 1 h to obtain a thin lipid film. The plates were then fixed, face-to-face, 1 mm apart, by sandwiching a Viton rubber spacer between them. The cell was filled with a 50 mM sucrose solution, after applying an 0.5 V, 10 Hz AC voltage (Hewlett Packard 332A Function Generator) and the aperture was sealed with Vitrex (Modulohm, Denmark). This sealed cell was placed in an oven (Memmert) at 72°C, during the process of electrical swelling. The alternating-current voltage was increased slowly from 0.5 to 1.5 V over 1 h. After 6 h the frequency was decreased to 5 Hz and the swelling was continued for an additional 4 h. In some cases, to enhance the optical contrast, 50 mM glucose solution was added to the resulting suspension just before viewing through the optical microscope.
4. Microscopic observation The effect of cooling on the morphology of the giant vesicles was observed by a reverse-phase microscope (Nikon Diaphot-TMD, 20×objective). The swelling cell, now containing giant vesicles with diameters up to 300 mm, was directly placed in a homemade temperature regulated support connected to a Lauda thermostated bath. The temperature changes were monitored by a microcomputer thermometer (Jenco). The thermocouple was inserted between the cell and the regulated heating support, which provided accurate readings of the cell temperature. The polymerization of GUVs of DC6,15PC was carried out by directly irradiating the sample in quartz covered cell with UV light from a mercury pen lamp. For temperature scans, giant vesicles made from DC6,15PC and DSPC were maintained above their chain melting phase transition temperature. They
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were then examined for morphological changes induced by lowering the temperature from 70 to 30°C.
5. Results and discussion The objective of the current study is to examine the influence of bilayer curvature on the morphology of self-organized structures. Since diacetylene may also function as a polymerizable unit, it is important to rule out any possibility of morphological changes induced by polymerization. This is particularly important for current experimental conditions where the temperature below Tm is favorable for diacetylene polymerization. Formation of non-vesicular, non-tubular toroidal structures is reported from diacetylenic lipid contaminated with partially polymerized lipid (Mutz and Bensimon, 1991). Before conducting temperature based studies influence of polymerization on GUV was probed by irradiating DC6,15PC vesicles with UV light and making a series of video micrographs, as shown in Fig. 2. In this experiment, there is sucrose inside the vesicles and added glucose with sucrose outside the vesicles to obtain a better contrast and to observe the permeability of the membrane. We did not observe any vesicle shrinkage after addition of 50 mM glucose solution to vesicles in 50 mM sucrose medium. The temperature was maintained at 50°C, far below Tm, so that the lipid would be in its low temperature ordered phase. At this temperature the acyl chains would be in the all-trans configuration, with their diacetylenic units packed close together, the favorable alignment required for topotactic polymerization (Tieke et al., 1979; Lever et al., 1983). As the polymerization progressed (2b), the membrane became porous and the contrast of the GUV in the optical images began to decrease due to rapid exchange between the inside and outside media. As shown in Fig. 2d, after about 30 s the contrast dropped substantially and the GUV became very difficult to distinguish from the surrounding medium. Polymerized GUV were found to be very stable at room temperature. TLC analysis (silica gel, chloroform:methanol:water 65:25:4) showed no
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A. Singh et al. / Chemistry and Physics of Lipids 94 (1998) 53–61
Fig. 2. A – D. Change in contrast due to photopolymerization of GUV from DC6,15PC: A, GUV before irradiation; B, GUV after 12 s; C, after 24 s; and D, after 31 s UV irradiation. Bar represents 15 mm.
monomeric lipid after UV irradiation. This experiment confirmed that morphological changes observed, as a result of cooling of the GUV are not caused by the polymerization process. For the temperature scan experiments, one giant vesicle was examined by optical microscopy as it was cooled from 70 to 30°C at a rate of 1°C/min. A Tm of 58.9°C reported here is an upscan value. The cooling Tm has been found to be 2°C lower than that of upscan transition. Fig. 3 shows a series of video images taken as a 260 mm diameter GUV of the diacetylenic lipid began to rupture and collapse. At 32.1°C a dark spot appeared at the equator of the spherical vesicle. This could be a defect in membrane structure. Disintegration of the membrane began at this point, spreading rapidly all around the circumference of the vesicle, resulting in a lipid aggregate of unknown morphology. In Fig. 4 in a similar
experiment, a vesicle of 180 mm diameter was observed to undergo the same phenomena beginning at 32.6°C but with two simultaneous burst locations, i.e. 2 loci of aggregation in the membrane. Fig. 5 shows a repeat of the temperature scan, this time using GUV from saturated 1,2 DSPC, Tm for DSPC has been reported to be 54.9°C (Marsh, 1990). In this case the temperature scan was from 60 to 42°C and rate of cooling was 1°C/min. The liquid to gel phase transition temperature occurred at 47.8°C. The sequence of video images shows a number of agglomerated vesicles, with gel formation at Tm indicated by angular shapes of the vesicles. Below 43.8°C, no further evolution of the structure was observed. No other obvious structures, such as tubules, ribbons or rods were observed. Only the original GUVs, somewhat shrunken in size, are seen.
A. Singh et al. / Chemistry and Physics of Lipids 94 (1998) 53–61
In an experiment described by Menger, a surfactant, sodium cholate, was added to a preparation of didodecyl dimethyl ammonium bromide vesicles observed by optical video microscopy (Menger and Lee, 1995). The vesicles fused under the influence of the cholate to form a GUV by a process termed ‘foraging’. Shortly after forming, the GUV disintegrated, its constituent lipid solubilizing in the surfactant medium. The GUV was
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in essence dissolved by the medium. In the present work, the diacetylenic GUV is not suspended in a solvent medium. Its disintegration appears to be driven by a mechanical stress related to cooling of the diacetylenic lipid far below its Tm. The lipid remains as amorphous material after the disintegration has occurred. A phenomenon that was noted in previous papers but not investigated in great detail was the
Fig. 3. A – F. Shrinkage of GUV made from DC6,15PC upon cooling: A, 32.6 C; B, 31.9; C, 31.8; D, 31.6; E, 31.55; and F, 31.5. Bar represents 50 mm.
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Fig. 4. A – F. Effect of cooling on GUV made from DC6,15PC. GUV in A are at 33°C and in frame F are at 32.65°C. Bar represents 50 mm.
occasional transformation of large diacetylenic lipid vesicles not into tubules but into ‘shards’ (Burke et al., 1988; Schoen et al., 1993). In this case vesicles were observed to cool a few degrees ( 5°C) below the acyl chain transition temperature without any initial change in morphology. The observed vesicle maintained its size and shape undisturbed, even though in its vicinity other
vesicles were transformed into tubules. Then after a few min the vesicle would appear to disintegrate, shattering almost like glass, leaving an aggregation of pieces of indeterminate shape, which were termed lipid shards. Subsequent freeze fracture/transmission electron microscopy suggests that the shards are pieces of tubules, i.e. curved fragments of the cylinders that would have
A. Singh et al. / Chemistry and Physics of Lipids 94 (1998) 53–61
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Fig. 5. A – D. Effect of cooling on the GUV made from DSPC: A, 50.4 C; B, 48.1; C, 47.5; and D, 43.8°C. Bar 50 mm.
formed if the shards had been larger (Schoen et al., 1993). Fig. 6 shows structures of diacetylenic lipid, formerly part of a GUV, which disintegrated as described above. These structures were observed within a min after vesicle disintegration has occurred. They were seen at the same temperature at which vesicle disintegration occurred, approximately at 31.5°C. It has the appearance of a mixture of morphologies, rod shapes combined with knots of lipid, sometimes arranged in branched, tree-like shapes. The nature of this latter morphology is not yet understood. DC6,15PC multilamellar vesicles (MLV) and large unilamellar vesicles (LUV) with diameters near 1 mm transform into tubules near Tm at 59°C. GUVs with diameters of 200 mm or more disintegrate at a temperature 26° cooler. Thus the large radius of curvature of the GUV accompa-
nies a larger temperature window of stability below Tm for the vesicular structure for this lipid. We note that for the diacetylenic lipid, LUVs and MLVs transform to the tubule morphology near Tm, although occasionally some of these vesicles will survive cooling several degrees below Tm. On the other hand, diacetylenic small unilamellar vesicles (SUV) remain stable at temperatures down to 2.4°C, almost 40° below Tm. Differential scanning calorimetric thermograms show a phase transformation event at this temperature (Burke et al., 1988). Freeze fracture transmission electron microscopy has shown that the new morphology formed by this transition is ‘stacked bilayer assemblies’, or bilayer sheets, a form with a radius of curvature which is very large, essentially infinite (Burke et al., 1988; Schoen et al., 1993). The infinite radius sheet
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Fig. 6. Formation of tubules, lipid knots with disintegrated GUV from DC6,15PC.
structure is stable at all temperatures below Tm and the small radius SUV morphology is stable from Tm down to 2.4°C, while intermediate radius vesicles are unstable below Tm. Large but finite radius GUVs are stable down to 26° below Tm. It appears that intermediate radius vesicles containing diacetylene do not form stable vesicles below Tm but that the stability may tend to increase, as the radius becomes very small or very large. Acknowledgements We thank Professor J.-M. Lehn for his interest in this work. Financial support from the Office of Naval Research is gratefully acknowledged (AS and PES). References Angelova, M.I., Dimitrov, D.S., 1987. Swelling of charged lipids and formation of liposomes on electrode surfaces. Mol. Cryst. Liq. Cryst. 152, 89–104. Bradley, J.C., Guedeau-Boudeville, M.-A., Jandeau, G., Lehn, J.-M., 1997. Toposomes bearing stable perforation by the action of an electric field on partially polymerized giant vesicles. Langmuir 13, 2457–2462.
Burke, T.G., Rudolph, A.S., Price, R.R., Sheridan, J.P., Dalziel, A.W., Singh, A., Schoen, P.E., 1988. Differential scanning calorimetric study of the thermotropic phase behavior of a polymerizable, tubule-forming lipid. Chem. Phys. Lipids 48, 215 – 230. Hub, H., Hupfer, B., Koch, H., Ringsdorf, H., 1980. Polymerizable phospholipid analogues. New stable biomembrane and cell models. Angew. Chem., Int. Ed. Engl. 19, 938 – 940. Lever, J., Alonso, A., Durrani, A., Chapman, D., 1983. The physical properties and photopolymerization of diacetylene-containing phospholipid liposomes. Biochim. Biophys. Acta 732, 210 – 218. Marsh, D., 1990. Handbook of Phospholipids. CRC Press, Boca Raton, FL. Menger, F.M., Gabrielson, K., 1994. Chemically induced birthing and foraging in vesicle systems. J. Am. Chem. Soc. 116, 1567 – 1568. Menger, F.M., Gabrielson, K., 1995. Cytomimetic organic chemistry: early developments. Angew. Chem., Int. Ed. Engl. 34, 2091 – 2106. Menger, F.M., Lee, S.J., 1995. Induced morphological changes in synthetic giant vesicles: growth, fusion, undulation, excretion, wounding and healing. Langmuir 11, 3685 – 3689. Mutz, M., Bensimon, D., 1991. Observation of toroidal vesicles. Phys. Rev. A 43, 4525 – 4527. Mutz, M., Bensimon, D., Brienne, M.J., 1991. Wrinkling transition in partially polymerized vesicles. Phys. Rev. Lett. 67, 923 – 926. Price, R.R., Schnur, J.M., Rudolph, A.S., Selinger, J., Singh, A., Gaber, B.P., Sustained delivery of pharmacologically
A. Singh et al. / Chemistry and Physics of Lipids 94 (1998) 53–61 active compounds from tubules with rational control. US Patent No. 5705191, January 6, 1998. Regen, S.L., Singh, A., Oehme, G., Singh, M., 1982. Polymerizable phosphatidylcholine vesicles: synthesis and characterization. J. Am. Chem. Soc. 104, 791–795. Schnur, J.M., 1993. Lipid tubules: A paradigm for molecularly engineered structures. Science 262, 1669– 1676. Schoen, P.E., Price, R.R., Schnur, J.M., Gulik, A., GulikKrzwicki, T., 1993. Formation of lipid tubule microstructures: time-resolved freeze-fracture electron microscopy and X-ray characterization. Chem. Phys. Lipids 65, 179– 191. Singh, A., 1990. An efficient synthesis of phosphatidylcholine. J. Lipid Res. 31, 1522–1525.
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Singh, A., Schnur, J.M., 1986. A general method for the synthesis of diacetylenic acids. Synth. Commun. 16, 847 – 852. Singh, A., Schnur, J.M., 1993. Polymerizable phospholipids. In: Cevc, G. (Ed.), Phospholipid Handbook. Marcel Dekker, New York, pp. 233 – 291. Tieke, B., Lieser, G., Wegner, G., 1979. Polymerization of diacetylenes in multilayers. J. Polym. Sci., Polym. Chem. Ed. 7, 631 – 1644. Yager, P., Schoen, P.E., 1984. Formation of tubules by a polymerizable surfactant. Mol. Cryst. Liq. Cryst. 106, 371 – 381. Zimmerman, U., 1983. Electric field-mediated fusion and related electrical phenomena. Biochim. Biophys. Acta 694, 227 – 277.
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Temperature dependent membrane phase reorganization in giant vesicles Alok Singh a,*, Paul E. Schoen a, Marie-Alice Guedeau-Boudeville b b
a Center for Bio/Molecular Science and Engineering, Na6al Research Laboratory, Washington DC 20375, USA Physique de la Matie`re Condense´e, Colle`ge de France, 11 Place Marcelin-Berthelot, 75231 Paris Ce´dex 05, France
Received 13 February 1998; received in revised form 6 April 1998; accepted 6 April 1998
Abstract Giant unilamellar vesicles (GUV) from diacetylenic phospholipid, 1,2 bis (heptacosa-8,10-diynoyl)-sn-glycero-3phosphocholine (DC6,15PC), have been prepared by applying electric field to the aqueous dispersion maintained above its chain melting transition temperature (Tm) of 58.9°C. Previous studies have shown that large vesicles ( 1 mm or greater diameter) of the diacetylenic lipid transform into the tubule morphology when the temperature of the dispersion medium is dropped below its Tm but these giant vesicles (200 mm diameter) are stable far below Tm. Then within a 2° temperature range at about 32°C (26° below Tm) the giant vesicles suddenly collapse, leaving oddly branched structures. Giant vesicles formed from the saturated lipid 1,2 distearoyl phosphatidylcholine, by contrast, remain intact as the temperature is lowered, eventually adhering to one another to form a gel. © 1998 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Giant vesicles; Diacetylene; Phospholipid; Tubule; Optical microscopy
1. Introduction A considerable amount of effort has been devoted to studying the different morphologies that can form with diacetylenic lipid membranes and the circumstances under which they can be transformed from one to another (Schnur, 1993; Singh and Schnur, 1993). Generally such lipids * Corresponding author. Tel.: + 1 202 4046060; fax: +1 202 7679594; e-mail: [email protected]
have a diacetylenic moiety inserted symmetrically into both acyl chains and behave much like nondiacetylenic lipids, in that they disperse into water, forming bilayer membrane vesicles in their high temperature phase. They display a phase transition, transforming to an ordered, low temperature phase below their main chain transition temperature (Tm). This phase transformation can be accompanied by remarkable morphological changes, including formation of helical and tubular shapes (Yager and Schoen, 1984). Lipid
0009-3084/98/$19.00 © 1998 Elsevier Science Ireland Ltd. All rights reserved. PII S0009-3084(98)00023-1
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A. Singh et al. / Chemistry and Physics of Lipids 94 (1998) 53–61
tubules are hollow, open ended cylinders formed from ribbons or sheets of bilayer membrane, about 0.5 mm in diameter and 10s to 100s of micrometers in length. They are of considerable technological interest because of their potential as templates for submicron high aspect ratio structures and as an encapsulating medium (Price et al., 1998). The presence of the diacetylenes in the lipids also allows polymerization to take place upon irradiation with UV light if the acyl chains are in their low temperature, ordered phase (Tieke et al., 1979; Hub et al., 1980; Lever et al., 1983). Polymerization in bilayer membranes was expected to provide a means for stabilization of membrane structures and to make them less permeable (Regen et al., 1982), but in fact polymerized diacetylenic vesicles turned leakier, i.e. their contents can exchange with the external medium (Lever et al., 1983). In addition, when partially polymerized diacetylenic lipid is used in membrane formation, a new, toroidal morphology is observed to form (Mutz and Bensimon, 1991; Mutz et al., 1991). In previous studies of temperature dependence, diacetylenic vesicles have demonstrated a variety of behaviors depending on their size. Large vesicles (\1 mm) usually, but not always, transform into tubules while smaller vesicles do not (Burke et al., 1988). To further probe the role of vesicle size on diacetylenic membrane transformation we have studied the giant unilamellar vesicle (GUV) morphology. The technique of forming GUVs, vesicles of 50 mm and greater diameter, was developed in the early 1980’s and has been used to study issues of vesicle stability (Menger and Gabrielson, 1994, 1995; Menger and Lee, 1995). GUVs could be observed individually and their behavior over time recorded by video microscopy. In this manner, for instance, GUVs were studied as reaction vials, as was the effect of injection of material into GUVs by micropipette (Menger and Lee, 1995) or by electroporation (Zimmerman, 1983; Bradley et al., 1997). In the present case, GUVs provided a system in which the stability and transformation of morphology of diacetylenic lipids could be studied when the
membrane was almost planar. In order to have a large range of observable temperatures, a diacetylenic lipid, 1,2 bis (heptacosa-8, 10diynoyl)-sn-glycero-3-phosphocholine (DC6,15PC), with 27 carbons in its acyl chains, was selected. Its main chain melting transition temperature of 58.9°C fell in the desired temperature range. The notation 6,15 refers to the number of methylenes above (proximal to the head group) and below the diacetylene group in the acyl chains, as shown in Fig. 1.
2. Experimental
2.1. Materials The lipid 1,2 bis (heptacosa-8, 10-diynoyl)-snglycero-3-phosphocholine (DC6,15PC) was prepared in 69% yield by following the literature procedure by reacting heptacosa- 8,10-diynoic anhydride (melting point 75°C) with glycerophosphoryl choline-CdCl2 (Singh and Schnur, 1986; Singh, 1990). Lipid gave satisfactory elemental analysis and its mass was determined FAB-MS (M + 1 = 1026). The purity of compound was maintained by intermittently checking chain melting transition temperature by DSC (58.9°C, DH 7.2 Kcal/mol). Distearoyl phosphatidylcholine (DSPC), sucrose and glucose were obtained from Sigma Chemical Company. Double distilled water was used in all preparations.
Fig. 1. Chemical structure of phospholipid DC6,15PC.
A. Singh et al. / Chemistry and Physics of Lipids 94 (1998) 53–61
3. Preparation of giant vesicles Unilamellar, spherical giant vesicles were obtained from DC6,15PC by modifying the technique described in the literature (Angelova and Dimitrov, 1987). A 10 ml chloroform/methanol (9:1) solution (10 mg/ml) of the lipid was spread on two conductive In— SnO2 — coated glass plates (3.5 × 6 cm) and then dried under high vacuum for 1 h to obtain a thin lipid film. The plates were then fixed, face-to-face, 1 mm apart, by sandwiching a Viton rubber spacer between them. The cell was filled with a 50 mM sucrose solution, after applying an 0.5 V, 10 Hz AC voltage (Hewlett Packard 332A Function Generator) and the aperture was sealed with Vitrex (Modulohm, Denmark). This sealed cell was placed in an oven (Memmert) at 72°C, during the process of electrical swelling. The alternating-current voltage was increased slowly from 0.5 to 1.5 V over 1 h. After 6 h the frequency was decreased to 5 Hz and the swelling was continued for an additional 4 h. In some cases, to enhance the optical contrast, 50 mM glucose solution was added to the resulting suspension just before viewing through the optical microscope.
4. Microscopic observation The effect of cooling on the morphology of the giant vesicles was observed by a reverse-phase microscope (Nikon Diaphot-TMD, 20×objective). The swelling cell, now containing giant vesicles with diameters up to 300 mm, was directly placed in a homemade temperature regulated support connected to a Lauda thermostated bath. The temperature changes were monitored by a microcomputer thermometer (Jenco). The thermocouple was inserted between the cell and the regulated heating support, which provided accurate readings of the cell temperature. The polymerization of GUVs of DC6,15PC was carried out by directly irradiating the sample in quartz covered cell with UV light from a mercury pen lamp. For temperature scans, giant vesicles made from DC6,15PC and DSPC were maintained above their chain melting phase transition temperature. They
55
were then examined for morphological changes induced by lowering the temperature from 70 to 30°C.
5. Results and discussion The objective of the current study is to examine the influence of bilayer curvature on the morphology of self-organized structures. Since diacetylene may also function as a polymerizable unit, it is important to rule out any possibility of morphological changes induced by polymerization. This is particularly important for current experimental conditions where the temperature below Tm is favorable for diacetylene polymerization. Formation of non-vesicular, non-tubular toroidal structures is reported from diacetylenic lipid contaminated with partially polymerized lipid (Mutz and Bensimon, 1991). Before conducting temperature based studies influence of polymerization on GUV was probed by irradiating DC6,15PC vesicles with UV light and making a series of video micrographs, as shown in Fig. 2. In this experiment, there is sucrose inside the vesicles and added glucose with sucrose outside the vesicles to obtain a better contrast and to observe the permeability of the membrane. We did not observe any vesicle shrinkage after addition of 50 mM glucose solution to vesicles in 50 mM sucrose medium. The temperature was maintained at 50°C, far below Tm, so that the lipid would be in its low temperature ordered phase. At this temperature the acyl chains would be in the all-trans configuration, with their diacetylenic units packed close together, the favorable alignment required for topotactic polymerization (Tieke et al., 1979; Lever et al., 1983). As the polymerization progressed (2b), the membrane became porous and the contrast of the GUV in the optical images began to decrease due to rapid exchange between the inside and outside media. As shown in Fig. 2d, after about 30 s the contrast dropped substantially and the GUV became very difficult to distinguish from the surrounding medium. Polymerized GUV were found to be very stable at room temperature. TLC analysis (silica gel, chloroform:methanol:water 65:25:4) showed no
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A. Singh et al. / Chemistry and Physics of Lipids 94 (1998) 53–61
Fig. 2. A – D. Change in contrast due to photopolymerization of GUV from DC6,15PC: A, GUV before irradiation; B, GUV after 12 s; C, after 24 s; and D, after 31 s UV irradiation. Bar represents 15 mm.
monomeric lipid after UV irradiation. This experiment confirmed that morphological changes observed, as a result of cooling of the GUV are not caused by the polymerization process. For the temperature scan experiments, one giant vesicle was examined by optical microscopy as it was cooled from 70 to 30°C at a rate of 1°C/min. A Tm of 58.9°C reported here is an upscan value. The cooling Tm has been found to be 2°C lower than that of upscan transition. Fig. 3 shows a series of video images taken as a 260 mm diameter GUV of the diacetylenic lipid began to rupture and collapse. At 32.1°C a dark spot appeared at the equator of the spherical vesicle. This could be a defect in membrane structure. Disintegration of the membrane began at this point, spreading rapidly all around the circumference of the vesicle, resulting in a lipid aggregate of unknown morphology. In Fig. 4 in a similar
experiment, a vesicle of 180 mm diameter was observed to undergo the same phenomena beginning at 32.6°C but with two simultaneous burst locations, i.e. 2 loci of aggregation in the membrane. Fig. 5 shows a repeat of the temperature scan, this time using GUV from saturated 1,2 DSPC, Tm for DSPC has been reported to be 54.9°C (Marsh, 1990). In this case the temperature scan was from 60 to 42°C and rate of cooling was 1°C/min. The liquid to gel phase transition temperature occurred at 47.8°C. The sequence of video images shows a number of agglomerated vesicles, with gel formation at Tm indicated by angular shapes of the vesicles. Below 43.8°C, no further evolution of the structure was observed. No other obvious structures, such as tubules, ribbons or rods were observed. Only the original GUVs, somewhat shrunken in size, are seen.
A. Singh et al. / Chemistry and Physics of Lipids 94 (1998) 53–61
In an experiment described by Menger, a surfactant, sodium cholate, was added to a preparation of didodecyl dimethyl ammonium bromide vesicles observed by optical video microscopy (Menger and Lee, 1995). The vesicles fused under the influence of the cholate to form a GUV by a process termed ‘foraging’. Shortly after forming, the GUV disintegrated, its constituent lipid solubilizing in the surfactant medium. The GUV was
57
in essence dissolved by the medium. In the present work, the diacetylenic GUV is not suspended in a solvent medium. Its disintegration appears to be driven by a mechanical stress related to cooling of the diacetylenic lipid far below its Tm. The lipid remains as amorphous material after the disintegration has occurred. A phenomenon that was noted in previous papers but not investigated in great detail was the
Fig. 3. A – F. Shrinkage of GUV made from DC6,15PC upon cooling: A, 32.6 C; B, 31.9; C, 31.8; D, 31.6; E, 31.55; and F, 31.5. Bar represents 50 mm.
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A. Singh et al. / Chemistry and Physics of Lipids 94 (1998) 53–61
Fig. 4. A – F. Effect of cooling on GUV made from DC6,15PC. GUV in A are at 33°C and in frame F are at 32.65°C. Bar represents 50 mm.
occasional transformation of large diacetylenic lipid vesicles not into tubules but into ‘shards’ (Burke et al., 1988; Schoen et al., 1993). In this case vesicles were observed to cool a few degrees ( 5°C) below the acyl chain transition temperature without any initial change in morphology. The observed vesicle maintained its size and shape undisturbed, even though in its vicinity other
vesicles were transformed into tubules. Then after a few min the vesicle would appear to disintegrate, shattering almost like glass, leaving an aggregation of pieces of indeterminate shape, which were termed lipid shards. Subsequent freeze fracture/transmission electron microscopy suggests that the shards are pieces of tubules, i.e. curved fragments of the cylinders that would have
A. Singh et al. / Chemistry and Physics of Lipids 94 (1998) 53–61
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Fig. 5. A – D. Effect of cooling on the GUV made from DSPC: A, 50.4 C; B, 48.1; C, 47.5; and D, 43.8°C. Bar 50 mm.
formed if the shards had been larger (Schoen et al., 1993). Fig. 6 shows structures of diacetylenic lipid, formerly part of a GUV, which disintegrated as described above. These structures were observed within a min after vesicle disintegration has occurred. They were seen at the same temperature at which vesicle disintegration occurred, approximately at 31.5°C. It has the appearance of a mixture of morphologies, rod shapes combined with knots of lipid, sometimes arranged in branched, tree-like shapes. The nature of this latter morphology is not yet understood. DC6,15PC multilamellar vesicles (MLV) and large unilamellar vesicles (LUV) with diameters near 1 mm transform into tubules near Tm at 59°C. GUVs with diameters of 200 mm or more disintegrate at a temperature 26° cooler. Thus the large radius of curvature of the GUV accompa-
nies a larger temperature window of stability below Tm for the vesicular structure for this lipid. We note that for the diacetylenic lipid, LUVs and MLVs transform to the tubule morphology near Tm, although occasionally some of these vesicles will survive cooling several degrees below Tm. On the other hand, diacetylenic small unilamellar vesicles (SUV) remain stable at temperatures down to 2.4°C, almost 40° below Tm. Differential scanning calorimetric thermograms show a phase transformation event at this temperature (Burke et al., 1988). Freeze fracture transmission electron microscopy has shown that the new morphology formed by this transition is ‘stacked bilayer assemblies’, or bilayer sheets, a form with a radius of curvature which is very large, essentially infinite (Burke et al., 1988; Schoen et al., 1993). The infinite radius sheet
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Fig. 6. Formation of tubules, lipid knots with disintegrated GUV from DC6,15PC.
structure is stable at all temperatures below Tm and the small radius SUV morphology is stable from Tm down to 2.4°C, while intermediate radius vesicles are unstable below Tm. Large but finite radius GUVs are stable down to 26° below Tm. It appears that intermediate radius vesicles containing diacetylene do not form stable vesicles below Tm but that the stability may tend to increase, as the radius becomes very small or very large. Acknowledgements We thank Professor J.-M. Lehn for his interest in this work. Financial support from the Office of Naval Research is gratefully acknowledged (AS and PES). References Angelova, M.I., Dimitrov, D.S., 1987. Swelling of charged lipids and formation of liposomes on electrode surfaces. Mol. Cryst. Liq. Cryst. 152, 89–104. Bradley, J.C., Guedeau-Boudeville, M.-A., Jandeau, G., Lehn, J.-M., 1997. Toposomes bearing stable perforation by the action of an electric field on partially polymerized giant vesicles. Langmuir 13, 2457–2462.
Burke, T.G., Rudolph, A.S., Price, R.R., Sheridan, J.P., Dalziel, A.W., Singh, A., Schoen, P.E., 1988. Differential scanning calorimetric study of the thermotropic phase behavior of a polymerizable, tubule-forming lipid. Chem. Phys. Lipids 48, 215 – 230. Hub, H., Hupfer, B., Koch, H., Ringsdorf, H., 1980. Polymerizable phospholipid analogues. New stable biomembrane and cell models. Angew. Chem., Int. Ed. Engl. 19, 938 – 940. Lever, J., Alonso, A., Durrani, A., Chapman, D., 1983. The physical properties and photopolymerization of diacetylene-containing phospholipid liposomes. Biochim. Biophys. Acta 732, 210 – 218. Marsh, D., 1990. Handbook of Phospholipids. CRC Press, Boca Raton, FL. Menger, F.M., Gabrielson, K., 1994. Chemically induced birthing and foraging in vesicle systems. J. Am. Chem. Soc. 116, 1567 – 1568. Menger, F.M., Gabrielson, K., 1995. Cytomimetic organic chemistry: early developments. Angew. Chem., Int. Ed. Engl. 34, 2091 – 2106. Menger, F.M., Lee, S.J., 1995. Induced morphological changes in synthetic giant vesicles: growth, fusion, undulation, excretion, wounding and healing. Langmuir 11, 3685 – 3689. Mutz, M., Bensimon, D., 1991. Observation of toroidal vesicles. Phys. Rev. A 43, 4525 – 4527. Mutz, M., Bensimon, D., Brienne, M.J., 1991. Wrinkling transition in partially polymerized vesicles. Phys. Rev. Lett. 67, 923 – 926. Price, R.R., Schnur, J.M., Rudolph, A.S., Selinger, J., Singh, A., Gaber, B.P., Sustained delivery of pharmacologically
A. Singh et al. / Chemistry and Physics of Lipids 94 (1998) 53–61 active compounds from tubules with rational control. US Patent No. 5705191, January 6, 1998. Regen, S.L., Singh, A., Oehme, G., Singh, M., 1982. Polymerizable phosphatidylcholine vesicles: synthesis and characterization. J. Am. Chem. Soc. 104, 791–795. Schnur, J.M., 1993. Lipid tubules: A paradigm for molecularly engineered structures. Science 262, 1669– 1676. Schoen, P.E., Price, R.R., Schnur, J.M., Gulik, A., GulikKrzwicki, T., 1993. Formation of lipid tubule microstructures: time-resolved freeze-fracture electron microscopy and X-ray characterization. Chem. Phys. Lipids 65, 179– 191. Singh, A., 1990. An efficient synthesis of phosphatidylcholine. J. Lipid Res. 31, 1522–1525.
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Singh, A., Schnur, J.M., 1986. A general method for the synthesis of diacetylenic acids. Synth. Commun. 16, 847 – 852. Singh, A., Schnur, J.M., 1993. Polymerizable phospholipids. In: Cevc, G. (Ed.), Phospholipid Handbook. Marcel Dekker, New York, pp. 233 – 291. Tieke, B., Lieser, G., Wegner, G., 1979. Polymerization of diacetylenes in multilayers. J. Polym. Sci., Polym. Chem. Ed. 7, 631 – 1644. Yager, P., Schoen, P.E., 1984. Formation of tubules by a polymerizable surfactant. Mol. Cryst. Liq. Cryst. 106, 371 – 381. Zimmerman, U., 1983. Electric field-mediated fusion and related electrical phenomena. Biochim. Biophys. Acta 694, 227 – 277.
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