Chemistry and Physics of Lipid& 54 (1990) 131--146 Elsevier Scientific Publishers Ireland Ltd.

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The formation of multilamellar vesicles from saturated phosphatidylcholines and phosphatidylethanolamines: morphology and quasi-elastic light scattering measurements Michael A. Singer a, L e o n a r d Finegold b, Paul R o c h o n c and T h o m a s J. Racey ~ *Department o f Medicine, Queen's University, Kingston, Ontario K7L 3N6 (Canada), bDepartment of Physics, Drexel University, Philadelphia, PA 19104 (U.S.A.) and ~Department o f Physics, Royal Military College of Canada, Kingston, Ontario K7K 5LO (Canada) (Received October 25th, 1989; revision received December 29th, 1989; accepted January 2nd, 1990)

The aggregation properties of diacyl phosphatidylcholines (PC) and phosphatidylethanolamines (PE), with linear symmetrical saturated chains, were characterized at temperatures below and above the lipid solid to fluid transition. PEs in the solid state form bundles of closely apposed flat bilayer stacks which at the solid to fluid transition temperature fold into closed multilamellar vesicles. On the other hand, PCs in the solid state form extended multilayer sheets. At the solid to fluid transition, multilamellar vesicles appear to "bud off" from the surface. Quasi-elastic light scattering (QELS) measurements irkiicated that for the PEs, bundle size is independent of acyl chain length n, but that the sizes of vesicles which form at the solid to fluid transition are positively correlated with n. The results of temperature jump experiments showed that once the transition temperature was reached, vesicle formation was largely complete within 30 s.

Keywords: phospholipid vesicle formation; light scattering.

Introduction Since the cardinal paper by Bangham et al. in 1965 [1], phospholipid vesicles or liposomes have served as a model system for cell membranes and their properties and more recently as delivery vehicles for drugs, and macromolecules in diverse pharmacological applications. Despite this extensive usage of vesicles, the mechanism of their formation is not yet well understood. As an initial step in this direction, we previously characterized the sizes of fluid phase multilamellar vesicles as a function of fatty acyl chain length and nature of head group [2]. Diacyl phospholipids were allowed to self aggregate in water under conditions which imposed as few constraints as possible on the aggregation process. We observed a positive correlation between

most probable vesicle size and acyl chain length, and for lipids with identical acyl chains there was an inverse correlation between vesicle size and effective head group cross-sectional area. Thus for a given phospholipid, the head group and acyl chain length set definite limits on the vesicle sizes which in essence minimize the system free energy. Multilamellar liposomes generally do not form if the phospholipid is below its transition temperature [3]. Using this observation, we have quenched phospholipid/water dispersions (phosphatidylcholines, CnPC and phosphatidylethanolamines, CnPE) from temperatures spanning the lipid solid to fluid transition and examined the bilayer topography of these samples by freeze fracture electron microscopy. Aggregation sizes at these same temperatures, as

0009-3084/90/$03.50 © 1990 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland

132 well as the time course of vesicle generation, were monitored using the technique of quasielastic light scattering (QELS).

Experimental procedures Materials The phospholipids used in this study were obtained from Avanti Polar Lipids (Pelham, AL). All the phospholipids gave single spots on heavily laden thin layer chromatography plates using two solvent systems. In addition, lipid purity was substantiated by the demonstration of narrow main transition endotherms (by differential scanning calorimetry) in liposomes formed from these lipids [4]. All other chemicals were of reagent grade. Twice distilled water was used for all experiments.

Lipid~water sample preparation The preparation of lipid/water dispersions was modified after the method described in Ref. 2. An appropriate aliquot of lipid (25 mg/ml in chloroform) was placed in a glass tube of inside dimensions 130 × 18 mm, partially dried on a rotary evaporator, and then dried for 24 h at 0.1 m Torr. The dried lipid (5 /~mol) was hydrated in excess water (1 ml) at 5°C, a temperature significantly below the solid to fluid transition of all of the lipids used. The mixture was then mechanically shaken as previously described (four cycles of 30 s vortexing separated by 2-min equilibration periods). The sample was maintained at 5 °C throughout and after this procedure until measurements were made (within 1--2 h of sample preparation).

Differential scanning calorimetry (dsc) Samples (20 /~l) were encapsulated in aluminum dsc pans and thermal measurements made on a Mettler TA 2000 B differential scanning calorimeter [5]. Scan rate was 1.2°C/min. Calibration was based on diphenyl ether [6] and water.

Freeze-fracture electron microscopy Freeze-fracture microscopy of the lipid dispersions was done on a Balzer's Freeze Etching Sys-

tem BAF (Model 400D) [2]. A gold grid was dipped into the lipid/water-mixture (at 5 °C) and then sandwiched between two gold supports. The sample was then incubated in air at the required temperature. Subsequently the samples were quickly immersed into a Freon slush and frozen. Frozen samples were transferred to a double replica specimen stage and introduced into the pre-cooled vacuum chamber. Fracturing was performed at - l l 0 ° C and the fractured surfaces were shadowed with platinum/carbon. Platinum evaporation was done from a 45 ° angle; carbon from a 90 ° angle. Replicas were examined in a Hitachi 500 electron microscope. Two types of freeze fracture experiments were performed in order to offset some of the inaccuracies in temperature control. As described above, a gold grid was dipped into the lipid/ water mixture at 5°C and then sandwiched between two gold supports. This sandwich was then incubated in a temperature controlled air chamber prior to freezing. Depending upon the final temperature required, equilibration from 5°C took from 15 to 45 s. In the first series of experiments samples were incubated at individual temperatures (spanning the phase transition) for 20 min prior to freezing. Preliminary temperature jump experiments (done with QELS) indicated that vesicle generation was well established within 60 s. Although the incubation medium had a temperature tolerance of 0.5 °C, a temperature error is introduced when the sample is removed from the air medium and frozen by immersion in Freon slush. Even though this operation was performed as quickly as possible (3--5 s) the sample temperature would change and our estimate is that the temperature could change by as much as one degree depending upon the differential between the incubated and ambient temperatures. Hence a conservative estimate of the temperature error for these experiments would be 1.5°C. In a second series of experiments, vesicle generation was monitored following a temperature jump. Data from QELS experiments (see later) indicated that the time course of vesicle generation was unaffected by the AT of the jump provided it spanned the transition temperature. We used an arbitrary AT of 20°C with the final temperature being 10°C

133 above the temperature peak of the phase transition as determined by dsc. Since temperature equilibration took from 15 to 45 s the shortest time sample was quenched one minute following the temperature jump, a time approximately 15 to 45 s after equilibration was reached.

Quasi-elastic light scattering (QELS) Quasi-elastic light scattering is a technique whereby the dynamics of laser light scattering by a solution of particles can be analyzed to calculate normalized autocorrelation functions which characterize the particular suspension of scattering particles ([2], and references therein). If the investigator can determine the form factor of the particular particle then it is possible in theory to calculate the size or shape distribution of the sample. In this case we did not know the form factor of the lipids at temperatures below the solid to fluid transition and therefore we used the autocorrelation function to characterize directly the physical state of the sample. Light scattering measurements were performed using a Lexel (model 65) argon laser as a source of incident light (4 = 0.488 /~m). The laser beam was operated at a power level of 40 mW and focused at the centre of a scattering cell containing the sample. The data was collected at a scattering angle of 90°and an autocorrelation function was determined using the Langley-Ford (model 1096) hardwired autocorrelator. The autocorrelation function was transferred to an IBM PC computer to be analyzed and hard copy plots were made on the HewlettPackard (model 7475A) digital plotter. Autocorrelation functions of QELS measurements were made on CnPC and CnPE lipids as they diffused through a water medium. The concentration of lipid in the scattering chamber was less than 0.05 mg/ml. The temperature of the water suspension, and thus the lipid, was incrementally increased and the autocorrelation function of the net movement of the particular sample was determined. The water bath was held at each temperature for ten minutes, a time interval that was more than sufficiently long to ensure the lipid was in equilibrium with the water bath. The time required had been previously determined through temperature

equilibrium studies with temperature probes in the bath and diffusion studies done with known diameter latex spheres. A delay time of 2.8 ms was chosen to characterize the autocorrelation functions for each sample and each lipid type. By this technique we could characterize the autocorrelation function with respect to temperature for the sample. Since the autocorrelation function would change as the shape or size distribution of the sample varied, we could monitor alterations in the size and shape distribution of the lipid, as a function of temperature, by its effect on the autocorrelation function. These characteristic values of the autocorrelation function can then be used, in conjunction with electron microscopy, to describe the effect of temperature on the state of the lipid preparation. For each sample, the lipid was warmed from well below to well above the phase transition. Some samples were subsequently cooled back down to the initial starting temperature, allowed to equilibrate at that temperature, and then reheated through the phase transition again. For some of the samples this was done several times to determine if repeated temperature cycling changed the QELS signature of the lipid. The lipids were also heated through the phase transition with large temperature gradients (temperature jump experiments) to determine if the dynamic thermal history affected the final QELS signature. The autocorrelation function of the sample was recorded at a predetermined temperature below the phase transition. The sample was then placed in an independent bath at this same temperature while the scattering chamber bath was heated to a temperature above the phase transition. The sample was reinserted into the scattering chamber and the autocorrelation functions recorded during appropriate time intervals (approximately 15 s) thereafter to examine the kinematics of the lipid's reaction to the temperature and to the temperature gradient. Results

(a) Differential scanning calorimetry Phospholipids, hydrated at a temperature at which the lipid is in a solid phase, do not form

134

characteristic liposomes until the temperature is raised to that of the solid to fluid transition. The actual solid phase formed when a phospholipid is hydrated at a low temperature (e.g. 5OC as in these experiments) will depend upon the of the lipid. Figure 1 illustrates nature representative dsc scans of the six lipids used in these experiments. As described under Experimental procedures, lipid/water dispersions were prepared at 5OC. In the case of each CnPE, the initial heating dsc scan showed a very energetic endotherm which was present only on the first scan. Second and subsequent scans showed a less energetic endotherm at a lower temperature. This pattern is typical for CnPEs hydrated

below the solid to fluid transition temperature [7 -101. The enthalpy changes and temperatures of maximum heat capacity for these transitions are summarized in Table I. For the CnPEs, the endotherm on the initial scan represents the well described L, to La transition where L, is a dehydrated crystalline packing arrangement. The endotherm on the second and subsequent scans represents an L, (gel) to Le transition. The important point is that these CnPEs dispersed in excess water at 5OC initially form the dehydrated crystalline Lc phase. The partially hydrated metastable La (gel) phase will only form in lipid/ water mixtures which have previously passed through the Lc to Lo transition. On the other

n

Cl2PE

Cl4PE

1

0

IO

20

30

40

TEMPERATURE

50

60

70

(“C)

Fig. 1. DSC scans of aqueous suspensions of CnPEs and CnPCs, where n refers to acyl chain length. All of the lipids were hydrated in excess water at 5OC. The first scan is shown as a solid line tracing while the second scan is depicted by the dotted hne (CnPEs). For C14PC and C16PC, first and second scans did not differ, hence only the first scan (solid line) is illustrated. Scan speed was 1.2V/min in all cases. The enthalpy changes and temperatures of maximum heat capacity for the various transitions are summarized in Table I.

135

TABLE I Transition temperatures (7") and enthalpy changes (AH) for the different lipids. All the lipids were hydrated at 5°C. The L c to L. transition was observed only on the initial dsc scan o f the CnPEs. Second (and subsequent) scans gave the L Dto Lo transition. The two C n P C s showed only an L# to L transition on the first and subsequent scans. T represents the temperature o f maxim u m heat capacity (°C) and A H is given in kcal/mol. Lipid

C10PE CI2PE CI4PE CI6PE CI4PC C16PC

L to L

26.6 44.5 57.6 67.3 ---

transition

L# to L

transition

AH

~

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11.92 I 1.55 17.02 19.0 ---

2.0 31.0 50.5 64.2 24.2 41.6

1.78 3.49 5.60 7.40 5.0 8.0

hand, the initial and subsequent dsc heating scans for CI4PC and C16PC dispersions (prepared at 5 °C) were essentially identical (Fig. 1 and Table I). Hence for these two CnPCs the La (gel) phase is formed as low as 5°C, i.e., no dehydrated L phase forms even when the lipid is hydrated at 5 °C. It is worth pointing out that these differences between the CnPEs and CnPCs probably result from the smaller size of the PE head group and the capacity of the CnPEs to engage in intermolecular hydrogen bonding.

(b) Freeze-fracture microscopy Although optical microscopy would have allowed us to follow vesicle generation continuously as a function of temperature, its spatial resolution was insufficient for our purposes. For all six lipids, vesicles began to form only when the temperature reached that of the given solid to fluid transition; Lc to Lo for the CnPEs, L# to Lo for the two CnPCs. As visualized by electron microscopy, the CnPEs and CnPCs displayed very different topographic features from each other, although within each group there was considerable uniformity. Figure 2 illustrates a composite of freeze fracture electron micrographs for the CnPEs. Below the L to Lo transition these lipids exist as bundles of closely apposed flat bilayer stacks. As the temperature approaches that of the L to L transition, these stacked bilayers appear to curvc l into a closed vesicular arrangement. Above the

phase transition temperature characteristic multilamellar liposomes are readily visible. Temperature jump experiments for the CnPEs indicated that for all four members a large number of liposomes were observed at the earliest time point (60 s). Figure 3 illustrates a composite of freeze fracture micrographs for C14PC and C16PC. The topographic features are very different from those of the CnPEs. Below the La to Lo transition, these lipids form extended multilamellar layers with both flat and undulating surfaces. On close examination, coarse ripples characteristic of the L# phase can be seen [11]. No structures similar to the bilayer stacks of the CnPEs were observed in these two CnPCs. As the temperature approaches that of the La to Lo phase transition, vesicles appear to "bud o f f " from the surface. This budding phenomenon is more apparent in the micrographs of C14PC, 60 s after a temperature jump. Although vesicle formation was well established by this time point, in these two pictures multilamellar vesicles can clearly be seen lifting off from the lipid surface. These vesicles are not simply embedded in the lipid background since one can easily follow continuous unbroken ripples from the lipid layer onto the contiguous surface of the forming vesicles. As the temperature exceeds that of the phase transition, characteristic multilamellar vesicles are readily seen. In summary, lipid topography and vesicle

136

137

edge to edge. CnPCs, on the other hand, form large multilameUar layers below the solid to fluid phase change, with vesicle formation occurring via a "budding" process at the transition.

(c) QELS measurements

Fig. 2. Freeze fracture micrographs of CnPE samples. The lipid samples were hydrated at 5°C and then incubated for 20 min at temperatures spanning the solid to fluid transition ( T ) . Morphological features were similar for all four CnPEs in both the solid and fluid states. Representative micrographs are illustrated to show the sequence of events when a CnPE is heated from a temperature below to a temperature above the solid to fluid transition. (A) CI4PE quenched from 50°C ( T - 7 ° C ) . Bundles of bilayer stacks can be seen edge on. (B) C10PE quenched from 22°C ( T - 5 ° C ) . Bilayer stacks are visible face on. (C) CI2PE quenched from 40°C ( T - 4 . 5 ° C ) . A bilayer stack appears to be folding (arrow). Several small vesicles are nearby. (D) CI2PE quenched from 42°C ( T - 2 . 5 ° C ) . Although this micrograph has some shadow artefact from too much carbon deposition, a bilayer stack is clearly seen to be folding (arrow). (E) CI2PE quenched from 55°C ( T + II°C). Typical multilamellar vesicles are visible. The bar represents 345 nm in length in all cases.

formation are quite different for CnPEs and CnPCs, when visualized by freeze fracture electron microscopy. Below the temperature of the solid to fluid transition, CnPEs form bundles of flat bilayer stacks which appear to fold into three dimensional vesicles at the transition i.e.

Latex spheres were used as a comparison standard for the lipid autocorrelation functions. A graph of temperature versus autocorrelation function for 2-/am spheres, is shown in Fig. 4(A). The value of the autocorrelation function decreases smoothly with temperature since the viscosity of water decreases as a function of temperature, thereby allowing the (constant shaped and sized) spheres to diffuse more rapidly. Two CnPCs, C16PC and C14PC were examined. C14PC was initially examined at 10°C to determine a base characteristic signature. It was then incrementally heated through the gel to liquid crystal phase transition and the autocorrelation function measured at each temperature. Four samples of C14PC were studied in this fashion and the averaged results are shown in Fig. 4(B). The value of the autocorrelation function decreases smoothly as the lipid approaches the phase transition. This decrease is consistent with the decrease in viscosity of water as a variation of temperature. However, as the lipid approaches its gel to liquid crystal phase transition at 24°C, the autocorrelation function undergoes a significant increase in value and then drops sharply as the phase transition is achieved. Above the transition temperature the value of the autocorrelation function continues to decrease smoothly, as if the previous changes had not occurred. Samples of CI4PC were recycled in temperature several times through the transition temperature and the results of the first and fourth cycles for the same sample are shown in Fig. 4(C). In each case there is a marked similarity between the values of the autocorrelation functions. The fourth cycle (Fig. 4C) exhibits the same decrease in value as the first cycle up to the transition temperature where the autocorrelation function then undergoes an increase in value followed by a decrease to a value that

138

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lation f u n c t i o n m e a s u r e d . F o u r sam p l es o f C 1 6 P C were e x a m i n e d in this f a s h i o n a n d t h e a v e r a g e d results, at each t e m p e r a t u r e , are s h o w n in Fig. 4(D). T h e v a l u e o f t h e a u t o c o r r e l a t i o n f u n c t i o n decreases s m o o t h l y o v e r a l l as the lipid a p p r o a c h e s the p h ase t r a n s i t i o n . Just b e l o w 4 2 ° C the a u t o c o r r e l a t i o n f u n c t i o n u n d e r g o e s a

w o u l d a p p e a r to be c o n t i n o u s w i t h the b e f o r e transition function. This e x p e r i m e n t was r e p e a t e d f o r t h e lipid C 1 6 P C . A baseline v a l u e o f th e a u t o c o r r e l a t i o n f u n c t i o n was o b t a i n e d at 15 ° C . T h e s a m p l e was t h e n i n c r e m e n t a l l y h e a t e d t h r o u g h the gel to liquid crystal p h as e t r a n s i t i o n a n d th e a u t o c o r r e -

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Fig. 4. Autocorrelation function value versus temperature for ]atex spheres and CnPCs. The autocorrelation function value at a delay time T = 2.8 ms is plotted on the ordinate and temperature (°C) is plotted on the abscissa. (A) Diffusing 2-~n latex spheres. The decrease in value is due to the decrease in water viscosity as a function of temperature. (B) C14PC. Each individual sample gave a very reproducible set of data points and an upward inflection near the temperature of the gel to liquid crystal transition. One such sample result is illustrated in C. The points here represent the averaged values at each temperature for four samples. Bars indicate the maximum range of values between samples. The arrow denotes the temperature of the gel to liquid crystal transition. (C) CI4PC. One sample was cycled through the full temperature range four times. The first cycle is denoted by the solid line, the fourth cycle by the dotted line. The arrow denotes the temperature of the gel to liquid crystal transition. (D) CI6PC. Each individual sample gave a very reproducible set of data points and an upward inflection near the temperature of the gel to liquid crystal transition. The points here represent the averaged values at each temperature for four samples. Bars indicate the maximum range of values between samples. The arrow denotes the temperature of the gel to liquid crystal transition.

142 significant increase in value as the lipid enters the phase transition followed by a decline to its previous value after the transition temperature is reached. The value of the autocorrelation function then smoothly decreases as a function of temperature, consistent with a particle of constant size diffusing in water. When samples of C16PC were recycled in temperature several times, the results of the first and the subsequent cycles were similar to each other and were similar to the functions shown in Fig. 4(C) for the C14PC sample. For both the CI4PC and the C16PC samples, the general relationship of the autocorrelation function values with respect to temperature were similar. Both lipids gave autocorrelation function-temperature curves that appeared to be relatively smoothly decreasing variations that could be part of the same baseline, except in the vicinity of the transition temperature. There the values increased significantly and then returned to the baseline. This pattern would appear to be a characteristic QELS signature of the CnPCs as they move through the gel to liquid crystal phase transition. Four CnPEs, (C10PE, C12PE, C14PE, C16PE) were examined in the same fashion. C10PE was initially measured at 10°C to determine a baseline signature. The averaged results for C10PE are shown in Fig. 5(A). The value of the autocorrelation function decreases smoothly as the lipid approaches the phase transition. Between 25°C and 27°C the value sharply decreases and then basically levels off as the phase transition is complete. The precipitous decrease in the value of the autocorrelation function at the transition is consistent with a major structural change in the lipid, such as the formation of vesicles from laminar sheets. Autocorrelation functions versus temperature were also measured for the other three CnPEs. The results of these temperature curves are shown in Fig. 5(B) (C12PE), Fig. 5(C) (C14PE), and Fig. 5(D) (C16PE). For each CnPE the results were similar. As each lipid was heated toward its crystal to liquid crystal phase transition temperature the values of its autocor-

relation function slowly decreased. As the lipid approached about 2°C below the phase transition temperature, the autocorrelation function sharply decreased and then stabilized at a lower value after the phase transition temperature was reached. One of the interesting features of these sharp falls in the value of the autocorrelation functions, is that the height of the fall appears to decrease as the length of the lipid acyl chain increases. One of the lipids, namely C14PE, was recycled in temperature. After it had been heated through the crystal to liquid crystal transition to produce the characteristic curve associated with this transition, it was then allowed to cool back through the phase transition to form the gel phase. The vesicles were then incrementally heated back through the full temperature range. The results are shown in Fig. 5(C). The autocorrelation functions did not show any change in value as the vesicles went through the gel to liquid crystal phase transition at 51°C but did show a very significant change in value as the lipid passed through the temperature range of 56 --59°C. This change at 56--59°C is very reminiscent of the phase transition signatures seen in Fig. 4 with the CnPCs. This signature was found in other C14PE samples and was observed for the other CnPEs cycled in the same fashion. In each case the value of the autocorrelation function decreased as the temperature of the vesicle suspension was heated. As the transition temperature at which vesicles originally formed was reached, the value of the function increased significantly and then returned to the previous baseline. Temperature jump experiments were performed to determine if the dynamic thermal history of the lipid affected its characteristic signature. A sample of CIOPE was put in the scattering cell (at 23 °C) and the autocorrelation function measured. The sample was then maintained at 23°C while the temperature of the scattering cell was increased to 40°C. The CIOPE sample was then exposed to this higher temperature and autocorrelation functions recorded during appropriate time intervals for the next 300 s.

143

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Fig. 5. Autocorrelation function value versus temperature for CnPEs. The autocorrelation function value at a delay time x = 2.8 ms is plotted on the ordinate and temperature (°C) is plotted on the abscissa. For all four lipids, individual samples gave very reproducible data points and a downward deflection near the temperature of the crystal to liquid crystal transition. In A, B, and D the points represent the averaged values at each temperature for four samples. Bars indicate the maximum range of values between samples. In C, one sample of CI4PE was cycled through the full temperature range twice. The first cycle is shown by the solid line, the second cycle is shown by the broken line, the shape of the curve for the second cycle resembles the curves obtained with the CnPCs (Fig. 4). The arrow (in each panel) denotes the temperature of the crystal to liquid crystal transition.

The values o f the a u t o c o r r e l a t i o n f u n c t i o n s are p l o t t e d in Fig. 6. R e o r g a n i z a t i o n o f the lipid within the first 30 s. A similar result was seen for the o t h e r C n P E s . C o m p a r a b l e results were o b t a i n e d with t e m p e r a t u r e g r a d ie n t s or j u m p s o f d i f f e r e n t m a g n i t u d e s p r o v i d e d t h a t the t e m p e r a ture j u m p s p a n n e d the solid to fluid t r a n s i t i o n .

Discussion O n e o f the m a i n p u r p o s e s o f these experim e n t s was to study the s e l f - a g g r e g a t i o n o f p h o sp h o l i p i d s into closed vesicular structures. L i p i d / w at er mixtures were s u b j e c t e d to m i n i m a l m e c h a n i c a l a g i t a t i o n as described in a p r e v i o u s

144 CCOPE TEMPERATURE JUMP 1.0

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-pO .5 ¢cI

o~

6b

i~o

18o

240

300

TIME AFTER 40'C IMMERSION ( SEC )

Fig. 6. Temperature jump experiment. A sample of C10PE, hydrated at 5°C was put into the scattering cell maintained at 23°C. The autocorrelation function was measured (time zero on graph). The sample was then maintained at 23°C while the temperature of the scattering cell was increased to 40°C. The CIOPE sample was then exposed to this higher temperature and autocorrelation functions rapidly measured for the next 300 s at about 30 s per correlation. The transformation shape for the sample appears to be complete in less than 30 s. The value of the autocorrelation function remained unchanged for at least 2 h (data not shown).

publication [2]. Multilamellar vesicles were observed to f o r m only when the lipids were in the fully disordered liquid crystal ( L ) phase. Lipids in a solid phase f o r m non-vesicular structures which have quite different m o r p h o l o g y depending upon whether the lipid is a C n P E or a CnPC. Before discussing the freeze fracture and light scattering data, we will review the different phase properties o f these two classes o f lipid (Fig. 1), especially those aspects relevant to the present experiments. All of the phospholipids were hydrated at 5°C, a temperature well below their respective solid to fluid phase transitions. Any hydration temperature could have been used provided it was below the temperature of this phase change. When hydrated at 5°C, the four C n P E s f o r m a well characterized (probably totally) dehydrated crystalline packing arrangement ( L ) [7--10]. At a temperature, individual for each C n P E , this L c phase converts to the fully hydrated disordered fluid phase (Lo). By

contrast, the two CnPCs, f o r m a partially hydrated gel (Lg) phase when hydrated at 5 °C. At a temperature characteristic of each C n P C , this gel phase converts to the fully hydrated L state. Hence vesicle formation will be occurring at the L c to L transition for the CnPEs and at the Lg to Lo transition for the CnPCs. The freeze fracture m o r p h o l o g y of the CnPEs below the phase change is quite different f r o m that of the CnPCs. This morphological difference could reflect either the difference between the L c and La packing arrangements or the structural differences between these two classes of lipid. We cannot discriminate between these two possibilities, although they are clearly inter-related i.e. CnPEs may form a different packing arrangement than the C n P C s when hydrated at 5°C because of the structural differences. The freeze fracture electron micrographs give a reasonably clear picture of liposome formation. CnPEs in the crystal ( L ) state form bundles of bilayer stacks which appear to fold into closed vesicles at the transition temperature. On the other hand, CnPCs f o r m extended multilayer sheets in the gel (Lg) state Which bud o f f vesicles at the transition temperature. For both classes of lipid two significant changes occur at the solid to fluid transition: an increase in the hydration state of the head groups and an increase in molecular motion of the acyl chains. Whether it is the change in hydration state a n d / o r acyl chain motion which allows vesicles to f o r m cannot be answered by these experiments. On the basis of temperature j u m p experiments, we can set an upper time limit for vesicle formation of about 30 s. The QELS data like the freeze fracture results showed that the C n P C s and CnPEs have different autocorrelation function versus temperature relationships as will now be discussed. The data shown in Fig. 4(A) indicate that the decrease in viscosity of the water medium suspending latex spheres (of constant size and shape) results in a relatively smoothly decreasing autocorrelation function as the temperature is increased. This smooth decrease occurs because the frequency shifting of the scattered light f r o m the spheres, which gives rise to decorrelation, is

145

due solely to their translational motion over the whole temperature range of study. Other factors which could cause decorrelation of the scattered signal, such as rotational motion, a shape change due to temperature, or effects such as absorption bands occurring at critical temperatures do not exist for the latex spheres. The temperature dependence of the autocorrelation functions for the CnPCs (Figs. 4B,C,D) was very different from that of latex spheres. This difference must be due to properties of the lipids themselves. C14PC and C16PC hydrated at 5 °C take on certain characteristic shapes (see Fig. 3) which will diffuse and rotate through the water medium. This motion will result in decorrelation of the scattered light from the (gel state) lipids. When these lipids pass from the gel to the liquid crystal state they form vesicles (Fig. 3). As the lipid approaches the gel to liquid crystal phase transition, the value of the autocorrelation function increases. This increase indicates that the scattered light decorrelates slower than at temperatures below or above this transition. Such a pattern is unusual since one would expect any additional motion to accelerate not slow the rate of decorrelation. As noted earlier, the rate of decorrelation before and after the phase transition is similar, indicating that the relative sizes of the lipid aggregrates before and after the phase transition are relatively comparable. For example, the lipid "sheets" in the gel state translate at about the same speed as the vesicles in the liquid crystal state above the phase transition temperature. It is this significant slowing in the rate of decorrelation as the lipid approaches its phase transition which is quite unusual. The exact cause of this slowing is unknown but it is obviously associated with events preceeding the phase transition since it not only appears for both lipids (C14PC, C16PC) when going from the gel to liquid crystal state the first time (during the formation of vesicles) but also with the passage of vesicles from the gel state to the liquid crystal state on subsequent occasions (Fig. 4C). This significant increase in value of the autocorrelation function can not be accounted for by motion since there is no reason to believe

the motion of the vesicles or sheets is any different in this temperature region than any other. In addition the slowing in the rate of decorrelation is not due to vesicle clumping at the transition since the concentration of vesicles in the scattering cell is so dilute that collisions between vesicles do not in essence occur. The answer must lie elsewhere. One speculation is that this result is due to some inherent change in the way that the lipid scatters light in the temperature region just preceding its phase transition. This change would be such that the motion of the vesicle on this time scale would be a smaller component of the scattering vector and would indicate that the scattering vector itself has changed. Since the angle of the detector has not moved, the change in the scattering vector would necessitate a change in the index of refraction of the lipid to a larger value. The index of refraction for light scattering is associated with the outer electronic levels of the molecules. A change in this index would indicate that the lipid has undergone changes in conductivity or shape of these outer electronic levels as it approaches the phase transition, such that the wave length of the scattered light is increased. The changes would be similar for both C16PC and C14PC. Appropriate experiments would have to be done to determine the exact cause for this change in wave length. The main conclusion to draw from the shape of the C14PC and C16PC curves is that the relative sizes of the "sheets" that the initial gel state lipid preferentially forms and of the vesicles that it forms after it is raised above the solid to fluid phase transition temperature are similar. This point will be more significant when contrasted with the case of the CnPEs. The change in configuration of the CnPEs when going from that in the initial crystalline state to the vesicle form in the liquid crystal state is very significant (Fig. 2). The values of the autocorrelation functions for the CnPEs (Fig. 5) show a precipitous decrease just as the phase transition temperature is achieved. This sharp decrease is consistent with a major shape change. CnPE vesicles diffuse much faster than the sheets of CnPE lipid in the crystalline state. Interestingly, the magnitude of the discontinuity

146 at or near the (dsc) transition temperature appears to decrease with increasing chain length (Fig. 5). F r o m these observations it would appear that the formation of vesicles at the phase transition is a far more dramatic event for the C n P E s than for the CnPCs. It is also interesting to note that the observed changes for both lipids take place several degrees below the actual (dsc) transition temperature. The changes are apparently complete by the time that this phase transition temperature is actually reached. When C14PE is cooled below its phase transition the lipid stays in a vesicle geometry but reverts to the gel state. In Fig. 5(C), when C14PE was reheated through the full temperature range, light scattering did not show any apparent difference at 50.5°C, at which temperature the lipid should have changed f r o m the gel to liquid crystal phase according to differential scanning calorimetry (dsc). C I 4 P E however, showed a significant change in light scattering (Fig. 5C) when it passed through the 57°C temperature that was associated with the previous crystal to liquid crystal transition. Dsc shows no transition at this temperature when C14PE is recycled in this manner. The molecular interpretation of this change in signal in the second cycle (Fig. 5C) is not clear, except to point out that the shape of this change is the same as for the gel to liquid crystal signal change of the CnPCs. It is tempting to speculate that this signal could be associated with a change in the index of refraction of the lipid at this particular temperature (57°C). This explantatien would imply that there might be two liquid crystal phases for C14PE, a liquid crystal phase that

extends f r o m 51°C to 57°C and another above 57°C that has the same index o f refraction change that the C n P C liquid crystal phase contains. This speculation would make the signatures between the two classes of lipids consistent. Acknowledgements

The authors would like to thank the Academic Research P r o g r a m o f the Department o f National Defence (T J R and PR) and the Medical Research Council (MAS) for financial support. References 1

A.D. Bangham, M.N. Standish and J.C. Watkins (1965) J. Mol. Biol. 13,238--252.

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T.J. Racey, M.A. Singer, L. Finegold and P. Rochon (1989) Chem. Phys. Lipids 49, 271--288. A.D. Bangham, M.W. Hill and N.G.A. Miller 0974) in: E.D. Korn 0Ed.), Methods in Membrane Biology, Vol. l, Plenum Press, N.Y. pp. 1--68. D.A. Wilkinson and J.F. Nagle (1984) Biochemistry 23, 1538--1541. L. Finegold, W.A. Shaw and M.A. Singer (1990) Chem. Phys. Lipids 53, 177--184. F.P. Schwarz (1986) Thermochim. Acta 107, 37--49. B.Z. Chowdhry, G. Lipka, A.W. Dalziel and J.M. Sturtevant (1984) Biophys. J. 45,901--904. H. Xu, F.A. Stephenson, H.-N. Lin and C.-H. Huang (1988) Biochim. Biophys. Acta 943, 63--75. H.H. Mantsch, S.C. Hsi, K.W, Butler and D.G. Cameron (1983) Biochim. Biophys. Acta 728, 325--330. H. Chang and R.M. Epand 0983) Biochim. Biophys. Acta 728, 319--324. E. Sackmann, D. Ruppel and C. Gebhardt 0980) in: W. Helfrich and G. Heppke (Eds.), Liquid crystals of one and two-dimensional order, Springer-Verlag, Berlin, pp. 309--326.

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