Aggregate Structure in Dilute Aqueous Dispersions of Phospholipids

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Aggregate Structure in Dilute Aqueous Dispersions of Phospholipids, Fatty Acids, and Lysophospholipids Nill Bergstrand* and Katarina Edwards Department of Physical Chemistry, Uppsala University, Box 532, S-751 21 Uppsala, Sweden Received January 8, 2001. In Final Form: March 9, 2001 Cryo-transmission electron microscopy (cryo-TEM) was employed to investigate the aggregate structure in dilute aqueous dispersions of egg-phosphatidylcholine (EPC), oleic acid (OA), and the lysophospholipid monooleoylphosphatidylcholine (MOPC). At physiological pH and salt concentration, a relatively monodisperse population of unilamellar liposomes was detected in mixtures containing equimolar concentrations of the three components. Threadlike micelles constituted the dominant aggregate structure in samples containing high concentrations of MOPC. Excess fatty acid forced, on the other hand, the system toward structures with net negative curvature. In the absence of phospholipid, cryo-TEM revealed bilayer fragments in coexistence with threadlike micelles in mixtures containing the same molar amount of MOPC and OA. External addition of MOPC to preformed EPC liposomes gave rise to a concentration dependent evolution of intermediate structures, including open liposomes and bilayer fragments. The structural rearrangements were found to be slow and permitted visualization of a number of interesting transition structures. In addition to the structural studies, static and time-resolved fluorescence measurements were employed to determine some fundamental parameters for MOPC micelles. The results indicate a critical micelle concentration of close to 5 µM and an aggregation number of approximately 142.

1. Introduction Many biological membranes contain small quantities of lysophospholipids and fatty acids. The fact that these components possess quite different physicochemical properties compared to those of typical membrane lipids makes them interesting from both a biological and a fundamental point of view. In dilute aqueous solution, neither lysophospholipids nor fatty acids form lamellar phases but organize into micelles and reverse hexagonal structures, respectively.1,2 When accumulated in high enough concentrations, they are therefore expected to destabilize lipid membranes. As a consequence, the membrane may become more sensitive to fusion processes3-9 or undergo morphological changes leading to increased permeability or complete membrane disruption.10-13 Physicochemical investigations in model systems may help in understanding, and possibly predicting the effect of, the interaction between lysophospholipids, fatty acids, and biological membranes. The phase behavior of phospholipid/lysophospholipid systems14-17 and of phospho(1) Arvidson, G.; Brentel, I.; Khan, A.; Lindblom, G.; Fontell, K. Eur. J. Biochem. 1985, 152, 753. (2) Edwards, K.; Silvander, M.; Karlsson, G. Langmuir 1995, 11, 2429. (3) Chernomordik, L. J. Membr. Biol. 1995, 146, 1. (4) Chernomordik, L.; Chanturiya, A.; Green, J.; Zimmerberg, J. Biophys. J. 1995, 69, 922. (5) Basa´nez, G.; Goni, F. M.; Alonso, A. Biochemistry 1998, 37, 3901. (6) Chernomordik, L.; Leikina, E.; Frolov, V.; Bronk, P.; Zimmerberg, J. J. Cell Biol. 1997, 136, 81. (7) Martin, I.; Ruysschaert, J. M. Biochim. Biophys. Acta 1995, 1240, 95. (8) Yeagel, P.; Smith, F.; Young, J.; Flanagan, T. Biochemistry 1994, 33, 1820. (9) Basanez, G.; Nieva, J.; Rivas, E.; Alonso, A.; Goni, F. Biophys. J. 1996, 70, 2299. (10) van Echteld, C.; de Kruijff, B.; Mandersloot, J.; de Gier, J. Biochim. Biophys. Acta 1981, 649, 211. (11) Han, H.; Kim, H. J. Biochem. 1994, 115, 26. (12) Hauser, H. Chem. Phys. Lipids 1987, 43, 283. (13) Mui, B.; Do¨bereiner, H.; Madden, T.; Cullis, P. Biophys. J. 1995, 69, 930. (14) McIntosh, T.; Kulkari, G.; Simon, S. Biophys. J. 1999, 76, 2090. (15) Zhelev, D. Biophys. J. 1998, 75, 321.

lipid/fatty acids systems2,14,18-20 has been investigated by a number of different techniques. Few studies have been made, however, on model systems consisting of lysophospholipid/fatty acid and, in particular, of systems containing all three components, phospholipid, lysophospholipid, and fatty acid. This is surprising, not least because the enzymemediated and the spontaneous hydrolysis of phospholipids, leading to a simultaneous accumulation of fatty acids and lysophospholipids, are important and well-studied phenomena.21-28 To gain further information about the effect of lysophospholipids and fatty acids on membrane structure, we have made a systematic study of the structural behavior in phospholipid/lysophospholipid/fatty acid mixtures. The lysophospholipid and fatty acid used were monooleoylphosphatidylcholine (MOPC) and oleic acid (OA), which are the most likely hydrolysis products of the used phospholipid, egg-phosphatidylcholine (EPC). Direct information about the aggregate structure was obtained by means of cryo-transmission electron microscopy, cryo-TEM. (16) Disalvo, E.; Viera, L.; Bakas, L.; Senisterra, G. J. Colloid Interface Sci. 1996, 178, 417. (17) Checchetti, A.; Golemme, A.; Chidichimo, G.; LaRosa, C.; Grasso, D.; Westerman, P. Chem. Phys. Lipids 1996, 82, 147. (18) Epand, R. M.; Epand, R. F.; Ahmed, N.; Chen, R. Chem. Phys. Lipids 1991, 57, 75. (19) Winter, R.; Erbes, J.; Templer, R.; Seddon, J.; Syrykh, A.; Warrender, N.; Rapp, G. Phys. Chem. Chem. Phys. 1999, 1, 887. (20) Templer, R.; Seddon, J.; Warrender, N.; Syrykh, A.; Huang, Z.; Winter, R.; Erbes, J. J. Phys. Chem. B 1998, 102, 7251. (21) Callisen, T. H.; Talmon, Y. Biochemistry 1998, 37, 10987. (22) Bent, E. D.; Bell, J. D. Biochim. Biophys. Acta 1995, 1254, 349. (23) Hayashi, K.; Arakane, K.; Naito, N.; Nagano, T.; Hirobe, M. Chem. Pharm. Bull. 1995, 43, 1751. (24) Arakane, K.; Hayashi, K.; Naito, N.; Nagano, T.; Hirobe, M. Chem. Pharm. Bull. 1995, 43, 1755. (25) Grit, M.; Crommelin, D. J. A. Chem. Phys. Lipids 1992, 62, 113. (26) Grit, M.; de Smidt, J. H.; Struijke, A.; Crommelin, D. J. A. Int. J. Pharm. 1989, 50, 1. (27) Zuidam, N. J.; Gouw, H. K. M. E.; Barenholtz, Y.; Crommelin, D. J. A. Biochim. Biophys. Acta 1995, 1240, 101. (28) Gerasimov, O. V.; Schwan, A.; Thompson, D. H. Biochim. Biophys. Acta 1997, 1324, 200.

10.1021/la010020u CCC: $20.00 © 2001 American Chemical Society Published on Web 05/04/2001

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Bergstrand and Edwards

2. Experimental Section 2.1. Materials. Egg yolk lecithin (EPC) of grade 1 was purchased from Lipid Products (Nutfield, U.K.). L-R-Lysophosphatidylcholine oleoyl (C18:1, [cis]-9) (MOPC) and oleic acid of 99%+ purity were purchased from Sigma-Aldrich. 1,6-Diphenyl1,3,5-hexatriene (DPH) was obtained from Fluka (Stockholm, Sweden) and used as received. Pyrene, of analytical grade >99%, was obtained from Serva (Feinbiochemica, Heidelberg, Germany). All other salts and reagents were of analytical grade and were used as received. 2.2. Preparation of Liposomes and Lipid Dispersions. Lipid mixtures were prepared by codissolving the lipids, lysophospholipids, and/or fatty acids in chloroform, removing the chloroform by evaporation under vacuum, and thereafter redissolving the dried lipid film in a buffer containing 150 mM NaCl and 20 mM Hepes (pH 7.4). For the turbidity measurements, the redissolved lipids were subjected to at least eight freeze-thaw cycles (including freezing in liquid nitrogen and heating to above 60 °C). Unilamellar liposomes were then produced by multiple extrusion through polycarborate filters (pore size 100 nm) mounted in a LiposoFast miniextruder from Avestin (Ottawa, Canada). The lipid dispersions used for cryo-TEM investigations were, unless otherwise stated, dispersed by vortexing and allowed to equilibrate at 25 °C overnight. All the samples had a final EPC or OA concentration of 3 mM, and the total lipid concentration was less than 1 wt %. 2.3. Steady-State Fluorescence Measurements. The critical micelle concentration of MOPC was determined by means of steady-state fluorescence utilizing the dye DPH. The fluorescence of the probe DPH is minimal in water, whereas in a hydrophobic environment the fluorescence is substantially enhanced. Samples were prepared by diluting a stock solution of MOPC (0.2 mM) in Hepes buffer (20 mM Hepes, 150 mM NaCl, pH 7.4) to desired concentrations within the range of 0.5-15 µM. A stock solution of DPH in methanol (0.32 mM) was prepared and added (measured with a Hamilton microsyringe) to the MOPC/Hepes samples. The final DPH concentration was 4 µM in all the samples. To make sure that the methanol did not affect the critical micelle concentration (cmc) value, the samples contained a final concentration of methanol less than 1% v/v. The samples were equilibrated, in the dark, for 8 h at 25 °C before measurements. The emission fluorescence spectra of the MOPC/DPH/Hepes buffer samples were obtained by a SPEX-fluorolog 1650 0.22-m double spectrometer from SPEX Industries Inc. (Edison, NJ). The excitation wavelength was set to 357 nm, and the solubilization of the DPH into MOPC micelles was determined from the emission intensity peak at 427 nm. 2.4. Time-Resolved Fluorescence Measurements. The micelle aggregation number (Nagg) was determined by a timeresolved quenching method.29 The method requires, for best results, a probe and a quencher that can be regarded as stationary inside the micelle. Pyrene excimer quenching was used, utilizing pyrene as both probe and quencher. Stock solutions of MOPC and pyrene were prepared. Pyrene was dissolved in ethanol at a concentration of 1.13 mM, and the MOPC was dissolved in Hepes buffer (150 mM NaCl, 20 mM Hepes, pH 7.4). From these stock solutions, three samples were prepared with the same MOPC concentration, 1.34 mM (above the cmc of MOPC), but with different pyrene concentrations (7.4, 10.6, and 14.9 µM). The samples were stirred continuously and left to equilibrate in the dark for 12 h at 25 °C. The time-resolved fluorescence decay data (pyrene emission detected at 397 nm) were collected at 25 °C with a single photon counting technique. More information about the measurements is given in the results section. A detailed description of the experimental technique and setup can be found elsewhere.29 2.5. Turbidity Measurements. The turbidity was determined by measuring the absorbance of the samples at 350 nm using a Hewlett-Packard 8453 UV-visible spectrophotometer. A stock solution of MOPC in Hepes buffer (20 mM Hepes, 150 mM NaCl, pH 7.4) was prepared. Samples containing different concentrations of MOPC were then prepared by diluting the liposomal dispersion and adding a proper amount of the stock solution. All (29) Almgren, M.; Hansson, P.; Mukhtar, E.; van Stam, J. Langmuir 1992, 8, 2405.

Figure 1. Fluorescence intensity (λex ) 357 nm, λem ) 427 nm) as a function of MOPC concentration. The break point at 5 µM indicates the critical micellar concentration of MOPC in Hepes buffer (pH 7.4) at 25 °C. the samples had a final EPC concentration of 3 mM and were left in the dark at 25 °C, both before and between the measurements. The total lipid concentration was less than 1 wt %. All samples were shaken before the measurements. 2.6. Cryo-Transmission Electron Microscopy. The technique, which has been described elsewhere,30,31 consists in short of the following. Thin (10-500 nm) sample films were prepared under controlled temperature (25 °C) and humidity conditions within a custom-built environmental chamber. The films were thereafter vitrified by quick freezing in liquid ethane and transferred to a Zeiss EM 902 transmission electron microscope for examination. To prevent sample perturbation and the formation of ice crystals, the specimens were kept cool (below 108 K) during both the transfer and viewing procedures. All observations were made in zero-loss bright-field mode and at an accelerating voltage of 80 kV. The electron exposure of the films varied between 5 and 15 e-/Å2, depending on the magnification.

3. Results 3.1. Critical Micelle Concentration of MOPC. The critical micelle concentration of pure MOPC was determined by steady-state fluorescence measurements of the hydrophobically solubilized dye DPH. The intensity of the emitted light (427 nm) from DPH was measured as a function of the MOPC concentration, as shown in Figure 1. At low concentrations of MOPC, no self-association takes place, and as a result the fluorescence intensities are very low. At higher concentrations, the MOPC forms micelles and upon solubilization of DPH in the hydrophobic core the intensity increases substantially. In Figure 1, the cmc value can be found at the point where the two linear curves cross, that is, where the intensity from the emitted light of the dye starts to increase. The cmc of MOPC in Hepes buffer (150 mM NaCl, 20 mM Hepes, pH 7.4) was found to be near 5 µM. Determination of the cmc by use of static light scattering measurements indicated a similar value (results not shown). 3.2. Aggregation Number of MOPC Micelles. The micelle aggregation number (Nagg) was determined by time-resolved measurements of pyrene excimer quenching. Pyrene molecules have very low aqueous solubility and may therefore be regarded as stationary inside a micelle. The unexcited molecules act as quenchers by forming dimers, excimers, with the excited pyrene molecules. The (30) Bellare, J. R.; Davis, H. T.; Scriven, L. E.; Talmon, Y. J. Electron Microsc. Tech. 1988, 10, 87. (31) Dubochet, J.; Adrian, M.; Chang, J. J.; Homo, J. C.; Lepault, J.; McDowall, A. W.; Schultz, P. Q. Rev. Biophys. 1988, 21, 129.

Aqueous Dispersions of EPC, OA, and MOPC

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Figure 2. Pyrene fluorescence decays recorded in solutions containing 1.34 mM MOPC, in Hepes buffer (pH 7.4) and 7.4 µM (9), 10.6 µM (b), and 14.9 µM (0) pyrene.

time evolution of the fluorescence signal, F(t), from a probe situated in small uniform micelles, can be described by the Infelta equation,32-36

F(t)/F(0) ) exp[-A2t + A3{exp(-A4t) - 1}]

Figure 3. Turbidity in samples containing EPC liposomes as a function of mol % externally added MOPC: 1 h (0), 16 h (9), 48 h (O), and 330 h (b) after addition of MOPC. All measurements were made at 25 °C and in Hepes buffer (pH 7.4).

(1)

with

A2 ) k0 + kqk-n/(kq + k-) A3 ) nkq2/(kq + k-)2 A 4 ) kq + kwhere F(t) is the fluorescence intensity at time t after excitation, F(0) is the fluorescence intensity at t ) 0, n is the average number of quenchers per micelle, kq is the first-order rate constant for quenching in a micelle with one quencher, k- is the exit rate constant for a quencher to leave the micelle, and k0 is the unquenched decay rate of the probe. The equation and the model behind it have been described more in detail elsewhere.32-36 All quenching curves were fitted to eq 1, and the parameter A3 was determined. The fluorescence decays and the fitted curves are shown in Figure 2. It should be mentioned that t ) 0 in Figure 2 is at 50 ns, where the laser pulse is positioned. If the quencher is stationary, eq 1 can be simplified, and A3 is equal to the average number of quenchers per micelle,

A3 ) [Q]micelleN/[S]micelle

(2)

Assuming that [S]micelle is equal to the concentration of the surfactant in the micellar phase and that all the pyrene molecules were situated in the micelles, that is, [Q]micelle ) [Q]tot ) [Pyrene]tot, the aggregation number (N) can be determined. Aggregation numbers determined at three different pyrene concentrations are presented in Table 1. Nagg,average was found to be 142. It should be noted that the decay rates at longer times are the same for all curves. (32) Infelta, P. P.; Gra¨tzel, M.; Thomas, J. K. J. Phys. Chem. 1974, 78, 190. (33) Infelta, P. P.; Gra¨tzel, M. J. Phys. Chem. 1983, 78, 5280. (34) Tachiya, M. Chem. Phys. Lett. 1975, 33, 289. (35) Tachiya, M. Chem. Phys. Lett. 1982, 76, 340. (36) Tachiya, M. Chem. Phys. Lett. 1983, 78, 5282.

Figure 4. Cryo-TEM pictures of pure EPC liposomes after (a) 14 days and (b) 23 days of incubation at 25 °C. Note the threadlike micelles (marked with an arrow) in (b). Bar ) 100 nm. Table 1. Parameters Estimated from Time-Resolved Excimer Quenching [Pyrene]tot/µM

A3

N

7.3 10.9 14.6

0.79 1.19 1.52

143 145 138 Naverage ) 142

This indicates that the pyrene is stationary and that the micelles are discrete, and thus the Infelta model is applicable. 3.3. Effect of Externally Added MOPC on the Structure of EPC Liposomes. Turbidity Measurements. To investigate the solubilization properties of MOPC, the

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Figure 5. Structures formed upon external addition of MOPC to EPC liposomes: (a) 30 mol % MOPC after 16 h; (b) 30 mol % MOPC after 330 h; (c) 50 mol % MOPC after 16 h. Note disklike structure (marked with a small-headed arrow) and an ice crystal deposited after vitrification of the sample (marked with a large-headed arrow); (d) 50 mol % MOPC after 330 h. Note open liposomes (marked with an arrowhead) and threadlike micelles growing from bilayer fragments (marked with an arrow); (e) 80 mol % after 48 h. Bar ) 100 nm.

absorbance of a series of samples containing EPC liposomes and different amounts of externally added MOPC was measured as a function of time. Between the turbidity measurements, the samples were stored in the dark at 25 °C. As shown in Figure 3, very little effect on the turbidity was observed 1 h after the addition of lysolecithin. After 16 h, the turbidity drops, however, with increasing MOPC concentration indicating that smaller structures are formed. At longer times, a dramatic change is observed in the turbidity within the range of 50-60 mol % MOPC. The large turbidity suggests that the structures are growing, or becoming more dense, with time. The turbidity of the sample containing 80 mol % decreases steadily with time and has, after about 24 h, reached an equilibrium value. The turbidity at longer than 14 days of incubation time is not shown here because at prolonged incubation

the hydrolysis of EPC becomes significant (see comment in next section). Cryo-Transmission Electron Microscopy. To identify the structural changes taking place in the above system, we investigated the samples by cryo-TEM. The pure EPC reference sample consisted of unilamellar liposomes, and no structural change was observed after 14 days of incubation at 25 °C, Figure 4a. Cryo-TEM pictures of the reference sample taken 23 days after the sample preparation showed, however, cylindrical micelles in coexistence with liposomes (Figure 4b). This result indicates a significant spontaneous hydrolysis of the lipids, and we therefore stopped the measurements of samples with externally added MOPC after 14 days. Parts a and c of Figure 5 present the structures observed in the samples containing 30 and 50 mol % MOPC,


respectively, after 16 h. The addition of MOPC decreases the size of the liposomes in both samples. Interestingly, the 50 mol % sample also contained some disklike structures. After 14 days, the liposomes containing 30 mol % MOPC have grown back to their original size of about 100 nm, Figure 5b. The size of the liposomes in the 50 mol % sample has also increased, and in addition to closed liposomes, the sample contains open structures and long threadlike cylindrical micelles, Figure 5d. Frequently the cylindrical micelles seem to be attached to, and originate from, a central bilayer fragment (marked with an arrow in Figure 5d). As the MOPC concentration is increased, cylindrical micelles become the dominating structures. Figure 5e shows that in samples containing 80 mol % MOPC the liposomes are completely solubilized into mixed cylindrical micelles. The micelles become shorter with increasing MOPC concentration, and in samples containing pure MOPC the micelles appear more or less spherical (results not shown). 3.4. Structures Obtained after Symmetric Distribution of MOPC. Upon external addition of MOPC, the distribution to the lipid bilayer becomes asymmetric. Initially, the majority of the added MOPC molecules will be located in the outer leaflet of the liposomes.37,38 A more symmetric distribution of MOPC between the outer and inner monolayer was achieved by mixing the MOPC and the EPC lipids before they were hydrated and dispersed in Hepes buffer. To make sure that freeze-thawing or extrusion would not affect the outcome of the experiments, only vortexing was used for the dispersion. As shown in Figure 6a, in the absence of MOPC the EPC lipid dispersions contain a polydisperse population of large and mostly multilamellar liposomes. When 30 mol % MOPC was added, the majority of the liposomes formed were no longer multilamellar but instead showed a nice spherical and unilamellar morphology (Figure 6b). Interestingly, at 50 mol % MOPC (Figure 6c,d) multilamellar structures were again observed. A typical feature was, however, the presence of long threadlike micelles which appeared to originate from the multilamellar liposomes. Upon inclusion of 80 mol % MOPC in the dispersions, only threadlike cylindrical micelles could be observed in the samples (result not shown but the structures in Figure 5e are very similar). 3.5. Mixtures of OA and EPC. Our investigations were made at pH 7.4, and at this pH pure oleic acid forms large aggregates composed of both lamellar and nonlamellar structures (Figure 7a). As shown in Figure 7b, the inclusion of a small amount of EPC gives rise to the formation of much smaller aggregates. In addition, the aggregate structure appears to become better defined, and particles of lamellar and nonlamellar structure can be clearly distinguished. On the basis of previous cryo-TEM pictures revealing a similar morphology in related amphiphilic systems,39 it is plausible to conclude that the nonlamellar structures represent dispersed particles of reversed hexagonal phase. The reversed phase particles disappear upon further addition of EPC, and in samples composed of equimolar amounts of OA and EPC the dominating structures are instead large, often invaginated, unilamellar liposomes (Figure 7c). Also, a different type of structure was observed at this sample composition. The inset in Figure 7c shows a complex assembly of bilayers connected via large interlamellar attachments or “fusion pores”. At high concentrations of EPC, multilamellar (37) Zhelev, D. V. Biophys. J. 1996, 71, 257. (38) Farge, E.; Devaux, P. F. Biophys. J. 1992, 61, 347. (39) Gustafsson, J.; Ljusberg-Wahren, H.; Almgren, M.; Larsson, K. Langmuir 1997, 13, 6964.

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Figure 6. Cryo-TEM pictures of structures formed in the MOPC/EPC system, dispersed by vortexing, at 25 °C: (a) 0 mol % MOPC, (b) 30 mol % MOPC, and (c and d) 50 mol % MOPC. Note the threadlike micelles (marked with an arrow) growing from large multilamellar liposomes. Bar ) 100 nm.

liposomes of essentially the same appearance as those shown in Figure 6a dominate the sample structure. 3.6. Mixtures of OA and MOPC. Figure 8 shows the structures appearing in mixtures of OA and MOPC at different molar ratios. Samples with an OA/MOPC molar ratio of 4:1 (mol/mol) contain liposomes and lamellar

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Figure 8. Cryo-TEM pictures of structures formed in the OA/ MOPC system, dispersed by vortexing, at 25 °C: (a) OA/MOPC 4:1 (mol/mol), (b) OA/MOPC 1:1 (mol/mol). Note the bilayer fragments (marked with an arrowhead) and threadlike micelles (marked with an arrow). Bar ) 100 nm.

Figure 7. Structures formed in the OA/EPC system, dispersed by vortexing, at 25 °C: (a) pure OA in Hepes buffer (pH 7.4); (b) OA/EPC 4:1 (mol/mol). Note the nonlamellar structures (marked with an arrow); (c) OA/EPC 1:1 (mol/mol). Bar ) 100 nm.

structures with a very heterogeneous morphology (Figure 8a). At equal molar amounts of OA and MOPC, the aggregate structure changes dramatically. As seen in Figure 8b, the samples now consist of cylindrical micelles and bilayer fragments in coexistence with small unilamellar liposomes. Cylindrical micelles completely dominate the aggregate structure when the amount of MOPC is increased to give a 1:4 molar ratio of OA and MOPC (results not shown). 3.7. Mixtures of EPC, MOPC, and OA. The last set of experiments was made in order to study the structural behavior in mixtures of all three lipids: lysophospholipid, fatty acid, and phosphatidylcholine. Figure 10 shows the structures obtained by mixing MOPC, OA, and EPC in different molar ratios and dispersing the samples in Hepes

Figure 9. Schematic diagram showing all the compositions that were investigated by cryo-TEM in the present study. The concentrations are given in mol % of the total lipid. The water content was above 99 wt % for all samples.

buffer (pH 7.4) by vortexing. A schematic diagram showing all the investigated sample compositions can be found in Figure 9. Parts a-c of Figure 10 represent samples having a fixed equimolar amount of EPC and MOPC but a varying amount of OA (compare the solid line in Figure 9). A polydisperse population of open and closed unilamellar liposomes was observed for compositions corresponding to EPC/MOPC/OA 4:4:1 (Figure 10a). Samples with composition EPC/MOPC/OA 1:1:1 contained a surprisingly monodisperse population of closed unilamellar liposomes (Figure 10b). Completely different structures were found in samples with high OA content. Figure 10c reveals that for samples with composition EPC/MOPC/OA 1:1:4 the majority of the lipid material is found in large dispersed particles of inversed lipid (see discussion). It is interesting to compare the aggregates in Figure 10c with the much denser aggregates observed in coexistence with lamellar


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Figure 10. Cryo-TEM pictures of structures formed in the OA/MOPC system, dispersed by vortexing, at 25 °C: (a) EPC/MOPC/OA 4:4:1 (mol/mol/mol), (b) EPC/MOPC/OA 1:1:1 (mol/mol/mol), (c) EPC/MOPC/OA 1:1:4 (mol/mol/mol), (d) EPC/MOPC/OA 4:1:4 (mol/ mol/mol), (e) EPC/MOPC/OA 1:4:1 (mol/mol/mol), and (f) EPC/MOPC/OA 1:4:4 (mol/mol/mol). Note the open liposomes and bilayer fragments (marked with arrowheads) in (a) and (f), respectively, and the threadlike micelles (marked with an arrow) in (f). Bar ) 100 nm.

material at compositions corresponding to OA/EPC 4:1 (Figure 7b). Figure 10d,e illustrates the change in aggregate structure upon variation of the MOPC amount in samples having a fixed and equimolar amount of OA and EPC (compare the dashed line in Figure 9). When the amount of MOPC is increased, the aggregate structure changes from large, mainly bilamellar liposomes (Figure 10d) via smaller unilamellar liposomes (Figure 10b) to long threadlike micelles (Figure 10e). The samples along the dotted line in Figure 9, finally, have a fixed and equimolar OA/MOPC ratio. A comparison between Figures 8b, 10f, and 10b shows how the number of liposomes increases at the expense of threadlike micelles when EPC is added to the samples.

4. Discussion 4.1. Critical Micelle Concentration and Aggregation Number for MOPC. Despite the numerous investigations published involving MOPC, we have found little data on micelle aggregation number and cmc value. We therefore began our investigations by determining these fundamental parameters. The cmc was found to be 5 µM in Hepes buffer at 25 °C. This value is in good agreement with the cmc of 3 µM reported by Needham et al.40 for MOPC in water. The average aggregation number Nagg was determined to be 142. A rough estimate of Nagg can (40) Needham, D.; Stoicheva, N.; Zhelev, D. Biophys. J. 1997, 73, 2615.

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be made based on Tanford’s formulas41 for the length (lmax) and volume (Vh) of a hydrocarbon chain:

lmax ) (1.5 + 1.265(nc - 1)) Å Vh ) (27.4 + 26.9(nc - 1)) Å3 where nc is the number of hydrocarbons in the chain. If we assume that the hydrophobic core of a MOPC micelle is strictly spherical and that there is no void volume in the center, simple geometric considerations show that each micelle should consist of approximately 105 monomers. Our experimentally determined value thus indicates that the micelle shape deviates from strictly spherical. In line with the idea of slightly elongated micelles, Needham et al.40 have in calculations where the comparably large headgroup area of MOPC was taken into account calculated Nagg for MOPC to be approximately 161. 4.2. Effects of MOPC on the Structure of EPC Liposomes. Although several investigations have been published concerning the phase behavior in lysophospholipid/phospholipid/water systems,14-17 the aggregate structure in very dilute solutions of lysophospholipids and phospholipids has not been thoroughly characterized. The solubilization curves, shown in Figure 3, were recorded in order to get a first clue to the changes in aggregate size and structure taking place upon external addition of MOPC to preformed EPC liposomes. A number of interesting observations can be made based on the results. The most obvious is the fact that the structural rearrangements take place on an extremely long time scale. The explanation for this is probably connected to the long times required to reach an equilibrium distribution of the MOPC monomers in the aggregates. Because of the large hydrophilic headgroup, the so-called flip-flop mechanism between the outer and inner monolayer of a lipid bilayer is slow for MOPC monomers. The distribution between the bulk and the bilayer is fast, however, and initially most of the added MOPC will thus be situated in the outer monolayer.37,38 The initial decrease in liposome size, observed by cryo-TEM in samples containing 30 mol % MOPC (Figure 5a,b), constitutes an interesting illustration of the effects caused by the uneven distribution of the MOPC. A similar effect has been observed also by van Echteld et al. and Hauser.10,12 The initial accumulation of MOPC in the outer monolayer appears to induce a budding process leading to fission of the large liposomes into much smaller, and highly curved, structures. With time, the small and very unstable liposomes fuse back into larger liposomes. A similar behavior was observed also for samples containing 50 mol % MOPC (Figure 5c,d), but at this composition cryo-TEM revealed bilayer disks in coexistence with the small liposomes initially formed. For comparison, we also investigated a series of samples where MOPC was not added to preformed liposomes but codissolved with EPC in the preparation mixture. It is important to note that these samples were neither freezethawed nor extruded; after hydration, the lipids were dissolved simply by vortexing. Comparing the cryo-TEM pictures shown in Figure 5b,d,e with those shown in Figure 6b,c,d, it is evident that similar types of structures eventually form irrelevant of the preparation procedure. The liposomes formed after codissolution of the two components are, however, significantly larger than those obtained at long times after external addition of MOPC. The solubilization curve in Figure 3 reveals another interesting aspect of the behavior in the EPC/MOPC (41) Tanford, C. The hydrophobic effect: formation of micelles and biological membranes, 2nd ed.; John Wiley & Sons: New York, 1980.


system. In the concentration regime between 50 and 60 mol % MOPC, extremely high turbidity values were recorded. A similar type of behavior has been observed by turbidity measurements also for EPC/MMPC.16 The huge increase in turbidity could theoretically be due to the formation of very large structures or to liposome aggregation. Alternatively, the phenomena could originate from a macroscopic phase separation, similar to what has been observed in a number of other EPC/nonionic surfactant systems.42,43 The cryo-TEM pictures, showing liposomes and bilayer fragments in coexistence with long threadlike micelles (Figures 5d and 6c), do not give an unambiguous answer to the question about the origin of the high turbidity. On the basis of the cryo-TEM results, the possibility of liposome aggregation may be ruled out, however, and the visualized aggregates do not appear large enough to explain the high turbidity values. The third alternative, that is, a macroscopic separation of the sample into two distinct phases, thus appears to be the most probable alternative, but the issue has to be investigated further. 4.3. Effects of OA on Aggregate Structure. Several different methods are available to explain and predict the preferred aggregate structure in lipid/surfactant systems. Israelachvili et al. have derived a relatively simple theory, based on the geometrical molecular shape, that often works remarkably well.44 An advantage of the model is that it does not demand information on mechanical properties of the bilayer and/or knowledge of the electrostatic parameters required for the use of more sophisticated models, such as those derived by Gruner and co-workers45,46 and Wennerstro¨m at al.47 According to the model stated by Israelachvili et al., addition of cone-shaped molecules to a lipid bilayer will induce a transformation into structures of higher curvature.44,48 This is precisely what we observe when liposomes in the presence of MOPC become solubilized into mixed micelles. When the shape of the incorporated molecule is switched from a cone to an inverted cone, the bilayer will according to the model instead be forced toward a more negative curvature. This effect is exemplified by the change in aggregate structure obtained upon addition of OA to dispersions of EPC. As seen in Figure 7, with increasing OA the multilamellar liposomes (compare Figure 6a) collapse into spongelike structures (inset of Figure 7c) and, finally, form dense particles of inverted, presumably hexagonal, phase. The large invaginated liposomes shown in Figure 7c deserve some special attention. At the lip of the invagination, the inner monolayer must adopt a negative curvature. It is thus plausible to assume that the presence of protonated fatty acids increases the probability for invagination to occur. Similarly, a local high negative curvature is required in the fusion pores observed in the particle shown in the inset of Figure 7c. It should be noted that the phase behavior in mixtures containing oleic acid is strongly dependent on the pH of the solution. At 25 °C and in the presence of 150 mM NaCl, a pKa value of between 7.2 and 8 may be expected for fatty acids situated in a lipid bilayer.2 Protonation of (42) Johnsson, M.; Edwards, K. Langmuir 2000, 16, 8632. (43) Almgren, M. Biochim. Biophys. Acta 2000, 1508, 146. (44) Israelachvili, J. N.; Marcelja, S.; Horn, R. G. Q. Rev. Biophys. 1980, 12, 121. (45) Gruner, S. M.; Tate, M. W.; Kirk, G. L.; So, P. T. C.; Turner, D. C.; Keane, D. T.; Tilcock, C. P. S.; Cullis, P. R. Biochemistry 1988, 27, 2853. (46) Kirk, G. L.; Gruner, S. M.; Stein, D. L. Biochemistry 1984, 23, 1093. (47) Jo¨nsson, B.; Wennerstro¨m, H. J. Phys. Chem. 1987, 91, 338. (48) Kumar, V. V. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 444.


the carboxyl group decreases the fatty acid headgroup area and, in addition, leads to a decrease in the electrostatic repulsion between the bilayers. The propensity for HII phase formation thus increases with decreasing pH. The structures formed by oleic acid/oleate in dilute aqueous solutions have been studied, with respect to concentration and pH, by Edwards et al.2 At high pH, where the fatty acid is essentially deprotonated, cylindrical micelles are formed. Lowering the pH to values around pH 9 gives rise to the formation of lamellar structures. At pH 7.4, aggregated lamellar and nonlamellar structures coexist (compare Figure 7a). 4.4. Mixtures of EPC, OA, and MOPC. The triangle diagram in Figure 9 presents the compositions that were investigated by means of cryo-TEM. Starting in point C, where the molar amounts of the three components are the same, we find large unilamellar liposomes (Figure 10b). A comparison with the aggregate structures found at compositions corresponding to point B (Figure 10a) and, in particular, point A (Figure 6c,d) clearly shows that the presence of OA prevents the formation of structures with high positive curvature. The decrease in curvature upon addition of OA is perhaps even more obvious if we move along the solid line in the diagram up to point D. At this composition, the cryo-TEM pictures (Figure 10c) reveal what appear to be dispersed particles of inverted, possibly cubic, phase. The true origin of the particles cannot be ascertained based solely on the cryo-TEM results, but similar structures have been observed in other systems and at compositions where the phase diagrams suggest the existence of a cubic phase in excess water.49,50 It is interesting to compare the structure shown in Figure 10c with the very material dense particles obtained at point E and shown in Figure 7b. On the basis of results from previous investigations of the EPC/OA system,2 we conclude that the latter most probably represent aggregates of dispersed inverted hexagonal (HII) phase.39 It seems reasonable that a structural change toward aggregates with higher net curvature, such as dispersed particles of cubic phase, takes place upon addition of MOPC, that is, upon moving from point E to point D in Figure 9. The strong influence of MOPC on the preferred aggregate structure is further demonstrated if we compare the structures formed as we move along the dashed line, starting in point F, toward the pure MOPC corner in Figure 9. A comparison of Figures 7c, 10d, 10b, and 10e shows how the invaginated liposomes and complex spongelike particles (inset of Figure 7c) transform into bi- and unilamellar liposomes and, finally, form threadlike micelles. In samples containing equimolar amounts of OA and MOPC, corresponding to point K in the diagram, we detect open and closed liposomes in coexistence with threadlike micelles (Figure 8b). Addition of EPC has the effect of stabilizing the bilayer arrangement, and from parts f and b of Figure 10 it is clear that the amount of micelles and open structures decreases as the EPC content increases. At compositions corresponding to point M in the diagram, the phase propensity of EPC dominates completely and the sample contains multilamellar liposomes of the same general appearance as those shown in Figure 6a. (49) Siegel, D.; Epand, R. Biophys. J. 1997, 73, 3089. (50) Johnsson, M.; Edwards, K. Biophys. J. 2001, 80, 313.

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The aggregate structures observed at different compositions along the dotted line in Figure 9 should be of particular interest to those concerned with the spontaneous or enzyme-mediated hydrolysis of phospholipids. The results presented in this study indicate that the small and equimolar amounts of fatty acid and lysophospholipid formed at early stages of the hydrolysis process would give little effect on the aggregate structure. The formation of unilamellar liposomes would be promoted when about 50 mol % of the initial phospholipid has been hydrolyzed. As complete hydrolysis is approached, bilayer fragments and cylindrical micelles would, finally, be expected. Quite extensive hydrolysis would thus be needed before any gross structural changes could be detected because of hydrolysis of phospholipid liposomes. The cylindrical micelles visualized in Figure 4b indicate that the spontaneous hydrolysis of pure EPC liposomes has proceeded quite far after 23 days at 25 °C. In this context, it is also interesting to compare the results of the present study with the evolution of aggregate structures observed by cryo-TEM during phospholipase A2 (PLA2)-mediated hydrolysis of DPPC liposomes. In accordance with our results, Callisen et al.21 recently showed that extensive PLA2 hydrolysis induces the formation of bilayer fragments and micelles. However, Callisen et al. observed a significant reduction in the liposome size at early stages during the hydrolysis process. Because in the above study the PLA2 was added to preformed liposomes, the enzyme acted primarily on the phospholipids in the outer monolayer. Similar to our studies of externally added MOPC, the resulting asymmetric distribution of the lysis products between the inner and outer monolayers may induce a budding-fission process. Furthermore, the PLA2-mediated in situ production of fatty acids and lysophospholipids will give rise to local variations in membrane composition. The evolution of regions rich in lysis products but poor in phospholipid may induce significant structural rearrangements of the original liposomes. 5. Conclusions The results of the present investigation show that the solubilization of phospholipid liposomes by lysophospholipids proceeds via the same general sequence of intermediate structures as observed for many conventional surfactants. The structural rearrangements are slow, however, and several days may be needed for equilibration. The cryo-TEM investigations presented in the study provide a guide to the aggregate structures formed in dilute aqueous dispersions of phospholipids, fatty acids, and lysophospholipids. The general trends observed may be satisfactorily explained in light of a balance between the intrinsic phase propensities of the three amphiphilic components. Furthermore, the phase behavior and aggregate structure can be rationalized in terms of simple considerations of geometrical molecular shape. Acknowledgment. Financial support from the Swedish Foundation for Strategical Research, the Swedish Research Council for Engineering Sciences, and the Swedish Cancer Foundation is gratefully acknowledged. We also thank Magnus Thelander, Jan Alsins, and Per Hansson for their assistance with the static and dynamic fluorescence measurements. LA010020U

Aggregate Structure in Dilute Aqueous Dispersions of Phospholipids

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