Structure of Mixed Micelles Formed in PEG-Lipid ... - ACS Publications

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Langmuir 2007, 23, 4192-4198

Structure of Mixed Micelles Formed in PEG-Lipid/Lipid Dispersions Maria C. Sandstro¨m,* Emma Johansson, and Katarina Edwards Department of Physical and Analytical Chemistry, Uppsala UniVersity, Box 579, 751 23 Uppsala, Sweden ReceiVed December 4, 2006. In Final Form: January 30, 2007 Polyethylene glycol (PEG)-conjugated lipids are commonly employed for steric stabilization of liposomes. When added in high concentrations PEG-lipids induce formation of mixed micelles, and depending on the lipid composition of the sample, these may adapt either a discoidal or a long threadlike shape. The factors governing the type of micellar aggregate formed have so far not been investigated in detail. In this study we have systematically varied the lipid composition in lipid/PEG-lipid mixtures and characterized the aggregate structure by means of cryo-transmission electron microscopy (cryo-TEM). The effects caused by adding sterols, phosphatidylethanolamines, and phospholipids with saturated acyl chains to egg phosphatidylcholine/1,2-distearoyl-sn-glycero-3-phosphatidylethanolamine-N[methoxy(polyethylene glycol)-2000 (EPC/DSPE-PEG2000) mixtures with a fixed amount (25 mol %) of DSPEPEG2000 was studied. Further, the aggregate structure in 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine/1,2dimyristoyl-sn-glycero-3-phosphatidylethanolamine-N-[methoxy(polyethylene glycol)-2000] (DMPC/DMPE-PEG2000) samples above and below the gel to liquid crystalline phase transition temperature (TC) was investigated. Our results revealed that lipid components, as well as environmental conditions, that reduce the lipid spontaneous curvature and increase the monolayer bending modulus tend to promote formation of discoidal micelles. At temperatures below the gel-to-liquid crystalline phase transition temperature reduced lipid/PEG-lipid miscibility, furthermore, likely contribute to the observed formation of discoidal rather than threadlike micelles.

Introduction Numerous reports have focused on the intermediate structures formed during vesicle-to-micelle transitions induced by conventional surfactants.1 Examples of common transition structures are open liposomes, perforated bilayers, and cylindrical micelles.2-9 Further, more or less circular bilayer fragments have been predicted on the basis of theoretical arguments10,11 and also documented in several experimental studies.3-5,7-9 The latter structures are normally very short lived; in order to minimize the unfavorable exposure of hydrocarbon chains to the polar environment the fragments tend to fuse and/or close upon themselves. This process appears to be prevented or slowed down, however, in the presence of certain surfactants. Particularly stable bilayer fragments have been found in systems containing polyethylene glycol (PEG)-grafted lipids.12-14 This class of polymer-conjugated lipids have received special attention due to their ability to increase the physical stability and blood circulation time of liposomes intended for drug delivery.15-17 * To whom correspondence should be addressed. Tel: (+46) 18 4713630. Fax: (+46) 18 4713654. E-mail: [email protected]. (1) Almgren, M. Biochim. Biophys. Acta 2000, 1508, 146-163. (2) Gustafsson, J.; Oradd, G.; Almgren, M. Langmuir 1997, 13, 6956-6963. (3) Edwards, K.; Gustafsson, J.; Almgren, M.; Karlsson, G. J. Colloid Interface Sci. 1993, 161, 299-309. (4) Johnsson, M.; Edwards, K. Langmuir 2000, 16, 8632-8642. (5) Edwards, K.; Almgren, M. J. Colloid Interface Sci. 1991, 147, 1-21. (6) Danino, D.; Talmon, Y.; Zana, R. J. Colloid Interface Sci. 1997, 185, 84-93. (7) Walter, A.; Vinson, P. K.; Kaplun, A.; Talmon, Y. Biophys. J. 1991, 60, 1315-1325. (8) Moschetta, A.; Frederik, P. M.; Portincasa, P.; vanBerge-Henegouwen, G. P.; van Erpecum, K. J. J. Lipid Res. 2002, 43, 1046-1053. (9) Silvander, M.; Karlsson, G.; Edwards, K. J. Colloid Interface Sci. 1996, 179, 104-113. (10) Lasic, D. D. Biochim. Biophys. Acta 1982, 692, 501-502. (11) Kozlov, M. M.; Lichtenberg, D.; Andelman, D. J. Phys. Chem. B 1997, 101, 6600-6606. (12) Johnsson, M.; Edwards, K. Biophys. J. 2001, 80, 313-323. (13) Edwards, K.; Johnsson, M.; Karlsson, G.; Silvander, M. Biophys. J. 1997, 73, 258-266. (14) Johnsson, M.; Edwards, K. Biophys. J. 2003, 85, 3839-3847. (15) Woodle, M. C.; Lasic, D. D. Biochim. Biophys. Acta 1992, 1113, 171199.

PEG-grafted distearoyl- and dipalmitoyl-phosphatidylethanolamine (DSPE-PEG, DPPE-PEG) with polymer molecular weights corresponding to 2000 are micelle forming surfactants.18 Consequently, these PEG-lipids are well suited to accumulate at and form a protective hemispherical cap around the hydrophobic rim of a membrane fragment or bilayer disc. Importantly, the large polymeric head-groups effectively prevent the discs from fusion or self-closure. Several studies have addressed the phase behavior in lipid/ PEG-lipid systems,19-25 and a few reports have, furthermore, focused on the structure of the aggregates formed.13,14,19,26 The latter investigations suggest that PEG-lipids induce the formation of open bilayer structures in various phospholipid systems.13,14,26 Interestingly, the nature of the bilayers varies considerably depending on the lipid composition. In egg phosphatidylcholine (EPC)/PEG-lipid systems large irregular bilayer fragments appear as intermediate structures prior to, or concomitantly with, the formation of long, threadlike, cylindrical micelles.13 Smaller and nearly circular discs, perhaps more accurately described as discoidal micelles, forms, on the other hand, when EPC is exchanged for 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (16) Papahadjopoulos, D.; Allen, T. M.; Gabizon, A.; Mayhew, E.; Matthay, K.; Huang, S. K.; Lee, K.-D.; Woodle, M. C.; Lasic, D. D.; Redemann, C.; Martin, F. J. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 11460-11464. (17) Blume, G.; Cevc, G. Biochim. Biophys. Acta 1990, 1029, 91-97. (18) Johnsson, M.; Hansson, P.; Edwards, K. J. Phys. Chem. B 2001, 105, 8420-8430. (19) Arleth, L.; Ashok, B.; Onyuksel, H.; Thiyagarajan, P.; Jacob, J.; Hjelm, R. P. Langmuir 2005, 21, 3279-3290. (20) Bedu-Addo, F. K.; Tang, P.; Xu, Y.; Huang, L. Pharm. Res. 1996, 13, 710-7. (21) Montesano, G.; Bartucci, R.; Belsito, S.; Marsh, D.; Sportelli, L. Biophys. J. 2001, 80, 1372-1383. (22) Belsito, S.; Bartucci, R.; Sportelli, L. Biophys. Chem. 2001, 93, 11-22. (23) Belsito, S.; Bartucci, R.; Montesano, G.; Marsh, D.; Sportelli, L. Biophys. J. 2000, 78, 1420-1430. (24) Kenworthy, A. K.; Simon, S. A.; McIntosh, T. J. Biophys. J. 1995, 68, 1903-1920. (25) Hristova, K.; Kenworthy, A.; McIntosh, T. J. Macromolecules 1995, 28, 7693-7699. (26) Johansson, E.; Engvall, C.; Arfvidsson, M.; Lundahl, P.; Edwards, K. Biophys. Chem. 2005, 113, 183-192.

10.1021/la063501s CCC: $37.00 © 2007 American Chemical Society Published on Web 03/08/2007

Structure of Mixed Micelles Formed in PEG-Lipid/Lipid Dispersions

(DPPC) or 1,2-distearoyl-sn-glycero-3-phosphatidylcholine (DSPC).14 Similarly, the vesicle-to-micelle transition appears to proceed via formation of circular discs, rather than large bilayer fragments and cylindrical micelles, in EPC/PEG-lipid systems supplemented with 40 mol % cholesterol.13,26 The reason for the observed differences in structural behavior has not been explored in detail. Further investigations concerning the aggregate structures formed in lipid/PEG-lipid systems are of value not only from a fundamental point of view. The PEG-stabilized bilayer discs formed in selected lipid systems may find important biotechnical and pharmaceutical applications. Recent studies show, for instance, that these open bilayer structures constitute an interesting, and sometimes superior, alternative to liposomes in studies of drug-membrane interactions.26-28 Further, the stable and welldefined discoidal aggregates have the potential to function well as model membranes in structure/function studies of membranebound proteins and as carriers of hydrophobic/amphiphilic drugs.26,28 In the present study we used cryo transmission electron microscopy (cryo-TEM) to systematically investigate and compare the aggregate structure in lipid/PEG-lipid systems of varying composition and a fixed amount of 25 mol % PEG-lipid. A main purpose of the investigations was to explore how various sample properties, such as bending modulus and spontaneous curvature, affect the structure in the lipid/PEG-lipid mixed systems.

Langmuir, Vol. 23, No. 8, 2007 4193 controlled humidity and temperature (25 °C if not otherwise stated). Immediately after film preparation the grid was plunged into liquid ethane held at a temperature just above its freezing point. The vitrified sample was then transferred to the microscope for analysis. During both the transfer and viewing procedure the temperature was kept below 108 K to prevent ice crystal formation and sample perturbations. A more detailed description of the cryo-TEM procedure can be found elsewhere.29,30 Lipid concentrations were 10 mM except for samples containing DMPC where the concentration was 3 mM.

Results

Materials. Egg phosphatidylcholine (EPC) was obtained from Lipid Products (Nutfield, UK). 1,2-Dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphatidylcholine (DSPC), 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC), 1,2-distearoyl-sn-glycero-3-phosphatidylethanolamine (DSPE), 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC), 1-stearoyl2-hydroxy-sn-glycero-3-phosphatidylcholine (MSPC), 1,2-distearoylsn-glycero-3-phosphatidylethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG2000), 1,2-dimyristoyl-sn-glycero-3-phosphatidylethanolamine-N-[methoxy(polyethyleneglycol)-2000](DMPEPEG2000), and 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamineN-[methoxy(polyethylene glycol)-2000] (DOPE-PEG2000) were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL). Cholesterol and Lanosterol were purchased from Sigma-Aldrich (Stockholm, Sweden). Octyl glucoside was obtained from Dojindo laboratories (Kumamoto, Japan) and Sephadex G50 from Amersham Biosciences (Uppsala, Sweden). Minicon-B15 concentrators were from Millipore (Bedford, MA). Spectra/Por 7 dialysis membrane was from SigmaAldrich (Stockholm, Sweden). All other salts and reagents were of analytical grade and were used as received. Sample Preparation. Lipids in desired amounts were co-dissolved in chloroform, and the solvent was thereafter removed in a gentle stream of N2 gas followed by further evaporation in vacuum over night. The dried lipid films were hydrated in HEPES-buffer (20 mM HEPES, 150 mM NaCl, pH 7.4) at 60 °C (80 °C for mixtures containing DSPE) for ∼30 min with intermittent mixing. Cryo-Transmission Electron Microscopy. Cryo-TEM images were obtained by use of a Zeiss EM 920 A transmission electron microscope (Carl Zeiss, Inc., Oberkochen, Germany) operating at 80 kV. A small drop of sample was placed on a copper grid coated with a perforated polymer film. Excess solution was thereafter removed by means of blotting with a filter paper. This procedure was performed in a custom-built environmental chamber under

EPC/Cholesterol/DSPE-PEG2000 Preparations. Figure 1a shows the aggregate structure in EPC/DSPE-PEG2000 (75:25 mol %) dispersions without cholesterol. The micrograph reveals unilamellar liposomes, ∼50-400 nm in diameter, in coexistence with micelles. The latter vary from small spherical and slightly elongated structures to long threadlike cylindrical aggregates. In addition to liposomes and micelles, large irregular bilayer flakes with edges growing into threadlike micelles were frequently observed in the cholesterol-free samples. Addition of 10 mol % cholesterol drastically reduced the amount, as well as the size, of the liposomes (Figure 1b). Further, long threadlike micelles were no longer observed and the short cylindrical micelles seemed to have adapted a more flattened shape. Irregular bilayer flakes were still present but did not display any threadlike protrusions. Upon addition of 20 mol % cholesterol a significant amount of circular or nearly circular discs appeared in the sample (Figure 1c). In addition to the discs, cryo-TEM revealed some large irregular flakes and a small population of liposomes. The flat thin “ribbons” observed at lower cholesterol content could, however, not be detected. Samples containing 30 mol % cholesterol were clearly dominated by circular discs in the size range 20-50 nm (Figure 1d). A small amount of larger circular discs, 150-300 nm in diameter, could also be observed, as well as a small number of liposomes and large irregular bilayer flakes. At a cholesterol content of 40 mol % the sample contained circular discs ranging from a few nanometers to ∼200 nm in diameter (Figure 1e). The large irregular flakes were absent, but a small population of unilamellar liposomes could still be found. For reference, the effect of cholesterol on aggregate structure in pure EPC samples was also investigated (results not shown). Samples containing up to 60 mol % cholesterol appeared homogeneous to the naked eye, and cryo-TEM investigations confirmed that liposomes of varying size and lamellarity constituted the only aggregates present. Visual inspection of samples containing 80 mol % cholesterol revealed, however, the presence of crystalline material. The crystals were too large to be captured by cryo-TEM, and only uni- and multilamellar liposomes were displayed in the micrographs. EPC/Lanosterol/DSPE-PEG2000 Preparations. The effect of lanosterol on pure EPC samples was first investigated. Inclusion of 20 mol % lanosterol had no visible effect on the aggregate structure (Figure 2a). The lanosterol-containing mixtures displayed, similar to pure EPC preparations (results not shown), intact liposomes with a broad size distribution. Upon increasing the amount of lanosterol to 40 mol % a new type of structures appeared in the samples. As shown in Figure 2b, bilayer bands (several micrometers long and ∼100-500 nm wide) were observed in coexistence with the liposomes. Samples containing 60 mol % lanosterol appeared inhomogeneous and large crystals

(27) Boija, E.; Lundquist, A.; Nilsson, M.; Isaksson, R.; Edwards, K.; Johansson, G. Manuscript in preparation. (28) Johansson, E.; Lundquist, A.; Zuo, S.; Edwards, K. Biochim. Biophys. Acta Submitted for publication.

(29) Almgren, M.; Edwards, K.; Karlsson, G. Colloids Surf. A 2000, 174, 3-21. (30) Dubochet, J.; Adrian, M.; Chang, J. J.; Homo, J. C.; Lepault, J.; McDowall, A. W.; Schultz, P. Q. ReV. Biophys. 1988, 21, 129-228.

Materials and Methods

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Figure 1. Cryo-TEM images of dispersions of (a) EPC/DSPE-PEG2000 (75:25 mol %), (b) EPC/cholesterol/DSPE-PEG2000 (65:10:25 mol %), (c) EPC/cholesterol/DSPE-PEG2000 (55:20:25 mol %), (d) EPC/cholesterol/DSPE-PEG2000 (45:30:25 mol %), and (e) EPC/cholesterol/ DSPE-PEG2000 (35:40:25 mol %). Scale bars indicate 100 nm.

Figure 2. Cryo-TEM images of samples containing (a) EPC/ lanosterol (80:20 mol %) and (b) EPC/lanosterol (60:40 mol %). Scale bars indicate 100 nm.

were visible to the naked eye. Cryo-TEM pictures showed for this sample a large amount of bilayer ribbons, together with liposomes and a few helical structures (compare helices in Figure 3b), (results not shown).

Figure 3. Cryo-TEM images of samples containing (a) EPC/ lanosterol/DSPE-PEG2000 (64:11:25 mol %) and (b) EPC/lanosterol/ DSPE-PEG2000 (35:40:25 mol %). Scale bars indicate 100 nm.

In sample preparations containing EPC/lanosterol/DSPEPEG2000 (64:11:25 mol %) small slightly enlongated micelles were found together with discs of varying size, many of which were circular in shape (Figure 3a). Flattened “cylinders” and large irregular bilayer flakes with extensions growing out from the edges were also present in the sample. Samples with 40 mol

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micellar extensions. Moreover, the sample contained a large population of slightly flattened threadlike micelles and numerous small circular or rise-shaped aggregates (Figure 6a). Pure DSPC/ DSPE-PEG2000 (75:25 mol %) samples displayed an aggregate structure similar to that observed for DSPE/DSPE-PEG2000 (75: 25 mol %) samples (Figure 6b). Aggregate Structure above and below the Gel-to-Liquid Crystalline Phase Transition Temperature. DMPC/DMPEPEG2000 samples equilibrated at 30 °C displayed mainly long threadlike cylindrical micelles (Figure 7a). Upon lowering the temperature to 5 °C the threadlike micelles disappeared, however, and the sample components rearranged into small discoidal aggregates (Figure 7b). In order to explore whether the structural change was reversible the sample was reheated to 30 °C, equilibrated for 24 h, and thereafter re-examined by cryo-TEM. The micrographs obtained after this temperature cycle were similar to that shown in Figure 7b and did not display any threadlike micelles (result not shown).

Discussion Figure 4. Cryo-TEM images of samples containing (a) DOPC/ DSPE-PEG2000 (75:25 mol %) and (b) DOPC/cholesterol/DSPEPEG2000 (45:30:25 mol %). Scale bars indicate 100 nm.

% lanosterol (EPC/Lanosterol/DSPE-PEG2000, 35:40:25 mol %) were dominated by circular discs with diameters in the range of 20-40 nm. As seen from the micrograph presented in Figure 3b, these samples also contained a fraction of long ribbons twisted into helical structures. DOPC/Cholesterol/DOPE-PEG2000 Preparations. DOPC/ DOPE-PEG2000 (75:25 mol %) samples contained threadlike micelles, liposomes, and both irregular and circular bilayer flakes (Figure 4a). DOPC/cholesterol/DOPE-PEG2000 (45:30:25 mol %) mixtures contained liposomes and a large amount of circular discs (Figure 4b). However, no threadlike micelles were displayed in the micrographs. EPC/DOPE/DSPE-PEG2000 Preparations. Inclusion of DOPE in the EPC/PEG-lipid mixtures reduced the number of threadlike micelles but did not lead to the formation of circular discs. As shown in Figure 5a, large irregular bilayers displaying threadlike protrusions were still frequently observed in samples containing as much as 54 mol % DOPE. Moreover, electron-dense particles characteristic of inverted lipid phase were occasionally observed in the samples (see inset of Figure 5a). EPC/DSPE/DSPE-PEG2000 and EPC/DSPC/DSPE-PEG2000 Preparations. As shown in Figure 5b, samples composed of EPC/DSPE/DSPE-PEG2000 (46:29:25 mol %) displayed numerous close-to-circular bilayer discs. In addition, the micrograph reveals some irregular bilayer flakes. Importantly, the latter display no threadlike protrusions, and no threadlike micelles were detected free in solution. The structural appearance was similar in samples containing 40 mol % DSPE (Figure 5c). In preparations composed of DSPE/DSPE-PEG2000 (75:25 mol %) circular discs with sizes ranging from a few nanometers up to around 200 nm in diameter constituted the only structures detected by cryo-TEM (Figure 5d). The aggregate structure changed markedly upon inclusion of lysolipids in the lipid mixture. As shown in Figure 5e threadlike micelles were frequently observed in EPC/DSPE/DSPE-PEG2000 (45:30:25 mol %) samples supplemented with 10 mol % monostearoylphosphatidylcholine (MSPC). Cryo-TEM investigation of EPC/DSPC/DSPE-PEG2000 (45: 30:25 mol %) revealed a polydisperse sample structure. Liposomes of varying size coexisted with bilayer flakes that exposed long

The PEG-conjugated lipid DSPE-PEG2000 has high positive spontaneous (or “intrinsic”) curvature and aggregates in aqueous solution into, more or less, spherical micelles.18,19 Similar to other micelle-forming surfactants, DSPE-PEG2000 has limited solubility in lipid bilayers. The bilayer saturation concentration varies depending on lipid composition, but lies typically in the range from about 4 to 10 mol %.13,14,21,22 PEG-lipid concentrations above this limiting value induce the formation of lipid/PEGlipid mixed micelles. Previous studies have shown that two fundamentally different types of mixed micelles may form as the concentration of PEG-lipid is increased above the bilayer saturation concentration. Accordingly, investigations based on cryo-TEM and different scattering techniques have revealed long, threadlike, cylindrical micelles in mixtures of EPC and DSPEPEG200013,14,19 whereas discoidal structures have been disclosed in mixtures of DPPC or DSPC and DSPE-PEG2000.13,14 Discoidal, rather than threadlike, micelles have furthermore been observed in EPC/DSPE-PEG2000 systems supplemented with 40 mol % cholesterol.13 The discoidal micelles, which most likely form as a consequence of partial component segregation and accumulation of PEG-lipids at the highly curved rim, are well described by an ideal disc model.14 The average diameter of the discoidal micelles increases with increasing lipid/PEG-lipid ratio and the large discs formed in samples with low PEG-lipid content are perhaps more accurately described as circular bilayer flakes. In both threadlike and disc-shaped mixed micelles the PEGlipids are permitted to associate into monolayers with high positive curvature. Irrelevant of whether cylindrical or discoidal aggregates form, the transformation into mixed micelles reduces the curvature free energy for the PEG layer. Formation of discoidal micelles is, however, associated with a significant energy penalty in the form of reduced mixing entropy. Therefore, discs should be favored over threadlike micelles only when the entropic loss due to component segregation is small compared to the energetic costs associated with forcing all the components in the lipid/ PEG-lipid mixture into the highly curved monolayer of a cylindrical micelle. On the basis of the above arguments, it appears logical that sample components, or environmental conditions, that reduce lipid/PEG-lipid miscibility should increase the tendency for disc formation. Further, properties of the lipid mixture such as monolayer bending modulus and spontaneous curvature should have a decisive influence on whether threadlike or discoidal mixed micelles form in the system. Effect of Cholesterol and Lanosterol. Cryo-TEM results presented in Figure 1 show that inclusion of cholesterol has a

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Figure 5. Cryo-TEM images of dispersions of (a) EPC/DOPE/DSPE-PEG2000 (21:54:25 mol %), inset show a particle with inverted structure, (b) EPC/DSPE/DSPE-PEG2000 (46:29:25 mol %), (c) EPC/DSPE/DSPE-PEG2000 (35:40:25mol%), (d) DSPE/DSPE-PEG2000 (75:25 mol %), (e) EPC/DSPE/DSPE-PEG2000/MSPC (40.5:26.8:22.7:10 mol %). Scale bars indicate 100 nm.

Figure 7. Cryo-TEM images of preparations of (a) DMPC/DMPEPEG2000 (75:25 mol %) at 30 °C and (b) DMPC/DMPE-PEG2000 (75:25 mol %) at 5 °C. Scale bars indicate 100 nm. Figure 6. Cryo-TEM images of samples containing (a) EPC/DSPC/ DSPE-PEG2000 (45:30:25 mol %) and (b) DSPC/DSPE-PEG2000 (75: 25 mol %). Scale bars indicate 100 nm.

prominent effect on the aggregate structure in mixtures of EPC and DSPE-PEG2000. The long threadlike micelles frequently observed in pure EPC/DSPE-PEG2000 mixtures are not detected in samples containing 10 mol %, or more, cholesterol. Further, with increasing cholesterol content the number of liposomes decreases and the bilayer fragments gradually adapt a more circular shape. Cholesterol is well known to increase lipid chain order when added to phosphocholine lipid bilayers in the liquid crystalline state. The chain order increases progressively with cholesterol content, and at concentrations around 25 mol % the sterol

eventually induces a transition from the liquid-disordered (ld) to the liquid-ordered (lo) phase.31-33 PEG-lipids could in theory be less soluble in the liquid ordered than in the liquid disordered phase. Importantly, lipid-cholesterol interactions take part mainly in the acyl chain region of the membrane, and possibly also involve hydrogen bonding in the vicinity of the hydrocarbon-water interface.34 It is hard to see how a hydrophilic very flexible PEG-polymer attached to the (31) Vist, M. R.; Davis, J. H. Biochemistry 1990, 29, 451-464. (32) Nielsen, M.; Thewalt, J.; Miao, L.; Ipsen, J. H.; Bloom, M.; Zuckermann, M. J.; Mouritsen, O. G. Europhys. Lett. 2000, 52, 368-374. (33) Mcmullen, T. P. W.; Mcelhaney, R. N. Biochim. Biophys. Acta 1995, 1234, 90-98. (34) Bhattacharya, S.; Haldar, S. Biochim. Biophys. Acta 2000, 1467, 39-53.

Structure of Mixed Micelles Formed in PEG-Lipid/Lipid Dispersions

distal end of the polar lipid headgroup would interfere with the lipid-cholesterol interactions. Reduced PEG-lipid/lipid miscibility in the lo phase would therefore only be expected in cases where the PEG-lipid anchor has a structure that is significantly different from that of the PC component. As shown in Figure 4, inclusion of cholesterol prevents the formation of threadlike micelles also in the DOPC/DOPE-PEG2000 system. Cholesterol is in this system not expected to have any important influence on lipid/PEG-lipid miscibility, and therefore reduced miscibility cannot explain why the sterol promotes component segregation and disc formation when added to the lipid/PEG-lipid mixture. A more plausible explanation for the observed behavior may be found if we consider the curvature-reducing effect of cholesterol. Simple geometrical considerations indicate that cholesterol is not well suited to associate into structures with positive curvature. In line with this, previous studies have shown that increasing concentrations of cholesterol progressively increase the bending modulus of phosphocholine membranes35 and, as expected, decrease phospholipid membrane curvature.36 The fact that cholesterol prevents formation of highly curved structures, such as threadlike micelles, is thus not suprising. The micrographs displayed in Figure 1 reveal that cholesterol, apart from inhibiting the formation of threadlike micelles, affects the shape and structure of the bilayer aggregates present in the mixtures. With increasing cholesterol content the large, open bilayer fragments become less irregular and tend toward a more circular shape. Since a perfectly circular shape minimizes the circumference of the fragments, the observed trend indicates that cholesterol tends to reduce the edge area of the individual fragments. In other words, the fraction of lipids situated in the “flat” part of the large fragments increases with increasing cholesterol/EPC ratio. Again, this fits nicely with the expected curvature-reducing effect of cholesterol. Moreover, the fact that cholesterol increases bending modulus of lipid membranes35,36 helps explain why the number of liposomes found in the samples decreases upon inclusion of cholesterol. Lanosterol, an evolutionary precursor to cholesterol,37,38 is similar to cholesterol known to increase lipid chain order in liquid crystalline membranes.39,40 The ordering effect of lanosterol is, however, markedly less than that of cholesterol. In line with this, several investigations have suggested that lanosterol is unable to promote and stabilize a pure liquid ordered phase.39-43 Recent measurements show, furthermore, that the effect of lanosterol and cholesterol on lipid chain order is closely correlated to their impact on bending rigidity.44 More specifically, both sterols increase the bending modulus of 1-palmitoyl-2-oleoyl-sn-glycero3-phosphatidylcholine membranes but the effect of cholesterol is more pronounced than that of lanosterol.35,44 Cryo-TEM results obtained in the present study reveal that the two sterols have a comparable effect on the aggregate structure in mixtures of EPC and DSPE-PEG2000. That is, lanosterol prevents formation of threadlike micelles and promotes disc formation to at least the same extent as documented for cholesterol (compare Figure 1 and 3). This fact suggests that bending modulus does not play a major role in determining the shape of the aggregates formed. (35) Henriksen, J.; Rowat, A. C.; Ipsen, J. H. Eur. Biophys. J. 2004, 33, 732741. (36) Chen, Z.; Rand, R. P. Biophys. J. 1997, 73, 267-276. (37) Bloch, K. E. CRC Crit. ReV. Biochem. 1983, 14, 47-92. (38) Bloch, K. Science 1965, 150, 19-28. (39) Mouritsen, O. G.; Zuckermann, M. J. Lipids 2004, 39, 1101-1113. (40) Miao, L.; Nielsen, M.; Thewalt, J.; Ipsen, J. H.; Bloom, M.; Zuckermann, M. J.; Mouritsen, O. G. Biophys. J. 2002, 82, 1429-1444. (41) Dahl, C. E.; Dahl, J. S.; Bloch, K. Biochemistry 1980, 19, 1462-1467. (42) Urbina, J. A.; Pekerar, S.; Le, H. B.; Patterson, J.; Montez, B.; Oldfield, E. Biochim. Biophys. Acta 1995, 1238, 163-176. (43) Xu, X.; London, E. Biochemistry 2000, 39, 843-849. (44) Henriksen, J.; Rowat, A. C.; Brief, E.; Hsueh, Y. W.; Thewalt, J. L.; Zuckermann, M. J.; Ipsen, J. H. Biophys. J. 2006, 90, 1639-1649.

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Rather, the propensity for disc formation appears to be modulated mainly by the change in lipid spontaneous curvature brought about by inclusion of the sterols. Data showing that lanosterol and cholesterol have a similar impact on spontaneous curvature are needed, however, in order to verify this hypothesis. The presence of helical bilayer bands in samples with high lanosterol content (Figure 3b) deserves some attention. Helical ribbons are well known to form as intermediates during cholesterol crystallization in bile45 and have, furthermore, been identified in a range of systems containing a micelle-forming surfactant, phosphatidylcholine, and a sterol.46 It is possible that the helical structures observed in the present study constitute precursors to the helical ribbons of much larger dimensions observed in previous studies. Should this be the case, it is noteworthy that helices were observed also in the pure EPC/lanosterol system, i.e., the presence of a micelle-forming component does not seem to be necessary. Effect of PE-Lipids. The results obtained with cholesterol and lanosterol suggest that curvature-reducing components other than sterols could prevent formation of threadlike micelles and perhaps promote formation of discoidal aggregates in mixtures of EPC and DSPE-PEG2000. The phospholipid DOPE has negative spontaneous radius of curvature47 and forms at 25 °C an inverted hexagonal (HII) phase.48 We hypothesized that DOPE, similar to cholesterol and lanosterol, would decrease the spontaneous curvature of the EPC/PEG-lipid mixture and thereby promote formation of discoidal structures. Our cryo-TEM investigations showed, however, that this was not the case. As shown in Figure 5a, irregular bilayer flakes with threadlike protrusions were observed in samples containing as much as 54 mol % DOPE. Moreover, dense particles likely representing HII phase were occasionally observed in the EPC/DOPE/DSPE-PEG2000 mixtures. The presence of these structures suggests that the lipid components are not evenly distributed in the sample. Instead, the HII particles likely consist of, more or less, pure DOPE whereas EPC and PEG-lipid constitute the major components in the lamellar and micellar aggregates. The structural behavior changed markedly when DOPE was exchanged for DSPE. As shown in Figure 5b, formation of threadlike micelles was now clearly inhibited and the overall aggregate structure resembled that observed for mixtures containing cholesterol (compare Figure 1d). DSPE differs from DOPE in that its two hydrocarbon chains are fully saturated. The absence of double bonds increases the chain melting temperature and, consequently, the liquid crystalline LR and HII phases formed by DSPE are stable only at high temperatures (above 74 and 100 °C, respectively).49 All samples depicted in Figure 5 were quenched from 25 °C and at this temperature DSPE is, in contrast to DOPE, not expected to drive lipid assemblies into structures with net negative curvature. Inclusion of DSPE, which has a small polar headgroup compared to EPC, would nevertheless tend to reduce the spontaneous curvature for the lipid mixture. It is in this context important to note that the cryo-TEM images disclosed threadlike micelles in EPC/DSPE/DSPE-PEG2000 samples supplemented with MSPC (Figure 5e). The inclusion of PC lysolipids, which have high positive curvature,47 obviously counteracts the curvature reducing effect of DSPE, and formation of threadlike micelles thus again becomes energetically favorable. (45) Donovan, J. M.; Carey, M. C. Gastroenterol. Clin. N. Am. 1991, 20, 47-66. (46) Zastavker, Y. V.; Asherie, N.; Lomakin, A.; Pande, J.; Donovan, J. M.; Schnur, J. M.; Benedek, G. B. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 7883-7887. (47) Fuller, N.; Rand, R. P. Biophys. J. 2001, 81, 243-254. (48) Ellens, H.; Bentz, J.; Szoka, F. C. Biochemistry 1986, 25, 285-294. (49) Cevc, G. Phospholipids handbook; Marcel Dekker, Inc.: New York, 1993.

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This observation constitutes further evidence for the impact of spontaneous curvature on aggregate shape. Noteworthy, EPC/DSPE/DSPE-PEG2000 (35:40:25 mol %) samples displayed a population of irregularly shaped discs in coexistence with the more circular bilayer objects (Figure 5c). When EPC was fully replaced by DSPE the vast majority of discs adapted, however, a close to perfectly circular shape (Figure 5d). It is here interesting to recall that the cryo-TEM analysis was carried out well below TC of DSPE. This fact likely contributes to the almost perfectly circular shape of the discs captured by cryo-TEM in the pure DSPE/DSPE-PEG2000 system (see further discussion below). Temperature Effects on Disc Formation. Results presented in Figure 6a show that inclusion of DSPC has little effect on the aggregate structure in EPC/DSPE-PEG2000 mixtures. Thus, threadlike micelles formed readily in samples containing 30 mol % DSPC, and the overall aggregate structure was similar to that observed for pure EPC/DSPE-PEG2000 samples. The hydrophobic moieties of DSPC and DSPE are identical, and the disc-promoting effect in EPC mixtures of the latter lipid thus stems from the properties of the PE headgroup rather than the saturated nature of the hydrocarbon chains. The result presented in Figure 6b shows, however, that discoidal rather than threadlike micelles form in the pure DSPC/PEGlipid system. Importantly, the discoidal structures were documented at 25 °C, i.e. at a temperature well below TC for DSPC. The bending modulus for phospholipid membranes is known to be about 10-fold higher in the gel compared to the liquid crystalline

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phase.50 Further, lipid/PEG-lipid miscibility is presumably reduced in the gel phase, and the energy loss due to component segregation is less at a lower temperature. Taken together these effects help explain why discs formed at temperatures below TC. In line with this reasoning the threadlike micelles observed at 30 °C in DMPC/DMPE-PEG2000 mixtures were found to transform into discoidal structures upon cooling of the sample to 5 °C (Figure 7), i.e. safely below TC (23.5 °C49). Noteworthy, the discoidal structure remained after reheating of the sample to 30 °C. Once formed the discs thus appear stabilized against the fusion and rearrangement events needed to convert them into threadlike micelles.

Conclusions The results presented in this study show that discoidal structures are preferred over cylindrical micelles when the lipid mixture contains components that reduce the spontaneous curvature and increase the monolayer bending modulus. Such components are, e.g., cholesterol, lanosterol, and DSPE. Discoidal structures are furthermore preferred at temperatures below TC of the lipid mixture. In this case, disc formation is likely promoted by a combination of high bending modulus and reduced lipid/PEGlipid miscibility. Acknowledgment. Financial support from The Swedish Research Council, and O. E. and Edla Johansson Science Foundation are gratefully acknowledged. LA063501S (50) Mecke, K. R.; Charitat, T.; Graner, F. Langmuir 2003, 19, 2080-2087.