Article pubs.acs.org/Langmuir
Cholesteryl Phosphocholine − A Study on Its Interactions with Ceramides and Other Membrane Lipids Max Lönnfors,† Otto Långvik,2 Anders Björkbom,† and J.Peter Slotte†,* † 2
Biochemistry, Department of Biosciences, Åbo Akademi University, Turku, Finland, and Laboratory of Organic chemistry, Department of Natural Sciences, Åbo Akademi University, Turku, Finland S Supporting Information *
ABSTRACT: We prepared cholesteryl phosphocholine (CholPC) by chemical synthesis and studied its interactions with small (ceramide and cholesterol) and large headgroup (sphingomyelin (SM) and phosphatidylcholine) colipids in bilayer membranes. We established that CholPC could form bilayers (giant uni- and multilamellar vesicles, as well as extruded large unilamellar vesicles) with both ceramides and cholesterol (initial molar ratio 1:1). The extruded bilayers appeared to be fluid, although highly ordered, even when the ceramide had an N-linked palmitoyl acyl chain. In binary systems containing CholPC and either palmitoyl SM or 1,2-dipalmitoyl-sn-glycero-3-phospholine, CholPC markedly destabilized the gel phase of the respective large headgroup lipid. In 1palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayers, CholPC was much less efficient than cholesterol in ordering the acyl chains. In complex bilayers containing POPC and cholesterol or palmitoyl ceramide, CholPC appeared to prefer interacting with the small headgroup lipids over POPC. When the degree of order in CholPC/PCer bilayers was compared to Chol/PSM bilayers, CholPC/PCer bilayers were more disordered (based on DPH anisotropy). This finding may result from different headgroup orientation and dynamics in CholPC and PSM. Our results overall can be understood if one takes into account the molecular shape of CholPC (large polar headgroup and modest size hydrophobic part) when interpreting molecular interactions between small and large headgroup colipids. The results are also consistent with the proposed umbrella model” for explaining cholesterol/colipid interactions.
1. INTRODUCTION Interactions of cholesterol with phospholipids in bilayer membranes are markedly affected by the headgroup properties of the phospholipid.1−3 Cholesterol has a relative preference for interacting with phospholipids having large head groups (e.g., phosphocholine) in both saturated sphingomyelin (SM) and phosphatidylcholine, whereas interactions with comparable molecules having increasingly smaller head groups (e.g., phosphatidylethanolamine and ceramide phosphoethanolamine) are less favored.1 When the methyl groups in the phosphocholine headgroup of SM were removed one at a time, the bilayer affinity of sterol decreased linearly with headgroup demethylation.4 It has been suggested that the phospholipid headgroup can act as an umbrella, shielding the underlying cholesterol molecule from unfavorable interactions with water, and thus increasing the membrane affinity of the sterol molecule.5−8 Cholesterol solubility studies in bilayer membranes also indicate that a bigger headgroup on the phospholipid allows for higher solubility compared to a situation with a smaller headgroup phospholipid.1 Ceramide is a hydrophobic sphingolipid having two free hydroxyls in the interfacial region of the molecule, one in position 1 (analogous to cholesterolś 3-hydroxyl), and one on carbon 3 in the long-chain base.9 Ceramide associates with saturated SMs, and is able to displace cholesterol from interacting with SM,10−12 suggesting that ceramide has a © 2013 American Chemical Society
higher affinity for saturated SM compared to cholesterol. It is safe to assume that ceramide, in a similar manner as cholesterol, needs protection from unfavorable exposure to water by the large headgroup of colipids. Indeed, Ali and co-workers showed that the maximum solubility of brain ceramides in 1-palmitoyl2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayers was 68 ± 2%,13 which is similar to the solubility limit of cholesterol in POPC (67 ± 1%). It is unclear how sensitive ceramide/ phospholipid interactions are to variations in headgroup size or properties because experimental data is lacking in the scientific literature. Both ceramide and cholesterol have small polar head groups making their mutual interactions unfavorable in the absence of large headgroup phospholipids. Other features in their molecular structure may also make ceramide/cholesterol interaction less favorable, such as the irregular beta-face of the sterol ring structure, and the extensive hydrogen-bonding network formed among ceramides. However, when ceramide gains a phosphocholine headgroup (becoming SM), their mutual interactions immediately become thermodynamically feasible. How would the interaction of ceramide with cholesterol change, if cholesterol gained a large phosphocholine Received: November 8, 2012 Revised: January 27, 2013 Published: January 28, 2013 2319
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residues of the product were identified. 1H NMR (600.13 MHz, CD3OD, 25 °C): 3.22 (s, 9 H); 3.60 − 3.64 (m, 2 H); 3.93 − 4.01 (m, 1 H); 4.22 − 4.27 (m, 2 H); 5.36 ± 5.39 (m, 1 H) ppm. 13C NMR (150.92 MHz, CD3OD, 25 °C): 54.7 (JC−N = 3.8 Hz); 60.3 (d, JC−P = 5.0 Hz); 67.4−67.6 (m); 77.2 (d, JC−P = 5.8 Hz); 123.2; 141.7 ppm. 31P NMR (242.93 MHz, CD3OD, 25 °C): −1.28 ppm. ESI-MS (MeOH): 574.45 [M+Na]+). 2.2. Preparation of Vesicles. Multilamellar lipid vesicles (MLVs) used in the study were prepared as described previously,20 to a lipid concentration of 0.05 for fluorescence studies, or to about 1.4 mM for DSC studies. Briefly, lipids from organic stock solutions were pipetted into glass tubes and mixed thoroughly before evaporation of the solvent under a stream of inert gas. The samples were kept under high vacuum for 1 h. Hydration was done with ultrapure water at 50 °C for 30 min (intermittent mixing with vortex). The samples were then subjected to a 10 min bath sonication at 50 °C. Extruded large unilamellar vesicles (LUVs) were prepared from CholPC and the indicated colipid by extrusion through filters with 200 nm pore size. Briefly, MLVs of the desired lipid compositions were extruded 11x at 50 °C with an Avanti syringe extruder. The size of the resulting LUVs was determined using a Malvern zeta-sizer instrument. Electroformed giant vesicles from CholPC and colipids (1:1 initial molar ratio) were prepared as described previously.21 In brief, 100 nmol of lipids with DiIC20 at a 1:400 molar ratio to lipids, were mixed and dissolved in 200 μL chloroform/ methanol (2:1 vol). The lipid mix was deposited (2.5 microL) on Pt wires in a GUV preparation chamber. The chamber was dried in vacuum for 1 h. The lipids on the Pt wires were hydrated with argon-purged buffer (10 mM HEPES, pH 7.4, 50 mM sucrose) at 50 °C. Electroformation was done at the following conditions: 50 Hz and 5 Vpp for 2 min at 50 °C, followed by 10 Hz and 3 Vpp for 90 min. The giant vesicles were slowly cooled to 23 °C (over a period of 3 h on a programmable termoblock). Visualization was performed with a Zeiss LSM 780 mounted on an inverted AxioObserver Z1 stand (Carl Zeiss Inc., Jena, Germany) and a Zeiss LSM510 META (Carl Zeiss Inc., Jena, Germany) with excitation at 488 nm and emission above 600 nm. 2.3. Determination of Steady-State DPH Anisotropy. The steady-state fluorescence anisotropy of DPH was measured in binary phospholipid/CholPC MLVs as described previously.15 Briefly, lipids and DPH were mixed, and the organic solvent evaporated under a stream of inert gas. Samples were hydrated with ultrapure water at 50 °C for 30 min with intermittent mixing. Samples were also subjected to a 5 min bath sonication at 50 °C before analysis. The DPH concentration was 1 mol %, and the temperature gradient during temperature ramping was 1 °C/min. 2.4. Differential Scanning Calorimetry. MLVs prepared from indicated amounts of phospholipids and CholPC were analyzed using a VP-DSC instrument (GE Healthcare Life Sciences). The temperature gradient was 1 °C/min, three upand down-scans were performed. 2.5. c-Laurdan Emission Spectra. c-Laurdan emission from extruded LUVs was measured at 23 °C (Ex 360 nm/ Em400−550 nm). c-Laurdan was included at 1 mol %, and measurements were performed at the indicated temperatures. The GP was calculated as follows: GP = (I440 − I480)/(I440 + I480). 2.6. Fluorescence Quenching Measurements. The fluorescence quenching of tPA by the quencher 7SLPC in
headgroup? A clue to this question was recently given in a study where cholesteryl-3-beta-phosphocholine (CholPC) was shown to form apparently stable bilayers with both dimyristoylglycerol and free cholesterol.14 It would be of interest to extend such a study to also include ceramides, since cholesterol/ceramide interaction in a system where cholesterol would carry the phosphocholine headgroup might reveal interesting information about their mutual interactions. Solubilizing ceramide with CholPC into stable bilayers could also prove to be a useful formulation by which ceramides could be transferred to cells in a solvent-free system. In this study, we have prepared CholPC by chemical synthesis, and studied its biophysical properties in binary and ternary bilayer systems. We have examined interactions with ceramides, and with saturated and unsaturated phospholipids. We found that CholPC formed giant uni- and multilamellar vesicles with both saturated and unsaturated ceramides, and also with cholesterol. Similarly, large unilamellar vesicles (initial 1:1 stoichiometry) could also be made by extrusion. Whereas CholPC could interact with small headgroup colipids (i.e., cholesterol or ceramides) in complex bilayers, interactions with large headgroup phospholipids were not favored.
2. MATERIALS AND METHODS 2.1. Material. High-purity POPC, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1-palmitoyl-2-(7-doxyl)stearoyl-sn-glycero-3-phosphocholine (7SLPC), N-oleoyl-Derythro-ceramide (OCer), N-palmitoyl-D-erythro-ceramide (PCer), and egg SM (99%+) were purchased from Avanti Polar Lipids (Alabaster, AL, USA) and used without further purification. DiIC20 was obtained from Molecular Targeting Technologies Inc. (USA). N-palmitoyl-D-erythro-sphingomyelin (PSM) was isolated to 99% purity from egg SM as described. 15 Cholesterol (99% pure) was from Sigma Chemicals (St. Louis, MO, USA). Stock solutions of phospholipids were prepared in methanol, whereas sterols and ceramides were dissolved in hexane/isopropanol (3:2 by vol), and kept at −20 °C. All lipid solutions were taken to ambient temperature before use. The concentration of all phospholipid solutions was determined by phosphate assay, subsequent to total digestion by perchloric acid. Diphenyl hexa-1,3,5-triene (DPH) was from Molecular Probes (Leiden, The Netherlands). c-Laurdan was a kind gift from professor Bong Rae Cho (Korea University) and was synthesized as described previously.16 trans-Parinaric acid (tPA) was synthesized in house from the methyl ester of alphalinolenic acid, as described by Kuklev and Smith.17 Fluorescent probes were stored under argon at −87 °C until dissolved in argon-purged methanol. The concentration of stock solutions of the fluorophores was determined with spectroscopy using their molar absorption coefficients (ε) values: 70 000 M−1cm−1 at 302 nm for tPA, 88 000 M−1cm−1 at 350 nm for DPH, and 20 000 M−1cm−1 at 365 nm for c-laurdan. Stock solutions of fluorescent reporter molecules were stored at −20 °C and used within a week. Water was purified by reverse osmosis followed by passage through a Millipore UF Plus water purification system having final resistivity of 18.2 MΩcm. CholPC was synthesized as described in refs 14, 18, and 19 with only minor practical modifications. CholPC was purified (mostly from unreacted cholesterol) by reverse-phase preparative HPLC (Supelco Discovery C18 column) using methanol as eluent.15 The identity of CholPC was verified by NMR and ESI-MS. Very distinct NMR signals from the phosphocholine 2320
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Figure 1. Giant vesicles made from CholPC and ceramide or cholesterol. CholPC with PCer (1:1 initial molar ratio, panels A and B); with OCer (1:1 initial molar ratio, panels C and D); or with cholesterol (1:1 initial molar ratio, panels E and F) formed giant vesicles, which were stable for hours but differed in size distribution and heterogeneity (scale bar is 10 µm). The brightness and contrast of each panel was slightly improved using Corel Photo-Paint X5.
(however, we did not perform systematic size measurements) than the CholPC/OCer vesicles (parts C and D of Figure 1), and many of the latter vesicles had complex internal structures. CholPC also formed giant uni- and multilamellar vesicles with cholesterol (parts E and F of Figure 1). To prepare LUVs, CholPC and other colipids were mixed in different molar ratios (1:3, 1:1, and 3:1 for CholPC and colipid, respectively) and hydrated. Lipid emulsions were then passed 11× through filters with 200 nm pores. For all LUVs, the diameter varied between 142 and 195 ± 25 nm, and the size did not appear to correlate with initial molar stoichiometry or with composition (Table S1 of the Supporting Information). Extruded LUVs prepared from 1:1 CholPC/PCer or CholPC/Chol were highly unilamellar (parts A and B of Figure S1 of the Supporting Information). To further study the interfacial characteristics of the extruded CholPC/colipid LUVs, we included c-laurdan (1 mol %) in the LUVs during the extrusion process, and determined the emission profile at different temperatures. c-Laurdan emission is sensitive to interfacial hydration, and thus indirectly reflects on interfacial packing. c-Laurdan emission is blue-shifted in gellike states, and red-shifted in the liquid-crystalline state.23 As shown in part A of Figure 2, the emission from c-laurdan was fairly blue-shifted at 20 °C for all three CholPC/colipid compositions, although CholPC/OCer bilayers also had a clear red-shifted shoulder, which was not seen at that temperature for the two other compositions. Increasing the temperature to 40 °C caused a marked red-shifted in the CholPC/OCer bilayers (part B of Figure 2). The emission from CholPC/PCer was slightly red-shifted (about 13 nm at a relative intensity or 0.6) by the 20 °C temperature increase, whereas CholPC/ cholesterol showed virtually no difference in c-laurdan emission at the two temperatures. The calculated GP values are shown in
complex ternary MLV membranes was measured as described previously.22 The F0 (unquenched) samples were prepared with 85 nmol POPC and indicated amounts of colipids (and 1 nmol tPA). The F (quenched) sample was identical to the F0 sample except that 30 nmol of POPC was substituted with the same amount of 7SLPC. The final sample volume was 2 mL. The fluorescence intensity in the F-sample was divided by the fluorescence intensity of the F0-sample giving the fraction of nonquenched CTL fluorescence plotted versus the temperature. 2.7. Lifetime Analysis of trans-Parinaric Acid. The lifetime analysis of tPA in different bilayer types was determined using a PicoQuant Fluotime 200 instrument (PicoQuant GmbH, Berlin, Germany) mainly as described previously.20 A PLS300 led laser (298 nm) was used for excitation. The tPA concentration was 1 mol % and the total lipid concentration 0.05 mM. Data analysis was performed using PicoQuant FluoFit software.
3. RESULTS 3.1. Formation of Giant Vesicles and Extruded LUVs by CholPC and Colipids. The study of Gotoh and co-workers showed that CholPC spontaneously formed multilamellar vesicles when comixed with dimyristoylglycerol or cholesterol.14 To study how CholPC formed vesicles with ceramides (or cholesterol), we prepared vesicles by electroformation, and extrusion (200 nm pore filters). Giant multilamellar and apparently unilamellar vesicles were reproducibly formed from CholPC and either PCer, OCer, or cholesterol (Figure 1). The electroformed vesicles often showed complex internal structures, and their apparent size varied between a few micrometers up to more than 30 µm. The CholPC/PCer vesicles (parts A and B of Figure 1) appeared in general to be smaller in size 2321
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interfacially hydrated and the lateral packing was apparently looser. Finally, we determined the lifetime components of tPA in CholPC/colipid LUVs prepared by extrusion. The lifetime of tPA is markedly affected the by the phase state of bilayers, with lifetimes being long (40−50 ns) in gel-like environments, and short in fluid environments (5−6 ns).20,24,25 The tPA emission decay could best be described by two lifetime components (τ1 and τ 2, Table 1). For CholPC/PCer LUVs at 20 °C, the long component was 29.8 ns (a1 = 17.7%) and the short one 10.2 ns (a2 = 82.3%). Both lifetime-components decreased with increasing temperature but their respective contribution to the emission was not dramatically changed. tPA emission lifetimes in CholPC/cholesterol and CholPC/OCer LUVs were shorter compared to CholPC/PCer, but the fractional distribution between the long and the short component was similar for all three systems. Assuming that tPA is influenced similarly by lateral packing in CholPC bilayers as it is in phospholipid bilayers, one can conclude that none of the studied CholPC bilayers exhibited clear gel-like characteristics (very long lifetimes). They also showed that CholPC/Chol and CholPC/OCer appeared to be in a similar state of order, as reported by tPA lifetime analysis (Table 1). 3.2. Properties of Binary Phospholipid Vesicles Containing Increasing Amounts of CholPC. To study a possible fluidizing (in gel phase) or ordering effect (in liquid crystalline phase) of CholPC on PSM or DPPC bilayer membranes, the steady-state anisotropy of DPH was measured over a temperature interval (10−60 °C). DPH anisotropy reports on changes in acyl chain packing since its excited state rotation will be affected by the order/disorder of neighboring acyl chains. As shown in parts A and B of Figure 3, CholPC clearly destabilized the gel phase of both PSM (part A of Figure 3) and DPPC (part B of Figure 3) vesicles. Such DPH-reported destabilization is normally not seen with cholesterol15 because DPH order is very restricted both in the gel phase as well as in the liquid-ordered phase formed by for example, PSM and cholesterol. One can therefore conclude that introduction of CholPC in a PSM or DPPC gel phase created packing defects (due to mismatch in hydrophobic volume between cholesterol and the ceramide or dipalmitoyl portion of the phosphocholine-containing lipids), which DPH sensed. To further determine the gel phase destabilization induced by CholPC, we analyzed the phospholipid/CholPC bilayers with high-sensitivity DSC (Figure 4). CholPC at 5 and 10 mol % had a marked effect on the gel−liquid crystalline phase transition for both PSM and DPPC. The transition was complex with multiple components, and extended over a temperature range of about 10 degrees. Comparable cholesterol/PSM and cholesterol/DPPC thermograms (at 5 and 10 mol %) show much narrower (higher co-operativity) transitions (cf., Figure 1 in ref 26 and Figure 3 in ref 27, respectively). The DPPC pretransition was seen at about 35 °C (Figure 4). It was still discernible with 5 mol % CholPC but was masked or eliminated at the higher CholPC concentration. The comparable pretransition of PSM was seen at 28−29 °C. Because of its much lower enthalpy (compared to the DPPC pretransition), it is difficult to judge what effects CholPC had on it. Cholesterol is known to order acyl chains of phospholipids in the liquid crystalline state. The ordering effect of cholesterol on POPC bilayers is shown in part A of Figure 5. Addition of CholPC to POPC bilayers failed to induce similar acyl chain ordering (part B of Figure 5) as seen with cholesterol addition.
Figure 2. Emission spectra and GP values of c-laurdan in LUVs made of CholPC and ceramide or cholesterol. Lipids (CholPC and PCer, OCer, or cholesterol) were mixed (1:1 initial molar ratio) from organic solvent and dried under high vacuum for 60 min. Hydration was done in water at 50 °C for 30 min (intermittent vigorous mixing). The MLVs formed were extruded 11x through filters with 200 nm pores. The final lipid concentration was 0.05 mM and c-laurdan was present at 1 mol %. Excitation was at 385 nm and emission was read between 400 and 550 nm. Panel A shows emission obtained at 20 °C, panel B emission at 40 °C, and panel C depicts the GP values calculated from emission spectra at different temperatures.
part C of Figure 2. The GP-values decreased markedly with temperature for ceramide-containing CholPC-bilayers, but much less so with cholesterol in CholPC. The blue-shifted claurdan emission and the high GP value suggest low interfacial hydration, and consequently high lateral packing density in CholPC/Chol and CholPC/PCer bilayers at ambient temperature, whereas the CholPC/OCer bilayers were much less 2322
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Table 1. Lifetime Analysis of tPA in 200 nm Bilayer Extruded LUVsa sample [1:1 ratio]
temperature [°C]
τ1
SD
τ2
SD
τav.
SD
a1 (%)
SD
a2 (%)
SD
CholPC/PCer
20 30 40 20 30 40 20 30 40
29.8 18.3 11.8 13.5 10.0 8.5 14.9 11.0 9.1
±0.9 ±0.1 ±0.1 ±0.3 ±0.1 ±0.1 ±1.0 ±0.2 ±0.1
10.2 7.0 5.0 6.3 4.9 4.1 7.0 5.3 4.4
±0.2 ±0.01 ±0.04 ±0.2 ±0.2 ±0.1 ±0.5 ±0.1 ±0.1
17.8 11.3 8.0 9.2 6.6 5.1 10.8 7.5 5.6
±0.2 ±0.1 ±0.1 ±0.6 ±0.2 ±0.1 ±1.3 ±0.3 ±0.7
17.7 19.3 24.4 24.1 19.7 12.1 30.4 23.3 18.5
±1.0 ±1.2 ±0.04 ±3.3 ±0.8 ±0.1 ±5.7 ±2.5 ±2.1
82.3 80.7 75.6 75.9 80.3 82.7 69.6 76.7 81.5
±1.0 ±1.2 ±0.04 ±3.3 ±0.8 ±7.4 ±5.7 ±2.5 ±2.1
CholPC/OCer
CholPC/Chol
τav values indicate intensity weighted average lifetime and a is the fractional amplitude of each lifetime (pre-exponential factor expressed as percentage). Values are averages from three different set of LUVs for each composition (±SD, n = 3). The lipid concentration was 0.05 mM and tPA was present at 1 mol %. a
Figure 3. Steady state anisotropy of DPH in PSM or DPPC bilayers containing increasing amounts of CholPC. Lipids were mixed from organic solvent and dried under high vacuum for 1 h. Hydration was done with water at 50 °C for 30 min (intermittent vigorous mixing), followed by 10 min bath sonication at 50 °C. The lipid concentration was 0.05 mM and DPH was added to give 1 mol %. Temperature was ramped at 1 °C/min. Panel A shows data for PSM/CholPC, and panel B for DPPC/CholPC bilayers. At least three experiments were performed with each composition, and a representative scan is shown.
Figure 4. DSC analysis of fully hydrated PSM or DPPC bilayers containing CholPC. Lipids were mixed in chloroform and dried under high vacuum for 12 h. Hydration was done in water at 50 °C for 30 min with intermittent vigorous mixing. Finally, the MLVs were bath sonicated for 10 min at 50 °C. After removal of dissolved air, the samples were loaded into the VP-DSC and run between 20 and 60 °C (1 °C/min) until reproducible thermograms were received (usually 3 up and 2 down scans were sufficient). Total lipid concentration varied between 1.33 and 1.48 mM. The 2nd upscan is shown.
Acyl chain ordering of saturated phospholipids above their Tm (Figure 3) was also weak. The degree of acyl chain ordering was much smaller with CholPC than it is with cholesterol in comparable bilayer systems.28 The weak ordering capacity of CholPC can be understood by considering its large headgroup, which prevents close interaction between the sterol ring system and the acyl chains of adjacent phospholipids.
To further compare CholPC/PCer with a system in which the phosphocholine headgroup is on PCer instead of cholesterol, equimolar mixed bilayers of PSM/cholesterol were prepared, and the steady-state DPH anisotropy was determined over a temperature range. At this composition (1:1), the PSM bilayer is in a liquid ordered phase. The DPH anisotropy function shows that acyl chain order was higher in PSM/cholesterol bilayers when compared to CholPC/PCer 2323
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Figure 6. Steady-state DPH anisotropy in equimolar bilayers containing CholPC/PCer or PSM/cholesterol. Lipids were mixed from organic solvent and dried under high vacuum for 1 h. Hydration was done with water at 50 °C for 30 min (intermittent vigorous mixing), followed by 10 min bath sonication at 50 °C. The lipid concentration was 0.05 mM and DPH was added to give 1 mol %. Temperature was ramped at 1 °C/min. Two experiments were performed with each composition, and a representative scan is shown.
bilayer systems, we prepared POPC bilayers and added PCer, cholesterol, and CholPC in different combinations to study the formation of ordered domains. PCer is not readily miscible with POPC, because of its saturated nature compared to POPC, and also because of extensive intermolecular hydrogen bonding among ceramides.9,29,30 Thus, PCer in known to form ceramide-rich ordered domains in the fluid bilayer. Such ordered domains can be determined using tPA quenching. tPA partitions into ordered domains, whereas the quencher (7SLPC) preferentially partitions into the disordered POPC phase.20,31,32 Therefore, at low temperatures, when ordered domains exist, tPA will be protected from quenching by 7SLPC (Figure 7). As the temperature is increased, ordered domains melt and the likelihood of interactions between tPA and 7SLPC increases (leading to more quenching). The PCer-rich domains shown in Figure 7 (bilayer composition: 85 nmol POPC and 15 nmol PCer) were fully melted at 35−36 °C. When cholesterol was added to this bilayer (15 nmol cholesterol, equimolar to PCer), the ordered domains formed by PCer were destabilized, and end melting was achieved already at 28−29 °C. This shows that cholesterol partitioned into the PCer-rich domain to some extent, and disorganized the lateral packing in the domain. It is also likely that some of the destabilization resulted from an ordering effect of cholesterol on POPC, which directly would reflect on the stability of the ordered domain. Interestingly, replacing cholesterol with CholPC (vesicle composition: 85 nmol POPC, 15 nmol PCer, and 15 nmol CholPC) also caused a reduction in the thermostability of the PCer-rich domain similarly as cholesterol did (Figure 7), suggesting that CholPC was able to mix into the PCer-rich domains and destabilize them. However, when bilayers were prepared to contain no PCer, but instead cholesterol and CholPC (15 nmol each, with 85 nmol POPC), tPA quenching did not reveal the presence of ordered domains that could protect tPA from becoming quenched by 7SLPC (Figure 7). Lifetime analysis of tPA present in similar mixed bilayers as shown in Figure 7 are given in Table 3 (measurement at 23 °C). The two lifetime components (τ1 and τ1) present in PCer-containing bilayers
Figure 5. Steady state anisotropy of DPH in POPC bilayers containing increasing amounts of either cholesterol (panel A) or CholPC (panel B). Lipids were mixed from organic solvent and dried under high vacuum for 1 h. Hydration was done with water at 50 °C for 30 min (intermittent vigorous mixing), followed by 10 min bath sonication at 50 °C. The lipid concentration was 0.05 mM and DPH was added to give 1 mol %. Temperature was ramped at 1 °C/min. Two experiments were performed with each composition, and a representative scan is shown.
bilayers (Figure 6). This was true for the whole temperature range investigated (10−70 °C). Consistent with this finding, also the lifetime analysis of tPA in such equimolar bilayers showed significantly shorter average lifetimes of tPA in CholPC/PCer bilayers when compared to cholesterol/PSM (Table 2). tPa showed three lifetime components for equimolar CholPC/PCer bilayers and two for all PSM/cholesterol stoichiometries (Table 2). At 23 °C the longest lifetime for CholPC/PCer was 43.0 ns (a1 = 2%), and the two shorter ones, 16.5 ns (a2 = 31.9%) and 6.4 ns (a3 = 66.2%). In the cholesterol/PSM (1:3) system at 23 °C, the longer lifetime component (43.7 ns, a1 = 37.1%) was more dominating compared to the 1:1 CholPC/PCer system suggesting the presence of a gel phase. Increasing cholesterol or temperature markedly shortened the long lifetime component in the cholesterol/PSM bilayers (Table 2). One can conclude that acyl chain order (based on DPH anisotropy, Figure 6) and lateral packing density (based on tPA lifetime analysis, Table 2) appears to be lower in the CholPC/PCer (1:1) complexes than in the liquid ordered cholesterol/PSM (1:1) bilayers under the conditions used. 3.3. Ordered Domain Formation in Ternary Bilayers. To further examine how CholPC behaves in more complex 2324
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Table 2. Lifetime Analysis of tPA in 200 nm Ternary Extruded LUVsa sample [ratio]
SD
a2 (%)
SD
a3 (%)
SD
2.0
±0.2
31.9
±1.4
66.2
±1.6
±0.1
12.0
±1.5
49.4
±4.2
38.6
±5.7
33.1
±0.1
37.1
±0.2
62.9
±0.2
±0.1
16.8
±0.1
30.8
±2.4
69.3
±2.4
15.5
±0.6
24.2
±0.2
31.5
±2.4
68.5
±2.4
±0.1
9.5
±0.03
±0.02
41.4
±0.9
58.6
±0.9
20.9
±0.01
9.9
±0.3
16.5
±0.1
40.9
±1.6
59.1
±1.6
13.0
±0.2
6.1
±0.2
10.8
±0.2
50.6
±0.7
49.4
±0.7
temperature [°C]
τ1
SD
τ2
SD
τ3
SD
τav.
SD
23
43.0
±0.2
16.5
±0.04
6.4
±0.1
14.5
±0.4
37
14.2
±0.2
6.6
±0.2
2.7
±0.2
8.1
23
43.7
±0.1
18.2
±0.2
37
23.3
±0.4
10.3
23
33.1
±0.9
37
18.9
23 37
CholPC/PCer [1:1]
CholPC/PSM [1:3]
CholPC/PSM [1:1]
CholPC/PSM [3:1]
15.0
a1 (%)
τav values indicate intensity weighted average lifetime and a is the fractional amplitude of each lifetime (pre-exponential factor expressed as percentage). Values are averages from three different set of LUVs for each composition (±SD, n = 3). Measurements were done at 23 and 37 °C. The lipid concentration was 0.05 mM and tPA was present at 1 mol %.
a
good agreement with the tPA quenching data (Figure 7) suggesting that packing differences in the ordered domains correlate with their thermal stability. The fluid nature of the POPC/CholPC/cholesterol system is shown both by the tPA quenching (no ordered domains detected) and lifetime (lowest average lifetime). 3.4. tPA Lifetime Analysis in Ternary Bilayers As a Function of Sterol Content. When cholesterol is added to a bilayer containing an unsaturated (e.g., POPC) and a saturated phospholipid (e.g., PSM or DPPC), it may form a liquidordered phase (possibly coexisting with a liquid disordered phase) above a certain concentration threshold, which depends both on the nature of the unsaturated and the saturated phospholipid and the temperature.20,33 Lifetime-analysis of tPA in complex bilayers may reveal very accurately the presence of different phases because the average lifetime of tPA increases substantially when going from disordered phases via the liquidordered phase to gel phases.20,24,25 We constructed bilayers which had 60 mol % POPC and 40 mol % of either PSM or DPPC. To these bilayers, we added on top cholesterol or CholPC (to yield final sterol concentrations of 5, 10, or 15 mol %). Then we determined the intensity weighed average lifetime of tPA at 23 °C (Figure 8). As cholesterol was added to the POPC/PSM system, the average lifetime of tPA increased from about 13 ns to about 19 ns. In the POPC/DPPC system, the average lifetimes decreased from 32 ns (zero cholesterol) to about 20 ns (15 mol % cholesterol). The different initial lifetime values for POPC/PSM or POPC/DPPC systems reflect differences in the gel phase properties of PSM or DPPC in the POPC environment due to different POPC solubility in
Figure 7. Formation of ordered domains in POPC bilayers. MLVs containing 85 nmol POPC (F 0 composition) and different combinations of small headgroup lipids (ceramide or cholesterol, 15 nmol) and CholPC (15 nmol) were made in water. tPA quenching was measured during temperature ramping (5 °C/min). The quencher was 7SLPC (replaced 30 nmol of POPC in F-samples). The ratio of F/ F0 was plotted against temperature and indicate the extent of quenching susceptibility of tPA. A high F/F0 ratio indicates poor quenching, whereas a lower F/F0 ratio shows more extensive quenching.
were fairly similar (37.7−39.7 ns and 5.5−8.3 ns, respectively), whereas the cholesterol system showed a markedly reduced longer lifetime component (Table 3). tPA lifetime data are in
Table 3. Lifetime Analysis of tPA in 200 nm Ternary Extruded LUVsa 85:15 or 85:15:15
τ1
SD
τ2
SD
τav.
SD
a1 (%)
SD
a2 (%)
SD
POPC/PCer POPC/PCer/Chol POPC/CholPC/PCer POPC/CholPC/Chol
39.6 37.7 39.7 9.9
±1.1 ±3.5 ±1.0 ±0.3
5.5 8.3 5.9 5.4
±0.4 ±0.6 ±0.1 ±0.02
30.7 23.3 24.7 7.6
±1.5 ±2.1 ±1.8 ±0.3
28.3 18.6 15.7 33.1
±2.5 ±0.2 ±1.6 ±2.4
71.7 81.4 84.3 66.9
±2.5 ±0.2 ±1.6 ±2.4
τav values indicate intensity weighted average lifetime and a is the fractional amplitude of each lifetime (pre-exponential factor expressed as percentage). Values are averages from three different set of LUVs for each composition (±SD, n = 3). Measurements were done at 23 °C. The lipid concentration was 0.05 mM and tPA was present at 1 mol %. a
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Ceramides are analogous molecules to diacylglycerides but can form more extensive hydrogen bonding networks in bilayers. The giant vesicles formed by CholPC and ceramides showed that bilayers were formed, and that the electroformed vesicles often had complex internal structures (Figure.1). It should be noted that neither cholesterol, CholPC, nor longchain ceramides can form bilayers alone. Clearly the combination of a large headgroup lipid (CholPC) with a small headgroup lipid (cholesterol or ceramides) made bilayer formation possible with these compounds. Large unilamellar vesicles could be formed from CholPC and cholesterol or ceramides by extrusion, and these were highly unilamellar (parts A and B of Figure S1 of the Supporting Information). The emission spectra of c-laurdan is sensitive to interfacial hydration, and thus to changed interfacial packing density.23,36 Analysis of c-laurdan emission in extruded LUVs prepared from CholPC and either cholesterol or PCer or OCer, suggested that interfacial hydration was very low at 20 °C for all three systems (part A of Figure 2). The blue-shifted emission of c-laurdan also implies very high packing density at this temperature for the three bilayers types. The GP value was close to 0.35 for CholPC/PCer and CholPC/Chol at this temperature, whereas that of CholPC/OCer was about 0.16 (part C of Figure 2). What is the phase state of such bilayers? Pure PSM bilayers at 20 °C (i.e., gel phase) have a laurdan GP value of about 0.42.37 For pure DPPC in the gel state, the laurdan GP was reported to be about 0.65 (at 20 °C,38). Thus, one could conclude that CholPC/PCer and CholPC/Chol bilayers could be in a phase state similar to the gel phase of e.g., PSM. However, the average lifetime of tPA in CholPC bilayers (9−18 ns) is markedly lower than reported for example a PSM gel phase (about 32 ns20) suggesting more disordered packing than in a gel state . The tPA average lifetimes for CholPC bilayers were actually more similar to lifetimes reported for a cholesterol/PSM liquid ordered phase20 suggesting a fluid but ordered nature of the CholPC bilayers. The fluid nature for the CholPC/PCer system likely stems from CholPC probably because of its rough surface (protruding methyl groups in the sterol skeleton), which prevents efficient intermolecular van der Waals interactions. In the CholPC/OCer and CholPC/cholesterol systems, fluidity could be conferred by both components. The finding that CholPC together with cholesterol or ceramides can form bilayers can be understood on the basis of the umbrella model, which stipulates that a lipid with a large headgroup can solubilize a colipid with a small headgroup (provided that the hydrophobic structures do not, for one or another reason, repel each other). The large headgroup lipid can protect the small headgroup lipid from exposing its hydrophobic part to unfavorable interactions with water, thus making association thermodynamically feasible. The model was first developed to explain differential solubility of cholesterol in phosphatidylcholine and phosphatidylethanolamine bilayers1,5 but has been extended to other systems as well.6,8,13,39,40 Because pure cholesterol and pure PCer cannot separately form bilayers, but can do so when one of them has a phosphocholine headgroup, we can also conclude that cholesterol/cholesterol interaction is possible if exposure of the hydrophobic parts of cholesterol to water can be avoided. Cholesterol/ceramide interactions have already been shown to be possible provided that ceramide has the phosphocholine headgroup (as in SM). The next case to study was the interaction between two large headgroup lipids (i.e., CholPC and PC or SM). PSM and saturated PC, which both had large head groups, do mix with
Figure 8. Lifetime analysis of tPA in different MLV systems. MLVs were prepared to contain 120 nmol POPC and 80 nmol of either PSM or DPPC. Cholesterol or CholPC were added on top of this (0−30 nmol). The tPA concentration was 0.5 mol % and the total lipid concentration 0.1 mM. The intensity weighted average lifetime of tPA in each system is plotted as a function of sterol concentration. Each value is the average from n = 3 ± SEM.
the respective ordered phase. At 23 °C, and a composition of 60% POPC and 40% PSM, the addition of cholesterol in excess of 5 mol % is known to induce the appearance of a liquidordered phase.33,34 A lifetime of about 20 ns for tPA is also diagnostic of a liquid-ordered microenvironment for tPA.20 Cholesterol was able to induce the formation of a liquidordered phase (or domains) in both PSM and DPPC systems (Figure 8). However, in the concentration range in which cholesterol induced the formation of a liquid ordered phase (or domains), CholPC clearly failed to form a similar phase. The initial tPA average lifetime in the two bilayer systems decreased with increasing CholPC (Figure 8) suggesting increased disordering but not the formation of a similar liquid-ordered phase (domains) as cholesterol formed with DPPC or PSM in the POPC matrix. These results agree with data presented in Figure 6 showing that equimolar bilayers of CholPC and cholesterol did not have the same degree of acyl chain order as compared to equimolar PSM/cholesterol bilayers (which are in a liquid ordered state). These data also suggest that CholPC most likely interacted with POPC and well as with the saturated phospholipids in the ternary system (Figure 8) because the gel phase destabilizing effect of CholPC was small.
4. DISCUSSION In this study, we have basically focused on two separate questions: (i) how can cholesterol (supplemented with the large headgroup, i.e., CholPC) interact with cholesterol or ceramides (lacking large head groups), and (ii) how will CholPC interact with unsaturated or saturated phospholipids in a bilayer. We hoped to get new information about how molecular shapes (ratio of headgroup volume to the volume of the hydrophobic moiety) of interacting molecules would influence molecular interactions.35 First, we started by confirming the results presented by Gotoh and co-workers,14 who showed that sterol phosphates and cholesteryl phosphocholine were able to form bilayers with cholesterol and a saturated diacylglycerol. In agreement with this study, we obtained bilayers with CholPC and cholesterol but we also extended our study to include ceramides. 2326
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Cholesterol is known to form a liquid ordered phase together with saturated SM and PC.45 In ternary bilayers, the formation of liquid ordered phases is dependent on temperature, and the concentration of all three lipids.20,33,34 Since the lifetime of tPA is very sensitive to the phase state of its microenvironment, we used lifetime analysis to address the question whether CholPC is able to form a liquid ordered phase with PSM or DPPC (Figure 6). Our results clearly indicate that, whereas tPA lifetime analysis reported the formation of the expected liquid ordered phase when cholesterol was used, it did not report typical lifetimes of the liquid ordered phase when CholPC was added. This finding is in agreement with the low ordering capacity of CholPC in the binary bilayers containing either PSM or DPPC (Figure 3) and suggests that the large headgroup in CholPC prevented it from interacting closely enough with the acyl chains of PSM or DPPC. Further, our experiments with equimolar bilayers were prepared from CholPC and PCer showed that the DPH anisotropy in these bilayers was markedly lower (at all temperatures examined, Figure 6) than in bilayers prepared from equimolar PSM and cholesterol (i.e., liquid ordered phase). This observation shows that bilayer packing is different for a ceramide/cholesterol system, depending on which molecule the phosphocholine headgroup is attached to. This difference may relate to the observation that the phosphocholine headgroup of PSM forms an intermolecular water bridge with the 3OH of the ceramide moiety.4,46 A similar interaction is not possible for CholPC, and hence headgroup orientation and dynamics are likely to be different in PSM and CholPC. To conclude, we have provided evidence showing that CholPC appears to have a relative preference for interacting with lipids having small head groups (i.e., cholesterol and ceramides), as compared to interactions with lipids with large head groups (POPC, PSM, DPPC). Our data can be fully explained by a decrease in the packing parameter and the umbrella model. Our finding that CholPC and PCer formed fluid bilayers may also have practical applications in the context of solvent-free delivery of ceramides to cells. Because ceramide is a potent bioactive lipid, which is involved in regulation of both cell proliferation and apoptosis,47 it is of great importance to have a good ceramide formulation, which allows for delivery of externally applied ceramides to cells in culture. Natural ceramides have long saturated acyl chains and will crystallize when added to aqueous cell growth medium. Therefore, it has become customary to deliver solvent-dissolved short-chain ceramides to cells. However, short chain ceramides do not behave similarly as long chain ceramides. We believe that bilayers of ceramides and CholPC could be a better way to deliver ceramides to cells in culture and would make obsolete the use of organic solvents. We have actually made CholPC/ C6-ceramide bilayers and used them to efficiently deliver this short-chain ceramide to cells (Slotte, Tö rnquist et al, unpublished observations), and this formulation is superior to the method of using DMSO as solvent.
each other, but the ideality of mixing is highly dependent on the hydrophobic interaction surface.41 When CholPC was mixed with PSM or DPPC, a dramatic destabilization of their gel phases was obvious (Figure.3 and 4). This strong disruption of gel phase packing of PSM or DPPC by CholPC is most likely explained by mismatch in the volumes of the headgroup versus the hydrophobic part of PSM and DPPC on the one hand and CholPC on the other. The volume of the phosphocholine group is similar for all three molecules, but the volume of the sterol body is much less than the volume of the ceramide of PSM. The volume per lipid has been estimated to about 1175 Å3 for PSM, and to about 990 Å3 for PCer.42 Cholesterolś molecular volume is about 630 Å3.43 One can therefore assume that the cholesterol part of CholPC cannot fill the intermolecular void created when it is mixed into the PSM gel phase, thus causing dramatic gel phase destabilization. An analogous situation would be expected for CholPC in the DPPC gel phase. For similar reasons, the large headgroup of CholPC prevented a close association of the sterol ring system with acyl chains of adjacent POPC, thus attenuating acyl chain ordering (Figure 5). Going for more complex ternary bilayer systems, we next examined how CholPC together with PCer or cholesterol behaved in a fluid POPC bilayer. PCer is known to form ordered domains in fluid bilayers, apparently because it prefers its own company over that of a disordered POPC. PCer interactions are stabilized not only by an entropic contribution but also by extensive hydrogen bonding involving the 2NH of the long chain base.44 It should be noted that the PCer gel domains cannot be pure ceramide domains (would melt at 90 °C) because that would expose the hydrophobic parts or ceramide to unfavorable interactions with water. The PCer gel domains must therefore include some POPC to provide the necessary large headgroup. The PCer domains can conveniently be detected by tPA quenching, as shown in Figure 5. When cholesterol was added to bilayers containing POPC and PCer (final composition 85:15:15 by mol), the PCer domains were destabilized (lower end melting temperature). This observation shows that cholesterol did partition into the PCer gel phase and caused a marked (about 10 degrees) destabilization of lateral packing. Although PCer can displace cholesterol from association with PSM in fluid bilayers when the sterol is equimolar to the PSM,10−12 in the present case cholesterol appeared to favor the more ordered environment created by PCer, over the more disordered phase created by the fluid POPC. Interestingly, CholPC behaved in this instance very similar to cholesterol, because it also destabilized the PCer gel domain in the POPC bilayer (Figure 5). In this scenario, it is likely that PCer chose to interact with CholPC instead of POPC because CholPC provided the needed large headgroup and also was more rigid compared to the flexible acyl chains of POPC. Lastly, it was observed that CholPC and cholesterol together did not provide an ordered domain in which tPA would be protected from quenching by 7SLPC in the fluid phase. Our data in Figure 5 do not reveal whether or not CholPC and cholesterol interacted with each other in the POPC environment but, based on all previous data in our study, we can assume that cholesterol preferred to interact with CholPC instead of POPC, because it provided the necessary headgroup, and was more rigid than the acyl chains of POPC. Our last set of experiments was performed to examine whether CholPC was able to create a liquid ordered phase together with PSM or DPPC in a fluid POPC bilayer.
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ASSOCIATED CONTENT
S Supporting Information *
Table of size distribution of extruded LUVs, figures of DPPENBD in POPC vesicles. This material is available free of charge via the Internet at http://pubs.acs.org. 2327
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affects acyl chain order and sterol interactions in bilayer membranes. Biochim. Biophys. Acta 2010, 1798, 1987−1994. (16) Kim, H. M.; Choo, H. J.; Jung, S. Y.; Ko, Y. G.; Park, W. H.; Jeon, S. J.; Kim, C. H.; Joo, T.; Cho, B. R. A two-photon fluorescent probe for lipid raft imaging: C-laurdan. Chembiochem. 2007, 8, 553− 559. (17) Kuklev, D. V.; Smith, W. L. Synthesis of four isomers of parinaric acid. Chem. Phys. Lipids 2004, 131, 215−222. (18) Bhatia, S. K.; Hajdu, J. Stereospecifi synthesis of ether and thioether phospholipids. The use of L-glyceric acid as a chiral phospholipid precursor. J. Org. Chem. 1988, 53, 5034−5039. (19) Roodsari, F. S.; Wu, D.; Pum, G. S.; Hajdu, J. a new approach to the stereospecific synthesis of phospholipids. The use of L-glycerid acid for the preparation of diacylglycerols, phosphatidylcholines, and related derivatives. J. Org. Chem. 1999, 64, 7727−7737. (20) Nyholm, T. K.; Lindroos, D.; Westerlund, B.; Slotte, J. P. Construction of a DOPC/PSM/cholesterol phase diagram based on the fluorescence properties of trans-parinaric acid. Langmuir 2011, 27, 8339−8350. (21) Angelova, M. I.; Dimitrov, D. S. Liposome electroformation. Faraday Discuss. Chem. Soc. 1−10−1986, 81, 303−312. (22) Bjorkqvist, Y. J.; Nyholm, T. K.; Slotte, J. P.; Ramstedt, B. Domain formation and stability in complex lipid bilayers as reported by cholestatrienol. Biophys. J. 2005, 88, 4054−4063. (23) Parasassi, T.; Krasnowska, E. K.; Bagatolli, L.; Gratton, E. LAURDAN and PRODAN as polarity-sensitive fluorescent membrane probes. Journal of Fluorescence 1998, 8, 365−373. (24) Castro, B. M.; de Almeida, R. F.; Silva, L. C.; Fedorov, A.; Prieto, M. Formation of ceramide/sphingomyelin gel domains in the presence of an unsaturated phospholipid. A quantitative multiprobe approach. Biophys. J. 2007, 93, 1639−1650. (25) Silva, L. C.; de Almeida, R. F.; Castro, B. M.; Fedorov, A.; Prieto, M. J. Ceramide-domain formation and collapse in lipid rafts: membrane reorganization by an apoptotic lipid. Biophys. J. 2006, 92, 502−516. (26) Nyholm, T. K.; Nylund, M.; Slotte, J. P. A calorimetric study of binary mixtures of dihydrosphingomyelin and sterols, sphingomyelin, or phosphatidylcholine. Biophys. J. 2003, 84, 3138−3146. (27) McMullen, T. P.; McElhaney, R. N. New aspects of the interaction of cholesterol with dipalmitoylphosphatidylcholine bilayers as revealed by high-sensitivity differential scanning calorimetry. Biochim. Biophys. Acta 1995, 1234, 90−98. (28) Lonnfors, M.; Engberg, O.; Peterson, B. R.; Slotte, J. P. Interaction of 3beta-amino-5-cholestene with phospholipids in binary and ternary bilayer membranes. Langmuir 2012, 28, 648−655. (29) Castro, B. M.; Silva, L. C.; Fedorov, A.; de Almeida, R. F.; Prieto, M. Cholesterol-rich fluid membranes solubilize ceramide domains: implications for the structure and dynamics of mammalian intracellular and plasma membranes. J. Biol. Chem. 2009, 284, 22978− 22987. (30) Hsueh, Y. W.; Giles, R.; Kitson, N.; Thewalt, J. The effect of ceramide on phosphatidylcholine membranes: A deuterium NMR study. Biophys. J. 2002, 82, 3089−3095. (31) Castanho, M.; Prieto, M.; Acuna, A. U. The transverse location of the fluorescent probe trans-parinaric acid in lipid bilayers. Biochim. Biophys. Acta 1996, 1279, 164−168. (32) de Almeida, R. F.; Loura, L. M.; Fedorov, A.; Prieto, M. Nonequilibrium phenomena in the phase separation of a twocomponent lipid bilayer. Biophys. J. 2002, 82, 823−834. (33) de Almeida, R. F.; Fedorov, A.; Prieto, M. Sphingomyelin/ phosphatidylcholine/cholesterol phase diagram: boundaries and composition of lipid rafts. Biophys. J. 2003, 85, 2406−2416. (34) Ionova, I. V.; Livshits, V. A.; Marsh, D. Phase diagram of ternary cholesterol/palmitoylsphingomyelin/palmitoyloleoyl-phosphatidylcholine mixtures: spin-label EPR study of lipid-raft formation. Biophys. J. 2012, 102, 1856−1865. (35) Israelachvili, J. N.; Mitchell, D. J. A model for the packing of lipids in bilayer membranes. Biochim. Biophys. Acta 1975, 389, 13−19.
AUTHOR INFORMATION
Corresponding Author
*Tel: +358-2-2154689, E-mail: jpslotte@abo.fi. Notes
Notes. The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank Dr. Patrik Eklund for valuable comments regarding the synthesis of CholPC and its NMR analysis, Dr. Helen Cooper for excellent help with confocal microscopy, and Dr. Tomi Airenne for valuable discussions regarding computationally obtained molecular volumes. This study was supported by generous grants from the Sigrid Juselius Foundation, the Åbo Akademi Foundation, and the Magnus Ehrnrooth Foundation.
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