Article pubs.acs.org/Langmuir
Formation and Diffusivity Characterization of Supported Lipid Bilayers with Complex Lipid Compositions Lisa Simonsson and Fredrik Höök* Department of Applied Physics, Chalmers University of Technology, Gothenburg, Sweden S Supporting Information *
ABSTRACT: The moving edge of a hydrodynamically manipulated supported lipid bilayer (SLB) can be used to catalyze SLB formation of adsorbed lipid vesicles that do not undergo spontaneous SLB formation upon adsorption on SiO2. By removing the lipid reservoir of an initially formed SLB, we show how a hydrodynamically moved SLB patch composed of POPC can be used to form isolated SLBs with compositions that to at least 95% represent that of the adsorbed lipid vesicles. The concept is used to investigate the diffusivity of lissamine rhodamine B 1,2-dihexadecanoyl-sn-glycero-3phosphoethanolamine (rhodamine−DHPE) in SLBs made from complex lipid compositions, revealing a decrease in diffusivity by a factor of 2 when the cholesterol content was increased from 0% to 50%. We also demonstrate how the concept can be used to induce stationary domains in SLBs containing 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3phosphoethanolamine (DOPE), and cholesterol (39:21:40 mol %, respectively). Because the method serves as a means to form SLBs with lipid compositions that hamper SLB formation via spontaneous rupture of adsorbed lipid vesicles, it opens up the possibility for new biophysical investigations of SLBs with more nativelike compositions.
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neuronal cells,20,21 and synaptical vesicles22 are particularly rich in cholesterol (up to 50 mol %) as well as DOPE lipids,19,23 also known to participate in lipid-domain formation.24 The existing knowledge regarding the influence of cholesterol on the fluidity of lipid membranes has been gained from studies of the diffusivity of alternative model membranes, such as suspended vesicles,25 lipid multilayers (see review15 and references therein), and black lipid membranes,26 using primarily NMR, 15,27 fluorescence recovery after photobleaching (FRAP),14,15,26 and fluorescence correlation spectroscopy.25 The investigation of the influence of cholesterol on the lipid diffusivity and domain formation in SLBs is limited to SLBs formed on quartz slides using the Langmuir−Blodgett/Schäfer method,14 while SLBs formed from lipid vesicle adsorption and rupture remain essentially unexplored. We recently contributed a method that enables SLB formation from vesicles that fail to spontaneously rupture into SLBs, including lipid vesicles containing high cholesterol concentrations and domain-inducing lipids and even vesicles derived directly from natural cell membranes.28 This method is based on the recently discovered possibility of using the hydrodynamic force from a flowing liquid in a microfluidic channel to drive a SLB and its molecular constituents in the direction of the liquid flow.29 By driving a preformed SLB front against adsorbed lipid vesicles we demonstrated that the latter were efficiently incorporated into the SLB, with the energetically unstable edge of the SLB acting as a catalyst for membrane
INTRODUCTION During the past decades, the supported lipid bilayer (SLB) has been established as one of the most important model systems of natural cell membranes.1,2 One of the most efficient and therefore most popular strategies to form SLBs is via lipid vesicle adsorption on solid substrates, which under certain conditions leads to spontaneous SLB formation.3−6 However, SLB formation tends to not occur spontaneously for vesicles of complex lipid compositions and is efficient on only a few selective surfaces (typically SiO2, TiO2, or mica). Although lipid vesicles with complex compositions can be forced to form SLBs by varying the external conditions, such as addition of divalent ions such as Ca2+,7 osmotic stress,8 raised temperature,7 and lowered pH,9 often in combination with very long (3−7 h) incubation times,7,8 there are many situations where SLB formation via lipid vesicle adsorption is prevented. Common situations include the presence of lipids in the gel phase,10 lipids with negative curvature,11 and incorporated membrane proteins.12 In addition, high cholesterol content (≥40 mol %) in phosphatidylcholine vesicles hamper the SLB formation on otherwise very potent SiO2 surfaces.13 This has in turn limited the use of SLBs to biophysical investigations of systems that lack several essential components of natural cell membranes. For instance, cholesterol is the single most common lipid species in mammalian cell membranes (25−50 mol % of the lipid in the plasma membrane, depending on cell type),14 where it is involved in regulating the physical properties of the cell membrane, such as fluidity,15 stiffness,16 and permeability.17 Cholesterol is also found in domains in both cell membranes and synthetic membranes.16,18 In particular, many natural membranes, such as those of red blood cells,19 myelin,19 © 2012 American Chemical Society
Received: May 8, 2012 Revised: June 14, 2012 Published: June 15, 2012 10528
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Figure 1. (a) Schematics of the different steps (i−iv) for forming a SLB patch. Green indicates NBD C12-HPC-labeled SLB, and red indicates rhodamine-labeled lipid vesicles adsorbed to the channel floor. (b) Micrographs from the microfluidic channel at two different stages of our experiments. At t = 0, the NBD-labeled POPC SLB (green) is removed exclusively from the left part of the microfluidic channel, representing step iv in part a. At t > 1 h, the micrograph shows part of a SLB patch (about 500 μm long in total) formed from rhodamine-labeled (red) lipid vesicles composed of DOPC/cholesterol, 60:40 mol %, as described in the text. A photobleached spot was used for FRAP analysis of the diffusivity of the formed SLB. The numbers (1−4) indicate the indexing of the in- and outlets of the four-arm channel. The micrographs are mergers of two pictures taken subsequently using a TRITC (red) and a FITC (green) filter cube, respectively.
on the part of the floor of channel arm 4 not yet covered by a SLB; see step iii in Figure 1a. Subsequently, the flow direction was reversed, now flowing from channel arm 4 to 1, and adjusted to a flow speed of 10 μL/min. At this stage, SDS was introduced from channel arm 1 to channel arms 2 and 3 at a flow speed of 50 μL/min, while the buffer flow from channel arm 4 was directed to 2 and 3 at a flow speed of 10 μL/min. In this way, local SDS-induced removal of the SLB could be achieved, leaving a SLB patch in the microfluidic channel arm 4, as illustrated in step iv in Figure 1a and 1b. The SLB patch was then driven toward the adsorbed and intact vesicles. Upon contact between the moving edge of the SLB and the adsorbed lipid vesicles, rupture and incorporation of the lipid vesicles into the SLB patch is expected. The feasibility of the concept was investigated using lipid vesicles with three different lipid compositions that do not spontaneously form SLBs upon adsorption on SiO2: (1) DOPC/ cholesterol (60:40 mol %), (2) DOPC/cholesterol (50:50 mol %) (see movie 1 in Supporting Information), and (3) DOPC/ DOPE/cholesterol (39:21:40 mol %) (see movie 2 in Supporting Information). The around 100 μm long POPC SLB patch was driven and fused with adsorbed lipid vesicles over at least 800 μm. The time evolution of the rhodamine−DHPE and NBD C12-HPC intensity profiles along the microfluidic channel when the SLB patch is driven toward lipid vesicles of composition (1) (Figure S1 in Supporting Information) shows accumulation of rhodamine−lipids and depletion of NBD−lipids at the front of the SLB, which is in agreement with efficient fusion.28 With the amount of lipid material per unit area of the adsorbed vesicles being approximately twice that of a SLB,30 6% of the lipids in the new SLB are expected to originate from the original POPC SLB. This is in fact a higher content than the around 2% that was estimated from the (total) intensity of the remaining NBD C12-HPC in the SLB. With fluorescence resonance energy transfer estimated to have a minor impact on the intensity (see Supporting Information), the reason for this difference is attributed to release of lipids from the NBD C12HPC-rich back of the SLB patch rather than, for example,
fusion.28 However, due to lipid diffusion, the lipid composition of a SLB formed in this way will spontaneously equilibrate into a mixture of the composition of the preformed SLB and that of the adsorbed lipid vesicles. This, in turn, may limit the applicability of the method in certain situations, since it is not suitable for studies in which it is desirable to precisely control the molecular composition of the SLB. In this work we demonstrate a means to circumvent this limitation by chemically removing the large lipid reservoir of the initially formed SLB. The remaining SLB patch is then driven against the adsorbed vesicles, leading to formation of a SLB with a composition determined to more than 95% by that of the adsorbed vesicles. The feasibility of the concept is demonstrated by investigating the influence on membrane fluidity of high cholesterol concentrations (≥40 mol %). Motivated by the ability of phosphoethanolamines to induce cholesterol-rich domains in homogeneous phosphatidylcholine/cholesterol SLBs,24 we also explore how the lateral fluidity and heterogeneity is influenced by addition of 1,2-dioleoyl-snglycero-3-phosphoethanolamine (DOPE) lipids.
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RESULTS AND DISCUSSION The concept of forming SLBs by driving a POPC bilayer toward adsorbed lipid vesicles which have not spontaneously ruptured upon adsorption on solid substrates was described in detail elsewhere.28 Here, this concept is extended to enable formation of a SLB with a lipid composition that is basically determined solely by the lipid composition of the adsorbed lipid vesicles. This was performed by first forming a dye-labeled (1 wt % nitrobenz-2-oxa-1,3-diazol-4-yl)amino)dodecanoyl-1hexadecanoyl-sn-glycero-3-phosphocholine [NBD C12-HPC]) SLB in channel arm 1 of a four-armed microfluidic crosschannel; see step i in Figure 1a. The SLB was then driven at a flow speed of 250 μL/min past the four-channel cross region until it reached around 100 μm into channel arm 4; see step ii in Figure 1a. Vesicles of complex lipid compositions labeled with lissamine rhodamineB 1,2-dihexadecanoyl-sn-glycero-3phosphoethanolamine (rhodamine−DHPE, 1 wt %) were then introduced at a flow speed of 20 μL/min, leading to adsorption 10529
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incomplete fusion. In fact, if fusion was not complete, i.e., if part of the vesicle lipid material was not incorporated into the SLB patch, the observed fraction of NBD C12-HPC in the SLB patch would rather be larger than the theoretically predicted fraction. Our observations thus suggest complete fusion, from which one can conclude that the lipid composition is to within at least 95% determined from the composition of the vesicles that do not spontaneously rupture into a SLB. Hence, the cholesterol content of compositions 1, 2, and 3 become at least 38, 47, and 38 mol %, respectively. It is also worthwhile to note that complete fusion between the SLB front and the adsorbed vesicles suggests that the lipid density in the SLB patch should increase locally. This is consistent with the occasional observations of transient lipid domains (see movie 3 in Supporting Information) in the front of SLB patches. These transient domains are tentatively attributed to a local increase in interfacial pressure of the SLB induced by a relatively high shear force combined with a local high lipid density originating from the high lipid vesicle coverage. Together, this leads to lipid phase separation, as previously observed for Langmuir−Blodgett films formed at a high interfacial pressure.31 The local and transient nature of the domain appearance is attributed to the generation of a lipid density gradient in the SLB patch, which equilibrates when the flow is stopped. Figure 2a shows the result from FRAP analyses32 of SLB patches of lipid composition 1 (see micrograph in Figure 1b) and 2 as well as a pure DOPC SLB. The resulting diffusivities were for DOPC: 2.5 ± 0.1 μm2 s−1 (n = 5), DOPC/cholesterol (60:40 mol %): 2.0 ± 0.1 μm2 s−1 (n = 4), DOPC/cholesterol (50:50 mol %): 1.2 ± 0.1 μm2 s−1 (n = 5), where n corresponds to the number of FRAP measurements performed per SLB composition. The average immobile fraction was 30 min, followed by extrusion through 30 nm polycarbonate membranes (Whatman, Maidstone, UK) 11 times. The total lipid concentrations were 0.5−3 mg/mL, depending on lipid composition. The lipid vesicle suspensions were stored at 4 °C. The lipid vesicles were diluted with buffer solution (10 mM Tris/HCl pH 7.4, 100 mM NaCl, 1 mM EDTA) to a total lipid concentration of 0.1 mg/mL prior to each experiment. Fluorescence Microscopy. The fluorescently labeled molecules were studied with an inverted Nikon Eclipse Ti-E microscope (Nikon Corporation, Tokyo, Japan), using an Andor iXon+ EMCCD camera (Andor Technology, Belfast, Northern Ireland) and a 60× magnification (NA = 1.49) oil immersion objective (Nikon Corporation). The acquired images consisted of 512 × 512 pixels with a pixel size of 0.38 × 0.38 μm. To monitor the fluorescent molecules, a mercury lamp connected to the microscope using an optical fiber (Intensilight C-HGFIE; Nikon Corporation) was used together with a TRITC (rhodamine−DHPE) or a FITC (NBD C12HPC) filter cube (Semrock, Rochester, NY), depending on the dye studied. All data generation was performed using time-lapse acquisition, with an exposure time of 100 ms. In the experiments with NBD C12-HPC in the SLB and rhodamine−DHPE in the adsorbed lipid vesicles, one image was first taken using a FITC filter cube, immediately followed by an image taken using a TRITC filter cube. Fluorescence Recovery after Photobleaching (FRAP). FRAP was used to determine the diffusivity of the labeled molecules in the lipid bilayer. All diffusivity measurements were performed with the bulk flow turned off. To create bleached areas, diode-pumped solid state lasers at either 475 nm (BWB-475-20E; B&W Tek Inc., Newark, DE) or at 532 nm (BWN-532-100E; B&W Tek Inc.) were used, depending on what dye the lipid bilayer contained. The acquired images were analyzed using the Hankel transform method, previously developed in the group.32 A single exponential with an offset was fitted to the data yielding the diffusivity and immobile fraction.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by the Swedish Research Council Grant-ID: 2010-5063 REFERENCES
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ASSOCIATED CONTENT
S Supporting Information *
Three videos recording fusion between the moving SLB patch edge and adsorbed lipid vesicles of different lipid compositions, the three cases (i) no visible domain formation (ii) stable domain formation, and (iii) transient domain formation are represented, details regarding temporal variation of the intensity profiles for analysis of the fusion process, details regarding the estimation of NBD C12-HPC remnants in the SLB patch and micrographs of the time evolution of stable domains. This material is available free of charge via the Internet at http://pubs.acs.org/. 10532
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