Monolayer behavior of pure F-DPPC and mixed films with DPPC studied by epifluorescence microscopy and infrared reflection absorption spectroscopy
Graphical abstract
Introduction
Lipid monolayers are used as model systems for lipid bilayers, because their state can be easily controlled by changing the molecular area of the lipids at the surface. In addition, the subphase composition can be changed at will (Maget-Dana, 1999; Brezesinski and Mohwald, 2003). Thus many problems which deal with the interaction of water soluble molecules with the surface of lipid bilayers can be addressed by using lipid monolayers. Upon compression of monolayers of many lipids with saturated acyl chains, a 2-D phase transition from the so-called liquid-expanded (LE) to the liquid-condensed (LC) state is observed. This transition can be visualized by epifluorescence microscopy using lipid fluorescent probes, which preferentially partition into the LE phase (Brezesinski and Mohwald, 2003; Lösche and Möhwald, 1984). Therefore, the LC domains formed upon compression appear black against a homogeneous light background of LE phase lipids. The shapes of these domains depend on the type of lipid and experimental conditions, such as compression speed. The quasi-equilibrium experienced in slowly compressed monolayers is quite different from substantially rapid compressions taking, for instance, place in lungs. Hence also out-of-equilibrium states in monolayers are biologically relevant (Smith et al., 2003).
F-DPPC is the mono fluorinated derivative of l-DPPC, where the sn-2 terminus bears a single fluorine atom. It is now well understood that the gel phase behavior of F-DPPC is governed by this fluorine atom. In the fluid phase, both DPPC and F-DPPC exist in the normal bilayer state, but the affinity of the fluorinated fatty acid chain for a polar environment leads to interdigitation in the gel phase of F-DPPC (Hirsh et al., 1998). This interdigitated phase of F-DPPC has been studied in more detail recently, and investigations have been performed on the effect of co-addition of other phospholipids (Smith et al., 2010; Smith et al., 2014; Smith and Dea, 2015), calcium (Toimil et al., 2014) and lanthanum (Toimil et al., 2012a) ions, and cholesterol (Smith et al., 2012) on the phase behavior. Interdigitation can lead to phase separation in mixed membranes, transmembrane coupling, decrease in surface charge density and change in the hydrophobic match in a membrane (Stillwell, 2013). F-DPPC has also been used to monitor the insertion of HIV fusion peptide into cell membranes (Qiang et al., 2009). The chemical structures of l-DPPC, d-DPPC and F-DPPC are shown below (see Fig. 1).
Selectively fluorinated amphiphiles have been used in the past for probing biological membranes using 19F-NMR (Gent et al., 1978; Cavanaugh et al., 1986). Fluorinated fatty acids are not widely distributed in nature (Carvalho and Oliveira, 2017) and they are known to cause heart impairment (Tosaki and Hearse, 1988). Perfluorinated amphiphiles have been employed for investigating lipid monolayer behavior (Matyszewska and Bilewicz, 2008), but they alter membrane properties (Matyszewska et al., 2007). For instance, perfluorinated alcohols due to their stiffness produce cholesterol like effects in phospholipid membranes (Jbeily et al., 2018). Perflorinated fatty acids and alcohols are widely used in industrial products. The chemical stability of these compounds presents a serious problem, as they have been characterized as bioaccumulative and toxic compounds. Thus they are considered as a potential risk to the environment and to humans (Brambilla et al., 2015). However, there is some hope that these compounds can be transformed into less toxic compounds. A recent study by Beškoski et al. (2018) showed that perfluoroalkyl acids can be transformed by certain microorganisms to monofluorinated fatty acids which are less toxic. Therefore, monofluorinated phospholipids, where only one of the two alkyl chains is fluorinated at the end of the alkyl chains is probably also less harmful (Beškoski et al., 2018). In any case, it is imperative to understand the physical properties of fluorinated phospholipids before using them for biological applications (Madsen et al., 2016).
The domain shapes in lipid monolayers reflect a trade-off between line tension and electrostatic dipolar forces (McConnell, 1991). When the line tension is dominating, circular shaped domains are observed, whereas the shapes undergo distortion when electrostatic dipolar forces prevail. According to Flores et al. (2007), the area density difference between LE and LC phases are responsible for different domain morphologies and shape transitions. In F-DPPC monolayers, the line tension as well as the dipolar forces are modified compared to the normal non-fluorinated DPPC. The question now arose, whether the single fluorine atom leads to a different morphology and size of the LC domains. There are only two available reports on the behavior of F-DPPC monolayers, one publication where co-spread mixtures with l-DPPC were investigated (Toimil et al., 2010) and the other on the influence of human serum albumin on the monolayer behavior of F-DPPC (Toimil et al., 2012b). The first study shows that the monolayer behavior of F-DPPC is slightly different from that of DPPC and that the size of fluorine atom plays a role, but a detailed analysis on the relationship between fluorine atom and the properties of LE and LC phases is still lacking. In a previous study from our group using F-DPPC monolayer, we investigated whether the terminal fluorine atom in F-DPPC had an effect on the incorporation of block copolymers with fluorinated chains into the monolayer and found that polymers with fluorinated chains were more stably anchored in F-DPPC monolayers up to higher surface pressure (Shah et al., 2017). In the present study, we try to answer the question whether the terminal fluorine atom imparts characteristic changes in properties to F-DPPC monolayers compared to DPPC monolayer, such as chain order or chain tilt in the LC-phase. In addition to the recording of pressure-area isotherms, the monolayers were therefore visualized by epifluorescence microscopy and also investigated by infrared reflection absorption spectroscopy (IRRAS). Finally, the effects of compression rate, pressure jumps, and dye-label type on the morphology of the LC domains and the morphological changes of the domains in mixtures with l-DPPC and d-DPPC were studied.
Section snippets
Material
1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (l-DPPC) and 2,3-Dihexadecanoyl-sn-glycero-1-phosphocholine (d-DPPC) were purchased from Sigma-Aldrich, whereas 1-palmitoyl-2-(16-fluoropalmitoyl)-sn-glycero-3-phosphocholine (F-DPPC) was acquired from Avanti Polar Lipids. The fluorescently labeled lipids 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl)(triethylammonium salt) (Rh-DHPE); 2-(12-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)dodecanoyl-1-hexadecanoyl-sn
Effect of dye-labels on condensed lipid domains
The surface pressure-area isotherms for F-DPPC and l-DPPC monolayers at the air-water interface are shown in Fig. 2, along with the derived compressibilities.
Compared to l-DPPC, F-DPPC has a higher lift-off area (111 Ų as compared to 92 Ų) and an increased LE→LC phase transition pressure (9.3 mN m−1 as compared to 4.9 mN m−1, see compressibility curves in Fig. 1 inset). Both values show that the fluid LE phase is stabilized by the fluorination of one acyl chain terminus on expanse of the
Conclusions
A single fluorine atom at the terminus of the sn-2 chain of the lipid F-DPPC strongly modulates the monolayer behavior at air-water interface. The end of the gas to LE phase transition occurs at larger molecular area compared to the values observed for DPPC monolayers. The LE phase is stable over a larger surface pressure range so that the LE-LC transition begins at higher surface pressure than the transition of a DPPC monolayer. The LC domains observed by epifluorescence microscopy had
Declaration of competing interest
The authors declare no conflict of interests.
Acknowledgments
This work was supported by a grant from the Deutsche Forschungsgemeinschaft FOR 1145, TP 5 (A.B. and C.S.) and a grant from the Higher Education Commission of Pakistan under the Faculty Development Program of the Hazara University, Mansehra (S.W.H.S.).
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