Comparison of Carboxybetaine with Sulfobetaine as Lipid Headgroup Involved in Intermolecular Interaction between Lipids in the Membrane

Abstract

Diacylglycerides (DAGs) constitute an important category of lipids owing to their ability to form a lipid membrane, which can be used in a wide variety of biomedical applications. DAGs often include a zwitterionic polar headgroup that can influence the properties of the lipid membrane (e.g., protein adsorption, ion binding, hydration, membrane fluidity, phase stability) and affect their applicability. To clarify the effect of the charge arrangement of zwitterionic headgroups on intermolecular interactions in the DAG bilayers, we investigated the intermolecular interaction between a naturally occurring DAG (1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)) and synthetic DAGs (which is called "inverse charge zwitterlipids (ICZLs)") whose headgroup charges were antiparallel with respect to those of DPPC. We used 1,2-dipalmitoyl-sn-glycero-3-carboxybetaine (DPCB) and 1,2-dipalmitoyl-sn-glycero-3-sulfobetaine (DPSB) as ICZLs and compared two combinations of the lipids (DPPC-DPCB and DPPC-DPSB). We obtained surface pressure-area (π-A) isotherms to elucidate the intermolecular interaction between the lipids in the monolayer at the air/water interface. We found shrinkage of the area per molecule in both lipid combinations, indicating that mixing DPPC with ICZLs results in an attractive intermolecular force. As an overall trend, the degree of shrinkage of the mixed monolayer and the thermodynamic favorability of mixing were greater in the DPPC-DPCB combination than in the DPPC-DPSB combination. These trends were also observed in the lipid bilayers, as determined from the gel-to-liquid crystal phase transition temperature (T _c) of the aqueous dispersion of the lipid vesicles. In the highly compressed lipid monolayers and vesicles (lipid bilayer), the molar fractions of ICZLs, in which the intermolecular interaction reached a maximum, were 0.6-0.8 for the DPPC-DPCB combination and 0.5 (equimolar composition) for the DPPC-DPSB combination. Therefore, in the compressed monolayers and bilayers, the mechanism of intermolecular interaction between DPPC and DPCB is different from that between DPPC and DPSB.

Figure 1

Chemical structures of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) and an inverse charge zwitterlipid (ICZL) and hypothesized intermolecular interaction between these lipids. The ICZLs used in this study had either carboxybetaine (1,2-dipalmitoyl-sn-glycero-3-carboxybetaine (DPCB)) or sulfobetaine (1,2-dipalmitoyl-sn-glycero-3-sulfobetaine (DPSB)) headgroups.

Figure 2

Surface pressure–area (π–A) isotherms of lipid monolayers of DPPC in the presence of either DPCB (solid lines) or DPSB (dotted lines) with different molar fractions (X_ICZL) (a). Total lipid amount used in the monolayer experiments was 9 nmol (DPPC + ICZL). The arrows in (a) indicate plateau regions for the DPPC–DPCB combinations where the tilt angle of the DPCB headgroups should change from parallel to perpendicular to the air/water interface (as shown in (b)). Comparison of the π–A isotherms of the equimolar mixtures composed of lipids whose headgroup charge arrangements were parallel or antiparallel (b). For all measurements of the isotherms, the subphase contained ultrapure water. The temperature of the subphase was 20 °C. The isotherms were from two or more independent measurements. Data for the DPPC–DPSB combination reprinted with permission from ref (17). Copyright 2017 American Chemical Society.

Figure 3

Extrapolated molecular area (A_L) of the lipid comprising the lipid monolayer. The open circles represent the A_L values of the DPPC and DPSB system. The filled symbols represent the A_L values of the DPPC and DPCB system. There were two series of A_L values in the DPPC–DPCB system, which were determined at high (filled circles) and low (triangles) surface pressures. The π–A isotherms for DPPC–DPSB mixtures do not have plateau regions (Figure 2a). This indicates that the tilt angle of DPSB does not change even though the lipid monolayer is laterally compressed. Thus, A_L for the DPPC–DPSB mixtures was determined as a single value at each lipid composition. The dotted lines represent the ideal additivity for both systems. The p-values for all series of A_L were < 0.01 (one-way ANOVA, n ≥ 3). Data for the DPPC–DPSB combination reprinted with permission from ref (17). Copyright 2017 American Chemical Society.

Figure 4

Representative plots of compressibility modulus (C_S^–1) as a function of the occupied molecular area for different molar fractions of ICZLs (filled circles for DPCB, open circles for DPSB) (a). Plots of the maximum C_S^–1 values as a function of X_ICZL (filled circles and triangles DPCB; open circles represent DPSB) (b). DPCB; p = 0.02 (high-pressure region), p > 0.05 (low-pressure region). DPSB; p > 0.05 (one-way ANOVA, n ≥ 3). Data for the DPPC–DPSB combination reprinted with permission from ref (17). Copyright 2017 American Chemical Society.

Figure 5

Excess free energy of mixing (ΔG^Exc) as a function of X_DPCB (a) and X_DPSB (b) at various surface pressure regions: 0–5 (open circles), 0–10 (filled circles), 0–20 (open triangles), 0–30 (filled triangles), and 0–40 mN/m (diamonds). The dashed line represents the ideal ΔG^Exc value for the binary system composed of DPPC and DPCB. The p-values for all surface pressure ranges were < 0.01 for the DPCB lipid mixture. For the DPSB mixture, the p-values for surface pressure ranges 0–10, 0–20, 0–30, and 0–40 mN/m were <0.01. For surface pressure range 0–5 mN, p > 0.05 (one-way ANOVA, n ≥ 3). Data for the DPPC–DPSB combination reprinted with permission from ref (17). Copyright 2017 American Chemical Society.

Figure 6

Representative DSC thermograms of an aqueous dispersion of DPPC in the presence of ICZL (plain lines: DPCB, dotted lines: DPSB) with the various molar fractions (a). Total lipid concentration was 20 mM. The thermograms for pure DPPC and DPCB are shown with downsized intensity (0.5×). Plots of the onset temperature of the gel-to-liquid crystal phase transition (T_c) as a function of X_ICZL (filled symbols: DPCB, open symbols: DPSB) (b). p < 0.01 for both lipid mixtures (one-way ANOVA, n ≥ 2). Comparison of DSC thermograms of lipid dispersions composed of the parallel arrangement of headgroup combination DPSB–DPCB (c). An equimolar amount of DPSB–DPCB mixture was used in this experiment (total lipid concentration = 20 mM). The significance of the A_L value between series of DPPC–DPSB and DPPC–DPSB was determined using Student’s t-test. As a result, p-values were less than 0.05 at all compositions of ICZLs except for X_ICZL = 0. The data for the DPPC–DPSB combination reprinted with permission from ref (17). Copyright 2016 American Chemical Society.