Interaction between artificial membranes and enflurane, a general volatile anesthetic: DPPC-enflurane interaction

Abstract

The structural modifications of the dipalmitoylphosphatidylcholine (DPPC) organization induced by increasing concentration of the volatile anesthetic enflurane have been studied by differential scanning calorimetry, small-angle, and wide-angle x-ray scattering. The interaction of enflurane with DPPC depends on at least two factors: the enflurane-to-lipid concentration ratio and the initial organization of the lipids. At 25 degrees C (gel state), the penetration of enflurane within the lipids induces the apparition of two different mixed lipid phases. At low anesthetic-to-lipid molar ratio, the smectic distance increases whereas the direction of the chain tilt changes from a tilt toward next-neighbors to a tilt between next-neighbors creating a new gel phase called L(beta')(2NNN). At high ratio, the smectic distance is much smaller than for the pure L(beta') DPPC phase, i.e., 50 A compared to 65 A, the aliphatic chains are perpendicular to the membrane and the fusion temperature of the phase is 33 degrees C. The electron profile of this phase that has been called L(beta)(i), indicates that the lipids are fully interdigitated. At 45 degrees C (fluid state), a new melted phase, called L(alpha)(2), was found, in which the smectic distance decreased compared to the initial pure L(alpha)(1) DPPC phase. The thermotropic behavior of the mixed phases has also been characterized by simultaneous x-ray scattering and differential scanning calorimetry measurements using the Microcalix calorimeter of our own. Finally, titration curves of enflurane effect in the mixed lipidic phase has been obtained by using the fluorescent lipid probe Laurdan. Measurements as a function of temperature or at constant temperature, i.e., 25 degrees C and 45 degrees C give, for the maximal effect, an enflurane-to-lipid ratio (M/M), within the membrane, of 1 and 2 for the L(alpha)(2) and the L(beta)(i) lamellar phase respectively. All the results taken together allowed to draw a pseudo-binary phase diagram of enflurane-dipalmitoylphosphatidylcholine in excess water.

FIGURE 1

Calorimetric curves of pure DPPC multilayers (lower trace) and of different enflurane/DPPC mixtures ([Lip]_tot = 20.3 mM). The R_tot are indicated on each curve. The dashed lines 1, 2, and 3 indicate the isothermal event detected on the calorimetric curves.

FIGURE 2

SAXS (a) and WAXS (b) patterns at 25°C for different DPPC-enflurane mixtures. The R_tot are indicated at the level of each trace: the superscript prime identifies traces measured on D43 line ([lip] = 61 mM). The others are those measured on D22 line ([lip] = 100 mM). Traces 0′ and 4′ are scale-expanded to make visible the higher order of Bragg peaks. (c and d) Long and short spacings as a function of R_tot. Unfilled and solid symbols correspond to measurements on D43 and D22 lines, respectively. (Circles, diamonds, and squares stand for L_β′^1NN, L_β′^2NNN, and L_βⁱ phases, respectively.)

FIGURE 3

Electron profiles calculated from the SAXS patterns of DPPC and Enf-DPPC (0′ and 4′ traces of Fig. 2). Electron profile of pure L_β′ DPPC lamellar phase (a) and of Enf-DPPC lamellar phase (b). A schematic representation of the lipid organization is also drawn on each panel. These schemes take into account the tilt orientation deduced from the WAXS patterns. The enflurane molecules are represented by a hatched box.

FIGURE 4

SAXS (a) and WAXS (b) patterns at 45°C on different DPPC-enflurane mixtures. The R_tot are indicated at the level of each trace. The superscript prime identifies traces measured on D43 line ([lip] = 61 mM). The others are those measured on D22 line ([lip] = 100 mM). (c) Long spacing as a function of R_tot. Unfilled and solid symbols correspond to measurements on D43 and D22 lines, respectively. (Circles and diamonds stand for L_α¹ and L_α² phases, respectively.)

FIGURE 5

SAXS, WAXS, and DSC of an enflurane-DPPC mixture at R_tot = 2.4 ([lip] = 100 mM).

FIGURE 6

SAXS (upper panels) and WAXS (lower panels) patterns of different enflurane/DPPC mixtures at different R_tot and temperatures. In both groups of panels, from left to right and top to bottom, the R_tot of the samples are 0, 0.4, 0.8, 1.2, 1.6, 2, 2.4, and 3.2. In each set of traces, the curves, from bottom to top, correspond to measurements at 15, 20, 25, 30, 35, 40, and 45°C. In some cases, inserts present a zoom on the first order of the Bragg peak to evidence coexistence of different lamellar structures.

FIGURE 7

Pseudobinary phase diagram built from x-ray and DSC data. The thermal events are represented by closed symbols whereas open symbols represent specific phases. The abscise of the phase diagram is the R_tot of the samples. ▾, T_on of the main transition; ▴, T_off of the main transition; ▪, chain melting temperature measured on WAXS patterns; and •, T_on of the L_β′–P_β′ transition. The structural information and symbolic representation of the different specific phases are given on the left of the diagram.

FIGURE 8

Evolution of the fluorescence intensity ratio I₄₉₀/I₄₃₅ of Laurdan. The different curves were obtained at constant [DPPC] = 0.85 mM and enflurane concentrations of 0 (+); 8.2 (▵); 13.7 (○); 27.5 (▴); 41.2 (+); 54.9 (♦); 68.7 (□); 82.4 (•); and 96.1 (▾) mM. The transition temperature is determined by the graphic tangent method as indicated on the figure for the curve obtained for pure DPPC liposomes. (Upper inset), Emission spectra of Laurdan (λ_ex = 370 nm) during an increase of temperature from 25 to 48°C at 0.22°C/min ([DPPC] = 0.85 mM and [Enf] = 13.7 mM). (Lower inset) Enflurane concentration-dependent shift of the T_on obtained from the fluorescence experiment at lipid concentrations of 1 mM (•), 2 mM (▾), and 7 mM (♦), and from DSC (▪, [Lip] = 20.3 mM).

FIGURE 9

(a) Fluorescence changes of Laurdan (expressed in percent of maximal change) as a function of [enflurane] at 25°C. The curves correspond to DPPC concentrations of 0.9 (•), 2.8 (▾), 5.8 (♦), 9.5 (□), and 12.5 (+) mM. (b) Relation between the enflurane concentrations required to reach 10, 20, 50, 70, and 90% of the maximum fluorescence change as a function of lipid concentration (see Table 4 and text for details).

FIGURE 10

(a) Fluorescence changes of Laurdan (expressed in percent of maximal change) as a function of [enflurane] at 45°C. The curves correspond to DPPC concentrations of 0.9 (•), 1 (▾), 5.7 (♦), and 9.7 (□) mM. (b) Relation between the enflurane concentrations required to reach 10, 20, 50, 70, and 90% of the fluorescence changes as a function of lipid concentration (see Table 4 and text for details).