Effects of Nicotine on the Thermodynamics and Phase Coexistence of Pulmonary Surfactant Model Membranes

Abstract

Phase separation is essential for membrane function, and alterations in phase coexistence by membrane-interacting molecules, such as nicotine, can impair membrane stability. With the increasing use of e-cigarettes, concerns have arisen about the impact of nicotine on pulmonary surfactants. Here, we used differential scanning calorimetry (DSC), molecular dynamics (MD) simulations, and electron spin resonance (ESR) to examine nicotine's effect on the phase coexistence of two surfactant models: pure DPPC and a DPPC/POPC/POPG mixture. Our DSC analysis revealed that nicotine interacts with both membranes, increasing enthalpy and entropy change during the phase transition. ESR revealed that nicotine affects membrane fluidity and packing of DPPC more effectively than the ternary mixture, especially near the surface. MD simulations showed that neutral nicotine resides in the mid-plane, while protonated nicotine remains near the surface. Nicotine binding to the membranes is dynamic, switching between bound and unbound states. Analysis via ESR/van't Hoff method revealed changes in the thermodynamics of phase coexistence, yielding distinct non-linear behavior. Nicotine altered the temperature dependence of the free energy, modifying the thermodynamic driving forces and the balance of non-covalent lipid interactions. These findings provide new insights into how nicotine influences pulmonary surfactant model membranes, with potential implications for surfactant function.

Keywords: DSC; ESR; MD simulations; drug-membrane interactions; nicotine; phase coexistence; pulmonary surfactant; van’t Hoff.

Figure 1

Thermotropic phase behavior of the surfactant model membranes as monitored by DSC. (A) Temperature dependence of the molar heat capacity of DPPC in the absence and presence of different concentrations of nicotine at pH 7.4. The inset illustrates the DPPC pretransition. (B) Effect of nicotine on the enthalpy change ($\Delta H$, circles) and the melting temperature ($T_m$, triangles) of DPPC lipid vesicles. (C) Temperature dependence of the molar heat capacity of DPPC/POPC/POPG in the absence and presence of 10 mol% of nicotine at different pH values. (D) Impact of nicotine on the enthalpy change ($\Delta H$) of the lipid mixture.

Figure 2

ESR spectral changes induced by nicotine. Representative ESR spectra of (A) 5-PCSL and (B) 16-PCSL in DPPC in the absence (black) and presence (red) of 10 mol% of nicotine at selected temperatures. The maximum hyperfine splitting, $2A_{max}$, is defined as the distance between the high-field minimum and the low-field maximum on the 5-PCSL spectrum. ESR samples were prepared using a pH 7.4 buffer.

Figure 3

NLLS spectral simulations of ESR spectra displaying characteristic two-component features. Experimental (black) and best-fit (red) ESR spectra of 16-PCSL in (A,B) DPPC and (C,D) DPPC/POPC/POPG at selected temperatures and at pH 7.4. The fitting of the signals used one (top) or two (bottom) spectral components, the latter shown in green and blue lines. Gray lines represent the difference between the experimental and best-fit spectra. Arrows point to spectral features characteristic of a second spin population displaying different ordering or mobility.

Figure 4

Thermotropic phase behavior of the membranes as monitored by ESR. Temperature dependence of the rotational diffusion rate $R_{\perp }$ (A,C,E) and the order parameter S (B,D,F) of 5-PCSL (A,B) and 16-PCSL (C–F) in DPPC (C,D) and DPPC/POPC/POPG (E,F) in the absence (full squares) and presence of nicotine at different concentrations: 2 mol% (empty circles), 4 mol% (full triangles), and 10 mol% (empty diamonds). Regions highlighted between dashed lines correspond to the phase coexistence. All experiments were performed using a pH 7.4 buffer.

Figure 5

Nonlinear van’t Hoff behavior of pulmonary surfactant model membranes. Van’t Hoff plots illustrating the thermodynamic profiles of (A) pure and nicotine-enriched DPPC, and (B) pure and nicotine-embedded DPPC/POPC/POPG. The solid lines best fit the van’t Hoff plots, yielding cubic functions for DPPC and quadratic functions for the ternary model membranes. The molar percentage of nicotine relative to the amount of lipids is indicated. The adjusted-R² values obtained from the non-linear least-squares fitting ranged from 0.985 to 0.997 for DPPC curves and from 0.991 to 0.999 for the ternary mixture curves.

Figure 6

Thermodynamics of the phase coexistence region of DPPC and DPPC/nicotine multilamellar vesicles. Temperature-dependence of the changes in the (A) Gibbs free energy, ΔG, (B) enthalpy, ΔH, (C) entropy, ΔS, and (D) heat capacity, ΔC for pure DPPC (full squares) and nicotine-containing membranes at two concentrations: 4 mol% (full triangles), and 10 mol% (empty diamonds). The solid lines are guides for the eye.

Figure 7

Thermodynamics of the phase coexistence region of nicotine-free and nicotine-containing DPPC/POPC/POPG multilamellar vesicles. Temperature-dependence of the changes in the (A) Gibbs free energy, ΔG, (B) enthalpy, ΔH, (C) entropy, ΔS, and (D) heat capacity, ΔC for pure DPPC/POPC/POPG (full squares) and nicotine-containing membranes at different concentrations: 2 mol% (empty circles), 4 mol% (full triangles), and 10 mol% (empty diamonds). The solid lines are guides for the eye.

Figure 8

Spatial distribution of nicotine in membranes revealed by molecular dynamics simulations. Symmetrized atom density profiles with respect to the lipid bilayer normal for lipids, water, and both neutral (NTN, left panels) and monoprotonated (NTH, right panels) nicotine. Panels (A,B) depict the results for DPPC at 37 °C, panels (C,D) at 45 °C, while panels (E,F) represent the data for the lung surfactant model membrane (DPPC/POPC/POPG) at 37 °C. The numbers 3, 7, and 13 following the designation of each system on the top of the panels refer to the number of nicotine molecules in the system. To accommodate the varying scales, the upper graph in each panels displays the density profile for lipids and water, while the lower graph illustrates the nicotine distribution.

Figure 9

Radial distribution functions between the pyrrolidyl nitrogen of nicotine and the phosphorus atoms of lipid phosphate groups in (A) DPPC and (B) DPPC/POPC/POPG systems. Panel (A) shows the RDFs for NTN and NTH in DPPC bilayers at 37 °C (left) and 45 °C (right). Panel (B) illustrates the RDFs for NTH (left) and NTN (right) in the lung surfactant model membrane, highlighting the contributions from the phosphate groups of DPPC, POPC, and POPG phosphate groups.