Dipalmitoylphosphatidylcholine (DPPC: purity ≥ 99%), power amphotericin B (AmB: purity > 75%), cholesterol (Chol: purity ≥ 99%) and Tris(hydroxymethyl) aminomethane (Tris: purity ≥ 99.9%) were purchased from Sigma, USA. The other chemicals were of analytical grade and were used without further purification. DPPC and Chol (7:3, mol/mol) were dissolved in a chloroform/methanol mixture (9:1, v/v) to give a final concentration of 0.64 mM lipid membrane-forming solution. AmB (0.66 mM) was dissolved to a concentration of approximately 6 × 10⁻⁴ M in a 3:1 (v/v) mixture of dimethylformamide and 1 M HCl (aq). A subphase buffer composed of 10 mM Tris-HCl buffer, pH 7.4, was prepared for the Langmuir experiment. High-purity water was obtained from a Milli-Q plus water purification system (18.2 MΩ/cm, Millipore, USA). The concentrations of different metal cations were respectively 140 mM Na⁺, 5 mM K⁺, and 2.5 mM Ca²⁺ ions, and pH was 7.4.

The Langmuir monolayer is a most useful model membrane, which is employed in the research of the interaction of amphipathic molecules at the air–water interface in the perspective of physical chemistry.^[37] It has mainly two unique features: 1) area per molecule can be controlled precisely; 2) simplicity of the physical model.

Langmuir monolayers are prepared by the Langmuir film balance. A Langmuir trough (KSV-Mini trough, Finland) was employed to study the monolayer behavior at the interface, and the surface pressure was measured with a Wilhelmy-type tensiometer using filter paper (10 mm × 30 mm × 0.15 mm). The accuracy of the sensor was 0.1 mN/m. Teflon barriers can symmetrically compress or expand the monolayer at a certain rate. For all the experiments, the temperature was maintained at 20 ± 1 °C.

Before each run, wash the Teflon trough (trough size 323 mm × 75 mm × 5 mm) with ethanol and rinse with purified water. The drug or lipid solutions are added at the air–water interface by a Hamilton microsyringe. Spread solvent evaporation over 10 min, then compress the monolayers at the speed of 75 cm²/min and obtain the surface pressure–area per molecule curves. Repeat the experiment three times.

Prepare Z-Langmuir film at a certain surface pressure and observe the structure of membrane by AFM (Shimadzu, Japan). In the contacting mode, the dipping rate for transfer was 5 mm/s. Use the silicon nitride pyramidal tip mounted on a 100-upmum-long cantilever with a force constant of 0.1 N/m.

Information on phase transition of lipid monolayer can be obtained by the surface pressure–area per molecule curve (π–A isotherm).^[38] The elasticity modulus is obtained from the data in the π–A isotherm,^[39] which is calculated according to its definition^[40,41]

Whether or not these metal cations are in water, the π–A isotherms of the Chol/DPPC monolayers have no obvious inflection point, which suggests that the process of phase transition has not occurred (Fig. 1). However, after the addition of AmB, the significant changes exist in the π–A isotherms. First of all, the point of transition from liquid expanded state to liquid condensed state exist obviously on the α–A curves of the AmB/Chol/DPPC mixed monolayer in water or in the environment of Na⁺ ions, which are at surface pressure of 14.49 and 14.53 mN/m, respectively. Thus in the environment of K⁺ or Ca²⁺ ions, the phase transition point is not seen clearly, which indicates that the K⁺ and Ca²⁺ ions may influence the interaction between amphotericin B and lipid molecules in the liquid phase. Secondly, for any environment involved in the experiment, the π–A curve of the AmB/Chol/DPPC monolayer intersects that of the Chol/DPPC monolayer at one point. The point is at the surface pressure of 12.47 mN/m, 9.81 mN/m, 13.37 mN/m or 14.78 mN/m in the environment of water, K⁺, Na⁺ and Ca²⁺ ions, respectively. At the same surface pressure under the point of intersection, the addition of AmB increases the area per molecule of lipid monolayer. On the contrary, at the same surface pressure above the point of intersection, the addition of AmB decreases the area per molecule of lipid monolayer.

AmB can influence the physical phase of the Chol/DPPC monolayer, which also can be seen in AFM images of Fig. 2 (in water). The binary lipid mixed monolayer is in liquid-gel state at 15 mN/m. The lipid molecules closely arrange to form flat microdomains of monolayer. From analysis of Fig. 2(a), the thickness of the microdomains is about 1.35 nm in gel state, thus is about 0.33 nm in liquid state (dark spaces in AFM images). Thus after addition of AmB, the monolayer is at liquid state at the same pressure, which contains the phase transition from liquid expanded to liquid condensed phase (Fig. 2(b)). The thickness of the AmB/Chol/DPPC monolayer reaches about 0.94 nm in liquid condensed phase thus about 0.34 nm in liquid expanded state. The drug AmB induces a liquid condensed phase, which may be relative to its cellular toxicity. The microdomains on the monolayer in the liquid state are uniformly distributed. The reduction of the monolayer’s thickness may indicate the stretching of hydrophobic chains. The hydrophobic chains are straight in the gel phase but are curving in the liquid phase. AmB may alter the physical phase of lipid monolayer, which causes the existence of the liquid condensed phase of the monolayer and the phase transition. This may be due to the interaction between AmB and Chol molecules or the aggregation of AmB molecules on the lipid monolayer.

Fig. 1.

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	Fig. 1. Surface pressure–area per molecule curves and of the Chol/DPPC monolayer with and without AmB in water (a) and in the presence of different metal cations ((b) K+ ions; (c) Na+ ions; (d) Ca2+ ions).

Fig. 2.

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	Fig. 2. AFM images and high analysis of the Chol/DPPC monolayer (a) and the AmB/Chol/DPPC monolayer (b) at the air–water interface without any metal cations at 15 mN/m.

After addition of K⁺, Na⁺, or Ca²⁺ ions into the subphase solution, the Chol/DPPC monolayer has a significantly different change in expansion of the α = 1 curve (Fig. 3). In the environment of K⁺ ions, at above 13 mN/m, the π−A curve extended outward compared with that in water. The area of each molecule increases at the same pressure caused by K⁺ ions. The similar phenomenon appears at the surface pressure below 30 mN/m for Na⁺ ions and at the surface pressure above 8 mN/m for Ca²⁺ ions. Different metal cations may influence the area per lipid molecule on the monolayer, which also may be relative to the surface pressure.

However, for the AmB/Chol/DPPC monolayer, its phase transition changes significantly after the addition of these metal cations (Fig. 4). In water and in the environment of Na⁺ ions, phase transition obviously exists. Thus there are no clear transition points on the π–A curves in the environment of K⁺ and Ca²⁺ ions. These results also have been analyzed using the data of (see Fig. 1). The changes of extending degree on the curves are similar to that of the Chol/DPPC monolayer. These phenomenon suggests that different metal cations can influence the interaction between AmB and lipid molecules on the monolayer, which mainly leads to the existence of phase transition.

Fig. 3.

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	Fig. 3. Surface pressure–area per molecule isotherms of the Chol/DPPC monolayer in the environment of different metal cations compared with that in water ((a) K+ ions, (b) Na+ ions, (c) Ca2+ ions).

Fig. 4.

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	Fig. 4. Surface pressure–per molecule curves of the AmB/Chol/DPPC monolayer in the environment of different metal cations compared with that in water ((a) K+ ions, (b) Na+ ions, (c) Ca2+ ions).

The information on the intermolecular interaction can be acquired from the ΔG^exc of monolayer. It was calculated by the following equation for binary Langmuir monolayer:^[42,43]

The ΔG^exc values of the mixed monolayers are negative at 15 mN/m, which is close to the surface pressure of phase transition point. This supports that the interaction between the molecules is attraction. After addition of AmB, the absolute value of ΔG^exc decreases, which indicates the weakening of the interaction on the monolayer. The phenomenon may be due to the fact that an AmB molecule combines with a Chol molecule to form the AmB-cholesterol complex,^[44] which causes the formation of pore on the monolayer. Also in the environment of different metal cations, the attraction between the molecules in the two monolayer systems are also all weakened. The head group of AmB combines with the hydrophilic head of lipid by a van der Waals force. These head groups have a hydrophilic interaction with water molecules. After addition of the metal cations, they are combined with these head groups through a weak ionic bond, which is weaker than the hydrophilic interaction. The decrease of the van der Waals force between AmB and lipid molecules at the air–water interface may be due to the weak ionic bond.

Fig. 5.

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	Fig. 5. The ΔGexc of the monolayers at about the surface pressure of phase transition (15 mN/m) in water and in the environment of different metal cations.

However, the strengths of the force between the head group and various metal cations have many differences. On the Chol/DPPC monolayer, the strength of intermolecular interaction in the environment of potassium ions is almost as strong as that in the presence of Na⁺ ions and Ca²⁺ ions. Significantly different, on the AmB/Chol/DPPC monolayer, the force is the strongest in the environment of K⁺ ions, thus is the weakest in the environment of calcium ions. Metal cations interact with −COO⁻ group of AmB molecules. The interaction may be due to the weak ion pairs. The differences of these metal cations’ reactions may be due to their atomic scales. On the other hand, a large number of K⁺ ions distribute inside of cells thus most of the Na⁺ and Ca²⁺ ions exist outside of the cells. So when AmB molecules are outside of the cells, the reaction between the drug and lipid molecules is weaker than that inside the cells.

The simplest equation of state for the Langmuir monolayer is the two-dimensional ideal gas law (Eq. (1)).

If the monolayer can be compressed to a finite area A₀, the equation of state is given as follows:^[47]

Thus, the following equation is the two-dimensional virial equation of state:^[48]

For a mixed monolayer, the second virial coefficient is as follows:^[48]

In Fig. 6, all of (b₁)^E values are positive, which indicates an attractive force between the molecules on the two monolayer systems. After the addition of AmB, or in the environment of these metal cations, values of (b₁)^E decrease, which indicates that the force is weakened. The phenomenon may be due to the fact that an AmB molecule combines with a Chol molecule to form the AmB-cholesterol complex, which causes the formation of pores on the monolayer. The head group of AmB combines with the hydrophilic head of the lipid by a van der Waals force. These head groups have a hydrophilic interaction with water molecules. After addition of the metal cations, they are combined with these head groups through a weak ionic bond, which is weaker than the hydrophilic interaction. The decrease of the van der Waals force between AmB and lipid molecules at air–water interface may be caused by the weak ionic bond. These results from theory analysis agree with that from ΔG^exc.

Fig. 6.

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	Fig. 6. Excess virial coefficients of the Chol/DPPC monolayer with and without AmB obtained using theoretic fitting.