A Study of the Molecular Structure of Phospholipids and the Aggregation of Liposomes Using the Molecular Orbital Method
Toshihiko OGIHARA, Hiroshi KAGAWA, Qi GAO and Kazuhide MORI
Liposomes are closed vesicles surrounded by a lipid bilayer membrane known as the lamellae. Lipids are major components of biological membranes among which phospholipids are ubiquitous in various types of biological membranes. As a result, phospholipid liposomes are used as functional liposomes in model cells and microcapsules. In functional liposomes, the aggregation or fusion of phospholipid liposomes is controversial in terms of effectiveness and safety [1 - 3]. Phospholipid liposomes that aggregate in a gel state disaggregate into a state of liquid crystal with an increase in temperature. This phenomenon is probably because phospholipid liposome membranes cause heat fluctuations that arise when phospholipid molecules undergo a phase transition from a gel state to a state of liquid crystal.
On the other hand, polar lipids such as phospholipids are lyotropic liquid crystals; they take various shapes and states when subjected to variables such as moisture content, pH, or temperature. Phospholipid molecules are known for changing their structure when subjected to different conditions [4, 5]. Phosphatidylcholines (PC), which are typical phospholipids, have a dipole with a local electric field on their hydrophilic group moiety. Therefore, we hypothesized that the aggregation-disaggregation phenomenon of phospholipid liposomes is influenced not only by heat fluctuations caused by phase transition, but also by the electric charge at the surface of the liposomal membrane. Taking the dipole into consideration, the three-dimensional conformation of dipalmitoylphosphatidylcholine (C42H80NO10P, DPPC) was examined using the molecular orbital method.
In the molecular orbital method, the total energy of the DPPC molecules was calculated, and 3 local optimized structures were obtained. A comparative examination between these and structures obtained in other experiments, showed a good correlation between the structures. Furthermore, we studied the interactions between membranes caused by electric forces between liposomes, with a focus on the changes of position and direction of the dipole on the hydrophilic group moiety.
On the basis of these facts, we concluded that the examination of local optimized structures by the molecular orbital method might contribute to the analysis of the molecular structure of phospholipids, the study of the interactions between membranes, and eventually the study of functional liposomes.
2 Materials and Methods
2. 1 Observation of the aggregation-disaggregation phenomena
To bring the lipid concentrations of DPPC (Sigma) to 10 mg/mL, water was added, and the dispersion liquids of the phospholipids were adjusted in test tubes. The dispersion liquids were placed in a waterbath for 15 min at a temperature of 50 °C, which was above the main transition temperature (Tc, 42 °C) , and multilamellar liposomes were prepared by performing ultrasonication treatment for 1 min at the same temperature. Subsequently, after they cooled to room temperature, the diameters of the multilamellar liposomes were measured with a microscope; the larger liposomes measured 1-3 mm whereas most were in the order of sub-mm.
Several drops of the preparation were dripped onto a hole slide glass, covered with a cover glass, and observed under an optical microscope (Olympus BH-2) at a controlled temperature. The temperature was increased and decreased at a speed of 1.0 °C/min, and the temperature range of observation was from below the subtransition temperature (Ts, 15-20 °C)  to above the main transition temperature (Tc).
2. 2 Calculations using the molecular orbital method
MOPAC input files of DPPC (C42H80NO10P, Figure 1) structure were created using FreeWheel.
Figure 1. Structural formula of phosphatidylcholines. In the case of DPPC, "..." indicates a chain of 12 CH2s.
The molecules had a complex structure which varied significantly from the optimized structure obtained under the initial conditions. Therefore, because the fatty acid moieties were linear and showed no significant variation, all atoms were optimized after rotating the A, B, and C axes of the glycerine moiety as shown in Figure 2; various structures were obtained through calculations using the AM1 method in MOPAC97 [8, 9], and the 3 lowest-energy structures (namely those with the maximum probability of presence) were determined.
Figure 2. Rotational axes A, B, and C for obtaining the lowest-energy structures on DPPC.
First, the phosphoric acid and choline moieties were rotated 360 ° by steps of 5 ° around the A axis (rotation was also performed in the reverse direction), and the total energy of the electrons in the optimized structure was calculated. A comparison with structures showing minimal values revealed that 2 low-energy structures were obtained; consequently, similar calculations were conducted using these as the starting structure around the B axis. Around the B axis, 3 types were chosen, on the basis of which similar calculations were conducted around the C axis, and their structures were examined.
3. 1 The aggregation-disaggregation phenomena
When the samples which aggregate at room temperature (Figure 3(a)) were placed at higher temperatures, a disaggregation of liposomes was observed at Tc (Figure 3(b)). When the temperature was decreased, reaggregation was observed at temperatures below Tc, and large colonies gradually formed again. These phenomena were reversible.
Figure 3. Light micrographs of dipalmitoylphosphatidylcholine (DPPC) liposomes in water (10 mg/mL) on heating (1.0 °C/min) from below Ts (subtransition temperature, approximately 15-20 °C) to above Tc (main transition temperature, 42 °C). (a) Colonies of liposomes formed by aggregation at 25 °C. (b) Disaggregation state at 43 °C. Size of images: 180 mm × 120 mm.
3. 2 Results of the calculations according to the molecular orbital method
As a result of the calculations, 3 lowest-energy structures were achieved, and the structures are shown in Figure 4 and Figure 5 according to the ascending order of energy values (637.341724, 636.469886 and 636.065059 kcal/mol).
PCs are typical phospholipids; it is known that depending on the temperature, they attain an Lc phase at temperatures below the Ts (subtransition temperature), attain an Lb' phase at temperatures above the Ts but below the Tp (pretransition temperature), and attain a Pb' phase at temperatures above the Tp but below the Tc (main transition temperature). At temperatures higher than the Tc, they change into a state of liquid crystal (La phase), and their acyl groups melt.
At temperatures above the Tc, the DPPC molecules change into a state of liquid crystal and do not have a stable structure; therefore, the 3 optimized structures that were found by molecular orbital calculations were considered to correspond to the Lc, Pb', and Lb' phases.
It has been reported  that in the Lc phase which occurs after a prolonged maintenance of PC at 0 °C, the long axis of the phospholipid molecules is slanted (at the inclined angle of hydrocarbon chains) relative to the lamellae which constitute the membranes. Among the structures found by using the molecular orbital method, the third lowest energy molecular structure, which had the biggest inclined angle of hydrocarbon chains (Figure 4(c)) was considered to correspond to this phase.
Figure 4. Representations in which the dipoles (red arrows) are turned sideways as seen longest and the acyl groups are turned perpendicularly. (a) 1st lowest structure, (b) 2nd lowest structure, (c) 3rd lowest structure. The inclined angle between hydrocarbon chains and dipole: (a) 97°, (b) 82°, (c) 121°. Strength of dipole: (a) 12.125 debye, (b) 12.680 debye, (c) 12.747 debye.
For the Lb' phase, the inclined angle of hydrocarbon chains was smaller than that of the Lc phase , therefore the first lowest-energy structure, which was found by using the molecular orbital method (Figure 4(a)) was considered to correspond to this phase.
Lamellae are known to increase in length in the Pb' phase ; however, if we look at the second lowest-energy structure (Figure 4(b)) among the 3 optimized structures, its hydrophilic group moieties increase in length longitudinally along the PC and elongate the PC molecules. This fact confirmed that the second lowest-energy structure (Figure 4(b), Figure 5(b)) corresponded to the Pb' phase.
Regarding the angle of inclination of the molecular chain in the optimized structures, it was hypothesized and found in Figure 3 that the dipole of the PC was parallel to the surface of the lamella, and the 3 lowest-energy optimized structures matched well with the results of other experiments.
Figure 5. Representations in which DPPCs of Figure 4 are rotated 90 degrees as the dipoles (red arrows) face in the front centering around the axes of the acyl groups keeping the acyl groups perpendicular. (a) 1st lowest structure, (b) 2nd lowest structure, (c) 3rd lowest structure.
Compared to the Lb' phase, the polar groups were more tightly bound in the Lc phase . This could also be confirmed from the strength of dipole (Figure 4(a),(c)). Therefore, the electric force at the surface of the molecules was stronger in the Lc phase, leading to a hypothesis that the aggregation force between the liposomes was also strong.
In the Pb' phase, there was a wavy lamellar structure which was said to change depending on the status of hydration . In the Pb' phase, the direction of the dipole became reversed (Figure 5 (b)). The fact that the structure allowed water molecules to penetrate not only from the top of the hydrophilic groups but also transversely, led to the supposition that it might be influenced by hydration.
The speculative theory about the variations of the surface electric charge caused by the difference of position and direction of the dipole in the optimized structures could also complement other findings.
From these facts, it appeared that examination of the molecular structure of phospholipids using the molecular orbital method is effective in the elucidation of the structure and functions of phospholipid liposomes. In future, we plan to build a model involving hydration to examine the relationship between the surface charge and the aggregation/disaggregation of phospholipid liposomes, in other words, the interactions between the membranes caused by the surface charge.
We are grateful to Prof. Kazuhide Mori who has provided us with much knowledge and instruction for a long time. He has recently taken an interest in biophysics and related fields, and we were blessed with the opportunity of researching and discussing with him. We have been pleased with the discussions we had with him. In our hearts, we have not yet accepted that we will no longer be able to spend time drinking and conversing with him. Even after this thesis, we would like to collaborate with his mind in our future work. We wish him happiness with all our heart.
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