Phase Behavior Of Binary Monolayers Consisting Of Betulinic Acid And Different Molecules At Interface

Betulinic acid (BA) is one of the compositions of various plant cuticular waxes and exhibits multiple biological activities, including anticancer and antimalarial activities. BA can directly induce mitochondrial membrane damage and trigger the mitochondrial pathway of apoptosis in cancer cells. The antimalarial activity of BA has been linked to its ability to cause alteration of erythrocyte membrane shape. The role of BA in plant cuticular waxes and the influence of BA on mammal mitochondrial membrane and erythrocyte membrane are strictly related with the phase behavior of its monolayer. Thus, the phase behaviors of BA monolayer and binary monolayers consisting of BA and different molecules from plant cuticular wax, mammal mitochondrial membrane and erythrocyte membrane have been studied for the first time in this paper. The main points are the interactions between BA and other molecules, and the effects of the structure of BA on the phase behaviors of these monolayers. The details are summarized as follows.(1) Using Langmuir technique, the phase behavior of BA monolayer has been studied by surface pressure-area (π-A) measurement, collapse analysis and compare of two compression before and after collapse at the air-water interface. The molecular arrangement of BA monolayer and the effect of the structure of BA on the phase behavior have been investigated through contrast with stearic acid (SA), octadecanol (OD) and cholesterol (Chol) and by ChemBio3D Ultra modeling. The results indicate that all BA molecules take tilted orientation with carboxylic group towards water without compression, and that the phase behavior of BA monolayer can be divided into three parts with compression. Part 1:With surface pressure increasing from 0, more and more BA molecules take nearly perpendicular orientation with hydroxyl group towards water instead of tilted orientation. Part 2:With surface pressure increasing from 30 mN/m, the ratio of BA molecules with nearly perpendicular orientation increases continually with BA monolayer compression and carboxylic groups of neighbour nearly perpendicular BA molecules start to be involved in hydrogen bonds to form dimers. Part 3:With surface pressure higher than 48 mN/m, almost all BA molecules take nearly perpendicular orientation and exist in dimers. BA monolayer collapses at about 50 mN/m. Besides, BA monolayer at the air-water interface has been transferred to mica substrates through Langmuir-Blodgett deposition for atomic force microscopy (AFM) observation. The coexistence of two distinct domains with differet thickness at lower surface pressure corresponds to the different orientations of BA molecules. The thicker domains appear in pairs at higher surface pressure might be related with the formation of dimers between neighbour nearly perpendicular BA molecules.(2) The phase behaviors of BA/SA monolayers and BA/OD monolayers have been studied byπ-A measurement, miscibility analysis, thermodynamic stability analysis and compressibility analysis at the air-water interface. The interactions between BA and SA or OD, and the change of the structure of these binary monolayers with different BA contents and different compression degrees have been emphasized in this research. The influences of the different hydrophilic groups of SA and OD on the phase behaviors of BA/SA monolayers and BA/OD monolayers have been compared. It has been suggested that BA molecules can interact with SA or OD molecules at the air-water interface, and these binary monolayers are miscible. With smaller BA content and at lower surface pressure, different components tend to attract with each other, and the binary monolayers tend to be condensed, more stable thermodynamically, and more ordered. With larger BA content and at higer surface pressure, different components tend to repulse with each other, phase separation occurs, and the binary monolayers tend to be expanded and more disordered. These results indicate that small amount of BA can integrate into SA or OD domains and different components attract with each other to form more ordered structures, especially at lower surface pressure; and that it's hard for SA or OD to be incorporated into BA domains, especially at higher surface pressure. Comparing with BA/SA monolayers, the increase in the ordering of BA/OD monolayers is smaller, and with compression expansion, phase separation and repulsion occur earlier and are more evident in BA/OD monolayers. These results indicate that BA tends to interact with the short-chain saturated aliphatic compound with carboxylic group to form more stable monolayer at lower surface pressure.(3) The phase behavior of binary monolayers consisting of BA and cardiolipin (CL, a unique phospholipid found only in mitochondrial membrane in mammal) has been studied byπ-A measurement and analyses at the air-water interface and by AFM observation transferred to mica substrates. Miscibility analysis and ChemBio3D Ultra modeling present that in BA/CL monolayers BA takes both tilted and nearly perpendicular orientations at surface pressure below 30 mN/m but only nearly perpendicular orientation at 30 mN/m. Thermodynamic stability analysis indicates that phase separation and repulsion occur in BA/CL monolayers. Compressibility analysis demonstrates that at 30 mN/m,20 mol% BA does markedly translate the liquid-condensed (LC) CL monolayer to BA/CL monolayer with the coexistence of LC and liquid-expanded (LE) phases. AFM observation shows that CL molecules form a homogeneous condensed monolayer at 30 mN/m; that 20 mol% BA leads CL domains to form dense net-like structures; and that CL domains transfer to branch-like structures and the branches become slimmer and thinner with BA content increasing. These results confirm that at high surface pressure corresponding to biologic situation, BA orients nearly perpendicularly, causes phase separation and decreases the ordering of CL monolayer, which is in agreement with the mitochondrial membrane damage and permeabilization induced by BA in vitro and in vivo.(4) The phase behavior of binary monolayers consisting of BA and sphingomyelin (SM, cell membrane phospholipid) has been studied byπ-A measurement and analyses at the air-water interface using Langmuir technique. The main points are the interaction between BA and SM, and the influence of different BA contents on BA/SM monolayers. The phase behavior of BA/SM monolayers has also been compared with BA/Chol monolayers from the molecular structure perspective. The results indicate that BA/SM monolayers are miscible. Only 40 mol% BA increases the thermodynamic stability of BA/SM monolayers and different conponents attract with each other. With other BA content, phase separation occurs and different components repulse with each other, especially with predominant BA content. At high surface pressure corresponding to biologic situation,20 mol% BA induces a slight decrease of the ordering of SM monolayer, and 40 mol% BA results in serious damage of the structure of SM monolayer. Comparing with the increase in the stability and the ordering of SM monolayer induced by Chol, BA tends to disrupt SM monolayer.Besides the study of binary monolayers consisiting of BA and different molecules, the phase behavior of binary monolayers consisiting of paclitaxel and dipalmitoyl phosphatidylcholine (DPPC, cell membrane phospholipid) has also been studied at the air-water interface. The absorption of paclitaxel through cell membrane and the preparation of paclitaxel liposome are closely related to the interaction between paclitaxel and lipids. The interaction between paclitaxel and DPPC has been studied byπ-A measurement and analyses using Langmuir technique, and the microstructure of the monolayers has been investigated by AFM observation. The results present that paclitaxel/DPPC monolayers are miscible, phase separation occurs and different components repulse with each other, which is consistent with the disturbance of cell membrane fluidity by paclitaxel. With paclitaxel content no more than 40 mol%, paclitaxel has a little influence on DPPC monolayer; more than 40 mol% paclitaxel results in disruption of the structure of DPPC monolayer. With 40 mol% paclitaxel, miscibility, phase separation and repulsion are most apparent and increase with monolayer compression. With other paclitaxel content, all these states increase at first and then decrease with monolayer compression.