Molecular Interactions between a Lipid and an Antineoplastic Drug

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Molecular Interactions between a Lipid and an Antineoplastic Drug Paclitaxel (Taxol) within the Lipid Monolayer at the Air/Water Interface Si-Shen Feng,*,†,‡ Ke Gong,† and Jolynn Chew† Department of Chemical and Environmental Engineering, and Division of Bioengineering, National University of Singapore, 10 Kent Ridge Crescent, Singapore 119260 Received October 12, 2001. In Final Form: February 4, 2002 The lipid monolayer at the air/water interface was used to simulate large bilayer vesicles and the cell membrane in characterization of molecular interactions between the phospholipid and an antineoplastic drug paclitaxel (taxol), which is one of the best anticancer drugs found from nature in the past decades. The mixed lipid/paclitaxel monolayers of various molar ratios were formed at the air/water interface in a Langmuir trough, and the surface pressure versus molecular area isotherms were measured upon compression of the monolayers. It was found from the analysis of membrane mechanics and thermodynamics that the two components were miscible and formed a nonideally mixed monolayer at the air/water interface. It was shown that introduction of paclitaxel into the dipalmitoyl phosphocholine (DPPC) monolayer caused instability of the monolayer, which was due to the molecular interactions between the two components. Similar results were also obtained by Fourier tranform infrared (FTIR) spectroscopy and atomic force microscopy (AFM) investigation of the mixed monolayers, which were deposited onto a solid surface at a desired surface pressure. The FTIR analysis showed that introduction of paclitaxel into the lipid monolayer increases the mobility of the acyl chains of the lipid, resulting a fluidizing effect on the monolayer. Paclitaxel increases the population of trans conformers at low surface pressures and the proportion of gauche conformers at high surface pressures. The AFM analysis demonstrated that introduction of paclitaxel into the lipid monolayer reduces the extent of liquid-condensed phases and causes a reorganization of microdomains of the monolayers.

Introduction Paclitaxel (Taxol) was first found as a natural product from the Western yew tree, Taxus brevifolia.1 It has been recognized as one of the most promising antineoplastic drugs for treatment of a wide spectrum of cancers such as breast, ovarian, colon, and lung cancer as well as melanoma and lymphoma.2 Paclitaxel is able to decrease the critical concentration and the induction time required for polymerization of tubulin to form microtubulin during cell multiplication, which leads to cell death.3 Paclitaxel is a complex diterpenoid, which consists of a rigid taxane ring and a flexible side chain (Figure 1). Because of its poor solubility in water and most pharmaceutical solvents, an adjuvant such as Cremophor EL (50% polyoxyethylated castor oil and 50% dehydrated alcohol) has to be used in current clinical administration of paclitaxel, which often causes serious side effects. Side effects caused by Cremophor EL include hypersensitivity reactions, nephrotoxicity, and neurotoxicity. Also, Cremophor EL has influence on the functions of endothelial and vascular muscle and causes vasodilation, labored breathing, lethargy, and hypotension.4-9 Alternative dosage forms have been developed to improve its clinical administration, * To whom correspondence should be addressed. Phone: (65) 68743835. Fax: (65) 67791936. E-mail: [email protected]. † Department of Chemical and Environmental Engineering. ‡ Division of Bioengineering. (1) Wani, M. C.; Taylor, H. L.; Wall, M. E.; Coggon, P.; McPhail, A. T. J. Am. Chem. Soc. 1971, 93, 2325. (2) Rowinsky, E. K.; Cazenave, L. A.; Donehower, R. C. J. Natl. Cancer Inst. 1990, 82, 1247. (3) Horwitz, S. B.; Fant, J.; Schiff, P. B. Nature 1979, 277, 665. (4) Weiss, R. B.; Donehower, R. C.; Wiernik, P. H.; Ohnuma, T.; Gralla, R. J.; Trump, D. L.; Baker, J. R., Jr.; Van Echo, D. A.; Von Hoff, D. D.; Leyland-Jones, B. J. Clin. Oncol. 1990, 8, 1263. (5) Rowinsky, E. K.; Onetto, N.; Canetta, R. M.; Arbuck, S. G. Semin. Oncol. 1992, 19, 646.

Figure 1. Molecular structure of paclitaxel.

which include liposomes,10 mixed micelles,11 polymeric microspheres12 and nanospheres,13 cyclodextrin complexes,14 and parenteral emulsions.15 Among these drug delivery systems, a liposomal formulation for controlled and targeted delivery of paclitaxel has aroused considerable interest. However, molecular interactions between paclitaxel and phospholipids within the lipid bilayer membrane have rarely been investigated in detail. Such interactions may lend insight not only into the therapeutic performance of the paclitaxel-liposome formulation but also into other novel mechanisms of action and cellular effects of paclitaxel.16 (6) Webster, L.; Linsenmeyer, M.; Millward, M.; Morton, C.; Bishop, J.; Woodcock, D. J. Natl. Cancer Inst. 1993, 85, 1685. (7) Fjallskog, M.; Frii, L. L.; Bergh, J. Lancet 1993, 342, 876. (8) Dorr, R. T. Ann. Pharmacother. 1994, 28, S11. (9) Kongshaug, M.; Cheng, L. S.; Moan, J.; Rimington, C. Int. J. Biochem. 1991, 23, 473. (10) Sharma, A.; Straubinger, R. M. Pharm. Res. 1994, 11, 889. (11) Alkan-Onyuksel, H.; Ramakrishnan, S.; Chai, H. B.; Pezzuto, J. M. Pharm. Res. 1994, 11, 206. (12) Wang, Y. M.; Sato, H.; Horikoshi, I. Chem. Pharm. Bull. 1996, 44, 1935. (13) Feng, S. S.; Huang, G. F. J. Controlled Release 2001, 71, 53. (14) Sharma, U. S.; Balasubramanian, S. V.; Straubinger, R. M. J. Pharm. Sci. 1995, 84, 1223. (15) Tarr, B. D.; Sanbandem, T. G.; Yalkowsky, S. H. Pharm. Res. 1987, 4, 62.

10.1021/la011545p CCC: $22.00 © 2002 American Chemical Society Published on Web 04/17/2002

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There are a few publications for the paclitaxel-lipid interactions investigated by various physical methods by using lipid bilayer vesicles (liposomes) as a model membrane.17,18 However, there has been no research on paclitaxel-lipid interactions by the lipid monolayer model. Compared with the bilayer, the monolayer at the airwater interface is easier to manipulate and experiments can be carried out at various conditions such as membrane composition, temperature, and ionic concentration. The Langmuir monolayer technique is powerful in conducting membrane compression and penetration analysis.19,20 Information obtained from the monolayer can then be interpreted by membrane mechanics and thermodynamics for the drug-lipid interaction properties within the bilayer membrane by considering the lipid monolayer-bilayer correspondence theory.21-24 Traditionally, the measurement of the surface pressure (π) versus molecular surface area (A) isotherm of the monolayer at the air/water interface has been applied to investigate the interfacial behavior and lipid/drug interactions.25,26 In this approach, the lipid/drug mixture is spread over a subphase (usually water or buffer) to form a monolayer in a Langmuir trough. The monolayer is then compressed, and the π-A isotherms of the monolayer are recorded. Various mechanochemical and thermodynamic properties can then be either directly measured (collapse pressure, phase transition temperature, compressibility or its reverse, area compression modulus, etc.) or theoretically deduced (free energy, chemical potential and surface activity of the lipid, etc.). Furthermore, the mixed monolayer formed at the air/water interface can be deposited by the Langmuir-Blodgett (LB) technique onto a molecularly smooth solid surface such as a mica surface. The supported mixed monolayer can then be investigated by various techniques to know its structure, morphology, surface chemistry, and so forth. The modern techniques used should include light/X-ray scattering, scanning electron microscopy (SEM), atomic force microscopy (AFM), transmission electron microscopy (TEM), Fourier transform infrared resonance (FTIR), X-ray photoelectron spectroscopy (XPS), and so forth. Unfortunately, no report on the application of them to the paclitaxel-lipid interaction can be found. In the present paper, the miscibility of paclitaxel with the phospholipid within the monolayer at the air/water interface was investigated by the Langmuir trough technique and other physical techniques. The stability of the mixed paclitaxel/lipid monolayers at various molar ratios was analyzed by applying membrane mechanics and thermodynamics. The effects of paclitaxel on the mechanochemical properties of the lipid monolayer such as the collapse pressure and the membrane compressibility were quantitatively obtained. The results were further revealed by FTIR and AFM investigation of the supported monolayers. (16) Ding, A. H.; Porteu, F.; Sanchez, E.; Nathan, C. F. Science 1990, 248, 370. (17) Balasubramanian, S. V.; Straubinger, R. M. Biochemistry 1994, 33, 8941. (18) Bernsdorff, C.; Reszka, R.; Winter, R. J. Biomed. Mater. Res. 1999, 46, 141. (19) Briggs, M. S.; Gierasch, L. M.; Zlotnick, A.; Lear, J. D.; DeGrado, W. F. Science 1985, 28, 1096. (20) Rosiliom, V.; Boissonnade, M. M.; Zhang, J.; Jiang, L.; Baszkin, A. Langmuir 1997, 13, 4669. (21) Nagle, J. F. Faraday Discuss. Chem. Soc. 1986, 81, 151. (22) Ja¨hnig, F. Biophys. J. 1984, 46, 687. (23) Marsh, D. Biochim. Biophys. Acta 1996, 1286, 183. (24) Feng, S. S. Langmuir 1999, 15, 998. (25) Dhathathreyan, A. Colloids Surf., A 1993, 81, 269. (26) Wiedmann, T. S.; Jordan, K. R. Langmuir 1991, 7, 318.

Feng et al.

Although only a specific drug, paclitaxel, and a specific lipid, DPPC (1,2-dipalmitoyl-sn-glycerol-3-phosphocholine), were concerned in the present paper, the methodology should be applicable to other drugs and other lipids of various chain length and chain unsaturation as well as membranes of more complicated structure such as the lipid/cholesterol and lipid/intramembrane protein monolayers and bilayers. Materials and Methods Materials. The lipid, DPPC, was purchased from Sigma (St. Louis, MO). The lipid was of highest available purity and used without further purification. The solvents used are chloroform and methanol of analytical grades, which are also purchased from Sigma. Paclitaxel, molecular weight 853.9, was obtained from Yunan Hande Biotech Inc., China (FDA approved). The purity is higher than 99.9%. The stock solution of lipid and paclitaxel in chloroform and methanol was conserved in a vacuumed desiccator at -20 °C. The phosphate buffer solution (PBS) of pH 7.4 was obtained from Sigma. Ultrapure water was obtained from a Milli Q Plus system (Millipore, France). Both surface pressure and surface tension were measured at 25 °C. The film spreading was carried out with the help of a microsyringe (Hamilton, Reno, NV). All glassware in contact with the samples was exhaustively rinsed with purified water. Langmuir Tough. The computer-controlled Langmuir film balance used in this research is a Nima 601M Langmuir-Blodgett trough manufactured by NIMA Technology Ltd. (Science Park, Coventry, U.K.). The measurement resolution is (0.1 mN/m, and the inaccuracy for surface area regulation is less than 1%. The Teflon trough (15 × 7 × 0.5 cm) with a 25 mm dipper stroke is mounted on an aluminum base plate with built-in water channels for temperature control of the subphase. Before each experiment, the trough was washed with ethanol and rinsed with purified water. In all experiments, the trough was filled with 60 mL of phosphate buffer solution as the subphase and the temperature was controlled at 25 ( 0.1 °C by an external circulator. The air/water interface was repeatedly compressed and expanded symmetrically at a desired rate by moving the two barriers. The cleanness of the trough and subphase was ensured before each run by cycling the full range of trough area and aspirating the air/water surface at the minimal surface area. When the surface pressure fluctuation was less than (0.2 mN/m during the compression of the entire surface area range, a sample was then spread on the water surface with a Hamilton microsyringe and 15 min was allowed for the solvent to evaporate before the experiment was started. The monolayer was spread by using chloroform as a solvent. Lipid and paclitaxel stock solutions were prepared at equimolar concentrations of 0.2 mM. The mixture of different molar ratios of paclitaxel/DPPC was prepared from the stock solution. In each measurement, an equal volume (60 µL) of solution was spread on the PBS subphase. Before each measurement, the trough was cleaned thoroughly with chloroform and Milli Q water. The compression rate was 1 Å2 molecule-1 min-1. FTIR Spectroscopy. After cleaning the trough, as described before, a clean CaF2 window was placed vertically into the trough. After adding PBS subphase, the window was then lowered to its maximum. The lipid monolayer was spread as before and compressed to a desired value of the surface pressure. After the formed monolayer has been stabilized, the CaF2 window was slowly withdrawn upward at a pulling rate of 5 mm/min. In this manner, a piece of monolayer could be transferred onto the hydrophilic CaF2 window. Freshly transferred films were immediately used for FTIR analysis. Infrared transmission spectra were collected by using a BioRad Fourier transform spectrometer (Galatic Industries Corp.) operating on Bio-Rad Win-IR Foundation version 4.14. A spectrum was obtained by coadding 50 interferograms at a spectral resolution of 4 cm-1 and triangular apodized. Individual peaks were correlated by IR Mentor Pro version 2.0 from BioRad (Sadtler Division). AFM. The atomic force microscope used is the MultiMode scanning probe microscope manufactured by Digital Instruments

Interactions between a Lipid and Paclitaxel

Langmuir, Vol. 18, No. 10, 2002 4063

can be calculated using the equation

Aπ,ideal ) X1(A1)π,ideal + (1 - X1)(A2)π,ideal

Figure 2. Surface pressure vs molecular area isotherms of the DPPC/paclitaxel monolayers of various molar ratios at 25 °C. Inc. (Santa Barbara, CA). The LB technique was employed to prepare the supported monolayer for AFM investigation. The procedure was similar to that for FTIR study. The difference was that the mica sheet had to be cleaved to obtain an atomically flat surface. The monolayer-coated mica chip was then trimmed to be 0.3 × 0.7 cm in size and mounted onto the atomic force microscope for scanning. Tapping mode was used, and typical scan rates ranged from 1 to 3 Hz, depending on the scan size. Scans were made at various parts of the mica surface. The experiment was then repeated with fresh samples.

Results and Discussion Miscibility of Paclitaxel with the DPPC Monolayer. The surface pressure versus molecular area isotherms for the DPPC/paclitaxel monolayers on phosphate buffer subphase at 25 °C are shown in Figure 2. The π-A isotherm for the DPPC monolayer has been studied extensively elsewhere.27 It is clearly characterized by four phases: gas, liquid-expanded (LE), liquidcondensed (LC), and solid state. Transition from LE to LC state for DPPC is known as a first-order transition and is marked by the appearance of a distinctly more ordered phase than the others. The collapse pressure of the DPPC monolayer is 53 mN/m. The isotherm of the paclitaxel monolayer shows a collapse point at 13 mN/m, which is much lower than that of the DPPC monolayer. This is quite reasonable since paclitaxel molecules have a few rigid carbon rings in their structures (Figure 1). Phase transition of the paclitaxel monolayers is not noticeable in the full compression range. The collapse surface pressure is one of the important parameters for a mixed monolayer. The isotherm of a monolayer consisting of two immiscible components will show two distinct collapse pressures corresponding to those of the two pure component monolayers, which are independent of the composition of the mixed monolayer.28 If the two components are miscible in the monolayer, however, the collapse surface pressure of the mixed monolayer is dependent on the composition of the mixed monolayer. From Figure 2, the collapse pressures of the DPPC/paclitaxel monolayers of molar ratios 8:2, 5:5, and 2:8 are 50, 28, and 19 mN/m, respectively. It is clear that the collapse pressure of the mixed monolayers with different molar ratios has a close relation to the composition. It can thus be concluded that paclitaxel is miscible in the DPPC monolayer. The ideal mean molecular area (Aideal) of a twocomponent monolayer at a given surface pressure (π) (27) Mingotaud, A.-F. Handbook of Monolayers; Academic Press: San Diego, 1993; pp 786-787. (28) Wu, S.; Huntsberger, J. R. J Colloid Interface Sci. 1969, 29, 138.

(1)

where X1 is the mole fraction of component 1 (DPPC) and (A1)π,ideal and (A2)π,ideal are the mean molecular areas of the pure monolayers of components 1 and 2 at an identical surface pressure. The Aπ actually measured from the π-A isotherm of the mixed monolayer at different mole fractions X1/X2 may be different from the calculated value of Aπ,ideal. Their difference defines either an area condensation or area expansion effect of component 2 (here paclitaxel) on the pure monolayer of component 1 (here DPPC). Such an analysis can be done if the measurement of the π-A isotherm of the mixed monolayer is more sensitive in molecular area than in surface pressure. For example, the area condensation effects of cholesterol on lipid monolayers were investigated in this way.29 However, if the measurement of the π-A isotherm of the mixed monolayer is more sensitive in surface pressure than in molecular area as in the present case, it is more practical to investigate the π-A behavior of the mixed monolayer from the pressure increment/decrement effect at given values of molecular area. The ideal value πA,ideal of surface pressure of the mixed monolayer at any given molecular area A can be calculated from those found from the two isotherms of the pure monolayers at the same given area A:

πA,ideal ) X1(π1)A,ideal + (1 - X1)(π2)A,ideal

(2)

where (π1)A,ideal and (π2)A,ideal are the surface pressures of the component 1 monolayer, that is, the pure DPPC monolayer, and the component 2 monolayer, that is, the pure paclitaxel monolayer, at a given molecular area A. The molecular interaction between paclitaxel and lipid can then be quantitatively analyzed by the difference between the calculated value πA,ideal and the actual value πA,exp measured from the mixed monolayer at the same given molecular area A:

∆π ) πA,exp - πA,ideal

(3)

The paclitaxel-induced surface pressure change at specific values of molecular area (90, 80, 70, 60, 50, 40, 30 Å2) was measured. The result is given in Figure 3, from which it can be found that the ability of paclitaxel to alter the lateral compressibility of the DPPC membrane depends on the monolayer phase state. At large molecular areas, for example, 90 and 80 Å2/molecule (LE phase) and 70, 60, and 50 Å2/molecule (LC phase), the experimental values of surface pressure of the mixed monolayers of various molar ratios of paclitaxel are higher than their corresponding ideal value. This means that paclitaxel produces a pressure increment effect on the DPPC monolayers in the domain of large molecular area. In the domain of small molecular areas, for example, 40 and 30 Å2/molecule, however, the measured surface pressure of the mixed monolayer is smaller than the ideal values. Therefore, paclitaxel produces a pressure decrement effect on the DPPC monolayer when closely packed at the interface. Another important parameter for a mixed monolayer is the excess area Aex, which can also indicate the miscibility of the two components in the mixed monolayer. The excess area is defined as the difference between the measured value of molecular area of the mixed monolayer (29) Smaby, J. M.; Brockman, H. L.; Brown, R. E. Biochemistry 1994, 33, 9135.

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measured molecular area is always larger than the ideal value for the mixed DPPC/paclitaxel monolayer. An areaexpanding effect of paclitaxel on the DPPC monolayer can thus be concluded. The possible reason for the areaexpanding effect of paclitaxel may be the bulky structure in the geometric conformation of its molecules. The excess area in all three cases of constant pressure of π ) 5, 10, and 12 mN/m has a maximum at paclitaxel molar fraction 0.5-0.6, for which the area expansion effect of paclitaxel on the DPPC monolayer is most significant. Compressibility of Paclitaxel/DPPC Monolayers. The two-dimensional compressibility Cs of a monolayer at a given molecular area, or equivalently at a given surface pressure, is defined as the partial change of the area strain with respect to the surface pressure, that is,

Cs ) (-1/A)(dA/dπ)

Figure 3. Surface pressure of the DPPC/paclitaxel monolayer versus percentage of paclitaxel at various molecular areas A (Å2).

(5)

Its reciprocal is the area modulus Cs-1, that is, the proportionality between the increase in the surface pressure and that in the area strain (area decrease per unit area). Both Cs and Cs-1 can be calculated directly from the slope of the π-A isotherms.32-35 For an ideal mixture, the compressibility is assumed to be additive with respect to the product (CsiAi) (i ) 1, 2, etc.). Thus, Cs,ideal is obtained as follows:

Cs,ideal ) (1/Aπ,ideal)[(Cs1Aπ,1)X1 + (Cs2Aπ,2)X2] (6) where Cs1 and Cs2 are the compressibility of the pure DPPC and the pure paclitaxel monolayer at a specific surface pressure, respectively. However, the experimental data Cs obtained from the π-A isotherm of the mixed monolayer is usually different from the ideal value. The change in compressibility due to the presence of paclitaxel (∆Cs,paclitaxel) is

∆Cs,paclitaxel ) Cs - Cs1

(7)

The change in compressibility due to interaction nonideality (∆Cs,real) is

∆Cs,real ) Cs - Cs,ideal Figure 4. Excess area of the DPPC/paclitaxel monolayers at various surface pressures.

and that averaged according to the molar ratio of the mixed monolayer at the same given surface pressure,30,31 that is,

Aex ) Aπ,exp - Aπ,ideal

(4)

If an ideally mixed monolayer is formed or the two components are completely immiscible, the excess area will be zero and a plot of A12 as a function of X1 or X2 at a given surface pressure would be a straight line. Any deviation from the straight line merely indicates miscibility and nonideality of mixing.30 Since our results showed no linear relationship, it can be confirmed that DPPC and paclitaxel are miscible and can form nonideal monolayers at the interface. In Figure 4, the excess area for the DPPC/ paclitaxel monolayers at π ) 5, 10, and 12 mN/m is plotted as a function of the molar ratio of paclitaxel. Positive values of the excess area are observed. This means that the (30) Gaines, G. L., Jr. Insoluble monolayers at liquid-gas interfaces; Wiley: New York, 1966. (31) Birdi, K. S. Lipid and biopolymer monolayers at liquid interface; Plenum: New York, 1989.

(8)

Area compressibility is a more sensitive means to determine how structural changes have been imparted to the acyl chains of DPPC during the course of physiological remodeling and might affect the interactions of DPPC with paclitaxel. The data of compressibility in the DPPC/ paclitaxel monolayers are shown in Table 1. The change in compressibility due to the presence of paclitaxel, ∆Cs,paclitaxel, is zero with the pure DPPC monolayer, which is indeed expected. We are comparing experimental values and subtracting that of the pure DPPC monolayer from them so that any change is due to the influence of the paclitaxel component. Figure 5 shows ∆Cs,paclitaxel and ∆Cs,real versus the mole fraction of paclitaxel in the mixed monolayers. There is a linear relationship between the changes of compressibility due to paclitaxel or interaction nonideality and the molar ratio of paclitaxel (in the range of e0.5). Measuring the change in compressibility due to interaction nonideality, ∆Cs,real, shows that the increment (32) Evans, E. A.; Skalak, R. Mechanics and thermodynamics of biomembranes; CRC Press: Boca Raton, FL, 1980; Chapter 4. (33) Smaby, J. M.; Momsen, M. M.; Brockman, H. L.; Brown, R. E. Biophys. J. 1997, 73, 1492. (34) Ali, S.; Smaby, J. M.; Momsen, M. M.; Brockman, H. L.; Brown, R. E. Biophys. J. 1998, 71, 338. (35) Ali, S.; Smaby, J. M.; Brockman, H. L.; Brown, R. E. Biochemistry 1994, 33, 2900.


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Figure 5. The change in compressibility of the DPPC/paclitaxel monolayers at 10 mN/m. Table 1. Parameters of Compressibility in DPPC/ Paclitaxel Monolayers Xpaclitaxel

Cs

Cs1

Cs,ideal

∆Cs,paclitaxel

∆Cs,real

0 0.1 0.2 0.3 0.4 0.5 1

0.0356 0.0306 0.0385 0.0444 0.0584 0.0611 0.022

0.022 0.012 0.0264 0.0227 0.0249

0.0352 0.0347 0.0369 0.0376 0.0342

0.0086 0.0165 0.0224 0.0364 0.0391

-0.0046 0.0038 0.0075 0.0208 0.0269

in the in-plane elasticity is not solely due to paclitaxel. It is likely to be attributable to energy minimization in the van der Waals contacts with the acyl chains of DPPC as well. It can be concluded that paclitaxel causes the monolayer to become more elastic. This may be due to the fact that paclitaxel affects the packing of the acyl chain domain of DPPC. It would require further studies to know in more detail the exact mechanism. Stability of Mixed Monolayers. The interaction (either repulsive or attractive) between the two components and the thermodynamic stability of a mixed monolayer can also be investigated from an evaluation of excess free energy, ∆Gex, or free energy of mixture, ∆Gmix.36 For a mixed monolayer composed of two components 1 and 2 at a given surface pressure π and temperature T, the excess free energy is

∆Gex )

∫0π [A12 - (X1A1 + X2A2)] dπ

(9)

The value of ∆Gex at a given surface pressure can be calculated from the π-A isotherms. ∆Gmix is then given by the relation

∆Gmix ) ∆Gex + ∆Gideal

(10)

where the ideal free energy of mixture, ∆Gideal, can be calculated from

∆Gideal ) RT(X1 ln X1 + X2 ln X2)

(11)

where R is the gas constant and T is the temperature. ∆Gex of the mixed DPPC/paclitaxel monolayers of various molar ratios at different surface pressures can be calculated from the corresponding π-A curves. Figure 6 shows ∆Gex values for the DPPC/paclitaxel monolayers in various compositions at surface pressures of 2, 5, 10, and 12 mN/ m. All of the values are positive and dependent on the (36) Pagano, R. E.; Gershfeld, N. L. J. Phys. Chem. 1972, 76, 1238.

Figure 6. Excess free energy (∆Gex) of the DPPC/paclitaxel monolayers at various surface pressures as a function of the molar ratio of paclitaxel.

Figure 7. Excess free energy (∆Fex) of the DPPC/paclitaxel monolayers at different molecular areas.

monolayer composition. The positive ∆Gex implies repulsive interactions between the DPPC and paclitaxel molecules. In other words, the monolayer becomes unstable after introduction of paclitaxel into the pure DPPC monolayer. Besides, our results show that the ∆Gex values become positively larger at high surface pressure, which suggests that molecular interactions between the DPPC and the paclitaxel are stronger and the DPPC/paclitaxel monolayer thus becomes less stable at higher surface pressure. The value of the excess free energy ∆Gex becomes maximum at the paclitaxel mole fraction of about 0.5. This result obtained from the excess free energy analysis agrees with the conclusion obtained from the analysis of the area expansion effect in the previous section. Since the collapse surface pressure of the paclitaxel monolayer is quite low (around 13 mN/m), an alternative form of analysis of the Gibbs free energy, the Helmholtz free energy (∆Fex), can be applied, which is

∆Fex ) -

∫AA [π12 - (X1π1 + X2π2)] dA 0

(12)

where π12 is the measured surface pressure at a given molecular area A. π1 and π2 are the surface pressures of the two pure component monolayers at the same molecular area, respectively. A0 is the molecular area where the phase transition from gas to liquid occurs during the compression. Figure 7 shows ∆Fex values for the DPPC/paclitaxel monolayers in various compositions at molecular areas of 80, 60, and 40 Å2. Almost all of the values are positive and

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Figure 8. Comparison of DPPC (top) and paclitaxel (bottom) FTIR spectra in a KBr matrix.

dependent on the monolayer composition. The larger the molecular area, the smaller the excess free energy, and the smaller the interaction between the two components. ∆Fex has a maximum at the paclitaxel mole fraction around 0.5. This result from the ∆Fex analysis agrees again with that obtained from the area expansion analysis as well as from the excess Gibbs free energy calculation. Our studies for the stability of the DPPC/paclitaxel monolayers suggest that molecular interactions between paclitaxel and DPPC are repulsive. It is probable that the interactions occur in the vicinity of the polar headgroups. Most likely, this may be due to the long carbon chains in the DPPC molecular structure in which paclitaxel cannot well orient into the lipid chain layer. The interactions between the paclitaxel and DPPC molecules become more evident when the mixed monolayer is highly compressed. To obtain further information on the interactions between lipid and paclitaxel molecules, the penetration behavior of paclitaxel into the DPPC monolayer and the relaxation of the DPPC/paclitaxel monolayers should also be investigated. These topics will be investigated and discussed elsewhere. The above thermodynamic and mechanical analyses have limitations of their own. It cannot be concluded simply from this paper what kind of actual molecular interactions exist between the two components. Further study is needed. However, the mechanochemical effects of such interactions can be known from the present study. FTIR Spectroscopy. We first prepared solid KBr matrixes containing DPPC and paclitaxel for FTIR analysis in the high wavenumber range of 2600-3200 cm-1. We did this in order to ensure that paclitaxel does not have transmittance in this region that would interfere with our later analysis. From Figure 8, it is clear that this is indeed so. We used DPPC and paclitaxel in solid form as the concentration is high enough to obtain complete spectra. We then prepared a piece of DPPC/paclitaxel LB monolayer transferred at π ) 28 mN/m and T ) 25 °C onto a CaF2 window and a bulk, polycrystalline DPPC solid pressed in a KBr matrix for FTIR analysis. Figure 9 shows the representative data of the DPPC in high wavenumbers. The spectra reveal the main two peaks, proving that LB transfer onto a solid surface at the condensed monolayer state did not noticeably perturb the essential inter- and intramolecular interactions.37 This also showed that the monolayer did not participate in any significant interaction with the CaF2 interface. At high (37) Oliver, R. Biochim. Biophys. Acta 1996, 1279, 5.

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Figure 9. Comparison of DPPC spectra using a transferred monolayer on CaF2 (above) and a KBr matrix (below).

wavenumbers, symmetric and antisymmetric modes of the methylene chain occur approximately at 2850 and 2920 cm-1, respectively. These wavenumbers are conformation sensitive and respond to temperature- and pressure-induced changes of the trans/gauche ratio in acyl chains.38 The position of the symmetric C-H stretching vibration is a measure of the number of gauche conformers in the acyl chains. When all methylene groups are in the trans conformation, the band is observed at around 2849 cm-1.39 Addition of gauche conformers results in a small shift to a higher wavenumber, which can be estimated to be 2 cm-1 from the main transition from gel to liquidcrystalline phase (note that the resolution is 4 cm-1). At pretransition, the magnitude of the wavenumber shift is even smaller as there are smaller conformational changes. The vibration mode (antisymmetric stretch) of terminal CH3 occurs at about 2960 cm-1. Bandwidths of the same bands also give dynamic information about the system. However, a bandwidth increment in the liquid-crystalline phase does not necessarily correspond to an increase in dynamics but may be an indication of subphases present in the membrane.39 We transferred the mixed monolayers with DPPC/ paclitaxel ratios at 10:0, 8:2, 5:5, 2:8, and 0:10 onto CaF2 windows at 10 mN/m and those with 10:0, 8:2, and 5:5 ratios at a higher surface pressure of 28 mN/m. The isotherm does not reach 28 mN/m for amounts of paclitaxel above 50%. On the basis of our data, we are unable to resolve the pretransition peak, as the band is not strong enough. However, the symmetric and antisymmetric modes of the methylene chain (at 2850 and 2920 cm-1) could be adequately resolved in most cases as seen in Figure 10. The FTIR spectra obtained for the monolayers transferred at lower surface pressures have lower relative intensity. This is related to the lower packing density and the accompanying increase of the average tilt angle between acyl chains and the surface normal, which gives rise to lower transmittance signals.39 The addition of paclitaxel also lowers the transmittance intensity as the proportion of lipid transmitting in the region decreases. At lower surface pressures (10 mN/m), the positions of the antisymmetric mode did not differ upon addition of paclitaxel to the DPPC monolayer. This implies that a majority of the methylene groups are in the trans conformation and this number increases with the presence (38) Johal, M. S. Langmuir 1999, 15, 1275. (39) Umemura, J. A. Biochim. Biophys. Acta 1980, 419, 206.


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Figure 10. Mixed DPPC/paclitaxel monolayer transmittance spectra at higher surface pressure: 5:5 (top); 8:2 (middle); 10:0 (bottom).

of paclitaxel. At higher pressures (28 mN/m), incorporation of paclitaxel shifts the symmetric mode to higher frequencies very slightly. This may be in conjunction with an increase in gauche conformers and disorder in the lipid arrangement.40 We pay more attention to the symmetric stretching mode because of its sensitivity to changes in mobility and in conformational disorder of the hydrocarbon chains, which gives a better reflection of changes in the system as compared to the antisymmetric stretching region.41 Bernsdorff has studied the effect of paclitaxel encapsulated in liposomes and the effect of temperature using FTIR.42 This author concluded that paclitaxel only causes minor changes in the phase transition of the lipid bilayer system, leading to a very slight increase in the wavenumber of the gel to liquid-crystalline state. This means that paclitaxel increases the chain flexibility and fluidizes the lipid bilayer. Our FTIR study on the monolayer model supported this conclusion. We found that paclitaxel does not induce considerable changes in the FTIR spectra of the mixed monolayers, indicating that lipid-drug interactions are not very significant. Paclitaxel does increase the mobility of the acyl chains for saturated lipids, which reflects a fluidizing effect on the monolayer. Paclitaxel increases the population of trans conformers at low surface pressures and increases the proportion of gauche conformers at higher surface pressures. AFM. We imaged the mixed DPPC/paclitaxel monolayers, which were deposited by the Langmuir-Blodgett technique onto a smooth mica surface at a given surface pressure at T ) 25 °C. Our main objective was to determine if paclitaxel would introduce changes in the microdomain formation of the DPPC monolayer. Figure 11 shows the AFM image of a pure DPPC monolayer deposited at 10 mN/m. There are various patches of light and dark regions, with the former forming islandlike structures. These “islands” are also known as surface islands and represent the existence of different phases due to packing differences. On the basis of the (40) Handan, B. J. Mol. Struct. 1997, 408/409, 269. (41) Severcan, F. Biosci. Rep. 1995, 15, 221. (42) Corinna, B. J. Biomed. Mater. Res. 1999, 46, 141.

Figure 11. AFM images of the DPPC monolayer at 10 mN/m: 4 µm × 4 µm (top), 1 µm × 1 µm with cross-sectional analysis (middle), and 1 µm × 1 µm topographical view (bottom).

π-A isotherm of DPPC, it is well-known that at such a surface pressure, the lipid molecules exist in either a liquid-expanded (LE) or liquid-condensed (LC) phase. We have assigned the brighter regions as belonging to lipids in a condensed state since its upright configuration would result in higher vertical heights and the darker regions to the liquid-expanded state. A cross-sectional analysis reveals that the height difference between the bright and dark regions is approximately 0.4 nm compared to the theoretical length of a DPPC molecule at about 2.8 nm.43 We can thus infer that at 10 mN/m, molecules in the liquidcondensed phase are standing straight up while those in the liquid-expanded phase are only slightly tilted from the normal, resulting a small height variation. Figure 12 shows a typical DPPC monolayer deposited at 28 mN/m. A majority of the lipids are in the liquidcondensed phase, separated by thin lines of liquidexpanded phase. The vertical height difference between the two phases is again about 0.4 nm. It is proposed that upon compression, the small solid phase domains grow in size from the liquid phase, changing from its initially loose packing to a more dense structure.45 Our AFM images support this theory as the thin edges of the liquid-expanded phase separating the condensed phase reveal the existence of islands. (43) Yang, X. M. Surf. Sci. 1994, 316, L1110. (44) Tomohiro, I. Colloids Surf., B 2000, 19, 81. (45) Yang, X. M. Appl. Surf. Sci. 1995, 90, 175.

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Figure 13. AFM images of mixed DPPC/paclitaxel monolayers at 10 mN/m: (a) pure DPPC, (b) DPPC/paclitaxel 8:2, and (c) DPPC/paclitaxel 5:5.

Figure 12. AFM images of the DPPC monolayer at 28 mN/m: 4 µm × 4 µm (top), 1 µm × 1 µm with cross-sectional analysis (middle), and 1 µm × 1 µm topographical view (bottom).

Upon further compression, at slightly higher pressures of 30 mN/m, other researchers have reported the DPPC monolayer to be entirely smooth, implying that the entire monolayer is in a liquid-condensed phase.44 This observation has been achieved at various surface pressures ranging from 25 to 37 mN/m as well.46,47 This disparity can be explained by the different operating conditions; for example, it is known that the compression rate of a monolayer pronouncedly influences the shape, microstructure, and roughness of solid phase domains.44 Other factors include deposition, temperature, subphase composition, substrate used for deposition, and trace impurities in the monolayer system. In Figures 11 and 12, we also notice the existence of pinholes and bright spots. Pinholes are thought to arise as a result of the mobility of phospholipid molecules during two-dimensional crystal formation.46 The smaller spots may be the result of dehydration of the monolayer after transfer onto the solid mica surface and subsequent exposure to air, resulting in the crystallization of some DPPC molecules, and the larger spots, which have a greater vertical height (up to 30 nm), are thought to be phospholipid liposomes that have lost their aqueous support.46,47 (46) Kim, K. J. Biomed. Mater. Res. 2000, 52, 836. (47) Vie, V. Langmuir 1998, 14, 4574.

Figure 13 shows images of the DPPC monolayer with increasing amount of paclitaxel at 10 mN/m. Again, we observe two phases, the LC and LE states of DPPC with a consistent height difference of ∼0.4 nm as before. The fact that we do not observe a third paclitaxel phase indicates that the drug is miscible in the lipid monolayer, an observation consistent with our Langmuir isotherm studies. Increasing the paclitaxel content reduces the extent of the LC phase (the brighter patches) such that it is no longer apparent at 50 mol % drug. Also, paclitaxel causes aggregation of the LC phase (middle figure) to form larger islands as compared with those in pure DPPC. Inspection of the mixed monolayers at higher magnification (Figure 14) reveals that the LC phases have become more “porous” upon addition of paclitaxel. It is obvious that the drug has disturbed the original compact packing typical of the lipid LC state. This is likely to further contribute to the overall fluidizing effect of paclitaxel on DPPC monolayers at low surface pressures. What these AFM images imply is that paclitaxel fluidizes the mixed monolayer at low surface pressures (e.g., 10 mN/m), causing more lipid molecules to exist in the expanded state rather than the rigid liquid-condensed phase. Furthermore, the existence of greater amounts of LE state would result in an area expansion, which is in line with the pressure increment observation in our Langmuir trough experiments. Incorporating paclitaxel into the lipid monolayer at higher surface pressures (e.g., 28 mN/m) causes a significant difference in monolayer packing (Figure 15). With DPPC alone, the liquid-condensed states exist mainly as circular or irregularly shaped domains thinly separated by lipids in the expanded state. Paclitaxel causes the condensed lipid molecules to form regions of linear


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Figure 15. AFM images of mixed DPPC/paclitaxel monolayers at 28 mN/m; (a) pure DPPC, (b) DPPC/paclitaxel 8:2, and (c) DPPC/paclitaxel 5:5.

Figure 14. AFM images of the DPPC/paclitaxel 8:2 monolayer at 10 mN/m: 4 µm × 4 µm (top), 1 µm × 1 µm with crosssectional analysis (middle), and 1 µm × 1 µm topographical view (bottom).

domains, separated at regular intervals. These linear microdomains coexist with portions of the original island formation. Furthermore, the packing is never as compact with paclitaxel and we observe greater portions of liquidexpanded (darker) regions. This can be clearly seen in the corresponding cross-sectional images, which reveal increasing distance between the taller condensed-state lipids with increasing paclitaxel content. At higher surface pressures, paclitaxel causes the reorganization of the monolayer from a thermodynamically stable structure (circular domains) to one that is inherently less stable (linear columns). This might have implications on stability issues of the mixed monolayer. We know that the corresponding surface pressure in a biological membrane (bilayer leaflet) is approximately 32 mN/m,46 which is close to our high surface pressure for imaging. The coexistence of LC-LE domains at this surface pressure, as opposed to the predominant LC phase without the drug, may have important consequences on the general stability of the lipid vesicle.47 One has to take extreme caution with our findings on the atomic force microscope. It is known that DPPC phase domains are susceptible to motion and aggregation under conditions of high humidity and that LC and LE lipid

domains evolve in an energetically favorable manner to reduce the number of boundary lipids, which are exposed to the coexisting lipid phase while maintaining the relative areas of the two phases.45 In our experiments, we minimized the occurrence of such phenomena by storing the lipid films in sealed containers before use, by reducing the time between film deposition and AFM scanning, and by using Millipore water as the subphase, which is found to slow the rearrangement process. Structural change might have occurred upon the deposition. Care must be exercised especially for the deposition rate. FTIR and AFM analysis should be conducted immediately after the deposition. The supported monolayers for FTIR and AFM analysis were all deposited at 25 °C. Information acquired at one temperature cannot be extended in general. Finally, the dry films used in this research have drawbacks. Hydrated films have advantages to preserve the structure of the monolayers at the airwater interface. However, we still have reasons to conduct the dry film experiment. Dry films make it possible to investigate the structure of the chain side of the monolayer, while the hydrated films allow investigation of the structure of the headgroup side of the monolayer membrane. Conclusions The compression isotherms of paclitaxel monolayers showed that paclitaxel is a surface-active agent. The surface activity of paclitaxel is not as high as that of lipids, which may be due to the bulky geometry of paclitaxel molecules. The present work showed that paclitaxel is miscible with the DPPC monolayer. The measurement of the excess area caused by molecular interactions between the lipid and the paclitaxel in the monolayer demonstrates that paclitaxel has an area expansion effect at a constant

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pressure for the DPPC monolayers and the magnitude of such an effect depends on the molar ratio of the paclitaxel. Thermodynamic analysis of the DPPC/paclitaxel monolayers showed that the values of the excess free energy of the DPPC/paclitaxel monolayers are positive, which implies a repulsive interaction between the paclitaxel and the lipid in the monolayer. This may due to the long carbon chain length of the DPPC molecules, which causes changes of the bulk geometric conformation of the paclitaxel in the lipid chain layer of the monolayers. FTIR analysis also showed that paclitaxel does not induce considerable changes in the FTIR spectra of the lipid monolayers, indicating that lipid-drug interactions are not very significant. However, paclitaxel did increase the mobility of the acyl chains for saturated lipids, which reflects a fluidizing effect on the monolayer. For saturated lipids, paclitaxel increases the population of trans conformers at low surface pressures and the proportion of gauche conformers at higher surface pressures.

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The conclusions obtained were also supported by AFM investigation. It was demonstrated that paclitaxel reduces the extent of liquid-condensed phases in the DPPC monolayers at low surface pressures, resulting in a fluidizing effect. At high surface pressures, paclitaxel causes a reorganization of microdomains, forming regions consisting of linear rows. AFM studies further revealed that the expulsion of paclitaxel molecules at high surface pressures occurs into the aqueous subphase and not on the top of the monolayer as originally thought. These AFM images further supported our conclusions from previous Langmuir film balance experiments. Acknowledgment. This work was supported by NUS Grants R-279-000-021-112 and R-279-000-052-112, National University of Singapore, Singapore. LA011545P

Molecular Interactions between a Lipid and an Antineoplastic Drug

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