UV Absorption Spectroscopic Analysis of the Molecular Orientation of

+

+

1566

Langmuir 1996, 12, 1566-1571

UV Absorption Spectroscopic Analysis of the Molecular Orientation of a Drug Penetrated into a DPPC Membrane Takeshi Hasegawa,* Yuko Ushiroda, Maki Kawaguchi, Yoshiko Kitazawa, Miho Nishiyama, Aya Hiraoka, and Jujiro Nishijo Kobe Pharmaceutical University, Motoyama-kita, Higashinada-ku, Kobe 658 Japan Received June 8, 1995. In Final Form: September 26, 1995X The present study quantitatively measured, via UV absorption spectroscopy, the molecular orientation of the drug chlorpromazine (CPZ) after spontaneous penetration into the gel phase of a phospholipid membrane. An L-R-dipalmitoylphosphatidylcholine (DPPC) Langmuir (L) film (a monolayer on an aqueous solution) was doped with CPZ and transferred onto a quartz substrate to form a Langmuir-Blodgett (LB) film. The transmission spectrum of the LB film was measured using a normal incident, nonpolarized UV beam. To calculate the theoretical absorbances, the extinction coefficients of oriented CPZ molecules in the DPPC LB film were deduced from the molar extinction coefficients of nonoriented, dispersed CPZ molecules in an aqueous solution. The anisotropic extinction coefficient of CPZ was determined with the uniaxial refractive index ellipsoid model as a function of orientation angle, using the extinction coefficient of CPZ in the bulk state. By comparing the theoretical absorbances with observed absorbances, the orientation angles from the surface normal of the LB film of transition moments along the molecular short and long axes were determined to be 17° and 85°, respectively. The results of the present study indicate that CPZ molecules penetrate deep into the DPPC membranes and that the molecular orientation of CPZ is determined by the surrounding DPPC molecules.

Introduction Sheetz and Singer1 first proposed the “bilayer couple hypothesis” to explain erythrocyte deformation caused by bound drugs. Subsequently, several studies investigated the interactions between various drugs and bilayer membranes, focusing on binding location and the number of drug molecules bound to the membranes.2-4 Few quantitative studies have analyzed the molecular orientation of a drug after it has penetrated into a membrane. Such studies may contribute to a better understanding of drug and phospholipid membrane interactions. In a previous investigation,5 we studied the aggregation of phospholipid liposomes and found that they localize in the gel phase of L-R-dipalmitoylphosphatidylcholine (DPPC, Figure 1) monolayers. The present study quantitatively evaluated the molecular orientation of the antidepressant drug chlorpromazine6 (CPZ, Figure 1) after penetration into a DPPC Langmuir (L) film. Spectroscopy is effectively used to study the molecular orientation of organic, ultrathin films.7,8 In particular, Fourier transform infrared (FT-IR) spectroscopy is a powerful and sensitive tool studying Langmuir-Blodgett (LB) films as demonstrated by Hasegawa et al.9 and Ozaki et al.10,11 In addition to LB films, this method has been adapted to study L films by using external reflection.12,13 * Author to whom correspondence should be sent. X Abstract published in Advance ACS Abstracts, March 1, 1996. (1) Sheetz, M. P.; Singer, S. J. Proc. Natl. Acad. Sci. U.S.A. 1974, 71, 4457. (2) Cater, B. R.; Chapman, D.; Hawes, S. M.; Saville, J. Biochim. Biophys. Acta 1974, 363, 54. (3) Breton, J.; Viret, J.; Leterrier, F. Archs. Biochem. Biophys. 1977, 179, 625. (4) Ro¨mer, J.; Bickel, M. H. Biochem. Pharmacol. 1978, 28, 799. (5) Hasegawa, T.; Ushiroda, Y.; Kawaguchi, M.; Kitazawa, Y.; Nishiyama, M.; Hiraoka, A.; Nishijo, J. Can. J. Chem., submitted. (6) Lee, A. G. Mol. Pharmacol. 1977, 13, 474. (7) Allara, D. L.; Nuzzo, R. G. Langmuir 1985, 1, 52. (8) Ishino, Y.; Ishida, H. Langmuir 1988, 4, 1341. (9) Hasegawa, T.; Umemura, J.; Takenaka, T. J. Phys. Chem. 1993, 97, 9009. (10) Ozaki, Y.; Uehara, K.; Araki, T.; Iriyama, K. Appl. Spectrosc. 1993, 47, 1509. (11) Ozaki, Y.; Iriyama, K. J. Phys. Chem. 1993, 97, 10445.

Figure 1. Structural formula for DPPC and CPZ with the directions of electron transition moments.

Furthermore, detailed analyses are now possible with the proposal of various analytical theories.14-17 Regarding optical constants for theoretical analysis, the anisotropic extinction coefficients are related to the orientation of the transition moment in a band. Since the direction of a transition moment is easily determined by analyzing a vibration mode, the molecular orientation of a substance (12) Dluhy, R. A. J. Phys. Chem. 1986, 90, 1373. (13) Gericke, A.; Michailov, A. V.; Hu¨hnerfuss, H. Vib. Spectrosc. 1993, 4, 335. (14) Umemura, J.; Kamata, T.; Kawai, T.; Takenaka, T. J. Phys. Chem. 1990, 94, 62. (15) Song, Y. P.; Petty, M. C.; Yarwood, J. Langmuir 1993, 9, 543. (16) Parikh, A. N.; Allara, D. L. J. Chem. Phys. 1992, 96, 927. (17) Hasegawa, T.; Takeda, S.; Kawaguchi, A.; Umemura, J. Langmuir 1995, 11, 1236.

+

+

UV Absorption Analysis

is easily derived from the FT-IR spectra. As such, most of these studies involve FT-IR spectroscopy. To analyze organic molecules that contain π-electrons, however, UV-visible spectroscopy or fluorimetry is more suitable, since the optical constants directly relate to the electron transitions in this wavelength region. In particular, fluorimetry is commonly used in the biological sciences because of its high level of sensitivity.18-20 Experimental results are often analyzed through the order parameter21 to quantitatively measure a change in molecular orientation. UV spectroscopy, on the other hand, is valuable in that the absolute intensities of each band can be theoretically analyzed to determine molecular orientation. Okamura et al. measured an L film of chlorophyll a utilizing a UV-visible polarized reflection technique involving optical fibers.22 From the reflection spectra, they were able to determine molecular orientation as a function of the surface pressure of the film. Their analysis was supported by Hansen’s optical approximation theory,23 which is useful in calculating the reflection and transmission coefficients of heterogeneous, ultrathin, stratified-layer films. This theory, however, assumes optically isotropic conditions in the film and an adequate film thinness. Recently, a simple extension from the isotropic theory to uniaxially anisotropic theory was proposed.17 The present study employed UV spectroscopy, using this extended theory, to quantitatively calculate the orientation angle of CPZ molecules in an LB film of DPPC. Because a UV beam with normal incident relative to the film surface was used, absorbances could have been calculated without the anisotropic theory. The anisotropic theory was used, however, because of the relative ease in which it allows molecular orientations to be derived from anisotropic optical constants. Prior to the present study, the anisotropic extinction coefficients for uniaxially oriented CPZ molecules in a phospholipid membrane were derived from the molar absorption coefficients obtained for CPZ molecules in an aqueous solution. Material and Methods Reagent grade (g99%) DPPC and CPZ hydrochloride were purchased from Sigma Chemical Co. and were used without further purification. Buffer solutions were modified with reagent grade monobasic potassium phosphate (KH2PO4), dibasic potassium phosphate (K2HPO4), and potassium chloride (KCl) purchased from Nacalai Tesque Inc. Purified water was obtained from a Millipore Co. Milli-Q Laboratory water purifier, after distillation by a Yamato Scientific Co. Ltd. Model WG-25 autodistiller equipped with ion exchange resin. LB films were made with a Kyowa Interface Science Co. Ltd. Model HBM LB film apparatus using previously described vertical dipping and horizontal lifting methods.24 The compressing barrier was made of Teflon-coated aluminum. The compressing speed was 14.0 cm2/min. The substrates for LB films were Model ESL-1 nonfluorescence quartz plates (15 × 50 × 2 mm) purchased from Daiko Seisakusyo, Kyoto. Before LB films were lifted, the plates were cleaned by sonication for 5 min in each of the following solutions: pure water, ethanol, acetone, and dichloromethane. The plate was transparent within the wavelength range 200-600 nm. The transmittance was flat and greater than 92% in the same wavelength range. (18) Rusinova, G. G.; Voloshin, V. N. Med. Radiol. 1986, 31, 58. (19) Li, K. P.; Glick, M. R.; Indralingam, R.; Winefordner, J. D. Spectrochim. Acta A 1989, 45A, 471. (20) Sepaniak, M. J.; Vo-Dinh, T. Philos. Trans. R. Soc. A 1990, 333, 85. (21) Yliperttula, M.; Lemmetyinen, H.; Mikkola, J.; Kinnunen, J. Chem. Phys. Lett. 1988, 152, 61. (22) Okamura, E.; Hasegawa, T.; Umemura, J. Biophys. J. 1995, 69, 1142. (23) Hansen, W. N. Symp. Faraday Soc. 1970, 4, 27. (24) Okamura, E.; Umemura, J.; Takenaka, T. Can. J. Chem. 1991, 69, 1691.

Langmuir, Vol. 12, No. 6, 1996 1567

Figure 2. The π-A isotherm of DPPC (a) on phosphate buffer, pH 6.7 at 25 °C and (b) before and after doping with CPZ at a. The point indicated by a is the corresponding surface pressure of the DPPC liposome. The dashed arrow indicates the sudden increase in surface pressure caused by doping with CPZ. The UV absorption spectra were obtained with a Beckman DU-600 UV-visible spectrophotometer. The spectra of LB films were measured by scanning 100 times.

Results and Discussion An L film of DPPC was considered to be a suitable membrane for studying the penetration of CPZ into DPPC liposomes. To determine the corresponding surface pressure of the DPPC liposomes, the π-A isotherm of DPPC was measured. The π-A isotherm of DPPC obtained on an aqueous subphase was adjusted to pH 6.7 at 25 °C (Figure 2a), which is consistent with other reports.25 The results obtained in the present study indicated that the limited surface area was 52.3 Å2/molecule. In a previous investigation, we studied the aggregation of a single-compartment DPPC liposome.5 In the study, the number of CPZ molecules penetrating into a DPPC membrane was determined as a function of surface area. To estimate the surface area or surface pressure of the microliposome, our results were compared to the number of CPZ molecules penetrating into DPPC liposomes, as determined by a previously described equilibrium dialysis technique.4 To study this, L films with six different-sized surface areas were doped with CPZ, and then each of them was compressed to obtain A*πf0. The asterisk (*) indicates that the limited surface area of DPPC is expanded by CPZ penetration. From the changes in area, ∆A ≡ A*πf0 Aπf0, the number of penetrated CPZ molecules in each L film was calculated and summarized versus the surface area at which the doping was done. The results indicated that the DPPC liposomes aggregate in the gel phase of a DPPC L film with the surface area found at point a in Figure 2a. At the surface pressure of point a, the membrane has molecular fluidity. To investigate drug penetration into DPPC liposomes, CPZ was applied to a DPPC L film at the surface pressure of point a in Figure 2a. Beginning with a wide surface area, the DPPC L film was compressed until the surface area was equivalent to that at point a. At this point, the CPZ solution was added to the trough. To obtain reliable surface pressure using a Wilhelmy balance, the volume of the subphase should remain unchanged during the doping procedure. To ensure this, the CPZ solution was prepared with the subphase taken from the trough. The solution was then poured back into the trough, causing the surface to fluctuate in pressure by (10 mN/m for about 10 min, as shown in Figure 2b. After about 1 h, the L film reached an equilibrium surface pressure with a fluctuation within (0.1 mN/m. The film was compressed again to obtain the isotherm above the dashed arrow in Figure 2b. The (25) Neuman, R. D.; Fereshtehkhou, S. J. Colloid Interface Sci. 1988, 125, 34.

+

1568

+

Langmuir, Vol. 12, No. 6, 1996

Hasegawa et al.

Figure 4. The UV absorption spectrum of CPZ in an aqueous solution at a concentration of 3 × 10-4 M.

Figure 3. The normal incident UV transmission spectrum of the DPPC LB film penetrated by CPZ. A schematic illustration of the measurement system is shown above the spectrum. The dashed arrows indicate the directions of electric field in the films.

observed limited surface area, A*πf0, was 57.0 Å2/molecule; this is greater than the value for DPPC alone (Aπf0 ) 52.3 Å2/molecule). This change represents the penetration of CPZ into the DPPC L film. The L films were transferred onto a quartz plate to generate a five-monolayer LB film on each side of the plate with a surface pressure of 40 mN/m. The first monolayer was transferred according to the conventional LB method of Blodgett et al.26 Using only the LB technique, the transfer ratio of DPPC onto solid substrates is known to be low, especially for the upper layers.24 Therefore, the successive layers were transferred according to a previously described horizontal lifting (HL) technique.27-29 Thus, LB films composed of a total of 10 monolayers were assembled. Next, the UV absorption spectrum of the LB film was measured. Although it is often effective to measure L films by direct UV reflection using an optical fiber, as in the work of Okamura et al.,22 in the presence study the proportion of CPZ molecules in the film was so low that greater sensitivity was required. Thus, the absorption spectrum of the LB film was measured with a normal incident nonpolarized UV beam. The electric field vectors of the beam were directed parallel to the LB film. The spectrum that was obtained is shown in Figure 3. Although there were 10 total layers and the spectrum was scanned 100 times, the observed intensity was weak and the signal-to-noise (S/N) ratio was low. Additional transfers of the L film onto the LB film were attempted to increase the intensity, but the number of layers reduced the transfer ratio. Thus, the spectrum of the 10-layer LB film was used for analysis. There were two major peaks at approximately 258 and 311 nm in the spectrum. The band at 258 nm was determined to be a π f π* transition, in which the transition moment was along the short axis of the phenothiazine group (tricyclic skeleton) in CPZ30 (Figure 1). The direction of the transition moment of the band at 311 nm, on the other hand, was normal to the (26) Blodgett, K. B. J. Am. Chem. Soc. 1935, 56, 495. (27) Langmuir, I.; Scha¨fer, V. J. J. Am. Chem. Soc. 1938, 60, 1351. (28) Iwahashi, M.; Naito, F.; Watanabe, N.; Seimiya, T. Chem. Lett. 1985, 187. (29) Kamata, T.; Umemura, J.; Takenaka, T. Chem. Lett. 1988, 1231. (30) Craig, D. P.; Hobbins, P. C. J. Chem. Phys. 1955, 24, 539.

Figure 5. The analysis scheme used for calculating molecular orientation angles using the optical constants obtained from the molar extinction coefficients of CPZ in an aqueous solution.

band at 258 nm.5 In the spectrum of the LB film, the intensity ratio of the band at 258 nm to that at 311 nm (I258/I311) was approximately 0.93. For comparison the spectra for various concentrations of CPZ in an aqueous solution were also measured. The spectrum measured at a CPZ concentration of 3 × 10-4 M is shown in Figure 4. In this spectrum, the band intensity ratio was approximately 9.5; this was much greater than the ratio of the LB film. This marked change in intensity ratio indicated that the penetrated CPZ molecules were oriented in the DPPC LB film, whereas the CPZ molecules were dispersed and nonoriented in aqueous solution. The uniaxial molecular orientation of penetrated CPZ molecules in the DPPC LB film was quantitatively analyzed. For accurate analysis, the transmission coefficient of the stratified layer for a normal incident UV beam should be calculated for various orientations of transition moments. Theoretically the calculated absorbance matches the experimental absorbance, and the orientation angle can be quantitatively determined. This theory is schematically summarized in Figure 5 and is described in detail later in this section. In an LB film in which the observable molecules are oriented, the optical constants are not isotropic. In addition, the extinction coefficient, which is the component of the complex refractive index, is related to the orientation of the transition moment at the absorbing wavelength. Thus, the calculation theory for anisotropic media appeared more suitable for the present investigation. Several different absorption spectra of one of the LB films were alternatively measured after rotating the LB film around its surface normal axis,

+

+

UV Absorption Analysis

Langmuir, Vol. 12, No. 6, 1996 1569

yielding nearly identical spectra. This indicated that the CPZ molecules in the LB film were oriented uniaxially, justifying the use of the calculation theory for uniaxial orientation.17 This theory was developed by expanding Hansen’s isotropic theory31 with Drude’s uniaxial anisotropic two-layer theory.32 Previously, this theory was used for the quantitative analysis of molecular orientation in LB films using FT-IR external reflection.17 Hansen’s isotropic theory can be expanded to an anisotropic theory by the transformation principle, described as follows:17

cos θ˜ j f

ξ˜ j

(1)

n˜ je

Here, θ˜ j is the refraction angle in the jth media, ξ˜ j is determined to be (n˜ je2 - n˜ 1o2 sin θ1)1/2, where n˜ je is the complex refractive index of the jth layer and the tilde indicates a complex value. The subscript o represents the surface parallel direction and the subscript e represents the surface normal direction. Other mathematical equations for calculating transmittance are given by

M ˜j)

[

[

i sin β˜ j qj -iq˜ j sin β˜ j cos β˜ j

cos β˜ j

-

]

(

)

(

) (

˜tHp )

)

)

2q˜ 1 (m ˜ 11 + m ˜ 12q˜ N) q˜ 1 + (m ˜ 21 + m ˜ 22q˜ N)

˜tEp )

n˜ 1o

˜tHp

n˜ No

]

(3)

(5)

and

Tp )

Re(q˜ N) |t˜ |2, δpt ) arg ˜tEp Re(q˜ 1) Hp

(6)

Here, all the notations are fundamentally the same as those of Hansen’s theory,31 except for the tilde mark. It should be noted that two parameters in these equations, q˜ j and β˜ j, have been transformed as follows:

q˜ j )

ξ˜ j n˜ jo n˜ je

(7)

(9)

4πkbulk λ

(10)

where λ (cm) is the wavelength. With eqs 9 and 10, we can calculate kbulk from the molar absorption coefficient by the equation

kbulk ) (4)

R ln 10C

where C (M) is the molar concentration. In this equation, R is related to the extinction coefficient in the bulk state, kbulk, by the equation18

R)

i 2π 2π sin ξ˜ (z - zk-1) ξ˜ k (z - zk-1) q ˜ λ k λ k 2π 2π iq˜ k sin ξ˜ (z - zk-1) cos ξ˜ (z - zk-1) λ k λ k

(

)

(2)

N ˜ k(z) ) cos

h4 ) 150 Å (30 Å × 5).33 The real number, n, of the complex refractive index for the films (CPZ) was taken to be 1.5. This is the accepted value for many cyclic compounds in the UV region,34 although the value has not been determined for CPZ. This real number may be affected by the molecular orientation of CPZ. The anisotropy of the real number was neglected in the present study, since molecular orientation has a greater influence on component at the absorbing wavelength, especially when studying permeable films. In order to obtain the components of the refractive indices, the following hypothesis was applied: According to Lambert-Beer’s law and Lambert’s law,35 the extinction coefficient  (M-1 cm-1) is related to the absorption coefficient R (cm-1) by the following equation:

ln 10 Cλ 4π

(11)

Since the obtained value, kbulk, is the extinction coefficient for pure, crystalline CPZ, the value must be transformed for CPZ in LB film, kbulk(LB). When the proportion of the oriented molecules in the film is less than unity, as in the present study, it is convenient to define the number of the penetrated drug molecules per 100 DPPC molecules in the LB film as N. Hence, a monolayer comprising 100 DPPC matrix membrane molecules with penetrated CPZ was used as a model (model monolayer I). The observed limited surface are expanded by penetration of drug, A*πf0 (Å2), multiplied by 100 gives the surface area of the model monolayer I. This surface area leads the volume of the model monolayer, Vfilm (Å3), with the thickness h (Å) of the film described as

Vfilm ) 100 A*πf0h

(12)

If the molecular volume of CPZ in the crystalline state is described as VCPZ (Å3), then the density of CPZ crystal, d (g/cm3), can be written as

and

β˜ j )

n˜ jo

2π ξ˜ h λ j n˜ je j

(8)

For the subsequent calculations, the complex refractive indices for each layer were required. In the present study, the five-layer (air-film-substrate-film-air) model was used. For the first and fifth layers (air), n˜ 1 ) n˜ 5 ) 1.0 were used. For the third layer (quartz substrate), h3 ) 2.0 × 107 Å and n˜ 3 ) 1.504 (at 257 nm) were used, per the manufacturer’s specifications. For the wavelength of 311 nm, n˜ 3 ) 1.485 was used. The film thickness was h2 ) (31) Hansen, W. N. J. Opt. Soc. Am. 1968, 58, 380. (32) Drude, P. Ann. Physik U. Chem. N. F. 1889, 32, 584.

1024MW d) VCPZNA

(13)

where MW is the molecular weight of CPZ, and NA is Avogadro’s number. The density is related to molar concentration C (M) by the following simple relation:

C)

1000d MW

(14)

(33) Reinhardt-Schlegel, H.; Kawamura, Y.; Furuno, T.; Sasabe, H. J. Colloid Interface Sci. 1991, 147, 295. (34) Erbelding, W. F. Anal. Chem. 1975, 47, 1983. (35) Milosevic, M.; Berets, S. L. Appl. Spectrosc. 1993, 47, 566.

+

1570

+

Langmuir, Vol. 12, No. 6, 1996

Hasegawa et al.

From eqs 12-14, the ratio of Vfilm to VCPZ is written as

Vfilm/VCPZ ) 10-25A*πf0 hCNA

(15)

This ratio represents the number of penetrated CPZ molecules, if they fully occupy the volume of model monolayer I (model monolayer II). A correlation factor, f, that relates kbulk to kbulk(LB) may be defined as

kbulk(LB) ) kbulk f

(16)

Both extinction coefficients in eq 16 correspond to the bulk state, without considering molecular orientations. The two coefficients depend on the number of molecules in the two model monolayers, I and II. As a result, if the number of CPZ molecules in the model monolayer I is defined as N, then the factor f may be expressed as

f)

N Vfilm/VCPZ

Figure 6. The relation between the molecular orientation and the calculated absorbance at 258 nm in the spectrum of the DPPC LB film penetrated by CPZ. The experimental absorbance is indicated in the figure by the line with an arrow.

(17)

From the above equations (eqs 11 and 15-17), the extinction coefficient of an oriented drug molecule in an LB film, kbulk, was determined to be

kbulk(LB) )

1025 ln 10 λN 4π A*πf0hNA

(18)

It should be noted that the resultant expression does not include molar concentration or density. To determine the molar extinction coefficients at the two bands, the UV spectra of various concentrations of CPZ aqueous solutions were measured. The spectra of CPZ showed fairly good linearity between absorbance and concentration, up to 5 × 10-4 M. The resulting molar extinction coefficients, 258 and 311, were 2.572 × 104 and 2.705 × 103 M-1 cm-1, respectively. From these values, kbulk for each band was calculated using eq 18, where h ) 30 Å.33 From our previous study,5 N at 70 Å2/molecule was determined to be 14. As a result, we calculated the extinction coefficients to be 0.0165 for kbulk(LB)258 and 0.002 04 for kbulk(LB)311. The anisotropic values of extinction coefficients, ko and ke, were determined according to a previously reported method that is based on the uniaxial ellipsoidal refractive indices.17 The mathematical expressions are as follows:

3 k (LB) sin2 φ 2 bulk

(19)

ke ) 3kbulk(LB) cos2 φ

(20)

ko ) and

In these equations, φ is the orientation angle of the transition moment from the surface normal of LB films. Thus, the complex refractive indices for CPZ in an LB film were determined to be n˜ o ) 1.5 + iko, n˜ e ) 1.5 + ike where i is the square root of negative one. The calculated absorbance is a function of the orientation angle φ; this is shown in Figure 6. Since the observed absorbance at 258 nm was 0.001 46, the orientation angle from the surface normal, derived from Figure 6, was 17°. Judging from the S/N ratio of the spectrum in Figure 3 (approximately 10%), the experimental error was estimated to be approximately (1°. This error is small because the

Figure 7. The relation between the molecular orientation and the calculated absorbance at 311 nm in the spectrum of the DPPC LB film penetrated by CPZ. The experimental absorbance is indicated in the figure by the line with an arrow.

Figure 8. An image model of the oriented CPZ in a compressed DPPC LB film.

deviation of experimental results does not significantly affect the orientation angle. This is evident in Figure 6, as the observed intensity for this band was in the lower region of the calculated absorbance axis. The orientation angle of the transition moment at 311 nm was analyzed in a similar manner. The relation between the calculated absorbance and the orientation angle for this band is shown in Figure 7. From the observed intensity of 0.001 57, the orientation angle was determined to be 85°, with an experimental error of approximately (5°. In this case, the observed intensity was in the higher region of the calculated absorbance axis; hence, the error was large because of the S/N ratio. From the results, schematic models of an oriented CPZ molecule in a DPPC LB film were drawn in Figure 8. We could not determine whether the tilting direction for the long axis (311 nm) of CPZ was right or left. The tilting direction of the short axis (258 nm), however, is likely as shown in the figure. If it were to tilt in the other direction, it would be inconsistent with

+

+

UV Absorption Analysis

the results obtained from the other band. The tilt angle of the short axis, 17°, is nearly identical to that of a hydrocarbon chain in compressed DPPC LB films, 19°,24,36 considering experimental error. The results of the present study indicated that CPZ spontaneously penetrated into the inside of the DPPC L film on an aqueous subphase due to the attractive forces between the hydrophobic groups. In addition, the results suggest that the tilt angle of CPZ was dictated by the tilt of surrounding DPPC molecules. Therefore, the drug CPZ can be said to penetrate deeply into a DPPC L film made at the beginning of the plateau region of the surface pressure curve (Figure 2). Conclusion The present study quantitatively measured, via UV absorption spectroscopy, the molecular orientation of CPZ (36) Akutsu, H.; Ikematsu, M.; Kyogoku, Y. Chem. Phys. Lipids 1981, 28, 149.

Langmuir, Vol. 12, No. 6, 1996 1571

molecules after spontaneous penetration into the gel phase of a DPPC monolayer. To calculate theoretical absorbances, the extinction coefficient of oriented CPZ molecules in an LB film was deduced from the molar extinction coefficient of CPZ in an aqueous solution. The obtained extinction coefficients correlated relatively well with the theoretical absorbances. The uniaxial anisotropic calculation theory was applied to the optical constants, yielding quantitative orientation angles. The results of the present study indicated that CPZ molecules spontaneously penetrated deep into the DPPC monolayer, drawn by the attractive forces between hydrophobic groups. Furthermore, the results suggest that the molecular orientation of CPZ was dictated by the surrounding DPPC molecules. The present study supports the theory that CPZ molecules penetrate deep into DPPC liposomes, possibly causing deformation of the membrane. LA950449J