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Stability and characterization of phospholipid Langmuir-Blodgett films
Thin Solid Films, 180 (1989) 123-127
123
STABILITY AND CHARACTERIZATION OF PHOSPHOLIPID LANGMUIR-BLODGETT FILMS SINZI MATUOKA,HARUMIASAMIAND ICHIROHATTA Department of Applied Physics, School of Engineering, Nagoya University, Nagoya 464-01 (Japan)
TOSHIOISHII School of Dental Medicine, Tsurumi University, Yokohama 230 (Japan)
KENICHIYOSHIKAWA College of General Education, Nagoya University, Nagoya 464-01 (Japan)
(ReceivedApril24, 1989;acceptedJune 1, 1989) The regularity of the bilayer structure was compared between dipalmitoylphosphatidic acid (DPPA) Langmuir-Blodgett (LB) films and "oriented multilayers" of DPPA, using X-ray diffraction and electron spectroscopy for chemical analysis (ESCA). The angular variation of the (001) Bragg spot due to the irregularity of the lamellar repeat was remarkable in the system of oriented multilayers. In contrast, the spot of DPPA LB films exhibited only a slight angular variation, suggesting a regular structure of this film. The take-off angle dependence of the phosphorus:carbon ratio obtained by ESCA indicates that the regular layered structure in DPPA LB film is maintained after 49 layers are deposited.
]. INTRODUCTION Phosphoiipids are amphiphilic molecules that form bilayer structures in water. For systems of multilamellar vesicles realized in excess water, these structures have been investigated intensively in connection with the structure of biomembranes 1. Well-arranged bilayers are very useful for studying their structures in detail. For this purpose so-called "oriented multilayers" have been used 2'3. The oriented multilayers have been constructed by putting the phospholipid solution onto a solid surface under a stream of nitrogen. Another excellent method for preparing wellarranged films is the Langmuir-Blodgett (LB) technique4. It has been reported that L B films of phosphatidylcholine molecules could not be constructedS. Hasmonay et al. 6 found, however, that it was possible to transfer LB multilayers of dipalmitoylphosphatidic acid (DPPA) with a divalent cation (Ca2+). Therefore the work reported here aims to investigate the difference in the regularity of the films between the oriented multilayers and the LB films constructed from DPPA (Fig. 1). 2. MATERIALSAND METHODS 2.1. Preparation o f oriented multilayers
DPPA (free acid, Sigma) was dissolved in chloroform-methanol (1:1 by volume) mixture (10 mg ml-1). 4 Ixl of the solution was put onto a glass slide and 0040-6090/89/$3.50
© ElsevierSequoia/Printedin The Netherlands
124
s. MATUOKAet al.
o,o Fig. 1. Structural formula for DPPA.
evaporated under a nitrogen stream. After the solvent was completely removed by drying in vacuum overnight, the sample was hydrated at 100~o relative humidity and 80 °C for 12 h.
2.2. Preparation of Langmuir-Blodgett films A D P P A multilayer was deposited on a glass substrate by the LB technique. D P P A (free acid, Sigma) was dissolved in chloroform. This solution was introduced onto the surface of a distilled water suophase containing ~aCl 2 (10-4mol 1-1) at pH 6.9. The monolayer of D P P A on the water subphase was compressed to pressure of 40 mN m-1. The glass slide substrate was inserted and successively pulled out vertically through the monolayer into the trough (Takahashi Seiki) at a rate of 10-15mmmin -I by manual operation. When the surface pressure was kept at 40 mN m - 1, multilayered Y-type films were formed and the deposition ratio (DR) was about 0.9-1.1. Hydrated LB films for X-ray diffraction measurements were prepared by incubating at 100~o relative humidity and 80 °C overnight.
2.3. X-ray diffraction measurements Nickel-filtered CuKQt X-ray from a 90kW high power X-ray generator (RU1500, Rigaku Denki) was used. LB films on the glass slides were set in brass holders. The relative humidity kept at 100~o. The temperatures of samples were controlled by circulating water supplied from a temperature constant bath.
2.4. Electron spectroscopy for chemical analysis Electron spectroscopy for chemical analysis (ESCA) data were obtained with a PHI 5100 ESCA (Perkin-Elmer) system using Mg K s radiation at 10- 8-10 9 Torr. Samples were mounted on a rotating stage (25 mm in diameter and 10 mm in height). 3.
RESULTS AND DISCUSSION
Y-type LB fihns were formed in the present preparation. The behaviour of the transferred D P P A LB multilayer exhibited a surface pressure dependence. When the surface pressure was less than 30 mN m 1, the DR was less than 0.5 on the down trip of the glass slide. As the surface pressure was increased to 4 0 m N m -1, tlae DR improved to 0.9-1.1 for the down trips after the fourth deposition. On the second and third down trips the DR was 0.7q9.8. In contrast, the deposition ratio on the up trip was 0.9-1.1 and exhibited no surface pressure dependence. This means that it is easy to deposit one hydrophilic surface onto another hydrophilic surface. The strong attractive interaction between them may arise from electrostatic interactions between head parts of D P P A bridged by Ca 2 +. X-ray diffraction patterns at low angles for a 49-layer (24.5 bilayers) LB film
PHOSPHOLIPID LB FILMS
125
deposited on a glass slide and oriented multilayers were recorded on the X-ray film. Both samples exhibited the low angle (001) X-ray diffraction peaks resulting from the lamellar structure. The lamellar spacing was 52.3/~ for the DPPA LB film at 20 °C and 100~o relative humidity. This spacing is consistent of the formation of a Ytype LB film because the length of the DPPA molecule is about 27 ~. The lamellar spacing decreased slightly with increasing temperature. At 73 °C the spacing was 51.0 A for the DPPA LB film. The above difference of the spacing is too small to be identified with a phase transition. Liao and Prestegard 7 reported that a 1:1 complex o f D P P A - C a 2 + exhibited no thermal phase transitions between temperatures of 25 and 90 °C, although a phase transition of Ca 2 +-free DPPA vesicles in excess water at pH 6.5 takes place at 67 + 1 °C 8. After the DPPA film had been dried in the atmosphere, the lamellar spacing at 20°C decreased to 50.8 A, suggesting the intercalation of water between hydrophilic surfaces at 100~o relative humidity. Figure 2 shows X-ray diffraction photographs of the DPPA LB film (Fig. 2(a)) and DPPA oriented multilayers (Fig. 2(b)). The line width along the diffraction angle 20 of oriented multilayers is narrower than that of LB films. The line width depends on the correlation length of the lamellar repeat in the direction normal to the layers. If all molecules in the oriented multilayers are used for the formation of bilayers, about 100 bilayers should be produced in this oriented multilayer, suggesting that the area per molecule is 40 A z. However, this LB film has only 25 bilayers. Thus it is reasonable that the correlation length of oriented multilayers is larger than that of LB films. From the spreads of the (001) diffraction band recorded on the X-ray film, the regularity was compared between LB films and oriented multilayers. The angular variation associated with the regularity was not significant for DPPA LB film; in contrast, it was about 30 ° for oriented multilayers of DPPA. The angular variation in oriented multilayers of dimyristoyl phosphatidylcholine is also as large as in oriented multilayers of DPPA. This suggests that the large angular variation depends on the process of construction of the oriented multilayers rather than the nature of the head-group. These facts indicate that the structure in the LB films is more regular than that in the oriented multilayers. Nevertheless, in the case of DPPA molecules it might be possible that the Ca 2 ÷ ion incorporated in the DPPA LB film plays some role in constructing the regular structure. To check the regularity of the DPPA LB film, ESCA was used. The chemical composition at various depths from the surface of the DPPA LB film could be estimated by the take-off angle dependence of the photoelectrons. As the take-off angle increases, the chemical composition in deeper parts of the film is observed. Figure 3 shows the take-off angle dependence of phosphorus:carbon ratio for 49 layers (24.5 bilayers) of DPPA film deposited on the glass slide. The high P:C ratio corresponds to the head parts of DPPA molecules because DPPA consists of a hydrophilic head part having a phosphorus atom and hydrophobic hydrocarbon chains containing carbon atoms. The P:C ratio gradually increases until it is 60 ° of the take-off angle 0t and then saturates. Since the mean free path 2 of the electron is 31 A, a take-off angle of 60 ° corresponds to a value of 27 ,~ of the sampling depth D = 2 sin 0t. This means that the region of high P:C ratio is located near 27 A. This is
s. MATUOKA et al.
126
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(b)
Fig. 2. (a) X-ray diffraction photograph ofDPPA LB film (49 layers) at 20 °C and 100% relative humidity. The orders of the Bragg spots due to the lamellar spacing are indicated. The spot S~ is a first-order Bragg spot from reflected Cu K[3 radiation. S 2 is a reflection from the glass slide observed without DPPA. S 3 is the shadow of the beam stop. (b) X-ray diffraction photograph of oriented multilayers of DPPA at 20 °C and 100% relative humidity. The spot S I is a first-order Bragg spot from reflected Cu K I~radiation. S 2 is a reflection from the glass slide observed without DPPA. $3 is the shadow of the beam stop.
3 o
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DPPA LB Film t
010
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I
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70
Take-Off Angle (deg)
Fig. 3 Take-offangle dependence of P:C ratio for 49 layers of DPPA LB film obtained by ESCA.
PHOSPHOLIPID LB FILMS
127
consistent with the result of X-ray diffraction by which the hydrophilic head part is expected to be located around 25/~ from the surface. It is worth pointing out that essentially the same take-off angle dependence of the P:C ratio was obtained for 5 layers of DPPA film. This fact indicates that the structures of the deposited LB films are similar between the 5th layer and the 49th layer. REFERENCES I 2 3 4 5 6 7 8
A. Tardieu, V. Luzzati and F. C. Reman, J. Mol. Biol., 75 (1973) 711. Y.K. Levine and M. H. F. Wilkins, Nature New Biol., 230 (1971) 69. M.R. Alecio, A. Miller and A. Watts, Biochim. Biophys. Acta, 815 (1985) 139. K. Yoshikawa, H. Hayashi, T. Shimooka, H. Terada and T. Ishii, Biochem. Biophys. Res. Commun., 145 (1987) 1092. D.M. Taylor and M. G. B. Mahboubian-Jones, Thin Solid Films, 87 (1982) 167. H. Hasmonay, M. Caillaud and M. Dupeyrat, Biochem. Biophys. Res. Commun., 89 (1979) 338. M.J. Liao and J. H. Prestegard, Biochim. Biophys. Acta, 645 (1981) 149. K. Jacobson and D. Papahadjopoulos, Biochemistry, 14 (1975) 152.
123
STABILITY AND CHARACTERIZATION OF PHOSPHOLIPID LANGMUIR-BLODGETT FILMS SINZI MATUOKA,HARUMIASAMIAND ICHIROHATTA Department of Applied Physics, School of Engineering, Nagoya University, Nagoya 464-01 (Japan)
TOSHIOISHII School of Dental Medicine, Tsurumi University, Yokohama 230 (Japan)
KENICHIYOSHIKAWA College of General Education, Nagoya University, Nagoya 464-01 (Japan)
(ReceivedApril24, 1989;acceptedJune 1, 1989) The regularity of the bilayer structure was compared between dipalmitoylphosphatidic acid (DPPA) Langmuir-Blodgett (LB) films and "oriented multilayers" of DPPA, using X-ray diffraction and electron spectroscopy for chemical analysis (ESCA). The angular variation of the (001) Bragg spot due to the irregularity of the lamellar repeat was remarkable in the system of oriented multilayers. In contrast, the spot of DPPA LB films exhibited only a slight angular variation, suggesting a regular structure of this film. The take-off angle dependence of the phosphorus:carbon ratio obtained by ESCA indicates that the regular layered structure in DPPA LB film is maintained after 49 layers are deposited.
]. INTRODUCTION Phosphoiipids are amphiphilic molecules that form bilayer structures in water. For systems of multilamellar vesicles realized in excess water, these structures have been investigated intensively in connection with the structure of biomembranes 1. Well-arranged bilayers are very useful for studying their structures in detail. For this purpose so-called "oriented multilayers" have been used 2'3. The oriented multilayers have been constructed by putting the phospholipid solution onto a solid surface under a stream of nitrogen. Another excellent method for preparing wellarranged films is the Langmuir-Blodgett (LB) technique4. It has been reported that L B films of phosphatidylcholine molecules could not be constructedS. Hasmonay et al. 6 found, however, that it was possible to transfer LB multilayers of dipalmitoylphosphatidic acid (DPPA) with a divalent cation (Ca2+). Therefore the work reported here aims to investigate the difference in the regularity of the films between the oriented multilayers and the LB films constructed from DPPA (Fig. 1). 2. MATERIALSAND METHODS 2.1. Preparation o f oriented multilayers
DPPA (free acid, Sigma) was dissolved in chloroform-methanol (1:1 by volume) mixture (10 mg ml-1). 4 Ixl of the solution was put onto a glass slide and 0040-6090/89/$3.50
© ElsevierSequoia/Printedin The Netherlands
124
s. MATUOKAet al.
o,o Fig. 1. Structural formula for DPPA.
evaporated under a nitrogen stream. After the solvent was completely removed by drying in vacuum overnight, the sample was hydrated at 100~o relative humidity and 80 °C for 12 h.
2.2. Preparation of Langmuir-Blodgett films A D P P A multilayer was deposited on a glass substrate by the LB technique. D P P A (free acid, Sigma) was dissolved in chloroform. This solution was introduced onto the surface of a distilled water suophase containing ~aCl 2 (10-4mol 1-1) at pH 6.9. The monolayer of D P P A on the water subphase was compressed to pressure of 40 mN m-1. The glass slide substrate was inserted and successively pulled out vertically through the monolayer into the trough (Takahashi Seiki) at a rate of 10-15mmmin -I by manual operation. When the surface pressure was kept at 40 mN m - 1, multilayered Y-type films were formed and the deposition ratio (DR) was about 0.9-1.1. Hydrated LB films for X-ray diffraction measurements were prepared by incubating at 100~o relative humidity and 80 °C overnight.
2.3. X-ray diffraction measurements Nickel-filtered CuKQt X-ray from a 90kW high power X-ray generator (RU1500, Rigaku Denki) was used. LB films on the glass slides were set in brass holders. The relative humidity kept at 100~o. The temperatures of samples were controlled by circulating water supplied from a temperature constant bath.
2.4. Electron spectroscopy for chemical analysis Electron spectroscopy for chemical analysis (ESCA) data were obtained with a PHI 5100 ESCA (Perkin-Elmer) system using Mg K s radiation at 10- 8-10 9 Torr. Samples were mounted on a rotating stage (25 mm in diameter and 10 mm in height). 3.
RESULTS AND DISCUSSION
Y-type LB fihns were formed in the present preparation. The behaviour of the transferred D P P A LB multilayer exhibited a surface pressure dependence. When the surface pressure was less than 30 mN m 1, the DR was less than 0.5 on the down trip of the glass slide. As the surface pressure was increased to 4 0 m N m -1, tlae DR improved to 0.9-1.1 for the down trips after the fourth deposition. On the second and third down trips the DR was 0.7q9.8. In contrast, the deposition ratio on the up trip was 0.9-1.1 and exhibited no surface pressure dependence. This means that it is easy to deposit one hydrophilic surface onto another hydrophilic surface. The strong attractive interaction between them may arise from electrostatic interactions between head parts of D P P A bridged by Ca 2 +. X-ray diffraction patterns at low angles for a 49-layer (24.5 bilayers) LB film
PHOSPHOLIPID LB FILMS
125
deposited on a glass slide and oriented multilayers were recorded on the X-ray film. Both samples exhibited the low angle (001) X-ray diffraction peaks resulting from the lamellar structure. The lamellar spacing was 52.3/~ for the DPPA LB film at 20 °C and 100~o relative humidity. This spacing is consistent of the formation of a Ytype LB film because the length of the DPPA molecule is about 27 ~. The lamellar spacing decreased slightly with increasing temperature. At 73 °C the spacing was 51.0 A for the DPPA LB film. The above difference of the spacing is too small to be identified with a phase transition. Liao and Prestegard 7 reported that a 1:1 complex o f D P P A - C a 2 + exhibited no thermal phase transitions between temperatures of 25 and 90 °C, although a phase transition of Ca 2 +-free DPPA vesicles in excess water at pH 6.5 takes place at 67 + 1 °C 8. After the DPPA film had been dried in the atmosphere, the lamellar spacing at 20°C decreased to 50.8 A, suggesting the intercalation of water between hydrophilic surfaces at 100~o relative humidity. Figure 2 shows X-ray diffraction photographs of the DPPA LB film (Fig. 2(a)) and DPPA oriented multilayers (Fig. 2(b)). The line width along the diffraction angle 20 of oriented multilayers is narrower than that of LB films. The line width depends on the correlation length of the lamellar repeat in the direction normal to the layers. If all molecules in the oriented multilayers are used for the formation of bilayers, about 100 bilayers should be produced in this oriented multilayer, suggesting that the area per molecule is 40 A z. However, this LB film has only 25 bilayers. Thus it is reasonable that the correlation length of oriented multilayers is larger than that of LB films. From the spreads of the (001) diffraction band recorded on the X-ray film, the regularity was compared between LB films and oriented multilayers. The angular variation associated with the regularity was not significant for DPPA LB film; in contrast, it was about 30 ° for oriented multilayers of DPPA. The angular variation in oriented multilayers of dimyristoyl phosphatidylcholine is also as large as in oriented multilayers of DPPA. This suggests that the large angular variation depends on the process of construction of the oriented multilayers rather than the nature of the head-group. These facts indicate that the structure in the LB films is more regular than that in the oriented multilayers. Nevertheless, in the case of DPPA molecules it might be possible that the Ca 2 ÷ ion incorporated in the DPPA LB film plays some role in constructing the regular structure. To check the regularity of the DPPA LB film, ESCA was used. The chemical composition at various depths from the surface of the DPPA LB film could be estimated by the take-off angle dependence of the photoelectrons. As the take-off angle increases, the chemical composition in deeper parts of the film is observed. Figure 3 shows the take-off angle dependence of phosphorus:carbon ratio for 49 layers (24.5 bilayers) of DPPA film deposited on the glass slide. The high P:C ratio corresponds to the head parts of DPPA molecules because DPPA consists of a hydrophilic head part having a phosphorus atom and hydrophobic hydrocarbon chains containing carbon atoms. The P:C ratio gradually increases until it is 60 ° of the take-off angle 0t and then saturates. Since the mean free path 2 of the electron is 31 A, a take-off angle of 60 ° corresponds to a value of 27 ,~ of the sampling depth D = 2 sin 0t. This means that the region of high P:C ratio is located near 27 A. This is
s. MATUOKA et al.
126
3rd
.~'~
2nd
1st
Ist
$1 ~
SI
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s/
S2
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S3
(a)
(b)
Fig. 2. (a) X-ray diffraction photograph ofDPPA LB film (49 layers) at 20 °C and 100% relative humidity. The orders of the Bragg spots due to the lamellar spacing are indicated. The spot S~ is a first-order Bragg spot from reflected Cu K[3 radiation. S 2 is a reflection from the glass slide observed without DPPA. S 3 is the shadow of the beam stop. (b) X-ray diffraction photograph of oriented multilayers of DPPA at 20 °C and 100% relative humidity. The spot S I is a first-order Bragg spot from reflected Cu K I~radiation. S 2 is a reflection from the glass slide observed without DPPA. $3 is the shadow of the beam stop.
3 o
O
2
"6 O t~
tv-
DPPA LB Film t
010
I
3o
I
I
5o
I
I
70
Take-Off Angle (deg)
Fig. 3 Take-offangle dependence of P:C ratio for 49 layers of DPPA LB film obtained by ESCA.
PHOSPHOLIPID LB FILMS
127
consistent with the result of X-ray diffraction by which the hydrophilic head part is expected to be located around 25/~ from the surface. It is worth pointing out that essentially the same take-off angle dependence of the P:C ratio was obtained for 5 layers of DPPA film. This fact indicates that the structures of the deposited LB films are similar between the 5th layer and the 49th layer. REFERENCES I 2 3 4 5 6 7 8
A. Tardieu, V. Luzzati and F. C. Reman, J. Mol. Biol., 75 (1973) 711. Y.K. Levine and M. H. F. Wilkins, Nature New Biol., 230 (1971) 69. M.R. Alecio, A. Miller and A. Watts, Biochim. Biophys. Acta, 815 (1985) 139. K. Yoshikawa, H. Hayashi, T. Shimooka, H. Terada and T. Ishii, Biochem. Biophys. Res. Commun., 145 (1987) 1092. D.M. Taylor and M. G. B. Mahboubian-Jones, Thin Solid Films, 87 (1982) 167. H. Hasmonay, M. Caillaud and M. Dupeyrat, Biochem. Biophys. Res. Commun., 89 (1979) 338. M.J. Liao and J. H. Prestegard, Biochim. Biophys. Acta, 645 (1981) 149. K. Jacobson and D. Papahadjopoulos, Biochemistry, 14 (1975) 152.