Langmuir 1994,10, 2272-2276
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Hydration Behavior of Phospholipid Langmuir-Blodgett (LB) Films Deposited on a Quartz-Crystal Microbalance Depending on Temperatures in Water1 Katsuhiko Arigat and Yoshio Okahata* Department of Biomolecular Engineering, Tokyo Institute of Technology, Nagatsuda, Midori-ku, Yokohama 227, Japan Received August 10, 1993. In Final Form: April 15, 1994@ Hydration behavior of phospholipid Langmuir-Blodgett (LB) films in water was studied by using a quartz-crystal microbalance (QCM) as a substrate. The hydration rate and amount were obtained from time-courses of frequency changes of the QCM deposited with LB films of various phospholipids such as phosphatidylethanolamine (DLPE,DMPE, and DPPE), phosphatidylcholine(DPPC),phosphatidylglycerol (DPPG),phosphatidylserine (DPPS),and cholesterol. The phosphatidylethanolamino(PE)LB films showed the large hydration (water penetration) only around their phase transition temperatures (T,), in which two domainsof crystal and fluid liquid-crystallinestates coexist. The acyl chain length affected the hydration rate but not the hydration amount. The LB film of DPPC and DPPG having relatively hydrophilic head groups existed as a stable membrane in the solid state below their T,;however, it gradually flaked from the substrate in the fluid state above their T,. On the other hand, the LB film of DPPS having relatively water-unacceptable head groups hardly hydrated at any temperatures both below and above T,.In the case of cholesterol LB films, water can penetrate into the structure defects of the membrane at low temperature, but water is removed at high temperature due to the aging effect.
Introduction Hydration of phospholipid head groups is a n essential property not only for stabilizing bilayer structures in an aqueous environment, but also for fusion or endocytosis of biological membranes including protein transfer^.^-^ Hydration or swelling behavior has only been studied by indirect methods such as X-ray diffracti~n,~ differential scanning calorimetry (DSC),6 and 2H-NMR.79s Recently we have reported that adsorption behavaior of various bioactive compounds to lipid membranes can be measured as a mass change by using a lipid-coated quartz-crystal microbalance (QCM)in an aqueous We have also reported that the QCM is useful to detect in situ a transfer ratio of LB films during a transfer process on the QCM late.'^-'^ QCMs are known to provide mass measuring devices because their resonance frequencies decrease upon a given mass on the electrode of the QCM as small as nanogram leve1.16-18 + Currentaddress;SupermoleculesProject, JRDC, 2432 Aikawacho, Kurume, Fukuoka 830, Japan. Abstract published in Advance ACS Abstracts, June 15,1994. (1) Characterization of Langmuir-Blodgett Films 16. For part 15 ofthis series, see Ebara, Y.; Ebato, H.; Ariga, K.; Okahata, Y. Langmuir @
1994, 10, 2267. (2) Rand, R. P. Annu. Reu. Biophys. Bioeng. 1981, 10, 277. (3) McIntosh, T. J.; Simon, S. A. Biochemistry 1986,25, 4058. (4) Cevc, G.; Marsh, D. Biophys. J . 1985,47, 21. (5) Ruocco, M. J.;Shipley, G. G. Biochim. Biophys. Acta 1982,691, 309. (6) Chapman, D. Forms and Function ofPhospho1zpid-s; Elsevier Scientific: Amsterdam, 1933; p 117. (7) Finer, E. G.; Darke, A. Chem. Phys. Lipids 1974,12, 1. (8) Borle, F.; Seelig, J. Biochim. Biophys. Acta 1983, 735, 131. (9)Okahata, Y.; Ye, X.; Shimizu, A.; Ebato, H. Thin Solid Films 1989, 180, 51. (10)Okahata, Y.; Ebara, Y. J . Chem. SOC.,Chem. Commun. 1992, 116. (11) Okahata, Y.; Ebato, H. Anal. Chem. 1991, 63, 203. Okahata, Y.; Ebato, H. J . Chem. SOC.,Perkin Trans. 2 1991, 457. (12) Okahata, Y.; Ariga, K. J. Chem. SOC.,Chem. Commun. 1987, 1535. (13) Okahata, Y.; Ariga, K.; Tanaka, K. Thin Solid Films 1992,210/ 211, 702. (14) Okahata, Y.; Ariga, K. Langmuir 1989,5, 1261. (15) Okahata,Y.;Kimura,K.;Ariga,K. J . A m . Chem.Soc.1989,111, 9190. (16) Sauerbrey, G. 2.Phys. 1959, 155, 206.
In this paper, we determined directly the hydration behavior and stability (flaking) of LB films of various naturally-occurring lipids such as dilauroyl-, dimyristoyl-, and dipalmitoylphosphatidylethanolamine(DLPE, DMPE, and DPPE, respectively), dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylglycerol (DPPG), dipalmitoylphosphatidylserine (DPPS), and cholesterol (see Figure 1). The frequency of the QCM deposited with phospholipid LB films is expected to decrease (mass increase), when hydration occurred at head groups of LB films on the QCM in water. The initial hydration rate and the hydration amount were obtained at various temperatures below and above T,, changing acyl chain length and hydrophilic head groups in phospholipids. Twodimensional morphology of phospholipid monolayers during hydration was also observed by fluorescent microscopy.
Experimental Section Materials. Lipid molecules, such as DLPE, DMPE, DPPE, DPPC, DPPG, DPPS, and cholesterol, were purchased as analytical grade chemicals. Benzene, chloroform, and ethanol for a stock solutionof an LB film studywere purchased as spectraanalyticalgrade chemicals. Octadecylrhodamine as a fluorescent probe was kindly offered from Prof. M. Shimomura (Hokkaido University,Sapparo). Water for the subphase was purified with Milli-QII system (Nippon Millipore Co., Tokyo) and poured directly in a trough. "he specific resistance ofthe water was c a . 18 MR cm. Langmuir-Blodgett (LB)Films. Measurements of pressure-area (n-A) isotherms and transfers of monolayers on a substrate (QCM plate) were carried out by using a computercontrolledfilm balance system(San-EsuKeisoku,Co., Fukuoka, Model FSD-20).l2-l6The concentrationof lipid solutions was ca. 1 mg/mL in chlorofodethanol and the spreading amount of lipid solutions was ca. 50-150 pL. After solvent evaporation,a monolayer was compressed at speed of 0.60 cm2s-l. "he surface of Ag electrodes on a QCM was turned to be hydrophobic by treating with 1,1,1,3,3,3-hexamethyldisilazane. l9 The optimum condition for the LB fdm transfer was determined by using a QCM as a substrate: transfer amounts of monolayers were (17) Ebersole, R.; Ward, D. M. J . Am. Chem. SOC.1988,110, 8623. (18) Thompson,M.;Arthur,C.L.;Dhaliwal,G. K A n a l . Chem. 1986, 58, 1206. (19) Farriss, G.; Lando, J.;Rickert, S. J . Mater. Sci. 1983,18,2603.
0743-7463/94/2410-2272$04.50/00 1994 American Chemical Society
Langmuir, Vol. 10,No. 7, 1994 2273
Hydration Behavior of Phospholipid LB Films Personal computer
Thermometer
LB Film-deposited Quartz-Crystal Microbalance (QCM)
II 0
LB Film-forming Lipid Molecules
DPPC CH~-(CH~)KCOO
I
0
CH3
6
OH
C H 3 - ( C H 2 ) 1 ~ C 0 ~-0 - o y N H : DPPS
'ud
HO
1
2 Time / hour
3
4
It is close to the theoretical equation calculated from eq 1 ( A m AF). The stability of the QCM frequency was = -1.30 x also examined. The standard deviations of frequencies in water at various temperatures were 2.5Hz a t 11.5 "C, 2.4 Hz at 29.9 "C, 7.4Hz at 39.3 "C, 8.2Hz at 51.0"C, and 15.9Hz a t 70.1 "C. When 10 layers of DPPE LB films were deposited on each side of the QCM, the mass on the QCM could be calculated to be 1130 f 5 ng from the frequency decrease of 889 f 5 Hz according to eq 2. This value was highly consistent with the theoretical mass of 20 dry layers of DPPE (1150ng).12-15 The resonance frequency is known to be affected not only by mass changes but also by changes of resonance resistance (R), especially when the depositing membrane exists in the fluid ~ t a t e . 1 ~The 9 ~resonance ~ resistance was also measured in water at various temperatures by an impedance analyzer (Yokogawa Hewlett-Packard, Co., Tokyo, Model 4192A). Observation of LB Films by Fluorescence Microscopy. The two-dimensional morphologyof transferred monolayers was observed by using a fluorescent microscope (Olympus,Co., Tokyo, Model BSH-RFK). The fluorescence image was detected with a highly sensitive SIT camera (Hamamatsu Photonics, Co., Tokyo, Model C2741)and an image processor (Hamamatsu Photonics, Co., model DVS-1000). The monolayer of DMPE with 2 mol % of octadecylrhodamine as a fluorescent dye was spread on water and transferred on to a slide glass plate (10mN m-l, 20 "C). Monolayer was immersed in water a t fixed temperature for 10 min. After drying the monolayer, the fluorescence image was observed with the fluorescence microscope. Octadecylrhodamine exists only in noncrystalline phase; therefore, the disturbed fluid region and the crystalline region are observed as a fluorescent and a dark image, respectively.22
(?:oyoH
C H 3 ~ ( c H 2 ~ 1 ~ c0~o DPPG
.
1
Figure 2. Typical time-courses of frequency changes when the QCM deposited with 10 layers of DMPE (T,= 49 "C) LB films on each side is immersed in water (a) at 25 "C, (b) a t 70 "C, and (c) at 50 "C.
0
DLPE (n = 12) DMPE (n = 14) DPPE (n = 16)
vn v
6
coo-
Choresterol
Figure 1. Apparatuses for frequency measurements of the LB film-deposited QCM in water and structures of lipid molecules. followed in situ from the frequency decrease (mass increase) of the QCM substrate.12J3 Although most phospholipid LB films could be transferred by a conventional vertical dipping method, the transfer of DPPC and DPPG monolayers having hydrophilic head groups was succeededonly by a horizontal lifting method.13 Quartz-Crystal Microbalance (QCM). QCMs (At-cut, 9 MHz, quartz plate area: 0.640cm2),on each side of which two Ag electrodes (area: 0.238cm2)were deposited, were purchased from Kyushu Dentsu Co., Tokyo. A homemade oscillator circuit specially designed to drive the quartz at its resonant frequency in aqueous phase was employed for frequencymeasurements. 12-15 The frequency of QCM was followed continuously by a universal frequency counter (Iwatsu,Co., Tokyo, Model SC 7201)attached to a microcomputer system (NEC,PC 9801Model). The following equation has been obtained for the AT-cut shear mode QCM:16
where AF is the measured frequency shift (Hz), F, the parent frequency of the QCM (9 x lo6 Hz),A m the mass change (g),A the electrode area (0.238cm2),,osthe density of quartz (2.65g ~ m - ~and ) , p, the shear modulus of quartz (2.95 x loll dyne cm-2). Calibration of the QCM used in our experiments by a polymer-casting method and an LB film transfer method gave the following e q ~ a t i o n . ~ - l ~ A m = -(1.27 f 0.01)x lo-' hF
(2)
Results and Discussions Hydration of PhosphatidylethanolamineLB Films. Ten layers of dimyristoylphosphatidylethanolamine (DMPE) LB films were deposited on the QCM. The QCM was immersed into temperature-controlled water phase, and the frequency was followed with time. Typical timecourses of frequency changes are shown in Figure 2. The phase transition temperature from solid to liquid crystalline states of DMPE membranes were determined to be T,= 49 "C from differential scanning calorimetry. The frequency hardly changed at 25 "C (below T,)and at 70 "C (above T,).On the contrary, the frequency largely decreased at 50 "C (near T,), which indicates the mass increase due to hydration around head groups of phospholipid LB films. After reaching the equilibrium of the frequency decrease, the QCM was picked up to the air phase and dried. The frequency was gradually reverted (20)Muramatsu, H.;Kimura, K. Anal. Chem. 1992, 64, 2502. (21)Muramatsu, H.;Tamiya, E.; Karube, I. Anal. Chem. 1988,60, 2142. (22) Shimomura,M.; Fujii, R;Shimamura,T.; Oguchi, M.; Shinohara, E.; Nagata, Y.; Matsubara, M.; Koshiishi, K. Thin Solid Films 1992, 2101211 , 98.
Ariga and Okahata
2274 Langmuir, Vol. 10, No. 7, 1994
Number of Layers
Temperature / "C Figure 3. Effect of temperatures on the hydration amount W, (0)and the initial hydration rate vo (A)of 10 layers of DMPE LB films. to the original mass of the dry LB films before immersing in water due to the evaporation of water in air. Thus, the frequency decrease indicates simply the mass increase due to hydration on LB films. The resonance frequency has been reported to be also affected with the change ofviscoelasticityofthe membrane on the QCM, especially when the membrane is thick and f l ~ i d . ~ The ~*~ resonance ~ r ~ ~ resistance reflecting viscoelasticityof the membrane was measured by an impedance analyzer to be constant (800f 50 SZ) during the large resonance frequency change (AF= -1400 f 10 Hz) at 50 "C (near T,). WeI6 and Muramatsu et aL20have already mentioned that the viscoelasticity change does not affect the frequency change when the membrane is very thin such as 10layers of LB flms even in the fluid state, because the energy loss in the thin film is very small. Therefore, the frequency decrease of 10 layers of DPPE LB films around their T, shown in curve c in Figure 2 is attributed mainly to the mass increase due to the hydration ( s w e b g ) of lipid membranes on the QCM. Twenty layers (1130f 5 ng) of DMPE LB films (each 10 layers on both sides of QCM electrodes)were observed to be hydrated with 1780 f 20 ng of water near T,a t 50 "C: 61 mol of HtO per 1 mol of DPPE. It has been reported that DPPE has 7-8 mol of non-frozen water molecules per lipid even in the dry state.24 The initial hydration rate v, and the equilibrium hydration amount W, were obtained as parameters reflecting the hydration behavior of LB films (see Figure 2). Temperature dependencies of the hydration behavior ( y o and W,)of 10 layers of DMPE (T, = 49 "C)LB films are shown in Figure 3. Large W, and v, values were observed only around the phase transition temperature (T,= 49 "C)of DMPE membranes. Thus, DMPE LB films were hydrated only near the T,, but not in the solid state below the T,and in the fluid state above the T,. This indicates that the coexistence of two phases (solid and fluid domains) near the T,caused the large hydration rate and amount in the LB films. Figure 4 shows effects of the membrane thickness of DMPE LB films on the hydration behavior a t three different temperatures. The hydration amount (WJ increased linearly with increasing the number of layers of LB films only around T,,but not temperatures below and above T,.This indicates that water molecules deeply penetrate into LB layers around T,. The hydration rate (Y,) was very large and hardly depended on the membrane (23)Jedrasiak, G.L.; Hasty, J. H. Biochim. Biophys.Acta 1974,337, 79. (24)Ladbrooke, B. D.;Chapman, D. Chem. Phys. Lipids 1969,3, 304.
Figure 4. Effect of the number of layers of DMPE LB films on each side of the QCM on the swelling behavior (W,and yo). (0) at 20 "C (below TA;(0)at 50 "C (nearTJ;(A) at 65 "C (above Tc).
X J0 c12
DCPE
c14
DMPE
cl6
DPPE
Acyl Chain Length Figure 6. Effect of acyl chain length of phospholipid (PE)LB films (10 layers on each side)on the swelling behavior (W,and vo)around each T,(DLPE: T,= 30 "C, DMPE: T,= 49 "C, and DPPE: T,= 63 "C).
thickness around T,. This means that water can penetrate from the top surface of the membrane, but not from the side part of LB films. Figure 5 shows effect of acyl chain length of phospholipid on the hydration behavior around the each T,of 10 layers of LB films of DLPE ((312, T,= 30 "C), DMPE (c14,T, = 40 "C), and DPPE (C16, T,= 63 "C). The hydration amount W, was constant and independent of acyl chain length, which indicates that the hydration water exists around hydrophilic head groups, but not in the hydrophobic acyl chain region. On the other hand, the hydration rate v, decreased sharply as the chain length increased. This means that water molecules penetrate through the defects of coexistingsolid and fluid states in the acyl chain regions near the T,,the hydration rates decreased largely in the long chain lipid membranes, and water molecules exist in the hydrophilic regions between layers. Hydration of Other Phospholipids and Cholesterol. Ten layers of DPPC (T, = 42 " C )LB films were deposited on a QCM plate by a horizontal lifting method on one side and immersed in water. Typical time-courses of frequency changes of the DPPC-deposited QCM are shown in Figure 6. At 25 "C in the solid state below the T,, DPPC LB films were stable and hardly swelled in water. However, the frequency gradually increased (mass decreased) at 50 "C (above the T,)and reached equilibrium a t AF = 450 f 50 Hz (-Am = 575 f 50 ng), which is equivalent to the loss of 10 layers mass of the dry LB films. Frequency measurements after drying in air indicated that most of LB films flaked from the QCM plate
Hydration Behavior of Phospholipid LB Films 500
Langmuir, Vol. 10,No. 7,1994 2275
1
a c
-s
2500.
2000.
-
5 1500m
c
io
at 25 "C
0
0
1
2
3
4
5
Time 1 hour Figure 6. Frequency changes of the QCM deposited with 10 layers of DPPC LB films when the QCM is immersed in water at 25 "C (below T,)and at 50 "C (around the T,of DPPC).
c
2500t
-.m
-200
2
-100
E
c
"
O
30
20
io
50
6'0
0
70
Temperature / "C Figure 8. Effect of temperature on the hydration amount W, (0) and the initial hydrationrate Y,(A)of 10layers of cholesterol LB films on each side of the QCM. without
Aging
t 15001
DU
Temperature / "C Figure 7. Effect of temperature on the hydration amount W, (0)and the initial hydration rate yo (A)of 10 layers of DPPS LB films on each side of the QCM.
into water. The similar flaking behavior in water at temperatures above the T,was also observed in the LB film of DPPG (T,= 42 "C). Thus, LB films of DPPC and DPPG having relatively hydrophilic head groups such as phosphocholine and phosphoglycerol are easily hydrated and then flaked in the fluid state above their T,in water. The hydration behavior of DPPS (T,= 55 "C) LB films is shown in Figureb 7. The DPPS LB film having phosphoserine head groups little hydrated (Am = 600 f 10 ng, 20 mol of water per lipid) even near the phase transition temperature. Hydration ability has been reported from adsorption experiments of water vapor to lipid powder to be in the order of PC > PE > PS lipids.23 It has been determined from calorimetry that the amount of nonfrozen water around lipid molecules is 10 mol, 7-8 mol, and 0 mol for 1 mol of PC, PE, and PS lipids, r e ~ p e c t i v e l yThis . ~ ~ tendency is consistent with our results that PC molecules are easily hydrated and flaked from the substrate, and PE and PS lipids are hydrated with 61 and 20 mol ofwater per lipid around their T,, respectively. The hydration behavior of LB films of cholesterol is shown in Figure 8. Interestingly both the hydration amount W, and the hydration rate v, decreased with increasing temperatures. Since cholesterol molecules are very hydrophobic and are thought to be hardly hydrated, the incorporated water might exist in the structure defects near the hydrophilic OH groups of LB films and these defects could disappear at high temperature by an annealing effect. After the aging of cholesterol LB films a t 70 "C for 1h in water, cholesterol membranes are hardly hydrated a t all temperatures (W,= 50 f 10 ng, 3-4 mol of HzO per cholesterol).
p1rr
\\
aged below T, (20 "C1
aged near T, (50 "C) Figure 9. Fluorescent images of the DMPE monolayer on a slide glass before and after aging at different temperatures. The details are described in the text.
Observation of LB Films by Fluorescence Microscopy. In order to confirm the large hydration behavior of phospholipid LB films only around their T,, twodimensional morphology of DMPE monolayers was observed by a fluorescence microscope. The DMPE monolayer containing 2 mol % of octadecylrhodamine as a fluorescent dye was transferred onto a slide glass and aged in water at different temperatures for 10min. After drying, the fluorescent image was observed and photographs are shown in Figure 9. A dark region represents the crystalline phase, because the fluorescent dye is excluded from the crystalline domain and exists in the disordered domain. The 10-20 pm size of crystalline domains were observed in the samples without aging and aged a t 20 "C (below T,).The sample after aging a t 60 "C (above T,)showed large crystalline (dark)domains due to a n annealing effect. On the other hand, when the sample was aged a t 50 "C (around T,),very small crystalline domains were observed, which indicates the microscopiccoexistenceof crystalline and liquid crystalline
2276 Langmuir, Vol. 10, No. 7, 1994
Ariga and Okahata
phases near T,.The total area of the disordered region (white region) after aging near T,was larger than those at any temperatures above and below T,.These observations are consistent with the large hydration behavior of DMPE LB films only around T,,which may occur in the defects between two domains. We also observed the morphological pictures of cholesterol LB films. The small disordered (white) area was frequently observed in the prepared cholesterol LB films and they were largely decreased after aging at 70 "C for 1h (photographs are not shown). This is consistent with the hydration behavior that the hydration of cholesterol LB films was largely decreased with increasing temperatures (see Figure 8).
lipid LB films can be classified into three types: (i) phospholipids having relatively hydrophilic head groups such as DPPC and DPPG are hydrated and then easily flaked from the substrate in the fluid liquid-crystalline state above T,;(ii) DPPE and DPPS having the less hydrophilic head groups are hydrated only near their T,; (iii) cholesterol LB films show relatively large hydration behavior even a t low temperatures due to the water penetration into the structure defects in the membrane. This is the first example to demonstrate directly and quantitatively the hydration behavior of phospholipid membranes in water. The combination of the QCM and the LB method is a useful tool for characterization of lipid membranes in water.
Conclusion From the frequency measurements of the LB-filmdeposited QCM plate in water, the behavior of phospho-
Acknowledgment. We thank Professor M. Shimomura (Hokkaido University) for the support of the fluorescence microscopy experiments.
