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Langmuir 1992,8, 2124-2729
Microdomain Structures in Polymerizable and Nonpolymerizable Diacetylenic Phosphatidylcholine Monolayers Sek Wen Hui' and Hao Yu Biophysics Department, Roswell Park Cancer Institute, Buffalo, New York 14263
Zhenchun Xu and Robert Bittman Department of Chemistry and Biochemistry, Queens College of the City University of New York, Flushing, New York 11367 Received November 18,1992. In Final Form: June 29, 1992 1,2-Diacyl-sn-glycero-3-phosphocholineswith diacetylenic acyl chains were synthesized with the diacetylenic bonds at the (4,64,6),(4,6;5,7),and (10,12;10,12) carbon positions. The molecular packing of these lipid monolayers at the air-water interface was studied by pressurearea measurements and by fluorescence microscopy. During first-time compression, an overshot or hump was detected in the F A isothermsof monolayers of the (4,6;4,6)diacetyleniclinked compound only. Thisphenomenon may indicate a lack of nucleation for the growth of domaine. On compression, dark domaine of this compound were observed from monolayers labeled with 1% phosphatidylethanolne-rhodamine. These domains are thin and long; severalof these domainsshare a common origin. These feather-likedomains have a tendency to bend counterclockwise. The domains became smaller and numerous upon subsequent recompression. After W exposure, the dark domains retained the shapes even after decompression. The long, thin domain shape indicates ordered molecular packing conetrains within the dark domains,possibly from the alignment of the diacetylenic moiety. The other two phospholipids and their corresponding fatty acids in their free form do not produce visible solid domains and do not become polymerized. The reasons for their different characteristics are discussed.
Introduction Photopolymerizable diacetylenic lipids have received much attention recently because they have potential biotechnological applications. These lipids, once polymerized, form extremely stable structures which may be used in surfacecoatingfor biocompatible materials, supporting matrices for biosensing molecules,and carrier vehicles for drugs, among other appli~ations.l-~ Their uses in surface coatings or in stabilizing matrices in biosensors involve the deposition of the polymerized lipids as monolayers on a solid substrate. The microstructure of the monolayers is very important with regard to their uniformity and stability. Although severaldetailedstructural studies have been reported on the tubules and bilayers of polymerized lipids,C7very little is known about the microstructure of monolayers formed by these polymerized lipids. Diacetylenicphospholipidsand their analogs,including phosphoesters, alcohols, acids, and amides containing diacetylenichydrocarbonchains, form monomolecularlayers on the air-water interface.8*@ The pressurearea curves of these analogs and some phospholipids (PC, PE, and PA) containing hexacoxa-l0,12-diynoic acids have been measured. It was reported that the diacetylenic phospholipTo whom correspondence should be addressed. (1)Yager, P. In Biotechnological Applications of Lipid Microstructures; Gaber, B. P., Schnur, J. M., Chapman, D., Eds.; Plenum Press: New York, 1988,p 257. (2)Regen,5.L. Ann. N.Y.Acad. Sei. 1986,445,296. (3)Johnston, D.S.; McLean, L. R.; Whittam, M. A,; Clark, A. D.; Chapman, D. Biochemistry 1989,22,3194. (4)Yager,P.; Schoen, P. E.; Davies, C.; Price, R.; Singh, A. Biophys. J. 1986,48,899. (5) Caffrey,M.; Hogan, J.;Rudolph,A. S.Biochemistry 1991,30,2134. (6) Plant, A.L.; Benson, D. M.; Truaty, G. L. Biophys. J. 1990,57,926. (7) Tremor, R.; Pace, M. D. Biochim. Biophys. Acta 1990,1046,1. (8) Hub, H. H.; Hupfer, B.; Koch, H.; R i o r f , H. J. Macromol. Sci. Chem. 1981,A15,701. (9)Hupfer, B.; Ringadorf, H. Chem. Phys. Lipids 1983,33,263.
ids behave similarly to their saturated chain counterparts but tend to have lower critical transition temperature and collapse at a larger area per molecule.B Recent advances on the use of fluorescence microscopy to study mono1ayer~lO-l~ enable UB to make a thorough study of the microstructure of polymerizable diacetylenic lipids on the air-water interface, prior to its deposition on a solid substrate. We report here our measurements of the pressurearea relationship of a series of diacetylenic phosphatidylcholines and their constituent fatty acids, before and after these lipids are exposed to UV light. We present our observations of unpolymerizable diacetylenic lipids, of reversible phase-separated domains of polymerizablebut unpolymerizedlipid monolayers at relatively high surface pressure, and of irreversible domains formed by polymerized lipidsat relativelyhigh surfacepressure. The factors governing the size and shape of domains are compared with previously published theories.lkl7
Materials and Methods Three kinds of dioctadecadiynoylphophatidylcholine (PC), named D C ~ ~ O PDCa,SC, C, and DC~,I~~,DPC (see Figure 11, with the diacetylenic unit varied along the hydrocarbon chains, were synthesized. The diacetylenicfatty acids were synthesized wing (10)von Tscharner, V.;McConnd, H. M. Biophys. J. 1981,36,409. (11)Peters, R.; Beck, K. R o c . Natl. Acad. Sci. 1988,80,7183. (12)k h e , M.; Sackmann, E.; Mahwald, H. Ber. Bunsen-&a. Phys. Chem. 1983,87,848. (13)McConneu, H.M.; Ta", L. K.;Weis, R. M. Proc. Natl. Acad. Sci. U.S.A. 1984,81,3249. (14)Weis, R. M.; McConnell, H. M. J. Phys. Chem. lS86,89,4453. (15)Fiecher, A,; Ihche, M.; M6hwald, H.;Sackmann, E. J.Phys.Lett. 1984,46L,785-L. (16)Keller, D. J.; McConnell, H. M.; Moy, V. T. J. Phys. Chem. 1986, 90,2311. (17)McConnell, H.M.; Moy, V. T. J. Phys. Chem. 1988,92,4520.
0143-1463/92/2408-2124$03.00/ 0 Q 1992 American Chemical Society
Langmuir, Vol. 8, No.11,1992 2126
Microdomain Structures in Phosphatidylcholine Monolayers 1
0 II R,-C-0-CH,
-
I R,-C-0-CH 0 I1 I I o CH,-0-P-0-CH,-CH,-N'-
II
(cy),
eEc
60-
50-
-
40-
t p!
30-
h
0'
U
Q%!OPC
R,=R,:
CH,- (CH,) , , - C E - C E -
(CH,),-
DC,,'PC
R,:R,:
CH,- (CH,) ,-CIC-CIC- (CH2),-
OC,,,, ,,pPC
R,:
CH,-(CH,),,-CIC-CIC-(CH,),-
p! a
n Q)
R,:
CH,- (CH,),-CE-C%-
Figure 1. Chemical structures of DCz,#C,
(CH2),-
DCs,rPC, and
DCz.io,s$'C.
f
20-
m 10-
butadiyne synthons as startingmaterials18and then were coupled to glycerol-3-phosphocholinecadmium-chloridecomplex in the presence of dicyclohexylcarbodiiiide and 4-(dimethylamino)pyridine. The purity of the lipids was indicated by a single spot in thin layer chromatography on silica gel GF glass plates (Analtech, Newark, DE). The producta had the appropriate lH nuclear magnetic resonance spectral characteristics and gave satisfactory combustion analyses for C, H, N, and P. The latter were carried by Desert Analytics (Tucson, AZ). The lipids were sealed in argon gas and stored at -70 OC prior to use. The fluoreacentprobe, phosphatidylethanolaminerhodamiie (PE-Rh), was purchased from Molecular Probes (Eugene, OR) and was mixed with the diacetylenic lipids at 1 mol %. The probe is known to preferentially partition in the fluid domain of phospholipid monolayers (Huiand Yu, unpublished results) and hae only a slight contribution to the pressure/area characteristics of the monolayer. AU solid powder lipid samples were dissolved in chloroform at a concentration of 0.5 mg/mL under a NZatmosphere just before spreading on a Langmuir trough. The custom-built, fourcompartment, environmentally controlled Langmuir trough and barriers were maintained within an enclosed chamber with thermostat wall, floor, and ceiling. The chamber was filled and continuously flushed with nitrogen gas. The size of the final trough where measurements were made was 15 X 15cm2. Surface preasure versus surface area per molecule of monomolecular films ( P A isotherms) were measured by a P A recording system. The area was adjusted by a motor-driven Teflon bar moving along the surface of the trough; the movement of the bar is either manual or servo-controlled (in the isobaric mode) by the output of the electrobalance. The position of the barrier, which moved at a rate of 1-10 mm2/s(0.18-1.8 (Az/molecule)/min),was converted to a voltage signal which was connected to the X-input of an X-Y recorder. Surface pressure measurements were made by a Wilhelmy plate. The change of surface tension was measured by a Cahn RG electric balance. The surface pressure (P)is defined
as r=
To-T
where TOis the surface tension of triple distilled water, and T is the surface tension of the f i i covering the water substrate. The amplified signal was connected to the Y-input of a Linseis LY-1600 X-Y recorder which plotted the a-A isotherms. Epifluorescence microscopic images were captured by an A 0 fluorescence microscope with a 40X (NA = 0.85) objective lens. A Teflon retaining ring with a small opening was attached to the objective lens, and immersed through the airwater interface. It was used to minimize monolayer drift. A Dage-MTI silicon intensified target (SIT)video camera MCP-SIT66was attached to the microscope. A Magnavox-80monitor was used to observe the image, which was recorded by a Panasonic A m 5 0 video recorder. The processing of image from the videotape cassette was carried out in an IBM-PC-ATcontrolled Datacube Maxvision system. Polymerization of the lipids in monolayers was done by illuminating the monolayer with an 18.4-W, 254-nm UV light (UVGL-58,UVP, San Gabriel, CA)for 15min. The light source (18) Xu,2.;Byun, H.-S.; Bittman, R. J. Org. Chem. 1991,56, 7193.
20
30 40 50 60
70 80 90 100
Area/Molecule
(A',
Figuret. Preasurearea ( P A ) isotherms of DC%1#C containing 1mol % PE-Rh, on the f i t compression at temperaturea of (a) 8.5, (b) 10.5, (c) 15.5, and (d) 21 OC. The pointa marked by arrowheads indicate the detectable onset of dark fluoreecence domaine, the corresponding area per molecule at the onset ie about 45 Az. was about 1cm from the monolayers. The temperature of the trough was maintained by circulating coolant during the W exposure.
Results 1. Pressure-Area (*-A)Characteristics. The F A isotherms of DCZJOPCwith 1 mol 7% of PE-Rh are presented in Figure 2. The addition of 1 mol % of the fluorescent dye has a minor effect on the P A characteristics. An "overshoot" transition at 50-55 A2/moleculeia observed at the onset of the LE/LC coexistence phase, which is normally seen as a plateau region in most phospholipids,such as dipalmitoylphosphatidylcholine(DPPC) monolayer^.'^ Thisovershoottransition existaup to h a t 21 OC when it is no longer observable (Figure 2). The critical temperature of DC2,loPCis considerablylower than that of the corresponding saturated PC, namely distearoylphoaphatidylcholine(DSPC),in agreementwith previous findingsmgThe transition from liquid condensed (LC) phase to solid condensed (SC)phase2O4linDCz,loPCmonolayers is not detected. The relatively incompressiblesolid phase increases with temperature, from 35 to 40 Azas the temperature increases from 8.5 to 21 OC (Figure 2). It is interesting to note that the overshoot hump ie observed only in monolayers at the low compression rate, about 1 mm2/s(0.18 (A2/molecule)/min)for the first time compression. Under fast compression,such as 100 mm2/s (18 (A2/molecule)/min),the hump is absent even if the compression is for the first time. The expansion curve after compression to 50 dyn/cm, and all subsequent compression curves, shows no humps but an onset of a relatively flat plateau (Figure 3, dashed lines) similar to the DPPC curves.19 Upon subsequent recompressions, after expansion to the equivalence of 100AYmolecule,the curves shift slightly to the left, indicating some loss of lipids from the monolayer at each cycle. Except for the 21 OC isotherm (curved in Figure 21,no monolayer collapse was observed. (19) Hui, S. W.; Cowden, M.; Papahadjopouloe,D.; Pareone,D.F.Biochim. Biophys. Acta 1976,382, 266. (20) MOhwald, H. Annu. Reu. Phys. Chem. 1990,41,441. (21) Weis, R. M. Chem. Phys. Lipids 1991,57,227.
Hui et al.
2726 Langmuir, Vol. 8, No. 11,1992
20
Area/Molecule (A2) Figure 3. Isotherms of DCZ,loPC/PE-Rh with the initial compression (-) and decompression at 10.5 "C with (- -) or without (- -) exposure to UV (underthe surface pressure at 50 dydcm).
-
40
60
80
100
120 (40 160
Area/Molecule (A2) Figure 5. Pressure-area ( P A ) isotherms of DCap@C/PERh at (a) 11, (b) 22, and (c) 32 O C on the f i t compression.
-E 50t
io
20
30
40
50
60
Area/Molecule (Az) Figure 4. Pressure-area ( P A ) isotherms of DCa,$C/PE-Rh at (a) 11, (b) 22, and (c) 32 "C on the f i t compression.
Area/Molecule (A2) Figure 6. Initial compression isotherms of the fatty acids (a) rl,&octadecadiynoic acid, (b) 6,7-octadecadiynoic acid, and (c) 10,12-octadecadiynoicacid. Each lipid contained 1mol % PERh.
UV-induced polymerization at the high pressure range (about 50 dyn/cm) results in rendering the monolayer extremely inelastic. An abrupt decline of pressure was observed upon a slight increase of area (Figure3, dots and dash lines). UV exposurehad no effect on the n-A curves if applied in the low pressure ranges where no domains were observed (see below). The PA isotherms of DCa,$C are shown in Figure 4. No transition was observed at temperatures as low as 11 "C, indicating that this lipid has a very low critical temperature. UV exposure had no observable effect on this lipid under our experimental conditions. At and below 22 "C, the T-A isotherms of DC2,lo;3,sPC show a kink-type transition (Figure 6); the hump-type transition is not detected. W exposurehad no effect on DC2,lo;3,sF'C monolayers. Otherwise,the n-A characteristics and the critical temperature are similar to those of DC2,loPC. At 26 "C, the monolayers of the diacetylenic odadecaynoic acids collapse at relatively low surface pressures (Figure 6). 2. FluorescenceMicroscopic Imagingof Domains. After the initial spreading at near zero pressure and prior to compression,the fluoreacencemicroscopicview of DC2,lr
PC monolayers containing 1%PE-Rh is homogeneous. Dark domain began to be recognized upon slow compreasion (0.18 (A2/molecule)/min)to around 46 A2/molecule (indicatedby arrows in Figure 2). The domain distribution in the monolayers is not uniform. The dark domains fmt appear as irregular dots and then quickly grow into strips. As the area is reduced,the domainstake on curved,featherlike shapes (Figure 7a). The thick ends of the feathers join together, and the thin ends invariably curve counterclockwise,resulting in a "phoenix tail" cluster. Aa the monolayer is compressed,the phoenix clusters are pushed together but the thickness of each feather remaine constant. During the expansion cycle, the feather-shape domains disperse and separate (Figure 7b) and finally merge with the uniform brightness of background, when the monolayer reaches a equivalent of >66 A2/molecule. The feather-like domains are longer at lower temperatures, as exemplified in Figures 8a,b and 9a,b, which represent the phoenix clusters seen at 10.6 and 8.6 "C, respectively, as compared to 16.6 OC in Figure 7a. Some feather-shape domains show branching at the thin end. If the monolayer pressure is reduced to near zero and the monolayer then recompressed,the feather-shapedomains
20
40
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io0
120
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Microdomain Structures in Phosphatidylcholine Monolayers
Langmuir, Vol. 8, No. 11, 1992 2727
Figure 7. Solid domains in DC2,loPC monolayer at 15.5 "C, as revealed from a higher fluorescentbackground in PE-Rh labeled fluid lipids. The s-urface pressures are (a) compressed to 30 dyn/ cm (41 A2/molecule)and (b) decompressed to 17 dyn/cm (52 A2/molecule).
become shorter and more numerous, and the phoenix clusters are much smaller (Figure 8c). When monolayers of DC~JOPCare exposed to UV while compressed to less than 40 A2/molecule (about 50 dyn/ cm), polymerization occurs as indicated by the inelastic behavior of the monolayers (Figure 3, dash and dot line). Upon decompression, polymerized phoenix clusters remain and become thickened (Figure 9b). Further decompression, even when the surface pressure is decreased to 0 dyn/cm (45-55 A2/molecule),leads to the breaking-up of some feathers into fragments, but each fragment retains the elongated or spindle shape. In some areas, these domain fragments coalesce into irregular shape patches (Figure 9c). Discussion 1. Monolayer Organization. In comparison to the well-studied saturated PC's, the monolayer F A characteristics of diacetylenic PC are distinct in that (1) the well-known LE/LC plateau region is replaced by a hump and steep incline and (2) the SC phase packing is very sensitiveto temperature. Previous monolayer 7r-A studies of diacetylenic included some measurement of hexacosa-l0,12-diynoyl-PC, but these characteristicswere not noted. Perhaps it is due to the difference in chain length and diacetylenic position of the lipids used. The LE/LC region in most saturated phospholipid monolayers represents the coexistence of fluid and solid phase below the critical temperature. In strict thermo-
Figure 8. Solid domains in DC2,loPC monolayer at 10.5 "C, as revealed from a higher fluorescentbackground of PE-Rh labeled fluid lipids. The surface pressures are (a) 16 dyn/cm (42A2/ molecule) and (b) 36 dyn/cm (38 A2/molecule) in first time compression and then the pressure is reduced to near zero and recompressed to (c) 16 dyn/cm (40 A2/molecule).
dynamic sense, this region should remain a constant pressure plateau in isotherms, as expected for a two-phase equilibrium. However, this plateau is missing in DC2,10PC, and the onset transition is replaced by a hump followed by a dip in the 7r-A curves. Similar features in other lipids and diacetylenic lipids have also been ~ b s e r v e d . ~ ~ - ~ ~ We interpret that the hump represents the extra energy needed to align the planes of diacetylenic bonds in neighboring acyl chains to a higher order than the
2728 Langmuir, Vol. 8,No.11,1992
Figure 9. Solid domains of DC2,loPC monolayers at 8.5 “C,as revealed from a higher fluorescentbackground of PE-Rh labeled fluid lipids. The surface pressures are (a) 23 dyn/cm (37 A2/ molecule) in first time compression. The monolayer is then exposed to UV at about 50 dyn/am (30 A2/molecule) and decompressedto (b) 5 dyn/cm (37 A2/molecule)and (c) 0 dyn/cm (45-55 A2/ molecule).
hexagonal-close-packarrangement of acyl chainsnormally expected in SC phase phospholipid monolayers. The rarity of these highly ordered nucleation sites during the first (22) Thuren, T.; Virtanen, J. A.; Vainio, P.; Kinnunen, P. K. J. J. Chem. Phys. Lipids 1983,33, 283. (23) Miller, P.;Peters, R.; Ringsdorf, H. Colloid Polym. Sci. 1989,267, 97. (24) Matuo, H.; Rice, D. K.; Balthasar, D. M.; Cadenhead,D. A. Chem. Phys. Lipids 1982,30, 367.
Hui et al.
compressionis believed to be responsible for this analogous supercooling phenomenon. These nucleation sites may not be large enough to be resolved by fluorescence microscopy; hence the onset of observing solid domains does not coincide with the hump. The nucleation hypothesis is supported by the fact that whenever visible, the first time “crystallized”domains are fewer but larger, and severalof these feather-shaped “crystals”are initiated from a single center, forming a phoenix cluster. After the first compression, apparently some molecules remain interlocked even after decompression and provide nucleation for subsequent domain formation on later compressions, which express no “supercooling” humps. The domains of recompressed monolayers are therefore more numerous and smaller. The nucleation hypothesis also agrees with our observation that “supercooling” is detectable only upon slow compression, since fast compression rates may exceed the slow rate of the formation of tightly locked nucleation sites, resulting in the failure of formation of tightly packed domains. The solid domains of molecules packed by tightly locked diacetylenic bonds are expected to occupy less area/ molecule than those by saturated phospholipids under the same condition. Indeed, the averaged molecular area of solid phase diacetyleniclipids (35-40 A2)is smallerthan DPPC (45-50 A2),given thatthe temperature dependence of the former is wide. The smaller molecular area may be accommodated only if the packing subcell is different. This is depicted by X-ray diffraction studies of liposomes and tubules of very long chain diacetylenic lipid^.^^^ For DC2,loPC, the molecular packing seems to be dominated by the alignment of the diacetylenicbonds near the head group. Apparently, the alignment of the diacetylenic moiety alone in the dark domains is sufficient to exclude the fluorescence probe from these domains. Domain shapes were suggested to be controlled by dipole-dipole interaction16J7as well as by molecular packing defects.25 The thin, elongated domain shape indicates strong constrains in molecular packing and/or dipole orientation in these domains againstthe line tension which tends to favor round domains. Strong interchain alignment of diacetylene bonds in DC2,laC may reduce packing defects to extend the correlation length in these large solid or hexatic domains. The fact that the domains remain thin and become even thinner at high surface pressure is in accordancewith the electrostatic theoretical prediction16J7 that the higher the dipole density, the stronger is the mutual repulsion which keeps domains narrow against line tension. Lower temperatures favor the stabilization of molecular and dipole alignment; therefore longer and thinner domains are found (Figures 7-9). However, the. orderly alignment of diacetylenic bonds does not imply the same for the rest of the chains. The critical temperature is lower for the diacetylenicPC (-21 OC for DC2,laC versus 41 OC for DPPC and even higher in DSPC), indicating the total intermolecular cohesive energy is still less than its saturated counterpart. Electron diffraction results (not shown) also indicate that most parts of the acyl chains in solid domains of DC2,roPC are not ordered. 2. Polymerizability. DC2,10;3,9PCdoes not polymerize under our experimental conditions. The structural difference between DC2,loPC and DC2,10;3,9PCis that in the latter, the diacetylenic bonds in the two adjacent chains are one carbon position apart, instead of being at the same (25) Sackmann, E.; Fiecher, A.; Frey, W. In Physics of AmphiphiZic Layers; Meunier, J., Langevin, D., Boccara, N., me.; Springer-Verb Berlin, 1987; p 25.
Microdomain Structures in Phosphatidylcholine Monolayers position. Apparently, the exact juxtaposition of diacetylenic bonds in adjacent chains, especially if these bonds are closeto the glycerol,is required for the specialmolecular alignmentof DC2,laC. The molecular structure of DPPC, as determined from X-ray crystallography,26depicts that the carbon atoms in the sn-2 chain are about one C-C bond away from their equivalence in the sn-1 chain; the nonequivalence in length arises from an abrupt bend at the C-2 atom of the sn-2 chain.27 If this configuration applies to DC2,1@3,9PC,it would bring the diacetylenic bonds in exact register between the two chains if they tilt at the same angle as in the crystalline state. Since staggering the diacetylenic units in the chains does not facilitate closer chain alignment nor polymerization, we conclude that the sn-2 chain conformation of DPPC structure does not apply to that of diacetylenic PC in the monolayer state. The diacetylenic bond positions of DCa,PC are further toward the terminal methyl group. The thermal motion of these lipids reduces the chance to become polymerized or even to become aligned with respect to each other. Indeed, the critical temperature is very low (
