Langmuir 1994,10, 1919-1925
1919
Structure of Mixed Monolayers of Dipalmitoylglycerophosphocholine and Polyethylene Glycol Monododecyl Ether at the Air/Water Interface Determined by Neutron Reflection and Film Balance Techniques C. Naumann,? C. Dietrich,? J. R. L u , ~R. K. Thomas,$ A. R. Rennie,% J. Penfold,l and T. M. Bayerr!? Physik Department E22, Technische Universitat Miinchen, 0-85748Garching bei Munchen, Physical Germany, Rutherford Appleton Laboratory, Chilton, Didcot, Oxon OX11 OQX, U.K., and Chemistry Laboratory, University of Oxford, South Parks Road, Oxford OX1 3QZ, U.K., Physical Chemistry Laboratory, University of Cambridge, Cambridge, U.K. Received January 14,1994. In Final Form: March 28,2994' The structure of mixed monolayers of the zwitterionic phospholipid dipalmitoylglycerophosphocholine (DPPC) containing nonionic surfactants of the polyethylene glycol monododecyl ether variety (&En with n = 2,4,6, 8) is studied at the air/water interface using a combination of neutron reflectivity with film balance techniques. The two methods applied simultaneously gave good agreement of the molecular area data using the most simple single-layer model for fitting the reflectivity curves. Isotopic substitution of both lipids and surfactants has been employed in order to determine structural parameters of both components separately. All measurements were done at lipid to surfactant molar ratios r L / S > 2 and at surfactant concentrationswell below their critical micelle concentration. Measurements were performed in the liquid-condensed (LC)and in the liquid-expanded (LE) phase states of the monolayer. The results show that all surfactants studied readily associate with the DPPC in the LE phase to a mixed monolayer but get completely transferred from the monolayer into the subphase at the transition to the LC phase. A determination of the surfactant partition between the mixed monolayer and subphase at the LE phase state provides evidence that virtually all surfactant is associatedwith the monolayer. The area per surfactant molecule in the mixed monolayer is found to increase rather linearly from 45 f 5 A2 (n = 2 ) to 165 f 5 A2 (n = 8) by an increment of 22 f 2.5 A2 per ethylene gl col group (film pressure 5 mN/m). The thickness of the surfactant alkyl chain was found to be 14 f 2 l f o r ClzE2 and 8 f 2 A for ClzEr which indicates a very high degree of chain disorder. The thickness of the fatty acyl region of the DPPC is not affected by the surfactant for n = 2 and 4. The headgroup region thickness of the mixed monolayer is 14 f 2 A for C12E2and 11 f 2 A for C12Ed. While the diethyleneglycol group exhibitsa rather extended conformation, longer surfactant headgroups are prone to a significantincidence of gauche conformations. Moreover, the data indicate that the interaction of C12Enwith the monolayer is markedly different for n = 2 compared to all other ethylene glycol groups (n = 4,6,s) studied.
Introduction Reflectivity measurements on monolayers at the air/ water interface employing neutrons have emerged as powerful tools for the investigation of their structural properties on a molecular scale.lF2 Monolayers of phospholipids and of surfactants have been studied by neutron reflectivity together with isotopic substitution and have drastically improved our knowledge about the microscopic interfacial structure of these systems. Among the lipid systems studied so far are mainly the zwitterionic lecithintype lipids dymyristoylglycerophosphocholine (DMPC) and dipalmitoylglycerophosphocholine(DPPC) while for surfactant studies both ionic and nonionic surfactants were used.- The results of these studies indicate significant structural differences between lipid and surfactant mono-
* To whom correspondence should be addressed at the Physik Department E22,Technische Universitiit MGnchen, James-FranckStraese, D-85748 Garching, Germany. Phone: 49 89 3209 2480. FAX: 49 89 3209 2469. e-mail:
[email protected]. + Technische Universitit M h c h e n . 1University of Oxford. f University of Cambridge. 1 Rutherford Appleton Laboratory. 0 Abstract published in Advance ACS Abstracts, May 15, 1994. (1)Penfold, J.;Thomas, R. K. J.Phys. Condens.Matter 1990,2,1369. (2) Ruasell, T. P. Mater. Sci. Rep. 1990, 5, 171. (3) Bayerl, T. M.; Thomas, R. K.; Penfold, J.; Rennie, A. R.; Sackmann, E.Biophys. J. 1990,57, 1095. (4) Vakinin,D.;Kjaer, K.; Als-Nielsen,J.; Lhche, M. Biophys. J.1991, 59, 1325.
layers at the air/water interface: (1)Lipids may exhibit a phase transition between a crystal-like liquid-condensed (LC) phase and a fluid-like liquid-expanded (LE) phase by variation of the lateral pressure of the monolayer. (2) Surfactant monolayers do not form any highly ordered monolayer phases comparable to the LC phase of lipids but exhibit a rather disordered fluid-like state. (3) At high surface coverage, the molecular area of nonionic surfactants of the polyethylene glycol monododecyl ether (C12En)variety tends to a limiting value which is mainly determined by steric interactions in the headgroup region. (4)Lipids exhibit remarkable effects of isotopic substitution on their phase behavior, particularly in the LC-LE coexistence range, whereas the nonionic surfactant behavior is insensitive to isotopic substitution. Although there is ample information available about the lipid-surfactant interaction in bilayer and membrane systems, virtually no studies have yet been devoted to this interaction in monolayers at the air/water interface.gJ0 The extreme sensitivity of lipid monolayer phase diagrams obtained by film balance measurements toward (5) B r u " , T.; Naumann, C.; Sackmann, E.; Rennie, A. R.; Thomas, R. K.; Kanellas, D.; Penfold, J.; Bayerl, T. M. Submitted for publication to Eur. Biophys. J. (6) Lee, E. M.;Thomas, R. K.; Penfold, J.; Ward, R. C. J.Phys. Chem. 1989, 93, 381.
(7)Lu, J. R.; Lee, E. M.; Thomas, R. K. Langmuir 1993,9,1352. (8) Lu, J. R.; Li, Z. X.; Su,T. J.;Thomas, R. K. Langmuir 1993,9,2408. (9) Eytan, G. D. Bmhem. Biophys. Acta 1982,694, 185.
0743-7463/94/2410-1919$04.50/00 1994 American Chemical Society
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1920 Langmuir, Vol. 10, No. 6, 1994
the presence of foreign molecules in the monolayer enables lipid-surfactant interaction studies at very low surfactant concentrations (well below their critical micelle concentration (cmc)). Thus, the very initial stage of a solubilization process can be explored. Combined film balance and neutron reflection measurements on the same monolayer improve the experimental options since both methods provide the area per molecule independently and thus enable a good check of the consistency. Moreover, the ability of the reflection technique to gain insight into the internal monolayer structure using isotopic substitution of either lipid or surfactant makes it possible to study lipid and surfactant structure separately in the mixed monolayer. We have chosen for the first study of lipid/surfactant mixed monolayers presented in this work those molecular species which have been studied extensively previously in pure monolayers by neutron reflection and which do not exhibit any significant intermolecular Coulomb interaction: the zwitterionic DPPC and the nonionic C12En (n = 2, 4, 6,8). Our experiments were done employing two isotopically distinguishable species of DPPC, abbreviated in the text as follows: (1)DPPC-d,s, perdeuterated tail (62 deuterons) and deuterated choline head (13 deuterons), the only protons are located in the glycerol backbone (3 protons); (2) DPPC-d~,selectively deuterated choline headgroup, the trimethyl group is deuterated. All measurements were done on a subphase of null-reflecting water (i.e., water contrast matched to air, CMW). For studies of the mixed DPPC/surfactant monolayers we used four isotopically distinguishable species of (n = 2, 4, 6, 81, denoted in the text as hC12hEn (fully protonated), dCladEn, (fully deuterated), dC&En (alkyl chain deuterated), and "C1ldEn (oxyethylene group deuterated). Combinations of these different lipid and surfactant isotopes in the mixed monolayer enable experimental setups where either lipid or surfactant dominates the reflectivity. Thus, the structure of each molecule in the mixed monolayer can be studied separately.
Materials and Methods
All lipids were obtained from Avanti Polar Lipids (Alabaster, AL). The protonated C12En (n = 2,4,6,8)were purchased from Nikkol Chemicals Inc. (Tokyo, Japan). Selectively and fully deuterated polyethylene glycol monododecylethers were prepared according to procedures described in detail elsewhere.' Fluorescence and Film Balance Measurements. The fluorescence measurements were performed with a film balance apparatus equipped with a fluorescence microscope mounted above the trough on a computer-controlled x-y translation stage. The size of the trough is 3 X 20 cm with a volume of about 29 mL. Peltier elements below the brass-plated base allow temperature regulation with an accuracy of about 0.2 OC. Most parts of the trough are covered by a glass slide t o protect the monolayer from impurities, air convection,and fluid condensation. Surface pressure of a solution in the trough is measured by a Wilhelmy plate system (accuracy 0.2 mN/m). Additionally, a thermostated film balance with a very good compression ratio was used to measure complete pressure-area diagrams (trough dimensions 9 X 32 cm with a volume of about 200 mL). The accuracyvalues for temperature stability and film pressure are the same as for the fluorescence setup described above. Neutron Reflection Measurements. Reflectivity measurements were done at the CRISP Spectrometer of the Rutherford (IO) Bayerl, T. M.; Werner, G.-D.; Sackmann, E. Biochem. Biophys. Acta 1989, 984, 214.
1
........ ........-._ .............. ........ -.--_. ......
lal 1.0
. . . . . . . . . . . . . . . . . . . . .
*.
0.9
....... .............
-pure lipid (DPPC-h) .............. ...... ........
..... .......,
......rm
i:
.'.....,.,
0.3
, - 0.6 0
5
10
15
20
25
30
35
time (min)
Figure 1. Time scan of mixed DPPC/ClZEz monolayers in the LE phase state at different lipid/surfactant molar ratios R / Sand at lateral pressures ?r as indicated. Appleton Laboratories (Didcot, U.K.)." A home-built, thermo. stated film balance with a trough size of 15 X 26 cm (volume400 mL), equipped with computer control for the barrier position and a Wilhelmy plate pressure detector (accuracy 0.1 mN/m), was used (measurement temperature 20 "C). The monolayers were prepared on ultrapure water by spreading a chloroform solution of the samples. The procedure for performing the measurements has been described in detail previously.BJ2 Reflectivity data were analyzed employing the optical matrix method.lI2 The incoherent background for each sample, which is uniform over the measured Q range, was obtained by extrapolation to high values of the momentum transfer Q and subtracted from the data. The stability of the surfactant/lipid mixed monolayers over the time range (1-4 h) of a typical reflectivity experiment was checked by a time scan on the film balance. In this mode, the lateral pressure is kept constant over time by adjusting the barrier position (i.e., the molecular area). A representative result of the time scan measurements is shown in Figure 1 for mixtures of fully protonated DPPC with bClzbEz. It clearly shows that the mixed monolayer exhibits negligible instability over the time range of the reflectivity experiment up to a lipid/surfactant molar ratio of FL/S = 2. In contrast, for < 2 there is significant instability of the monolayer, limiting the concentration range where reflectivity measurements are applicable. A similar result regarding the limiting -1s values was obtained for hClzhE4 and "C12"Ee.
Results Film Balance Measurements. Pressurearea isotherms (compression mode) of protonated DPPC-h/ hClzhE4mixtures are shown in Figure 2A. The compression curves clearly indicate increasing surfactant incorporation into the monolayer for decreasing Surfactant partition into the monolayer causes a drastic increase of the area per molecule. The transition region between the LE and LC phases is shifted toward a higher area per molecule due to the surfactant incorporation, and the plateau region typical for pure DPPC is diminished. Interestingly, the limiting area per molecule to which the monolayer can be compressed remainsunchanged at the value of 50 A2,which is comparable with that of pure DPPC.13 This suggests that a t the transition to the LC phase there is increasing surfactant depletion in the monolayer, leaving a t highest (11)Penfold,J.;Ward,R.C.;Wdiams,W.G.J.Phys.E.: Sci.Imtrum.
1987,20, 1411. (12) Johnson, S.J.; Bayerl, T. M.; Weihan, W.; Noack, H.; Penfold, J.;
Thomas, R. K.; Kanellas, D.; Rennie, A. R.; Sackmann, E. Biophys. J. 1991,60, 1017. (13)Albrecht, 0.; Gruler, H.; Sackmann, E. J.Phys. (Paris) 1978,39, 301.
-
Mixed Monolayers of DPPC and C l a n 40
Langmuir, Vol. 10, No. 6,1994 1921
....?.___._...--A -
150 area per molecule A,
200
100
250
DPPC-h I C12E2-h (r, = 3)
(ryg = 3)
.--. DPPC-hlC12E6-h (ryg = 3)
60
SO
100
120
DPPC-h I C12E8-h
140
area per molecule A,
160
I
I
2
1
Figure3. Film balance adsorption scau for a DPPC/C&mixed monolayer. The variation of the molecular area AMas defined in eq 1 vemu the amount of C i a in the subphase is shown at lateral pressures as indicated.
......DPPC-h I C12E4-h
I.,...
I
3
lipid I surfactant molar ratio rus
- DPPC-h --
4
[A2]
--------4-----u
)(.
50
50
A
180
200
[A2]
Figure 2. (A, top) Pressure area diagrams of pure DPPC and of mixed monolayers of DPPC and C12E4 at rt/s = 4 and rr, s = 1as indicated. (B,bottom) Pressure area diagramsof pure D6PC and of mixed monolayers of DPPC and CI~E,for n = 2,4,6, and 8 (as indicated) at a fixed -1s = 2.
pressure a rather pure DPPC monolayer. A likely explanation is an extremely low surfactant partition in the crystalline (LC phase) domains formed. This may cause an expulsion of the surfactant into the subphase with increasing lateral pressure over the transition region. The virtually complete removal of surfactant from the LC phase monolayer becomes obvious by measuring the values. Such isotherms in expansion mode for different n~/s measurements provide similar isotherms for all -1s values, identical to the expansion isotherm obtained for pure DPPC (data not shown). Figure 2B demonstrates the effect of the length of the ethylene glycol group on the isotherms at a fixed -1s = 3. The rather parallel shift of the LE part of the curves toward a higher area per molecule in distinct steps with n indicates that the ethylene glycol chain length mainly controls the total area the surfactant occupies in the mixed monolayer. A remarkable feature of these data is the different behavior of the DPPC/CuE:! mixture at the transition to the LC phase. A much lower lateral pressure is required t o squeeze the into the subphase than for all other surfactants studied, indicating a peculiar structure of this mixed monolayer (Figure 2B). Figure 3 shows the variation of the molecular area AM (cf. eq 1for a definition of A M )with the molar ratio of lipid and surfactant p ~ as p measured with the film balance at different lateral pressures r. The slope of the lines connecting the data points indicates a rather pressure independent surfactant incorporation into the DPPC monolayer in the LE phase state (up to a = 10 mN/m).
Surfactant incorporation is markedly reduced at pressures corresponding to the coexistence range (a = 16 mN/m). At the LC phase state (a = 35 mN/m) there is no measurable surfactant incorporation a t all as indicated by AM = 50 A2 (which corresponds to that of pure DPPC at this a), independent of ~ 1 s . Fluorescence film balance measurements using the LC phase insoluble fluorescence probe Texas Red were used for the assessment of possible structural changes of the crystalline domains due to the surfactant. The left image in Figure 4 shows the domain structure typically observed in the LE-LC transition region for pure L-wDPPC. The propeller-like shape of the domains is well known and caused by the chirality of the DPPC.14 The right image in Figure 4 shows the corresponding domains for a DPPCh/hC12hE4mixture = 2). The presence of surfactant causes the propeller-like structure to disappear, but the average size of the domains is retained. One possible explanation of this finding is a nonzero partition of surfactant in the crystalline domains in the transition region (which might get squeezed out at higher lateral pressure). Another, more likely explanation is that the crystalline domains indeed contain virtually no surfactant but that the change of the domain shape is a response to the obviously different line tension between the LE and LC phases (compared to the control) owing to the surfactant partitioned in the LE phase. This explanation is supported by the above-mentioned results of the expansion isotherm measurements and describes the shape change in terms of a minimization of the total free energy of the mixed monolayer. Neutron Reflectivity Measurements. 1. DPPC/ Surfactant Mixed Monolayer Structure. According to the film balance results reported above regarding the stability of the monolayer in the presence of surfactant, reflectivity measurements were restricted to a lipid/ surfactant ratio of ~ 1 2s 2. Moreover, since the film balance results suggest that the surfactant interacts predominantly with DPPC in the LE phase state of the monolayer, most measurements of mixed monolayers were done on this phase. The measurements were performed on null-reflecting water (CMW), and the DPPC species used were DPPCd75 and DPPC-dS. The interaction with C12En was studied for different lengths of the hydrophilic ethylene glycol ~~
(14) Web,
~
R. M.; M c Connell, H.M. Nature
1984, 310,47.
1922 Langmuir, Vol. 10, No. 6, 1994
Naumann et al.
Figure 4. Fluorescence film balance images using a LC phase insoluble fluorescence probe of the coexistence range of a pure DPPC = 2 (right) a t a temperature of 20 "C. monolayer (T = 6 mN/m) (left) and of a mixed DPPC/C12EI monolayer (T = 16 mN/m) a t The size of each picture is 150 pm X 150 pm. 44
i
r DPPC475on CMW m DPPCd75 I C12E2-h on CMW I DPPC475I Cl2E4-h on CMW A
Table 1. Optical Matrix One-Layer Fitting Results (Layer Thickness d and Scattering Length Density p ) for Mixed DPPC-d7#ClzhE, (n = 2,4,8) Monolayers at the LE Phaae State ( a i s - 2.3) and Molecular Area AM Determined from the Reflectivity Measurements and from Film Balance Measurements
DPPGd75I Cl2E8-h on CMW
A-2)
4.34 21.0 ~ d A (neutron 2 88.7 reflectivity) AdA*(film 88.7 balance) pl(10-B
dlA
0.b
o.bs
0.iO
0.k
o.;o
0.b
o.bo
momentumtransfer Q [ k']
Figure 5. Neutron reflectivity profiles of a mixed DPPC-&/ Cl2E,,(n = 2,4) monolayer in the LE phase state (T = 5 mN/m) together with single-layer model fits to the data ( h i s = 2). For comparison, the data and fit for a pure DPPC-& monolayer a t the corresponding lateral pressure are shown.
chain (n = 2,4,8). For studying the effect of surfactant on the lipid structure in the monolayer, fully deuterated DPPC-d75 and protonated surfactant were used: DPPCd75/hC12hEn(n= 2,4,8). The structure of the surfactant in the mixed monolayer and its partition between the monolayer and subphase were measured using headgroup deuterated DPPC-d~and EO group perdeuterated thC12dEn),alkyl chain perdeuterated (dC12hEn),or totally deuterated (dC12dE,) surfactant (n = 2, 4). Figure 5 shows reflectivity curves obtained from mixed monolayers in the LE phase (a = 5 mN/m) for three different ethylene glycol chain lengths (n= 2,4,8). Since DPPC-d75and protonated hC12hEnwere used, the reduction in reflectivity is a clear indication that (a) hCIZhEnassociates with the DPPC in the monolayer for all ethylene glycol chain lengths (n= 2,4,8) studied in the LE phase and (b) the reduction of the reflectivity increases with n and is highest for n = 8. Model fits to the data in Figure 5 in terms of singlelayer models (represented by the full lines in Figure 5 ) show that the surfactant incorporation changes drastically the scattering length density p of the monolayer while the total layer thickness d remains for n = 2 and 4 close to the
3.83 19.66 106.8
3.09 20.39 127.7
2.97 16.26 169.68
110.9
122.1
175.8
value of &+LE = 21 A obtained for pure DPPC-d75 (cf. Table 1). This indicates that the association of (n = 2, 4) with DPPC reduces essentially the monolayer density but does not change significantly the order of the lipid with respect to the surface normal. For n = 8, however, a significantreduction of d by 5A can be observed. Measuring the reflectivity of the mixed monolayer after compression into the LC phase state (at a = 35 mN/m) gives a profile very similar to that of pure DPPC-& for all n (data not shown). The fitting parameters d and p obtained by analyzing the data in terms of a single-layer model are within the experimental error the same as for pure DPPC-d75. The combination of film balance and reflectivity measurements enables now a very sensitive reliability check of the fitting models to be applied to the data. This is because both methods allow a calculation of the average area per molecule AMto which both surfactant and lipid contribute for a mixed monolayer:
AL and As are the lipid and surfactant molecular areas, bL and bs are their respective coherent scattering lengths, d and p are the thicknessand the scattering length density of the layer obtained by a one-layer model fit of the data, and e is the lipid/surfactant molar ratio in the monolayer. It should be noted that E 1 ~ L / Sas E refers solely to the monolayer while r ~ includes p a possible surfactant subphase partition (see below). Since bL >> bs for a mixed
Langmuir, Vol. 10, No. 6, 1994 1923
Mixed Monolayers of DPPC and C l a n 40x1Oa
----model:r,=2
35
8 DPPGd9 I Cl2E2-h on CMW
x DPPCd9I C12E2d8 on CMW
2-
a
.-.-*w ”
i
L
model: r,
=3
-model: r,
=4
.......
DPPCd9 I C12E2d25 on CMW
u nflectivkydata n
A DPPCd9I C12E2d33 on CMW
molar ratio (weighed)
c
0
3
E
U
25-
Q
L
20 10-50.
/.z
.- .-
-
15-
8.
m
7. I
I
I
I
I
I
I
7
0.06
0.08
0.10
0.12
0.14
0.16
0.18
0.20
10-
1
momentumtransfer Q [ A-‘ 1
5x;04
scatteringlength of C,,E,
Figure 6. Neutron reflectivity profiles of a mixed DPPC-dsI C12& monolayer in the LE phase state (a= 5 mN/m) a t = 2 along with one-layer model fits to the data. Protonated ( K ~ E z )ethylene , glycol chain perdeuterated (WlzdE,), alkyl chain perdeuterated (dC12hE2),and fully deuterated (dC12dE2) surfactant was used as indicated.
monolayer of DPPC-d,s with protonated surfactant, the latter scattering length can be neglected for the determination of AM. Table 1givesthe molecular area obtained for both methods, the very satisfactory agreement between both &values justifying not only the above approximation but incidating that the simple single-layer model used for fitting the reflectivity data is indeed appropriate. It is interesting to note that the increase of AM as a function of the number of ethylene glycolgroups (cf. Table 1)is surprisin ly linear with an average increase of UM = 21.7 f 2.5 per 2 groups ( at A = 5 mN/m). 2. Partitionof Surfactant between Monolayer and Subphase. Neutron reflection in combination with contrast variation has the potential to determine for the first time the partition of surfactant molecules between the monolayer and its subphase in the LE phase state. Deuterated surfactant is a prerequisite for such measurements in order to give a strong contribution to monolayer reflectivity. We used mixtures of DPPC-dg with either of the above-mentioned three selectively deuterated C12En (n = 2,4) species for the determination of this partition. Representative data are shown in Figure 6 for C12E2 along with fits to the data according to single-layer models. The fitting results are summarized in Table 2. The values obtained for the total layer thickness d depend on the degree of deuteration of the surfactant. However, totally deuterated (dC12dE,)and alkyl chain deuterated (dC12hE,) surfactants give the same values for d within experimental error. The partition of surfactant between bilayer and subphase can be estimated by writing eq 1as
Figure 7. Surfactant partition between the DPPC monolayer and the subphase: e values as given in Table 2 in terms of pd versus the scattering length of the three selectively deuterated species of ClzE4 (*). For comparison, the squares represent the corresponding value of r ~ / sfrom Table 2. The straight lines were obtained from calculations according to eq 2 for different values of the lipid/surfactant molar ratio e in the monolayer using the AMvalue from film balance measurements.
q...?... water I
7)
.............................
...........
II
.....................................
......................................
r
l2
AM
[A]
air Figure 8. Schematic depiction of the structure of a mixed monolayer of DPPC with C12& (left side) and with C1& (right side) in the LE phase state (I,hydrophobic headgroup region; 11, hydrophobic chain region).
and plotting pd versus the scattering length of the surfactant (for different degreesof deuteration) for various e values, using the AMvalues obtained from film balance measurements. This is shown for the case of Cl2E4 in Figure 7. The straight lines obtained by this procedure reflect the behavior when all surfactant would be partitioned in the monolayer. Introducing now into this plot the values of pd obtained experimentally (Table 2) for the differently deuterated surfactants as well as the actual ~ L / Svalues (obtained by weighing the two components) enables the assessment of the surfactant partition. The rather good agreement of pd from weighing and from reflectivity measurements indicates a very low partition of surfactant in the subphase. Hence, e = Q s in the LE phase. A similar result was obtained for Clz 2 (data.not shown). 3. Surfactant Molecular Area in the Mixed Monolayer. With the knowledge about the surfactant partition between monolayer and subphase it now becomes possible to determine the area per surfactant molecule As in the
A
Table 2. Optical Matrix Fitting Results (One-Layer Model Fit) for Mixed DPPC-ds/C12& and D P P C - ~ S / C ~Monolayere ~E~ in the LE Phase State (T = 5 mN/m)a pI(1O-g A-2)
dlA Q/S
c
0.62 7.0 2.3 2.5
0.47 7.4 2.3 2.5
1.1 13.7 2.3 2.5
1.25 11.17
2.3 2.5
0.93 20.95 2.3 2.5
0.88
1.1
19.2 2.3 2.7
21.1 2.0 2.1
1.27 19.4 2.0 2.1
The values p and d were used to determine the lipidlsurfactant molar ratio c in the monolayer according to eq 2 as represented in thia table. For comparison, the ratio ~ 1 (as s determined by weighing) is given. Q
1924 Langmuir, Vol. 10, No. 6, 1994 Table 3. Area per hCnhEm (n = 2, 4,6,8) Molecule in a Mixed DPPC-& Monolayer at the LE Phaw State and at -1s = 2.3, Obtained from Neutron Reflectivity and from Film Balance Measurements DPPC-d?s+ DPPC-d76 + DPPC-d7s + DPPC-d7s + C12&-h C1zE4-h C12&h 160 AdAz (neutron 42 94 reflectivity) 52 90 129 171 ~ sA=l(film balance)
mixed monolayer using As = (AM- AL)/e for both methods. The values of AMwere taken from Table 1, and AL was obtained from the film balance and reflectivity measurements of DPPC-d76. Table 3 shows the corresponding As values obtained with both methods in the LE phase state, and the agreement is very good. It is obvious from these data that the length of the ethylene glycol segment determines to a large extent the value of As. This can be seen from the rather linear increase of As with n (n = 2, 4, 6, 8) by 40 f 2 A2 (at ?r = 5 mN/m) and from the fact that Cl& with its single alkyl chain requires an area similar to that of DPPC with its two palmitoyl fatty acyl chains at the same lateral pressure. The increase of As with n compares well with the above-mentioned increase of AMwith n by & 4 ~ = 21.7 A2 (Table 1) at the same value of rLp. Discussion Effect of the Monolayer Phase State. One of the most remarkable findings of this study is the drastic dependence of the association behavior of the surfactant with the DPPC monolayer on the phase state of the latter. While the surfactant is incorporated into the monolayer in the LE phase (with virtually no partition into the subphase), compression toward the LC phase leads to surfactant depletion over the coexistencerange and finally to its complete removal from the monolayer at highest lateral pressure (LC phase). It should be noted that all our measurements were made at surfactant concentrations far below their critical micellization concentrations (cmc), with the highest surfactant concentration used being about O.l(cmc). This may explain why all surfactant associates with the DPPC in the monolayer at the LE phase. The partition within an amphiphilic layer at the aidwater interface is energetically much more favorable than the monomeric state in water where the hydrophobic alkyl chain is exposed to the solvent. This situation might change for ethylene glycol chains with more than eight oxyethylene segmenta, which were not covered by this study. Basically,the interactionbetween amphiphilic molecules in the monolayer (at constant lateral pressure) is determined by the balance between attractive van der Waals interaction in the hydrophobic region, repulsive steric interaction (including hydration repulsion) in the headgroup region, and repulsive or attractive forces in the interface region. The finding that the partition of surfactant between monolayer and water is similar for E2 and E4 (Table 2) indicates that the hydrophobic interaction dominates the other forces for short ethylene glycol chains in DPPC bilayers at the LE phase state. Increasing the lateral pressure of the mixed monolayer over the coexistence region causes surfactant depletion in the monolayer as shown in Figure 3. Spatial fluctuations of the surfactant concentration over the monolayer due to the lateral diffusion of both DPPC and surfactant could give rise to temporary surfactant depletion in small regions of the monolayer, which can be seen as a prerequisite for the formation of crystalline domains.
Naumann et al. Further compression increases the proportion of crystalline domains in the monolayer area, thereby reducing the surfactant partition in the monolayer. Since the final LC phase at high lateral pressure does not contain any surfactant, a direct surfactant transfer from the LE phase into the subphase is likely. The driving force is most likely a sterical repulsion between the headgroups of lipid and surfactant which becomes a dominating feature at higher lateral pressure and ultimately prevents a closer packing of the mixed monolayer. This repulsion orginates from the fact that both DPPC and C12Enare molecules which exhibit a larger cross section of the headgroup region than of the tail region so that their effective molecular shape can be considered as a cone. While DPPC has a limiting molecular area of about 50 A2 (Figure 2), a value of 43 A2 has been reported for lamellar phases of C12E4 and a value of 44 A2 for monolayers of the same surfactant (at the cmc).8J6 Considering now that the limiting area of an alkyl chain is 20 A2 and that the surfactant has one single chain while the DPPC has two may demonstrate the remarkable mismatch between the area and tail cross section of the surfactant.16J7 This mismatch is by far too large to allow compensation by acollectivetilt of the chains as observed for the case of pure DPPC in the LC phase, where the chains are tilted by 30 f 5°.495 Hence, LC phase formation for the mixed monolayer is very unlikely until the surfactant is completely removed. An additional contribution to the surfactant depletion at high pressure may arise from the length mismatch between the tails of surfactant and DPPC by four methylene groups. From phospholipid mixtures it is well known that such a mismatch causes a demising in the gel phase.18 In the LE phase the mismatch can be compensated by the chain disorder of both molecules. We find surfactant molecular areas As in the DPPC bilayer which are significantlyhigher than the limitingvalues mentioned above (cf. Table 3). At a lateral pressure of 5 mN/m, where most of our mixed monolayer experiments were done, As = 70 f 5 A2 is reported for a pure m~nolayer.~ This compares well with our value of As = 94 A2for in the DPPC monolayer (~L/s = 2), taking into account that we found an increase of AAs = 20 A2 per ethylene glycol group (Table 3). It should be noted that the area of a DPPC molecule at this pressure lies in the same range (AL = 89 &) although the lipid has two fatty acyl chains. This indicates that even in the LE phase of the mixed monolayer the repulsion between the bulky hydrated ethylene glycol groups determines the lateral packing and that the surrounding DPPC molecules do not induce any ordering in the surfactant alkyl chains. Hence, increasing the surfactant concentration in the mixed monolayer shifts the balance between attractive dispersion forces in the chain region and repulsive sterical forces in the headgroup region in favor of the latter until the monolayer becomes unstable, as observed experimentally at p ~ / s< 2 (Figure 1). The energy required for the expulsion of surfactant into the subphase is presumably provided by the lateral pressure. This surmise is supported by the finding that a mixed monolayer of C12E4 and DMPC (dimyristoylglycerophosphocholine)shows a similar surfactant expulsion at pressures above 20 mN/m, although DMPC does (16) Carvell, M.; Hall,D. G.; Lyle,J. G.; Tiddy, G. T.FaradayDiscuss. Chem. SOC.1986,81,223. (16) Kjaer, K.; Ale-Nieleen, J.; Helm, C. A.; Tippmann-Krayer, P.; Mbhwald, H. J. Phys. Chem. 1989,93,3200. (17) Wiener, M. C.; Suter,R. M.; Nagle, J. F.Biophys. J.1989,55,309. (18)Knoll, W.; Schmidt, G.;Riitzer, H.; Henkel, T.; Pfeiffer, W.; Sackmann, E.;Mittler-Neher,S. Chem. Phys. Lipids 1991,57, 363.
Mixed Monolayers of DPPC and C1p?3, not even form a LC phase under the experimental conditions (C. Naumann and C. Dietrich, unpublished results). DPPC Fatty Acyl Chain Order. A further interesting point is the observation that the layer thickness d of the mixed monolayer is similar to that of pure DPPC for C12E2 and C12E4 but that a remarkable decrease can be observed for C12Es (Table 1). It should be emphasized that attempts to fit the C12Es data with a fixed d value of 20 A provided no acceptable fits. Obvious1 the area requirements of a C12E8 molecule of 160-170 (Table 3) which is about twice that of a DPPC molecule causes a drastic area mismatch between the head and tail regions of the mixed monolayer. To compensate this mismatch, the DPPC chains must spread out horizontally, thereby reducing d. For the E2 and E4 this effect is unlikely to occur since their area does not exceed that of DPPC (Table 3). Headgroup Structure of the Mixed Monolayer. Information about the headgroup conformation of the E, groups can be drawn from the results presented in Table 2. The addition of hC12dE, (n = 2, 4) to DPPC-d9 monolayers nearly doubles both the layer thickness d and scattering length density p as compared to pure DPPC-d9. The length of a fully extended ethylene glycol group is 3.6 A so the increase of d from 7 8, (pure DPPC-ds) to 14 A (mixed monolayer with hC12dE2at n t / s = 2) would indicate that the E2 group must be fully extended and the center of the distributions of the E2 group and of the trimethyl group of DPPC is unlikely to be located in the same plane. A rather extended conformation of the E2 group is also indicated by the molecular area As = 42 A2for C12E2 (Table 3) which is close to the value of 36 A2 reported for C12E3 under conditions of fully extended headgroups.7 Generally, two arrangements of lipid and surfactant headgroups in the mixed monolayer are conceivable. Either (a) the ethylene glycol groups protrude significantly from the DPPC headgroup region toward the subphase or (b) the former extend from the choline group down toward the hydrophobic region. Both arrangements are energetically rather unfavorable since they require a certain exposure of hydrophobic alkyl chain groups to water or of ethylene glycol groups to hydrophobic regions. Our data indicate that case (a) is the likely arrangement for Cl2E2 for the following reasons. The thickness d of the layer does increase by 7 A for dC12dE2compared with hC12dE2(Table 2) so that the alkyl chain region thickness would be 7 8, only compared to a theoretical length of 15 A for an all-trans conformation. However, the nearly is indicated on the basis extended conformation of of its molecular area (42 A2) and by the thickness of 21 A obtained for dC12dE2 (Table 2). For comparison, the theoretical length of the fully extended C12E2 is 22-23 A. Hence, up to four methylene groups of the alkyl chain must extend into the interface region where they overlap with the DPPC headgroup region in order to explain the above-mentioned 6-7-A thickness increase for DPPC-d9 without and with hC12dE,. For C12E4 the headgroup structure differs significantly from that of C12E2. First, its molecular area (94 A2) and the thickness of the layer for DPPC-d9with dC12dE4of 19 A (compared to 29-30 A for the theoretical length of the
K
Langmuir, Vol. 10, No. 6, 1994 1925 fully extended molecule) suggest significant deviations from the extended conformation. Second, the length of the E4 group is sufficient to protrude from the lipid headgroup region toward the subphase by about the same amount as for EZand to span the whole lipid headgroup region, preventing the exposure of alkyl chain groups to water. Our data in Table 2 indicate that the protrusion into the subphase might be even less than for the case of ClzE2 (Table 2) where an increase of d by 7 A was observed (compared to 5 A for C12E4). Considering now that there are no significant changes in the total thickness (DPPCd9 with dC12dEn)for n = 2 and 4, the assumption seems justified that the longer E4 group is considerablydisordered or folded around the plane of the DPPC headgroup. Together with the strong hydration of the ethylene glycol groups, this must cause a significant increase of the molecular area, as is experimentally observed (Table 3). The further increase of AS with n suggests that also for n = 6 and 8 the ethylene glycol groups essentially spread in a random conformation around the plane of the DPPC headgroup. The above-suggested structure for the DPPC/C12E2 headgroup with its exposure of hydrophobic groups to water must render this mixed monolayer far more unstable than for the longer ethylene glycol groups. This instability is indeed evident from the film balance measurements. The data in Figure 2B clearly show that the expulsion of C12Ez from the monolayer into the subphase occurs at a nearly 50% lower lateral pressure than for all other surfactants studied. This finding would be hard to understand without a monolayer instability, since ClzE2 is the surfactant with the lowest hydrophilicity. We can conclude that C12E2 exhibits a rather fully extended headgroup in the mixed monolayer while surfactants with longer headgroups are prone to a significant incidence of gauche conformations which increases the molecular area As. Surfactant Alkyl Chain Structure. The measurements of DPPC-d~with fully deuterated C12En (n = 2,4) enable the assessment of the degree of disorder in the alkyl chain region of the surfactant by comparison with the above results (cf. Table 2). The substitution of hC12dE2 by the fully deuterated analog increases the value of d from 14 to 21 A. Since the ethylene glycol length is 7 A, the alkyl chain thickness is 14 A. For &E4 the alkyl chain thickness can be approximated as the difference thickness for dC12dE4(19.2 A) and hC12dE4 (11.2 A), giving8 f 2 A. Moreover, a chain region thickness Of 7 A is reported for a pure C12E3monolayer at ita highest measurable dilution (0.0182(~mc)).~This indicates a remarkable disorder of the Cl2E4 alkyl chain by numerous gauche conformers along the chain or even by a backfolding of the chain. The latter is well conceivable particularly for the surfactants with longer ethylene glycol groups for the drastic mismatch between headgroup and chain areas.
Acknowledgment. This work was supported by grants from the Deutsche Forschungsgemeinschaft and the
BMFT.
