Langmuir 1999, 15, 2431-2434
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Synthetic Lecithin Monolayers on Hydrophobized Silica Supports Interdigitate with the Surface-Attached Alkyl Chains under Gel Phase Conditions M. Ka¨sbauer and T. M. Bayerl* Universita¨ t Wu¨ rzburg, Physikalisches Institut EP-5, D-97074 Wu¨ rzburg, Germany Received October 26, 1998. In Final Form: December 10, 1998 The phase transition behavior of spherical supported lipid monolayers (SSMs) of symmetric-chain phosphatidylcholines of different chain length was studied by a combination of differential scanning calorimetry (DSC) and deuterium nuclear magnetic resonance (2H-NMR). The SSMs are self-assembled lipid monolayers on silica gels, which were functionalized by an alkyl chain layer. We find that the chain melting temperature Tm,m of the monolayer differs from the main phase transition temperature Tm,b of a bilayer of the corresponding lipid in a systematic way. The difference temperature, Tm,m - Tm,b, shows a linear dependence on the lipid chain length for a given surface-attached all-trans alkyl chain, with a zero crossing for the case of when the chain lengths of lipid and attached alkyl match. This behavior suggests that interdigitation between lipid and attached alkyl chains occurs under gel phase conditions. A comparison of 2H-NMR gel phase line shapes of SSMs and of multilamellar vesicles (MLV) at different temperatures further supports the existence of an interdigitation in SSMs. A phenomenological equation is suggested that describes the dependence of Tm,m on the lipid chain length for a given all-trans alkyl chain. It is based on the established Tm,b dependence on lipid chain length in MLVs but has been modified by a term that accounts for the chain length mismatch between lipid and attached alkyl.
Introduction Monolayers of alkyl chains covalently attached to silicate have far ranging applications from sensors to chromatography.1, 2 Recently, we have shown that zwitterionic phospholipids such as synthetic lecithins can self-assemble on top of a previously formed octadecyl-dimethyl-monochlorosiloxane (OMS) monolayer that is covalently attached to spherical silica gel surfaces.3 The result is a spherical supported lipid monolayer (SSM) that mimicks in many aspects a supported lipid bilayer4 but shows higher mechanical stability. These superior mechanical properties together with a drastically improved control of the surface charge density of the silica support give SSMs a potential advantage for applications in bioseparation and in biosensors. The phase transition behavior of SSMs, studied in detail so far only for the synthetic lecithin dipalmitoyl-phosphatidylcholine (DPPC),3 shows significant differences compared with that of bilayers, in particular with respect to the melting temperature of the lipid chains.4 The latter is several degrees Celsius higher for the SSM than for the corresponding supported bilayer. Another yet unresolved question is how the SSM responds to the lateral stress that builds up in the lipid monolayer at the transition from the fluid to the gel state of the lipid. This stress arises due to the reduction of the surface area per lipid molecule at the transition while the organosiloxane monolayer below it remains unchanged. A conceivable * To whom correspondence should be addressed. Telephone: 49931-888-5863. Fax: 49-931-888-5851. E-mail:
[email protected]. (1) Feng, X.; Fryxell, G. E.; Wang, L.-Q.; Kim, A. Y.; Liu, J.; Kemner, K. M. Science 1997, 276, 923. (2) Wirth, M. J.; Fairbank, R. W. P.; Fatunmbi, H. O. Science 1997, 275, 44. (3) Linseisen, F. M.; Hetzer, M.; Brumm, T.; Bayerl, T. M. Biophys. J. 1997, 72, 1659. (4) Naumann, C.; Brumm, T.; Bayerl, T. M. Biophys. J. 1992, 63, 1314.
response would be an interdigitation between lipid and organosiloxane monolayers, leading to a gel phase structure dominated by the packing of the organosiloxane. The optimized filling of the spaces between the (all-trans) siloxane chains by the lipid chains would be approximately achieved for the case in which lipids and siloxane feature the same chain length. In contrast, for a length mismatch between both chains, the van der Waals interaction potential between them can be expected to change in a predictable way, leading to a systematic variation of the lipid chain melting temperature with the chain length at their transition from the gel to the fluid state. To test this hypothesis, we studied the SSM phase transition temperatures of synthetic lecithins on octadecylfunctionalized (C18) silica gels for lipid chain lengths ranging from C12 to C20 and compared them with those measured for bilayers. To obtain additional information about the molecular order in the putative interdigitated phase, we compared the temperature dependencies of the deuterium nuclear magnetic resonance (2H-NMR) line shapes for gel phase SSMs with those of bilayers. Materials and Methods Selectively chain-deuterated dipalmitoyl-7,8-d4-phosphatidylcholine (DPPC-d8), chain-perdeuterated dimyristoyl-phosphatidylcholine-d54 (DMPC-d54), dilauroyl-phosphatidylcholine (DLPC), dimyristoyl-phosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), distearoyl-phosphatidylcholine (DSPC), and diarachidoyl-phosphatidylcholine (DAPC) were obtained from Avanti Polar Lipids (Alabaster, AL). Porous silica gels (Nucleosil 4000-7 C18, with a pore size of 400 nm and a bead diameter of 7 µm) functionalized with octadecyl-monochloride (in the following referred to as hydrophobized silica gel or attached alkyl respectively) were purchased from Macherey-Nagel (Du¨ren, Germany). Deuterium depleted water used in 2H-NMR experiments was from Isotec Inc. (Miamisburg, OH). The water used for all other experiments and preparations was purified with a Milli-Q-System (Millipore Corp., Bedford, MA). The SSMs were prepared by a self-assembly procedure described in detail previously.3 Dry lipid was dissolved in
10.1021/la981500x CCC: $18.00 © 1999 American Chemical Society Published on Web 03/10/1999
2432 Langmuir, Vol. 15, No. 7, 1999
Figure 1. DSC endotherms and exotherms (dotted) of spherical supported lecithin monolayers (SSMs) for (a) DLPC (C12), (b) DMPC (C14), (c) DPPC (C16), (d) DSPC (C18), and (e) DAPC (C20). The spherical support consists of a silica gel with covalently attached octadecyl chains (C18). methylenchloride along with the hydrophobized silica gel. By adding polar solvent (first methanol, then water) in a stepwise manner and simultaneously evaporating the less polar solvent, the SSMs dissolve in buffer A (20 mM Hepes; 0.5 mM EDTA; pH 7.0). Excess lipids were removed by up to four washing steps using buffer A. For NMR experiments, the water in the final SSM dispersion was replaced by deuterium-depleted water in another washing step. Differential scanning calorimetry (DSC) measurements were performed using a Microcal MC-2 (Microcal Inc., North Hampton, MA). Both ascending and descending temperature modes were done at a scan rate of 20 °C/h. Slower scan rates (10 °C/h) gave identical results. All samples were measured twice in succession, and the second heating and cooling scans were used after baseline correction using the Origin software (Microcal Inc., North Hampton, MA). Deuterium NMR experiments were performed at different temperatures using a Bruker AMX-500 spectrometer equipped with a 10-mm solid-state probe. The spectra were obtained by applying a quadrupolar echo (QE) pulse sequence with two 90° pulses of 5.5 µs duration, a pulse separation of τ ) 35 µs, and a CYCLOPS5 phase cycling sequence. The repetition time was 150 ms. The 4096 complex data points were collected in quadrature with a dwell time of 1 µs, and the number of scans was 100 000. The spectra were obtained by a one-dimensional Fourier transform starting at the top of the echo of the corresponding free induction decays after zeroing the imaginary part to improve the quality of the spectra. Sample temperature was controlled within the error bars (as shown in Figure 5) using a Bruker temperature control unit with liquid nitrogen as coolant. Second moments were calculated numerically from the corresponding NMR line shape f(ω) according to M2 ) 2‚∫∞0 (ω ∞ f(ω)dω where ω0 is the resonance frequency.6 ω0)2‚f(ω)dω/∫-∞
Results and Discussion 1. DSC. Figure 1 shows the DSC endotherms and exotherms obtained for SSMs of different synthetic lecithins on hydrophobized silica substrates: (a) DLPC (C12:0), (b) DMPC (C14:0), (c) DPPC (C16:0), (d) DSPC (C18:0), and (e) DAPC (C20:0). All samples exhibit rather sharp chain melting transitions of the monolayer lipids at the temperature Tm,m and additionally (except for DLPC), a secondary, much weaker peak at Tm,b (arrows). The temperatures Tm,b agree with the main phase transition temperatures of the corresponding lipids that we measured in fully hydrated bilayers: for DMPC at 23.7 °C, DPPC at 40.8 °C, DSPC at 54.4 °C, and for DAPC at 64.5 °C. For DLPC-MLV, the main phase transition temperature is at (5) Rance, M.; Byrd, A. J. Magnet. Reson. 1983, 52, 221. (6) Davis, J. H. Biophys. J. 1979, 27, 339.
Ka¨ sbauer and Bayerl
Figure 2. Plot of the transition temperatures Tm,m of SSM (4) and Tm,b of multilamellar vesicles (MLV) (O) as a function of the number of carbon atoms of the lecithin chains. The fitted curves were obtained by using eqs 2 and 3 (see text for details). Table 1. Thermodynamic Properties of the Lipids (in °C) lipid parameter
DLPC
DMPC
DPPC
DSPC
DAPC
# of carbons (n) Tm,b Tm,m fwhm (Tm,m) hysteresis (Tm,m) Tm,m - Tm,b
C12 -1.0 17.1 3.0 1.1 18.1
C14 23.8 35.0 4.7 1.1 11.2
C16 40.9 46.5 4.5 1.1 5.6
C18 54.4 53.1 2.6 1.3 -1.3
C20 64.8 56.2 1.6 1.4 -8.6
-1 °C which explains the absence of this peak in the temperature range chosen for the representation of Figure 1. The exact origin of the weak peaks is not clear. Contamination of the samples by low amounts of bilayer structures (e.g.; small vesicles) cannot be excluded. However, multiple washings of the samples in excess buffer did not cause a reduction of these peaks, suggesting that these bilayer structures must be tightly associated with the SSMs. Some additional features of the DSC curves from Figure 1 are noteworthy (see also Table 1): (a) none of the samples shows a pre-transition that can be observed for bilayers; (b) the full width at half-maximum (fwhm) of the monolayer melting transition at Tm,m ranges from about 2 to 5 °C; and (c) the hysteresis observed between the Tm,m values of heating and cooling scans (i.e.; endo- and exotherms) is a factor of 1.5-2 larger than measured for bilayers (multilamellar vesicles, MLV). It should be noted that the C18 chains of the attached alkyl layer do not contribute to the DSC results, Fourier transform infrared (FT-IR) measurements of the hydrophobized silica gels used in this work clearly showed that these chains are in an all-trans conformation at all relevant temperatures (asymmetric CH2-stretching mode at 2918.5 cm-1 and the symmetric one at 2848.5 cm-1). To be sure, a DSC scan of the dry hydrophobized silica gel did not show any thermodynamic event in the range 2-80 °C. Plotting the temperatures Tm,m and Tm,b versus the number of carbons of the corresponding lipid chains (Figure 2) shows that the difference between both temperatures depends in a systematic way on the lipid chain length. At short chain lengths of the lipids, the monolayer melting (Tm,m) occurs well above the corresponding event in the bilayer (Tm,b), but the situation is reversed for long chain lipids. The zero crossing (i.e.; Tm,m ) Tm,b) occurs around the C18 chain length (DSPC) of the lipids. It is interesting to note that the zero crossing takes place at that lipid chain length that matches (under all-trans conditions) the length of the attached C18 chains. Such a (7) Cevc, G. Phospholipids Handbook; Marcel Dekker: New York, 1993.
Interdigitation of Lecithin Monolayers under Gel Phase Conditions Table 2. Phase Transition Temperatures (°C) of Asymmetric PCs According to Ref 8
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Table 3. Comparison of FiT-Results According to Eqs 2 and 3 for MLV- and SSM-Systems
# of carbons (n)
Tm,b(C18:Cn-PC)
Tm,b(Cn:C18-PC)
Tm,bav a
system
nm
nh
c
χ2
12 14 16 18
18 30 45 55
23 38 49 55
20.5 34 46.5 55
MLV MLVa SSM
-3.0 -3.2 -3.1
-12.8 -10.6 -12.8
0 0 3.4
0.25
a
a
0.20
Data taken from ref 10.
Tm,bav is the average of the two values for a given n.
behavior is hardly conceivable under conditions that the chains of the lipid monolayer on one side and of the attached alkyl chains on the other side are separated from each other within the confines of their respective layers. There must exist some direct contact between both types of chains. The most likely arrangement would be a partial or full interdigitation between lipid and attached alkyl chains under gel phase conditions of the lipid (note that the attached alkyl chains are at all relevant temperatures in all-trans conformation). An interesting analogue to the situation just presented is the phase transition behavior of asymmetric chain lecithins where the sn-1 chain is approximately up to twice as long as the sn-2 chain and vice versa.8,9 For these types of lipids, chain interdigitation is a common feature of the gel phase. For example, 1-acyl-2-stearoyl-PC (Cn:C18-PC) and 1-stearoyl-2-acyl-PC (C18:Cn-PC) exhibit different Tm,b values in bilayers8 (see Table 2) and the difference between both goes to zero with n approaching 18. An even closer correspondence to our case is obtained by using an averaged temperature
Tav m,b(asym.,n,18)
1 ) (Tm,b(C18:Cn) + Tm,b(Cn:C18)) (1) 2
the values of which are listed in Table 2 as well. A phenomenological model for the thermodynamic behavior of MLV shown in Figure 2 is obtained by expressing the chain melting phase transition temperature of an arbitrary symmetric phospholipid as a function of the number of carbons per chain as a polynomial expansion:10
(
Tm,b(n) ) Tm(∞) 1 +
)
nm n h + 2 + ... n n
(2)
Here, nm is the length of the shortest segment for which a first-order chain-melting phase transition is possible11 and nh accounts phenomenologically for the headgroup and other effects.10 The parameter Tm(∞) refers to the chain melting transition temperature of a hypothetical lipid with infinitely long chains. Using Tm(∞) ) 414 K11 and fitting eq 2 to our bilayer data (Tm,b) yields the dashed line in Figure 2 and provides values for nm and nh that are listed in Table 3. The data agree well with those published previously by Cevc,10 which are also listed in Table 3 for comparison. In contrast, attempts to fit this equation to our monolayer values (Tm,m) did not yield any satisfactory fit of the data. To obtain a satisfactory fit, we also had to consider the difference in chain length between the surfaceattached C18 chains and that of the lipid for each sample. Assuming interdigitation, we can expect the van der Waals attraction between both alkyl chains to depend on the (8) Mattai, J.; Sripada, P. K.; Shipley, G. G. Biochemistry 1987, 26, 3287. (9) Lewis, R. N. A. H.; McElhaney, R. N.; O ¨ sterberg, F.; Gruner, S. M. Biophys. J. 1994, 66, 207. (10) Cevc, G. Biochemistry 1991, 30, 7186. (11) Nagle, J. F.; Wilkinson, D. A. Biophys. J. 1978, 23, 159.
Figure 3. Plot of the difference temperature Tm,m - Tm,b as a function of the number of carbon atoms in the lecithin chains. The straight line was included to guide the eye.
length mismatch between the two chains, with a maximum interaction for the case of perfect match. This dependence is phenomenologically expressed by adding a term to eq 2 which accounts for this difference in chain lengths, giving
(
Tm,m(n) ) Tm(∞) 1 +
)
nm nh + 2 + c(n - 18) n n
(3)
where c is a constant. Equation 3 yielded a very good fit (dotted line in Figure 2) to the Tm,m versus chain length data, and the values obtained for nm and nh are given in Table 3. They are very similar to those obtained for bilayers (MLV), and the squared residuals χ2 are in both cases very small. Another interesting feature of the SSM phase transition behavior is the finding that a plot of the difference Tm,m - Tm,b versus the chain length is linear (Figure 3), and the zero crossing occurs in the region where the length of the attached alkyl chains matches that of the lipid chains (n ) 18). It would be interesting to investigate if the point of zero crossing varies with the length of the attached alkyl chains. Unfortunately, the only other hydrophobized beads species commercially available carries an octyl chain. These chains are fluid at all temperatures above zero (established by IR measurements; asymmetric CH2-stretching mode at 2922.5 cm-1 and symmetric at 2853.5 cm-1) and thus cannot be used for comparison because an all-trans state of the attached alkyl chains is a prerequisite for the coating with the lipid monolayer. Summing up the DSC results suggests that lecithin monolayers on C18-functionalized silica undergo interdigitation of their chains with those of the attached alkyl under gel phase conditions. This process causes a significant increase of Tm,m compared with bilayer main phase transition Tm,b for lecithins with chain lengths shorter than C18 and a decrease for those with longer chain lengths. The latter is probably a result of an incomplete interdigitation due to sterical reasons. A characteristic feature of the process is a linear dependence of Tm,m - Tm,b on the chain length, with the zero crossing under conditions of matching chain lengths. It is noteworthy that SSMs do not show any stress-related reduction of their phase
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Figure 4. Temperature dependence of the 2H-NMR line shape of (a) DPPC-d8 MLV and of (b) DPPC-d8 SSM in the region of 20 to -20 °C.
transition temperatures (compared with that of MLVs), as observed previously for supported lipid bilayers. 4 2. 2H-NMR. To obtain further support for the existence of interdigitation, we performed 2H-NMR experiments on SSMs with a selectively deuterated lecithin (DPPC-d8) for the monolayer. Ruocco et al.12 have previously demonstrated the potential of 2H-NMR for analyzing chain interdigitation in bilayers. They concluded that axial chain mobility on the 2H-NMR time scale, which affects in turn the 2H-NMR line shape, persists under interdigitation conditions down to very low temperatures because of the much higher surface area per molecule. This result is in contrast to noninterdigitated bilayers of DPPC where the lower surface area of ≈50 Å2 per molecule13 virtually stops these motions on the NMR time scale at a significantly higher temperature, rendering the asymmetric contributions more pronounced at comparable temperatures. Therefore, we compared the temperature dependence of MLV and SSM gel-phase line shapes for the case of selectively chain deuterated 7,7,8,8- DPPC-d8 (Figure 4). For both samples, a decrease of axial-symmetric contributions to the line shape with decreasing temperature is obvious. However, for the SSM sample, this temperature dependence is significantly less pronounced in the 20 to -20 °C range than in the case of the bilayer sample. This difference is most striking at temperatures of -10 and -20 °C (Figure 4). A quantitative measure of the differences is provided by a comparison of the second moments,6 which are particularly sensitive to intensity contributions at the wings of the spectra (Figure 5). For more details on 2H-NMR line shape in ordered hydrocarbon chain environments we refer the reader to the literature.14,15 In comparison with the work of Ruocco12 the differences between the line shapes of interdigitated and noninterdigitated systems are still less pronounced for the SSMs. (12) Ruocco, M. J.; Makriyannis, A.; Siminovitch, D. J.; Griffith, R. G. Biochemistry 1985, 24, 4844. (13) Mo¨hwald, H. Annu. Rev. Phys. Chem. 1990, 41, 441. (14) Davis, J. H.; Jeffrey, K. R.; Bloom, M.; Valic, M. I.; Higgs, T. P. Chem. Phys. Lett. 1976, 42, 390. (15) Davis, J. H. Biochim. Biophys. Acta 1983, 737, 117.
Ka¨ sbauer and Bayerl
Figure 5. Plot of the second moments calculated from the spectra in Figure 4 as a function of temperature of MLV (O) and SSM (4).
However, there is one essential difference that has to be considered; that is, in the Ruocco work, interdigitated bilayers of C16-ether-linked lecithins were studied, but in our case, only the lecithin monolayer shows chain melting while the monolayer of the surface-attached C18 chains remain in all-trans conformation. Hence, an optimized interdigitated packing, which may occur in the bilayer (simultaneous transition of both monolayers to the gel phase) is prevented for the SSMs. For the latter case, the DPPC chains have to accommodate to the given packing of the (two methylenes longer) attached alkyl chains upon interdigitation, rendering this arrangement less efficient in packing density. Conclusions Both DSC and NMR results indicate a gel phase interdigitation between lipid and surface-attached alkyl chains in SSMs. The chain length dependence of the lipid chain melting transition in SSMs can be satisfactorily explained by a phenomenological equation suggested previously by Cevc10 for bilayers, with an extra term that accounts for the length mismatch between lipid and attached alkyl chains. In a previous work3 we concluded that the lipid density in the SSM is crucial for its phase transition temperature. In light of the results just presented, our earlier conclusion is equivalent to the notion that depending on the structure and density of the surfaceattached alkyl layer, interdigitation may or may not occur. In the latter case, the SSM shows transition temperatures similar to those of MLVs.3 However, under interdigitation conditions, one can assume that the gel phase lateral lipid spacing in SSMs and possibly also the headgroup conformation will differ from those observed in the gel phase of a bilayer. Acknowledgment. The help of M. Hetzer and M. Junglas in performing the NMR experiments and data analysis is gratefully acknowledged. This work was supported by a grant from the Deutsche Forschungsgemeinschaft (DFG). LA981500X
