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Two-Dimensional Physical Networks of Lipopolymers at the Air/Water Interface: Correlation of Molecular Structure and Surface Rheological Behavior C. A. Naumann,*,†,‡ C. F. Brooks,† G. G. Fuller,† T. Lehmann,§ J. Ru¨he,§ W. Knoll,†,§ P. Kuhn,| O. Nuyken,| and C. W. Frank*,† Department of Chemical Engineering, Stanford University, Stanford, California 94305-5025, Chemistry Department, IUPUI, 402 N. Blackford Street, Indianapolis, Indiana 46202-3274, MPI f. Polymerforschung, Mainz, Germany, and Lehrstuhl f. Makromolekulare Stoffe, Technische Universita¨ t Mu¨ nchen, Mu¨ nchen, Germany Received June 1, 2000. In Final Form: November 29, 2000 Recent surface rheology and film balance experiments on monolayers of PEG lipopolymers and phospholipid/PEG lipopolymer mixtures at the air-water interface have revealed a new class of quasi two-dimensional physical networks. Two different kinds of associative interactions are necessary to form the network: microcondensation of alkyl chains of lipopolymers to form small clusters and water molecule mediation of the interaction between adjacent PEG clusters via hydrogen bonding. In the experiments presented here, we are interested to learn whether the physical gelation is PEG specific or whether it is a more general characteristic of lipopolymers at the air-water interface. To address this topic, we have expanded our surface rheology and film balance experiments to poly(oxazoline) lipopolymers. Our experiments indicate the occurrence of a rheological transition if the poly(oxazoline) lipopolymers consist of a dioctadecylglycerol anchor. This shows that the physical gelation among lipopolymers is not a PEGspecific phenomenon. No physical gelation is found, however, if the dioctadecylglycerol anchor of the lipopolymer is replaced by a dioctadecylamine anchor. The observed importance of the hydrophobic anchor supports our previous findings that the alkyl chain condensation should be seen as one of two kinds of physical junctions necessary for the formation of the physical network.
Introduction Lipopolymers are molecules in which one terminus of a single hydrophilic polymer chain is covalently attached to the headgroup of a phospholipid-like molecule. Because of their unique properties, they have been used for several applications within the biosciences and life sciences, such as the following: (1) the engineering of complex tethered biomembranes on solid substrates;1-3 (2) model systems to study the properties of end-grafted polymers;4-6 (3) the study of two-dimensional physical gelation phenomena;6,7 (4) the engineering of drug delivery liposomes.8 Most of these applications rely on lipopolymer molecules carrying PEG chains, because PEG not only is very hydrophilic in a polar solution (e.g. water) but also shows a specific biological inertness. The hydrophilic character of PEG, which is unique for polyethers, is related to a * Corresponding authors. E-mail: C.W.F., curt@chemeng. stanford.edu; C.A.N. (at IUPUI),
[email protected]. † Stanford University. ‡ IUPUI. § MPI f. Polymerforschung. | Technische Universita ¨ t Mu¨nchen. (1) Spinke, J.; Yang, J.; Wolf, J.; Liley, M.; Ringsdorf, H.; Knoll, W. Biophys. J. 1992, 63, 167. (2) Sackmann, E. Science 1996, 271, 43. (3) Naumann, C.; Lehmann, T.; Prucker, O.; Ruehe, J.; Knoll, W.; Frank, C. W. To be submitted for publication. (4) Baekmark, T. R.; Elender, G.; Lasics, D. D.; Sackmann, E. Langmuir 1995, 11, 3975. (5) Baekmark, T. R.; Wiesenthal, T.; Kuhn, P.; Albersdoerfer, A.; Nuyken, O.; Merkel, R. Langmuir 1999, 15, 3616. (6) Naumann, C. A.; Brooks, C. F.; Fuller, G. G.; Knoll, W.; Frank, C. W. Langmuir 1999, 15, 7752. (7) Naumann, C. A.; Brooks, C. F.; Wyatno, W.; Fuller, G. G.; Knoll, W.; Frank, C. W. Macromolecules, submitted for publication. (8) Lasic, D. D.; Papahadjopoulos, D. Curr. Opin. Solid State Mater. Sci. 1996, 1, 392.
specific structuring of water molecules along the polymer chain and the formation of H-bonds between ether oxygens and surrounding water molecules.9,10 The observed PEGspecific behavior is, therefore, linked to a complex interplay between hydrophobic interactions, H-bonding between water and PEG, and PEG-PEG interactions.11-13 In contrast to Langmuir monolayers of nonionic surfactants carrying PEG chains, which already tend to submerge into the subphase at moderate film pressures,14 lipopolymers may form stable monolayers at the air-water interface until the collapse pressure of the monolayer is reached. This water insolubility of lipopolymer monolayers enables the controlled configurational change of “quasi”grafted polymer chains between pancakelike, mushroomlike, and brushlike polymer configurations by simply compressing or expanding the monolayer. The corresponding π/A isotherms of lipopolymers typically exhibit two transition plateaus-a low film pressure transition at about 10 mN/m, which is related to the submerging of polymer chains from the air-water interface into the subphase,4 and a high-film-pressure transition, which was found to be related to a first-order-like alkyl chain condensation.15 Recent surface rheology experiments on PEG lipopolymers at the air-water interface revealed a remarkable (9) Kjellander, R.; Florin, E. J. Chem. Soc., Faraday Trans. 1981, 177, 2053. (10) Blandamer, M. J.; Fox, M. F.; Powell, E.; Stafford, J. W. Makromol. Chem. 1969, 124, 222. (11) Amiji, M.; Park, K. J. Biomater. Sci., Polym. Ed. 1993, 4, 217. (12) Lee, J. H.; Lee, H. B.; Andrade, J. Prog. Polym. Sci. 1995, 20, 1043. (13) Elbert, L.; Hubbell, J. A. Annu. Rev. Mater. Sci. 1996, 26, 365. (14) Naumann, C.; Dietrich, C.; Lu, J. R.; Thomas, R. K.; Rennie, A. R.; Penfold, J.; Bayerl, T. M. Langmuir 1994, 10, 1919. (15) Wiesenthal, T.; Baekmark, T. R.; Merkel, R. Langmuir 1999, 15, 6837.
10.1021/la000778y CCC: $20.00 © 2001 American Chemical Society Published on Web 04/06/2001
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change of the viscoelastic properties in the range of the high-film-pressure transition at which the polymer chains are forced into a highly stretched brushlike polymer configuration.6 The observed rheological transition can be linked to the formation of a quasi two-dimensional physical network. Additional information about the nature and the structural behavior of the gelation phenomenon could be obtained from experiments on phospholipid/ PEG-lipopolymer mixtures.7 For example, we found that the high-film-pressure transition is a necessary precursor for the existence of the rheological transition. Nevertheless, both transitions describe completely different phenomena affecting different moieties of the lipopolymer molecule. Two different kinds of associative interactions are found to be necessary to form the network-the microcondensation of alkyl chains of lipopolymers to small clusters and the water molecule mediation of the interaction between adjacent PEG clusters via hydrogen bonding.7 Furthermore, in agreement with Flory’s model of physical gelation, the rheological transition itself cannot be described by a thermodynamic transition of first, second, or third order.16,17 Still, there is no conclusive answer as to whether the observed two-dimensional gelation phenomena are limited to PEG lipopolymers or whether we are observing a more general behavior of lipopolymers at the air-water interface even if they consist of different kinds of polymer chains. Therefore, one aim of our experiments is to address the question about the PEG specificity of the rheological transition behavior of lipopolymers at the air-water interface. To evaluate the effect of the polymer moiety on the rheological transition behavior, we have extended surface rheology and film balance experiments to poly(ethyl oxazoline) and poly(methyl oxazoline) lipopolymers. By performing IR experiments on selectively deuterated lipopolymers at the air-water interface, Wiesenthal et al. showed that the high-film-pressure transition is related to a first-order-like alkyl chain condensation.15 The same authors observed earlier that only lipopolymers carrying a hydrophobic anchor of saturated alkyl chains exhibit a high-film-pressure transition, whereas those with unsaturated chains show no transition.5 The latter behavior was, for example, also described for polystyrene/PEG diblock copolymers.18 Recent surface rheology and film balance experiments on mixtures of PEG lipopolymers and phospholipids have suggested that physical gelation cannot be expected without the appearance of the highfilm-pressure transition.7 A further aim of our experiments is, therefore, to investigate the link between the molecular structure of the hydrophobic anchor and its surface rheological behavior by replacing the phospholipid anchor with dioctadecylamine.
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Figure 1. Molecular structures of the lipopolymers investigated: DSPE-EO45; DODA-E35; DC18Gly-E31; DC18Gly-M35.
Materials. The PEG lipopolymer, 1,2-distearoyl-sn-glycero3-phosphoethanolamine-N-[poly(ethylene glycol) 2000] (DSPEEO45), was purchased from Avanti Polar Lipids (Alabaster, AL). The poly(ethyl oxazoline) lipopolymer carrying a dioctadecylamine anchor, dioctadecylamine [poly(ethyloxazoline) 4032] (DODA-E35), was synthesized by following the procedure described recently.19 The poly(ethyl oxazoline) lipopolymer, dioctadecanoylglycerol-[poly(2-ethyl-2-oxazoline) 3710] (DC18Gly-E31), and the poly(methyl oxazoline) lipopolymer, diocta-
decanoylglycerol-[poly(2-methyl-2-oxazoline) 3640] (DC18GlyM35), which both carry a dioctadecanoylglycerol as anchor, were synthesized by following the procedure described recently.20,21 The corresponding chemical structures are summarized in Figure 1. Chloroform was used as a spreading solvent for preparing the monolayers at the air-water interface. Milli-Q Water (pH ) 5.5, 18 MΩ cm resistivity) was used as a subphase material for all film balance experiments. Methods. To investigate the viscoelastic properties of Langmuir films, we used an interfacial stress rheometer described recently.22 In short, a magnetized rod, which is kept at the airliquid interface due to the surface tension, was subjected to an oscillatory force generated by a pair of Helmholtz coils in a welldefined flow geometry between two glass walls. A mini-Langmuir trough (KSV Instruments) was used to change the surface concentration and detect the film pressure of the monolayer. The resulting motion of the needle was detected using an optical microscope and a photodiode array that detected the shadow of the needle behind the light source. The oscillating magnetic field on the rod induced a sinusoidal motion at the same frequency but different in phase. From the rod’s position (strain, γs) in relation to the applied magnetic field (stress, σs), we could determine the delay, δ, between the strain and the stress and the ratio of their amplitudes, AR. Such stress-strain experiments provided information about the material property of the dynamic modulus, Gs*. Because the delay, δ, is known, we were able to determine both the storage modulus, Gs′, which represents the elastic properties, and the loss modulus, Gs′′, describing the viscous behavior. To determine the dynamic moduli, we used the same experimental strategy already described in the case of experiments on PEG lipopolymers and phospholipid-lipopolymer mixtures.6,7 All the films investigated were independent of strain (0.0050.07), indicating linear viscoelasticity. In the case of frequencydependent experiments, the frequency was varied from 0.5 to 10 rad/s. Most of the surface-rheological experiments were, however, conducted as a function of film pressure (or equivalently, area per molecule), where the frequency was kept constant at 0.92 rad/s. At least five measurements were performed at each film pressure for statistical confidence (sampling time for each pressure point: 4 min). Because the compression range of the ISR setup is limited due to the presence of the glass walls, we used a KSV5000 Langmuir trough for measurements of the complete isotherms (π/A diagrams). In this case, the compression speed of the barrier was set to 10 mm/min.
(16) Flory, P. J. J. Am. Chem. Soc. 1941, 63, 3083. (17) Flory, P. J. J. Am. Chem. Soc. 1941, 63, 3088. (18) Gonc¸ alves da Silva, A. M.; Filipe, E. J. M.; d’Oliveira, J. M. R.; Martinho, J. M. G. Langmuir 1996, 12, 6547. (19) Lehmann, T. Ph.D. Thesis, University Mainz, 1998.
(20) Kuhn, P. Ph.D. Thesis, TU Munich, 1997. (21) Hommes, P.; Jordan, R.; Persigehl, P.; Nuyken, O. Manuscript in preparation. (22) Brooks, C. F.; Fuller, G. G.; Robertson, C. R.; Frank, C. W. Langmuir 1999, 15, 2450.
Materials and Methods
2D Physical Networks of Lipopolymers
Figure 2. (A, B) Comparison of the π/A isothermal behavior of the lipopolymers DSPE-EO45, DC18Gly-M35, and DC18GlyE31, around their high-film-pressure transition at T ) 20 °C (A). The dotted line indicates the minimum area/molecule, Amin(lipid), typically found for phospholipids at the air-water interface. (B) Surface rheological behavior of the lipopolymers DSPE-EO45, DC18Gly-E31, and DC18Gly-M35 in the vicinity of their high-film-pressure transitions and the corresponding transition behavior of DMPE (Figure 2B). Both the storage modulus Gs′ and the loss modulus Gs′′ are depicted as a function of the area/molecule A. The area/molecule at the gel point, where Gs′ ) Gs′′, is named Arheo. The lipopolymers show a more viscous behavior at A > Arheo but become remarkably elastic for A < Arheo. In contrast to the lipopolymers, which show a gel point at Arheo (Gs′ ) Gs′′), the phospholipid DSPE shows a broader rheological transition, thereby never reaching the gel point.
Results Figure 2A,B compares the isothermal and surface rheological behavior of the phospholipid DPPE and the lipopolymers DSPE-EO45, DC18Gly-M35, and DC18Gly-E31. They represent molecules with comparable alkyl chain lengths, but different polymer moieties, thus providing information about the influence of the polymer chain on both the film balance and surface rheological behavior at the air-water interface. Figure 2A shows the π/A isothermal behavior in the vicinity of the high film pressure transition at T ) 20 °C (compression speed of the barrier: 10 mm/min). As was already shown by Baekmark et al.,5 not only DSPE-EO45 but also DC18Gly-M35 and DC18Gly-E31 exhibit the characteristic high-film-pressure transition. They have interpreted this transition on the basis of the interplay of two opposing interactions: (1) the poor solubility of hydrocarbon chains in air favors their condensation if spread at the air-water interface; (2) the configurational entropy of the hydrophilic polymer chains leads to a repulsion among adjacent polymer chains, thus preventing
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alkane chain condensation if the polymer size is large enough. Upon condensation, the conformational freedom of the alkane chains is drastically reduced.3 By considering the area range at which the plateaus of the high-filmpressure transitions can be observed (∆Afb), we find for DSPE-EO45 a value of ∆Afb ∼ 150-200 Å2. This is significantly higher than the values found for the two poly(oxazoline) lipopolymers, which are in the range of ∆Afb ∼ 80-120 Å2. Although this is a remarkable change, the values found for poly(oxazoline) lipopolymers are still significantly higher than the value typically found for the formation of a continuous phospholipid monolayer in the liquid condensed phase, which typically occurs at an area/ molecule of 40-50 Å2 (see also isotherm of the phospholipid DPPE in Figure 2A). As already reported by Baekmark et al.,3 Figure 2A also shows that the PEG lipopolymer and its poly(oxazoline) analogues differ not only in their transition areas but also in their transition pressures at the high-film-pressure transition, πhigh (T ) 20 °C). While the plateau of DSPE-EO45 can be found at πhigh ) 19 mN/m, those of DC18Gly-M35 and DC18Gly-E31 are at πhigh ) 28 mN/m and πhigh ) 33 mN/m, respectively. The corresponding surface rheological behavior is shown in Figure 2B, where both Gs′′ and Gs′ are depicted as a function of the area/molecule. Despite the differences of the isothermal behavior described above, we observe for DC18Gly-M35 and DC18Gly-E31 the same characteristic rheological transition already described in the case of DSPE-EO45.6 For A > Arheo, the response of the needle cannot be distinguished from that without a monolayer at the air-water interface (Gs′′ > Gs′). At Arheo, however, an increase of the dynamic moduli by 3-4 orders of magnitude can be observed. For A < Arheo, the monolayer behaves like an elastic material with Gs′ > Gs′′. The only notable difference can be found if the area values at the rheological transition, Arheo, are compared. While the rheological transition of DSPE-EO45 occurs at Arheo ) 160 Å2, those of DC18Gly-M35 and DC18Gly-E31 can be found at Arheo ) 90 Å2. In contrast to the lipopolymers, the phospholipid DMPE shows a broader rheological transition, thereby never reaching the gel point. Figure 3A,B compares the isothermal and surface rheological behavior of DODA-E35 and DC18Gly-E31 in order to investigate the link between the molecular structure of the hydrophobic anchor and the surface rheological response. Both lipopolymers consist of a poly(ethyl oxazoline) polymer chain of similar chain length but differ regarding their hydrophobic anchors. While a dioctadecylamine anchor was used in the case of DODAE35, DC18Gly-E31 consists of a dioctadecanoylglycerol moiety. A comparison of both lipopolymers allows studies of how the surface rheological response might be influenced by the molecular design of the hydrophobic anchor. Figure 3A shows the π/A isothermal behavior for DODAE35 and DC18Gly-E31 in the range of the high-film-pressure transition at T ) 20 °C. Both lipopolymers show the characteristic high-film-pressure transition within an area range/lipopolymer of ∆Afb ∼ 75-100 Å2. Again, as already described in Figure 2A, the transition plateaus can be found at different film pressures. While the transition plateau of DC18Gly-E31 occurs at πhigh ) 33 mN/m, DODAE35 exhibits its plateau at πhigh ) 41 mN/m (at T ) 20 °C). The corresponding surface rheological behavior is shown in Figure 3B, where both Gs′′ and Gs′ are illustrated as a function of the area/molecule. While DC18Gly-E31 shows the PEG-typical rheological transition behavior with a very sharp transition at Arheo ) 90 Å2, DODA-E35 is rather characterized by a gradual change of its dynamic moduli
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Figure 4. Relationship between the compressibility and the average area density per molecule for DSPE-EO45, DC18GlyE31, DC18Gly-M35, and DODA-E35 in the range of their highfilm-pressure transitions. The peaks represent a measure for the strength of the first-order-like alkyl chain condensation.
and DC18Gly-M35 are far smaller. Another interesting finding is the observed difference between DC18Gly-E31 or DC18Gly-M35 and DODA-E35, which shows almost no transition peak. Discussion
Figure 3. (A, B) Comparison of the π/A isothermal behavior of the lipopolymers DC18Gly-E31 and DODA-E35 around their high-film-pressure transition at T ) 20 °C (A). The dotted line indicates the minimum area per molecule, Amin(lipid), typically found for phospholipids at the air-water interface. (B) Comparison of the surface rheological behavior of DC18Gly-E31 and DODA-E35 in the vicinity of the high-film-pressure transition and corresponding transition behavior of the phospholipid DMPE (B). Both the storage modulus, Gs′, and the loss modulus, Gs′′, are depicted as a function of the area/molecule, A. The crossover point (where Gs′ ) Gs′′), or the gel point, is labeled as Arheo. While DC18Gly-E31 shows the same sharp viscoelastic transition already found for PEG lipopolymers, DODA-E35 lipopolymers exhibit rather broad transitions, thereby never reaching the gel point.
over an area range of 70-100 Å2. This remarkably different rheological transition behavior resembles some phospholipids, such as DMPE or DPPC, above the first-order-like phase transition of their alkyl chains rather than a physical gelation transition typically found in the case of PEG lipopolymers.6 The main difference can be found if we consider the range at A < Arheo. While lipopolymers of the PEG-type clearly show elastic behavior, with Gs′ > Gs′′, DODA-E35 remains rather viscous, with Gs′ < Gs′′. Figure 4 compares the compressibility behavior of DSPE-EO45, DC18Gly-E31, DC18Gly-E31, and DODA-E35 in the range of the film-pressure transition. The compressibility, C, is defined as
1 dA C)A dπ
( )
(1)
where A is the area/molecule and π is the film pressure. The presentation of the area compressibility as a function of the area density, as shown in Figure 4, not only provides clear evidence for the first-order-like nature of the high film pressure transition but also reveals remarkable differences regarding the strength of this transition among the lipopolymers compared. While DSPE-EO45 shows quite a pronounced transition peak, those of DC18Gly-E31
Influence of the Polymer Moiety. Recent surface rheology and film balance experiments on pure PEG lipopolymers at the air-water interface have provided experimental evidence for the existence of a quasi twodimensional physical gelation in the vicinity of the highfilm-pressure transition.6 The physical gelation can be seen as an indication that the polymer chains are here in a fibrillar state, where the chains appear rather rigid.23 The rigidity can be caused by either an intrinsic chain stiffness or a helical structure stabilized by solvent molecules.24 The network is formed by fibrillar junction zones where each junction is held together by weak molecular interactions of just a few kT. The weakness of those junctions leads to the reversible character of the physical network formation, because their nonpermanent character is related to a specific lifetime.25 Additional experiments on PEG lipopolymer-phospholipid mixtures showed that the physical network consists of two different kinds of junction points: (1) alkyl chain condensation to small clusters of only a few lipopolymer molecules; (2) junctions between PEG chains of neighboring clusters mediated by water molecules via hydrogen bonding.7 The experiments have, indeed, verified that there is only a physical gelation if the lipopolymers show the high-filmpressure transition caused by the condensation of their alkyl chains to small domains of lipopolymers. Not understood, so far, is the question of whether the observed two-dimensional physical gelation is either a PEG-specific phenomenon or a more general effect observed for a broader class of amphiphilic molecules. To address this question, we compare in Figure 2A,B π/A isotherms and rheological properties of DSPE-EO45, DC18Gly-M35, and DC18Gly-E31, which represent lipopolymers mainly distinguished by their polymer chains. Baekmark et al. (1999) already verified the existence of a high-film(23) Guenet, J.-M. Thermoreversible gelation of polymers and biopolymers; Academic Press: London, 1992. (24) Guenet, J.-M. In The Wiley Polymer Networks Group Review Series Vol. 1; te Nijenhuis, K., Mijs, W. J., Eds.; John Wiley: Chichester, U.K., 1998. (25) Marrucci, G.; Van den Brule, B. H. A. A. In The Wiley Polymer Networks Group Review Series Vol. 1; te Nijenhuis, K., Mijs, W. J., Eds.; John Wiley: Chichester, U.K., 1998.
2D Physical Networks of Lipopolymers
pressure transition in the case of DC18Gly-M35, and DC18Gly-E31 as shown in Figure 2A.5 In agreement with our earlier predictions that a highfilm-pressure transition is usually accompanied by a physical gelation transition,7 we observe in Figure 2B for DC18Gly-M35 and DC18Gly-E31 the same kind of rheological transition already found in the case of DSPE-EO45. Obviously, our data show that the rheological transition is not limited to PEG lipopolymers. It can also be found for several other lipopolymers. A rather unexpected result, however, is the observation that both PEG and poly(oxazoline) lipopolymers are very similar in respect to their rheological transition behavior, despite the remarkable difference in Arheo (or Afb), which indicates a significantly different polymer-water complex between the PEG lipopolymer and the poly(oxazoline) analogues. The observed difference could also be caused by different polymer chain lengths, as recent measurements on DSPE-EO45 and DSPE-PEG5000 have shown.6 However, it should be emphasized that all three lipopolymers compared here not only have the same alkyl chain length but also polymer chain lengths of 45, 35, and 31 monomer units, which are too similar to explain the observed differences. Therefore, we assume that the observed changes in Arheo cannot be explained by variations of the polymer chain length but should rather be linked to a different structure of the polymer-water complex. It should be mentioned that DSPE-EO45, DC18Gly-M35, and DC18Gly-E31 show almost the same surface rheological behavior if Gs′ and Gs′′ are considered on a reduced area scale, Ar ) (Arheo- A)/Arheo (data not shown). Most notably, each monolayer compared behaves at A < Arheo like an elastic material (Gs′ > Gs′′). This brings us to the assumption that different polymerwater complexes can lead to very similar surface rheological properties. Guenet and co-workers first proposed the possibility of the formation of a physical gel by polymer-solvent complexes.26,27 Following Guenet’s approach, we were able to explain our recent surface rheological experiments on PEG lipopolymers by assuming that water intercalates lead to physical gelation.6 The similarity of both the molecular structure and the surface rheological behavior among DSPE-EO45, DC18Gly-M35, and DC18Gly-E31 described above suggests that the basic molecular interaction scenario should be very similar among those lipopolymers. It is, therefore, very likely that the scenario recently described for the rheological transition of PEG lipopolymers should also be valid for DC18Gly-M35 and DC18GlyE31-the formation of a two-dimensional physical network between small lipopolymer clusters of lipopolymers mediated by water molecules between polymer chains of adjacent clusters via hydrogen bonding.6,7 As found in the case of PEG lipopolymers, the formation of the physical network requires (1) a highly stretched brushlike configuration of the polymer chains and (2) the “squeezing out” of hydrated water molecules from the polymer-water complex. Regarding the first requirement, it has been reported that lipopolymers show different structural configurations (pancake, mushroom, or brush) depending on their corresponding area/lipopolymer.28 As neutron reflectivity experiments on phospholipids at the air-water interface have verified, configurational changes within the hydrophilic moiety of amphiphilic molecules as a result (26) Franc¸ ois, J.; Gan, J. Y. S.; Guenet, J.-M. Macromolecules 1986, 19, 2755. (27) Gan, J. Y. S.; Franc¸ ois, J.; Guenet, J.-M. Macromolecules 1986, 19, 173. (28) Majewski, J.; Kuhl, T. L.; Gerstenberg, M. C.; Israelachvili, J. N.; Smith, G. S. J. Phys. Chem. B 1997, 101, 3122.
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of the compression of the monolayer should be seen as a rather general phenomenon among such molecules.29 The submerging of water molecules out of the hydrophilic moieties of amphiphiles, as a result of the compression of the monolayer, is the second requirement for the formation of a physical network. This is a well-known phenomenon found for amphiphiles at the air-water interface, as neutron reflectivity measurements on phospholipids and nonionic surfactants have shown.30 Influence of the Lipidlike Moiety. Keeping in mind that the physical network formation of lipopolymers at the air-water interface requires a previous microphase separation via alkyl chain condensation, we should also ask how molecular modifications of the hydrophobic anchor affect the film balance and surface rheological behavior. Meanwhile, it is well established that structural modifications of the hydrophobic anchor may lead to changed isothermal properties. Baekmark et al. (1999) showed, for example, that there is no high-film-pressure transition if the lipopolymers consist of unsaturated alkyl chains.5 Similarly, experiments on polystyrene/PEG diblock copolymers also verified the absence of a high-film-pressure transition.18 Obviously, we may expect changes in the isothermal properties due to modifications of the hydrophobic anchor. By keeping in mind the strong relationship between the existence of a high-film-pressure transition and the occurrence of a PEG-like rheological transition, we might expect that molecular modifications within the hydrophobic anchor of the lipopolymer molecule should lead to changed surface rheological properties. Indeed, as recent experiments on DSPE-EO45 and DPPE-PEG2000 have shown, both the film balance and surface rheological behavior are affected if the alkyl chain length is varied.6 In this case, we observed a value of Arheo ) 160 Å2 for DSPE-EO45, which is slightly higher than the value of Arheo ) 152 Å2 found for DPPE-PEG2000 with the shorter alkyl chains. To obtain a better understanding of how the surface rheological properties of lipopolymers are affected if the hydrophobic anchors are modified, we have performed comparison experiments on two different poly(ethyloxazoline) lipopolymers, one carrying a dioctadecylamine (DODA-E35) and the other carrying a dioctadecanoylglycerol lipidlike moiety (DC18Gly-E31). As illustrated in Figure 3A, both lipolymers show a high-film-pressure transition, although the transition plateau of DODA-E35 is less pronounced than that of DC18Gly-E31. Most surprisingly, DODA-E35 shows a rheological transition behavior remarkably different from that of DC18Gly-E31. While DC18Gly-E31 shows a PEG-like behavior with Gs′ > Gs′′ at A < Arheo, which has been interpreted as twodimensional physical gelation, DODA-E35 reveals rather a transition behavior recently found in the case of some phospholipids, such as DMPE and DPPE.6 In agreement with earlier findings on several phospholipids, we assume that the surface rheological transition in the case of DODA-E35 should be rather linked to the friction between interacting alkyl chains than to a physical gelation because the film remains rather viscous with Gs′′ > Gs′, even at A < Arheo.6 So far, we have argued that a high-film-pressure transition is necessary in order to find a rheological transition. Now we have found a situation where this conclusion is in question. One possible explanation for (29) Brumm, T.; Naumann, C.; Sackmann, E.; Rennie, A. R.; Thomas, R. K.; Kanellas, D.; Penfold, J.; Bayerl, T. M. Eur. Biophys. J. 1994, 23, 289. (30) Naumann, C.; Brumm, T.; Rennie, A. R.; Penfold, J.; Bayerl, T. M. Langmuir 1995, 11, 3948.
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this is related to the stability of the monolayer because we are dealing with relatively high film pressures of more than 40 mN/m. Here, we should, however, mention that the rheology experiments have been performed by keeping the pressure constant for each data point. Two other possible explanations for the unusual behavior of DODAE35 are (1) the DODA-alkyl chains “feel” a different microenvironment strongly affecting the strength of the alkyl chain condensation as one form of junction points necessary for the formation of a physical network and (2) DODA-E35 and DC18Gly-E31 are attached reversed regarding their lipid heads (see Figure 1), which might lead to a different polymer-water complex. To get a better understanding about the transition behavior at the high-film-pressure transition, Figure 4 compares the compressibility behavior of DSPE-EO45, DC18Gly-M35, DC18Gly-E31, and DODA-E35. The peak areas can be seen as a measure of the strength of the alkyl chain condensation at the high-film-pressure transition. Indeed, we observe remarkable differences with respect to those integrated areas. DSPE-EO45 shows quite a pronounced transition peak. DC18Gly-E31 and DC18GlyE31, on the other hand, show significantly smaller peak areas indicating weaker phase transitions. The peak area is even more diminished in the case of DODA-E35. Obviously, there is no direct correlation between the strength of the alkyl chain condensation and the strength of the physical network because DC18Gly-E31 and DC18Gly-E31 showed the same surface rheological behavior despite a significantly weaker alkyl chain condensation found in Figure 4. However, as Figure 4 also indicates, one might expect that there is no possible formation of a physical network if the strength of the alkyl chain condensation is below a characteristic threshold value. Unfortunately, our experimental data do not provide accurate thermodynamic parameters such as the transition enthalpy. Nevertheless, we can expect a reasonable threshold value of the order of the thermal energy of only a few kT. Concluding Remarks The experiments described have verified that the formation of a physical network is not limited to PEG lipopolymers. Our experiments have shown that lipopolymers carrying either a poly(methyloxazoline) or a poly(ethyloxazoline) moiety are also able to form physical networks. Furthermore, the observed difference of Arheo for different lipopolymers indicates that even differences in hydration properties do not lead to a measurable change of the surface rheological transition behavior. From measurements on DODA-E35 we found conditions where a high-film-pressure transition could be observed even if no rheological transition was detected. In this case, we should expect that the strength of the alkyl chain condensation does not exceed the thermal energy of a few kT. On the experimental time scale of our surface rheology experiment, which is about 0.1-2 s, this is too weak to create rather permanent junctions. A likely scenario to describe the network formation is explained in Figure 5. For Alipo > Afb, we expect no
Naumann et al.
Figure 5. Schematic of a possible molecular model to explain the observed physical network formation among poly(methyl oxazoline) lipopolymers. As the lipopolymer molecules are compressed to a brushlike configuration beyond A< Arheo, two regions should be distinguished: (A) the chain is hydrated with water molecules at locations where there are vacant H-bonding sites exposed to bulk water; (B) the hydrated water molecules act as intercalates, linking two neighboring chains via hydrogen bonding. These physical junction points are not permanent, leading to the observed reversible character of the rheological transition. The model for the physical gelation of poly(ethyl oxazoline) lipopolymers is assumed to be similar (not shown).
microphase separation (alkyl chain condensation) because the lipopolymer alkyl chains are too far away to aggregate. At Alipo ) Afb, the center of the high-film-pressure transition, small clusters of alkyl chains of lipopolymers (circle at the top). The area mismatch of lipopolymers between polymer and alkyl chain moiety does not allow macroscopic phase separation. As the lipopolymers are compressed beyond A < Arheo, the surface-rheological transition, two regions should be distinguished: (A) the hydrated chain exposes vacant H-bonding sites to bulk water; (B) the hydrated water molecules act as mediators, linking two adjacent polymer chains via hydrogen bonding. Currently, we are focusing on related experiments that go beyond the mere surface-rheological characterization. These experiments concentrate more on structural (neutron reflectivity), thermodynamic (temperature-dependent experiments), and dynamic aspects (lifetimes of physical junctions) of the two-dimensional network formation among lipopolymers. Acknowledgment. This work was supported by the Deutsche Forschungsgemeinschaft (DFG) and the NSF Materials Research Science and Engineering Center Program under Grants DMR 94-00354 and DMR 98-08677 through the Center on Polymer Interfaces and Macromolecular Assemblies (CPIMA). LA000778Y
