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Giant Free-Standing ABA Triblock Copolymer Membranes Corinne Nardin,† Mathias Winterhalter,‡ and Wolfgang Meier*,† Department of Physical Chemistry, University of Basel, Klingelbergstrasse 80, CH-4056, Basel, Switzerland, and IPBS-CNRS UPR 9062, University Paul Sabatier, 31077 Toulouse, France Received February 14, 2000. In Final Form: July 20, 2000
For the first time giant free-standing monomolecular films of a functionalized poly(2-methyloxazoline)block-poly(dimethylsiloxane)-block-poly(2-methyloxazoline), PMOXA-PDMS-PMOXA, triblock copolymer were prepared. Stable films with areas up to about 1 mm2 and thickness of 10 nm were achieved. This triblock copolymer carries polymerizable methacrylate groups at both chain ends. These end groups could be polymerized by UV light after the formation of the self-assembled structure. The mechanical properties of these films were characterized by applying short electric field pulses. In comparison to lipid bilayers, the polymer films were significantly more cohesive, seen by higher critical voltages required for rupture. Moreover, polymerization increased the stability significantly.
Introduction Amphiphilic molecules such as surfactants or lipids aggregate in dilute aqueous solution into micelles or vesicles of various shapes. Similar and even larger superstructures are formed by higher molecular weight analogues such as block copolymers consisting of hydrophilic and hydrophobic blocks.1-5 The shape of the resulting aggregates depends according to the current understanding, mainly on the molecular geometry of the underlying amphiphiles.6 For example, amphiphilic block copolymers may form spontaneously vesicular structures in water.3 Rather short hydrophilic blocks compared to the hydrophobic ones typically characterize the suitable block length ratio. The vesicle-forming block copolymer membranes appeared significantly more stable than those formed by conventional lipids. The larger size of the hydrophobic part and the slower dynamics of the underlying copolymer molecules likely cause this. Similar to lipid vesicles, these aggregates are held together by hydrophobic and van der Waals interactions. Hence, the formation of such vesicles is a reversible process; i.e., the vesicles may disintegrate upon dilution or in the presence of detergents. The structure of such aggregates is considerably strengthened if the individual block copolymers within the vesicle-forming bilayer are interconnected via covalent bonds forming a giant “supermacromolecule”. For example, recently it was shown that a cross-linking polymerization in giant wormlike poly(ethylene oxide)-block-poly(butadiene) micelles4 and in vesicular aggregates formed by a reactive poly(2-methyloxazoline)-block-poly(dimethyl†
University of Basel. University Paul Sabatier. * Corresponding author: Tel +41-61-267 3835; Fax ++41-61267 3855; E-mail wolfgang.meier@ unibas.ch. ‡
(1) Zhang, L.; Eisenberg, A. Science 1995, 268, 1728. (2) Jenekhe, S. A.; Chen, X. L. Science 1998, 279, 1903. (3) Nardin, C.; Hirt, T.; Leukel, J.; Meier, W. Langmuir 2000, 16, 1035. (4) Won, Y.-Y.; Ted Davis, H.; Bates, F. S. Science 1999, 283, 960. (5) Disher, B. M.; Won, Y.-Y.; Ege, D. S.; Lee, J. C. M.; Bates, F. S.; Disher, D. E.; Hammer, D. A. Science 1999, 284, 1143. (6) Israelachvili, J. N.; Mitchell, D. J.; Horn, R. J. Chem. Soc., Faraday Trans. 2 1976, 72, 1525.
siloxane)-block-poly(2-methyloxazoline), PMOXA-PDMSPMOXA, triblock copolymer3 can be used to freeze permanently their self-assembled superstructure. In the latter case the triblock copolymer had been functionalized with polymerizable methacrylate end groups which did not disturb the aggregation behavior of the polymer. The triblock copolymer forms small unilamellar vesicles in dilute aqueous solution with a controlled size in the range 50-500 nm. These vesicles were converted into stable internally cross-linked nanocapsules that were able to preserve their morphological integrity even after their extraction from the aqueous phase. This clearly demonstrates their enormous stabilization. A first test on the mechanical properties of diblock copolymer membranes in giant unilamellar vesicles was made by micromanipulation with a micropipet.5 The block copolymer membranes were considerably tougher and had a reduced permeability compared to those of conventional lipid membranes. Here in this work we focus on an investigation of the mechanical and viscoelastic properties of the chemically different PMOXA-PDMS-PMOXA triblock copolymer membranes and their corresponding polymerized counterparts. A well-established technique to quantify viscoelastic properties of planar lipid membranes (so-called “black lipid membranes”) is to apply controlled forces, e.g., via a short electric field pulse.7 Electric field pulses are used to charge the membrane, causing an electric stress inside the bilayer. Above a critical voltage rupture of the membrane is induced, and a fast discharge across the defect is observed. An analysis of the critical voltage gives information about the energy barrier of the membrane against rupture. An analysis of the discharge kinetics allows conclusion on the kinetics of defect widening and the underlying physical forces. Although it is obvious that bilayer-forming block copolymers could be used to prepare analogue planar freestanding films, no such “black polymer membranes” (“BPM”) have been reported. Hence, we describe in this paper the preparation of giant planar PMOXA-PDMS(7) Diederich, A.; Strobel, M.; Meier, W.; Winterhalter, M. J. Phys. Chem. B 1999, 103, 9, 1402.
10.1021/la000204t CCC: $19.00 © 2000 American Chemical Society Published on Web 08/31/2000
ABA Triblock Copolymer Membranes
Figure 1. Schematic representation of a giant planar poly(2-methyloxazoline)-block-poly(dimethylsiloxane)-block-poly(2methyloxazoline), PMOXA-PDMS-PMOXA, triblock copolymer membrane.
PMOXA triblock copolymer membranes (see Figure 1). In a second step the reactive methacrylate end groups are polymerized after formation of a film. The physical properties of the polymer films before and after polymerization are characterized, and the results are compared with conventional black lipid membranes. Experimental Section The synthesis of the PMOXA-PDMS-PMOXA copolymer molecules carrying methacrylate end groups used in this study has been described previously.3 The chemical structure is shown in Figure 2. The molecular weight (Mw) is 9000 g mol-1, i.e., a flexible hydrophobic PDMS middle block of 5400 g mol-1 and of two hydrophilic PMOXA blocks each of 1800 g mol-1. The polydispersity was determined to be Mw/Mn ) 1.7. Formation of Black PMOXA-PDMS-PMOXA Triblock Copolymer Membranes. Free-standing films from these macromolecules could be made adapting the standard procedure applied with conventional low molecular weight lipids.8 The end group functionalized triblock copolymer molecules were dissolved in chloroform (2 wt % polymer). This solution was diluted with toluene in order to obtain a clear, homogeneous 1 wt % polymer solution. About 1 µL of this solution was used for prepainting the Teflon surrounding the hole in the septum dividing the cuvette into two compartments (cis and trans) (Figure 3). After 20 min allowing for drying, the two (cis and trans) chambers of the (8) Mueller, P.; Rudi, D. P.; Tien, H. T.; Wescott, W. C. J. Phys. Chem. 1963, 67, 534.
Langmuir, Vol. 16, No. 20, 2000 7709 Teflon cuvette (Figure 3) were filled with a standard buffer containing 1 M KCl, 1 mM CaCl2, and 10 mM tris(hydroxymethyl)aminomethane (Tris) adjusted to pH 7.4. The freestanding film was made using the membrane forming solution of the triblock copolymer by adding about 2 µL on a Teflon loop and smearing it across the prepainted hole. Within a few seconds the film started to thin out. The polymerization of the black copolymer membranes could be achieved by irradiating the free-standing film during 5 min with an UV lamp. Previous investigations on the polymerization of the functionalized triblock copolymers in vesicles3 and lyotropic liquid crystalline phases9,10 revealed a conversion of more than 90% of the methacrylate end groups under these conditions. All experiments were performed at room temperature (T ) 22 ( 2 °C). Electroporation Technique (Figure 3). To control the quality of the PMOXA-PDMS-PMOXA triblock copolymer freestanding film, the capacitance was determined by charging the membrane via Ag/AgCl electrodes and recording the relaxation across a defined 10 MΩ resistance. Here we applied a rectangular pulse of 500 mV amplitude and 10 µs duration using a fast pulse generator (DS 345, 30 MHz synthesized function generator, Stanford Research System). It has to be emphasized that this amplitude is below the critical one leading to rupture. The value of the capacitance of the free-standing film is calculated from the RC time constant of the exponential discharge process of the membrane across the 10 MΩ resistance of the passive oscilloscope probe (Figure 3). To observe the widening of a single defect and not an undefined number of defects, we carefully approached the critical voltage leading to rupture. In these experiments we started at 500 mV. After application of about 10 pulses at each level we increased the voltage by 20 mV steps. The presented technique charges the membrane capacitor, and after the voltage pulse, the transmembrane voltage discharges immediately which avoids high field strengths. The time course of the transmembrane voltage during electric field induced irreversible rupture is shown in Figure 4. These data correspond to one single representative experiment. The applied rectangular voltage pulse (duration 10 µs) charged the membranes to a transmembrane voltage of about 1 or 1.5 V, respectively. This stage is followed by a steep not well understood voltage drop.7 This nonideal region is followed by an exponential decay of the voltage due to the discharging of the membrane that gives information on the capacitance. The defect formation is seen by the sharp kink after about 40 µs (nonpolymerized and polymerized membrane) that allows evaluating the widening velocity of defects. A more detailed description of this technique is given in ref 7. Theoretical Background. Carefully raising the voltage amplitude of the pulses leads to a sudden drop in the transmembrane voltage (see Figure 4). Initially the defect area is negligible compared to the total area, and the membrane capacitance can be considered as constant. A defect-free membrane is a perfect insulator. Under the chosen conditions, the measured conductance is due to the 10 MΩ resistor of the passive oscilloscope probe and possible defects in the membrane occurring after rupture. After initiating the irreversible rupture, the defect formation becomes the major source of conductance. This yields a simple relation for the membrane conductance G(t):7
G(t) ) -C
d ln U(t) dt
(1)
where C is the membrane capacitance and U(t) the transmembrane voltage. All irreversible ruptures of triblock copolymer membranes and their polymerized counterparts yielded a linear increase of the membrane conductance in time in agreement with previous measurements on pure lipid membranes (Figure 5).7,11 The (9) Hirt, T.; Baron, R. C.; Lohman, D.; Meier, W. WO99/12059. (10) Meier, W. Macromolecules 1998, 31, 2212. (11) Wilhelm, C.; Winterhalter, M.; Zimmermann, U.; Benz, R. Biophys. J. 1993, 64, 121.
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Figure 2. Structure of the functionalized poly(2-methyloxazoline)-block-poly(dimethylsiloxane)-block-poly(2-methyloxazoline), PMOXA-PDMS-PMOXA, triblock copolymer molecule.
Figure 3. Scheme of the charge pulse instrumentation used to induce and record irreversible rupture of black (polymerized) poly(2-methyloxazoline)-block-poly(dimethylsiloxane)-block-poly(2-methyloxazoline), PMOXA-PDMS-PMOXA, triblock copolymer membranes. The free-standing films are obtained by smearing an organic polymer solution across the hole in the septum dividing the Teflon cuvette into two compartments (cis and trans). The set up allows applying fast rectangular pulses of varying amplitude and 10 µs duration.
Figure 4. Time course of the transmembrane voltage during electric field induced irreversible rupture. Data correspond to one single representative experiment. Full line, nonpolymerized membrane; dash line, polymerized membrane. observed conductance after initialization of a defect is due to one or a few pores. These have a large radius in comparison to the
Figure 5. Representative time course of the conductance during rupture of a nonpolymerized and a polymerized membrane obtained by analyzing the voltage versus time curves according to eq 1. Full line, not polymerized membrane; dash line, polymerized membrane. membrane thickness, which simplifies the relation between the pore radius and the pore conductance. For thin large holes the resistance is given by the access resistance which reflects the
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Table 1. Results from Rupture Experiments Performed on Nonpolymerized and Polymerized Membranesa ABA triblock copolymer membranes
C (nF)
da/dt (ms-1)
Vc (V)
∆t (µs)
n
nonpolymerized 0.05 ( 0.02 1.0 ( 0.2 1.4 ( 0.8 51 ( 45 115 polymerized 0.04 ( 0.02 1.5 ( 0.2 3(1 40 ( 20 12 a C ) capacitance; V ) breakdown voltage; da/dt ) rupture c velocity; ∆t ) delay time; n ) number of experiments.
compression of the electric field lines from the electrode to the hole. The conductance Gpore(t) is given by the inverse access resistance Rpore7,11
Gpore(t) )
1 ) 2κa(t) Rpore
(2)
where κ is the specific conductance of the electrolyte. In the case of a single pore, a(t) corresponds to the pore radius or else to the sum of all the radii. From the observed linear increase, a constant radial pore widening velocity is concluded, suggesting inertiadriven rupture kinetics.12,13 In the case of a single pore a combination of eqs 1 and 2 yields the following expression for the radial rupture velocity:
da ) dt
-C d ln U(t) 2κ dt
(
)
(3)
Results and Discussion Free-standing films of functionalized PMOXA-PDMSPMOXA copolymer molecules (see Figure 1 for a schematic representation) were obtained adapting the preparation protocol of conventional low molecular weight lipid membranes.8 The block copolymer macromolecules are soluble in a few organic solvents including chloroform. However, a chloroform-based membrane-forming solution phase separates in our case too fast. Addition of toluene slowed the process and yielded the reproducible formation of giant stable free-standing films. Depending on the setup, stable films with areas up to about 1 mm2 can be obtained. The present experiments yielded typically an area of 0.02 mm2. This smaller area has the advantage that the thinning of the films, observed through the quasiinstantaneous evanescence of interference colors, is faster. The capacitance C is proportional to the area A of a plate condenser and to the reciprocal value of the membrane thickness d
C ) 01A/d
(4)
where 0 is the absolute permittivity of vacuum and 1 the relative permittivity of the triblock copolymer molecules. Equation 4 has often been used to determine the thickness of conventional lipid membranes which has been found to be typically around 5 nm.11 The triblock copolymer membranes gave a mean value of C ) 0.05 nF (see Table 1). With an area of A ) 0.02 mm2 and the relative dielectric constant of the PDMS hydrophobic middle block of 1 ) 2.7,14 eq 4 yields a hydrophobic thickness of about 10 nm. This value is significantly larger compared to the thickness d ) 5 nm of a typical lipid membrane.15,16 Nevertheless, (12) Frankel, S.; Mysels, K. J. J. Phys. Chem. 1969, 73, 9, 3028. (13) Winterhalter, M. Liposomes in Electric Fields. In Handbook of Nonmedical Applications of Liposomes; Lasic, D. D., Barenholz, Y. M., Eds.; CRC Press: Boca Raton, FL, 1996; p 285. (14) Sauer, R. O.; Mead, D. J. J. Am. Chem. Soc. 1946, 68, 1794. (15) Benz, R.; Janko, K. Biochem. Biophys. Acta 1976, 455, 721. (16) Dilger J. P.; Benz, R. J. Membr. Biol. 1985, 85, 181.
taking into account the size of the triblock copolymer macromolecules, this thickness seems to be reasonable. This value is supported by cryo-TEM investigations performed on polymerized PMOXA-PDMS-PMOXA block copolymer vesicles which revealed also a mean lamellar thickness of about 10 nm.17 Similarly, membranes assembled from poly(ethylene oxide)-block-poly(ethylethylene) diblock copolymers of comparable molecular weight yielded a mean lamellar thickness of about 8 nm.5 Each triblock copolymer molecule carries polymerizable groups at both chain ends. Hence, it is possible to achieve a cross-linking polymerization of the individual triblock copolymer molecules by UV irradiation of the membrane. In the course of the free radical polymerization of the methacrylate end groups, each triblock copolymer molecule is covalently attached to its neighbors. Recently, it was observed that vesicles formed from PMOXA-PDMSPMOXA triblock copolymers contracted upon polymerization.3 This had been attributed to a closer packing of the triblock copolymer molecules within the membrane due to the new covalent bonds between them. Hence, the polymerization process must induce a stretching of the individual triblock copolymer molecules which where initially in a more coil-like conformation, i.e., an increase of the hydrophobic thickness of the membrane. However, as the capacitance of the polymerized membranes is identical to the one of their nonpolymerized precursors within the experimental error, the effect seems to be too small to be resolved by our technique (see Table 1). Since the bond formation occurs at the extremities of the long hydrophilic parts the effect should depend, however, on the length of the PMOXA hydrophilic blocks. Systematic studies on this topic will be performed in the future. Because of the finite contact angle at the edge of the Teflon rim, the interface is submitted to a surface tension that will favor opening of pores in the membrane. However, to create a pore requires the formation of an edge. This will provide an energy barrier. Because of the longer hydrophobic part in comparison to lipids, the cohesion energy between the polymerizable triblock copolymer molecules is expected to be higher than the one between conventional low molecular weight lipids. To quantify this effect, a set of experiments was devoted to characterize the stability of the triblock copolymer membranes. Figure 4 shows typical time courses of the transmembrane voltage after short rectangular charge pulses of 10 µs duration. It has to be emphasized that the curves were obtained from single experiments performed on defect-free membranes. The transmembrane voltage shows an exponential decay caused by the discharge through the 10 MΩ resistor of the passive oscilloscope probe. This relaxation is followed by a second decay which is faster than an exponential indicating the onset of the irreversible rupture of the membrane. A transmembrane electric field lowers the energy barrier for pore formation13,18
γ ) γ0 - 0wU2/2π
(5)
with γ0 the edge energy in absence of an external electric field and w the relative dielectric constant of the aqueous phase. The critical voltage, Vc, leading to rupture, defined as the magnitude of the voltage when the voltage decay (17) Nardin, C.; Thoeni, S.; Widmer, J.; Winterhalter, M.; Meier, W. Chem. Commun., in press. (18) Lindemann, M.; Steinmetz, M.; Winterhalter, M. Prog. Colloid Polym. Sci. 1997, 105, 209.
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starts to follow the RC behavior, is related to the energy required for defect formation and to the cohesive energy within the membrane. Consequently, it reflects directly the mechanical stability of the free-standing films.19 For nonpolymerized triblock copolymer membranes the breakdown voltage is unusually high, i.e., 1.0 ( 0.2 V (see Table 1), compared to 0.5 V for conventional black lipid membranes.11 That means that the energy barrier for pore formation is higher by a factor of 4. It has to be expected that the polymerization of the reactive triblock copolymer molecules within these films enforces considerably the mechanical stability of the membranes originally formed via noncovalent interactions and reduces the mobility of the individual triblock copolymer molecules. Indeed, the critical voltage increased after polymerization to 1.5 ( 0.2 V (see Table 1); i.e., the energy barrier for pore formation is now more than 9 times higher than that of a conventional lipid membrane.11 A further parameter revealed by the applied technique is the rupture velocity, da/dt, obtained via eq 3. Assuming the formation of a single pore of circular shape, the analysis of the conductance curves (Figure 5) yields fast rupture velocities of about 1.4 ( 0.8 ms-1 (Table 1) for the nonpolymerized membranes. This value is an order of magnitude higher than that observed in lipid membranes.11 However, the triblock copolymer films are considerably thicker than conventional low molecular weight lipid membranes. A similar increase of the defect growth velocity with increasing thickness of the film has been observed for Newton black films of comparable thickness.20 This behavior has been attributed to the elasticity of the films, which can be expected to play also a significant role in our polymer membranes. After the initial defect is formed, the pore area starts to increase. Because of the fast relaxation of the membrane potential, the contribution of the electrical field is negligible, and the kinetics is determined by the material properties of the membrane only.11,13 The widening of the pore is driven by the finite surface tension and controlled by the inertia of the film. Interestingly, the velocity of pore growth for the polymerized triblock copolymer membranes has been found to be 3 ( 1 ms-1 (Table 1), i.e., a factor of 3 higher than for their nonpolymerized counterparts. This may be a result of the cross-linking polymerization that has probably an additional effect on elastic properties of the membrane and on the dynamics of the triblock copolymer molecules. Depending on the length of the hydrophilic PMOXA parts, it has to be expected that the mobility of (19) Winterhalter, M.; Klotz, K.-H.; Benz, R. High Electric Field Effects on Artificial Lipid Bilayer Membranes. In Electromanipulation of Cells; Zimmermann, U., Neil, G. A., Eds.; CRC Press: Boca Raton, FL, 1995; p 137. (20) Evers, L. J.; Shulepov, S. Y.; Frens, G. Faraday Discuss. 1996, 104, 335, 209.
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the triblock copolymer molecules slows down because of their binding to each other. An additional information is the delay time, ∆t, between the pulse and the beginning of the membrane rupture. This delay time may reflect the ability of the macromolecules to rearrange prior to the start of the pore widening. The delay time of 50 ( 45 µs (Table 1) for the triblock copolymer membranes was comparable to those found in lipid membranes.11 After polymerization the delay time appeared slightly less with 40 ( 20 µs (Table 1). This would reflect a lower ability of the macromolecules to self-heal a defect as a result of their covalent binding to each other. However, this apparent trend has to be regarded cautiously because of the considerable scattering of the experimental values. Although the capacitance measurements indicate that single, defect-free triblock copolymer membranes are formed in a reproducible manner, there may be local (e.g., conformational) disorder in these membranes responsible for the wide range of observed delay times. This will be clarified in the near future. Conclusion For the first time free-standing films of large size were achieved from amphiphilic copolymers. We adapted the preparation procedure for conventional low molecular weight lipid membranes to an ABA triblock copolymer composed of a hydrophobic middle block B and hydrophilic side blocks A. The PMOXA-PDMS-PMOXA copolymer molecules used in this study yield stable giant membranes with a mean hydrophobic thickness of 10 nm and a surface area up to 1 mm2. Furthermore, these triblock copolymer molecules carry polymerizable methacrylate groups at both chain ends. Hence, similar to an analogous polymerization of this PMOXA-PDMS-PMOXA copolymer molecules in vesicular structures,4 the free radical polymerization within the planar triblock copolymer membranes leads to a considerable mechanical stabilization. After the cross-linking polymerization, the individual triblock copolymer molecules are covalently linked together. Because of this linkage, the edge energy of the macromolecules in the membrane is considerably enhanced. We expect such polymerized triblock copolymer membranes to possess great potential for applications as a matrix for biosensors because of their enormous mechanical stability. Acknowledgment. We thank Dr. P. van Gelder and Dr. F. Dumas for bright and helpful discussions and T. Haefele for his contribution to the experimental part. The Swiss National Science Foundation is gratefully acknowledged for financial support. LA000204T
