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pH Responsiveness of Block Copolymer Vesicles with a Polypeptide Corona Reinhard Sigel, Magdalena Łosik, and Helmut Schlaad* Max Planck Institute of Colloids and Interfaces, Colloid Chemistry, Research Campus Golm, 14424 Potsdam, Germany ReceiVed February 13, 2007. In Final Form: April 5, 2007 The aggregation behavior of polybutadiene165-block-poly(L-lysine)88 in saline solution was studied by combined static and dynamic light scattering analyses. Vesicles were observed if the polypeptide segment was in a 100% coil conformation (pH 7.0) or in an 80% R-helical conformation (pH 10.3). At the higher pH, aggregates were smaller in size (hydrodynamic radius: 364 nm f 215 nm) and chains were more densely packed at the core-corona interface (interchain distance: 3.2 nm f 2.4 nm). Changes in size and structure could be explained in basic terms of colloid stabilization without considering a secondary structure effect.
Introduction Water-soluble amphiphilic block copolymers have gained much attention in recent years because of their ability to generate highly organized, self-assembled structures in biologically relevant environments.1,2 Particularly interesting are block copolymers comprising polypeptide or protein segments, so-called “molecular chimeras”,3 because of their exceptionally rich phase behavior, including the formation of stimuli-responsive and hierarchical structures,4 and their potential use in biomedical applications.5 Although polypeptide-based block copolymers have been available since the 1970s,6 not much work has been devoted to their aggregation behavior in dilute aqueous solution. Just very recently, Schlaad et al.7 and Lecommandoux et al.8,9 described the self-assembly of anionic 1,2-polybutadiene-block-poly(Lglutamate) (PB-b-PLGlu) in dilute saline solution. The micellar and unilamellar vesicular aggregates observed consisted of a soft hydrophobic PB core (glass transition temperature, Tg ≈ -10 °C) and a hydrophilic PLGlu corona, the secondary structure of which (random coil or R-helix) could be triggered by the pH of the solution in the presence of salt. It was found that a change in the conformation of PLGlu from coil to helix did not have a serious impact on the shape or morphology of aggregates, but might have one on their size (-20%).9 The main parameter determining the shape appeared to be the chemical composition of the copolymer (fLGlu: mole fraction of LGlu); small spherical micelles (hydrodynamic radius, Rh ≈ 10 nm) were formed if fLGlu ) 0.70-0.75, and vesicles (Rh > 50 nm) were formed if fLGlu ) 0.17-0.54. Cylindrical micelles have not yet been observed. Spherical and nonspherical micelles have been observed for poly(L-lysine) (PLLys)-based block copolymers with fLLys > * Corresponding author. E-mail:
[email protected]. Phone: ++49.(0)331.567.9514, Fax: ++49.(0)331.567.9502. (1) Discher, D. E.; Eisenberg, A. Science 2002, 297, 967-973. (2) Fo¨rster, S.; Konrad, M. J. Mater. Chem. 2003, 13, 2671-2688. (3) Schlaad, H.; Antonietti, M. Eur. Phys. J. E 2003, 10, 17-23. (4) Schlaad, H. AdV. Polym. Sci. 2006, 202, 53-73. (5) Osada, K.; Kataoka, K. AdV. Polym. Sci. 2006, 202, 113-153. (6) Perly, B.; Douy, A.; Gallot, B. C. R. Acad. Sci., Ser. C 1974, 279, 11091111. (7) Kukula, H.; Schlaad, H.; Antonietti, M.; Fo¨rster, S. J. Am. Chem. Soc. 2002, 124, 1658-1663. (8) Che´cot, F.; Lecommandoux, S.; Gnanou, Y.; Klok, H.-A. Angew. Chem., Int. Ed. 2002, 41, 1340-1343. (9) Che´cot, F.; Bruˆlet, A.; Oberdisse, J.; Gnanou, Y.; Mondain-Monval, O.; Lecommandoux, S. Langmuir 2005, 21, 4308-4315.
0.4.10-12 However, data are not conclusive to allow for mapping in a “phase diagram”. For spherical micelles of 3,4-polyisopreneblock-poly(L-lysine), Lecommandoux et al.10 recognized a decrease in the size by 50% upon switching the PLLys segment from a coiled to a helical conformation. This effect has been attributed to the differences in contour length (lc) between a stretched chain and an R-helix. Whether or not the aggregation number () number of polymer chains building an aggregate) and/or the packing density of chains were subject to change has not been elucidated in detail. As claimed by Lecommandoux et al.,9 the aggregation number should remain constant when switching the conformation of the polypeptide. In the present work, we report on a detailed static and dynamic light scattering (SLS and DLS) study of the pH-dependent behavior of PB165-b-PLLys88 (fLLys ) 0.35, see the structure in Figure 1) vesicles in dilute saline solution (0.15 wt % NaCl). The major task is to address the role of the secondary structure for the aggregation behavior of polypeptide-based systems. Experimental Section Materials. All chemicals were purchased from Aldrich-Fluka with the highest purity grade available and, unless otherwise noted, used as received. (Z)-L-lysine-N-carboxyanhydride (ZLLys-NCA) was prepared by refluxing a solution of ZLLys and triphosgene (0.36 equiv) in ethyl acetate in the presence of R-pinene as an HCl trap.13 Primary amine-functionalized polybutadiene (PB165-NH2, microstructure: 93% 1,2, 7% 1,4-trans; polydispersity index, PDI ) 1.09) was synthesized via anionic polymerization as reported elsewhere.14 Polymer Synthesis and Characterization. PB165-b-PZLLys88 was synthesized by a conventional ring-opening polymerization of ZLLys-NCA using PB165-NH2 as the macroinitiator.15 The reaction was performed in a CHCl3/DMF (1:1 v/v) mixed solvent at 60 °C for 3 days. The product was precipitated in petrolether (thus removing any traces of PB homopolymer), filtered, and dried at 35 °C in a (10) Babin, J.; Rodrı´guez-Herna´ndez, J.; Lecommandoux, S.; Klok, H.-A.; Achard, M.-F. Faraday Discuss. 2005, 128, 179-192. (11) Gebhardt, K. E.; Ahn, S.; Venkatachalam, G.; Savin, D. A. Langmuir 2007, 23, 2851-2856. (12) Lu¨bbert, A.; Castelletto, V.; Hamley, I. W.; Nuhn, H.; Scholl, M.; Bourdillon, L.; Wandrey, C.; Klok, H.-A. Langmuir 2005, 21, 6582-6589. (13) ISOCHEM. French Patent EP 1 201 659 A1, 2002. (14) Kukula, H.; Schlaad, H.; Falkenhagen, J.; Kru¨ger, R.-P. Macromolecules 2002, 35, 7157-7160. (15) Dimitrov, I.; Kukula, H.; Co¨lfen, H.; Schlaad, H. Macromol. Symp. 2004, 215, 383-393.
10.1021/la7004248 CCC: $37.00 © 2007 American Chemical Society Published on Web 05/22/2007
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Figure 1. Chemical structure of the polymer investigated: PB165b-PLLys88. vacuum. The chemical structure of the copolymer was confirmed by 1H NMR and Fourier transform infrared (FT-IR) spectroscopy. According to size exclusion chromatography, the sample exhibited a monomodal molecular weight distribution with PDI ∼ 1.5 (see Supporting Information). The protecting groups were removed by stirring the sample with a solution of 30 wt % HBr in glacial acid for 1 h at room temperature. The mixture was then diluted with water, neutralized with NaOH, dialyzed against water, acidified with HCl, once again dialyzed against water, and freeze-dried. FT-IR analysis indicated that the product PB165-b-PLLys88 (hydrochloride) contained no residual protecting groups (see Supporting Information). Sample Preparation. Dissolution of the block copolymer in a 0.15 wt % aqueous NaCl solution occurred during several days with the aid of ultrasonication. The opalescent solutions containing 0.047 wt % polymer were titrated against 0.1 N NaOH employing a standard pH electrode and were passed through 5 µm filters (SchleicherSchu¨ll). Solutions of lower polymer content were diluted from this stock solution with water adjusted to the same pH value. For transmission electron microscopy (TEM) analysis, a solution of 0.2 wt % of the polymer in water was prepared. Analytical Instrumentation. Circular dichroism (CD) analyses were done at room temperature with a JASCO J 715 spectrometer employing quartz cells with a 1 mm optical path length. TEM images were taken with a Zeiss Omega 912 electron microscope operating at an acceleration voltage of 120 kV. Freeze-fractured specimens were prepared with a Balzers BAF 400. DLS/SLS experiments were carried out at 25 °C with a helium-neon laser light source (λ0 ) 633 nm; intensity: 34 mW; Polytec PL3000), an ALV goniometer, and an ALV-5000 multiple-tau digital correlator (ALV GmbH, Langen, Germany). Measurements were performed at scattering angles from 20° to 150°. Refractive index increments were measured using an NFT-Scanref differential refractometer operating at λ ) 633 nm.
Figure 2. (a) CD spectra of PB165-b-PLLys88 in saline solution at pH 7.0 (dashed line) and pH 10.3 (solid line). Location of the isodichroistic point at λ ) 204 nm indicates that the PLLys segment contains only R-helix and random coil; percentages can be estimated on the basis of calculated CD spectra (Greenfield and Fasman).16 (b) TEM image of a freeze-fractured specimen of PB165-b-PLLys88 aggregates (0.2 wt %) in water at neutral pH.
Results and Discussion CD spectroscopy (Figure 2a) indicated that the peptide segment of PB165-b-PLLys88 adopts a coiled conformation at pH 7.0 and a partially disordered R-helical conformation (∼80% R-helix and 20% random coil) at pH 10.3.16 Considering that PLLys has an average pKa of 10,17 the degrees of protonation of LLys units should be close to 100% and about 35%, respectively. Accordingly, the block copolymer becomes less hydrophilic the higher the pH of the solution, and precipitation occurs above pH 10.5. Stable colloidal solutions of aggregates with a purely R-helical polypeptide corona could therefore not be prepared. Freezefracture TEM reveals that, in aqueous solution, there exist large spherical objects, most likely vesicles, that are more than 200 nm in diameter (Figure 2b). SLS/DLS experiments were performed on dilute saline solutions containing 0.011-0.047 wt % PB165-b-PLLys88 (four different concentrations); experimental static and dynamic Zimm plots are provided in the Supporting Information. In the following, we focus on the values obtained by extrapolation to concentration c ) 0, where effects of particle interactions vanish. SLS data are displayed in Figure 3a as a Kratky plot, in which q is the modulus (16) Greenfield, N.; Fasman, G. D. Biochemistry 1969, 8, 4108-4116. (17) Harada, A.; Cammas, S.; Kataoka, K. Macromolecules 1996, 29, 61836188.
Figure 3. (a) Kratky plot of SLS data and (b) apparent diffusion coefficients determined by DLS for saline solutions of PB165-bPLLys88 at pH 7.0 (open spheres) and pH 10.3 (solid spheres); data are extrapolated values to c ) 0. Lines are simultaneous fits of DLS and SLS data to a model of polydisperse vesicles (see text for details).
of the scattering vector, RΘ is the absolute scattering intensity (called the Rayleigh ratio, obtained with toluene as a reference and its known scattering power),18 and K ∼ (dn/dc)2 is a contrast (18) Bender, T. M.; Lewis, R. J.; Pecora, R. Macromolecules 1986, 244-245.
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Table 1. Results of the Simultaneous Fit of SLS and DLS Data of Saline Solutions of PB165-b-PLLys88 at pH 7.0 and pH 10.3 Fd/(g mol-1 nm-2) R0/nm σ C
pH 7.0
pH 10.3
3970 364 0.9 0.004
6950 215 1.0 0.005
parameter.19 The refractive index increment of the polymer solutions at pH 7.0 and pH 10.3 was found to be same, that is, dn/dc ) 0.140 mL/g. The plot reaches a plateau at large q values, indicating the presence of particles with a fractal dimension of D ) 2; such particles could be vesicles or Gaussian coils (e.g., wormlike micelles).20 It should be emphasized that the experimental window covers the Porod regime where qRg . 1 (Rg: average radius of gyration of particles). Data at much lower values of q (Guinier regime) would be needed for a reliable determination of Rg and the molecular weight of particles. For the characterization of aggregates, we performed a simultaneous fit of SLS data and the apparent diffusion coefficient Dapp measured by DLS (Figure 3b). As suggested by TEM (Figure 2b), an appropriate fitting model would be that of a vesicle. The pronounced minima of the form factor of a vesicle were smeared out through the implementation of a size distribution. We chose a log-normal distribution with a maximum at a radius R0 and a width σ. The shell thickness d of the vesicles has only a very small impact on the fitting function; it was kept fixed at d ) 30 nm. An important parameter is the mass per unit area Fd, which reflects the height of the plateau in the Kratky plot. Since plateau values are well established, as seen in Figure 3a, the determination of Fd is reliable and independent of the other details of the applied model. The fitting function for the apparent diffusion constant was calculated from an intensity-weighted superposition of the initial slopes for particles with the assumed size distribution. A significant improvement of the fit of Dapp at large q was achieved by allowing for a q-dependent apparent diffusion constant Dapp(R) ) D0(R)(1 + Cq2R2) for the fraction of particles with the radius of gyration R. This functional form is common for soft particles, where C characterizes the contribution of shape fluctuations to the relaxation of the correlation function measured in DLS.21 Typical values are C ) 0 for hard spheres and 0.2 for Gaussian chains with a PDI of 2. In fact, it is the relatively weak q-dependency of Dapp data that excludes a consistent evaluation of data with the model of Gaussian chains. In contrast, the fitting lines included in Figure 3b show that the data are compatible with the assumed model of polydisperse unilamellar vesicles. The results of the fit are listed in Table 1. Although the extrapolations to q ) 0 in Figure 3b should not be overinterpreted, the difference in size R0 seems significant, since it reflects the difference in magnitude of the measured values of Dapp. Within experimental uncertainty, the particles show a similar distribution width σ and similar softness C. The most striking and most reliable distinction is the difference in area mass density Fd. Increasing pH and changing the conformation of the polypeptide coronal chains from coil to helix causes a considerable shrinkage of the aggregates by ∼40% and an increase of the area mass density or packing density by 75%. The packing density of chains and the size of aggregates are connected with each (19) Schmidt, M. In Dynamic Light Scattering; Brown, W., Ed.; Clarendon: Oxford, 1993. (20) Higgins, J. S.; Benoit, H. C. Polymers and Neutron Scattering; Clarendon Press: Oxford, 1994. (21) Burchard, W.; Schmidt, M.; Stockmayer, W. H. Macromolecules 1980, 13, 1265-1273.
Figure 4. Tentative structures of the bilayered membrane of PB165b-PLLys88 vesicles at different pH; b denotes the average distance of chains at the core-corona interface.
other; for instance, doubling the packing density of chains while keeping the aggregation number constant (see above)9 affords a decrease in the vesicle size by a factor of 2-0.5 ≈ 0.71. The large change in size observed here (∆R ∼ 150 nm) cannot be explained in terms of different contour lengths of polypeptide helices and all-trans chains, as proposed by Klok and Lecommandoux.10 A detailed interpretation of data is somewhat complicated by the fact that two parameters are subject to change at the same time, namely, the secondary structure and the degree of ionization of PLLys chains, and both contribute to the packing density of chains (see Klok and Lecommandoux et al.10). Screening of repulsive electrostatic interactions by the addition of salt can lead to a decrease in the average distance between polymer chains at the core-corona interface by ∼20% (b: 2.2 nm f 1.8 nm), as observed for poly(2-ethylethylene)-poly(styrenesulfonic acid) block copolymer micelles by Fo¨rster et al.22 For the unilamellar vesicles with a bilayered membrane made of PB165-b-PLLys88 (‚HCl) at pH 7.0 (see Figure 4), it is b ) (2Mn/Fd)0.5 ∼ 3.4 nm, with Mn being the molecular weight of polymer chains. The value of b drops to about 2.4 nm at pH 10.3 (-33%), and, in the same course, the core-forming PB segments must get more stretched. The interchain distance is somewhat larger than the diameter of a polypeptide helix () 1.5 nm),23 which can be explained by the existence of repulsive forces and/or back-folding of helices; the latter seems unlikely because polylysine helices in the absence of solvent are not folded, but rather fully stretched.24 Repulsive forces can be due to electrostatics (∼35% of LLys units are charged) and local dipole-dipole interactions between helices that are oriented in the same direction, that is, perpendicular to the core-corona interface, as illustrated in Figure 4. The energy between two PLLys88 helices with a dipole moment of |µ b| ) 3.5 D per monomer unit25 is equal to the thermal energy at a distance of 3 nm.26 The experimental “equilibrium distance” is shorter because PLLys88 helices are partially disordered (see above). In addition, the dipole moments are screened by a suitable arrangement of ions in solution. (22) Fo¨rster, S.; Hermsdorf, N.; Bo¨ttcher, C.; Lindner, P. Macromolecules 2002, 35, 4096-4105. (23) Stryer, L. Biochemistry; W. H. Freeman & Company: New York, 1988. (24) Losik, M.; Kubowicz, S.; Smarsly, B.; Schlaad, H. Eur. Phys. J. E 2004, 15, 407-411. (25) Hol, W. G. J.; Vanduijnen, P. T.; Berendsen, H. J. C. Nature 1978, 273, 443-446. (26) Israelachvili, J. N. Intermolecular and Surface Forces, 2nd ed.; Academic Press: Amsterdam, 2002.
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The pH-induced changes of the vesicle size and structure can thus be well explained in basic terms of colloid stabilization.26 A major impact of the polypeptide secondary structure on the aggregation behavior cannot be recognized. Nevertheless, the observed change in curvature or packing parameter may induce a transition in the morphology of aggregates. But such a transition may only happen in close proximity to a boundary in the “phase diagram”. It is worth mentioning that Savin et al.11 found a rodsphere transition for PB-PLLys block copolymer assemblies (fLLys ) 0.45-0.48) at about pH 4. The origin of this transition, which cannot be correlated to a helix-coil transition, is not yet known.
Conclusion The vesicles formed by PB165-b-PLLys88 in aqueous solution at pH 7.0 (PLLys in 100% coil conformation) and pH 10.3 (PLLys in 80% R-helical conformation) were characterized by combined SLS and DLS analyses. Aggregates at higher pH were considerably smaller, and chains were more densely packed at the corecorona interface, which could be explained in basic terms of
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colloid stabilization. Any direct impact of the secondary structure on vesicle size and structure is not evident. Finally, it is worth mentioning that this mechanism may be interesting for drug delivery purposes because packing density should correlate with the permeability of the membrane and thus pharmacokinetics. Acknowledgment. Markus Antonietti, Ines Below, Olaf Niemeyer, Marlies Gra¨wert, Andreas Erbe, Birgit Schonert, Erich C., and Brigitte Tiersch (U Potsdam) are thanked for their contributions to this work. Financial support was given by the Max Planck Society and the German Research Foundation (Sfb 448: “Mesoscopically organized composites”). The Hermann Schnell Foundation is also gratefully acknowledged. Supporting Information Available: Spectroscopic and chromatographic data for the polymer and light scattering data (SLS and DLS) data of aqueous solutions of PB185-b-PLLys88. This information is available free of charge via the Internet at http://pubs.acs.org. LA7004248
