Thermoreversible Gel−Sol Behavior of Rod−Coil−Rod Peptide-Based Triblock Copolymers

A series of peptide-based triblock copolymers cons i s t ing of poly(γ -benzy l -L -g lu tamate) -b -po ly (dimethylsiloxane)-b-poly(γ-benzyl-L-glutamate) [PBLG-bPDMS-b-PBLG] were synthesized using ring-opening polymerization (ROP). The chemical structure and degree of polymerizations were evaluated by H NMR. These triblock copolymers form thermoreversible gels in toluene with critical gel concentration as low as 1.5 wt % and following trends which correlate directly with the secondary structure of the peptidic block. Fourier transform infrared spectroscopy (FTIR) studies indicate that the α-helical content is increased while β-sheets and random coil contents are systematically decreased with increasing volume fraction of the PBLG blocks. The gel−solution transition behavior of the triblock copolymers was examined using modulated dynamic light scattering (MDLS). It was observed that all the gels undergo gel−solution transition around 50 °C and revert back to its original state when cooling down to room temperature. Dye diffusion and diffusing wave spectroscopy (DWS) experiments showed a reduced mobility of both the dye molecules and tracer particles in the gels compared to that in solution state. The rheological studies on the organogels indicate that increasing molecular weight of the PBLG blocks or concentration of the triblock copolymers increase the gel strength considerably. Using transmission electron microscopy (TEM), the morphology of the organogels was shown to be prevalently formed by nanofibrils, with an average thickness in the range of 6−12 nm. ■ INTRODUCTION The self-assembly behavior of rod−coil block copolymers is different from the common coil−coil block copolymers due to the presence of the rigid segment in their architectures and has attracted great attention in recent years. When dissolved in solution, in some rod−coil block copolymer systems, the polymers can form nanostructured gels in solution instead of micelles. Generally, the network formation in polymers can occur through chemical cross-linking between polymer chains or physical interactions through the entanglements or selfassembled supramolecular structures. These physical gels can exhibit reversible sol−gel transitions by changing temperature, pH, ionic strength, or concentration of the solution. Gels are a class of soft materials composed of a confined phase in a continuous three-dimensional network. On the basis of the solvent media, gels are classified in two kinds of systems: hydrogels (in aqueous medium) and organogels (in organic medium). Gels are predominantly composed of liquids; nevertheless, in terms of their mechanical response, they behave like a solid material. Moreover, in peptide-based block copolymers, gels can be further tuned by ionic interactions, coil−coil interactions, or hydrophobic association. In the case of peptide-based hydrogel systems, the gel formation is based on hydrophobic interactions due to the hydrophobicity of some amino acid residues (i.e., valine or leucine), which maintains the α-helix as a supramolecular selfassembled structure. Small peptide-based gels have been shown to undergo into molecular fibril networks and showed thermoreversibility and pH responsiveness. Peptide-based rod−coil diblock copolymers can form hydrogels with various potential applications in biotechnology. Organogels have unique applications, which are not possible for hydrogels, and these organic-based gels are currently an area of active research. The peptide-based block copolymer organogel formation is similar to that of hydrogels, typically through noncovalent interactions such as hydrogen bonding, Received: December 5, 2011 Revised: February 1, 2012 Published: February 15, 2012 Article pubs.acs.org/Macromolecules © 2012 American Chemical Society 1982 dx.doi.org/10.1021/ma2026379 | Macromolecules 2012, 45, 1982−1990 π−π stacking, van der Waals interactions, and solvophobic interactions. Poly(γ-benzyl-L-glutamate) (PBLG) is a synthetic peptide, which forms α-helices at high degree of polymerization by intramolecular hydrogen bonding. This rigid-rod-like structure of PBLG shows thermotropic liquid-crystalline order in bulk and thermoreversible gelation behavior in solution. Indeed, the synthesis and self-assembly of PBLGbased rod−coil block copolymers have been studied extensively due to its biocompatibility for several decades, and the resulting functional materials have several applications in the biomedical and tissue engineering field. The pure PBLG homopolymer form thermoreversible gels in solvents such as toluene or benzyl alcohol passing through a lyotropic liquid-crystalline phase before gelation, and fibrillike network structures were found in pure PBLG homopolymer gels. Recently, PBLG-based rod−coil diblock copolymers have been prepared, and their solutions showed thermoreversible gel formation in toluene. The mechanism for the self-assembly behavior of the PBLG-based rod−coil block copolymers suggests that a wide variety of this kind of systems with novel architectures could generate supramolecular structures in solution. Very recently, the synthesis and gel formation in toluene of PBLG−POSS (polyhedral oligomeric silsesquioxane) diblock copolymers have been reported, where the POSS block protrusion from the ribbons prevented the aggregation of the nanoribbons and allowed the formation of clear gels. Block copolymers of poly(Z-lysine) with different coil blocks showed an increase in the gel strength with increasing the molecular weight of the peptide block. Peptide-based rod−coil−rod triblock copolymers have been synthesized and studied in bulk state, but the thermoreversible gel−sol behavior of such systems has never been explored before. For this purpose, we synthesized and characterized a series of six rod−coil−rod triblock copolymers consisting of PBLG-b-PDMS-b-PBLG, where the degree of polymerization of the peptide-based sequences was systematically modified. The choice for these polymers (PBLG and PDMS) was based on their biocompatibility, the rigidity and self-assembly features of the PBLG block, and the low Tg and high solubility in toluene of the PDMS segment. By controlling the degree of polymerization and the α-helical content of the peptide blocks, we were able to control the self-assembly behavior of PBLG rods and the gel formation of the triblock copolymers into fibril-like networks. ■ EXPERIMENTAL PART Materials. L-Glutamic acid γ-benzyl ester (Fluka, ≥99.0%), triphosgene (Aldrich, 98%), N,N-dimethylformamide, DMF (SigmaAldrich, ≥99.8%, over molecular sieve), dichloromethane, DCM (Acros, 99.99%), methanol (Fluka, 99.8%), and toluene (Fluka, ≥99.7%) were used as received. Ethyl acetate (Sigma-Aldrich, ≥99.9%) and cyclohexane (Sigma-Aldrich, ≥99.9%) were dried and distilled over CaH2 (Fluka, >97.0%) at normal pressure. Poly(dimethylsiloxane) (PDMS), diaminopropyl-terminated, was purchased from ABCR Chemicals, Germany. The (E)-4,4′-bis(hex-5-en1-yloxy)azobenzene dye was synthesized accordingly to the literature. Dried silica microspheres (540 nm diameter, SS03N, Bangs Laboratories) were dispersed in toluene at 10 wt % concentration. Synthesis of (BLG-NCA) Monomer. The BLG-NCA monomer (γ-benzyl L-glutamate N-carboxyanhydride) was synthesized as published in our previous work. Briefly, 15 g (63.2 mmol, 1 equiv) of γ-benzyl L-glutamate and 8.13 g (27.4 mmol, 0.43 equiv) of triphosgene were placed in a 500 mL two-necked round-bottomed flask equipped with a magnetic stirrer, condenser, and nitrogen inlet. The system was purged with nitrogen for 10 min, 250 mL of freshly distilled ethyl acetate over CaH2 was added, and the reaction mixture was brought to 145 °C. After several hours (5−6 h), the reactants were completely soluble, and the reaction was cooled down to room temperature. The monomer was obtained by recrystallization from ethyl acetate/cyclohexane. Yield: 15.3 g (91%). H NMR (360 MHz, CDCl3): δ = 7.10−7.42 (5H, m, Ar), 6.62 (1H, s, NH), 5.04 (2H, s, Ar−CH2), 4.29 (1H, t, α-CH, J = 6.0 Hz), 2.50 (2H, t, γ-CH2, J = 6.6 Hz), 2.16 (1H, m, β-CH), 2.03 (1H, m, β-CH) ppm. Synthesis of PBLG-b-PDMS-b-PBLG Triblock Copolymers. All triblock copolymers were obtained by using the procedure described in what follows. As a representative example, the synthesis of the triblock copolymer P25 (PBLG24-PDMS314-PBLG24) is described. In a 25 mL round-bottomed flask, the BLG-NCA monomer was dissolved in dry DMF under a nitrogen atmosphere. In another 10 mL roundbottomed flask, the diamino-terminated poly(dimethylsiloxane) (H2NPDMS-NH2) macroinitiator was dissolved in dry DMF under a nitrogen atmosphere. The monomer solution was transferred to the polymer solution by syringe. The resulting mixture was stirred at room temperature for 5 days under a nitrogen atmosphere. Afterward, the solvent was removed under vacuum, and the residue was dissolved in DCM. The resulting triblock copolymer was obtained as a white solid after reprecipitation with cold methanol followed by centrifugation (3000 rpm, 0 °C). The supernatant was then removed, and this process was repeated three times. Yield: 85−90%. H NMR (360 MHz, CDCl3): δ = 9.00−7.70 (43H, NH), 7.60−6.90 (241H, Ar), 5.50−4.60 (91, Ar−CH2), 4.50−3.60 (47H, α-CH), 2.85−1.40 (212H, β-CH2 and γ-CH2), 1.00−0.70 (26H, Si−β-CH2), 0.65−0.40 (10H, Si−α-CH2), 0.35−0.00 (1874H, Si−CH3) ppm. Preparation of the Organogels. The gels of PBLG-b-PDMS-bPBLG triblock copolymers were prepared in toluene according to the published procedure. The required amounts of polymer and toluene were placed together in a sample vial. Afterward, the sealed vial was heated until the mixture became homogeneous. The polymer solution was brought to room temperature and maintained at this temperature for 48 h. Gelation was monitoredat firstby the test tube invert method. The lowest gelation concentration was identified as the critical concentration for the gelation (cgel). The gel−sol transition temperature (Tgel) was measured by the procedure reported by Hirst et al. All cgel and Tgel values were measured in triplicates. Techniques and Apparatus. H NMR measurements were carried out at room temperature on a Bruker DPX-360 spectrometer operating at 360 MHz and using CDCl3 as solvent. Fourier transform infrared (FTIR) spectra of solid samples were measured at room temperature using a Bruker Tensor 27 FTIR spectrometer in attenuated total reflection (ATR) mode. For the gel samples, the FTIR measurements were performed on a Varian 640 instrument, placing the sample between two CaF2 windows in transmission mode. Modulated dynamic light scattering (MDLS) measurements were performed using a LS Instruments apparatus with a He−Ne laser beam (632.8 nm). The instrument is equipped with advanced modulated 3D cross-correlation technology, which suppresses the multiple scattering. Samples were prepared in glass cuvettes of 10 mm thickness. The scattered intensity fluctuations were collected at a fixed angle of 90° and averaged from three runs of 600 s each. The measurements were performed on selected samples at different temperature conditions ranging from 25 to 60 °C with 5 °C steps. Temperatures were controlled by a thermostat within an error of 0.1 °C. Diffusing wave spectroscopy (DWS) measurements were carried out in a transmission mode using a commercial DWS apparatus (LS Instruments) with a He−Ne laser beam (λ = 632.8 nm). Samples were prepared and placed in quartz cuvettes with a 2 mm optical path length. The silica tracer nanoparticles (∼20 μL) were added to the polymer solution, and the cuvette temperature was controlled with a Peltier temperature controller (±0.2 °C), keeping the samples 10 min at the required temperature before the measurement started. The observed autocorrelation function was determined with a digital Macromolecules Article dx.doi.org/10.1021/ma2026379 | Macromolecules 2012, 45, 1982−199