Thermally and photochemically triggered self-assembly of peptide hydrogels.

Self-assembling systems present attractive platforms for engineering stimulus-responsive materials with controlled nanoand microstructures. Recent efforts to design such systems include synthetic block copolymers,1a proteins that self-assemble into hydrogels via leucine zipper motifs,1b cyclic peptides that selfassemble into nanotubes,1c-e and peptides that form fibrillar â-sheet networks.1f-i Here we demonstrate systems that selfassemble from fluid precursors into highly cross-linked peptide hydrogels in response to thermal or photochemical triggering. Our system takes advantage of stimuli-responsive release of encapsulated salts from liposomes to trigger the self-assembly of a 16amino acid peptide. We show that the induction of self-assembly by gentle warming or by exposure to near-infrared light results in rapid gelation of peptide/liposome suspensions into highly cross-linked, fibrillar, â-sheet hydrogels. The ability to trigger rapid sol-gel transformations of peptide solutions via physiologically benign stimuli (temperature, light) may lead to the development of new injectable materials for drug delivery, wound healing, and tissue engineering applications. The 16-amino acid peptide consists of alternating hydrophobic and hydrophilic residues: H2N-(FEFEFKFK)2-COOH, (FEK16). We chose to investigate FEK16 because several related peptides with alternating hydrophobic-hydrophilic residues are known to self-assemble into â-sheet structures, often in an ionic-strengthdependent manner.1f,2 â-sheet structure is favored by the strictly alternating hydrophobic-hydrophilic primary structure of the peptide, which positions all hydrophobic side chains on one side of the â-sheet and all hydrophilic side chains on the other.3 Saltinduced self-assembly of this peptide may be driven by the shielding of electrostatic repulsive forces with increasing ion concentrations, allowing attractive hydrophobic and van der Waals forces to dominate.2d The self-assembly characteristics of FEK16 were found to resemble those of other peptides with similar alternating structures.1f,1i,2b FEK16 was found to be highly soluble in pure H2O but formed self-assembled aggregates (gels at FEK16 concentrations >10 mg/mL) in the presence of mM concentrations of NaCl, KCl, and CaCl2. FTIR spectroscopy of FEK16 (30 mg/ mL) in D2O and CaCl2/D2O revealed strong amide I absorption peaks at 1622 and 1694 cm-1 and an amide II peak at 1525 cm-1 (see Supporting Information for IR spectra), indicating a high content of â-sheet structure.4a-b A weak band at 1694 cm-1 suggested that the â-sheet was antiparallel, although it is also possible for this peak to arise from â-turn or disordered structures.4b More detailed structural analysis such as solid-state NMR spectroscopy will be necessary to confirm the antiparallel conformation.4c The IR spectra of self-assembled FEK16 induced by CaCl2 addition was nearly identical to that of FEK16 in pure D2O, suggesting that FEK16 exists predominantly as small â-sheet multimers in the liquid state and that gel formation involves coalescence/aggregation of these preformed â-sheet domains. CD was utilized to assess the concentration dependence of â-sheet formation in H2O. It was confirmed that FEK16 adopted a â-sheet conformation at concentrations above 10 μM, whereas below this concentration the peptide adopted an R-helical conformation. At low peptide concentrations (<10 μM), addition of CaCl2 induced an R-helix-to-â-sheet transition in FEK16. (See Supporting Information for CD spectra). When concentrated aqueous solutions of FEK16 (40 mg/mL) were extruded from a micropipet into Dulbecco’s phosphate buffered saline (PBS) (Gibco), aqueous NaCl (200 mM) or aqueous CaCl2 (7.5-560 mM), hydrogels were formed within a few minutes. These assemblies maintained the shape of the extruded filament and were strongly stained by Congo red, an indicator of aggregated â-sheet structure.5 Furthermore, once assembled, these structures did not disassemble upon the addition of excess buffer or upon the exchange of buffer with water. In contrast to its behavior in salt solutions, FEK16 did not form hydrogels or stain with Congo red in water or aqueous sucrose. Triggered assembly of peptide gels was accomplished by utilizing stimuli-responsive liposomes designed to release salts such as CaCl2 at a specific temperature6 or in response to nearinfrared (NIR) light exposure.7 CaCl2-containing temperaturesensitive liposomes were prepared by the interdigitation-fusion technique8 from 9:1 dimyristoylphosphatidylcholine (DMPC): dipalmitoylphosphatidylcholine (DPPC) as described.6 Lightsensitive liposomes were prepared from 6:4 distearoylphosphatidylcholine (DSPC):diplasmenylcholine (DPPlsCho) containing bacteriochlorophyll as described.7b Both lightand temperaturesensitive liposomes were stable for long periods of time at room temperature. Extravesicular sucrose was used to osmotically balance the CaCl2-containing liposomes. 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