Mixed thermoreversible gels were successfully fabricated by the addition of a thermosensitive polymer, poly­(N-isopropylacrylamide) (PNIPAM), to fibrillar nanostructures self-assembled from a short peptide I3K. When the temperature was increased above the lower critical solution temperature of the PNIPAM, the molecules collapsed to form condensed globular particles, which acted as cross-links to connect different peptide nanofibrils and freeze their movements, resulting in the formation of a hydrogel. Since these processes were physically driven, such hydrogels could be reversibly switched between the sol and gel states as a function of temperature. As a model peptide, I3K was formulated with PNIPAM to produce a thermoreversible sol–gel system with a transition temperature of ∼33 °C, which is just below the body temperature. The antibacterial peptide of G­(IIKK)3I-NH2 could be conveniently encapsulated in the hydrogel by the addition of the solution at lower temperatures in the sol phase and then increasing the temperature to be above 33 °C for gelation. The hydrogel gave a sustained and controlled linear release of G­(IIKK)3I-NH2 over time. Using the peptide nanofibrils as three-dimensional scaffolds, such thermoresponsive hydrogels mimic the extracellular matrix and could potentially be used as injectable hydrogels for minimally invasive drug delivery or tissue engineering.

Hydrogels offer great potential for many biomedical and technological applications. For clinical uses, hydrogels that act as scaffold materials for cell culture, regenerative medicine, and drug delivery are required to have bactericidal properties. The amphiphilic peptide A9K2 was designed to effectively inhibit bacterial growth via a mechanism of membrane permeabilization. The present study demonstrated that addition of fetal bovine serum (FBS) or plasma amine oxidase (PAO) induced a sol-gel transition in A9K2 aqueous solutions. The transformation of A9K2 molecules catalyzed by lysyl oxidase (LO) in FBS or PAO accounted for the hydrogelation. Importantly, the enzymatic A9K2 hydrogel displayed high antibacterial ability against both Gram-negative and Gram-positive bacterial strains while showing extremely low mammalian cell cytotoxicity, thus demonstrating good biocompatibility. Under established coculture conditions, the peptide hydrogel showed excellent selectivity by favoring the adherence and spreading of mammalian cells, while killing pathogenic bacteria, thus avoiding bacterial contamination. These advantages endow the enzymatic A9K2 hydrogel with great potential for biomedical applications.

Self-assembling peptides have the ability to spontaneously aggregate into large ordered structures. The reversibility of the peptide hydrogen bonded supramolecular assembly make them tunable to a host of different applications, although it leaves them highly dynamic and prone to disassembly at the low concentration needed for biological applications. Here we demonstrate that a secondary hydrophobic interaction, near the peptide core, can stabilise the highly dynamic peptide bonds, without losing the vital solubility of the systems in aqueous conditions. This hierarchical self-assembly process can be used to stabilise a range of different β-sheet hydrogen bonded architectures.

Peptide self-assembly is a hierarchical process, often starting with the formation of α-helices, β-sheets or β-hairpins. However, how the secondary structures undergo further assembly to form higher-order architectures remains largely unexplored. The polar zipper originally proposed by Perutz is formed between neighboring β-strands of poly-glutamine via their side-chain hydrogen bonding and helps to stabilize the sheet. By rational design of short amphiphilic peptides and their self-assembly, here we demonstrate the formation of polar zippers between neighboring β-sheets rather than between β-strands within a sheet, which in turn intermesh the β-sheets into wide and flat ribbons. Such a super-secondary structural template based on well-defined hydrogen bonds could offer an agile route for the construction of distinctive nanostructures and nanomaterials beyond β-sheets.

Super-resolution fluorescence microscopy, specifically stochastic reconstruction microscopy (STORM), and atomic force microscopy (AFM) were used to image the self-assembly processes of the peptide surfactant IK. The peptide surfactants self-assembled into giant helical fibrils with diameters between 5 and 10 nm with significant helical twisting. The resolution of the STORM images was 30 nm, calculated using the Fourier ring correlation method. STORM compares favorably with AFM for the calculation of contour lengths (∼6 μm) and persistence lengths (10.1 ± 1.2 μm) due to its increased field of view (50 μm), and its ability to image bulk morphologies away from surfaces under ambient solution conditions. Two-color STORM experiments were performed to investigate the dynamic process of self-assembly after mixing of two separately labeled samples, and the results revealed the formation of long nanofibers via end-to-end connections of short ones. No evidence was found for significant monomer exchange between the samples, and the self-assembled structures were very stable and long-lived.

The self-assembly of short peptides is a promising route to the creation of smart biomaterials. To combine peptide self-assembly with enzymatic catalysis, we design an amphiphilic short peptide I3QGK that can self-assemble into long nanoribbons in aqueous solution. Upon addition of transglutaminase (TGase), the peptide solution undergoes a distinct sol-gel transition to form a rigid hydrogel, which shows strong shear-thinning and immediate recovery properties. Transmission electron microscopy (TEM) and atomic force microscopy (AFM) measurements indicate the occurrence of considerable nanofibers in addition to the original nanoribbons. Liquid chromatography and mass spectrometry analyses reveal the enzymatic formation of peptide dimers from monomers through intermolecular ε-(γ-glutamyl)lysine isopeptide bonding. The dimers rapidly self-assemble into flexible and entangled nanofibers, and the coexistence of the original nanoribbons and the newly created nanofibers is responsible for hydrogelation. Factor XIII in blood is converted by thrombin to an active TGase (Factor XIIIa) during bleeding, so the peptide solution shows a more rapid and effective hemostasis via a combination of gelling blood and promoting platelet adhesion, relative to other hemostasis methods or materials. These features of I3QGK, together with its low cytotoxicity against normal mammalian cells and noninduction of nonspecific immunogenic responses, endow it with great potential for future clinical hemostasis applications.

Due to their structural simplicity and robust self-assembled nanostructures, short peptides prove to be an ideal system to explore the physical processes of self-assembly, hydrogels, semi-flexible polymers, quenched disorder and reptation. Rational design in peptide sequences facilitates cost-effective manufacturing, but the huge number of possible peptides has imposed obstacles for their characterisation to establish functional connections to the primary, secondary and tertiary structures. This review aims to cover recent advances in the self-assembly of designed short peptides, with a focus on physical driving forces, design rules, characterisation methods and exemplar applications. Super-resolution microscopy in combination with modern image analysis have been applied to quantify the structure and dynamics of peptide hydrogels whilst SANS and ssNMR continue to provide valuable information on structures over complementary lengths. Short peptides are attractive in biomedicine and nanotechnology, e.g., as antimicrobials, anticancer agents, vehicles for controlled drug release, peptide bioelectronics and responsive cell culture materials.

Mixed thermoreversible gels were successfully fabricated by the addition of a thermosensitive polymer, poly(N-isopropylacrylamide) (PNIPAM), to fibrillar nanostructures self-assembled from a short peptide I 3 K. When the temperature was increased above the lower critical solution temperature (LCST) of the PNIPAM, the molecules collapsed to form condensed globular particles, which acted as cross-links to connect different peptide nanofibrils and freeze their movements, resulting in the formation of a hydrogel. Since these processes were physically driven, such hydrogels could be reversibly switched between the sol and gel state as a function of temperature. As a model peptide, I 3 K was formulated with PNIPAM to produce a thermoreversible sol-gel system with a transition temperature of ~33 °C, which is just below the body temperature. The antibacterial peptide of G(IIKK) 3 I-NH 2 could be conveniently encapsulated in the hydrogel by addition of the solution at lower temperatures in the sol phase and then increasing the temperature to be above 33 °C for gelation. The hydrogel gave a sustained and controlled linear release of G(IIKK) 3 I-NH 2 over time. Using the peptide nanofibrils as 3D scaffolds, such thermoresponsive hydrogels mimic the extracellular matrix and could potentially be used as injectable hydrogels for minimally invasive drug delivery or tissue engineering.