Aline F. Miller

Self-assembly and gelation properties of α-helix versus β-sheet forming peptides

We have investigated the self-assembly and gelation properties of a set of four octa-peptides: AEAEAKAK, AEAKAEAK, FEFEFKFK and FEFKFEFK. The phenylalanine based peptides adopt β-sheet conformations in solution and the alanine based peptides form α-helices. No self-assembly in solution was observed for AEAKAEAK but AEAEAKAK was found to self-assemble forming thick, rigid fibres with a diameter of ∼6 nm. These fibres were composed of two fibrils aggregating side by side to form “pearl-necklace” morphologies. No gelation was observed for AEAEAKAK in the concentration range investigated (0 to 100 mg ml−1). In contrast, both phenylalanine based peptides were found to self-assemble in solution and to form hydrogels at an initial concentration of ∼8 mg ml−1. Similar morphologies were observed for both peptides corresponding to a relatively homogeneous dense network of semi-flexible fibres with a mesh size of ∼15 to 30 nm depending on the concentration. The fibre diameter was found to be ∼4 nm in good agreement with models found in the literature. TEM micrographs clearly showed that these fibres have a helicoidal or twisted structure. Comparison of TEM with AFM data highlighted the influence of substrate chemistry on the macromolecular assembly of small peptides. In contrast small angle neutron scattering (SANS) approaches, which allow for the probing of hydrogel morphology and structure without the need for sample preparation on solid substrates, provide vital data on hydrogel morphology in solution.

Enzymatic catalyzed synthesis and triggered gelation of ionic peptides

We investigate the possibility of using the protease thermolysin to drive the synthesis and gelation of ionic-complementary peptides from nongelling precursors. In this system, short peptide fragments are continuously interconverted to form a dynamic peptide library, which eventually favors synthesis of peptides that are thermodynamically stabilized by molecular self-assembly. Thermolysin was added at a fixed concentration (0.3 mg mL(-1)) to solutions (0-300 mg mL(-1)) of the short tetrapeptide FEFK. Initially, the protease partially hydrolyzed the tetrapeptide into dipeptides in all samples. Subsequently, longer peptide sequences were found to form through reverse-hydrolysis. The stability of the different sequences was found to be dependent on their self-assembling properties. The sequences that self-assembled into antiparallel beta-sheet rich fibers became the stable products for the reverse hydrolysis reaction, while the others formed were unstable and disappeared with increasing incubation time. Ultimately, the main product of the system was octapeptide, which suggests that it represents the thermodynamically favored product of this dynamic library. Its concentration dictated the gelation behavior of the sample, and gels with moduli up to 25 kPa where obtained depending on the initial concentration of tetrapeptide.

Self-assembled octapeptide scaffolds for in vitro chondrocyte culture

Nature has evolved a variety of creative approaches to many aspects of materials synthesis and microstructural control. Molecular self-assembly is a simple and efficient way to fabricate complex nanostructures such as hydrogels. We have recently investigated the gelation properties of a series of ionic-complementary peptides based on the alternation of non-polar hydrophobic and polar hydrophilic residues. In this work we focus on one specific octapeptide, FEFEFKFK (F, phenylalanine; E, glutamic acid; K, lysine). This peptide was shown to self-assemble in solution and form β-sheet-rich nanofibres which, above a critical gelation concentration, entangle to form a self-supporting hydrogel. The fibre morphology of the hydrogel was analysed using transmission electron microscopy and cryo-scanning electron microscopy illustrating a dense fibrillar network of nanometer size fibres. Oscillatory rheology results show that the hydrogel possesses visco-elastic properties. Bovine chondrocytes were used to assess the biocompatibility of the scaffolds over 21 days under two-dimensional (2-D) and three-dimensional (3-D) cell culture conditions, particularly looking at cell morphology, proliferation and matrix deposition. 2-D culture resulted in cell viability and collagen type I deposition. In 3-D culture the mechanically stable gel was shown to support the viability of cells, the retention of cell morphology and collagen type II deposition. Subsequently the scaffold may serve as a template for cartilage tissue engineering.

Effect of peptide and guest charge on the structural, mechanical and release properties of β-sheet forming peptides.

The effect of peptide charge on the self-assembly, gelation behavior, and model drug release profiles has been explored here for three octa-peptides, VEVKVEVK (VEK2), VKVKVEVK (VEK3), and VEVEVKVE (VEK1), that carry a net charge of 0, +2, and -2 at neutral pH, respectively. Transparent, self-supporting hydrogels were found to form above a critical concentration when the peptide charge modulus was >1 and this was independent of the sign of the charge. TEM, SAXS, and shear rheology revealed that there were no differences in hydrogel structure or mechanical properties when the peptides were at the same concentration and carried the same charge modulus. All peptides were found to form dense fibrillar networks formed by β-sheet rich single fibers where lateral aggregation of the fibers occurred and increased with decreasing charge modulus. Such behavior was found to correlate with an increase in hydrogel mechanical properties, demonstrating that fiber lateral aggregation is inextricably linked with the mechanical properties of these hydrogels. Two hydrophilic model drug molecules, namely napthol yellow (NY) and martius yellow (MY), were subsequently incorporated within the VEK1 and VEK3 hydrogels at pH 7 and although they did not effect the self-assembly of the peptide at a molecular level, they did effect the level of lateral fiber aggregation observed and, therefore, the mechanical properties of the hydrogels. The release of each molecule from the hydrogels was monitored over time and shown to be controlled by Fickian diffusion where the diffusion rate, D, was dependent on the ratio between the overall effective charges carried by the peptide, i.e., the fibrillar network, and the overall charges carried by the guest molecules, but independent from the hydrogel concentration and mechanical properties within the ranges investigated. This work highlights the possibility of controlling the rate of release of small drug molecules by manipulating the charges on the guest molecules as well as the charged state of the self-assembling peptide.

Selective synthesis of double temperature-sensitive polymer-peptide conjugates

A novel synthetic route has been developed to couple selectively a modified octa-peptide, that is able to gel at low temperature, to the prototypical thermoresponsive polymer poly(N-isopropylacrylamide) to give a bioconjugate that exhibits double thermoresponsiveness.

Effect of Enzyme Concentration of the Morphology and Properties of Enzymatically Triggered Peptide Hydrogels

We have recently shown that thermolysine, a protease enzyme obtained from Bacillus thermoproteolyticus rokko , can be used to trigger the gelation of FEFK (F, phenylalanine; E, glutamic acid; K, lysine) tetrapeptides through reverse hydrolysis and formation of longer peptide sequences, mainly octapeptides, that self-assemble readily. In this article we investigate the effect of enzyme concentration on the morphology and properties of enzymatically triggered peptide hydrogels using HPLC, FTIR, real-time SAXS, TEM, and shear rheology. We have shown that the enzyme concentration, Cenz, does not affect the final composition of the samples. Instead, this is dictated by the initial tetrapeptide concentration, C0, suggesting the existence of a chemical equilibrium. We went on to show that Cenz does not affect the self-assembly of these peptides at a molecular level either nor the structure of the fibrillar network formed at the nanometer scale. Interestingly, the mechanical properties were found to be affected by Cenz, where the shear moduli of the hydrogels were found to increase with increasing Cenz. These results suggest that morphological differences between the hydrogels at the microscale are at the origin of their difference in mechanical properties. In this paper, we propose a morphological model in which denser network regions are found around the enzymes, resulting in the creation of heterogeneous networks. These were confirmed by TEM measurements. The existence of these denser network regions will result in the reinforcement of the hydrogels, thus, explaining the high shear moduli obtained increasing Cenz.

Human osteoblasts within soft peptide hydrogels promote mineralisation in vitro

Biomaterials that provide three-dimensional support networks for the culture of cells are being developed for a wide range of tissue engineering applications including the regeneration of bone. This study explores the potential of the versatile ionic-complementary peptide, FEFEFKFK, for such a purpose as this peptide spontaneously self-assembles into β-sheet-rich fibres that subsequently self-associate to form self-supporting hydrogels. Via simple live/dead cell assays, we demonstrated that 3 wt% hydrogels were optimal for the support of osteoblast cells. We went on to show that these cells are not only viable within the three-dimensional hydrogel but they also proliferate and produce osteogenic key proteins, that is, they behave like in vivo bone cells, over the 14-day period explored here. The gel elasticity increased over time when cells were present – in comparison to a decrease in control samples – indicating the deposition of matrix throughout the peptide scaffold. Moreover, significant quantities of calcium phosphate were deposited. Collectively, these data demonstrate that ionic-complementary octapeptides offer a suitable three-dimensional environment for osteoblastic cell function.

Osteogenic differentiation of human mesenchymal stem cells promotes mineralization within a biodegradable peptide hydrogel

An attractive strategy for the regeneration of tissues has been the use of extracellular matrix analogous biomaterials. Peptide-based fibrillar hydrogels have been shown to mimic the structure of extracellular matrix offering cells a niche to undertake their physiological functions. In this study, the capability of an ionic-complementary peptide FEFEFKFK (F, E, and K are phenylalanine, glutamic acid, and lysine, respectively) hydrogel to host human mesenchymal stem cells in three dimensions and induce their osteogenic differentiation is demonstrated. Assays showed sustained cell viability and proliferation throughout the hydrogel over 12 days of culture and these human mesenchymal stem cells differentiated into osteoblasts simply upon addition of osteogenic stimulation. Differentiated osteoblasts synthesized key bone proteins, including collagen-1 (Col-1), osteocalcin, and alkaline phosphatase. Moreover, mineralization occurred within the hydrogel. The peptide hydrogel is a naturally biodegradable material as shown by oscillatory rheology and reversed-phase high-performance liquid chromatography, where both viscoelastic properties and the degradation of the hydrogel were monitored over time, respectively. These findings demonstrate that a biodegradable octapeptide hydrogel can host and induce the differentiation of stem cells and has the potential for the regeneration of hard tissues such as alveolar bone.

Enzymatically triggered peptide hydrogels for 3D cell encapsulation and culture

We have investigated the possibility of using enzymatically triggered peptide hydrogels for the encapsulation and culture of cells. Based on recent work done on the enzymatically triggered gelation of FEFK (F, phenylalanine; E, glutamic acid; K, lysine) using thermolysin, a protease enzyme from Bacillus Thermoproteolyticus Rokko, we have investigated the possibility of using this gelation triggering mechanism to encapsulate cells within a 3D hydrogel matrix. First, the properties of enzymatically triggered hydrogels prepared in phosphate buffer solution were investigated and compared with the properties of hydrogels prepared in HPLC grade water from our previous work. We showed that the use of phosphate buffer solution allowed the production of hydrogels with very high shear moduli (>1 MPa). The gelation kinetics was also investigated, and the mechanical properties of the system were shown to closely follow the synthesis of the octapeptide by the enzyme through reverse hydrolysis. In a second phase, we developed, on the basis of information acquired, a facile protocol for the encapsulation of cells and plating of the hydrogel. Human dermal fibroblasts were then used to exemplify the use of these materials. FEFEFKFK octapeptide hydrogels prepared under the same conditions and with the same mechanical properties were used as a control. We showed that no significant differences were observed between the two systems and that after a decrease in cell number on day 1, cells start to proliferate. After 5 days of culture, the cells can be seen to start to adopt a stretched morphology typical of fibroblasts. The results clearly show that the protocol developed minimises the potential detrimental effect that thermolysin can have on the cells and that these enzymatically triggered hydrogels can be used for the 3D encapsulation and culture of cells. Copyright

Peptide Hydrogels as Mucoadhesives for local drug delivery

We have investigated the possibility of using self-assembling peptide-based viscous solutions and hydrogels as mucoadhesives for the improved delivery of drugs to local mucosal surfaces. The stability of the samples under flow after deposition on a mucosal surface mimic was studied using a simplified in vitro model. Subsequently lidocaine and flurbiprofen, two commercial drugs, were incorporated into the viscous solutions and hydrogels and their release properties investigated using the same model. Peptide-based hydrogels showed a good resistance to erosion under flow conditions. Addition of the soluble drug (lidocaine at low pH) resulted in a stiffening of the samples but did not affect the overall peptide release. Although for this drug the conditions were not favourable, improved retention of the drug was observed for the stiffest samples tested. In the case of the insoluble drug (flurbiprofen) the samples mechanical properties were not altered when the drug was incorporated, however the sample stability and peptide release were. For mechanically weaker samples the presence of the drug as insoluble small particles resulted in an increase in their susceptibility to physically erode when a flow of medium was applied over its surface. On the other hand mechanically stronger samples showed an improved resistance to erosion, which resulted in enhanced drug retention.