Functionalised peptide hydrogel for the delivery of cardiac progenitor cells.

Heart failure (HF) remains one of the leading causes of death worldwide; most commonly developing after myocardial infarction (MI). Since adult cardiomyocytes characteristically do not proliferate, cells lost during MI are not replaced. As a result, the heart has a limited regenerative capacity. There is, therefore, a need to develop novel cell-based therapies to promote the regeneration of the heart after MI. The delivery and retention of cells at the injury site remains a significant challenge. In this context, we explored the potential of using an injectable, RGDSP-functionalised self-assembling peptide - FEFEFKFK - hydrogel as scaffold for the delivery and retention of rat cardiac progenitor cells (CPCs) into the heart. Our results show that culturing CPCs in vitro within the hydrogel for one-week promoted their spontaneous differentiation towards adult cardiac phenotypes. Injection of the hydrogel on its own, or loaded with CPCs, into the rat after injury resulted in a significant reduction in myocardial damage and left ventricular dilation.

In vivo characterisation of a therapeutically relevant self-assembling 18 F-labelled ß-sheet forming peptide and its hydrogel using positron emission tomography.

Positron emission tomography (PET) and fluorescence labelling have been used to assess the pharmacokinetics, biodistribution and eventual fate of a hydrogel-forming nonapeptide, FEFKFEFKK (F9), in healthy mice, using 18 F-labelled and fluorescein isothiocyanate (FITC)-labelled F9 analogues. F9 was site-specifically radiolabelled with 2-[18 F]fluoro-3-pyridinecarboxaldehyde ([18 F]FPCA) via oxime bond formation. [18 F]FPCA-F9 in vivo fate was evaluated both as a solution, following intravenous administration, and as a hydrogel when subcutaneously injected. The behaviour of FITC-F9 hydrogel was assessed following subcutaneous injection. [18 F]FPCA-F9 demonstrated high plasma stability and primarily renal excretion; [18 F]FPCA-F9 when in solution and injected into the bloodstream displayed prompt bladder uptake (53.4 ± 16.6 SUV at 20 minutes postinjection) and rapid renal excretion, whereas [18 F]FPCA-F9 hydrogel, formed by co-assembly of [18 F]FPCA-F9 monomer with unfunctionalised F9 peptide and injected subcutaneously, showed gradual bladder accumulation of hydrogel fragments (3.8 ± 0.4 SUV at 20 minutes postinjection), resulting in slower renal excretion. Gradual disaggregation of the F9 hydrogel from the site of injection was monitored using FITC-F9 hydrogel in healthy mice (60 ± 3 over 96 hours), indicating a biological half-life between 1 and 4 days. The in vivo characterisation of F9, both as a gel and a solution, highlights its potential as a biomaterial.

Peptide hydrogel in vitro non-inflammatory potential.

Peptide-based hydrogels have attracted significant interest in recent years as these soft, highly hydrated materials can be engineered to mimic the cell niche with significant potential applications in the biomedical field. Their potential use in vivo in particular is dependent on their biocompatibility, including their potential to cause an inflammatory response. In this work, we investigated in vitro the inflammatory potential of a ß-sheet forming peptide (FEFEFKFK; F: phenylalanine, E: glutamic acid; K: lysine) hydrogel by encapsulating murine monocytes within it (3D culture) and using the production of cytokines, IL-ß, IL-6 and TNFα, as markers of inflammatory response. No statistically significant release of cytokines in our test sample (media + gel + cells) was observed after 48 or 72 h of culture showing that our hydrogels do not incite a pro-inflammatory response in vitro. These results show the potential biocompatibility of these hydrogels and therefore their potential for in vivo use. The work also highlighted the difference in monocyte behaviour, proliferation and morphology changes when cultured in 2D vs. 3D. © 2016 The Authors Journal of Peptide Science published by European Peptide Society and John Wiley & Sons Ltd.

Self-assembling peptide/thermoresponsive polymer composite hydrogels: effect of peptide-polymer interactions on hydrogel properties.

We have investigated the effect of doping the self-assembling octapeptide FEFEFKFK (F, phenylalanine; E, glutamic acid; K, lysine) hydrogels with various amounts of thermoresponsive conjugate of FEFEFKFK and poly(N-isopropylacrylamide) (PNIPAAm) in order to create novel hydrogels. The samples were characterized using a range of techniques including microdifferential scanning calorimetry (µDSC), oscillatory rheology, transmission electron microscopy (TEM), atomic force microscopy (AFM), and small angle neutron scattering (SANS). The peptide from the conjugate was shown to be incorporated into the peptide fiber, resulting in the polymer being anchored to the peptide fiber. The conjugation of the polymer to the peptide and its anchoring to the peptide fibers did not affect its lower critical solution temperature (LCST). On the other hand, it did result in a decrease in the LCST enthalpy and a significant increase in the G' of the hydrogels, suggesting the presence of hydrogen bond interactions between the peptide and the polymer. As a result, the polymer was found to adopt a fibrillar arrangement tightly covering the peptide fiber. The polymer was still found to go through a conformational change at the LCST, suggesting that it collapses onto the peptide fiber. On the other hand, the fibrillar network was found to be mainly unaffected by the polymer LCST. These changes at the LCST were also found to be fully reversible. The nature of the interaction between the polymer and the peptide was shown to have a significant effect on the conformation adopted by the polymer around the fibers and the mechanical properties of the hydrogels.

Immobilization of cell-binding peptides on poly-ε-caprolactone film surface to biomimic the peripheral nervous system.

Cell-material interactions are crucial for cell adhesion and proliferation on biomaterial surfaces. Immobilization of biomolecules leads to the formation of biomimetic substrates, improving cell response. We introduced RGD (Arg-Gly-Asp) sequences on poly-ε-caprolactone (PCL) film surfaces using thiol chemistry to enhance Schwann cell (SC) response. XPS elemental analysis indicated an estimate of 2-3% peptide functionalization on the PCL surface, comparable with carbodiimide chemistry. Contact angle was not remarkably reduced; hence, cell response was only affected by chemical cues on the film surface. Adhesion and proliferation of Schwann cells were enhanced after PCL modification. Particularly, RGD immobilization increased cell attachment up to 40% after 6 h of culture. It was demonstrated that SC morphology changed from round to very elongated shape when surface modification was carried out, with an increase in the length of cellular processes up to 50% after 5 days of culture. Finally RGD immobilization triggered the formation of focal adhesion related to higher cell spreading. In summary, this study provides a method for immobilization of biomolecules on PCL films to be used in peripheral nerve repair, as demonstrated by the enhanced response of Schwann cells.

Effect of peptide and guest charge on the structural, mechanical and release properties of ß-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 ß-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.

Density, DSC, X-ray and NMR measurements through the gel and lamellar phase transitions of 1-myristoyl-2-stearoyl-sn-glycero-3-phosphatidylcholine (MSPC) and 1-stearoyl-2-myristoyl-sn-glycero-3-phosphatidylcholine (SMPC): observation of slow relaxation processes and mechanisms of phase transitions.

Dialkyl lecithin dispersions in water exhibit two phase transitions upon cooling from the lamellar phase (L(α)). At the main transition (T(M)) the L(α) phase changes to a ripple (gel) phase (P(ß')) which then transforms to a second gel phase (L(ß')) at the "pretransition" (T(P)). We have made accurate density measurements through the various phases for two lecithins having unequal chains: 1-myristoyl-2-stearoyl-sn-glycero-3-phosphatidylcholine (MSPC) and 1-stearoyl-2-myristoyl-sn-glycero-3-phosphatidylcholine (SMPC). The measurements were carried out over five heat/cool cycles from 5 to 55 °C, followed by cooling back to 5 °C. The samples were then held at 50 °C for 24 hours, followed by a further three cool/heat cycles. For SMPC we observe an increase in density of the gel phases over the first 5 cycles, followed by much smaller changes after incubation at 50 °C. The lamellar phase also shows an increase in density, albeit much smaller. This parallels the behaviour of 1,2-di-palmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) and 1,2-di-myristoyl-sn-glycero-3-phosphatidylcholine (DMPC) reported earlier (Jones et al., Liquid Crystals 32, 1465 (2005)). For MSPC we observe a decrease in density within the gel phases while T(P) almost disappears after the first cycle. The lamellar phase shows little evidence of any change with each cycle. Within the lamellar phases there is a marked reduction in density on approaching T(M), which is attributed to the formation of transitory gel phase domains. Additional measurements by DSC and X-ray diffraction show that the changes in densities are not accompanied by large changes in transition enthalpies or phase structures. NMR data indicate that the pretransitional event within the L(α) phase is accompanied by ordering of the alkyl chains. The results indicate that the exact nature of the lipid alkyl chains could play a key role in the formation of gel phase patches within membrane bilayers. Their detailed chemical structures merit more attention than by simply assuming a uniform "bending energy" to describe the behaviour.

Thermoreversible lysozyme hydrogels: properties and an insight into the gelation pathway.

The gelation behaviour of aqueous solutions of hen egg white lysozyme (HEWL) in the presence of 20 mM DTT in the concentration range 0.7 to 4.0 mM has been investigated using microDSC, FTIR, cryoTEM, SANS and oscillatory rheology. The macroscopic critical gelation concentration, Cgel, was found to be ∼ 3.0 mM. The disruption of the disulfide bonds by the DTT and the destabilisation of the protein were found to be a prerequisite for the formation of ß-sheet rich fibrils under the mild conditions used in this work. Using our methodology the hydrogels obtained have a pH of 7, hence are suitable for cell culture, and are also thermoreversible. The hydrogel melting temperature was found to increase with increasing concentration and a similar structure was observed across the concentration range investigated. Our results suggest these systems are composed of a well defined regular network where single ß-sheet rich fibrils (∼ 3 nm diameter) form initially, then two of these fibrils associate two-by-two to form junctions (∼ 6 nm diameter) and then on cooling further aggregate to form larger bundles of fibres. The network mesh size was found to decrease with increasing concentration. Our results suggest that below Cgel small unconnected gel-like aggregates exist that have a similar structure to the hydrogels obtained above Cgel. Using our data we propose a model for the denaturation and gelation behaviour of our system. During the first heating an α-helix to ß-sheet molecular transition for the protein conformation occurs resulting in ß-sheet rich fibrils forming through the self-assembly of ß-sheet rich denaturated proteins. At high temperature the solution contains ß-sheet rich fibrils with dissolved protein. On cooling an increase in the amount of ß-sheet was observed via FTIR suggesting that as the temperature is decreased more and more protein forms ß-sheet rich fibrils. At the gelation temperature these fibrils associate two-by-two to form the network junctions resulting in the macroscopic gelation of the sample. Our results suggest the network junctions are formed via specific hydrophobic interactions. The hydrogels elastic modulus was found to scale as C2.45 for C > Cgel.