A switch in N-terminal capping of ß-peptides creates novel self-assembled nanoparticles.

Small tripeptides composed entirely of ß3-amino acids have been shown to self-assemble into fibres following acylation of the N-terminus. Given the use of Fmoc as a strategy to initiate self-assembly in α-peptides, we hypothesized that the acyl cap can be replaced by an Fmoc without perturbation to the self-assembly and enable simpler synthetic protocols. We therefore replaced the N-acyl cap for an Fmoc group and herein we show that these Fmoc-protected ß3-peptides produce regular spherical particles, rather than fibrous structures, that are stable and capable of encapsulating cargo. We then demonstrated that these particles were able to deliver cargo to cells without any obvious signs of cytotoxicity. This is the first description of such regular nanoparticles derived from Fmoc-protected ß3-peptides.

Elucidating the cell penetrating properties of self-assembling ß-peptides.

Self-assembling lipopeptide hydrogels have been widely developed for the delivery of therapeutics due to their rapid gelation, injectability, and highly controlled physicochemical properties. Lipopeptides are also known for their membrane-associating and cell penetrating properties, which may impact on their application in cell-encapsulation. Self-assembling lipidated-ß3-peptide materials developed in our laboratory have previously been used in cell culture as 2D substrates, thus as a continuation of this work we aimed to encapsulate cells in 3D by forming a hydrogel. We therefore assessed the self-assembling lipidated-ß3-peptides for cell-penetrating properties in mesenchymal stems cells (MSC) using fluorescence microscopy and membrane association with surface plasmon resonance spectroscopy (SPR). The results demonstrated that lipidated ß3-peptides penetrate the MSC plasma membrane and localise to the mitochondrial network. While self-assembling lipopeptide hydrogels have shown tremendous potential for delivery of therapeutics, further optimisation may be required to minimise the membrane uptake of the lipidated-ß3-peptides for cell encapsulation applications.

A comparison of fixation methods for SEM analysis of self-assembling peptide hydrogel nanoarchitecture.

Determining the porosity of hydrogels is an important component of material characterisation. While scanning electron microscopy (SEM) is a widely used method to study hydrogel nanoarchitecture, it is well-established that SEM sample preparation methods can alter the structure of hydrogels. Herein we describe the impact of sample preparation on the SEM analysis of self-assembling ß-peptide hydrogels. Three methods of hydrogel preparation for SEM were compared, and each method preserved distinctly different nanoarchitecture, specifically, different levels of fibre alignment and porosity. Comparison of conventional SEM preparation and our hybrid method, which comprises high pressure freezing, freeze substitution without fixative and critical point drying, showed a high degree of similarity at the nanometre scale and diverging architecture at the micron scale. This study quantified the impact of chemical fixation versus high pressure freezing on self-assembling ß3-peptide hydrogels, demonstrated the effect of sample preparation on fibre alignment and porosity, and presents a novel hybrid preparation method where chemical fixation can be avoided when conventional SEM is desired.

Esterase-Mediated Sustained Release of Peptide-Based Therapeutics from a Self-Assembled Injectable Hydrogel.

A synthetic strategy for conjugating small molecules and peptide-based therapeutics, via a cleavable ester bond, to a lipidated ß3-tripeptide is presented. The drug-loaded ß3-peptide was successfully co-assembled with a functionally inert lipidated ß3-tripeptide to form a hydrogel. Quantitative release of lactose from the hydrogel, by the action of serum esterases, is demonstrated over 28 days. The esterase-mediated sustained release of the bioactive brain-derived neurotrophic factor (BDNF) peptide mimics from the hydrogel resulted in increased neuronal survival and normal neuronal function of peripheral neurons. These studies define a versatile strategy for the facile synthesis and co-assembly of self-assembling ß3-peptide-based hydrogels with the ability to control drug release using endogenous esterases with potential in vivo applications for sustained localized drug delivery.

Staphylococcus aureus entanglement in self-assembling ß-peptide nanofibres decorated with vancomycin.

The increasing resistance of pathogenic microbes to antimicrobials and the shortage of antibiotic drug discovery programs threaten the clinical use of antibiotics. This threat calls for the development of new methods for control of drug-resistant microbial pathogens. We have designed, synthesised and characterised an antimicrobial material formed via the self-assembly of a population of two distinct ß-peptide monomers, a lipidated tri-ß-peptide (ß3-peptide) and a novel ß3-peptide conjugated to a glycopeptide antibiotic, vancomycin. The combination of these two building blocks resulted in fibrous assemblies with distinctive structures determined by atomic force microscopy and electron microscopy. These fibres inhibited the growth of methicillin resistant Staphylococcus aureus (MRSA) and associated directly with the bacteria, acting as a peptide nanonet with fibre nucleation sites on the bacteria observed by electron microscopy and confocal microscopy. Our results provide insights into the design of peptide based supramolecular assemblies with antibacterial activity and establish an innovative strategy to develop self-assembled antimicrobial materials for future biomedical application.

Enhancement of glioblastoma multiforme therapy through a novel Quercetin-Losartan hybrid.

Glioblastoma multiforme (GBM) is the most common and aggressive primary malignant brain tumor. Maximal surgical resection followed by radiotherapy and concomitant chemotherapy with temozolomide remains the first-line therapy, prolonging the survival of patients by an average of only 2.5 months. There is therefore an urgent need for novel therapeutic strategies to improve clinical outcomes. Reactive oxygen species (ROS) are an important contributor to GBM development. Here, we describe the rational design and synthesis of a stable hybrid molecule tethering two ROS regulating moieties, with the aim of constructing a chemopreventive and anticancer chemical entity that retains the properties of the parent compounds. We utilized the selective AT1R antagonist losartan, leading to the inhibition of ROS levels, and the antioxidant flavonoid quercetin. In GBM cells, we show that this hybrid retains the binding potential of losartan to the AT1R through competition-binding experiments and simultaneously exhibits ROS inhibition and antioxidant capacity similar to native quercetin. In addition, we demonstrate that the hybrid is able to alter the cell cycle distribution of GBM cells, leading to cell cycle arrest and to the induction of cytotoxic effects. Last, the hybrid significantly and selectively reduces cancer cell proliferation and angiogenesis in primary GBM cultures with respect to the isolated parent components or their simple combination, further emphasizing the potential utility of the current hybridization approach in GBM.

Transition of Nano-Architectures Through Self-Assembly of Lipidated ß3-Tripeptide Foldamers.

ß3-peptides consisting exclusively of ß3-amino acids adopt a variety of non-natural helical structures and can self-assemble into well-defined hierarchical structures by axial head-to-tail self-assembly resulting in fibrous materials of varying sizes and shapes. To allow control of fiber morphology, a lipid moiety was introduced within a tri-ß3-peptide sequence at each of the three amino acid positions and the N-terminus to gain finer control over the lateral assembly of fibers. Depending on the position of the lipid, the self-assembled structures formed either twisted ribbon-like fibers or distinctive multilaminar nanobelts. The nanobelt structures were comprised of multiple layers of peptide fibrils as revealed by puncturing the surface of the nanobelts with an AFM probe. This stacking phenomenon was completely inhibited through changes in pH, indicating that the layer stacking was mediated by electrostatic interactions. Thus, the present study is the first to show controlled self-assembly of these fibrous structures, which is governed by the location of the acyl chain in combination with the 3-point H-bonding motif. Overall, the results demonstrate that the nanostructures formed by the ß3-tripeptide foldamers can be tuned via sequential lipidation of N-acetyl ß3-tripeptides which control the lateral interactions between peptide fibrils and provide defined structures with a greater homogeneous population.

Renal functional effects of the highly selective AT2R agonist, ß-Pro7 Ang III, in normotensive rats.

Recently, we designed a group of peptides by sequential substitution of the naturally occurring α-amino acid throughout the Ang III peptide sequence with the corresponding ß-amino acid. ß-Amino acid substitution at the proline residue of Ang III (ß-Pro7-Ang III) resulted in a highly selective AT2R ligand, demonstrating remarkable selectivity for the AT2R in both binding and functional studies. To provide additional functional evidence for the suitability of ß-Pro7 Ang III as a novel AT2R agonist, we tested effects of acute systemic administration of ß-Pro7-Ang III on renal hemodynamic and excretory function in anesthetized normotensive male and female rats. We also compared the natriuretic effects of acute intrarenal administration of native Ang III and ß-Pro7-Ang III in the presence of systemic AT1R blockade in anesthetized female rats to allow for the differentiation of systemic versus direct intrarenal natriuretic actions of ß-Pro7-Ang III. In both male and female rats, acute systemic administration of ß-Pro7-Ang III elicited renal vasodilatation and natriuresis. Notably, greater renal vasodilatory effects were observed in female versus male rats at the highest dose of ß-Pro7-Ang III administered. Moreover, intra-renal administration of ß-Pro7-Ang III produced significant natriuretic effects in female rats and, like Ang III, evoked AT2R translocation to the apical plasma membrane in renal proximal tubular cells. Taken together, our findings support the use of ß-Pro7-Ang III as a novel AT2R agonist and experimental tool for exploring AT2R function and its potential as a therapeutic target. Furthermore, our findings provide further evidence of a sex-specific influence of AT2R stimulation on renal function.

Migration and Differentiation of Neural Stem Cells Diverted From the Subventricular Zone by an Injectable Self-Assembling ß-Peptide Hydrogel.

Neural stem cells, which are confined in localised niches are unable to repair large brain lesions because of an inability to migrate long distances and engraft. To overcome these problems, previous research has demonstrated the use of biomaterial implants to redirect increased numbers of endogenous neural stem cell populations. However, the fate of the diverted neural stem cells and their progeny remains unknown. Here we show that neural stem cells originating from the subventricular zone can migrate to the cortex with the aid of a long-lasting injectable hydrogel within a mouse brain. Specifically, large numbers of neuroblasts were diverted to the cortex through a self-assembling ß-peptide hydrogel that acted as a tract from the subventricular zone to the cortex of transgenic mice (NestinCreERT2:R26eYFP) in which neuroblasts and their progeny are permanently fluorescently labelled. Moreover, neuroblasts differentiated into neurons and astrocytes 35 days post implantation, and the neuroblast-derived neurons were Syn1 positive suggesting integration into existing neural circuitry. In addition, astrocytes co-localised with neuroblasts along the hydrogel tract, suggesting that they assisted migration and simulated pathways similar to the native rostral migratory stream. Lower levels of astrocytes were found at the boundary of hydrogels with encapsulated brain-derived neurotrophic factor, comparing with hydrogel implants alone.

3D bioprinted endometrial stem cells on melt electrospun poly ε-caprolactone mesh for pelvic floor application promote anti-inflammatory responses in mice.

Endometrial mesenchymal stem/stromal cells (eMSCs) exhibit excellent regenerative capacity in the endometrial lining of the uterus following menstruation and high proliferative capacity in vitro. Bioprinting eMSCs onto a mesh could be a potential therapy for Pelvic Organ Prolapse (POP). This study reports an alternative treatment strategy targeting vaginal wall repair using bioprinting of eMSCs encapsulated in a hydrogel and 3D melt electrospun mesh to generate a tissue engineering construct. Following a CAD, 3D printed poly ε-caprolactone (PCL) meshes were fabricated using melt electrospinning (MES) at different temperatures using a GMP clinical grade GESIM Bioscaffolder. Electron and atomic force microscopies revealed that MES meshes fabricated at 100 °C and with a speed 20 mm/s had the largest open pore diameter (47.2 ±â€¯11.4 µm) and the lowest strand thickness (121.4 ±â€¯46 µm) that promoted optimal eMSC attachment. An Aloe Vera-Sodium Alginate (AV-ALG) composite based hydrogel was optimised to a 1:1 mixture (1%AV-1%ALG) and eMSCs, purified from human endometrial biopsies, were then bioprinted in this hydrogel onto the MES printed meshes. Acute in vivo foreign body response assessment in NSG mice revealed that eMSC printed on MES constructs promoted tissue integration, eMSC retention and an anti-inflammatory M2 macrophage phenotype characterised by F4/80+CD206+ colocalization. Our results address an unmet medical need highlighting the potential of 3D bioprinted eMSC-MES meshes as an alternative approach to overcome the current challenges with non-degradable knitted meshes in POP treatment. STATEMENT OF SIGNIFICANCE: This study presents the first report of bioprinting mesenchymal stem cells derived from woman endometrium (eMSCs) to boost Pelvic Organ Prolapse (POP) treatment. It impacts over 50% of elderly women with no optimal treatment at present. The overall study is conducted in three stages as fabricating a melt electrospun (MES) mesh, bioprinting eMSCs into a Ca2+ free Aloe Vera-Alginate (AV-Alg) based hydrogel and in vivo study. Our data showed that AV-ALG hydrogel potentially suppresses the foreign body response and further addition of eMSCs triggered a high influx of anti-inflammatory CD206+ M2 macrophages. Our final construct demonstrates a favourable foreign body response to predict expected tissue integration, therefore, provides a potential for developing an alternative treatment for POP.

Novel Materials From the Supramolecular Self-Assembly of Short Helical ß3-Peptide Foldamers.

Self-assembly is the spontaneous organization of small components into higher-order structures facilitated by the collective balance of non-covalent interactions. Peptide-based self-assembly systems exploit the ability of peptides to adopt distinct secondary structures and have been used to produce a range of well-defined nanostructures, such as nanotubes, nanofibres, nanoribbons, nanospheres, nanotapes, and nanorods. While most of these systems involve self-assembly of α-peptides, more recently ß-peptides have also been reported to undergo supramolecular self-assembly, and have been used to produce materials-such as hydrogels-that are tailored for applications in tissue engineering, cell culture and drug delivery. This review provides an overview of self-assembled peptide nanostructures obtained via the supramolecular self-assembly of short ß-peptide foldamers with a specific focus on N-acetyl-ß3-peptides and their applications as bio- and nanomaterials.

The use of hydrogels for cell-based treatment of chronic kidney disease.

Chronic kidney disease (CKD) is a major and growing public health concern with increasing incidence and prevalence worldwide. The therapeutic potential of stem cell therapy, including mesenchymal stem cells (MSCs) and endothelial progenitor cells (EPCs) holds great promise for treatment of CKD. However, there are significant bottlenecks in the clinical translation due to the reduced number of transplanted cells and the duration of their presence at the site of tissue damage. Bioengineered hydrogels may provide a route of cell delivery to enhance treatment efficacy and optimise the targeting effectiveness while minimising any loss of cell function. In this review, we highlight the advances in stem cell therapy targeting kidney disease and discuss the emerging role of hydrogel delivery systems to fully realise the potential of adult stem cells as a regenerative therapy for CKD in humans. MSCs and EPCs mediate kidney repair through distinct paracrine effects. As a delivery system, hydrogels can prolong these paracrine effects by improving retention at the site of injury and protecting the transplanted cells from the harsh inflammatory microenvironment. We also discuss the features of a hydrogel, which may be tuned to optimise the therapeutic potential of encapsulated stem cells, including cell-adhesive epitopes, material stiffness, nanotopography, modes of gelation and degradation and the inclusion of bioactive molecules. This review concludes with a discussion of the challenges to be met for the widespread clinical use of hydrogel delivery system of stem cell therapy for CKD.

Identifying the Coiled-Coil Triple Helix Structure of ß-Peptide Nanofibers at Atomic Resolution.

Peptide self-assembly represents a powerful bottom-up approach to the fabrication of nanomaterials. ß3-Peptides are non-natural peptides composed entirely of ß-amino acids, which have an extra methylene in the backbone, and we reported fibers derived from the self-assembly of ß3-peptides that adopt 14-helical structures. ß3-Peptide assemblies represent a class of stable nanomaterials that can be used to generate bio- and magneto-responsive materials with proteolytic stability. However, the three-dimensional structure of many of these materials remains unknown. To develop structure-based criteria for the design of ß3-peptide-based biomaterials with tailored function, we investigated the structure of a tri-ß3-peptide nanoassembly by molecular dynamics simulations and X-ray fiber diffraction analysis. Diffraction data was collected from aligned fibrils formed by Ac-ß3[LIA] in water and used to inform and validate the model structure. Models with 3-fold radial symmetry resulted in stable fibers with a triple-helical coiled-coil motif and measurable helical pitch and periodicity. The fiber models revealed a hydrophobic core and twist along the fiber axis arising from a maximization of contacts between hydrophobic groups of adjacent tripeptides on the solvent-exposed fiber surface. These atomic structures of macroscale fibers derived from ß3-peptide-based materials provide valuable insight into the effects of the geometric placement of the side chains and the influence of solvent on the core fiber structure which is perpetuated in the superstructure morphology.

Exploring Molecular-Biomembrane Interactions with Surface Plasmon Resonance and Dual Polarization Interferometry Technology: Expanding the Spotlight onto Biomembrane Structure.

The molecular analysis of biomolecular-membrane interactions is central to understanding most cellular systems but has emerged as a complex technical challenge given the complexities of membrane structure and composition across all living cells. We present a review of the application of surface plasmon resonance and dual polarization interferometry-based biosensors to the study of biomembrane-based systems using both planar mono- or bilayers or liposomes. We first describe the optical principals and instrumentation of surface plasmon resonance, including both linear and extraordinary transmission modes and dual polarization interferometry. We then describe the wide range of model membrane systems that have been developed for deposition on the chips surfaces that include planar, polymer cushioned, tethered bilayers, and liposomes. This is followed by a description of the different chemical immobilization or physisorption techniques. The application of this broad range of engineered membrane surfaces to biomolecular-membrane interactions is then overviewed and how the information obtained using these techniques enhance our molecular understanding of membrane-mediated peptide and protein function. We first discuss experiments where SPR alone has been used to characterize membrane binding and describe how these studies yielded novel insight into the molecular events associated with membrane interactions and how they provided a significant impetus to more recent studies that focus on coincident membrane structure changes during binding of peptides and proteins. We then discuss the emerging limitations of not monitoring the effects on membrane structure and how SPR data can be combined with DPI to provide significant new information on how a membrane responds to the binding of peptides and proteins.

ß3-tripeptides act as sticky ends to self-assemble into a bioscaffold.

Peptides comprised entirely of ß3-amino acids, commonly referred to as ß-foldamers, have been shown to self-assemble into a range of materials. Previously, ß-foldamers have been functionalised via various side chain chemistries to introduce function to these materials without perturbation of the self-assembly motif. Here, we show that insertion of both rigid and flexible molecules into the backbone structure of the ß-foldamer did not disturb the self-assembly, provided that the molecule is positioned between two ß3-tripeptides. These hybrid ß3-peptide flanked molecules self-assembled into a range of structures. α-Arginlyglycylaspartic acid (RGD), a commonly used cell attachment motif derived from fibronectin in the extracellular matrix, was incorporated into the peptide sequence in order to form a biomimetic scaffold that would support neuronal cell growth. The RGD-containing sequence formed the desired mesh-like scaffold but did not encourage neuronal growth, possibly due to over-stimulation with RGD. Mixing the RGD peptide with a ß-foldamer without the RGD sequence produced a well-defined scaffold that successfully encouraged the growth of neurons and enabled neuronal electrical functionality. These results indicate that ß3-tripeptides can form distinct self-assembly units separated by a linker and can form fibrous assemblies. The linkers within the peptide sequence can be composed of a bioactive α-peptide and tuned to provide a biocompatible scaffold.

ß3-Tripeptides Coassemble into Fluorescent Hydrogels for Serial Monitoring in Vivo.

ß3-peptides uniquely form shear thinning hydrogels which are proteolytically stable and biocompatible. Herein we describe the synthesis, material and optical characterization of a new class of fluorescently labeled hydrogelators based on a helical N-acetylated ß3-peptide backbone. The resulting hydrogels were analyzed using fluorescence microscopy to confirm successful incorporation of the fluorophore within the fiber matrix without compromising the ß3-peptide self-assembly. Serial, noninvasive conscious animal imaging was used to monitor the injected hydrogel, delivered via subcutaneous injection, while tracking their degradation patterns in real-time. The hydrogels demonstrated persistent, high-intensity fluorescence when monitored over a 14-day period.

Shortened Penetratin Cell-Penetrating Peptide Is Insufficient for Cytosolic Delivery of a Grb7 Targeting Peptide.

Delivery across the cell membrane is of critical importance for the development of therapeutics targeting intracellular proteins. The use of cell-penetrating peptides (CPPs), such as Penetratin (P16), has facilitated the delivery of otherwise cell-impermeable molecules allowing them to carry out their biological function. A truncated form of Penetratin (RRMKWKK) has been previously described as the minimal Penetratin sequence that is required for translocation across the cell membrane. Here, we performed a detailed comparison of cellular uptake by Penetratin (P16) and the truncated Penetratin peptide (P7), including their ability to deliver G7-18NATE, a cyclic peptide targeting the cytosolic cancer target Grb7-adapter protein into cells. We identified that both P16 and P7 were internalized by cells to comparable levels; however, only P16 was effective in delivering G7-18NATE to produce a biological response. Live-cell imaging of fluorescein isothiocyanate-labeled peptides suggested that while P7 may be taken up into cells, it does not gain access to the cytosolic compartment. Thus, this study has identified that the P7 peptide is a poor CPP for the delivery of G7-18NATE and may also be insufficient for the intracellular delivery of other bioactive molecules.

Using β-Amino Acids and β-Peptide Templates to Create Bioactive Ligands and Biomaterials.

β-Amino acids are being increasingly used in the design of bioactive ligands and more recently in the generation of novel biomaterials. Peptides containing either individual β-amino acid substitutions or peptides comprised entirely of ß-amino acids, display unique properties in terms of their structural and/or chemical characteristics. β-Peptides form well-defined secondary structures that exhibit different geometries compared to the corresponding α-peptides. β-Peptides, including α-peptides containing only one or two β-amino acids, can be easily modified with different functional groups and are metabolically stable and, together with the predictable side chain topography, have led to the design of a growing number of bioactive β-peptides with a range of biological targets and therapeutic applications. More recently, our understanding of the folding and self-assembly of β-peptides has resulted in the generation of novel biomaterials. The focus of this review is to examine how the structural and chemical properties of β-peptides have been exploited in the design of bioactive peptides and selfassembled nanomaterials.

Unexpected involvement of staple leads to redesign of selective bicyclic peptide inhibitor of Grb7.

The design of potent and specific peptide inhibitors to therapeutic targets is of enormous utility for both proof-of-concept studies and for the development of potential new therapeutics. Grb7 is a key signaling molecule in the progression of HER2 positive and triple negative breast cancers. Here we report the crystal structure of a stapled bicyclic peptide inhibitor G7-B1 in complex with the Grb7-SH2 domain. This revealed an unexpected binding mode of the peptide, in which the staple forms an alternative contact with the surface of the target protein. Based on this structural information, we designed a new series of bicyclic G7 peptides that progressively constrain the starting peptide, to arrive at the G7-B4 peptide that binds with an approximately 2-fold enhanced affinity to the Grb7-SH2 domain (KD = 0.83 µM) compared to G7-B1 and shows low affinity binding to Grb2-, Grb10- and Grb14-SH2 domains (KD > 100 µM). Furthermore, we determined the structure of the G7-B4 bicyclic peptide in complex with the Grb7-SH2 domain, both before and after ring closing metathesis to show that the closed staple is essential to the target interaction. The G7-B4 peptide represents an advance in the development of Grb7 inhibitors and is a classical example of structure aided inhibitor development.

Orthogonal strategy for the synthesis of dual-functionalised ß(3)-peptide based hydrogels.

We have described a new class of hydrogelator based on helical ß(3)-peptides carrying a bioactive payload. The ß(3)-peptides self-assemble in aqueous solution to form a nanofibrous mesh resulting in a stable hydrogel. The simple design provides the versatility for attaching different functional payloads to the ß(3)-peptide scaffold to develop new materials.