B21-High Throughput SAXS
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Diamond Proposal Number(s):
[24693]
Abstract: Tuning the enzymatic degradation and disassembly rates of polymeric amphiphiles and their assemblies is crucial for designing enzyme-responsive nanocarriers for controlled drug delivery applications. The common methods to control the enzymatic degradation of amphiphilic polymers are to tune the molecular weights and ratios of the hydrophilic and hydrophobic blocks. In addition, to these approaches, the architecture of the hydrophilic block can also serve as a tool to tune enzymatic degradation and disassembly. To gain deeper understanding of the effect of the molecular architecture of the hydrophilic block we prepared two types of well-defined PEG-dendron amphiphiles bearing linear or V-shaped PEG chains as the hydrophilic blocks. The high molecular precision of these amphiphiles, which emerges from the utilization of dendrons as the hydrophobic blocks allowed us to study the self-assembly and enzymatic degradation and disassembly of the two types of amphiphiles with high resolution. Interestingly, the micelles of the V-shaped amphiphiles were significantly smaller and disassembled faster than those of the amphiphiles based on linear PEG. However, the complete enzymatic cleavage of the hydrophobic end-groups was significantly slower for the V-shaped amphiphiles. Our results show that the V-shape architecture can stabilize the unimer state and hence plays a double role in the enzymatic degradation and the induced disassembly and how it can be utilized to control the release of encapsulated or bound molecular cargo.
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Aug 2020
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I22-Small angle scattering & Diffraction
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Diamond Proposal Number(s):
[17102]
Open Access
Abstract: Understanding peptide self-assembly mechanisms and stability of the formed assemblies is crucial for development of functional nanomaterials. Herein, we have adopted rational design approach to demonstrate how minimal structural modification to a non-assembling ultra-short ionic self-complementary tetrapeptide FEFK (Phe4) remarkably enhanced stability of self-assembly into β-sheet nanofibres and induced hydrogelation. This was achieved by replacing flexible phenylalanine residue (F) by the rigid phenylglycine (Phg) resulting in constrained analogue PhgEPhgK (Phg4), which positioned aromatic rings in an orientation favourable for aromatic stacking. Phg4 self-assembly into stable β-sheet ladders was facilitated by π-staking of aromatic sidechains alongside hydrogen bonding between backbone amides along the nanofibre axis. The contribution of these non-covalent interactions in stabilising self-assembly was predicted by in silico modelling using molecular dynamics simulations and semi-empirical quantum mechanics calculations. In aqueous medium, Phg4 β-sheet nanofibres entangled at a critical gelation concentration > 20 mg/mL forming a network of nanofibrous hydrogel. Phg4 also demonstrated unique surface activity in presence of immiscible oils and was superior to commercial emulsifiers in stabilising oil-in-water emulsions. This was attributed to interfacial adsorption of amphiphilic nanofibrilles forming nanofibrillised microspheres. To our knowledge, Phg4 is the shortest ionic self-complementary peptide rationally designed to self-assemble into stable β-sheet nanofibres capable of gelation and emulsification. Our results suggest that Ultra-short Ionic-complementary Constrained Peptides or UICPs have significant potential for the development of cost-effective, sustainable and multifunctional soft bionanomaterials.
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May 2020
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I22-Small angle scattering & Diffraction
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Diamond Proposal Number(s):
[12950, 15246]
Abstract: Hydrogels’ hydrated fibrillar nature makes them the material of choice for the design and engineering of 3D-scaffolds for cell culture, tissue engineering and drug delivery applications. One particular class of hydrogels that has been the focus of significant research is self-assembling peptide hydrogels. In the present work we were interested in exploring how fibre-fibre edge interactions affect the self-assembly and gelation properties of amphipathic peptides. For this purpose we investigated two β-sheet forming peptides, FEFKFEFK (F8) and KFEFKFEFKK (KF8K), the latter one having the fibre edges covered by lysine residues. Our results showed that the addition of the two lysine residues did not affect the ability of the peptides to form β-sheet rich fibres provided that the overall charge carried by the two peptides was kept constant. It did though significantly reduce edge driven hydrophobic fibre-fibre associative interactions resulting in a reduced tendency for KF8K fibres to associate / aggregate laterally and form large fibre bundles and consequently network crosslinks. This effect resulted in the formation of hydrogels with lower moduli but faster dynamics. As a result KF8K fibres could be aligned only under high shear and at high concentration while F8 hydrogel fibres were found to align readily at low shear and low concentration. In addition F8 hydrogels were found to fragment at high concentration due to the high aggregation state stabilising the fibre bundles resulting in fibre breakage rather than disentanglement and alignment.
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Apr 2020
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I22-Small angle scattering & Diffraction
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Diamond Proposal Number(s):
[17580]
Abstract: Surface hydrophobization of cellulose nanomaterials has been used in the development of nanofiller reinforced polymer composites and formulations based on Pickering emulsions. Despite well known effect of hydrophobic domains on self assembly or association of water soluble polymer amphiphiles, very few studies have addressed the behavior of hydrophobized cellulose nanomaterials in aqueous media. In this study, we investigate the properties of hydrophobized cellulose nanocrystals (CNCs) and their self assembly and amphiphilic properties in suspensions and gels. CNCs of different hydrophobicity were synthesized from sulfated CNCs by coupling primary alkylamines of different alkyl chain lengths (6, 8 and 12 carbon atoms). The synthetic route permitted the retention of surface charge, ensuring good colloidal stability of hydrohobized CNCs in aqueous suspensions. We compare surface properties (surface charge, Zeta potential), hydrophobicity (water contact angle, microenvironment probing using pyrene fluorescence emission) and surface activity (tensiometry) of different hydrophobized CNCs and hydrophilic CNCs. Association of hydrophobized CNCs driven by hydrophobic effects is confirmed by X ray scattering (SAXS) and autofluorescent spectroscopy experiments. As a result of CNC association, CNCs suspensions/gels can be produced with a wide range of rheological properties depending on the hydrophobic/hydrophilic balance. In particular, sol gel transitions for hydrophobized CNCs occur at lower concentrations then hydrophilic CNCs and more robust gels are formed by hydrophobized CNCs. Our work illustrates that amphiphilic CNCs can complement associative polymers as modifiers of rheological properties of water based systems.
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Jan 2020
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I07-Surface & interface diffraction
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Diamond Proposal Number(s):
[13569]
Abstract: The biopolymer cellulose is investigated in terms of the crystallographic order within thin films. The films were prepared by spin coating of a trimethylsilyl cellulose precursor followed by an exposure to HCl vapors; two different source materials were used. Careful pre-characterization of the films was performed by infrared spectroscopy and atomic force microscopy. Subsequently, the films were investigated by grazing incidence X-ray diffraction using synchrotron radiation. The results showed broad diffraction peaks, indicating a rather short correlation length of the molecular packing in the range of a few nm. The analysis of the diffraction pattern was based on the known structures of crystalline cellulose, since the observed peak pattern was comparable to cellulose phase II and phase III. The dominant fraction of the film is formed by two different types of layers which are oriented parallel to the substrate surface. The stacking of the layers results in a one-dimensional crystallographic order with a defined interlayer distance of either 7.3 Å or 4.2 Å. As a consequence, two different preferred orientations of the polymer chains are observed. In both cases, polymer chain axes are aligned parallel to the substrate surface, and the orientation of the cellulose molecules are concluded to be either edge-on or flat-on. A minor fraction of the cellulose molecules form nanocrystals that are randomly distributed within the films. In this case, the molecular packing density was found to be smaller in comparison to the known crystalline phases of cellulose.
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Nov 2019
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B21-High Throughput SAXS
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Diamond Proposal Number(s):
[14684, 17118]
Abstract: The gastric peptide hormone human PYY3-36 is a target for the development of therapeutics, especially for treatment of obesity. The conformation and aggregation behaviour of PEGylated and lipidated derivatives of this peptide is examined using a combination of fluorescence dye assays, circular dichroism (CD) spectroscopy, analytical ultracentrifugation (AUC) measurements, small-angle x-ray scattering (SAXS) and cryogenic-transmission electron microscopy (cryo-TEM). The behaviour of two PYY3-36 derivatives lipidated (with octyl chains) in different positions is compared to that of two derivatives with PEG attached at different residues and to that of the native peptide. We find that, unexpectedly, PYY3-36 forms amyloid fibril structures above a critical aggregation concentration. Formation of these structures is suppressed by PEGylation or lipidation. PEGylation significantly reduces the (reversible) loss of α-helix content observed on heating PYY3-36. The PEG conjugates form mainly monomeric structures in solution, coiled coil formation and other aggregation presumably being sterically hindered by swollen PEG chains. However, some small aggregates are detected by AUC. In complete contrast, both of the two lipidated peptides show the formation of spherical micelle-like structures which are small oligomeric aggregates. Our findings show that PEGylation and lipidation are complementary strategies to tune the conformation and aggregation of the important gastric peptide hormone human PYY3-36.
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Sep 2018
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B21-High Throughput SAXS
I22-Small angle scattering & Diffraction
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Diamond Proposal Number(s):
[17902, 15778, 17118]
Abstract: The activity of antimicrobial peptides stems from their interaction with bacterial membranes, which are disrupted according to a number of proposed mechanisms. Here, we investigate the interaction of a model antimicrobial peptide that contains a single arginine residue with vesicles containing model lipid membranes. The surfactant-like peptide Ala6-Arg (A6R) is studied in the form where both termini are capped (CONH-A6R-NH2, capA6R) or uncapped (NH2-A6R-OH, A6R). Lipid membranes are selected to correspond to model anionic membranes (POPE/POPG) resembling those in bacteria or model zwitterionic membranes (POPC/DOPC) similar to those found in mammalian cells. Viable antimicrobial agents should show activity against anionic membranes but not zwitterionic membranes. We find, using small-angle X-ray scattering (SAXS) and cryogenic-TEM (transmission electron microscopy) that, uniquely, capA6R causes structuring of anionic membranes due to the incorporation of the peptide in the lipid bilayer with peptide β-sheet conformation revealed by circular dichroism spectroscopy (CD). There is a preferential interaction of the peptide with POPG (which is the only anionic lipid in the systems studied) due to electrostatic interactions and bidentate hydrogen bonding between arginine guanidinium and lipid phosphate groups. At a certain composition, this peptide leads to the remarkable tubulation of zwitterionic phosphatidylcholine (PC) vesicles, which is ascribed to the interaction of the peptide with the outer lipid membrane, which occurs without penetration into the membrane. In contrast, peptide A6R has a minimal influence on the anionic lipid membranes (and no β-sheet peptide structure is observed) but causes thinning (lamellar decorrelation) of zwitterionic membranes. We also investigated the cytotoxicity (to fibroblasts) and antimicrobial activity of these two peptides against model Gram positive and Gram negative bacteria. A strong selective antimicrobial activity against Gram positive Listeria monocytogenes, which is an important food-borne pathogen, is observed for capA6R. Peptide A6R is active against all three studied bacteria. The activity of the peptides against bacteria and mammalian cells is related to the specific interactions uncovered through our SAXS, cryo-TEM, and CD measurements. Our results highlight the exquisite sensitivity to the charge distribution in these designed peptides and its effect on the interaction with lipid membranes bearing different charges, and ultimately on antimicrobial activity.
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May 2018
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I22-Small angle scattering & Diffraction
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Diamond Proposal Number(s):
[12950, 15246]
Abstract: A recent strategy that has emerged for the design of increasingly functional hydrogels is the incorporation of nanofillers in order to exploit their specific properties to either modify the performance of the hydrogel or add functionality. The emergence of carbon nanomaterials in particular has provided great opportunity for the use of graphene derivatives (GDs) in biomedical applications. The key challenge when designing hybrid materials is the understanding of the molecular interactions between the matrix (peptide nanofibers) and the nanofiller (here GDs) and how these affect the final properties of the bulk material. For the purpose of this work, three gelling β-sheet forming self-assembling peptides with varying physiochemical properties and five GDs with varying surface chemistries were chosen to formulate novel hybrid hydrogels. First the peptide hydrogels and the GDs where characterised, subsequently the molecular interaction between peptides nanofibres and GDs where probed before formulating and mechanically characterising the hybrid hydrogels. We show how the interplay between electrostatic interactions, that can be attractive or repulsive, and hydrophobic (and π-π in the case of peptide containing phenylalanine) interactions, that are always attractive, play a key role on the final properties of the hybrid hydrogels. The shear modulus of the hydrid hydrogels is shown to be related to the strength of fibre adhesion to the flakes, the overall hydrophobicity of the peptides as well as the type of fibrillar network formed. Finally the cytotoxicity of the hybrid hydrogel formed at pH 6 was also investigated by encapsulating and culturing human mesenchyme stem cells (hMSC) over 14 days. This work clearly shows how interactions between peptides and GDs can be used to tailor the mechanical properties of the resulting hydrogels allowing the incorporations of GD nanofillers in a controlled way and opening the possibility to exploit their intrinsic properties to design novel hybrid peptide hydrogels for biomedical applications.
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Apr 2018
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B21-High Throughput SAXS
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Diamond Proposal Number(s):
[14684, 15778]
Abstract: We investigate the self-assembly of two telechelic star polymer-peptide conjugates based on poly(ethylene oxide) (PEO) four-arm star polymers capped with oligotyrosine. The conjugates were prepared via N-carboxy anhydride (NCA)-mediated ring-opening polymerization from PEO star polymer macroinitiators. Self-assembly occurs above a critical aggregation concentration determined via fluorescence probe assays. Peptide conformation was examined using circular dichroism spectroscopy. The structure of self-assembled aggregates was probed using small-angle X-ray Scattering (SAXS) and cryogenic transmission electron microscopy (Cryo-TEM). In contrast to previous studies on linear telechelic PEO-oligotyrosine conjugates which show self-assembly into -sheet fibrils, the star architecture suppresses fibril formation and instead micelles are generally observed, a small population of fibrils only being observed upon pH adjustment. Hydrogelation is also suppressed by the polymer star architecture. However, hydrogels can be produce by addition of sodium alginate with calcium chloride. These peptide-functionalized star polymer solutions are cytocompatible at sufficiently low concentration. These polymers present tyrosine at high density and may be useful in the development of future enzyme or pH-responsive biomaterials.
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Dec 2017
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Abstract: Polymer stabilized nanodiscs are self-assembled structures composed of a polymer belt that wraps around a segment of lipid bilayer, and as such are capable of encapsulating membrane proteins directly from the cell membrane. To date, most studies on these nanodiscs have used poly(styrene-co-maleic acid) (SMA) with the term SMA-lipid particles (SMALPs) coined to describe them. In this study, we have determined the physical and thermodynamic properties of such nanodiscs made with two different SMA copolymers. These include a widely used and commercially available statistical poly(styrene-co-maleic acid) copolymer (coSMA) and a RAFT-synthesized copolymer with narrow molecular weight distribution and alternating styrene and maleic acid groups with a polystyrene tail, (altSMA). We define phase diagrams for each polymer, and show that, regardless of polymer topological structure, self-assembly is driven by the free energy change associated with the polymers. We also show that nanodisc size is polymer dependent, but can be modified by varying polymer concentration. The thermal stability of each nanodisc type is similar and both can effectively solubilize proteins from the E. coli membrane. These data show the potential for the development of different SMA polymers with controllable properties to produce nanodiscs that can be optimized for specific applications and will enable more optimized and widespread use of the SMA-based nanodiscs in membrane protein research.
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Dec 2017
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