B21-High Throughput SAXS
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Jacek K.
Wychowaniec
,
Ezgi Irem
Bektas
,
Marcia
Muerner
,
Jiranuwat
Sapudom
,
Martin
Šrejber
,
Marielle
Airoldi
,
Roland
Schmidt
,
Andrea J.
Vernengo
,
Charlotte J. C.
Edwards-Gayle
,
Paul Sean
Tipay
,
Michal
Otyepka
,
Jeremy
Teo
,
David
Eglin
,
Matteo
D'Este
Diamond Proposal Number(s):
[29767]
Open Access
Abstract: Self-assembling peptides (SAPs) are fully defined nanobiomaterials offering unprecedented opportunities to control nanostructure and chemical attributes to investigate and manipulate cellular signals. To investigate the influence of chemical and morphological characteristics on inflammatory signaling in native immunity, we designed five β-sheet SAPs: EFEFKFEFK (EF8), YEFEFKFEFK (YEF8), EFEFKFEFKY (EF8Y), YEFEFKFEFKY (YEF8Y), and EYEFKFEFK (EYF8) (F: phenylalanine; E: glutamic acid; K: lysine, Y: tyrosine). The position of tyrosine in the peptide sequence dictated the self-assembly into nanostructures, with all SAPs self-assembling into thin constituent nanofibers with d ≈ 3.8 ± 0.4 nm, and sequences YEF8 and EF8 showing a propensity for associative bundling. These distinct SAPs induced contrasting inflammatory responses of monocytic model THP-1 cells-derived macrophages (MΦs). Presence of soluble EF8 nanofibers (at 2 mM) induced an anti-inflammatory response and polarization toward an M2 state, whereas YEF8 (at 2 mM) displayed a tendency for inducing a pro-inflammatory response and polarization toward an M1 state. EF8Y, YEF8Y, and EYF8 SAPs did not induce an inflammatory response in our models. These results were validated using peripheral blood mononuclear cells (PBMCs)-derived MΦs from human donors, confirming the critical role of EF8 and YEF8 SAPs as possible orchestrators of the repair of tissues or inducers of pro-inflammatory state, respectively. The same MΦs polarization responses from THP-1-derived MΦs cultured on 20 mM hydrogels were obtained. These findings will facilitate the utilization of this family of SAPs as immunomodulatory nanobiomaterials potentially changing the course of inflammation during the progression of various diseases.
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Apr 2025
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B21-High Throughput SAXS
I22-Small angle scattering & Diffraction
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Mohamed A. N.
Soliman
,
Abdulwahhab
Khedr
,
Tarsem
Sahota
,
Rachel
Armitage
,
Raymond
Allan
,
Katie
Laird
,
Natalie
Allcock
,
Fatmah I.
Ghuloum
,
Mahetab H.
Amer
,
Reem
Alazragi
,
Charlotte J. C.
Edwards-Gayle
,
Jacek K.
Wychowaniec
,
Attilio V.
Vargiu
,
Mohamed A.
Elsawy
Diamond Proposal Number(s):
[28287, 28806]
Open Access
Abstract: Guiding molecular assembly of peptides into rationally engineered nanostructures remains a major hurdle against the development of functional peptide-based nanomaterials. Various non-covalent interactions come into play to drive the formation and stabilization of these assemblies, of which electrostatic interactions are key. Here, the atomistic mechanisms by which electrostatic interactions contribute toward controlling self-assembly and lateral association of ultrashort β-sheet forming peptides are deciphered. Our results show that this is governed by charge distribution and ionic complementarity, both affecting the interaction patterns between charged residues: terminal, core, and/or terminal-to-core attraction/repulsion. Controlling electrostatic interactions enabled fine-tuning nanofiber morphology for the 16 examined peptides, resulting into versatile nanostructures ranging from extended thin fibrils and thick bundles to twisted helical “braids” and short pseudocrystalline nanosheets. This in turn affected the physical appearance and viscoelasticity of the formed materials, varying from turbid colloidal dispersions and viscous solutions to soft and stiff self-supportive hydrogels, as revealed from oscillatory rheology. Atomistic mechanisms of electrostatic interaction patterns were confirmed by molecular dynamic simulations, validating molecular and nanoscopic characterization of the developed materials. In essence, detailed mechanisms of electrostatic interactions emphasizing the impact of charge distribution and ionic complementarity on self-assembly, nanostructure formation, and hydrogelation are reported.
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Jan 2025
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I22-Small angle scattering & Diffraction
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Diamond Proposal Number(s):
[17102]
Open Access
Abstract: Self-assembling peptides remain persistently interesting objects for building nanostructures and further assemble into macroscopic structures, e.g. hydrogels, at sufficiently high concentrations. The modulation of self-assembling β-sheet-forming peptide sequences, with a selection from the full library of amino acids, offers unique possibility for rational tuning of the resulting nanostructured morphology and topology of the formed hydrogel networks. In the present work, we explored how a known β-sheet-disassembling amino acid, proline (P), affects the self-assembly and gelation properties of amphipathic peptides. For this purpose, we modified the backbone of a known β-sheet-forming peptide, FEFKFEFK (F8, F = phenylalanine, E = glutamic acid, and K = lysine), with P to form three sequences: FEFKPEFK (FP), FEFKPEFKF (KPE) and FEFEPKFKF (EPK). The replacement of F by P in the hydrophobic face resulted in the loss of the extended β-sheet conformation of the FP peptide and no gelation at concentration as high as 100 mg mL−1, compared to typical 5 mg mL−1 concentration corresponding to F8. However, by retaining four hydrophobic phenylalanine amino acids in the sequences, hydrogels containing a partial β-sheet structure were still formed at 30 mg mL−1 for KPE (pH 4–10) and EPK (pH 2–5). TEM, AFM, small-angle X-ray scattering (SAXS) and wide-angle X-ray scattering (WAXS) revealed that KPE and EPK peptides self-assemble into nanoribbons and twisted nanofibers, respectively. Molecular dynamics confirmed that the single amino acid replacement of F by P prevented the assembly of the FP peptide with respect to the stable β-sheet-forming F8 variant. Moreover, additional prolongation by F in the KPE variant and shuffling of the polar amino acid sequence in the EPK peptide supported aggregation capabilities of both variants in forming distinct shapes of individual aggregates. Although the overall number of amino acids is the same in both KPE and EPK, their shifted charge density (i.e., the chemical environment in which ionic groups reside) drives self-assembly into distinct nanostructures. The investigated structural changes can contribute to new material designs for biomedical applications and provide better understanding in the area of protein folding.
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Nov 2024
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I22-Small angle scattering & Diffraction
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Diamond Proposal Number(s):
[28806]
Open Access
Abstract: The influence of surfactant, cross-linker, and initiator on the final structure and thermoresponse of poly(N-isopropylmethacrylamide) (pNIPMAM) microgels was evaluated. The goals were to control particle size (into the nanorange) and transition temperature (across the physiologically accessible range). The concentration of the reactants used in the synthesis was varied, except for the monomer, which was kept constant. The thermoresponsive suspensions formed were characterized by dynamic light scattering, small-angle X-ray scattering, atomic force microscopy, and rheology. Increasing surfactant, sodium dodecyl sulfate content, produced smaller microgels, as expected, into the nanorange and with greater internal entanglement, but with no change in phase transition temperature (LCST), which is contrary to previous reports. Increasing cross-linker, N,N-methylenebis acrylamide, content had no impact on particle size but reduced particle deformability and, again contrary to previous reports of decreases, progressively increased the LCST from 39 to 46 °C. The unusual LCST trends were confirmed using different rheological techniques. Initiator, potassium persulfate, content was found to weakly influence the outcomes. An optimized content was identified that provides functional nanogels in the 100 nm (swollen) size range with controlled LCST, just above physiological temperature. The study contributes chemistry-derived design rules for thermally responsive colloidal particles with physiologically accessible LCST for a variety of biomedical and soft robotics applications.
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Aug 2024
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B21-High Throughput SAXS
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Diamond Proposal Number(s):
[28287]
Abstract: A library of composite polymer networks (CPNs) were formed by combining Pluronic F127, as the primary gelator, with a range of di-acrylate functionalised PEG polymers, which tune the rheological properties and provide UV crosslinkability. A coarse-grained sol–gel room temperature phase diagram was constructed for the CPN library, which identifies PEG-dependent disruption of micelles as leading to liquefication. Small angle X-ray scattering and rheological measurements provide detailed insight into; (i) micelle-micelle ordering; (ii) micelle-micelle disruption, and; (iii) acrylate-micelle disruption; with contributions that depend on composition, including weak PEG chain length and end group effects. The influence of composition on 3D extrusion printability through modulation of the cohesive/hydrophobic interactions was assessed. It was found that only micelle content provides consistent changes in printing fidelity, controlled largely by printing conditions (pressure and feed rate). Finally, the hydrogels were shown to be UV photo-crosslinkable, which further improves fidelity and structural integrity, and usefully reduces the mesh size. Our results provide a guide for design of 3D-printable CPN inks for future biomedical applications.
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Jan 2024
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B21-High Throughput SAXS
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Diamond Proposal Number(s):
[29767]
Open Access
Abstract: Sterilisation of implantable biomaterials such as hydrogels remains a key step towards their clinical translation. Standard sterilisation methods can significantly alter hydrogels' physicochemical and biological performance. Previously, we developed composite hydrogels based on ε-polylysine (ε-PL) and hyaluronic acid (HA). The developed hydrogels demonstrated promising antibacterial activity and in vitro cell viability and their variable properties depending on the chosen ε-PL to HA ratio. In this study, we fabricated a series of chemically cross-linked ԑ-PL/HA hydrogels with expanded ԑ-PL to HA mass ratios.
Using small-angle X-ray scattering (SAXS), we unravelled the topological differences between physically and chemically crosslinked hydrogels. We then selected the chemically crosslinked hydrogel ԑ-PL/HA series of 60:40 wt%, 70:30 wt%, and 80:20 wt% ratios, with similar network topologies, to evaluate the impact of steam sterilisation on their physicochemical and viscoelastic properties. The antibacterial activity of the sterilized hydrogels was also evaluated against Gram-negative and Gram-positive bacteria. Our results showed that steam sterilisation minimally affects structure and physicochemical properties of ԑ-PL/HA hydrogels. Furthermore, the developed hydrogel ԑ-PL/HA series of 60:40 wt%, 70:30 wt%, and 80:20 wt% ratios showed pronounced antibacterial activity against Gram-negative and Gram-positive pathogenic bacteria. We expect our results will contribute to the growing understanding of using sterilisation methods for antibacterial hydrogels that have the potential for wider tissue engineering applications.
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Dec 2023
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B21-High Throughput SAXS
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Diamond Proposal Number(s):
[26998]
Abstract: Topographical cues on materials can manipulate cellular fate, particularly for neural cells that respond well to such cues. Utilizing biomaterial surfaces with topographical features can effectively influence neuronal differentiation and promote neurite outgrowth. This is crucial for improving the regeneration of damaged neural tissue after injury. Here, we utilized groove patterns to create neural conduits that promote neural differentiation and axonal growth. We investigated the differentiation of human neural stem cells (NSCs) on silicon dioxide groove patterns with varying height-to-width/spacing ratios. We hypothesize that NSCs can sense the microgrooves with nanoscale depth on different aspect ratio substrates and exhibit different morphologies and differentiation fate. A comprehensive approach was employed, analyzing cell morphology, neurite length, and cell-specific markers. These aspects provided insights into the behavior of the investigated NSCs and their response to the topographical cues. Three groove-pattern models were designed with varying height-to-width/spacing ratios of 80, 42, and 30 for groove pattern widths of 1 μm, 5 μm, and 10 μm and nanoheights of 80 nm, 210 nm, and 280 nm. Smaller groove patterns led to longer neurites and more effective differentiation towards neurons, whereas larger patterns promoted multidimensional differentiation towards both neurons and glia. We transferred these cues onto patterned polycaprolactone (PCL) and PCL-graphene oxide (PCL-GO) composite ‘stamps’ using simple soft lithography and reproducible extrusion 3D printing methods. The patterned scaffolds elicited a response from NSCs comparable to that of silicon dioxide groove patterns. The smallest pattern stimulated the highest neurite outgrowth, while the middle-sized grooves of PCL-GO induced effective synaptogenesis. We demonstrated the potential for such structures to be wrapped into tubes and used as grafts for peripheral nerve regeneration. Grooved PCL and PCL-GO conduits could be a promising alternative to nerve grafting.
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Oct 2023
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Open Access
Abstract: In this chapter, we will thoroughly discuss characterization techniques used to elucidate the exact structure and define properties of peptide-based nanomaterials. In particular we divide methods into: 1.
Quality control performance (mass spectroscopy and high-performance liquid chromatography.
2.
Spectroscopy (Fourier transform infrared spectroscopy, Raman spectroscopy, circular and linear dichroism, nuclear magnetic resonance and fluorescence spectroscopy).
3.
Microscopy (scanning and transmission electron microscopies, atomic force microscopy, optical and polarized light microscopy).
4.
Scattering (small angle X-ray and neutron scattering, X-ray diffraction).
5.
Bulk structures (mainly hydrogels) rheological characterization. The methodology is described for molecular structures, self-assembled nanostructures and aggregates, as well as hybrid, composite and/or conjugated nanomaterials and their bulk forms. Both common, as well as more exotic versions of all methods are presented in the context of peptide-based nanomaterials. Where utilized, examples of combinatorial use of techniques are demonstrated. Representative studies accompany the discussion and usefulness of all presented methods.
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Aug 2023
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B21-High Throughput SAXS
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Diamond Proposal Number(s):
[28806, 28287]
Abstract: Bioinspired de novo self-assembling peptides have been widely used for the development of soft biomaterials for a wide variety of biomedical and pharmaceutical applications, such as cell scaffolding for tissue engineering1, controlled and localised drug delivery2, biosensing3, and many others. The meticulous control of peptide-based nanomaterial properties over the length scale, by molecular design, remains the main challenge for tailoring biomaterials properties to meet the application needs. In our group, we have recently adopted a minimalistic molecular engineering approach for the development of Ultrashort Ionic-complementary Constrained Peptides (UICPs), which were rationally designed to self-assemble into amphiphilic β-sheet nanofibers with unique hydrogelation properties and surface activity.4 We have previously demonstrated the crucial role played by aromatic stacking for the formation and thermodynamic stabilisation of UICP β-sheet structures. Herein, we will show how charge interactions can be manipulated for fine tuning molecular self-assembly, morphology and size of nanofibrous structures formation and viscoelasticity of UICP hydrogels.
A library of 18 peptide sequences (4-5 residues long) was developed to study the effect of the sequence net charge, charge density distribution, reversal of charge order and ionic self-complementarity on their propensity towards self-assembly and gelation. Interestingly, 12 of these peptides self-assembled into β-sheet nanofibrous structures forming hydrogels at pH 4.5-5, as confirmed by ATR-FTIR, SEM, TEM, SAXS and oscillatory rheology. Full control over β-sheet content (ranging from ~30-80%), fibre morphology (thin fibrils, thick straight fibre bundles, twisted helical nanofibres, flat nanoribbons and nanotubes) and sizes (~4-67 nm in diameter), as well as gelation (critical gelation concentrations ranging from <7.5 to >100 mM) and viscoelastic properties (storage moduli G’ ~0.1-100 KPa) was achieved by the careful positioning of both Glu and Lys residues at both C- and N-termini, in the sequence core and on both the hydrophilic and hydrophobic faces of the peptide chain. In essence, this design approach enabled/disabled lateral growth along the β-sheet ladder via electrostatic attraction (counter charge, anion-pi and cation-pi)/repulsion, hence controlling fibre thickness, morphology, entanglement, and the resulting viscoelasticity of the system. Our UICPs platform thus provides the flexibility in peptide molecular design for the manufacturing of soft biomaterials with versatile properties that can be in future tailored to the relevant biomedical application.
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Apr 2023
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I22-Small angle scattering & Diffraction
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Diamond Proposal Number(s):
[15246, 15478]
Open Access
Abstract: The current work investigates the effect of the addition of graphene nanoplatelets (GNPs) and graphene oxide (GO) to high hard-segment polyurethane (75% HS) on its thermal, morphological, and mechanical properties. Polyurethane (PU) and its nanocomposites were prepared with different ratios of GNP and GO (0.25, 0.5, and 0.75 wt. %). A thermal stability analysis demonstrated an enhancement in the thermal stability of PU with GNP and GO incorporated compared to pure PU. Differential Scanning Calorimetry (DSC) showed that both GNP and GO act as heterogeneous nucleation agents within a PU matrix, leading to an increase in the crystallinity of PU. The uniform dispersion and distribution of GNP and GO flakes in the PU matrix were confirmed by SEM and TEM. In terms of the mechanical properties of the PU nanocomposites, it was found that the interaction between PU and GO was better than that of GNP due to the functional groups on the GO’s surface. This leads to a significant increase in tensile strength for 0.5 wt. % GNP and GO compared with pure PU. This can be attributed to interfacial interaction between the GO and PU chains, resulting in an improvement in stress transferring from the matrix to the filler and vice versa. This work sheds light on the understanding of the interactions between graphene-based fillers and their influence on the mechanical properties of PU nanocomposites.
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Oct 2022
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