I22-Small angle scattering & Diffraction
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Diamond Proposal Number(s):
[33542]
Open Access
Abstract: We report on the lyotropic phase behaviour of fully-hydrated mixtures of α-tocopherol (α-toc) with the unsaturated phospholipid dioleoyl phosphatidylcholine (DOPC), as studied by synchrotron small-angle x-ray diffraction. Increasing amounts of α-toc progressively swell the layer spacing of the fluid lamellar Lα phase of DOPC, and then induce a transition to an inverse hexagonal HII phase. Low-resolution electron density profiles show that this increase is largely due to an increased thickness of the bilayer, with little change in the water layer thickness. In the range 30 – 50 mol% α-toc, additional weak low-angle peaks were observed, whose characteristic ratios are in agreement with the presence of swollen inverse bicontinuous cubic phases of spacegroups Im3m / Pn3m. This research has applications both in the biological field and for industrial product development. We show that the effect of α-toc addition in DOPC membranes has some similarities to that of cholesterol by stabilizing inverse curvature structures, which play crucial roles in cell division, membrane trafficking and endocytosis. Concerning industrial applications, the stabilization of inverted hexagonal (HII) and swollen bicontinuous cubic phases offers the opportunity to develop new delivery systems.
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Aug 2025
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B21-High Throughput SAXS
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Diamond Proposal Number(s):
[38885]
Open Access
Abstract: Cholesterol is an essential sterol in cell membranes that regulates organization and fluidity. This biomolecule has been identified as one of the critical factors in the internalization process of various viruses in human cells. Therefore, understanding these mechanisms is crucial for a deeper comprehension of viral pathogenicity in the search for practical therapeutic approaches against viral diseases. The biochemical and biophysical processes related to these diseases are highly complex. For this reason, studying model systems capable of mimicking the interaction of lipid membranes with cholesterol and proteins is fundamental. In this work, we propose to study the structural and elastic changes in mono-, bi-, and tridimensional lipid systems composed of dipalmitoylphosphatidylcholine (PC) with varying amounts of cholesterol in the presence and absence of the S protein (Spike) and its receptor-binding domain (RBD) from SARS-CoV-2. To characterize these systems, we used both experimental and theoretical approaches such as Langmuir trough, atomic force microscopy (AFM), small-angle X-ray scattering (SAXS), electrochemical methods, and molecular dynamics (MD) simulations. With the interpretation of all results obtained in this work, it was possible to propose a structural model of the membrane in the presence of cholesterol and the interaction with the Spike protein and RBD. The behavior of the adsorption isotherm and SAXS data, together with the results provided by MD simulations, led us to conclude that cholesterol in PC monolayers promotes local alterations, inducing the formation of more rigid membrane regions. More importantly, cholesterol plays a crucial role in facilitating the allocation of SARS-CoV-2 proteins in lipid systems. This is especially true for the Spike protein, which displayed a non-ACE2 mediated stable binding to the lipid membrane with high internalization.
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Jun 2025
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I22-Small angle scattering & Diffraction
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Diamond Proposal Number(s):
[29532]
Open Access
Abstract: Ultrasound standing waves (USW) produce a force capable of displacing micrometer-sized free-flowing particles in a fluid, wherein this phenomenon is also referred to as acoustophoresis. However, the effect of acoustophoresis on dynamically changing and growing crystal networks is unclear. An example of such a system are monoglyceride (MG)-based oleogels, which are free-flowing lipids (e.g., vegetable oils) structured with a lipid-crystal network. In this work, we use MG oleogels as an example system to investigate the acoustophoretic effect on the structuration of a growing crystal network. For this purpose, multifaceted characterization is conducted utilizing optical and coded excitation scanning acoustic microscopy as well as small-angle X-ray scattering, respectively. The optical microscopy results show that USW produces local density differences of the structuring crystalline material and induces the orientation of the MG platelets. X-ray diffraction measurements confirm these findings and show a 23% average increase in MG platelet correlation length, which can be linked to platelet thickness, as well as an increase in the MG nanoplatelet surface smoothness. These findings produce a foundation for better understanding the effect of acoustophoresis in dynamically developing lipid-based materials and illuminate the mechanical changes that arise because of USW treatment.
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Jun 2025
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B21-High Throughput SAXS
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Diamond Proposal Number(s):
[38885]
Open Access
Abstract: This study reports the preparation and characterization of zeolitic imidazolate framework-67 (ZIF-67) through the recycling of spent synthesis liquor containing unreacted 2-methylimidazole. The recycling enhances the yield of ZIF-67 while maintaining the dodecahedral rhombic morphology of the particles. In the first synthesis, a molar ratio of 1:26 (Co2:2-methylimidazole) results in particles with an average diameter of 230 nm and a surface area of 1374 m2 g−1. The first recycling step produces ZIF-67 particles that doubl in diameter and surface area, reaching 520 nm and 1690 m2 g−1, respectively. In the second recycling step, the particles further increase to 1040 nm in diameter and 1806 m2 g−1 in surface area. This increase in diameter is attributed to changes in the metal-to-ligand ratio, which affects the nucleation and growth rates. Increased surface area is linked to a reduction in the average micropore diameter, which decreases from 1.42 nm (first synthesis) to 1.37 nm (second recycling step). There is a 6 m2 g−1 increase in surface area for every 0.001 cm3 g−1 increase in the volume of micropores. This indicates that the spent liquor can be utilized in consecutive batches to produce ZIF-67, minimizing reagent waste.
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Jun 2025
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labSAXS-Offline SAXS and Sample Environment Development
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Abstract: Adequate drug solubility in the gastrointestinal tract is essential for systemic therapy of orally administered medications. In order to measure the solubility of poorly soluble drugs in vitro, simulated intestinal fluid (SIF) is often used in place of human intestinal fluid (HIF). A suite of fasted state SIF, based on variability observed in a range of fasted state HIF samples was designed and used to study the relationship between the solubility of eight poorly soluble biopharmaceutics classification system class II drugs and the particle size of the colloidal structures formed by the drugs in the fluid. The drugs of interest included three acidic drugs (naproxen, indomethacin and phenytoin), three neutral drugs (felodipine, fenofibrate, griseofulvin) and two basic drugs (carvedilol and tadalafil). The overall aim of this research is to work towards a better understanding of the colloidal structures formed in SIF. Solubility was measured using high performance liquid chromatography and results indicated that the solubility was typically greater in the acidic drugs than in the neutrals or bases and that the solubility tended to increase with increasing media point (pH × [TAC]). Particle size was determined using both dynamic light scattering and nanoparticle tracking analysis. Dynamic light scattering data confirmed the polydispersity of size distribution within the samples analysed. Typically, as the concentration of amphiphiles (total amphiphile concentration ([TAC])) is increased, the particle size of the structures measured decreases. A comparison with the solubility data revealed that the general trend indicated that while solubility is to some extent affected by pH and [TAC] or (pH × [TAC]), the relationship between solubility and particle size is linked with [TAC]. Small angle X-ray scattering (SAXS) analysis was carried out at the Diamond Light Source national facility, using the laboratory SAXS (labSAXS) beamline on drug and drug free SIF samples and the data was processed at the University of Strathclyde. Unfortunately, there was no significant scattering measured in the sample fluids which is thought to be a result of samples that are too weakly scattering to be detected by a labSAXS instrument. The data obtained serves as excellent preliminary data for a future beamtime application using a synchrotron beamline. The final work explores a model, using simple mathematics, to estimate the number of drug molecules per colloid or mixed micelle structure in a series of SIF. The experimental data, collected in earlier chapters, was applied in both this calculation and a calculation to estimate the solubility enhancement provided in the SIF media. Analysis of the data and results indicates that there is a direct relationship between particle size of the colloidal structures and the number of estimated drug molecules per structure. As expected, as the particle size decreases, as does the estimated number of drug molecules per micelle. The larger structures can accommodate a greater number of drug molecules per micelle. Solubility enhancement was also calculated, with the acidic drugs, naproxen and indomethacin proving to be most solubility enhanced in the suite of simulated intestinal fluid.
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Jun 2025
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B21-High Throughput SAXS
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Alexander
Leithner
,
Oskar
Staufer
,
Tanmay
Mitra
,
Falk
Liberta
,
Salvatore
Valvo
,
Mikhail
Kutuzov
,
Hannah
Dada
,
Jacob
Spaeth
,
Weijie
Zhou
,
Felix
Schiele
,
Sophia
Reindl
,
Herbert
Nar
,
Stefan
Hoerer
,
Maureen
Crames
,
Stephen
Comeau
,
David
Young
,
Sarah
Low
,
Edward
Jenkins
,
Simon J.
Davis
,
David
Klenerman
,
Andrew
Nixon
,
Noah
Pefaur
,
David
Wyatt
,
Omer
Dushek
,
Srinath
Kasturirangan
,
Michael L.
Dustin
Open Access
Abstract: Bispecific T cell engagers (TcEs) link T cell receptors to tumor-associated antigens on cancer cells, forming cytotoxic immunological synapses (IS). Close membrane-to-membrane contact (≤13 nm) has been proposed as a key mechanism of TcE function. To investigate this and identify potential additional mechanisms, we compared four immunoglobulin G1-based (IgG1) TcE Formats (A–D) targeting CD3ε and Her2, designed to create varying intermembrane distances (A < B < C < D). Small-angle X-ray scattering (SAXS) and modeling of the conformational states of isolated TcEs and TcE–antigen complexes predicted close contacts (≤13 nm) for Formats A and B and far contacts (≥18 nm) for Formats C and D. In supported lipid bilayer (SLB) model interfaces, Formats A and B recruited, whereas Formats C and D repelled, CD2–CD58 interactions. Formats A and B also excluded bulky Quantum dots more effectively. SAXS also revealed that TcE–antigen complexes formed by Formats A and C were less flexible than complexes formed by Formats B and D. Functional data with Her2-expressing tumor cells showed cytotoxicity, surface marker expression, and cytokine release following the order A > B = C > D. In a minimal system for IS formation on SLBs, TcE performance followed the trend A = B = C > D. Addition of close contact requiring CD58 costimulation revealed phospholipase C-γ activation matching cytotoxicity with A > B = C > D. Our findings suggest that when adhesion is equivalent, TcE potency is determined by two parameters: contact distance and flexibility. Both the close/far-contact formation axis and the low/high flexibility axis significantly impact TcE potency, explaining the similar potency of Format B (close contact/high flexibility) and C (far contact/low flexibility).
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Jun 2025
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B21-High Throughput SAXS
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Diamond Proposal Number(s):
[34289]
Open Access
Abstract: Pseudomonas aeruginosa employs the Type VI secretion system (T6SS) to outcompete other bacteria in its environment. Among the effectors secreted by the T6SS of P. aeruginosa PAO1, Tse4 is known for its potent antibacterial activity. This study elucidates the molecular function of Tse4, which promotes cell depolarization in competing bacteria. Our results show that Tse4 spontaneously incorporates into lipid monolayers and forms multiionic channels in planar bilayers, with either ohmic conduction or diode-like rectifying currents and a preference for cations over anions. These observations allow us to propose a model of action whereby Tse4 channels couple cell depolarization with K+ efflux. These insights into Tse4’s pore-forming activity enhance our understanding of bacterial competition and exemplify a finely tuned antibacterial strategy, coupling its ability to cause membrane depolarization with potassium efflux that synergises with other T6SS effectors. These results highlight the sophistication of Pseudomonas aeruginosa’s competitive arsenal.
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Jun 2025
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I22-Small angle scattering & Diffraction
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Diamond Proposal Number(s):
[33006]
Open Access
Abstract: Understanding the assembly of small molecules in aqueous media is crucial for the development of adaptive biomaterials. The mechanical properties of supramolecular networks, including stiffness and stress relaxation, play a key role in cellular spreading and can be tuned via formulation strategies or monomer design. Here, we demonstrate the modulation of supramolecular polymerization and cellular response of ureidopyrimidinone (UPy) monomers in water by tailoring the length of the alkyl spacer within the monomer structure. A library of four UPy derivatives with varying hydrophilic–hydrophobic balances was synthesized by using an optimized synthetic approach. The assembly behavior and dynamics of the supramolecular polymers were investigated both in solution and gel states using a wide range of techniques. The results revealed that the alkyl spacer length significantly affects the supramolecular polymer dynamics, kinetics, and stability. Monomers with 6 and 8 methylene units formed dynamic elongated structures, while those with 10 and 12 units yielded robust and stable bundled fibers. In the gel state, a physical cross-linker was required for gel formation. The gels formed by the monomers featuring 8 and 10 methylene units exhibited optimal mechanical properties, promoting the spreading of human normal dermal fibroblasts in both 2D and 3D cultures. These findings highlight the impact of the monomer architecture on the properties of UPy supramolecular systems, paving the way for the rational design of biomaterials with tunable properties.
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Jun 2025
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B21-High Throughput SAXS
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Diamond Proposal Number(s):
[31952]
Abstract: Chirality and non-covalent chemistry are essential features in the construction of complex molecular systems that constitute living organisms. The specific interactions between molecular components with defined geometric orientations enable nature’s dynamism, responsiveness, specificity, and selection. The precision with which nature controls its homochirality and functional structures inspired the design of synthetic supramolecular systems. These systems have aimed at understanding the origins of these properties and mimicking their complexity. Research in the last decades has led to many insights into synthetic and natural supramolecular polymerization processes and principles of amplification of asymmetry. However, the complexity observed in nature remains far from being understood. In particular, detailed studies of assembly mechanisms and amplification of asymmetry principles in water-compatible supramolecular systems are scarce due to the challenging contribution of hydrophobic effects leading to non-equilibrium states of supramolecular assemblies. Valuable contributions to this field have been made in recent years, including the introduction of in situ chemical modifications and dissipative non-equilibrium assembly states, leading to evolutionary features such as self-replication. This chapter introduces the concepts of supramolecular polymerization and shows the analogies between natural and synthetic systems. In particular, the influence of homochirality in natural macroscopic structures is emphasized, showing the importance of this property in water-compatible supramolecular systems. Therefore, key developments in research on the origin of homochirality and challenges for future research to understand this essential feature of life are outlined.
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May 2025
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B21-High Throughput SAXS
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Diamond Proposal Number(s):
[39167]
Open Access
Abstract: We report the first reversible addition–fragmentation chain transfer polymerisation-induced self-assembly (RAFT-PISA) in ionic liquid (IL) that proceeds under emulsion conditions. Moreover, this formulation exploits refractive index contrast matching to generate highly transparent nanoparticle dispersions. Specifically, 1-ethyl-3-methyl-imidazolium ethylsulfate, [EMIM][EtOSO3], was used as the solvent for the chain extension of poly(2-hydroxyethyl methacrylate) (PHEMA) macromolecular chain transfer agents (macro-CTAs) using n-butyl methacrylate (BuMA) via RAFT emulsion polymerisation. Two series of PHEMAx-b-PBuMAy diblock copolymers with target PBuMA degrees of polymerisation (DPs) varying from 50 to 1000 were synthesised using either a PHEMA21 or PHEMA77 macro-CTA. All resulting nanoparticle dispersions yielded highly transparent dispersions, even when nanoparticle diameters exceeded 100 nm, due to the closely matched refractive index values of the [EMIM][EtOSO3] solvent and PBuMA nanoparticle core. Detailed analysis using small-angle X-ray scattering (SAXS) and transmission electron microscopy (TEM) confirmed the presence of spherical nanoparticles. Furthermore, the synthesis of PHEMA-b-PBuMA via this new PISA formulation was directly compared to equivalent block copolymer syntheses conducted in N,N-dimethylformamide (DMF) or ethanol/water mixtures. It was found that syntheses conducted in [EMIM][EtOSO3] resulted in the highest monomer conversions (up to >99%) and lowest dispersity (ĐM) values (as low as 1.16) in the shortest reaction times (2 hours) compared to the other solvent systems. This work demonstrates the use of ILs as a more sustainable and effective solvent for RAFT–PISA via the development of the first emulsion PISA formulation in IL.
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May 2025
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