Time-resolved Small-angle Neutron Scattering Research Articles

Time-resolved small-angle neutron scattering for characterization of molecular exchange in lipid nanoparticle therapeutics

HypothesisNano-scale dynamics of self-assembled therapeutics play a large role in their biological function. However, assessment of such dynamics remains absent from conventional pharmaceutical characterization. We hypothesize that time-resolved small-angle neutron scattering (TR-SANS) can reveal their kinetic properties. For lipid nanoparticles (LNP), limited molecular motion is important for avoiding degradation prior to entering cells while, intracellularly, enhanced molecular motion is then vital for effective endosomal escape. We propose TR-SANS for quantifying molecular exchange in LNPs and, therefore, enabling optimization of opposing molecular behaviors of a pharmaceutical in two distinct environments. ExperimentsWe use TR-SANS in combination with traditional SANS and small-angle x-ray scattering (SAXS) to experimentally quantify nano-scale dynamics and provided unprecedented insight to molecular behavior of LNPs. FindingsLNPs have molecular exchange dynamics relevant to storage and delivery which can be captured using TR-SANS. Cholesterol exchanges on the time-scale of hours even at neutral pH. As pH drops below the effective pKa of the ionizable lipid, molecular exchange occurs faster. The results give insight into behavior enabling delivery and provide a quantifiable metric by which to compare formulations. Successful analysis of this multi-component system also expands the opportunities for using TR-SANS to characterize complex therapeutics.

Skyrmion lattice formation and destruction mechanisms probed with TR-SANS.

Magnetic skyrmions are topologically protected, nanoscale whirls of the spin configuration that tend to form hexagonally ordered arrays. As a topologically non-trivial structure, the nucleation and annihilation of the skyrmion, as well as the interaction between skyrmions, varies from conventional magnetic systems. Recent works have suggested that the ordering kinetics in these materials occur over millisecond or longer timescales, which is unusually slow for magnetic dynamics. The current work investigates the skyrmion ordering kinetics, particularly during lattice formation and destruction, using time-resolved small angle neutron scattering (TR-SANS). Evaluating the time-resolved structure and intensity of the neutron diffraction pattern reveals the evolving real-space structure of the skyrmion lattice and the timeframe of the formation. Measurements were performed on three prototypical skyrmion materials: MnSi, (Fe,Co)Si, and Cu2OSeO3. To probe lattice formation and destruction kinetics, the systems were prepared in the stable skyrmion state, and then a square-wave magnetic field modulation was applied. The measurements show that the skyrmions quickly form ordered domains, with a significant distribution in lattice parameters, which then converge to the final structure; the results confirm the slow kinetics, with formation times between 10 ms and 99 ms. Comparisons are made between the measured formation times and the fundamental material properties, suggesting the ordering temperature, saturation magnetization and magnetocrystalline anisotropy may be driving the timeframes. Micromagnetic simulations were also performed and support a scaling of the kinetics with sample volume, a behavior which is caused by the reconciling of misaligned domains.

Dynamic Exchange of Amphiphilic Random Copolymers between Micelles in Water: Kinetics and Mechanism Analyzed by TR-SANS

Amphiphilic random copolymers bearing poly(ethylene glycol) (PEG) and alkyl groups as side chains are intermolecularly self-assembled into size-controlled multichain micelles in water. The random copolymer micelles are known to induce the exchange of their polymer chains, whereas the details of the kinetics and mechanism have not been elucidated yet. Herein, we investigated the exchange kinetics and mechanism of the random copolymer chains between their micelles by time-resolved small-angle neutron scattering (TR-SANS). For this purpose, random copolymers carrying PEG and deuterated butyl or dodecyl groups were designed for deuterated micelles. After mixing deuterated and non-deuterated micelle solutions, the resulting mixtures were monitored by TR-SANS at various concentrations and temperatures. The scattering intensity of the micelle mixtures decayed with time, indicating that deuterated copolymers were gradually mixed with non-deuterated copolymers via chain exchange between their micelles to form micelles consisting of both deuterated and non-deuterated copolymers. The kinetic analysis revealed that the exchange of their polymer chains involved two mechanisms: A unimer release and insertion pathway was dominant in diluted conditions, whereas the contribution of a micelle collision pathway increased with increasing total polymer concentration and temperature. The activation energy of the polymer exchange process was dependent on the hydrophobic alkyl groups and larger than that of a related surfactant micelle.

Determination of water permeability through stress-free lipid membranes using time-resolved small angle neutron scattering.

The lipid bilayer, the basic unit of biological membranes, is an essentially impermeable barrier. Because of this, the environment inside the cell can be significantly different from the environment surrounding it, such as pH, ion compositions and biomolecular concentrations. Since several cellular functions require the fast transport of large or charged molecules through the membrane, the cell membrane contains specialized proteins that specifically transport these across the membrane. Notwithstanding permeation happens via passive diffusion as well. This type of transport, we imagine, is slow, even for small molecules, such as water. Indeed, the cell has active water transporters (aquaporin) to move water through its membranes faster. The time and energy required to move water across the lipid bilayer passively is still debated, however. Several techniques have been used to measure water permeation such as NMR, fluorescence, and micropipetting. Here we take advantage of contrast between H2O and D2O to investigate, through time-resolved SANS, the water permeability of stress-free membranes composed of different saturated lipids. The principle is to follow the scattering intensity of a hydrogenated vesicle solution, initially prepared in H2O, when diluted with D2O. The resulting changes in scattering are a result of the intake of D2O into the vesicle's interior without any induced stress on the membrane. Our results show that water permeation in membranes in the gel state is faster, by at least an order of magnitude, than previously reported. The water permeation changes due to cholesterol in liquid ordered phases is also discussed.

Investigating the cut-off effect of n-alcohols on lipid movement: a biophysical study.

Cellular membranes are responsible for absorbing the effects of external perturbants for the cell's survival. Such perturbants include small ubiquitous molecules like n-alcohols which were observed to exhibit anesthetic capabilities, with this effect tapering off at a cut-off alcohol chain length. To explain this cut-off effect and complement prior biochemical studies, we investigated a series of n-alcohols (with carbon lengths 2-18) and their impact on several bilayer properties, including lipid flip-flop, intervesicular exchange, diffusion, membrane bending rigidity and more. To this end, we employed an array of biophysical techniques such as time-resolved small angle neutron scattering (TR-SANS), small angle X-ray scattering (SAXS), all atomistic and coarse-grained molecular dynamics (MD) simulations, and calcein leakage assays. At an alcohol concentration of 30 mol% of the overall lipid content, TR-SANS showed 1-hexanol (C6OH) increased transverse lipid diffusion, i.e. flip-flop. As alcohol chain length increased from C6 to C10 and longer, lipid flip-flop slowed by factors of 5.6 to 32.2. Intervesicular lipid exchange contrasted these results with only a slight cut-off at alcohol concentrations of 30 mol% but not 10 mol%. SAXS, MD simulations, and leakage assays revealed changes to key bilayer properties, such as bilayer thickness and fluidity, that correlate well with the effects on lipid flip-flop rates. Finally, we tie our results to a defect-mediated pathway for alcohol-induced lipid flip-flop.

New approach for SANS measurement of micelle chain mixing during size and morphology transitions.

Chain exchange in amphiphilic block polymer micelles is measurable with time-resolved small-angle neutron scattering (TR-SANS) where contrast-matched conditions reveal chain mixing as reduced intensity. However, analyzing chain mixing on short time scales e.g. during micelle transformations remains challenging. SANS model fitting can quantify chain mixing during size and morphology changes, however short acquisition times lead to lower data statistics (higher error). Such data are unsuitable for form factor fitting, especially with polydisperse and/or multimodal scenarios. An integrated-reference approach, R(t), is compatible with such data by using fixed reference patterns for the unmixed and fully mixed states that are each integrated to improve data statistics (lower error). Although the R(t) approach is tolerant of low data statistics, it remains incompatible with size and morphology changes. A new shifting references relaxation approach, SRR(t), is proposed where reference patterns are acquired at each time point to enable mixed state calculations regardless of short acquisition times. The additional experimental measurements needed are described which provide these time-varying reference patterns. The use of reference patterns makes the SRR(t) approach size/morphology-agnostic, allowing for the extent of micelle mixing to be directly calculated without this knowledge. SRR(t) is thus compatible with arbitrary levels of complexity and can provide accurate assessment of the mixed state which could support future model analysis. Calculated scattering datasets were used to demonstrate the SRR(t) approach during multiple size, morphology, and solvent conditions (scenarios 1-3). The mixed state calculated from the SRR(t) approach is shown to be accurate for all three scenarios.

Observing protein degradation in solution by the PAN-20S proteasome complex: Astate-of-the-art example of bio-macromolecular TR-SANS.

In the present book chapter we illustrate the state-of-the-art of time-resolved small-angle neutron scattering (TR-SANS) by a concrete example of a dynamic bio-macromolecular system, i.e., regulated protein degradation by the archaeal PAN-20S proteasome complex. We present the specific and unique structural information that can be obtained by this approach, in combination with bio-macromolecular deuteration and online spectrophotometric measurements of a fluorescent substrate (GFP). The complementarity with atomic-resolution structural biology techniques (SAXS, NMR, crystallography and cryo-EM) and with the advent of atomic structure prediction are discussed, as well as the respective limitations and future perspectives.

Dynamics and Equilibration Mechanisms in Block Copolymer Particles.

Self-assembly of block copolymers into interesting and useful nanostructures, in both solution and bulk, is a vibrant research arena. While much attention has been paid to characterization and prediction of equilibrium phases, the associated dynamic processes are far from fully understood. Here, we explore what is known and not known about the equilibration of particle phases in the bulk, and spherical micelles in solution. The presumed primary equilibration mechanisms are chain exchange, fusion, and fragmentation. These processes have been extensively studied in surfactants and lipids, where they occur on subsecond time scales. In contrast, increased chain lengths in block copolymers create much larger barriers, and time scales can become prohibitively slow. In practice, equilibration of block copolymers is achievable only in proximity to the critical micelle temperature (in solution) or the order-disorder transition (in the bulk). Detailed theories for these processes in block copolymers are few. In the bulk, the rate of chain exchange can be quantified by tracer diffusion measurements. Often the rate of equilibration, in terms of number density and aggregation number of particles, is much slower than chain exchange, and consequently observed particle phases are often metastable. This is particularly true in regions of the phase diagram where Frank-Kasper phases occur. Chain exchange in solution has been explored quantitatively by time-resolved SANS, but the results are not well captured by theory. Computer simulations, particularly via dissipative particle dynamics, are beginning to shed light on the chain escape mechanism at the molecular level. The rate of fragmentation has been quantified in a few experimental systems, and TEM images support a mechanism akin to the anaphase stage of mitosis in cells, via a thin neck that pinches off to produce two smaller micelles. Direct measurements of micelle fusion are quite rare. Suggestions for future theoretical, computational, and experimental efforts are offered.

The effect of PEO homopolymers on the behaviours and structural evolution of Pluronic F127 smart hydrogels for controlled drug delivery systems

Understanding the structure-property relationships of drug delivery system (DDS) components is critical for their development and the prediction of bodily performance. This study investigates the effects of introducing polyethylene oxide (PEO) homopolymers, over a wide range of molecular weights, into Pluronic injectable smart hydrogel formulations. These smart DDSs promise to enhance patient compliance, reduce adverse effects and dosing frequency. Pharmaceutically, Pluronic systems are attractive due to their unique sol-gel phase transition in the body, biocompatibility, safety and ease of injectability as solutions before transforming into gel matrices at body temperature. This paper presents a systematic and comprehensive evaluation of gelation and the interplay of microscopic and macroscopic properties under both equilibrium and non-equilibrium conditions in controlled environments, as measured by rheology in conjunction with time-resolved Small Angle Neutron Scattering (SANS). The non-equilibrium conditions investigated in this work offer a better understanding of the two polymeric systems’ complex interactions affecting the matrix thermo-rheological behaviour and structure and therefore the future release of an active pharmaceutical ingredient from the injectable DDS.

SANS characterization of time dependent, slow molecular exchange in an SDS micellar system.

We have investigated the molecular exchange of sodium dodecyl sulfate (SDS) micelles in aqueous solution by time-resolved small angle neutron scattering (TR-SANS) measurements as a function of the surfactant and salt concentration. Starting with deuterated (d-SDS) and protonated (h-SDS) SDS micelles, surfactant exchange across the micelles leads to a randomized distribution of d-SDS and h-SDS within each micelle. By employing the contrast matching technique, we have studied this randomization process which is a direct measure of the molecular exchange of this system. Our results show that the randomization of the pure h-SDS and d-SDS micelles occurs in two steps: first, an almost instantaneous drop in the scattering intensity is observed where ∼80% of the micelles are randomized (contrast matched). After this, micelle randomization progresses slowly spanning over ∼100 hours. Importantly, we show that the kinetics in the second step are dominated by the formation of domains rich in either h-SDS, d-SDS and randomized (50 : 50 h-SDS : d-SDS). The slow exchange step is modeled via a phenomenological approach by drawing analogy to the Langmuir adsorption theory. Finally, the effects of the surfactant and salt concentrations on the instantaneous, and the time dependent randomization of SDS micelles are discussed.

Molecular Transport and Growth of Lipid Vesicles Exposed to Antimicrobial Peptides.

It is well-known that lipids constituting the cytoplasmic membrane undergo continuous reorganization to maintain the appropriate composition important for the integrity of the cell. The transport of lipids is controlled by mainly membrane proteins, but also spontaneous lipid transport between leaflets, lipid “flip–flop”, occurs. These processes do not only occur spontaneously under equilibrium, but also promote structural rearrangements, morphological transitions, and growth processes. It has previously been shown that intravesicular lipid “flip–flop” and intervesicular lipid exchange under equilibrium can be deduced indirectly from contrast variation time-resolved small-angle neutron scattering (TR-SANS) where the molecules are “tagged” using hydrogen/deuterium (H/D) substitution. In this work, we show that this technique can be extended to simultaneously detect changes in the growth and the lipid “flip–flop” and exchange rates induced by a peptide additive on lipid vesicles consisting of DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine), d-DMPC (1,2-dimyristoyl-d54-sn-glycero-3-phosphocholine), DMPG (1,2-dimyristoyl-sn-glycero-3-phospho-(1′-rac-glycerol)), and small amounts of DMPE-PEG (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]). Changes in the overall size were independently monitored using dynamic light scattering (DLS). We find that the antimicrobial peptide, indolicidin, accelerates lipid transport and additionally induces limited vesicular growth. Moreover, in TR-SANS experiments using partially labeled lipid mixtures to separately study the kinetics of the lipid components, we show that, whereas peptide addition affects both lipids similarly, DMPG exhibits faster kinetics. We find that vesicular growth is mainly associated with peptide-mediated lipid reorganization that only slightly affects the overall exchange kinetics. This is confirmed by a TR-SANS experiment of vesicles preincubated with peptide showing that after pre-equilibration the kinetics are only slightly slower.

Soft Matter Sample Environments for Time-Resolved Small Angle Neutron Scattering Experiments: A Review

With the promise of new, more powerful neutron sources in the future, the possibilities for time-resolved neutron scattering experiments will improve and are bound to gain in interest. While there is already a large body of work on the accurate control of temperature, pressure, and magnetic fields for static experiments, this field is less well developed for time-resolved experiments on soft condensed matter and biomaterials. We present here an overview of different sample environments and technique combinations that have been developed so far and which might inspire further developments so that one can take full advantage of both the existing facilities as well as the possibilities that future high intensity neutron sources will offer.

Light Scattering and Absorption Complementarities to Neutron Scattering: In Situ FTIR and DLS Techniques at the High-Intensity and Extended Q-Range SANS Diffractometer KWS-2

Understanding soft and biological materials requires global knowledge of their microstructural features from elementary units at the nm scale up to larger complex aggregates in the micrometer range. Such a wide range of scale can be explored using the KWS-2 small-angle neutron (SANS) diffractometer. Additional information obtained by in situ complementary techniques sometimes supports the SANS analysis of systems undergoing structural modifications under external stimuli or which are stable only for short times. Observations at the local molecular level structure and conformation assists with an unambiguous interpretation of the SANS data using appropriate structural models, while monitoring of the sample condition during the SANS investigation ensures the sample stability and desired composition and chemical conditions. Thus, we equipped the KWS-2 with complementary light absorption and scattering capabilities: Fourier transform infrared (FTIR) spectroscopy can now be performed simultaneously with standard and time-resolved SANS, while in situ dynamic light scattering (DLS) became available for routine experiments, which enables the observation of either changes in the sample composition, due to sedimentation effects, or in size of morphologies, due to aggregation processes. The performance of each setup is demonstrated here using systems representative of those typically investigated on this beamline and benchmarked to studies performed offline.

Structural and Biophysical Properties of Supercharged and Circularized Nanodiscs.

Nanodiscs based on membrane scaffold proteins (MSPs) and phospholipids are used as membrane mimics to stabilize membrane proteins in solution for structural and functional studies. Combining small-angle X-ray scattering (SAXS), differential scanning calorimetry (DSC), and time-resolved small-angle neutron scattering (TR-SANS), we characterized the structure and lipid bilayer properties of five different nanodiscs made with dimyristoylphosphatidylcholine and different MSPs varying in size, charge, and circularization. Our SAXS modeling showed that the structural parameters of the embedded lipids are all similar, irrespective of the MSP properties. DSC showed that the lipid packing is not homogeneous in the nanodiscs and that a 20 Å wide boundary layer of lipids with perturbed packing is located close to the MSP, while the packing of central lipids is tighter than in large unilamellar vesicles. Finally, TR-SANS showed that lipid exchange rates in nanodiscs decrease with increasing nanodisc size and are lower for the nanodiscs made with supercharged MSPs compared to conventional nanodiscs. Altogether, the results provide a thorough biophysical understanding of the nanodisc as a model membrane system, which is important in order to carry out and interpret experiments on membrane proteins embedded in such systems.

Chain Exchange Kinetics of Bottlebrush Block Copolymer Micelles

Chain exchange kinetics of the bottlebrush core micelles formed by poly(5-perfluorooctyl-2-norbornene-block-N-cyclohexyl-exo-norbornene-5,6-dicarboxyimide) (PF-b-PC) block copolymer in tetrahydrofuran (THF), selective for PC blocks, were investigated by time-resolved small-angle neutron scattering (TR-SANS). The bottlebrush PF core blocks are densely tethered with rigid fluoroalkyl side chains. Despite the unfavorable interaction between the PF cores and the medium, TR-SANS revealed that the chain exchange between the bottlebrush core micelles occurs within a measurable timescale (∼1 h) at 40 °C. The measured exchange kinetics were successfully described by the model based on the thermodynamic barrier for the core block extraction and the solvent plasticizing effect associated with partial swelling of the PF cores. This chain exchange rate is also partly attributed to the bottlebrush architecture of PF blocks, as supported by lower melt viscosities of PF homopolymers than those of the analogous fluorine-containing linear polymers. These results are discussed in terms of current understanding of molecular exchange between block copolymer micelles, and particular attention is paid to the issue of polymer architecture, given the bottlebrush type of the core block.

Deciphering lipid transfer between and within membranes with time-resolved small-angle neutron scattering.

This review focuses on time-resolved neutron scattering, particularly time-resolved small angle neutron scattering (TR-SANS), as a powerful in situ noninvasive technique to investigate intra- and intermembrane transport and distribution of lipids and sterols in lipid membranes. In contrast to using molecular analogues with potentially large chemical tags that can significantly alter transport properties, small angle neutron scattering relies on the relative amounts of the two most abundant isotope forms of hydrogen: protium and deuterium to detect complex membrane architectures and transport processes unambiguously. This review discusses advances in our understanding of the mechanisms that sustain lipid asymmetry in membranes-a key feature of the plasma membrane of cells-as well as the transport of lipids between membranes, which is an essential metabolic process.

Contrast-Variation Time-Resolved Small-Angle Neutron Scattering Analysis of Oil-Exchange Kinetics Between Oil-in-Water Emulsions Stabilized by Anionic Surfactants.

Contrast-variation time-resolved small-angle neutron scattering (CV-SANS) was used to examine oil-exchange kinetics between identical mixtures of hydrogenated/deuterated hexadecane emulsion systems. Oil-exchange rates were estimated by transforming recorded scattering profiles to a relaxation function and by fitting to exponential decay models. We find that the oil-exchange process was accelerated when the droplets were stabilized by anionic surfactants even at concentrations well below the surfactant critical micelle concentration. Moreover, the exchange rate was not significantly accelerated when surfactant micelles were present. This suggests that micellar-mediated transport mechanisms do not play the dominant role in these systems. Screening electrostatic repulsion by increasing the ionic strength of the medium also had a negligible effect on oil-exchange kinetics. In contrast, the use of oils with shorter alkane chain lengths (e.g., dodecane), having a higher solubility in water, significantly accelerated rates of oil transport between droplets. Oil-transport rates for hexadecane were also found to increase with temperature and to follow Arrhenius behavior. These results were rationalized as an increase in the droplet-collision frequency due to Brownian motion that results in direct oil transport without irreversible coalescence. Thus, primary mechanisms for oil exchange in insoluble anionic surfactant-stabilized emulsion systems are hypothesized to be through direct emulsion contact, reversible coalescence, and/or direct oil permeation through thin liquid films. CV-SANS is also demonstrated as a powerful technique for the study of transport kinetics in all kinds of emulsion systems.

Kinetic Analysis of Ultrasound-Induced Oil Exchange in Oil-in-Water Emulsions through Contrast Variation Time-Resolved Small-Angle Neutron Scattering.

Ultrasound is one of the most commonly used methods for synthesizing and processing emulsion systems. In this study, the kinetics of acoustically induced emulsion oil exchange was examined using contrast variation time-resolved small-angle neutron scattering (CV-SANS). A custom-built sample environment was used to deliver acoustic forces while simultaneously performing CV-SANS experiments. It was observed that the oil exchange rate was significantly accelerated when sonicating at high acoustic pressures, where violent cavitation events can induce droplet coalescence and breakup. No significant oil exchange occurred at acoustic pressures below the cavitation threshold within the short time scales of the experiments. It was also observed that the oil exchange kinetics was deterred when emulsions were stabilized by surfactants. In addition, oil exchange rates varied nonlinearly with the concentration of surfactant, and exchange was slowest when the emulsions were stabilized by an intermediate concentration. It is hypothesized that emulsion size, electrostatic repulsion, and Gibbs elasticity of the oil-water interface play significant roles in the observed trends. The observed trends in oil exchange rates versus surfactant concentration coincide well with theoretical models for the fluctuation of the elasticity of the interface. Acoustically induced oil exchange was most inefficient when the interfacial elasticity was at its maximum value.

Measuring the formation energy barrier of skyrmions in zinc-substituted Cu2OSeO3

We report small-angle neutron scattering (SANS) measurements of the skyrmion lattice in ${({\mathrm{Cu}}_{0.976}{\mathrm{Zn}}_{0.024})}_{2}{\mathrm{OSeO}}_{3}$ under the application of electric and magnetic fields. These measurements show an expansion of the skyrmion lattice stability region with electric field. Furthermore, using time-resolved SANS, we observe the slow formation of skyrmions after an electric or magnetic field is applied, which has not been observed in pristine ${\mathrm{Cu}}_{2}{\mathrm{OSeO}}_{3}$ crystals. The measured formation times are dramatically longer than the corresponding skyrmion annihilation times after the external field is removed, and increase exponentially from 100 s at 52.5 K to 10 000 s at 51.5 K. This thermally activated behavior indicates an energy barrier for skyrmion formation of 1.57(2) eV, the size of which demonstrates the huge cost for creating these complex chiral objects.

Evaluation of Interbilayer and Transbilayer Transfer Dynamics of Phospholipids Using Time-Resolved Small-Angle Neutron Scattering.

The bilayer structure of biomembranes consists of thousands of lipids, the composition of which is different for each organelle. Since most lipids are synthesized in the endoplasmic reticulum, subsequent distribution to each organelle determines the composition and function of the biomembranes. Thus, interbilayer transfer and transbilayer movement (flip-flop) of phospholipids play important roles in maintaining homeostasis. A crucial task in biophysics and cell biology is to understand how rapidly lipids migrate between bilayers spontaneously or through proteins and to control these lipid dynamics. Time-resolved small-angle neutron scattering (TR-SANS) is a powerful technique to determine the intervesicular exchange and flip-flop rates of lipids in situ and real time. In this review, I explain how TR-SANS detects the interbilayer and transbilayer transfer of phospholipids and introduce recent progress of my group on the evaluation of spontaneous and protein- (or peptide-)mediated lipid transfer in several phospholipid dispersion systems.