Small-angle X-ray scattering unveils the internal structure of lipid nanoparticles.

Lipid nanoparticles own a remarkable potential in nanomedicine, only partially disclosed. While the clinical use of liposomes and cationic lipid-nucleic acid complexes is well-established, liquid lipid nanoparticles (nanoemulsions), solid lipid nanoparticles, and nanostructured lipid carriers have even greater possibilities. However, they face obstacles in being used in clinics due to a lack of understanding about the molecular mechanisms controlling their drug loading and release, interactions with the biological environment (such as the protein corona), and shelf-life stability. To create effective drug delivery carriers and successfully translate bench research to clinical settings, it is crucial to have a thorough understanding of the internal structure of lipid nanoparticles. Through synchrotron small-angle X-ray scattering experiments, we determined the spatial distribution and internal structure of the nanoparticles' lipid, surfactant, and the bound water in them. The nanoparticles themselves have a barrel-like shape that consists of coplanar lipid platelets (specifically cetyl palmitate) that are covered by loosely spaced polysorbate 80 surfactant molecules, whose polar heads retain a large amount of bound water. To reduce the interface cost of bound water with unbound water without stacking, the platelets collapse onto each other. This internal structure challenges the classical core-shell model typically used to describe solid lipid nanoparticles and could play a significant role in drug loading and release, biological fluid interaction, and nanoparticle stability, making our findings valuable for the rational design of lipid-based nanoparticles.

Small-angle scattering from flat bilayers containing correlated scattering length density inhomogeneities.

Model lipid bilayers have been widely employed as a minimal system to investigate the structural properties of biological membranes by small-angle X-ray (SAXS) and neutron scattering (SANS) techniques. These have nanometre resolution and can give information regarding membrane thickness and scattering length densities (SLDs) of polar and apolar regions. However, biological membranes are complex systems containing different lipids and protein species, in which lipid domains can be dynamically assembled and disassembled. Therefore, SLD variations can occur within the biomembrane. In this work, a novel method has been developed to simulate SAXS and SANS profiles obtained from large unilamellar vesicles containing SLD inhomogeneities that are spatially correlated over the membrane surface. Such inhomogeneities are represented by cylindrical entities with equivalent SLDs. Stacking of bilayers is also included in the model, with no correlation between horizontal and vertical order. The model is applied to a lipid bilayer containing SLD inhomogeneities representing pores, lipid domains, and transmembrane, partially immersed and anchored proteins. It is demonstrated that all the structural information from the host lipid bilayer and from the SLD inhomogeneity can be consistently retrieved by a combined analysis of experimental SAXS and SANS data through the methodology proposed here.

Developing advanced models of biological membranes with hydrogenous and deuterated natural glycerophospholipid mixtures.

Cellular membranes are complex systems that consist of hundreds of different lipid species. Their investigation often relies on simple bilayer models including few synthetic lipid species. Glycerophospholipids (GPLs) extracted from cells are a valuable resource to produce advanced models of biological membranes. Here, we present the optimisation of a method previously reported by our team for the extraction and purification of various GPL mixtures from Pichia pastoris. The implementation of an additional purification step by High Performance Liquid Chromatography-Evaporative Light Scattering Detector (HPLC-ELSD) enabled for a better separation of the GPL mixtures from the neutral lipid fraction that includes sterols, and also allowed for the GPLs to be purified according to their different polar headgroups. Pure GPL mixtures at significantly high yields were produced through this approach. For this study, we utilised phoshatidylcholine (PC), phosphatidylserine (PS) and phosphatidylglycerol (PG) mixtures. These exhibit a single composition of the polar head, i.e., PC, PS or PG, but contain several molecular species consisting of acyl chains of varying length and unsaturation, which were determined by Gas Chromatography (GC). The lipid mixtures were produced both in their hydrogenous (H) and deuterated (D) versions and were used to form lipid bilayers both on solid substrates and as vesicles in solution. The supported lipid bilayers were characterised by quartz crystal microbalance with dissipation monitoring (QCM-D) and neutron reflectometry (NR), whereas the vesicles by small angle X-ray (SAXS) and neutron scattering (SANS). Our results show that despite differences in the acyl chain composition, the hydrogenous and deuterated extracts produced bilayers with very comparable structures, which makes them valuable to design experiments involving selective deuteration with techniques such as NMR, neutron scattering or infrared spectroscopy.

Interaction of a Histidine-Rich Antimicrobial Saliva Peptide with Model Cell Membranes: The Role of Histidines.

Histatin 5 is a histidine-rich, intrinsically disordered, multifunctional saliva protein known to act as a first line of defense against oral candidiasis caused by Candida albicans. An earlier study showed that, upon interaction with a common model bilayer, a protein cushion spontaneously forms underneath the bilayer. Our hypothesis is that this effect is of electrostatic origin and that the observed behavior is due to proton charge fluctuations of the histidines, promoting attractive electrostatic interactions between the positively charged proteins and the anionic surfaces, with concomitant counterion release. Here we are investigating the role of the histidines in more detail by defining a library of variants of the peptide, where the former have been replaced by the pH-insensitive amino acid glutamine. By using experimental techniques such as circular dichroism, small angle X-ray scattering, quartz crystal microbalance with dissipation monitoring, and neutron reflectometry, it was determined that changing the number of histidines in the peptide sequence did not affect the structure of the peptide dissolved in solution. However, it was shown to affect the penetration depth of the peptide into the bilayer, where all variants except the one with zero histidines were found below the bilayer. A decrease in the number of histidine from the original seven to zero decreases the ability of the peptide to penetrate the bilayer, and the peptide is then also found residing within the bilayer. We hypothesize that this is due to the ability of the histidines to charge titrate, which charges up the peptide, and enables it to penetrate and translocate through the lipid bilayer.

Dark peptide discs for the investigation of membrane proteins in supported lipid bilayers: the case of synaptobrevin 2 (VAMP2).

Supported lipid bilayers (SLBs) are commonly used as model systems mimicking biological membranes. Recently, we reported a new method to produce SLBs with incorporated membrane proteins, which is based on the application of peptide discs [Luchini et al., Analytical Chemistry, 2020, 92, 1081-1088]. Peptide discs are small discoidal particles composed of a lipid core and an outer belt of self-assembled 18A peptides. SLBs including membrane proteins can be formed by depositing the peptide discs on a solid support and subsequently removing the peptide by buffer rinsing. Here, we introduce a new variant of the 18A peptide, named dark peptide (d18A). d18A exhibits UV absorption at 214 nm, whereas the absorption at 280 nm is negligible. This improves sample preparation as it enables a direct quantification of the membrane protein concentration in the peptide discs by measuring UV absorption at 280 nm. We describe the application of the peptide discs prepared with d18A (dark peptide discs) to produce SLBs with a membrane protein, synaptobrevin 2 (VAMP2). The collected data showed the successful formation of SLBs with high surface coverage and incorporation of VAMP2 in a single orientation with the extramembrane domain exposed towards the bulk solvent. Compared to 18A, we found that d18A was more efficiently removed from the SLB. Our data confirmed the structural organisation of VAMP2 as including both α-helical and ß-sheet secondary structure. We further verified the orientation of VAMP2 in the SLBs by characterising the binding of VAMP2 with α-synuclein. These results point at the produced SLBs as relevant membrane models for biophysical studies as well as nanostructured biomaterials.

Strikingly Different Roles of SARS-CoV-2 Fusion Peptides Uncovered by Neutron Scattering.

Coronavirus disease-2019 (COVID-19), a potentially lethal respiratory illness caused by the coronavirus SARS-CoV-2, emerged in the end of 2019 and has since spread aggressively across the globe. A thorough understanding of the molecular mechanisms of cellular infection by coronaviruses is therefore of utmost importance. A critical stage in infection is the fusion between viral and host membranes. Here, we present a detailed investigation of the role of selected SARS-CoV-2 Spike fusion peptides, and the influence of calcium and cholesterol, in this fusion process. Structural information from specular neutron reflectometry and small angle neutron scattering, complemented by dynamics information from quasi-elastic and spin-echo neutron spectroscopy, revealed strikingly different functions encoded in the Spike fusion domain. Calcium drives the N-terminal of the Spike fusion domain to fully cross the host plasma membrane. Removing calcium, however, reorients the peptide back to the lipid leaflet closest to the virus, leading to significant changes in lipid fluidity and rigidity. In conjunction with other regions of the fusion domain, which are also positioned to bridge and dehydrate viral and host membranes, the molecular events leading to cell entry by SARS-CoV-2 are proposed.

Interaction of Caffeine with Model Lipid Membranes.

Caffeine is not only a widely consumed active stimulant, but it is also a model molecule commonly used in pharmaceutical sciences. In this work, by performing quartz-crystal microbalance and neutron reflectometry experiments we investigate the interaction of caffeine molecules with a model lipid membrane. We determined that caffeine molecules are not able to spontaneously partition from an aqueous environment, enriched in caffeine, into a bilayer. Caffeine could be however included in solid-supported lipid bilayers if present with lipids during self-assembly. In this case, thanks to surface-sensitive techniques, we determined that caffeine molecules are preferentially located in the hydrophobic region of the membrane. These results are highly relevant for the development of new drug delivery vectors, as well as for a deeper understanding of the membrane permeation role of purine molecules.

Lipid bilayer degradation induced by SARS-CoV-2 spike protein as revealed by neutron reflectometry.

SARS-CoV-2 spike proteins are responsible for the membrane fusion event, which allows the virus to enter the host cell and cause infection. This process starts with the binding of the spike extramembrane domain to the angiotensin-converting enzyme 2 (ACE2), a membrane receptor highly abundant in the lungs. In this study, the extramembrane domain of SARS-CoV-2 Spike (sSpike) was injected on model membranes formed by supported lipid bilayers in presence and absence of the soluble part of receptor ACE2 (sACE2), and the structural features were studied at sub-nanometer level by neutron reflection. In all cases the presence of the protein produced a remarkable degradation of the lipid bilayer. Indeed, both for membranes from synthetic and natural lipids, a significant reduction of the surface coverage was observed. Quartz crystal microbalance measurements showed that lipid extraction starts immediately after sSpike protein injection. All measurements indicate that the presence of proteins induces the removal of membrane lipids, both in the presence and in the absence of ACE2, suggesting that sSpike molecules strongly associate with lipids, and strip them away from the bilayer, via a non-specific interaction. A cooperative effect of sACE2 and sSpike on lipid extraction was also observed.

Structural Characterization of Natural Yeast Phosphatidylcholine and Bacterial Phosphatidylglycerol Lipid Multilayers by Neutron Diffraction.

Eukaryotic and prokaryotic cell membranes are difficult to characterize directly with biophysical methods. Membrane model systems, that include fewer molecular species, are therefore often used to reproduce their fundamental chemical and physical properties. In this context, natural lipid mixtures directly extracted from cells are a valuable resource to produce advanced models of biological membranes for biophysical investigations and for the development of drug testing platforms. In this study we focused on single phospholipid classes, i.e. Pichia pastoris phosphatidylcholine (PC) and Escherichia coli phosphatidylglycerol (PG) lipids. These lipids were characterized by a different distribution of their respective acyl chain lengths and number of unsaturations. We produced both hydrogenous and deuterated lipid mixtures. Neutron diffraction experiments at different relative humidities were performed to characterize multilayers from these lipids and investigate the impact of the acyl chain composition on the structural organization. The novelty of this work resides in the use of natural extracts with a single class head-group and a mixture of chain compositions coming from yeast or bacterial cells. The characterization of the PC and PG multilayers showed that, as a consequence of the heterogeneity of their acyl chain composition, different lamellar phases are formed.