There is an urgent need for novel inhalable drug carriers to fight respiratory infections. Lipid-coated mesoporous silica particles (LC-MSPs) combine the biocompatibility of lipids with the aerosolization properties of micronized low-density MSPs. In this study, the abundant lung surfactant phospholipid dipalmitoylphosphatidylcholine (DPPC) was used to coat disordered MSPs by means of two methods: vesicle fusion (VF) and spray-drying (SD). FT-IR and TGA analyses indicated the presence of the lipid coating, while SEM images revealed spherical particles with a smooth, homogenous surface and no detectable lipid aggregates. Both the VF and SD methods resulted in full phospholipid coverage on the outer silica surface (>100 %). However, the VF method produced a more homogeneous coating across particles and achieved a higher lipid content compared to SD (7.0 vs 3.0 % w/w). The resulting LC-MSPs exhibited favorable aerosolization properties, enabling efficient pulmonary delivery of clofazimine, a lipophilic antitubercular drug. The DPPC coating promoted interaction with endogenous lung surfactant, which enhanced the dispersion of the particles in the alveolar environment and significantly increased drug dissolution (from 35 to 75 %). Lipid coating significantly enhances particle adhesion and penetration across the human bronchial mucus layer and into the underlying tissue. Overall, our study presents a refined formulation strategy using phospholipid-coated MSPs as a single-component dry powder carrier, offering targeted lung deposition, enhanced drug dissolution, mucoadhesion, and tissue penetration.

Natural occurring deep eutectic solvents (NADES) are solvents made of metabolites occurring in living organisms, and are thought to play a special role in plants especially given that some of these metabolites exist in concentrations as high as 1 M. NADES have properties similar to ionic liquids, and have been shown to protect enzymes against loss of activity as well as proteins against thermal denaturation. Here, we explore the structure of lipid vesicles in NADES rich aqueous solutions and compare to concentrated saline aqueous solutions matching the various NADES osmolarity. The vesicle structure was analysed by small angle X-ray scattering (SAXS) and dynamic light scattering (DLS). Two types of NADES were prepared using choline chloride and glucose or maleic acid at a molar ratio of 1 : 1 giving the solvents a neutral or an acidic nature, respectively. The stability of the vesicles in the various solvents was measured against time and temperature. The results of this work demonstrate that lipid bilayers retain their structure in NADES rich aqueous solutions as compared to pure water, in contrast to high saline aqueous solutions. Moreover, the vesicles are more stable against sedimentation and aggregation in NADES than in water.

Lipoproteins, key mediators of lipid transport, facilitate the bidirectional transfer of lipids such as fatty acids, triglycerides, and cholesterol between soluble particles and cell membranes. High-density lipoproteins (HDL) primarily engage in reverse cholesterol transport, while low-density lipoproteins (LDL) predominantly deposit lipids, affecting cardiovascular health with a well-known role in the formation of the atherosclerotic plaque. In addition, lipoproteins play an important role in neutralizing bacterial lipopolysaccharides (LPS), the major component of Gram-negative bacterial outer membranes, which act as potent TLR4 agonists and can trigger severe immune responses. Lipoproteins bind LPS in plasma, with HDL showing strong binding affinity and LDL contributing to LPS clearance under specific conditions. Here, we explore the interaction of LDL and human serum albumin (HSA), another serum lipid-binding protein, with model lipid bilayers containing either phospholipids or LPS. Using neutron reflectometry and attenuated total reflection infrared spectroscopy, we characterize lipid transfer processes influenced by calcium levels and lipid composition. Calcium plays a key role in receptor-mediated LDL binding, but less is known on its effect on LDL-mediated lipid transfer in the absence of LDL receptors. Our results show that elevated calcium levels enhance stable LDL adsorption onto mammalian phospholipid-cholesterol membranes, promoting lipid cargo deposition despite the absence of specific LDL-receptors. Conversely, LDL showed no stable binding to LPS reconstituted in asymmetric outer membrane models but was able to deposit phospholipids in the membrane. In contrast, HSA removed lipids from mammalian membranes and exhibited minimal interaction with LPS-containing models. The findings elucidate the distinct lipid exchange mechanisms of LDL and HSA and their roles in modulating lipid transfer at membrane interfaces. Receptor-free enhanced LDL lipid deposition in calcium-enriched environments may have implications for cardiovascular disease progression. Conversely, the minimal interaction of LDL with bacterial LPS suggests a limited ability to extract LPS from membrane environments. This study provides structural insights into the interplay between lipoproteins, calcium, and membrane composition, with relevance to atherosclerosis and systemic endotoxemia.

High-density lipoproteins (HDLs) are responsible for removing cholesterol from arterial walls, through a process known as reverse cholesterol transport. The main protein in HDL, apolipoprotein A-I (ApoA-I), is essential to this process, and changes in its sequence significantly alter HDL structure and functions. ApoA-I amyloidogenic variants, associated with a particular hereditary degenerative disease, are particularly effective at facilitating cholesterol removal, thus protecting carriers from cardiovascular disease. Thus, it is conceivable that reconstituted HDL (rHDL) formulations containing ApoA-I proteins with functional/structural features similar to those of amyloidogenic variants hold potential as a promising therapeutic approach. Here we explored the effect of protein cargo and lipid composition on the function of rHDL containing one of the ApoA-I amyloidogenic variants G26R or L174S by Fourier transformed infrared spectroscopy and neutron reflectometry. Moreover, small-angle x-ray scattering uncovered the structural and functional differences between rHDL particles, which could help to comprehend higher cholesterol efflux activity and apparent lower phospholipid (PL) affinity. Our findings indicate distinct trends in lipid exchange (removal vs. deposition) capacities of various rHDL particles, with the rHDL containing the ApoA-I amyloidogenic variants showing a markedly lower ability to remove lipids from artificial membranes compared to the rHDL containing the native protein. This effect strongly depends on the level of PL unsaturation and on the particles' ultrastructure. The study highlights the importance of the protein cargo, along with lipid composition, in shaping rHDL structure, contributing to our understanding of lipid-protein interactions and their behavior.

Peptide-based liquid droplets (coacervates) produced by spontaneous liquid-liquid phase separation (LLPS), have emerged as a promising class of drug delivery systems due to their high entrapping efficiency and the simplicity of their formulation. However, the detailed mechanisms governing their interaction with cell membranes and cellular uptake remain poorly understood. In this study, we investigated the interactions of peptide coacervates composed of HB pep —peptide derived from the histidine-rich beak proteins (HBPs) of the Humboldt squid—with model cellular membranes in the form of supported lipid bilayers (SLBs). We employed quartz crystal microbalance with dissipation monitoring (QCM-D), neutron reflectometry (NR) and atomistic molecular dynamics (MD) simulations to reveal the nature of these interactions in the absence of fluorescent labels or tags. HB pep forms small oligomers at pH 6 whereas it forms µm-sized coacervates at physiological pH. Our findings reveal that both HB pep oligomers and HB pep -coacervates adsorb onto SLBs at pH 6 and 7.4, respectively. At pH 6, when the peptide carries a net positive charge, HB pep oligomers insert into the SLB, facilitated by the peptide’s interactions with the charged lipids and cholesterol. Importantly, however, HB pep coacervate adsorption at physiological pH, when it is largely uncharged, is fully reversible, suggesting no significant lipid bilayer rearrangement. HB pep coacervates, previously identified as efficient drug delivery vehicles, do not interact with the lipid membrane in the same manner as traditional cationic drug delivery systems or cell-penetrating peptides. Based on our findings, HB pep coacervates at physiological pH cannot cross the cell membrane by a simple passive mechanism and are thus likely to adopt a non-canonical cell entry pathway.

Neutrons are characterized by a low absorption in many engineering materials. At the same time the scattering cross section of light elements, such as hydrogen and deuterium, may be large. These properties make neutron scattering experiments performed under grazing incidence geometry an excellent tool for the study of solid–liquid interfaces. In this review we describe the basic concepts of neutron reflection and grazing incidence scattering experiments as well as experimental procedures and sample cells. The full power of the method is exemplified on a range of science areas, including polymers, bio- and ionic liquid lubricants, electrolytes as well as bio-membranes or magnetic liquids.

There is a close relationship between the SARS-CoV-2 virus and lipoproteins, in particular high-density lipoprotein (HDL). The severity of the coronavirus disease 2019 (COVID-19) is inversely correlated with HDL plasma levels. It is known that the SARS-CoV-2 spike (S) protein binds the HDL particle, probably depleting it of lipids and altering HDL function. Based on neutron reflectometry (NR) and the ability of HDL to efflux cholesterol from macrophages, we confirm these observations and further identify the preference of the S protein for specific lipids and the consequent effects on HDL function on lipid exchange ability. Moreover, the effect of the S protein on HDL function differs depending on the individuals lipid serum profile. Contrasting trends were observed for individuals presenting low triglycerides/high cholesterol serum levels (LTHC) compared to high triglycerides/high cholesterol (HTHC) or low triglycerides/low cholesterol serum levels (LTLC). Collectively, these results suggest that the S protein interacts with the HDL particle and, depending on the lipid profile of the infected individual, it impairs its function during COVID-19 infection, causing an imbalance in lipid metabolism.

Lipid nanoparticles (LNPs) are the most versatile and successful gene delivery systems, notably highlighted by their use in vaccines against COVID-19. LNPs have a well-defined core-shell structure, each region with its own distinctive compositions, suited for a wide range of in vivo delivery applications. Here, we discuss how a detailed knowledge of LNP structure can guide LNP formulation to improve the efficiency of delivery of their nucleic acid payload. Perspectives are detailed on how LNP structural design can guide more efficient nucleic acid transfection. Views on key physical characterization techniques needed for such developments are outlined including opinions on biophysical approaches both correlating structure with functionality in biological fluids and improving their ability to escape the endosome and deliver they payload.

Arrays of nanoparticle-supported lipid bilayers (nanoSLB) are lipid-coated nanopatterned interfaces that provide a platform to study curved model biological membranes using surface-sensitive techniques. We combined scattering techniques with direct imaging, to gain access to sub-nanometer scale structural information on stable nanoparticle monolayers assembled on silicon crystals in a noncovalent manner using a Langmuir-Schaefer deposition. The structure of supported lipid bilayers formed on the nanoparticle arrays via vesicle fusion was investigated using a combination of grazing incidence X-ray and neutron scattering techniques complemented by fluorescence microscopy imaging. Ordered nanoparticle assemblies were shown to be suitable and stable substrates for the formation of curved and fluid lipid bilayers that retained lateral mobility, as shown by fluorescence recovery after photobleaching and quartz crystal microbalance measurements. Neutron reflectometry revealed the formation of high-coverage lipid bilayers around the spherical particles together with a flat lipid bilayer on the substrate below the nanoparticles. The presence of coexisting flat and curved supported lipid bilayers on the same substrate, combined with the sub-nanometer accuracy and isotopic sensitivity of grazing incidence neutron scattering, provides a promising novel approach to investigate curvature-dependent membrane phenomena on supported lipid bilayers.

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 alpha-helical and beta-sheet secondary structure. We further verified the orientation of VAMP2 in the SLBs by characterising the binding of VAMP2 with alpha-synuclein. These results point at the produced SLBs as relevant membrane models for biophysical studies as well as nanostructured biomaterials.