搜索结果：Chemistry and physics of lipids

Sphingolipids constitute a class of bioactive lipids essential for the structural and functional integrity of milk fat globule membrane (MFGM). Milk sphingomyelin (milk-SM), as a key component of MFGM, contributes to the stability of milk fat emulsions. Milk-SM and other sphingolipids, like glycosphingolipids (GSL), coexist in the same outer bilayer of MFGM, suggesting significant role of their interaction in shaping the structural properties and functions of MFGM. In this study, using an in-vitro model membrane system, we investigated the impact of various GSLs, including cerebrosides and gangliosides, on the lateral segregation and phase behavior of milk-SM in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine bilayers. We also incorporated N-palmitoyl-D-erythro-ceramide for a comparative analysis of the impacts of sphingolipid head groups. The lateral segregation of sphingolipid gel phases was assessed using trans-parinaric acid (tPA) fluorescence lifetime analysis, and their thermostability was examined using steady-state fluorescence anisotropy of tPA. Additionally, we assessed the binary interactions between milk-SM and GSLs using the steady-state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene (DPH). The results indicate that GSLs promote the lateral segregation and stabilization of milk-SM-rich gel phases in the membrane bilayers. The size of the GSL head groups significantly influenced the degree of this stabilization, with larger head groups demonstrating diminished interactions with milk-SM. Our results provide valuable insights into the role of various sphingolipid structures in membrane phase behavior and organization. Comprehensive understanding of the interactions of these important sphingolipids in MFGM environment is crucial due to their structural and functional importance in dairy and nutritional applications.

In this work, the effects of two UV filters - avobenzone and oxybenzone - on the membranes of fibroblasts and keratinocytes in ex vivo model systems (Langmuir monolayers) and cell line experiments were examined. The goal of these studies was to analyze the significance of lipid structures in the mechanism of UV filter-induced toxicity to skin cells. Monolayers composed of lipids characteristic of mammalian cell membranes - namely phosphatidylcholine (SOPC), sphingomyelin (SM), cholesterol (Chol) and ceramides (Ceramide 22 and Ceramide 17) - were used as model systems. Both mixed monolayers mimicking fibroblast and keratinocyte membranes and one-component lipid films were investigated. The surface pressure/area measurements and penetration studies were done, and Brewster angle microscopy was applied to verify the morphology of the studied systems. It was found that avobenzone has a stronger impact on molecular organization of skin cell model membranes than oxybenzone; however, its effect is concentration-limited. Both UV filters exhibited stronger affinity to Chol, SM and SOPC monolayers than to ceramides, which are the lipids characteristic for skin cells. Therefore, it can be suggested that ceramides may hinder the penetration of UV filter molecules into the interior of skin cells. Cell line model studied with SEM microscopy suggested that UV filters alter the skin cell membrane. Finally, it was summarized that the mechanism of UV filter toxicity is complex, but one of its important elements is the impact on the organization of lipid structures.

The persistent global burden of viral infections, compounded by the emergence of resistance and suboptimal therapeutic efficacy, underscores the urgency for innovative treatment strategies. Recent viral outbreaks such as COVID-19, Human metapneumovirus (HMPV), Zika, Ebola, Nipah, and various influenza viral strains have highlighted the limitations of conventional antivirals. This necessitates the need for targeted, adaptable, and innovative drug delivery platforms. In light of this, LNCs have emerged as versatile systems capable of enhancing drug stability, biodistribution, and cellular uptake. With their tunable architecture and ability to encapsulate diverse antiviral agents, these nanocarriers offer a promising avenue to overcome pharmacological barriers, improve therapeutic efficacy, and enable effective intervention against both established and emerging viral pathogens. To gather supporting evidence, publications were identified on Google Scholar, PubMed, and ScienceDirect with specific search terms such as "antivirals", "drug loading", "encapsulation efficiency", "lipid nanocarriers", "liposomes", "solid lipid nanoparticles (SLNs)", "nanostructured lipid carriers (NLCs)", "cubosomes", "virus", "viral disease", and "resistance". We did not impose any restrictions on the publication date during the selection of papers. However, it is imperative to highlight that the initial reports containing specified keywords began publication in 1964; it is noteworthy that a majority of these publications were 2000 or beyond. LNCs, including SLNs, NLCs, liposomes, and cubosomes, etc, demonstrated improved antiviral efficacy by enhancing drug stability, targeted delivery, and bioavailability. Several formulations showed superior pharmacokinetics and reduced toxicity compared to conventional therapies. Additionally, in vivo studies supported enhanced lymphatic uptake and therapeutic outcomes across multiple viral models. Despite notable progress, challenges in scalability, stability, and regulatory compliance limit their clinical translation. Hence, techniques such as microfluidics and other continuous manufacturing approaches improve reproducibility and process control. Moreover, artificial intelligence is revolutionizing LNC development by enabling rapid optimization, in silico prediction of pharmacokinetics, and real-time quality monitoring. Incorporating AI-enabled quality-by-design frameworks with state-of-the-art analytics may streamline regulatory approval. Moving forward, translating LNC technologies from bench to bedside will require scalable production methods, standardized characterization, and regulatory alignment.

The selection of lipids and their ratios play a critical role in determining drug loading capacity and the structural properties of nanostructured lipid carriers (NLCs), directly impacting their stability. Among liquid lipids, vegetable oils have been explored both as active pharmaceutical ingredients (APIs) and as excipients in NLCs intended for topical use. The pulp oil of Tucumã, derived from Brazilian biodiversity, is known for its anti-inflammatory and antioxidant properties, attributed to its high content of carotenoids. This study focused on evaluating the compatibility of Tucumã oil with various solid lipids (SLs) commonly used in NLC production, developing an optimized NLC formulation containing this oil, and monitoring its stability over a 28-days' period. Lipid screening was performed to assess the compatibility of Tucumã oil with a series of SLs, followed by preliminary formulations to determine the type of SL and surfactant for the experimental design. A 22 experimental factorial design was used to understand and identify the significant effects and interactions of lipid phase and surfactant concentrations on Tucumã oil-loaded NLCs, and the stability of the optimized formulation was monitored by determining the mean particle size (z-Ave), polydispersity index (PI), zeta potential (ZP), and recrystallization index (RI%) over 28 days. Compritol® was identified as the most suitable SL, resulting in round shaped NLCs with z-Ave of 309 nm, PI of 0.23 and high ZP (-25.5 mV). The RI% was shown to be influenced by the storage time and temperature. The optimal formulation contained 8 % of lipid phase (at a 20:80 ratio of oil to SL) and 3 % of Tween® 80 as surfactant, showing stability at 5ºC, 25ºC and 40ºC. The experimental factorial design revealed a positive effect of surfactant concentration on z-Ave and PI, with no significant impact on ZP. Over time, NLCs exhibited a gradual color loss (becoming whiter), with no other signs of instability. These findings support the potential use of Tucumã oil for producing stable NLCs suitable for topical delivery.

Prostate cancer (PC) is one of the most prevalent malignancies among men, with a staggering 1.5 million new cases and 350,000 deaths reported globally in 2022. Conventional treatment methods, including chemotherapy, radiation therapy, surgery, and hormonal therapy, often encounter significant challenges such as systemic toxicity and diminished efficacy, particularly in the advanced stages of the disease. Treatment of prostate cancer remains a formidable challenge because of the poor water solubility of many chemotherapeutic agents, which severely limits their bioavailability. However, the rise of targeted therapies has catalyzed the development of innovative drug delivery systems designed to enhance the bioavailability and precision of therapeutic agents. Solid lipid nanoparticles (SLNs) are a promising solution that can effectively encapsulate chemotherapeutic agents and genetic materials. Their unique attributes, such as biocompatibility, controlled release profile, and customizable surface properties, make them advantageous alternatives to conventional treatment strategies, effectively addressing the inherent limitations of prostate cancer therapy. To provide the context, relevant publications were searched on Google Scholar, PubMed, Science Direct, Dimension AI, and EBSCO Host using specific keywords such as controlled drug release, cationic surfactants, drug delivery, drug loading, drug encapsulation lipid, prostate cancer, surface modification, solid lipid nanoparticles (SLNs), tumor microenvironment, to list a few. We did not add any limits to the publication date during the inclusion of papers. However, it is noteworthy that the initial reports including the aforementioned keywords have been published starting from 2010. SLNs demonstrate substantial potential as effective nanocarriers for precise delivery of chemotherapeutic agents and genetic materials to prostate cancer cells. Their targeted delivery to these cells by surface modification with suitable ligands, antigens, peptides, and other recognition molecules has enhanced therapeutic efficacy. Further research on the interaction of SLN with the tumor environment is imperative to fully comprehend the uptake pathways. Extensive translation and preclinical studies are required to determine the safety and efficacy of SLNs before their use in clinical settings.

Researchers have explored and cultivated suitable membrane mimetics to preserve a physiological solvent condition for membrane protein functions. This involves emulating the properties of lipid bilayers, particularly within the hydrophobic core. Membrane mimetics exist in diverse forms, such as micelles, bicelles, liposomes, and nanodiscs. Polymers, such as styrene-maleic acid (SMA), have been found to offer a potentially suitable means to solubilize membrane proteins without resorting to detergents. It is widely recognized that various membrane mimetics yield distinct structural and dynamic configurations in membrane proteins. Styrene-maleic acid derivatives (SMADs) are of particular significance in this study; they are known for their ability to generate lipid nanoparticles. It has been hypothesized that using SMA derivatives with the same charge as the target membrane protein preserves the protein's structural and dynamic attributes compared to other bilayer membrane mimetics. This study explores the impact of different charges of SMA derivatives on two bacteriophage-encoded peptides explicitly focusing on their influence as charged peptides. Positively charged, neutral, and negatively charged SMA derivatives interactions with pinholin S21 and the phage-encoded cationic antimicrobial peptide gp28 lipid vesicles were assessed. These interactions were characterized using dynamic light scattering (DLS) techniques and continuous wave electron paramagnetic resonance (CW-EPR) spectroscopy. From our DLS results, we observed a reduction in size compared to the vesicle control, which is consistent with the formation of SMADLPs (styrene maleic acid derivative lipid nanoparticles). The key outcome was in the identification of how various SMA derivatives affect the interaction of gp28 and pinholin membrane peptides, which is useful when trying to understand how the different SMA polymers can influence the behavior and stability of protein complexes. For gp28 peptide, CW-EPR spectral analysis indicates no line broadening in its profile, suggesting that binding interactions with SMA derivatives do not significantly disrupt the structural integrity or dynamic behavior of the gp28 peptide. SMA-Pos interaction with pinholin shows some minimal perturbation, confirming that it is not as compatible compared to SMA-Neut and SMA-Glu. This study will provide insights into the optimal conditions for studying membrane protein interactions, focusing on the structural dynamics of gp28 and pinholin in the presence of different SMA derivatives.

The intercellular lipid matrix of stratum corneum is composed of many lipid components, including ceramides, free fatty acids, and cholesterol, which nonetheless form regularly arranged structures; short- and long-period lamellar structures, and hexagonal and orthorhombic hydrocarbon-chain packing structures. From the viewpoint of compatibility among the structures, there should be a correlation between a lamellar structure arranged periodically along the long axis of the lipid molecules and a packing structure of the hydrocarbon chains in the plane orthogonal to the long axis. To address this issue, differential scanning calorimetry and temperature-dependent X-ray diffraction experiment were performed on human stratum corneum. A detailed comparative analysis of the results on phase transitions obtained by the two methods revealed that the short-period lamellar structure has the orthorhombic hydrocarbon-chain packing structure that is normally observed, while the long-period lamellar structure has another orthorhombic hydrocarbon-chain packing structure hidden in the normal structure, and furthermore, the hexagonal hydrocarbon-chain packing structure does not appear to form a corresponding multilamellar structure and start to undergo a phase transition to the liquid state approximately at 33 °C. The domain constructed with the short-period lamellar structure and the orthorhombic hydrocarbon-chain packing structure is important in considering the water regulation mechanism in stratum corneum, since it provides evidence not only of the water layer of the former but also of changes in the head group of the latter.

A variety of studies published in the last decades in the field of lipid biophysics deal with the puzzle regarding the relationship between the signaling power of bioactive lipids (sphingolipids) and their capacity to induce lipid membrane heterogeneity (domains). Advances in technology, particularly Atomic Force Microscopy (AFM), have provided a solid contribution in this regard. Moreover, supported planar bilayers (SPB) have become an established membrane model in the study of lipid-lipid interactions. However, in spite of the large amount of published results in this field, the data remain scattered, and a coherent collection that allows easy access to the investigator is missing. This review summarizes the relevant results obtained in our laboratory through the use of AFM under comparable experimental conditions, offering a collection of data on supported lipid bilayer thicknesses and breakthrough forces. An extensive list of lipid compositions including phospholipids, cholesterol and sphingolipids (sphingomyelins, ceramides), at varying molecular ratios, has been considered.

Silver nanoparticles (AgNPs) are extensively used in healthcare, medicine, and environmental fields owing to their strong antiviral and antibacterial properties. Although these NPs interact with cellular biomembranes or vesicular lipid membranes, their mechanisms of interaction under physiological conditions using cell-mimetic giant unilamellar vesicles (GUVs) have been rarely investigated. In this study, we focused on the interaction of AgNPs with cell-sized GUVs and investigated deformation, membrane permeation, and change in membrane area under 0.3 - 5.0 μg/mL concentrations of AgNPs. The synthesized particles exhibited an average size of 58.1 nm and a zeta potential of -6.9 mV. The deformation of GUV and the fraction of deformation increase with the increase of AgNPs concentration. The encapsulating calcein of GUVs leaked out through the membranes while interacting the AgNPs, indicated the nano-sized pore formation in the membranes of vesicles. The leakage constant increased with the increase of NPs concentration, as well as the pore size. The membrane area of a GUV measured by micropipette technique exhibits a dynamic response: an initial rapid expansion, followed by a gradual contraction, and a subsequent slight increase over time. This provides insights into AgNPs-GUVs binding dynamics. These investigations help to understand the mechanism of interaction of AgNPs in the cell membranes which might be used in several biophysical and biomedical applications.

Dental fluorosis, as a common chronic fluoride toxicity oral disease, is mainly caused by long-term excessive intake of fluoride, which seriously affects the aesthetics and function of patients' teeth. In recent years, with the rapid development of metabolomics technology, lipidomics, as an important means to study the changes in lipid metabolism in organisms, has shown great potential in revealing the mechanisms of disease development. As a major component of cell membranes and a signaling molecule, metabolic disorders of lipids are closely related to a variety of diseases, but the specific mechanism of action in dental fluorosis is still unclear. Therefore, the present study aimed to systematically analyze the differences in lipid profiles between dental fluorosis patients and healthy populations by using broad-based targeted lipidomics technology to provide new perspectives on the pathogenesis of dental fluorosis. To this end, the researchers compared the salivary lipidome of healthy participants with the salivary micro lipidome of dental fluorosis patients. Their saliva samples were collected, and advanced broad-based targeted lipidomics technology, combined with a high-performance liquid chromatography-mass spectrometry (LC-MS) system, was used to comprehensively detect and quantify the lipids in the samples. The lipid data were processed and analyzed by bioinformatics to identify the unique patterns of changes in the lipid profiles of dental fluorosis patients and to verify the significance of these changes using statistical methods. Several glycerophospholipids, fatty acyls, and sphingolipids exhibited marked alterations in dental Among these, glycocholic acid, LPA (18:4), taurolithocholic acid-3-sulfate, lithocholic acid-3-sulfate, and taurochenodeoxycholic acid-3-sulfate were observed between dental fluorosis patients and healthy controls. taurochenodeoxycholic acid was significantly decreased, while PA (12:0_12:0) levels were significantly elevated. These findings suggest that These findings suggest that disturbances in lipid metabolism play a crucial role in developing dental fluorosis.

The role of cholesterol in the organization and ordering of membrane domains has been well established over the past decades. However, the involvement of cholesterol precursors and byproduct sterols in modulating the physicochemical properties of cell membranes remains less thoroughly explored. In this study, we investigated the effects of cholesterol, two hydroxylated catabolites (24-hydroxycholesterol and 25-hydroxycholesterol), and two biosynthesis precursors (desmosterol and lanosterol) on model of liquid-ordered (Lo) and liquid-disordered (Ld) membrane domains. Membrane ordering and molecular mobility were assessed using two fluorescent probes; Laurdan, which senses polarity near the membrane aqueous interface and cholesterol-pyrene, which senses ordering closer to the center of the membrane bilayer. The results showed that Laurdan can distinguish between environmental polarity and the contribution of membrane domains. The probe mobility varied depending on the sterol and did not strictly correlate with membrane order. Cholesterol-pyrene revealed that the sterols induce varying degrees of ordering around the bilayer center. A notable observation in Ld membranes using different probes was that the ordering effect of sterols was similar near the lipid head groups and at the center of the bilayer. Hydroxycholesterols exhibited a low ordering effect, whereas cholesterol and desmosterol induced a strong effect. In contrast, in Lo membranes, hydroxycholesterols produced a strong ordering effect near the head groups but a reduced effect near the bilayer center.

Two procedures are compared for the isolation of detergent-resistant membranes (DRMs) from the HeLa model cell line. The isolation was based on application of Triton X-100 followed by 4 or 18 h ultracentrifugation in sucrose (5-42.5, % w) or Optiprep™ (10-25, % w) gradients. In the fractions obtained, the total amount of protein, cholesterol, and free thiols was evaluated using spectrophotometry. Increased protein as well as free thiol contents were demonstrated in higher density fractions. In contrast, the highest cholesterol levels were observed in light or medium heavy fractions with a low proportion of sucrose or Optiprep, especially after 18 h of centrifugation. For the sucrose gradient, we used voltammetric determination of the catalytic hydrogen evolution reaction at the Hg-electrode for individual fractions. The catalytic response, expressed as the height of the presodium wave, increased from light to heavy fractions corresponding to the protein content and/or other catalytically active species. The size of the DRMs or their associates ranged from 20 to 1000 nm, independently of the isolation protocol used. Proteins typically associated with DRMs such as caveolin and flotillin and characteristic for light and medium heavy gradient fractions, were determined using immunochemistry. We studied the subcellular localization of caveolin, flotillin, raftlin and transferrin, a control protein found intracellularly in the cytoplasm. Using confocal fluorescence microscopy, we confirmed the presence of caveolin and flotillin in the cytoplasmic membrane of HeLa cells. Raftlin was identified in both the membrane, and as part of the cell nucleus. We also performed untargeted lipidomic LC-MS analysis of the individual fractions of sucrose ultracentrifugation gradient obtained after 18 h. The predominant lipid subclasses were phosphatidylcholines and diacylglycerols. Apart from cholesterol and its ester, the rest of identified lipid classes was similar to that found in full HeLa cell lysates. The presented findings could be important for interpreting interlaboratory results and may be used as a guide for further studies on DRMs.

The triggering receptor expressed on myeloid cells 2 (TREM2) is an immunoreceptor that interacts with a wide range of non-protein ligands, and it has been implicated in infectious and non-infectious diseases. However, there is a limited understanding on how this receptor interacts with non-protein ligands and the potential of such information to develop new therapeutic drugs. Therefore, our study aimed to elucidate the interactions between TREM2 and its non-protein ligands. First, we searched PubChem and Protein Data Bank (PDB) for TREM2 structures and their corresponding non-protein ligands. Subsequently, these structures were employed in molecular docking and MM/GBSA simulations with the Maestro software and molecular dynamics in GROMACS software. TREM2 was subsequently subjected to druggable site prediction using CavityPlus and receptor-based drug repositioning via the DrugRep server. TREM2 interacts with high affinity with its 12 non-protein ligands, with affinity values ranging from -33.01 kcal/mol for phosphatidylserine to -80.87 kcal/mol for cardiolipin (CLP). In molecular dynamics simulations, homodimeric TREM2 bound more stably to its lipid ligands, such as CLP and PSF, whereas it was unstable when unbound. The interactions between the receptor and its non-protein ligands were driven by the complementarity determining regions (CDR) 1 and 2, that are present in the hydrophobic and positively charged regions, highlighting that the Y38-R98 region is fundamental for drugs targeting TREM2. Our data underscore the significance of TREM2's CDRs in recognizing its ligands, suggesting they as promising targets for prospective drug design studies.

Hinokitiol (β-thujaplicin) is a natural antimicrobial agent used in cosmetics. The aim of presented studies was to gain insight into the interactions of hinokitiol with lipids in model membranes and to correlate this with the selective effect of hinokitiol on cells. To reach this goal, the toxicity of hinokitiol was evaluated using keratinocyte and fibroblast cell lines, and studies were performed on lipid monolayers (both one component and mixed systems). During investigations the surface pressure - area measurements, penetration studies and Brewster angle microscopy experiments were done. The analysis of the parameters calculated from the experimental data and the comparison of BAM images evidenced that, at membrane - related surface pressure, hinokitiol does not insert into model keratinocyte and fibroblast membranes and its impact on these systems is very weak. This important conclusion correlates with the in vitro experiments. The results for one component systems evidenced that the effect of hinokitiol on mammalian lipid films depends on the monolayer organisation and the lipid structure (especially the lipid polar head). In consequence, the type and proportion of lipids determines the effect of hinokitiol on the mixed films. The latter corroborates with the differences in the influence of hinokitiol on bacteria compared to mammalian lipids. It was concluded that hinokitiol exhibits selective activity toward bacterial cells compared to mammalian cells and their corresponding model membranes. Thus, the predominance of hinokitiol's antibacterial properties over its toxicity to skin cells may therefore be related to interactions of this compound with membrane lipids.

Monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) are the main lipids present in thylakoids, site of photosynthesis in chloroplasts. In this work we are interested in the behavior of highly unsaturated MGDG and DGDG, studied by surface pressure measurements. Compression isotherms of these lipids show that they are able to form Langmuir monolayers, remaining in liquid expanded phase during all the compression. However, measurements at constant surface pressure and compression modulus suggest that these monolayers are not stable, even if increasing the number of spread molecules seems to increase the stability, with a particularly noticeable effect in the case of MGDG as compared to DGDG. Monolayers are then transferred on mica supports and resulting Langmuir-Blodgett films are imaged by Atomic Force Microscopy. Images and height profiles reveal the presence of thicker areas showing that some molecules are ejected outside the monolayer's plane, with a higher propensity in the case of MGDG monolayers. We discuss these results taking into account two mechanisms, which may occur jointly: the oxidation of lipid hydrophobic chains and the desorption of lipids from the interface. The desorption amplitude would be higher in the case of MGDG, due to its propensity to organize in non-bilayer nature.

Nitro-fatty acids (NO2-FAs) are endogenous electrophilic signalling modulators, and some of them have been proposed as drug candidates. The main ones include nitro-oleic acid (NO2-OA) and other derivatives of unsaturated fatty acids such as nitro-linoleic acid (NO2-LA). In this study, we describe the behavior of 9/10-NO2-OA, 10-NO2-LA and the conjugated nitro-linoleic acid (9/12-NO2-cLA) in a model POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) membrane using molecular dynamics and selected experimental approaches. We showed that when loaded in liposomes, NO2-FAs undergo degradation (a decay reaction) to a very limited extent, in contrast to the free molecular form in an aqueous environment. This was confirmed by the electron paramagnetic resonance spectroscopic analysis of NO radical release. In general, NO2-FAs suppress membrane hydration, especially in the segment where the ester groups are located. Further, in the presence of NO2-FAs, there is increased membrane fluidity and a decrease in the degree of lipid order. These effects are greater for NO2-FAs than for their non-nitrated versions. The presence of a nitro group in close contact with the polar head groups was confirmed. This drives the tilt of the lipid chain which in turn induces membrane disorder. Protonated NO2-FAs penetrated more easily/deeper into the membrane structure than the dissociated forms and this makes the membrane bilayer surface more negatively charged based on zeta potential measurement. We also found that NO2-FAs incorporated into POPC liposomes retained their ability to activate the Nrf2 pathway. This was documented by an increased expression of heme oxygenase-1 at the level of mRNA, with a parallel decrease in protein levels of Keap1, in murine macrophage RAW264.7 cells. The NO2-FAs treatment resulted in an increase in intracellular NO level in vitro as determined by a genetically encoded G-geNOp sensor. This was confirmed at statistically significant level only for NO2-OA, not for NO2-LA or NO2-cLA. The results indicate that biologically relevant NO release may be strictly dependent on which NO2-FA is investigated. This study supports the hypothesis that NO2-FAs are distributed (co-localized) in cells and tissues in the lipid or aqueous phase, which affects whether they are mobile, stable, and thus biologically active.

Model lipid bilayers, reconstituted by using bacterial lipid extracts, are reliable systems to investigate the physical properties of bacterial membranes, and can be used, for example, to aid the design of new antibiotics. Here, we discuss the optimisation of a protocol for the production of hydrogenous and deuterated glycerophospholipid (GPL) extracts from Escherichia coli, and their reconstitution into model membranes. This protocol stands apart from state-of-the-art methods by introducing an additional purification step, which ensures a better separation of the GPL molecules from other membrane components such as neutral lipids. The composition of these extracts was characterised with different analytical methods. Experimental conditions were optimised for producing bacterial membrane models in the form of vesicles, lipid monolayers at the air/water interface and supported lipid bilayers. A combination of biophysical techniques, including Langmuir isotherms, neutron reflectometry, quartz crystal microbalance with dissipation monitoring, and small angle X-ray scattering provided detailed information on the self-assembled structures, and highlighted interesting differences between hydrogenous and deuterated extracts. Altogether, we report a detailed description of extraction and characterisation of hydrogenous and deuterated E. coli GPL extracts. The study of such complex lipid mixtures is important to recreate highly biologically relevant bacterial membrane models for studies aimed at understanding the biological function of bacterial membranes.

Lipid Nanoparticles (LDE) have been used as a drug delivery vehicle to treat various diseases. LDEs resemble the structure of human low-density lipoprotein (LDL), but lack apoliprotein B (apo-B). The aim of this study was to determine whether changes in the proportion of unesterified cholesterol (UC) or triacylglycerols (TG) affect the physical stability of LDE in aqueous solutions over a six-month observation period, as analysed by Ultra Small-angle X-ray Scattering (USAXS), Dynamic Light Scattering (DLS) and zeta potential measurements. It was shown that variations in UC or TG content in the initial lipid mixture did not alter the size of the resulting LDE nanoparticles, which remained within the 30-35 nm range. This particle size was maintained for up to three months in formulations with varying TG content and up to four months in those with varying UC content. Thereafter, a progressive increase in nanoparticle size was observed, which suggests enhanced aggregate formation and reduced of LDE stability between 3 and 6 months of storage. This loss of stability did not appear to be directly related to changes in UC or TG composition. Notably, USAXS and DLS measurements yielded comparable results, which reinforces the reliability of the data. In addition, the zeta potential remained close to zero for all seven nanoparticle compositions throughout the six months, indicating that all LDE formulations had electrostatic neutral potential and remain so when they progressively aggregate with time. Complementary analyses also showed that LDE particles are, on average, spherical in shape. Overall, these findings provide relevant insights for the rational design of lipid mixtures in the preparation of nanoemulsions for drug delivery applications.

The properties of phospholipid bilayers, which are important in various biophysical and biomedical studies, critically depend on the hydration of the lipid bilayer. Interbilayer water in multilamellar vesicles or planar multilayers is a very convenient object for studying the interfacial lipid-water interaction. However, many parameters of the interbilayer water remain incompletely studied, and in some cases different experimental methods yield different parameters of interbilayer water. Here, we developed a Raman spectroscopy method for characterizing interbilayer water in multilayer phospholipid samples. This method was applied to one saturated (DPPC) and one unsaturated (DOPC) phospholipid hydrated at high relative humidity and studied over a wide temperature range. It was found that although above the freezing point of water the OH stretching spectra of interbilayer water were similar to those of bulk water, only about one-fifth of the interbilayer water crystallized at the lowest experimental temperature (110 K). In combination with Raman spectra of aqueous suspensions of phospholipids of known compositions, the number of interbilayer H2O molecules per lipid molecule (hydration number) was determined. The hydration number was found for the ordered (gel) and disordered (fluid) phases of hydrated phospholipid bilayers at different temperatures and several relative humidities. The results were compared with values of the hydration number obtained by other methods, and an interpretation was proposed that takes into account the fractions of the free and non-free (perturbed) interbilayer water.