Phosphatidylcholine Headgroup Research Articles

Solid-State NMR Validation of OPLS4: Structure of PC-Lipid Bilayers and Its Modulation by Dehydration.

Atomistic molecular dynamics (MD) simulations are a much-used tool for investigating the structure and dynamics of biomembranes with atomic resolution. The validity of the representations obtained is determined by the accuracy and realism of the MD model (force field). Here, we evaluated the proprietary OPLS4 force field of Schrödinger, Inc. against atomic-resolution experimental data, and compared its performance to CHARMM36, one of the best-performing openly available force fields. As a benchmark, we used high-resolution nuclear magnetic resonance (NMR) order parameters for C-H bonds─directly and reliably calculable from MD simulations─measured in phosphatidylcholine (PC) lipid bilayers under varying hydration conditions. Comparisons were made with two dehydration data sets: for saturated (1,2-dimyristoylphosphatidylcholine, DMPC) lipid bilayers from the literature and for unsaturated (1-palmitoyl-2-oleoylphosphatidylcholine, POPC) lipid bilayers measured here. Our findings indicate that OPLS4 reproduces the structure and dehydration response of PC-lipid bilayers fairly well, even slightly outperforming CHARMM36. Both models' main inaccuracies appear in (1) the order parameter magnitudes in the glycerol backbone and unsaturated carbon segments and (2) the qualitatively differing structural response of the PC headgroup to dehydration compared to experiments. In summary, this work underscores the importance of independent validation for (proprietary) force fields and highlights the striking similarities and nuanced differences between OPLS4 and CHARMM36 in describing biomembranes.

Imidazole Headgroup Phospholipid Shows Asymmetric Distribution in Vesicles and Zinc-Dependent Esterase Activity.

Artificial lipids have become increasingly important in generating novel nanoenzymes and nanoparticles. Imidazole has been well established as a versatile catalyst in synthetic chemistry and through its related amino acid histidine in enzymes. By exploiting the transphosphatidylation reaction of phospholipase D, the choline headgroup of phosphatidyl choline was exchanged for the imidazole moiety containing histidinol. Here, we introduce a novel phosphatidylhistidinol (PtdHisOH) lipid and characterise it with respect to its catalytic abilities and its ability to modulate vesicle size. Our data reveal a zinc-dependent esterase activity that was strongest in vesicles and micelles, with slower catalytic rates being observed in flat lipid presentation systems and two-phase systems, indicating the importance of surface presentation and curvature effects on the catalytic activity of PtdHisOH. Such lipids offer the opportunity to impart de novo catalytic functionality to self-assembled lipid systems such as synthetic cells, leading to the development of new technologies for biocatalysis applications.

Phosphatidylcholine head group chemistry alters the extrahepatic accumulation of lipid-conjugated siRNA

Small interfering RNAs (siRNAs) are revolutionizing the treatment of liver-associated indications. Yet, robust delivery to extrahepatic tissues remains a challenge. Conjugating lipids (e.g., docosanoic acid, DCA) to siRNA supports extrahepatic delivery, but tissue accumulation remains lower than that achieved in liver by approved siRNA therapeutics. Early evidence suggests functionalizing DCA with a headgroup (e.g., phosphatidylcholine, PC) may enhance delivery to certain tissues. Here, we report the first systematic evaluation of the effect of PC headgroup chemistry on the extrahepatic distribution of DCA-conjugated siRNAs. We show that functionalizing DCA with a PC headgroup enhances siRNA accumulation in heart, muscle, lung, pancreas, duodenum, urinary bladder, and fat. Varying the size of the linker between the phosphate and choline moiety of the PC headgroup altered the extrahepatic accumulation of siRNA, with optimal linker length being different for different tissues. Increasing PC headgroup valency also improved extrahepatic accumulation in a tissue-specific manner. This study demonstrates the structural impact of the PC moiety on the biodistribution of lipid-conjugated siRNA and introduces multiple novel phosphocholine linker variants for chemical optimization of DCA-conjugated siRNA. These chemical variants can be used in the context of other lipids to increase the repertoire of conjugates for extrahepatic distribution of siRNAs.

Antivesiculation and Complete Unbinding of Tail-Tethered Lipids.

We report the effect of tail-tethering on vesiculation and complete unbinding of bilayered membranes. Amphiphilic molecules of a bolalipid, resembling the tail-tethered molecular structure of archaeal lipids, with two identical zwitterionic phosphatidylcholine headgroups self-assemble into a large flat lamellar membrane, in contrast to the multilamellar vesicles (MLVs) observed in its counterpart, monopolar nontethered zwitterionic lipids. The antivesiculation is confirmed by small-angle X-ray scattering (SAXS) and cryogenic transmission electron microscopy (cyro-TEM). With the net charge of zero and higher bending rigidity of the membrane (confirmed by neutron spin echo (NSE) spectroscopy), the current membrane theory would predict that membranes should stack with each other (aka "bind") due to dominant van der Waals attraction, while the outcome of the nonstacking ("unbinding") membrane suggests that the theory needs to include entropic contribution for the nonvesicular structures. This report pioneers an understanding of how the tail-tethering of amphiphiles affects the structure, enabling better control over the final nanoscale morphology.

Effect of invasome composition on membrane fluidity, vesicle stability and skin interactions

Invasomes have been widely exploited to enhance the percutaneous permeation of drugs. On the other hand, few studies have been dedicated to evaluating how their composition impacts the interaction with the skin, vesicle rigidity and stability, which was the focus of this investigation. Light scattering and spectroscopic techniques were considered for vesicle characterization. The addition of cholesterol (CHOL) into the phosphatidylcholine (PC) vesicles led to increased membrane rigidity (from PC:CHOL 5:0.5) and a concentration-dependent disorder effect on skin domains. Nevertheless, these vesicles were showed to be less stable. Ethanol, in turn, resulted in larger and more flexible vesicles, which can be attributed to its preferential distribution in headgroups of PC. The effect of limonene on membrane rigidity was dependent on the vesicle composition. It reduced the rigidity when few constituents were considered, but an opposite effect was observed for vesicles containing PC, CHOL, ethanol and limonene. Competitive effects of limonene and CHOL by the same domains in PC could explain these findings. Limonene was crucial to obtaining more monodisperse vesicles and it showed a synergistic action with CHOL in the disruption of lipid domains in the skin. Invasomes were more stable than liposomes. CHOL-free invasomes showed to be stable for up to 40 days at room temperature.

Synthesis of a photocleavable bola-phosphatidylcholine

Phosphatidylinositol transfer proteins (PITPs) are ubiquitous in eukaryotes and are involved in the regulation of phospholipid metabolism, membrane trafficking, and signal transduction. Sec14 is a yeast PITP that has been shown to transfer phosphatidylinositol (PI) or phosphatidylcholine (PC) from the endoplasmic reticulum to the Golgi. It is now believed that Sec14 may play a greater role than just shuttling PI and PC throughout the cell. Genetic evidence suggests that retrieval of membrane-bound PI by Sec14 also manages to present PI to the phosphatidylinositol-4-kinase, Pik1, to generate phosphatidylinositol-4-phosphate, PI(4)P. To test this hypothetical model, we designed a photocleavable bolalipid to span the entire membrane, having one phosphatidylcholine or phosphatidylinositol headgroup on each leaflet connected by a photocleavable diacid. Sec14 should not be able to present the bola-PI to Pik1 for phosphorylation as the head group will be difficult to lift from the bilayer as it is tethered on the opposite leaflet. After photocleavage the two halves would behave as a normal phospholipid, thus phosphorylation by Pik1 would resume. We report here the synthesis of a photocleavable bola-PC, a precursor to the desired bola-PI. The mono-photocleavable bola-PC lipid was designed to contain two glycerol molecules with choline head groups connected through a phosphodiester bond at the sn3 position. Each glycerol was acylated with palmitic acid at the sn1 position. These two glycerol moieties were then connected through their respective sn2 hydroxyls via a photocleavable dicarboxylic acid containing a nitrophenyl ethyl photolabile protecting group. The bola-PC and its precursors were found to undergo efficient photocleavage when irradiated in solution or in vesicles with 365 nm light for two minutes. Treatment of the bola-PC with a mutant phospholipase D and myo-inositol produced a mono-inositol bola-PC-PI.

Key Role of Choline Head Groups in Large Unilamellar Phospholipid Vesicles for the Interaction with and Rupture by Silica Nanoparticles.

For highly abundant silica nanomaterials, detrimental effects on proteins and phospholipids are postulated as critical molecular initiating events that involve hydrogen-bonding, hydrophobic, and/or hydrophilic interactions. Here, large unilamellar vesicles with various well-defined phospholipid compositions are used as biomimetic models to recapitulate membranolysis, a process known to be induced by silica nanoparticles in human cells. Differential analysis of the dominant phospholipids determined in membranes of alveolar lung epithelial cells demonstrates that the quaternary ammonium head groups of phosphatidylcholine and sphingomyelin play a critical and dose-dependent role in vesicle binding and rupture by amorphous colloidal silica nanoparticles. Surface modification by either protein adsorption or by covalent coupling of carboxyl groups suppresses the disintegration of these lipid vesicles, as well as membranolysis in human A549 lung epithelial cells by the silica nanoparticles. Furthermore, molecular modeling suggests a preferential affinity of silanol groups for choline head groups, which is also modulated by the pH value. Biomimetic lipid vesicles can thus be used to better understand specific phospholipid-nanoparticle interactions at the molecular level to support the rational design of safe advanced materials.

Momentum-dependent sum-frequency vibrational spectroscopy of bonded interface layer at charged water interfaces.

Interface-specific hydrogen (H)-bonding network of water directly controls the energy transfer and chemical reaction pathway at many charged aqueous interfaces, yet to characterize these bonded water layer structures remains a challenge. We now develop a sum-frequency spectroscopic scheme with varying photon momenta as an all-optic solution for retrieving the vibrational spectra of the bonded water layer and the ion diffuse layer and, hence, microscopic structural and charging information about an interface. Application of the method to a model surfactant-water interface reveals a hidden weakly donor H-bonded water species, suggesting an asymmetric hydration-shell structure of fully solvated surfactant headgroups. In another application to a zwitterionic phosphatidylcholine lipid monolayer-water interface, we find a highly polarized bonded water layer structure associating to the phosphatidylcholine headgroup, while the diffuse layer contribution is experimentally proven to be negligible. Our all-optic method offers an in situ microscopic probe of electrochemical and biological interfaces and the route toward future imaging and ultrafast dynamics studies.

Location of the TEMPO moiety of TEMPO-PC in phosphatidylcholine bilayers is membrane phase dependent

The (2,2,6,6-tetramethylpiperidin-1-yl)oxyl (TEMPO) moiety tethered to the headgroup of phosphatidylcholine (PC) lipid is employed in spin labeling electron paramagnetic resonance spectroscopy to probe the water dynamics near lipid bilayer interfaces. Due to its amphiphilic character, however, TEMPO spin label could partition between aqueous and lipid phases, and may even be stabilized in the lipid phase. Accurate assessment of the TEMPO-PC configuration in bilayer membranes is essential for correctly interpreting the data from measurements. Here, we carry out all-atom molecular dynamics (MD) simulations of TEMPO-PC probe in single-component lipid bilayers at varying temperatures, using two standard MD force fields. We find that, for a dipalmitoylphosphatidylcholine (DPPC) membrane whose gel-to-fluid lipid phase transition occurs at 314 K, while the TEMPO spin label is stabilized above the bilayer interface in the gel phase, there is a preferential location of TEMPO below the membrane interface in the fluid phase. For bilayers made of unsaturated lipids, 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), which adopt the fluid phase at ambient temperature, TEMPO is unequivocally stabilized inside the bilayers. Our finding of membrane phase-dependent positioning of the TEMPO moiety highlights the importance of assessing the packing order and fluidity of lipids under a given measurement condition.

Capability of Polyunsaturated Phosphatidylcholine for Non-raft Domain Formation in Cholesterol-containing Lipid Bilayers

Polyunsaturated lipids are one of major components in cell membranes, but have attracted less interests compared to lipids with saturated or monosaturated acyl chains. A recent study reported that polyunsaturated phosphatidylethanolamine (PE) induces the non-raft domain formation in lipid bilayers comprising fluid phosphatidylcholine (PC) and cholesterol (Chol) via the segregation of polyunsaturated PE. In this study, we investigated the effects of polyunsaturated PC on the non-raft domain formation. We prepared supported lipid bilayers (SLBs) comprising polyunsaturated PC, monounsaturated PC (POPC), and Chol. Observation by fluorescence microscopy and atomic force microscopy showed that polyunsaturated PCs caused the domain formation, depending on their concentration and the degree of unsaturation. However, their effectivity was less than polyunsaturated PEs. Intermolecular interaction at the hydrophobic part induced the segregation of polyunsaturated PC from the Chol-containing region, although competing with the umbrella effect due to the hydrophilic headgroup of PC which favors the association with Chol.

The effect of dopamine on model lipid membrane structure

Dopamine is a neurotransmitter released by neurons to send signals to other nerve cells. Previous work on the interaction of dopamine with model lipid bilayers made using solution-state NMR spectroscopy has shown that dopamine interacts with phosphatidylcholine (PC) and phosphatidylserine (PS) lipid headgroups primarily through the aromatic side opposite to the hydroxyl groups [1]. Here we use low-angle x-ray scattering (LAXS), solid-state deuterium NMR, as well as electron paramagnetic resonance (EPR) to determine the effect of dopamine on the physical properties of lipid membranes.

The location of TEMPO moiety of TEMPO-PC is membrane phase-dependent

TEMPO-PC is a nitroxide spin-labeled lipid in which the 2,2,6,6-tetramethyl piperidine-1-oxyl (TEMPO) group is covalently attached to the choline head group of phosphatidylcholine (PC). TEMPO-PC has been utilized to interpret the dynamics of water molecules near membrane surfaces in electron resonance spectroscopy as well as to assess the penetration depth of water molecules through the membranes. Because of an amphiphilic character of TEMPO moiety, and the flexibility of lipid molecules, the depth distribution of TEMPO moiety is potentially affected by the lipid type and the phase of bilayer membranes.

Mycobacterium abscessus cell wall and plasma membrane characterization by EPR spectroscopy and effects of amphotericin B, miltefosine and nerolidol

Spin label electron paramagnetic resonance (EPR) spectroscopy was used to characterize the components of the Mycobacterium abscessus massiliense cell envelope and their interactions with amphotericin B (AmB), miltefosine (MIL), and nerolidol (NER). Spin labels analogous to stearic acid and phosphatidylcholine (PC) were distributed on an envelope layer with fluidity comparable to other biological membranes, probably the mycobacterial cell wall, because after treatment with AmB a highly rigid spectral component was evident in the EPR spectra. Methyl stearate analogue spin labels found a much more fluid membrane and did not detect the presence of AmB, except for at very high drug concentrations. Unlike other spin-labeled PCs, the TEMPO-PC spin probe, with the nitroxide moiety attached to the choline of the PC headgroup, also did not detect the presence of AmB. On the other hand, the steroid spin labels were not distributed across the membranes of M. abscessus and, instead, were concentrated in some other location of the cell envelope. Both MIL and NER compounds at 10 μM caused increased fluidity in the cell wall and plasma membrane. Furthermore, NER was shown to have a remarkable ability to extract lipids from the mycobacterial cell wall. The EPR results suggest that the resistance of mycobacteria to the action of AmB must be related to the fact that this drug does not reach the bacterial plasma membrane.

The tertiary structure of the human Xkr8\u2013Basigin complex that scrambles phospholipids at plasma membranes

Xkr8–Basigin is a plasma membrane phospholipid scramblase activated by kinases or caspases. We combined cryo-EM and X-ray crystallography to investigate its structure at an overall resolution of 3.8 Å. Its membrane-spanning region carrying 22 charged amino acids adopts a cuboid-like structure stabilized by salt bridges between hydrophilic residues in transmembrane helices. Phosphatidylcholine binding was observed in a hydrophobic cleft on the surface exposed to the outer leaflet of the plasma membrane. Six charged residues placed from top to bottom inside the molecule were essential for scrambling phospholipids in inward and outward directions, apparently providing a pathway for their translocation. A tryptophan residue was present between the head group of phosphatidylcholine and the extracellular end of the path. Its mutation to alanine made the Xkr8–Basigin complex constitutively active, indicating that it plays a vital role in regulating its scramblase activity. The structure of Xkr8–Basigin provides insights into the molecular mechanisms underlying phospholipid scrambling.

The Role of Pulmonary Surfactant in COVID-19 Understanding

Background: In Covid-19 the virus infects the respiratory tract in human. When lung tissue becomes diseased, the walls and lining of the alveoli and capillaries are damaged. At this point lung compliance and ventilation decrease. Pulmonary surfactant that is produced and dispersed into alveolar space, has a significant role in understanding how heavily covid-19 interferes and infects lung cells. The importance of pulmonary surfactant in alveoli is to lower surface tension at air/liquid interface in the lung. This is achieved by reducing the work of breathing and preventing alveolar collapse. The main constituent of pulmonary surfactant is dipalmitoylphosphatidylcholine (DPPC) (C40H80NO8P). It is a phospholipid containing two non polar palmitic acid C16 chains as hydrophobic tails linked to a polar head group of a phosphatidylcholine (also known as lecithin).
 Rationale of the Review and Objective Method: When DPPC molecules are in contact with a polar solvent, micelles which grow further into bilayers are formed considering their cylindrical structures. This trait makes the whole structure of pulmonary surfactant as amphipathic and surface active molecules. The head group of phosphatidylcholine in the pulmonary surfactant is attracted by polar liquid molecules causing a reduction of the liquid surface tension.
 Conclusion: This review complements the quoted information analysing them theoretically and integrates recent advances in pulmonary surfactant research with the global pandemic.

Rotational Dynamics of Water at the Phospholipid Bilayer Depending on the Head Groups Studied by Molecular Dynamics Simulations

Hydration states are a crucial factor that affect the self-assembly and properties of soft materials and biomolecules. Although previous experiments have revealed that the hydration state strongly depends on the chemical structure of lipid molecules, the mechanisms at the molecular level remain unknown. Classical and density-functional tight-binding (DFTB) molecular dynamics (MD) simulations are employed to determine the mechanisms underlying dissimilar water dynamics between lipid membranes with phosphatidylcholine (PC) and phosphatidylethanolamine (PE) head groups. The classical MD simulation shows that rotational relaxations of water are faster on the PE lipid than on the PC lipid, which is consistent with a previous experimental study using terahertz spectroscopy. Furthermore, DFTB-MD simulation of N(CH3)4+ and NH4+ ions, which correspond to the different head groups in PC and PE, respectively, shows qualitative agreement with the classical MD simulation. Remarkably, the PE lipids and the NH4+ ions break hydrogen bonds between water molecules in the secondary hydration shell. In contrast, the PC lipids and the N(CH3)4+ ions bind water molecules weakly in both the primary and secondary hydration shells and increase hydrogen bonds between water. Our atomistic simulations show that these changes in the hydrogen-bond network of water molecules cause the different rotational relaxation of water molecules between the two lipids.

Impact of Liposomal Spherical Nucleic Acid Structure on Immunotherapeutic Function.

Liposomal spherical nucleic acids (L-SNAs) show significant promise as cancer immunotherapeutics. L-SNAs are highly modular nanoscale assemblies defined by a dense, upright radial arrangement of oligonucleotides around a liposomal core. Herein, we establish a set of L-SNA design rules by studying the biological and immunological properties of L-SNAs as a function of liposome composition. To achieve this, we synthesized liposomes where the lipid phosphatidylcholine headgroup was held constant, while the diacyl lipid tail chain length and degree of saturation were varied, using either 1,2-dioleylphosphatidylcholine (DOPC), 1,2-dimyristoyl-phosphatidylcholine (DMPC), 1,2-dipalmitoylphosphatidylcholine (DPPC), or 1,2-distearoyl-phosphatidylcholine (DSPC). These studies show that the identity of the constituent lipid dictates the DNA loading, cellular uptake, serum stability, in vitro immunostimulatory activity, and in vivo lymph node accumulation of the L-SNA. Furthermore, in the 4T1 mouse model of triple-negative breast cancer (TNBC), the subcutaneous administration of immunostimulatory L-SNAs synthesized with DPPC significantly decreases the production of lung metastases and delays tumor growth as compared to L-SNAs synthesized using DOPC, due to the enhanced stability of L-SNAs synthesized with DPPC over those synthesized with DOPC. Moreover, the inclusion of cell lysates derived from Py8119 TNBC cells as antigen sources in L-SNAs leads to a significant increase in antitumor efficacy in the Py8119 model when lysates are encapsulated in the cores of L-SNAs synthesized with DPPC rather than DOPC, presumably due to increased codelivery of adjuvant and antigen to dendritic cells in vivo. This difference is further amplified when using lysates from oxidized Py8119 cells as a more potent antigen source, revealing synergy between the lysate preparation method and liposome composition in synthesizing immunotherapeutic L-SNAs. Together, this work shows that the biological properties and immunomodulatory activity of L-SNAs can be modulated by exchanging liposome components, providing another handle for the rational design of nanoscale immunotherapeutics.

Plasmalogen Lipids Influence Lateral and Transverse Organization in Model Membranes

Plasmalogens are a class of glycerophospholipids found in many different organisms. In mammalian plasma membranes, they are primarily located in the cytosolic leaflet, where they comprise as much as 20 mol% of the total phospholipids. While most biologically relevant phospholipids are diacylglycerols with ester linkages between the glycerol backbone and the hydrocarbon chains, plasmalogens are distinguished by a cis-vinyl ether linked fatty alcohol at the sn-1 position. Most plasmalogens possess either a phosphatidylcholine (PC) or phosphatidylethanolamine (PE) headgroup and an ester linked polyunsaturated fatty acid (either 20:4 or 22:6) at the sn-2 position.

Using Open Data to Rapidly Benchmark Biomolecular Simulations: Phospholipid Conformational Dynamics.

Molecular dynamics (MD) simulations are widely used to monitor time-resolved motions of biomacromolecules, although it often remains unknown how closely the conformational dynamics correspond to those occurring in real life. Here, we used a large set of open-access MD trajectories of phosphatidylcholine (PC) lipid bilayers to benchmark the conformational dynamics in several contemporary MD models (force fields) against nuclear magnetic resonance (NMR) data available in the literature: effective correlation times and spin–lattice relaxation rates. We found none of the tested MD models to fully reproduce the conformational dynamics. That said, the dynamics in CHARMM36 and Slipids are more realistic than in the Amber Lipid14, OPLS-based MacRog, and GROMOS-based Berger force fields, whose sampling of the glycerol backbone conformations is too slow. The performance of CHARMM36 persists when cholesterol is added to the bilayer, and when the hydration level is reduced. However, for conformational dynamics of the PC headgroup, both with and without cholesterol, Slipids provides the most realistic description because CHARMM36 overestimates the relative weight of ∼1 ns processes in the headgroup dynamics. We stress that not a single new simulation was run for the present work. This demonstrates the worth of open-access MD trajectory databanks for the indispensable step of any serious MD study: benchmarking the available force fields. We believe this proof of principle will inspire other novel applications of MD trajectory databanks and thus aid in developing biomolecular MD simulations into a true computational microscope—not only for lipid membranes but for all biomacromolecular systems.

Effect of Oleic Acid, Cholesterol, and Octadecylamine on Membrane Stability of Freeze-Dried Liposomes Encapsulating Natural Antimicrobials

Liposomes have been broadly studied as delivery systems for bioactive compounds, although its relatively low stability remains a limitation for commercial application. In this study, phosphatidylcholine (PC) liposomes were prepared entrapping a mixture of garlic extract (GE) and nisin (Nis) using cholesterol (CHO), oleic acid (OA), or octadecylamine (ODA) as membrane stabilizers to evaluate their physical, chemical, bioactive, and stability properties, in fully hydrated state and after freeze-drying. GE/Nis-loaded liposomes presented hydrodynamic diameter below 200 nm and polydispersity index below 0.30, typical for small unilamellar vesicles produced by thin film method. Under induced oxidation, the PC-OA-GE/Nis liposomes presented 91% less lipid peroxidation compared with the unloaded PC liposomes. The Fourier transform infrared spectroscopy (FTIR) analysis revealed a high level of hydrogen bonds in the polar head group of PC after addition of GE/Nis in all liposome formulations, in agreement to the high values of water activity and hygroscopicity found in the samples after freeze-drying. During 5 months storage at 4 °C, fully hydrated and lyophilized liposomes showed an increment in their average size and polydispersity index, but these values were reduced by the trehalose addition as lyoprotector. All liposome preparations maintained 100% activity against Listeria monocytogenes; nevertheless, a gradual reduction of activity against Salmonella enterica serovar Enteritidis was observed, suggesting a partial loss of GE active compounds. Despite some physical modifications, freeze-dried liposomes containing OA as stabilizer showed best antimicrobial properties and high lipid oxidation resistance, constituting a promising approach to stabilize GE/Nis for long-term storage.