Oxidation of Membrane Lipids Alters the Activity of the Human Serotonin 1A Receptor.

Lipid oxidation has significant effects on lipid bilayer properties; these effects can be expected to extend to interactions between the lipid bilayer and integral membrane proteins. Given that G protein-coupled receptor (GPCR) activity is known to depend on the properties of the surrounding lipid bilayer, these proteins represent an intriguing class of molecules in which the impact of lipid oxidation on protein behavior is studied. Here, we study the effects of lipid oxidation on the human serotonin 1A receptor (5-HT1AR). Giant unilamellar vesicles (GUVs) containing integral 5-HT1AR were fabricated by the hydrogel swelling method; these GUVs contained polyunsaturated 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine (PLinPC) and its oxidation product 1-palmitoyl-2-(9'-oxo-nonanoyl)-sn-glycero-3-phosphocholine (PoxnoPC) at various ratios. 5-HT1AR-integrated GUVs were also fabricated from lipid mixtures that had been oxidized by extended exposure to the atmosphere. Both types of vesicles were used to evaluate 5-HT1AR activity using an assay to quantify GDP-GTP exchange by the coupled G protein α subunit. Results indicated that 5-HT1AR activity increases significantly in bilayers containing oxidized lipids. This work is an important step in understanding how hyperbaric oxidation can change plasma membrane properties and lead to physiological dysfunction.

Mechanisms of aggregation and fibril formation of the amyloidogenic N-terminal fragment of apolipoprotein A-I.

The N-terminal (1-83) fragment of the major constituent of plasma high-density lipoprotein, apolipoprotein A-I (apoA-I), strongly tends to form amyloid fibrils, leading to systemic amyloidosis. Here, using a series of deletion variants, we examined the roles of two major amyloidogenic segments (residues 14-22 and 50-58) in the aggregation and fibril formation of an amyloidogenic G26R variant of the apoA-I 1-83 fragment (apoA-I 1-83/G26R). Thioflavin T fluorescence assays and atomic force microscopy revealed that elimination of residues 14-22 completely inhibits fibril formation of apoA-I 1-83/G26R, whereas Δ32-40 and Δ50-58 variants formed fibrils with markedly reduced nucleation and fibril growth rates. CD measurements revealed structural transitions from random coil to ß-sheet structures in all deletion variants except for the Δ14-22 variant, indicating that residues 14-22 are critical for the ß-transition and fibril formation. Thermodynamic analysis of the kinetics of fibril formation by apoA-I 1-83/G26R indicated that both nucleation and fibril growth are enthalpically unfavorable, whereas entropically, nucleation is favorable, but fibril growth is unfavorable. Interestingly, the nucleation of the Δ50-58 variant was entropically unfavorable, indicating that residues 50-58 entropically promote the nucleation step in fibril formation of apoA-I 1-83/G26R. Moreover, a residue-level structural investigation of apoA-I 1-83/G26R fibrils with site-specific pyrene labeling indicated that the two amyloidogenic segments are in close proximity to form an amyloid core structure, whereas the N- and C-terminal tail regions are excluded from the amyloid core. These results provide critical insights into the aggregation mechanism and fibril structure of the amyloidogenic N-terminal fragment of apoA-I.

Optimization of the Inverted Emulsion Method for High-Yield Production of Biomimetic Giant Unilamellar Vesicles.

In the field of bottom-up synthetic biology, lipid vesicles provide an important role in the construction of artificial cells. Giant unilamellar vesicles (GUVs), due to their membrane's similarity to natural biomembranes, have been widely used as cellular mimics. So far, several methods exist for the production of GUVs with the possibility to encapsulate biological macromolecules. The inverted emulsion-based method is one such technique, which has great potential for rapid production of GUVs with high encapsulation efficiencies for large biomolecules. However, the lack of understanding of various parameters that affect production yields has resulted in sparse adaptation within the membrane and bottom-up synthetic biology research communities. Here, we optimize various parameters of the inverted emulsion-based method to maximize the production of GUVs. We demonstrate that the density difference between the emulsion droplets, oil phase, and the outer aqueous phase plays a crucial role in vesicle formation. We also investigated the impact that centrifugation speed/time, lipid concentration, pH, temperature, and emulsion droplet volume has on vesicle yield and size. Compared to conventional electroformation, our preparation method was not found to significantly alter the membrane mechanical properties. Finally, we optimize the parameters to minimize the time from workbench to microscope and in this way open up the possibility of time-sensitive experiments. In conclusion, our findings will promote the usage of the inverted emulsion method for basic membrane biophysics studies as well as the development of GUVs for use as future artificial cells.

Mechanism of Initial Stage of Pore Formation Induced by Antimicrobial Peptide Magainin 2.

Antimicrobial peptide magainin 2 forms pores in lipid bilayers, a property that is considered the main cause of its bactericidal activity. Recent data suggest that tension or stretching of the inner monolayer plays an important role in magainin 2-induced pore formation in lipid bilayers. Here, to elucidate the mechanism of magainin 2-induced pore formation, we investigated the effect on pore formation of asymmetric lipid distribution in two monolayers. First, we developed a method to prepare giant unilamellar vesicles (GUVs) composed of dioleoylphosphatidylglycerol (DOPG), dioleoylphosphatidylcholine (DOPC), and lyso-PC (LPC) in the inner monolayer and of DOPG/DOPC in the outer monolayer. We consider that in these GUVs, the lipid packing in the inner monolayer was larger than that in the outer monolayer. Next, we investigated the interaction of magainin 2 with these GUVs with an asymmetric distribution of LPC using the single GUV method, and found that the rate constant of magainin 2-induced pore formation, kp, decreased with increasing LPC concentration in the inner monolayer. We constructed a quantitative model of magainin 2-induced pore formation, whereby the binding of magainin 2 to the outer monolayer of a GUV induces stretching of the inner monolayer, causing pore formation. A theoretical equation defining kp as a function of magainin 2 surface concentration, X, reasonably explains the experimental relationship between kp and X. This model quantitatively explains the effect on kp of the LPC concentration in the inner monolayer. On the basis of these results, we discuss the mechanism of the initial stage of magainin 2-induced pore formation.

Light-Controlled Membrane Mechanics and Shape Transitions of Photoswitchable Lipid Vesicles.

Giant unilamellar vesicles (GUVs) represent a versatile model system to emulate the fundamental properties and functions associated with the plasma membrane of living cells. Deformability and shape transitions of lipid vesicles are closely linked to the mechanical properties of the bilayer membrane itself and are typically difficult to control under physiological conditions. Here, we developed a protocol to form cell-sized vesicles from an azobenzene-containing phosphatidylcholine (azo-PC), which undergoes photoisomerization on irradiation with UV-A and visible light. Photoswitching within the photolipid vesicles enabled rapid and precise control of the mechanical properties of the membrane. By varying the intensity and dynamics of the optical stimulus, controlled vesicle shape changes such as budding transitions, invagination, pearling, or the formation of membrane tubes were achieved. With this system, we could mimic the morphology changes normally seen in cells, in the absence of any molecular machines associated with the cytoskeleton. Furthermore, we devised a mechanism to utilize photoswitchable lipid membranes for storing mechanical energy and then releasing it on command as locally usable work.

Cations induce shape remodeling of negatively charged phospholipid membranes.

The divalent cation Ca2+ is a key component in many cell signaling and membrane trafficking pathways. Ca2+ signal transduction commonly occurs through interaction with protein partners. However, in this study we show a novel mechanism by which Ca2+ may impact membrane structure. We find an asymmetric concentration of Ca2+ across the membrane triggers deformation of membranes containing negatively charged lipids such as phosphatidylserine (PS) and phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2). Membrane invaginations in vesicles were observed forming away from the leaflet with higher Ca2+ concentration, showing that Ca2+ induces negative curvature. We hypothesize that the negative curvature is produced by Ca2+-induced clustering of PS and PI(4,5)P2. In support of this notion, we find that Ca2+-induced membrane deformation is stronger for membranes containing PI(4,5)P2, which is known to more readily cluster in the presence of Ca2+. The observed Ca2+-induced membrane deformation is strongly influenced by Na+ ions. A high symmetric [Na+] across the membrane reduces Ca2+ binding by electrostatic shielding, inhibiting Ca2+-induced membrane deformation. An asymmetric [Na+] across the membrane, however, can either oppose or support Ca2+-induced deformation, depending on the direction of the gradient in [Na+]. At a sufficiently high asymmetric Na+ concentration it can impact membrane structure in the absence of Ca2+. We propose that Ca2+ works in concert with curvature generating proteins to modulate membrane curvature and shape transitions. This novel structural impact of Ca2+ could be important for Ca2+-dependent cellular processes that involve the creation of membrane curvature, including exocytosis, invadopodia, and cell motility.

Monodisperse Uni- and Multicompartment Liposomes.

Liposomes are self-assembled phospholipid vesicles with great potential in fields ranging from targeted drug delivery to artificial cells. The formation of liposomes using microfluidic techniques has seen considerable progress, but the liposomes formation process itself has not been studied in great detail. As a result, high throughput, high-yielding routes to monodisperse liposomes with multiple compartments have not been demonstrated. Here, we report on a surfactant-assisted microfluidic route to uniform, single bilayer liposomes, ranging from 25 to 190 µm, and with or without multiple inner compartments. The key of our method is the precise control over the developing interfacial energies of complex W/O/W emulsion systems during liposome formation, which is achieved via an additional surfactant in the outer water phase. The liposomes consist of single bilayers, as demonstrated by nanopore formation experiments and confocal fluorescence microscopy, and they can act as compartments for cell-free gene expression. The microfluidic technique can be expanded to create liposomes with a multitude of coupled compartments, opening routes to networks of multistep microreactors.

Giant Host Red Blood Cell Membrane Mimicking Polymersomes Bind Parasite Proteins and Malaria Parasites.

Malaria is an infectious disease that needs to be addressed using innovative approaches to counteract spread of drug resistance and to establish or optimize vaccination strategies. With our approach, we aim for a dual action with drug- and 'vaccine-like' activity against malaria. By inhibiting entry of malaria parasites into host red blood cells (RBCs) - using polymer vesicle-based (polymersome) nanomimics of RBC membranes - the life cycle of the parasite is interrupted and the exposed parasites are accessible to the host immune system. Here, we describe how host cell-sized RBC membrane mimics, formed with the same block copolymers as nanomimics, also bind the corresponding malaria parasite ligand and whole malaria parasites, similar to nanomimics. This was demonstrated using fluorescence imaging techniques and confirms the suitability of giant polymersomes (GUVs) as simple mimics for RBC membranes.

Quantitative analysis of the lamellarity of giant liposomes prepared by the inverted emulsion method.

The inverted emulsion method is used to prepare giant liposomes by pushing water-in-oil droplets through the oil/water interface into an aqueous medium. Due to the high encapsulation efficiency of proteins under physiological conditions and the simplicity of the protocol, it has been widely used to prepare various cell models. However, the lamellarity of liposomes prepared by this method has not been evaluated quantitatively. Here, we prepared liposomes that were partially stained with a fluorescent dye, and analyzed their fluorescence intensity under an epifluorescence microscope. The fluorescence intensities of the membranes of individual liposomes were plotted against their diameter. The plots showed discrete distributions, which were classified into several groups. The group with the lowest fluorescence intensity was determined to be unilamellar by monitoring the exchangeability of the inner and the outer solutions of the liposomes in the presence of the pore-forming toxin α-hemolysin. Increasing the lipid concentration dissolved in oil increased the number of liposomes ∼100 times. However, almost all the liposomes were unilamellar even at saturating lipid concentrations. We also investigated the effects of lipid composition and liposome content, such as highly concentrated actin filaments and Xenopus egg extracts, on the lamellarity of the liposomes. Remarkably, over 90% of the liposomes were unilamellar under all conditions examined. We conclude that the inverted emulsion method can be used to efficiently prepare giant unilamellar liposomes and is useful for designing cell models.

Self-assembled vesicles with functionalized membranes.

Biological membranes play a key role for the function of living organisms. Thus, many artificial systems have been designed to mimic natural cell membranes and their functions. A useful concept for the preparation of functional membranes is the embedding of synthetic amphiphiles into vesicular bilayers. The dynamic nature of such noncovalent assemblies allows the rapid and simple development of bio-inspired responsive nanomaterials, which find applications in molecular recognition, sensing or catalysis. However, the complexity that can be achieved in artificial functionalized membranes is still rather limited and the control of their dynamic properties and the analysis of membrane structures down to the molecular level remain challenging.

Asymmetric GUVs prepared by MßCD-mediated lipid exchange: an FCS study.

We report a simple method to obtain stable asymmetric giant unilamellar vesicles (GUVs). Fluorescence correlation spectroscopy was used to quantitatively characterize vesicle properties. After brain sphingomyelin (bSM) was exchanged into dioleoylphosphatidylcholine (DOPC) GUVs, lateral diffusion in the bSM-containing outer leaflet decreased, whereas that in the DOPC-containing inner leaflet was largely unchanged, confirming asymmetry and a lack of coupling between the physical states of the inner and outer leaflets. In contrast, after bSM was exchanged into brain phosphatidylcholine vesicles, lateral diffusion decreased in both leaflets. Thus, asymmetric GUVs should be useful for investigating the molecular mechanisms behind interleaflet coupling.

Stepwise synthesis of giant unilamellar vesicles on a microfluidic assembly line.

Among the molecular milieu of the cell, the membrane bilayer stands out as a complex and elusive synthetic target. We report a microfluidic assembly line that produces uniform cellular compartments from droplet, lipid, and oil/water interface starting materials. Droplets form in a lipid-containing oil flow and travel to a junction where the confluence of oil and extracellular aqueous media establishes a flow-patterned interface that is both stable and reproducible. A triangular post mediates phase transfer bilayer assembly by deflecting droplets from oil, through the interface, and into the extracellular aqueous phase to yield a continuous stream of unilamellar phospholipid vesicles with uniform and tunable size. The size of the droplet precursor dictates vesicle size, encapsulation of small-molecule cargo is highly efficient, and the single bilayer promotes functional insertion of a bacterial transmembrane pore.

Giant vesicles: preparations and applications.

There is considerable interest in preparing cell-sized giant unilamellar vesicles from natural or nonnatural amphiphiles because a giant vesicle membrane resembles the self-closed lipid matrix of the plasma membrane of all biological cells. Currently, giant vesicles are applied to investigate certain aspects of biomembranes. Examples include lateral lipid heterogeneities, membrane budding and fission, activities of reconstituted membrane proteins, or membrane permeabilization caused by added chemical compounds. One of the challenging applications of giant vesicles include gene expressions inside the vesicles with the ultimate goal of constructing a dynamic artificial cell-like system that is endowed with all those essential features of living cells that distinguish them from the nonliving form of matter. Although this goal still seems to be far away and currently difficult to reach, it is expected that progress in this and other fields of giant vesicle research strongly depend on whether reliable methods for the reproducible preparation of giant vesicles are available. The key concepts of currently known methods for preparing giant unilamellar vesicles are summarized, and advantages and disadvantages of the main methods are compared and critically discussed.

ESR and NMR studies provide evidence that phosphatidyl glycerol specifically interacts with poxvirus membranes.

BACKGROUND: The lung would be the first organ targeted in case of the use of Variola virus (the causative agent of smallpox) as a bioweapon. Pulmonary surfactant composed of lipids (90%) and proteins (10%) is considered the major physiological barrier against airborne pathogens. The principle phospholipid components of lung surfactant were examined in an in vitro model to characterize their interactions with VACV, a surrogate for variola virus. One of them, Dipalmitoyl phosphatidylglycerol (DPPG), was recently shown to inhibit VACV cell infection. RESULTS: The interactions of poxvirus particles from the Western Reserve strain (VACV-WR) and the Lister strain (VACV-List) with model membranes for pulmonary surfactant phospholipids, in particular DPPG, were studied by Electron Spin Resonance (ESR) and proton Nuclear Magnetic Resonance (1H-NMR). ESR experiments showed that DPPG exhibits specific interactions with both viruses, while NMR experiments allowed us to deduce its stoichiometry and to propose a model for the mechanism of interaction at the molecular level. CONCLUSIONS: These results confirm the ability of DPPG to strongly bind to VACV and suggest that similar interactions occur with variola virus. Similar studies of the interactions between lipids and other airborne pathogens are warranted.

Drug-cyclodextrin-vesicles dual carrier approach for skin targeting of anti-acne agent.

In the present study attempt was made for preparation of isotretinoin-hydroxypropyl beta cyclodextrin (HP-beta-CD) inclusion complex and encapsulate this complex in elastic liposomes to study the effect of dual carrier approach on skin targeting of isotretinoin. The isotretinoin HP-beta-CD complex was prepared by freeze-drying method and characterized by IR spectroscopy. The drug and drug-CD complex loaded elastic liposomal formulation were prepared and characterized in vitro, ex-vivo and in vivo for shape, size, entrapment efficiency, no. of vesicles per cubic mm, in vitro skin permeation and deposition study, photodegradation and skin toxicity assay. The transdermal flux for different vesicular formulations was observed between 10.5 +/- 0.5 to 13.9 +/- 1.6 microg/cm(2)/h. This is about 15-21 folds higher than that obtained from drug solution (0.7 +/- 0.1 microg/cm(2)/h) and 4-5 folds higher than obtained with drug-CD complex solution (2.7 +/- 0.1 microg/cm(2)/h). The amount of drug deposit was found to increase significantly (p < 0.05) by cyclodextrin complexation (30.1 +/- 0.1 microg). The encapsulation of this complex in elastic liposomal formulation further increases its skin deposition (262.2 +/- 21 microg). The results of skin irritation study using Draize test also showed the significant reduction in skin irritation potential of isotretinoin elastic liposomal formulation in comparison to free drug. The results of the present study demonstrated that isotretinoin elastic liposomal formulation possesses great potential for skin targeting, prolonging drug release, reduction of photodegradation, reducing skin irritation and improving topical delivery of isotretinoin.

Starch aided synthesis of giant unilamellar vesicles.

Synthesis of giant unilamellar vesicles (GUVs) of charged and uncharged lipids at physiological salt concentration is presented using the starch hydrogel method as an example of the gel assisted synthesis method. The swelling of the gel is assisted by the presence of a high amount of amylopectin in starch and yields giant-sized vesicles, which are unilamellar in nature. This method holds promise since starch is a commonly available cheap bio-compatible material. This work indicates that native starch yields vesicles of better size range as compared to the acid-treated starch. It is demonstrated that contrary to the common belief, pre-hydration of bilayers is not critical to the success of this method. The synthesis of GUVs in physiological salt concentrations is possible since the salt does not produce any osmotic effect on its own. At low starch concentration, the size of the vesicles is found to correlate with the swelling factor. The conjugate effect of the starch concentration and ion leads to the change in the swelling factor of the gel and thereby influence the size and architecture of the vesicles. Also, interactions between starch and lipid play an important role in the formation of the giant vesicles.

Electrostatic effects on the electrical tension-induced irreversible pore formation in giant unilamellar vesicles.

Irreversible electroporation (IRE) is a new technique in which a series of short pulses with high frequency electrical energy is applied on the targeted regions of cells or vesicles for their destruction or rupture formation. IRE induces lateral tension in the membranes of vesicles. We have investigated the electrostatic interaction effects on the constant electrical tension-induced rate constant of irreversible pore formation in the membranes of giant unilamellar vesicles (GUVs). The electrostatic interaction has been varied by changing the salt concentration in buffer and the surface charge density of membranes. The membranes of GUVs are synthesized by a mixture of negatively charged lipid dioleoylphosphatidylglycerol (DOPG) and neutral lipid dioleoylphosphatidylcholine (DOPC) using the natural swelling method. The rate constant of pore formation increases with the decrease of salt concentration in buffer along with the increase of surface charge density of membranes. The tension dependent probability of pore formation and the rate constant of pore formation are fitted to the theoretical equation, and obtained the line tension of membranes. The decrease in energy barrier of a prepore due to electrostatic interaction is the key factor causing an increase of rate constant of pore formation.

Lipid monolayer as a simple model membrane for comparative assessment of the photodynamic therapy photosensitizer efficiency via macroscopic measurements.

Cellular membrane is one of the main targets of photodynamic therapy. Its high complexity has led to the study of the efficiency of photosensitizers on artificial lipid systems mimicking membranes. However, the preliminary analysis of this efficiency remains limited due to difficulty of the model construction and/or implementation of the required measurement techniques. Hereby, we propose a quite simple way for the rapid comparative assessment of novel photosensitizers in terms of membrane photodegradation, based on simple and fast measurements, such as wetting angle and surface plasmon resonance spectroscopy. As a proof of concept, we applied this methodology to two bacteriopurpurinimide derivatives. We have shown in particular that such complementary techniques can be employed not only for the multiparametric monitoring of the kinetics of the photodegradation, but also for the comparison of the damaging efficiency of the photosensitizers in the lipid structures as well.

Mechanical and transport properties of chitosan-zwitterionic phospholipid vesicles.

Chitosan is a polysaccharide that has shown promise in liposomal drug delivery because of certain desirable properties such as muco-adhesivity, biodegradability and low toxicity. In this study, chitosan-bearing 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine giant unilamellar vesicles were prepared using inverse phase precursor method to measure their mechanical and transport properties. We show that while an increase in chitosan: lipid molar ratio in the vesicle bilayer at pH 7 led to a substantial increase in its bending modulus, chitosan-mediated change in bending modulus was diminished at pH 4.5. Water permeability across the vesicle bilayer, as well as phospholipid diffusivity within supported lipid bilayers, were also found to decrease with increasing chitosan: lipid molar ratio. Together, these findings demonstrate that incorporation of chitosan in phospholipid bilayers modulates the mechanical and transport properties of liposomes which may affect their in vivo circulation time and drug release rate.

Label-Free Surface-Enhanced Raman Spectroscopy Biosensor for On-Site Breast Cancer Detection Using Human Tears.

Surface-enhanced Raman scattering (SERS) is an ultrasensitive molecular screening technique with greatly enhanced Raman scattering signals from trace amounts of analytes near plasmonic nanostructures. However, research on the development of a sensor that balances signal enhancement, reproducibility, and uniformity has not yet been proposed for practical applications. In this study, we demonstrate the potential of the practical application for detecting or predicting asymptomatic breast cancer from human tears using a portable Raman spectrometer with an identification algorithm based on multivariate statistics. This potentiality was realized through the fabrication of a plasmonic SERS substrate equipped with a well-aligned, gold-decorated, hexagonal-close-packed polystyrene (Au/HCP-PS) nanosphere monolayer that provided femtomole-scale detection, giga-scale enhancement, and <5% relative standard deviation for reliability and reproducibility, regardless of the measuring site. Our results can provide a first step toward developing a noninvasive, real-time screening technology for detecting asymptomatic tumors and preventing tumor recurrence.