Engineering asymmetric vesicles (original) (raw)
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Preparation of asymmetric phospholipid vesicles for use as cell membrane models
Nature protocols, 2018
Freely suspended liposomes are widely used as model membranes for studying lipid-lipid and protein-lipid interactions. Liposomes prepared by conventional methods have chemically identical bilayer leaflets. By contrast, living cells actively maintain different lipid compositions in the two leaflets of the plasma membrane, resulting in asymmetric membrane properties that are critical for normal cell function. Here, we present a protocol for the preparation of unilamellar asymmetric phospholipid vesicles that better mimic biological membranes. Asymmetry is generated by methyl-β-cyclodextrin-catalyzed exchange of the outer leaflet lipids between vesicle pools of differing lipid composition. Lipid destined for the outer leaflet of the asymmetric vesicles is provided by heavy-donor multilamellar vesicles containing a dense sucrose core. Donor lipid is exchanged into extruded unilamellar acceptor vesicles that lack the sucrose core, facilitating the post-exchange separation of the donor an...
Preparation of Artificial Plasma Membrane Mimicking Vesicles with Lipid Asymmetry
PLoS ONE, 2014
Lipid asymmetry, the difference in lipid distribution across the lipid bilayer, is one of the most important features of eukaryotic cellular membranes. However, commonly used model membrane vesicles cannot provide control of lipid distribution between inner and outer leaflets. We recently developed methods to prepare asymmetric model membrane vesicles, but facile incorporation of a highly controlled level of cholesterol was not possible. In this study, using hydroxypropyl-a-cyclodextrin based lipid exchange, a simple method was devised to prepare large unilamellar model membrane vesicles that closely resemble mammalian plasma membranes in terms of their lipid composition and asymmetry (sphingomyelin (SM) and/or phosphatidylcholine (PC) outside/phosphatidylethanolamine (PE) and phosphatidylserine (PS) inside), and in which cholesterol content can be readily varied between 0 and 50 mol%. We call these model membranes ''artificial plasma membrane mimicking'' (''PMm'') vesicles. Asymmetry was confirmed by both chemical labeling and measurement of the amount of externally-exposed anionic lipid. These vesicles should be superior and more realistic model membranes for studies of lipid-lipid and lipid-protein interaction in a lipid environment that resembles that of mammalian plasma membranes.
Effect of leaflet asymmetry on mechanical properties of lipid bilayers with phosphatidic acid
ABSTRACTAsymmetry in membranes strongly affects their biophysical characteristics and behaviour. It is a very important factor to consider for understanding the functioning of membranes in biological systems, especially in regards to lipid molecules that are known to localize in one leaflet over the other. Phosphatidic acid is one representative of this family of molecules. Reported to play a crucial role in lipid metabolism and suspected to participate in cellular signalling, its specific nature might only be exhibited in asymmetric systems. Here, using giant unilamellar vesicles, we studied two asymmetric phosphocholine-based bilayer systems with 1-palmitoyl-2-oleoyl phosphatidic acid (POPA) localized in either the inner or the outer membrane leaflet. We systematically characterized and compared the investigated systems to their symmetric counterpart. To verify and quantify the presence of POPA in the membrane, α-synuclein-mEGFP was used. We report an increase in the area compress...
Applied Sciences
Lipid vesicles (liposomes) are a unique and fascinating type of polymolecular aggregates, obtained from bilayer-forming amphiphiles—or mixtures of amphiphiles—in an aqueous medium. Unilamellar vesicles consist of one single self-closed bilayer membrane, constituted by the amphiphiles and an internal volume which is trapped by this bilayer, whereby the vesicle often is spherical with a typical desired average diameter of either about 100 nm or tens of micrometers. Functionalization of the external vesicle surface, basically achievable at will, and the possibilities of entrapping hydrophilic molecules inside the vesicles or/and embedding hydrophobic compounds within the membrane, resulted in various applications in different fields. This review highlights a few of the basic studies on the phase behavior of polar lipids, on some of the concepts for the controlled formation of lipid vesicles as dispersed lamellar phase, on some of the properties of vesicles, and on the challenges of eff...
Langmuir
Hybrid, i.e. intimately mixed polymer/phospholipid vesicles can potentially marry in a single membrane the best characteristics of the two separate components. The ability of amphiphilic copolymers and phospholipids to selfassemble into hybrid membranes has been studied until now at the sub-micron scale using optical microscopy on Giant Hybrid Unilamellar Vesicles (GHUVs), but limited information is available on Large Hybrid Unilamellar Vesicles (LHUVs). In this work, copolymers based on poly(dimethyl siloxane) and poly(ethylene oxide) with different molar masses and architectures (graft, triblock) were associated with 1,2-di-palmitoyl-sn-glycero-3-phosphocholine (DPPC). Classical protocols of LUV formation were used to obtain nano-sized self-assembled structures. Using Small Angle Neutron Scattering (SANS), Time Resolved Förster Resonance Energy Transfer (TR-FRET) and Cryo-Transmission Electron Microscopy (Cryo-TEM), we show that copolymer architecture and molar mass have a direct consequence on the formation of hybrid nanostructures that can range from worm-like hybrid micelles to hybrid vesicles presenting small lipid nanodomains. Hybrid polymer/phospholipid vesicles are emerging structures that combine the biocompatibility and biofunctionality of liposomes, with the robustness, low permeability, and functional variability conferred by copolymer chains. This should become of great interest in pharmaceutical applications for which a few formulations based on liposomes are commercially available despite decades of research, viz. DaunoXome ® , Doxil ® /Caelyx ® , Thermodox ® , Visudyne ® .
Lipid vesicles: applications, principal components and methods used in their formulations: A review
Acta Biológica Colombiana
Liposomes and niosomes are currently the most studied lipid vesicles in the nanomedicine field. The system formed by a phospholipid bilayer in aqueous medium allows these vesicles to carry both hydrophilic and lipophilic compounds, providing an increase in solubility of drugs lready used in conventional therapy. The focus on the development of these vesicles should be directed to determining the ideal composition, with low toxicity, biocompatibility and which remains stable for long periods. These characteristics are related to the components used for formulation and the substances that will be encapsulated. Another important point relates to the methods used during formulation, which are important in determining the type of vesicle formed, whether these be large or small, unilamellar or multilamellar. Because of the deliberate actions applied in the development of these vesicles, this review sought to gather updated information regarding the different methods used, including their ...
Hybrid polymer/lipid vesicles: state of the art and future perspectives
Materials Today, 2013
Hybrid vesicles resulting from the combined self-assembly of both amphiphilic copolymers and lipids have attracted particular interest from chemists and (bio)physicists over the last five years. Such assemblies may be viewed as an advanced vesicular structure compared to their liposome and polymersome forerunners as the best characteristics from the two different systems can be integrated in a new, single vesicle. To afford such a design, the different parameters controlling both self-assembly and membrane structure must be tuned. This highlight aims to present a comprehensive overview of the fundamental aspects related to these structures, and discuss emerging developments and future applications in this field of research.
Production of Unilamellar Vesicles Using an Inverted Emulsion
Langmuir, 2003
We investigate a method for the controlled assembly of unilamellar vesicles consisting of bilayers assembled one leaflet at a time. We use water-in-oil emulsions stabilized by the material for the inner leaflet and produce vesicles by passing the water droplets through a second oil-water interface, where they become coated with the outer leaflet. We have used this technique to form vesicles from lipids, mixed lipid and surfactant systems, and diblock copolymers. The stability of lipid-stabilized emulsions limits the range of sizes that can be produced and the vesicle yield; nevertheless, there are several advantages with this emulsion-based technique: It is possible to make unilamellar vesicles with sizes ranging from 100 nm to 1 µm. Moreover, the process allows for efficient encapsulation and ensures that the contents of the vesicles remain isolated from the continuous aqueous phase. To illustrate possible applications of this technique, we demonstrate the use of vesicles as microreactors where we polymerize actin through the addition of magnesium and show that the polymerization kinetics are unaffected by the encapsulation.
Langmuir, 1999
The physical conjugation of (tri-) block copolymer molecules to phospholipid vesicle bilayers in order to construct sterically stabilized vesicles can be carried out in two different ways: by allowing the copolymer molecules to freely participate in the small unilamellar vesicle (SUV) formation process along with the lipids or by adding the copolymer molecules to pre-formed small unilamellar liposomes. Structurally and morphologically different copolymer coated vesicle systems occur. The effect on the mean vesicle diameter and the vesicle surface characteristics is monitored by dynamic light scattering and laser Doppler electrophoresis techniques for a wide variety of block copolymer molecules of the PEO-PPO-PEO type (PEO is poly(ethylene oxide); PPO poly(propylene oxide)). Systematic investigations as a function of copolymer added concentration and molecular structure were undertaken throughout. The results indicate a dramatic increase in mean vesicle diameter when the polymer molecules are present during vesiculation, while in the case of copolymer addition to already formed liposomes the mean vesicle size follows a classic Langmuirian-type adsorption curve as a function of copolymer concentration. The -potential values obtained decrease in a very similar pattern irrespective of the way of addition for the large PF127 (PEO99-PPO65-PEO99) molecule, illustrating the presence of polymer chains at the vesicle surface. For the small, more hydrophobic L61 (PEO10-PPO16-PEO10) molecule, the reduced -potential value is maintained only when the copolymer molecules participate in bilayer formation, indicating absence of interaction between the polymer and the lipids when added to preformed liposomes, due to the preferred copolymer tendency to aggregate into micelles separate from the lipid bilayer particles (that eventually leads to phase separation). According to the molecular models proposed to describe the occurring lipid-copolymer interactions, addition of copolymer molecules after liposomes have been formed leads to their adsorption onto the outer liposome surface, its effectiveness being dependent on the influence that the hydrophilic (PEO) and hydrophobic (PPO) blocks exert on the copolymer molecular behaviour. Copolymer-lipid coparticipation toward bilayer formation, at low added polymer concentrations, leads to PPO block protection by arranging along with the lipids as integral parts of the vesicle bilayer, hence anchoring the PEO chains that dangle in the aqueous solution onto the vesicles. Simple geometrical considerations are also included, reinforcing the theoretical feasibility of the described models. The latter type of physically conjugating polymer chains onto vesicle surfaces is proposed as an improved alternative to the weak adsorption of amphiphilic molecules and the cumbersome chemical modification of the lipid polar headgroups to confer steric protection to liposomal surfaces.
Biomacromolecules, 2002
Networks of N-isopropylacrylamide (NIPAM) copolymers, coupled to spherical phospholipid bilayers, are suitable as a model for the study of the interaction between the cytoskeleton and cellular membranes, as well as for promising new drug delivery systems with triggerable drug release properties and improved stability. In this article, we describe a simple preparation technique for liposomes from egg phosphatidyl choline (EPC) encapsulating a cross-linked NIPAM-TEGDM copolymer skeleton (tetraethylene glycol dimethacrylate, TEGDM) which is coupled only to the inner monolayer by a novel membrane anchor monomer. Polymerization in the lipid vesicles was initiated at the inner membrane surface by the radical initiator 2,2-diethoxy-acetophenone (DEAP) permeating through the membrane from the outside. The effects of photopolymerization and polymer formation on vesicle shape and membrane integrity were studied by transmission electron microscopy (TEM), cryo-TEM, and atomic force microscopy (AFM). Upon UV irradiation, approximately 100% of the vesicles contained a polymer gel and only occasional changes in the spherical shape of the liposomes were observed. The architecture of the polymer network inside the liposomal compartment was determined by the conditions of the photopolymerization. Composite structures of polymer hollow spheres or solid spheres, respectively, tethered to spherical membrane vesicles were produced. The increased stability of the polymer-tethered lipid bilayers against solubilization by sodium cholate, compared to pure EPC vesicles, was determined by radiolabeling the lipid membrane.