Asymmetric Liposomes Research Articles

Studying lipid flip-flop in asymmetric liposomes using 1H NMR and TR-SANS.

The specific spatial and temporal distribution of lipids in membranes play a crucial role in determining the biochemical and biophysical properties of the system. In nature, the asymmetric distribution of lipids is a dynamic process with ATP-dependent lipid transporters maintaining asymmetry, and passive transbilayer diffusion, that is, flip-flop, counteracting it. In this chapter, two probe-free techniques, 1H NMR and time-resolved small angle neutron scattering, are described in detail as methods of investigating lipid flip-flop rates in synthetic liposomes that have been generated with an asymmetric bilayer composition.

Inner leaflet cationic lipid increases nucleic acid loading independently of outer leaflet lipid charge in asymmetric liposomes.

Use of cationic lipid vesicles (liposomes) can yield large amounts of nucleic acid entrapped inside the vesicles and/or bound to the external surface of the vesicles. To show a method to prepare asymmetric lipid vesicles (liposomes) with high amounts of entrapped nucleic acid is possible, symmetric and asymmetric lipid vesicles composed of mixtures of neutral (zwitterionic), anionic, and/or cationic phospholipids were formed in the presence of oligo DNA. For symmetric large unilamellar vesicles nucleic acid association with vesicles was roughly 100 times greater for vesicles with a net cationic charge than for vesicles having a net neutral or anionic net charge. A high degree of association between nucleic acid and lipid was also achieved using asymmetric large unilamellar vesicles with a net cationic charge in their inner leaflet, even when they had an anionic charge in their outer leaflet. In contrast, asymmetric vesicles in which only the outer leaflet had a net cationic charge had only low amounts of vesicle-associated nucleic acid, similar in amount to the amount of nucleic acid associated with asymmetric vesicles with an outer leaflet having a net anionic charge. These results indicate that in asymmetric vesicles with cationic lipid enriched inner leaflets nucleic acid is largely entrapped inside the vesicle lumen rather than bound to their external surface, and that asymmetric vesicles can be used to trap high amounts of nucleic acid even when using a lipid composition in the outer leaflet of a lipid vesicle that does not associate with nucleic acids. Such asymmetrically charged vesicles should have applications in studies of membrane protein-nucleic acid interactions as well as in studies of how membrane charge asymmetry can influence membrane protein structure, orientation, and function.

The rise of FTIR spectroscopy in the characterization of asymmetric lipid membranes

In contrast to symmetric unilamellar liposomes (sLUVs) prepared from a mixture of different lipids, asymmetric ones (aLUVs) with different lipid composition in the inner and outer membrane leaflets are more suitable model systems of eukaryotic plasma membranes. However, apart from the challenging preparation of asymmetric liposomes and small amounts of obtained asymmetric unilamellar liposomes (aLUVs), a major drawback is the qualitative characterization of asymmetry, as each of the techniques used so far has certain limitations. In this regard, we prepared aLUVs composed dominantly of DPPC(out)/DPPS(in) lipids and, along with 1H NMR and DSC characterization, we showed for the first time how FTIR spectroscopy can be used in the presence of (a)symmetry between DPPC/DPPS lipid bilayers. Using second derivative FTIR spectra we demonstrated not only that the hydration of lipids glycerol backbone and choline moiety of DPPC differs in s/aLUVs, but in addition that the lateral interactions between hydrocarbon chains during the phase change display different trend in s/aLUVs. Molecular dynamics simulations confirmed different chain ordering and packing between s/a bilayers, with a significant influence of temperature, i.e. membrane phase.

Asymmetric Lipid Vesicles: Techniques, Applications, and Future Perspectives as an Innovative Drug Delivery System.

Novel lipid-based nanosystems have been of interest in improving conventional drug release methods. Liposomes are the most studied nanostructures, consisting of lipid bilayers ideal for drug delivery, thanks to their resemblance to the cell plasma membrane. Asymmetric liposomes are vesicles with different lipids in their inner and outer layers; because of this, they can be configured to be compatible with the therapeutic drug while achieving biocompatibility and stability. Throughout this review, topics such as the applications, advantages, and synthesis techniques of asymmetric liposomes will be discussed. Further, an in silico analysis by computational tools will be examined as a helpful tool for designing and understanding asymmetric liposome mechanisms in pharmaceutical applications. The dual-engineered design of asymmetric liposomes makes them an ideal alternative for transdermal drug delivery because of the improved protection of pharmaceuticals without lowering adsorption rates and system biocompatibility.

Enzyme mediated production of asymmetrically charged liposomes.

In asymmetric liposomes, the inner and outer leaflets of the lipid bilayer have different compositions. As such, these liposomes are appealing platforms for modeling and studying membrane asymmetry in biological membranes. In addition, asymmetric liposomes can serve as innovative drug delivery vehicles in which, the inner leaflet can be designed for maximal cargo encapsulation while the outer leaflet can be optimized for minimal toxicity. This study aims to explore the use of a membrane-active enzyme, phospholipase D (PLD), to produce liposomes with asymmetrically-charged membranes. PLD is an interfacial enzyme that catalyzes the hydrolysis of zwitterionic phosphatidylcholine (PC) lipids to produce choline and negatively-charged phosphatidic acid (PA) lipids. For this work, we introduced PLD to pre-formed PC liposomes and monitored the zeta potential of these liposomes at different time points. Zeta potential of these liposomes showed a time-dependent drop, presumably due to PLD activity and production of negatively-charged PA lipids in the membrane. Using a calibration curve, the zeta potential values were converted to the amount of PA formed in the outer leaflet of liposomes. Notably, the use of heat-deactivated PLD enzyme did not have an impact on liposomal zeta values. In addition, the results of a choline assay that was used to quantify the produced choline and thus PA in the system were comparable to those from zeta measurements. Furthermore, performing these studies with different PLD concentrations showed that the rate of PA production varied with the concentration of enzyme. Lastly, experiments of dye leakage from liposomes suggested that this enzymatic activity did not have a significant impact on liposomal membrane integrity. Together, these findings indicate that PLD enzymatic activity can be utilized to generate liposomes with asymmetrically charged leaflets, providing exciting opportunities for membrane biophysical studies as well as drug delivery applications.

Insights into Asymmetric Liposomes as a Potential Intervention for Drug Delivery Including Pulmonary Nanotherapeutics

Liposome-based drug delivery systems are nanosized spherical lipid bilayer carriers that can encapsulate a broad range of small drug molecules (hydrophilic and hydrophobic drugs) and large drug molecules (peptides, proteins, and nucleic acids). They have unique characteristics, such as a self-assembling bilayer vesicular structure. There are several FDA-approved liposomal-based medicines for treatment of cancer, bacterial, and viral infections. Most of the FDA-approved liposomal-based therapies are in the form of conventional "symmetric" liposomes and they are administered mainly by injection. Arikace® is the first and only FDA-approved liposomal-based inhalable therapy (amikacin liposome inhalation suspension) to treat only adults with difficult-to-treat Mycobacterium avium complex (MAC) lung disease as a combinational antibacterial treatment. To date, no "asymmetric liposomes" are yet to be approved, although asymmetric liposomes have many advantages due to the asymmetric distribution of lipids through the liposome's membrane (which is similar to the biological membranes). There are many challenges for the formulation and stability of asymmetric liposomes. This review will focus on asymmetric liposomes in contrast to conventional liposomes as a potential clinical intervention drug delivery system as well as the formulation techniques available for symmetric and asymmetric liposomes. The review aims to renew the research in liposomal nanovesicle delivery systems with particular emphasis on asymmetric liposomes as future potential carriers for enhancing drug delivery including pulmonary nanotherapeutics.

Open-channel structure of a pentameric ligand-gated ion channel reveals a mechanism of leaflet-specific phospholipid modulation

Pentameric ligand-gated ion channels (pLGICs) mediate synaptic transmission and are sensitive to their lipid environment. The mechanism of phospholipid modulation of any pLGIC is not well understood. We demonstrate that the model pLGIC, ELIC (Erwinia ligand-gated ion channel), is positively modulated by the anionic phospholipid, phosphatidylglycerol, from the outer leaflet of the membrane. To explore the mechanism of phosphatidylglycerol modulation, we determine a structure of ELIC in an open-channel conformation. The structure shows a bound phospholipid in an outer leaflet site, and structural changes in the phospholipid binding site unique to the open-channel. In combination with streamlined alchemical free energy perturbation calculations and functional measurements in asymmetric liposomes, the data support a mechanism by which an anionic phospholipid stabilizes the activated, open-channel state of a pLGIC by specific, state-dependent binding to this site.

Interdigitation-Induced Order and Disorder in Asymmetric Membranes

We studied the transleaflet coupling of compositionally asymmetric liposomes in the fluid phase. The vesicles were produced by cyclodextrin-mediated lipid exchange and contained dipalmitoyl phosphatidylcholine (DPPC) in the inner leaflet and different mixed-chain phosphatidylcholines (PCs) as well as milk sphingomyelin (MSM) in the outer leaflet. In order to jointly analyze the obtained small-angle neutron and X-ray scattering data, we adapted existing models of trans-bilayer structures to measure the overlap of the hydrocarbon chain termini by exploiting the contrast of the terminal methyl ends in X-ray scattering. In all studied systems, the bilayer-asymmetry has large effects on the lipid packing density. Fully saturated mixed-chain PCs interdigitate into the DPPC-containing leaflet and evoke disorder in one or both leaflets. The long saturated acyl chains of MSM penetrate even deeper into the opposing leaflet, which in turn has an ordering effect on the whole bilayer. These results are qualitatively understood in terms of a balance of entropic repulsion of fluctuating hydrocarbon chain termini and van der Waals forces, which is modulated by the interdigitation depth. Monounsaturated PCs in the outer leaflet also induce disorder in DPPC despite vestigial or even absent interdigitation. Instead, the transleaflet coupling appears to emerge here from a matching of the inner leaflet lipids to the larger lateral lipid area of the outer leaflet lipids.Graphical abstract

Using cyclodextrins to produce asymmetric liposomes, quantify lipid-lipid interactions and rates of transbilayer lipid flip-flop

Due to their bilayer structure, liposomes are prevalent model systems for studies of biophysical processes on plasma membranes, which naturally show lipid asymmetry. Phosphatidylethanolamine (PE), for example, occurs mainly in the inner leaflet and phosphatidylglycerol (PG) in the outer leaflet of prokaryotic membranes. Cyclodextrins (cyd) are widely used to extract, insert, or exchange lipids from or into the outer leaflet of liposomes or cell membranes. This strategy is, for example, playing a key role in several protocols to produce lipid-asymmetric liposomes.

Preparation and Drug Entrapment Properties of Asymmetric Liposomes Containing Cationic and Anionic Lipids.

We have developed cyclodextrin-catalyzed lipid exchange methods to prepare large unilamellar vesicles (LUVs) with asymmetric charge distributions, i.e., with different net charges on the lipids in the inner and outer leaflets. LUVs contained a mixture of a zwitterionic lipid (phosphatidylcholine), cholesterol, and various cationic lipids (O-ethyl phosphatidylcholine or dioleoyl-3-trimethylammonium propane) or anionic lipids (phosphatidylglycerol, phosphatidylserine, or phosphatidic acid). Symmetric and asymmetric LUVs with a wide variety of lipid combinations were prepared. The asymmetric LUVs contained cationic or anionic outer leaflets and inner leaflets that had either the opposite charge or were uncharged. The behavior of symmetric LUVs prepared with zwitterionic, anionic, or cationic leaflets was compared to those of asymmetric LUVs. Lipid exchange was confirmed by quantitative thin-layer chromatography, and lipid asymmetry by a novel assay measuring binding of a cationic fluorescent probe to the LUV outer leaflet. For both symmetric and asymmetric LUVs, the level of entrapment of the cationic drug doxorubicin was controlled by the charge on the inner leaflet, with the greatest entrapment and slowest leakage in vesicles with an anionic inner leaflet. This shows that it is possible to choose inner leaflet lipids to maximize liposomal loading of charged drugs independently of the identity of outer-leaflet lipids. This implies that it should also be possible to independently vary outer-leaflet lipids to, for example, impart favorable bioavailability and biodistribution properties to lipid vesicles.

Lipid Scrambling of Asymmetric Liposomes Induced by Membrane Active Substances

Membrane leakage assays based on vesicles loaded with self quenching dyes have been widely used for quantifying the activity of membrane active substances that induce membrane pores or leaks. Time-resolved fluorescence decays of calcein-loaded liposomes allow a parallel quantification of the free and entrapped dye fractions and their effective local concentrations.1 Thus, different types and mechanisms of membrane leakage to aqueous solutes can be distinguished by this method. However, much less is known about another way of membrane permeabilization: the enhancement of lipid flip flop that causes a scrambling of the lipids between the leaflets. Since virtually all biological membranes show an asymmetric distribution of lipids between the two leaflets, such a scrambling should have massive impact on the cell even if it proceeds without leakage to aqueous solutes. In order to assess such activity, we utilized a recent protocol2 to produce asymmetric vesicles with anionic diacyl phosphatidyl glycerol (POPG) in the outer layer exclusively. Addition of lipid scrambling agents permits the POPG to flip to the inner leaflet, which can be monitored in terms of a less negative zeta potential of the liposomes. The study compares the activities of nonionic detergents including n-Dodecyl β-D-maltoside, n-Octylglucoside and C12EO8 to induce lipid scrambling and pore formation, respectively. References 1 H. Patel, C. Tscheka, H. Heerklotz; Soft Matter, 2009, 5, 2849-2851. 2 M. Markones, C. Drechsler, M. Kaiser, L. Kalie, H. Heerklotz, S. Fiedler; Langmuir. 2018, 34(5), 1999-2005.

Stairway to Asymmetry: Five Steps to Lipid-Asymmetric Proteoliposomes

Membrane proteins are embedded in a complex lipid environment that influences their structure and function. One key feature of nearly all biological membranes is a distinct lipid asymmetry. However, the influence of membrane asymmetry on proteins is poorly understood, and novel asymmetric proteoliposome systems are beneficial. To our knowledge, we present the first study on a multispanning protein incorporated in large unilamellar liposomes showing a stable lipid asymmetry. These asymmetric proteoliposomes contain the Na+/H+ antiporter NhaA from Salmonella Typhimurium. Asymmetry was introduced by partial, outside-only exchange of anionic phosphatidylglycerol (PG), mimicking this key asymmetry of bacterial membranes. Outer-leaflet and total fractions of PG were determined via ζ-potential (ζ) measurements after lipid exchange and after scrambling of asymmetry. ζ-Values were in good agreement with exclusive outside localization of PG. The electrogenic Na+/H+ antiporter was active in asymmetric liposomes, and it can be concluded that reconstitution and generation of asymmetry were successful. Lipid asymmetry was stable for more than 7 days at 23°C and thus enabled characterization of the Na+/H+ antiporter in an asymmetric lipid environment. We present and validate a simple five-step protocol that addresses key steps to be taken and pitfalls to be avoided for the preparation of asymmetric proteoliposomes: 1) optimization of desired lipid composition, 2) detergent-mediated protein reconstitution with subsequent detergent removal, 3) generation of lipid asymmetry by partial exchange of outer-leaflet lipid, 4) verification of lipid asymmetry and stability, and 5) determination of protein activity in the asymmetric lipid environment. This work offers guidance in designing asymmetric proteoliposomes that will enable researchers to compare functional and structural properties of membrane proteins in symmetric and asymmetric lipid environments.

Peptide-Induced Lipid Flip-Flop in Asymmetric Liposomes Measured by Small Angle Neutron Scattering.

Despite the prevalence of lipid transbilayer asymmetry in natural plasma membranes, most biomimetic model membranes studied are symmetric. Recent advances have helped to overcome the difficulties in preparing asymmetric liposomes in vitro, allowing for the examination of a larger set of relevant biophysical questions. Here, we investigate the stability of asymmetric bilayers by measuring lipid flip-flop with time-resolved small-angle neutron scattering (SANS). Asymmetric large unilamellar vesicles with inner bilayer leaflets containing predominantly 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and outer leaflets composed mainly of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) displayed slow spontaneous flip-flop at 37 ◦C (half-time, t1/2 = 140 h). However, inclusion of peptides, namely, gramicidin, alamethicin, melittin, or pHLIP (i.e., pH-low insertion peptide), accelerated lipid flip-flop. For three of these peptides (i.e., pHLIP, alamethicin, and melittin), each of which was added externally to preformed asymmetric vesicles, we observed a completely scrambled bilayer in less than 2 h. Gramicidin, on the other hand, was preincorporated during the formation of the asymmetric liposomes and showed a time resolvable 8-fold increase in the rate of lipid asymmetry loss. These results point to a membrane surface-related (e.g., adsorption/insertion) event as the primary driver of lipid scrambling in the asymmetric model membranes of this study. We discuss the implications of membrane peptide binding, conformation, and insertion on lipid asymmetry.

Automatic generation system of cell-sized liposomes

Artificial cell membranes such as liposomes and lipid vesicles have been developed using microfluidic technologies. These technologies have advantages for generating cell-sized liposomes, including a monodispersed and asymmetric lipid composition. Cell-sized asymmetric liposomes with various combinations of asymmetric lipid leaflets are sequentially produced using an improved pulsed-jetting device with the six wells arranged on a circular rotation table by manual manipulation. In this study, we reveal optimal parameters of rotation speed and jetting timing for the cell-sized liposome formation using an automatic sequential liposome generation system driven by a stepping motor. First, we optimize the rotation speed of the stepping motor for forming the planar lipid bilayer under control of a microcomputer. Next, we generate cell-sized liposomes using this automated system at the optimized rotation speed. This automatic system will contribute to high-throughput investigations of membrane protein activities and functions using the electrophysiological or the asymmetric liposome analyses.

Liposomes with asymmetric bilayers produced from inverse emulsions for nucleic acid delivery

Asymmetrical lipid nanoparticles are interesting nanocarriers for charged molecules, like nucleic acids. They promise control over inner and outer charge. High charge density on the inside is favourable for efficient condensation and charge neutralisation of highly charged biopharmaceuticals, while a neutral or slightly negative outer layer promotes biocompatibility. The main goal of this work was the development and characterisation of asymmetric liposomes, prepared using water-in-oil (w/o) nanoemulsions of phospholipids (PLs) and squalene in a centrifugal field. This method enables the control over the lipid composition of each monolayer.Liposomes were prepared by passing PL w/o nanoemulsions through an oil–water interface previously saturated with PLs. We used N-(7-Nitrobenz-2-Oxa-1,3-Diazol-4-yl)-1,2-Dihexadecanoyl-sn-Glycero-3-Phosphoethanolamine (NBD-PE) or N-(7-Nitrobenz-2-Oxa-1,3-Diazol-4-yl)-1,2-Dihexadecanoyl-sn-Glycero-3- phosphocholine (NBD-PC) as a fluorescent marker for either the inner or outer lipid layer and plasmid DNA (pDNA) as nucleic acid payload. The final liposomes had sizes below 200 nm and polydispersity indexes of 0.3 and had a bilayer asymmetry of 70%, thus shielding the charge of positive PLs in the inner bilayer leaflet. Final formulations were examined using negative staining transmission electron microscopy (TEM). Plasmid encapsulation efficiency of the method was 10–15%. Our results indicate that the w/o nanoemulsion-centrifugation method allows the successful production of liposomes with tailored features for encapsulation of nucleic acid therapeutics.

Preparation of Asymmetric Liposomes Using a Phosphatidylserine Decarboxylase

Lipid asymmetries between the outer and inner leaflet of the lipid bilayer exist in nearly all biological membranes. Although living cells spend great effort to adjust and maintain these asymmetries, little is known about the biophysical phenomena within asymmetric membranes and their role in cellular function. One reason for this lack of insight into such a fundamental membrane property is the fact that the majority of model-membrane studies have been performed on symmetric membranes. Our aim is to overcome this problem by employing a targeted, enzymatic reaction to prepare asymmetric liposomes with phosphatidylserine (PS) primarily in the inner leaflet. To achieve this goal, we use a recombinant version of a water soluble PS decarboxylase from Plasmodium knowlesi, which selectively decarboxylates PS in the outer leaflet, converting it to phosphatidylethanolamine. The extent of decarboxylation is quantified using high-performance thin-layer chromatography, and the local concentration of anionic PS in the outer leaflet is monitored in terms of the ζ potential. Starting, for example, with 21 mol % 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine sodium salt, the assay leads to liposomes with 21 mol % in the inner and 6 mol % PS in the outer leaflet. This asymmetry persists virtually unchanged for at least 4 days at 20°C and at least 2 days at 40°C. The use of a highly specific enzyme carries the advantage that a minor component such as PS can be adjusted without affecting or being affected by the other lipid species present in the model membrane. The phenomena governing the residual outside PS content are addressed but warrant further study.

Investigating the Transbilayer Distribution of Cholesterol in Asymmetric Unilamellar Vesicles using Small-Angle Scattering

Cholesterol is the most abundant component of eukaryotic plasma membranes (PMs), accounting for 30-50% of the total lipid. In addition to modulating the structural and mechanical properties of lipid bilayers, cholesterol promotes the formation of ordered+disordered fluid phase separation in model membranes, and may similarly contribute to microdomain or “raft” formation in the PM. A major unresolved question is how PM properties and functions are influenced by the asymmetric transbilayer distribution of lipids, including cholesterol. While the distribution of most phospholipids and sphingolipids is known, determining the average transbilayer distribution of cholesterol has been challenging due to its rapid translocation between the PM leaflets. Previous attempts to probe the cholesterol distribution in cell membranes using fluorescence-based methods have yielded conflicting results. Moreover, studies using model vesicles have been limited due to the difficulty of preparing asymmetric liposomes. Making use of recent advancements enabling the production of asymmetric large unilamellar vesicles (aLUVs) via cyclodextrin-mediated lipid exchange, we prepared cholesterol-containing aLUVs in which one leaflet was enriched in egg sphingomyelin (eSM), and the other in phosphatidylethanolamine (POPE). We then used small-angle neutron and x-ray scattering combined with isotopic labeling strategies to probe the average cholesterol location within these bilayers. An analysis of scattering data, together with complementary Molecular Dynamics simulations, provides insight into the transbilayer distribution of cholesterol in asymmetric membranes.

Engineering Asymmetric Lipid Vesicles: Accurate and Convenient Control of the Outer Leaflet Lipid Composition.

The asymmetric distribution of lipids between the two bilayer leaflets represents a typical feature of biological membranes. The loss of this asymmetry, for example the exposure of negatively charged lipids on the extracellular membrane leaflet of mammalian cells, is involved in apoptosis and occurs in tumor cells. Thus, the controlled production of asymmetric liposomes helps to better understand such crucial cellular processes. Here, we present an approach that allows us to design asymmetric model-membrane experiments on a rational basis and predict the fraction of exchanged lipid. In addition, we developed a label-free and nondestructive assay to quantify the asymmetric uptake of negatively charged lipids in terms of the zeta potential. This significantly enhances the applicability, impact, and predictive power of model membranes.

Asymmetric cationic liposomes designed for heat-activated association with cells

Improved anticancer drugs and drug carriers are needed in combination therapies, such as hyperthermia-assisted chemotherapy. Liposomal drug carriers with advanced functions are attractive candidates for targeted accumulation and drug release in response to heat stimulus. We report on the design of liposomes with a heat-activated surface function. Our design is based on asymmetric lipid membranes with a defined gel to liquid-crystalline phase-transition temperature around 41°C. Asymmetry between the inner and the outer membrane leaflets was generated through selective PEGylation of cationic lipids in the outer membrane leaflet. In a physiological buffer, the PEGylated asymmetric liposomes had a neutral zeta potential and did not bind to planar anionic model membranes. In contrast, following upon heat-activation, binding of liposomes to the model membranes occurred. Release of a hydrophilic dye encapsulated in the asymmetric liposomes occurred at 40°C. Enhanced uptake of the asymmetric liposomes by hypopharyngeal carcinoma cells (FaDu cells) was observed when hyperthermia was applied compared to experiments performed at 37°C. These results show the potential of asymmetric liposomes for localized delivery of drugs into cells in response to (external) temperature stimulus.

Tuning Membrane Asymmetry

The basic building block of any organism is the cell, a self-contained unit enveloped by a semi-permeable plasma membrane. This small structural unit in turn is further compartmentalised in eukaryotic organisms, containing several organelles many of whom possess their own membranes. The classic Singer-Nicholson model presented the structure of these membranes as fluid assemblies of lipids, in a bilayer functionally. Whilst a landmark contribution in the field of lipidomics, the model omitted a key feature of the membrane, asymmetry. Bilayers are found to be transversely asymmetrical, with anionic lipids dominating the inner, cytosolic leaflet, whilst zwitterionic and sphingophospholipids are predominately found in the extra-cellular leaflet.Classically, membranes mimics employed in-vitro have largely been transversely symmetric with the exception of supported lipid bilayers, which allow greater control of the composition of each leaflet. However issues with support interaction, impairing features of the bilayer, affecting membrane insertion and diffusion rates of bilayer components both laterally and transversely. Only in the past decade have asymmetric bilayer synthetic methodologies began to emerge, including, droplet interface bilayers, inverted emulsion techniques and methyl-β-cyclodextrin catalysed exchange. Each technology is in its infancy, with limitations on the properties of the bilayers, as well as their applications. The latter method is the only method capable of forming asymmetric liposomes in a broad range of diameters. A new methodology to produce transversely asymmetric liposomes will be presented utilising synthetic lipids capable of molecular recognition.