Mixed Lipid Monolayers Research Articles

Interaction of DNA with Cationic Lipid Mixtures-Investigation at Langmuir Lipid Monolayers.

Four different binary lipid mixtures composed of a cationic lipid and the zwitterionic colipids DOPE or DPPC, which show different DNA transfer activities in cell culture models, were investigated at the soft air/water interface to identify transfection efficiency determining characteristics. Langmuir films are useful models to investigate the interaction between DNA and lipid mixtures in a two-dimensional model system by using different surface sensitive techniques, namely, epifluorescence microscopy and infrared reflection-absorption spectroscopy. Especially, the effect of adsorbed DNA on the properties of the lipid mixtures has been examined. Distinct differences between the lipid composites were found which are caused by the different colipids of the mixtures. DOPE containing lipid mixtures form fluid monolayers with a uniform distribution of the fluorescent probe in the presence and absence of DNA at physiologically relevant surface pressures. Only at high nonphysiological pressures, the lipid monolayer collapses and phase separation was observed if DNA was present in the subphase. In contrast, DPPC containing lipid mixtures show domains in the liquid condensed phase state in the presence and absence of DNA in the subphase. The adsorption of DNA at the positively charged mixed lipid monolayer induces phase separation which is expressed in the morphology and the point of appearance of these domains.

Targeted delivery of nano-PTX to the brain tumor-associated macrophages

Nanoparticles containing mixed lipid monolayer shell, biodegradable polymer core and rabies virus glycoprotein (RVG) peptide as brain targeting ligand, were developed for brain targeted delivery of paclitaxel (PTX) to treat malignant glioma. RVG conjugated PTX loaded NPs (RVG-PTX-NPs) had the desirable size (~140 nm), narrow size distribution and spherical shape. RVG-PTX-NPs showed poor uptake by neurons and selective targeting to the brain tumor associated macrophages (TAMs) with controlled release and tumor specific toxicity. In vivo studies revealed that RVG-PTX-NPs were significant to cross the blood-brain barrier (BBB) and had specific targeting to the brain. Most importantly, RVG-PTX-NPs showed effectiveness for anti-glioma therapy on human glioma of mice model. We concluded that RVG-PTX-NPs provided an effective approach for brain-TAMs targeted delivery for the treatment of glioma.

Phosphatidylserine Allows Observation of the Calcium-Myristoyl Switch of Recoverin and Its Preferential Binding.

Recoverin undergoes a calcium-myristoyl switch during visual phototransduction. Indeed, calcium binding by recoverin results in the extrusion of its myristoyl group, which allows its membrane binding. However, the contribution of particular lipids and of specific amino acids of recoverin in its membrane binding has not yet been demonstrated. In the present work, the affinity of recoverin for the negatively charged phosphatidylserine has been clearly shown to be governed by a cluster of positively charged residues located in its N-terminal segment. Moreover, the calcium-myristoyl switch of recoverin was only observed upon binding onto monolayers of phosphatidylserine and not in the case of other anionic phospholipids. Fluorescence microscopy experiments with mixed lipid monolayers allowed confirmation of the specific binding of myristoylated recoverin to phosphatidylserine, whereas the extent of penetration of recoverin in phosphatidylserine monolayers was estimated by ellipsometry. A model has thus been proposed for the membrane binding of myristoylated recoverin in the presence of calcium.

Exploring the Impact of Tail Polarity on the Phase Behavior of Single Component and Mixed Lipid Monolayers Using a MARTINI Coarse-Grained Force Field.

Coarse-grained molecular dynamics simulations have been used to investigate the effect of dipalmitoylphosphatidylcholine (DPPC) tail group polarity on the structural and phase behavior of both single component and binary mixed monolayers using the MARTINI force field. Surface pressure-area isotherms of single component systems indicate that DPPC monolayers become more expanded as a function of increasing tail group polarity, as observed in experimental measurements in the literature. A combination of radial distribution functions and tilt angle measurements indicate that increasing tail group polarity results in the formation of increasingly disordered monolayers. For the mixed monolayer systems, the time dependence of the radial distribution function as well as average cluster size measurements indicate that phase separation takes place between components of different tail group polarity when the monolayers undergo phase transition into disordered configurations.

Cholesterol Depletion from a Ceramide/Cholesterol Mixed Monolayer: A Brewster Angle Microscope Study.

Cholesterol is crucial to the mechanical properties of cell membranes that are important to cells’ behavior. Its depletion from the cell membranes could be dramatic. Among cyclodextrins (CDs), methyl beta cyclodextrin (MβCD) is the most efficient to deplete cholesterol (Chol) from biomembranes. Here, we focus on the depletion of cholesterol from a C16 ceramide/cholesterol (C16-Cer/Chol) mixed monolayer using MβCD. While the removal of cholesterol by MβCD depends on the cholesterol concentration in most mixed lipid monolayers, it does not depend very much on the concentration of cholesterol in C16-Cer/Chol monolayers. The surface pressure decay during depletion were described by a stretched exponential that suggested that the cholesterol molecules are unable to diffuse laterally and behave like static traps for the MβCD molecules. Cholesterol depletion causes morphology changes of domains but these disrupted monolayers domains seem to reform even when cholesterol level was low.

The effective combination therapy against human osteosarcoma: doxorubicin plus curcumin co-encapsulated lipid-coated polymeric nanoparticulate drug delivery system

Objective: To overcome both the dose-limiting side effects of conventional chemotherapeutic agents and the therapeutic failure incurred from multidrug resistant (MDR) in osteosarcoma (OS), biodegradable lipid-coated polymeric nanoparticles (LPNs) were explored for the loading of doxorubicin (DOX) and curcumin (CUR).Methods: DOX plus CUR co-encapsulated LPNs (DOX + CUR LPNs) of mixed lipid monolayer shell and biodegradable polymer core were prepared. The cytotoxicity effect of DOX + CUR LPNs, single drug loaded LPNs, and free drug solutions were evaluated on human OS cell line KHOS cells and mice KHOS cells xenograft in vivo.Results: DOX + CUR LPNs displayed a curative effect on OS cell lines than the free drug counterparts. Also, best anti-OS effects were observed on the animal model compared with other groups tested.Conclusion: This promising dual drugs co-encapsulated lipid-coated polymeric nanoparticulate drug delivery system enhanced the cell delivery and activity of drugs against human OS cancer cell lines and in cancer bearing mice. This research may offer new options for the treatment of OS.

Phase Behavior of Mixed Lipid Monolayers on Perfluorocarbon Nanoemulsions and its Effect on Acoustic Contrast.

Lipid-stabilized nanoemulsions containing a volatile liquid perfluorocarbon core have been studied as ultrasound contrast agents owing to their ability to transform into high-contrast microbubbles when subjected to high intensity focused ultrasound (HIFU). However, while there have been several studies on the effect of acoustic parameters on contrast, the effect of the droplet's stabilizing shell has not been studied as extensively. Inspired by previous studies showing lateral phase separation in microbubbles and vesicles, nanodroplets were formulated with a perfluorohexane core and a shell composed of varying amounts of saturated (DPPC) phospholipids, unsaturated (DOPC) phospholipids, and cholesterol, which were fractionated to obtain nanodroplets of mean diameter 300-400 nm and were stable over one week. When the DOPC content was increased to 40 mol%, ultrasound contrast increased by about one order of magnitude over DPPC-only droplets. Based on fluorescence microscopy results of lateral lipid phase separation on the droplet surface, the various combinations of DPPC, DOPC, and cholesterol were assigned to three regimes on the ternary phase diagram: solid-liquid ordered (low contrast), liquid ordered-liquid disordered (medium contrast), and solid-liquid disordered (high contrast). These regimes were confirmed by TEM analysis of nanoscale droplets. Droplets containing mixed lipid monolayers were also found to produce a significantly greater yield than single-component droplets. The discovery of the dependence of acoustic response on lipid phase separation will help to understand the formulation, behavior, and vaporization mechanism of acoustically-responsive nanoemulsions.

Auxins action on Glycine max secretory phospholipase A2 is mediated by the interfacial properties imposed by the phytohormones

Secretory phospholipase A2 (sPLA2) are soluble enzymes that catalyze the conversion of phospholipids to lysophospholipids and free fatty acids at membrane interfaces. The effect of IAA and IPA auxins over the activity of recombinant sPLA2 isoforms from Glycine max was studied using membrane model systems including mixed micelles and Langmuir lipid monolayers. Both phytohormones stimulate the activity of both plant sPLA2 using DLPC/Triton mixed micelles as substrate.To elucidate the mechanism of action of the phytohormones, we showed that both auxins are able to self-penetrate lipid monolayers and cause an increment in surface pressure and an expansion of lipid/phytohormone mixed interfaces. The stimulating effect of auxins over phospholipase A2 activity was still present when using Langmuir mixed monolayers as organized substrate regardless of sPLA2 source (plant or animal). All the data suggest that the stimulating effect of auxins over sPLA2 is due to a more favorable interfacial environment rather to a direct effect over the enzyme.

Stability and softening of a lipid monolayer in the presence of a pain-killer drug

The aim of this study is to investigate the interaction of a drug (Piroxicam, 4-hydroxy-2-methyl-N-(2-pyridinyl)-2H-1,2-benzothiazine-3-carboxamide 1,1 dioxide) with a lipid (DMPC) monolayer used as a membrane-mime in terms of drug-induced changes in stability and compressibility with variation in temperature, surface-pressure, drug-dose and ionic states of the monolayers. Drug-induced fluidization is noticed in the π−A isotherms through increase in phase-transition pressure at constant temperature. The long-term dynamics of the lipid-monolayer is characterized by algebraic decays in surface-energy E with time t, E∼t−p1,2,3, with an initial decay exponent p1 that changes to p2 after ∼1000s, and, at high surface pressures and/or drug-dose, to a third exponent p3 after ∼3500s, suggesting structural reorganizations in the monolayer. With increasing drug–lipid ratio (D/L), p1 shows a decrease ending at an almost constant value after 0.05, p2 shows an almost negligible lowering while p3 shows a monotonic and considerable increase. The reorganization is summarized by proposing two mechanisms: (a) ‘charging–discharging’ where drug-molecules sitting parallel to the interface increase headgroup separations and (b) ‘discharging–charging’ where drug-molecules sitting roughly perpendicular to the interface bring headgroups closer. Drug-induced softening of lipid-monolayers is characterized by the compressibilites of pure and mixed lipid monolayers. Compressibility-change (i.e., compressibility difference between drug/lipid and pure lipid monolayer) with pressure is maximum in the LE–LC transition zone and compressibility-change with drug-dose reveals an optimum dose of drug for maximum increase in compressibility. Molecular dynamics simulation shows that the ordering in the different parts of the lipid chains is changed to different extents in the presence of drugs with maximum change near the headgroups and again points to an optimum dose for maximum disorder.

Effects of Chain Rigidity on the Adsorption of a Polyelectrolyte Chain on Mixed Lipid Monolayer: A Monte Carlo Study

We apply Monte Carlo simulation to explore the adsorption of a positively charged polyelectrolyte on a lipid monolayer membrane, composed of electronically neutral, monovalent anionic and mulvitalent anionic phospholipids. We systematically assess the influence of various factors, including the intrinsic rigidity of the polyelectrolyte chain, the bead charge density of the polyelectrolyte, and the ionic strength of the saline solution, on the interfacial structural properties of the polyelectrolyte/monolayer complex. The enhancement of the polyelectrolyte chain intrinsic rigidity reduces the polyelectrolyte conformational entropy loss and the energy gains in electrostatic interaction, but elevates the segregated anionic lipid demixing entropy loss. This energy-entropy competition results in a nonmonotonic dependence of the polyelectrolyte/monolayer association strength on the degree of chain rigidity. The semiflexible polyelectrolyte, i.e., the one with an intermediate degree of chain rigidity, is shown to associate onto the ternary membane below a higher critical ionic concentration. In this ionic concentration regime, the semiflexible polyelectrolyte binds onto the monolayer more firmly than the pancake-like flexible one and exhibits a stretched conformation. When the chain is very rigid, the polyelectrolyte with bead charge density Zb = +1 exhibits a larger tail and tends to dissociate from the membrane, whereas the one with Zb= +2 can still bind onto the membrane in a bridge-like conformation. Our results imply that chain intrinsic rigidity serves as an efficient molecular factor for tailoring the adsorption/desorption transition and interfacial structure of the polyelectrolyte/monolayer complex.

Folate-modified lipid-polymer hybrid nanoparticles for targeted paclitaxel delivery.

The purpose of this study was to develop a novel lipid–polymer hybrid drug carrier comprised of folate (FA) modified lipid-shell and polymer-core nanoparticles (FLPNPs) for sustained, controlled, and targeted delivery of paclitaxel (PTX). The core-shell NPs consist of 1) a poly(ε-caprolactone) hydrophobic core based on self-assembly of poly(ε-caprolactone)–poly(ethylene glycol)–poly(ε-caprolactone) (PCL-PEG-PCL) amphiphilic copolymers, 2) a lipid monolayer formed with 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy (polyethylene glycol)-2000] (DSPE-PEG2000), 3) a targeting ligand (FA) on the surface, and were prepared using a thin-film hydration and ultrasonic dispersion method. Transmission electron microscopy and dynamic light scattering analysis confirmed the coating of the lipid monolayer on the hydrophobic polymer core. Physicochemical characterizations of PTX-loaded FLPNPs, such as particle size and size distribution, zeta potential, morphology, drug loading content, encapsulation efficiency, and in vitro drug release, were also evaluated. Fluorescent microscopy proved the internalization efficiency and targeting ability of the folate conjugated on the lipid monolayer for the EMT6 cancer cells which overexpress folate receptor. In vitro cytotoxicity assay demonstrated that the cytotoxic effect of PTX-loaded FLPNPs was lower than that of Taxol®, but higher than that of PTX-loaded LPNPs (without folate conjugation). In EMT6 breast tumor model, intratumoral administration of PTX-loaded FLPNPs showed similar antitumor efficacy but low toxicity compared to Taxol®. More importantly, PTX-loaded FLPNPs showed greater tumor growth inhibition (65.78%) than the nontargeted PTX-loaded LPNPs (48.38%) (P<0.05). These findings indicated that the PTX loaded-FLPNPs with mixed lipid monolayer shell and biodegradable polymer core would be a promising nanosized drug formulation for tumor-targeted therapy.

Kinetic characterization, optimum conditions for catalysis and substrate preference of secretory phospholipase A2 from Glycine max in model membrane systems.

Two secretory phospholipase A2 (sPLA2s) from Glycine max, GmsPLA2-IXA-1 and GmsPLA2-XIB-2, have been purified as recombinant proteins and the activity was evaluated in order to obtain the optimum conditions for catalysis using mixed micelles and lipid monolayers as substrate. Both sPLA2s showed a maximum enzyme activity at pH 7 and a requirement of Ca(2+) in the micromolar range. These parameters were similar to those found for animal sPLA2s but a surprising optimum temperature for catalysis at 60 °C was observed. The effect of negative interfacial charges on the hydrolysis of organized substrates was evaluated through initial rate measurements using short chain phospholipids with different head groups. The enzymes showed subtle differences in the specificity for phospholipids with different head groups (DLPC, DLPG, DLPE, DLPA) in presence or absence of NaCl. Both recombinant enzymes showed lower activity toward anionic phospholipids and a preference for the zwitterionic ones. The values of the apparent kinetic parameters (Vmax and KM) demonstrated that these enzymes have more affinity for phosphatidylcholine compared with phosphatidylglycerol, in contrast with the results observed for pancreatic sPLA2. A hopping mode of catalysis was proposed for the action of these sPLA2 on mixed phospholipid/triton micelles. On the other hand, Langmuir-monolayers assays indicated an optimum lateral surface pressure for activity in between 13 and 16 mN/m for both recombinant enzymes.

Analytic Model for the Dipole Potential of a Lipid Layer.

The larger permeability of anions than cations through a lipid bilayer can be rationalized by the positive sign of the bilayer's dipole potential. That is, upon crossing the lipid headgroups toward the hydrocarbon chain region, the electrostatic potential increases by several hundred millivolts. We derive an analytic expression for the dipole potential of a single lipid layer using an electrostatic model that is based on an extended version of the linearized Poisson-Boltzmann theory. The model highlights the ability of the lipid headgroups to render the dipole potential positive by inducing an orientational ordering of the solvent molecules. The positive contribution of the solvent overcompensates the negative dipole potential due to the bare lipids. Our theoretical prediction compares accurately with measurements of the dipole potential that we have conducted for mixed anionic-zwitterionic lipid monolayers at the air-water interface.

Calcium-mediated binding of DNA to 1,2-distearoyl-sn-glycero-3-phosphocholine-containing mixed lipid monolayers

The calcium-mediated interaction of DNA with monolayers of the non-toxic, zwitterionic phospholipid, 1,2-distearoyl-sn-glycero-3-phosphocholine when mixed with 50 mol% of a second lipid, either the zwitteronic 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine or neutral cholesterol was investigated using a combination of surface pressure-area isotherms, Brewster angle microscopy, external reflectance Fourier transform infrared spectroscopy and specular neutron reflectivity in combination with contrast variation. When calcium and DNA were both present in the aqueous subphase, changes were observed in the compression isotherms as well as the surface morphologies of the mixed lipid monolayers. In the presence of calcium and DNA, specular neutron reflectivity showed that directly underneath the head groups of the lipids comprising the monolayers, DNA occupied a layer comprising approximately 13 and 18% v/v DNA for the 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine and cholesterol-containing monolayers, respectively. The volume of the corresponding layer for 1,2-distearoyl-sn-glycero-3-phosphocholine only containing monolayers was ∼15% v/v DNA. Furthermore regardless of the presence and nature of the second lipid and the surface pressure of the monolayer, the specular neutron reflectivity experiments showed that the DNA-containing layer was 20-27 Å thick, suggesting the presence of a well-hydrated layer of double-stranded DNA. External reflectance Fourier transform infrared studies confirmed the presence of double stranded DNA, and indicated that the strands are in the B-form conformation. The results shed light on the interaction between lipids and nucleic acid cargo as well as the role of a second lipid in lipid-based carriers for drug delivery.

Frequency Modulated Kelvin Probe Force Microscopy to map surface potential of mixed lipid monolayers with and without cholesterol

Extended abstract of a paper presented at Microscopy and Microanalysis 2012 in Phoenix, Arizona, USA, July 29 – August 2, 2012.

Langmuir Balance Investigation of Superoxide Dismutase Interactions with Mixed-Lipid Monolayers

Higher than theoretical encapsulation efficiencies in liposomes of the cytoplasmic protein, superoxide dismutase (SOD), were previously observed. The high encapsulation of SOD led to the consideration of lipid-protein interactions and the embedding of SOD in the lipid bilayer. Difficulty in other methods such as dynamic scanning calorimetry due to cholesterol obscuring the measurements brought about the interest for a modified Langmuir monolayer relaxation study. A novel method was devised to distinguish between different lipid compositions that formed either a favorable or an unfavorable environment for SOD. Normalized monolayer relaxations with SOD were compared between mixed-lipid compositions containing 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), and cholesterol (Chol). Lipid-monolayer relaxation with and without SOD in the subphase was plotted over 30 min to determine if the protein was altering the lipid-monolayer relaxation. The monolayer relaxation with SOD was normalized to the monolayer relaxation without SOD over the 30 min period. The results indicated that lipid length and mole percent of cholesterol were important parameters that must be adjusted in order to support a favorable environment for SOD interaction with the lipid. It was determined that hydrophobic interactions were dominant over electrostatic forces; thus, SOD was embedding into the lipid monolayer. Additionally, this study was correlated to a previous liposome study and proved that lipid-protein interactions were the reason for the higher encapsulation efficiencies. The significance of this method is that it (1) provides a connection between lipid-protein interactions observed in monolayers and bilayers and (2) establishes a simple and effective manner to test lipid compositions for lipid-protein interaction that will aid in optimization of liposome encapsulation efficiency.

Effect of the β-Propiolactone Treatment on the Adsorption and Fusion of Influenza A/Brisbane/59/2007 and A/New Caledonia/20/1999 Virus H1N1 on a Dimyristoylphosphatidylcholine/Ganglioside GM3 Mixed Phospholipids Monolayer at the Air–Water Interface

The production protocol of many whole cell/virion vaccines involves an inactivation step with β-propiolactone (BPL). Despite the widespread use of BPL, its mechanism of action is poorly understood. Earlier work demonstrated that BPL alkylates nucleotide bases, but its interaction with proteins has not been studied in depth. In the present study we use ellipsometry to analyze the influence of BPL treatment of two H1N1 influenza strains, A/Brisbane/59/2007 and A/New Caledonia/20/1999, which are used for vaccine production on an industrial scale. Analyses were conducted using a mixed lipid monolayer containing ganglioside GM3, which functions as the viral receptor. Our results show that BPL treatment of both strains reduces viral affinity for the mixed monolayer and also diminishes the capacity of viral domains to self-assemble. In another series of experiments, the pH of the subphase was reduced from 7.4 to 5 to provoke the pH-induced conformational change of hemagglutinin, which occurs following endocytosis into the endosome. In the presence of the native virus the pH decrease caused a reduction in domain size, whereas lipid layer thickness and surface pressure were increased. These observations are consistent with a fusion of the viral membrane with the lipid monolayer. Importantly, this fusion was not observed with adsorbed inactivated virus, which indicates that BPL treatment inhibits the first step of virus-membrane fusion. Our data also indicate that BPL chemically modifies hemagglutinin, which mediates the interaction with GM3.

Structural Studies of the Monolayers and Bilayers Formed by a Novel Cholesterol-Phospholipid Chimera

Langmuir isotherm, neutron reflectivity, and small angle neutron scattering studies have been conducted to characterize the monolayers and vesicular bilayers formed by a novel chimeric phospholipid, ChemPPC, that incorporates a cholesteryl moeity and a C-16 aliphatic chain, each covalently linked via a glycerol backbone to phosphatidylcholine. The structures of the ChemPPC monolayers and bilayers are compared against those formed from pure dipalmitoylphoshatidylcholine (DPPC) and those formed from a 60:40 mol % mixture of DPPC and cholesterol. In accord with previous findings showing that very similar macroscopic properties were exhibited by ChemPPC and 60:40 mol % DPPC/cholesterol vesicles, it is found here that the chimeric lipid and lipid/sterol mixture have very similar monolayer structures (each having a monolayer thickness of ∼26 Å), and they also form vesicles with similar lamellar structure, each having a bilayer thickness of ∼50 Å and exhibiting a repeat spacing of ∼65 Å. The interfacial area of ChemPPC, however, is around 10 Å(2) greater than that of the combined DPPC/cholesterol unit in the mixed lipid monolayer (viz., 57 ± 1 vs 46 ± 1 Å(2), at 35 mN·m(-1)), and this difference in area is attributed to the succinyl linkage which joins the ChemPPC steroid and glyceryl moieties. The larger area of the ChemPPC is reflected in a slightly thicker monolayer solvent distribution width (9.5 vs 9 Å for the DPPC/cholesterol system) and by a marginal increase in the level of lipid headgroup hydration (16 vs 13 H(2)O per lipid, at 35 mN·m(-1)).

Using Phospholipids To Control Interparticle Distance in SERS-Active Substrates

Control over interparticle distance while creating effective SERS substrates is crucial to exploit an enhanced electromagnetic field. In the present study, phase behaviors of zwitterionic phospholipid 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE) and of a 1:1 (w/w) mixture of DPPE with DPPE analogue labeled with Rhodamine B (DPPE-RhB) were used to control optical properties of silver nanocube substrates prepared by the Langmuir monolayer technique. A more homogeneous distribution of the nanocubes was observed in the DPPE/DPPE-RhB mixture, compared to DPPE at all surface pressures due to a more fluid nature of the mixed lipid monolayer. As a result, interparticle coupling and corresponding optical properties can be effectively controlled with the DPPE/DPPE-RhB monolayer. Enhancement of the RhB Raman signal is detected and found to increase exponentially with increasing surface pressure due to the creation of an intense electromagnetic field in interparticle regions. Results of the study show that the phospholipid matrix enables effective control over the interparticle spacing in the monolayer.

The application of monolayer studies in the understanding of liposomal formulations

The study of surfactant monolayers is certainly not a new technique, but the application of monolayer studies to elucidate controlling factors in liposome design remains an underutilised resource. Using a Langmuir–Blodgett trough, pure and mixed lipid monolayers can be investigated, both for their interactions within the monolayer, and for interfacial interactions with drugs in the aqueous sub-phase. Despite these monolayers effectively being only half a bilayer, with a flat rather than curved structure, information from these studies can be effectively translated into liposomal systems. Here we outline the background, general protocols and application of Langmuir studies with a focus on their application in liposomal systems. A range of case studies are discussed which show how the system can be used to support its application in the development of liposome drug delivery. Examples include investigations into the effect of cholesterol within the liposome bilayer, understanding effective lipid packaging within the bilayer to promote water soluble and poorly soluble drug retention, the effect of alkyl chain length on lipid packaging, and drug-monolayer electrostatic interactions that promote bilayer repackaging.