Lipid Hydration Research Articles

An aptamer ligand based liposomal nanocarrier system that targets tumor endothelial cells

The objective of this study was to construct our recently developed aptamer-modified targeted liposome nano-carrier (Apt-PEG-LPs) system to target primary cultured mouse tumor endothelial cells (mTEC), both in vitro and in vivo. We first synthesized an aptamer-polyethylene glycol 2000-distearoyl phosphoethanolamine (Apt-PEG2000-DSPE). The conjugation of the Apt-PEG2000-DSPE was confirmed by MALDI-TOF mass spectroscopy. A lipid hydration method was used to prepare Apt-PEG-LPs, in which the outer surface of the PEG-spacer was decorated with the aptamer. Apt-PEG-LPs were significantly taken up by mTECs. Cellular uptake capacity was observed both quantitatively and qualitatively using spectrofluorometry, and confocal laser scanning microscopy (CLSM), respectively. In examining the extent of localization of aptamer-modified liposomes that entered the cells, approximately 39% of the Apt-PEG-LPs were not co-localized with lysotracker, indicating that they had escaped from endosomes. The uptake route involved a receptor mediated pathway, followed by clathrin mediated endocytosis. This Apt-PEG-LP was also applied for in vivo research whether this system could target tumor endothelial cells. Apt-PEG-LP and PEG5000-DSPE modified Apt-PEG-LP (Apt/PEG5000-LP) were investigated by human renal cell carcinoma (OS-RC-2 cells) inoculating mice using CLSM. Apt-PEG-LP and Apt/PEG5000-LP showed higher accumulation on tumor vasculature compared to PEG-LP and the co-localization efficacy of Apt-PEG-LP and Apt/PEG5000-LP on TEC were quantified 16% and 25% respectively, which was also better than PEG-LP (3%). The findings suggest that this system is considerable promise for targeting tumor endothelial cells to deliver drugs or genes in vitro and in vivo.

Systematic implicit solvent coarse graining of dimyristoylphosphatidylcholine lipids.

We have used systematic structure-based coarse graining to derive effective site-site potentials for a 10-site coarse-grained dimyristoylphosphatidylcholine (DMPC) lipid model and investigated their state point dependence. The potentials provide for the coarse-grained model the same site-site radial distribution functions, bond and angle distributions as those computed in atomistic simulations carried out at four different lipid-water molar ratios. It was shown that there is a non-negligible dependence of the effective potentials on the concentration at which they were generated, which is also manifested in the properties of the lipid bilayers simulated using these potentials. Thus, effective potentials computed at low lipid concentration favor to more condensed and ordered structure of the bilayer with lower average area per lipid, while potentials obtained at higher lipid concentrations provide more fluid-like structure. The best agreement with the reference data and experiment was achieved using the set of potentials derived from atomistic simulations at 1:30 lipid:water molar ratio providing fully saturated hydration of DMPC lipids. Despite theoretical limitations of pairwise coarse-grained potentials expressed in their state point dependence, all the resulting potentials provide a stable bilayer structure with correct partitioning of different lipid groups across the bilayer as well as acceptable values of the average lipid area, compressibility and orientational ordering. In addition to bilayer simulations, the model has proven its robustness in modeling of self-aggregation of lipids from randomly dispersed solution to ordered bilayer structures, bicelles, and vesicles.

Nanometer-resolved radio-frequency absorption and heating in biomembrane hydration layers.

Radio-frequency (RF) electromagnetic fields are readily absorbed in biological matter and lead to dielectric heating. To understand how RF radiation interacts with macromolecular structures and possibly influences biological function, a quantitative description of dielectric absorption and heating at nanometer resolution beyond the usual effective medium approach is crucial. We report an exemplary multiscale theoretical study for biomembranes that combines (i) atomistic simulations for the spatially resolved absorption spectrum at a single planar DPPC lipid bilayer immersed in water, (ii) calculation of the electric field distribution in planar and spherical cell models, and (iii) prediction of the nanometer resolved temperature profiles under steady RF radiation. Our atomistic simulations show that the only 2 nm thick lipid hydration layer strongly absorbs in a wide RF range between 10 MHz and 100 GHz. The absorption strength, however, strongly depends on the direction of the incident wave. This requires modeling of the electric field distribution using tensorial dielectric spectral functions. For a spherical cell model, we find a strongly enhanced RF absorption on an equatorial ring, which gives rise to temperature gradients inside a single cell under radiation. Although absolute temperature elevation is small under conditions of typical telecommunication usage, our study points to hitherto neglected temperature gradient effects and allows thermal RF effects to be predicted on an atomistically resolved level. In addition to a refined physiological risk assessment of RF fields, technological applications for controlling temperature profiles in nanodevices are possible.

Construction of an aptamer modified liposomal system targeted to tumor endothelial cells.

We describe herein the development of a high affinity and specific DNA aptamer as a new ligand for use in liposomal nanoparticles to target cultured mouse tumor endothelial cells (mTECs). Active targeted nanotechnology based drug delivery systems are currently of great interest, due to their potential for reducing side effects and facilitating the delivery of cytotoxic drugs or genes in a site specific manner. In this study, we report on a promising aptamer candidate AraHH036 that shows selective binding towards mTECs. The aptamer does not bind to normal cells, normal endothelial cells or tumor cells. Therefore, we synthesized an aptamer-polyethylene glycol (PEG) lipid conjugate and prepared aptamer based liposomes (ALPs) by the standard lipid hydration method. First, we quantified the higher capacity of ALPs to internalize into mTECs by incubating ALPs containing 1 mol%, 5 mol% and 10 mol% aptamer of total lipids and compared the results to those for unmodified PEGylated liposomes (PLPs). A confocal laser scanning microscope (CLSM) uptake study indicated that the ALPs were taken up more efficiently than PLPs. The measured Kd value of the ALPs was 142 nM. An intracellular trafficking study confirmed that most of the rhodamine labeled ALPs were taken up and co-localized with the green lysotracker, thus confirming that they were located in lysosomes. Finally, using an aptamer based proteomics approach, the molecular target protein of the aptamer was identified as heat shock protein 70 (HSP70). The results suggest that these ALPs offer promise as a new carrier molecule for delivering anti-angiogenesis drugs to tumor vasculature.

Lipid Flip-Flop in Binary Membranes Composed of Phosphatidylserine and Phosphatidylcholine

The kinetics and thermodynamics of lipid flip-flop in bilayers composed of 1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine (DPPS) and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) were studied using sum-frequency vibrational spectroscopy. The kinetics of DSPC and DPPS flip-flop were examined as a function of temperature and bilayer composition. The rate of DSPC flip-flop did not exhibit any significant dependence on bilayer composition while the rate of DPPS flip-flop was inversely dependent on the mole fraction of DPPS. The transition-state thermodynamics for DSPC and DPPS lipids in these mixed bilayers were determined in order to identify the energetic impact of the phosphatidylserine headgroup on lipid flip-flop. The thermodynamics for the DSPC component remained statistically identical to bilayers composed entirely of DSPC. The activation energy for the DPPS component showed a linear correlation with the mole fraction of DPPS for all bilayer compositions. The enthalpy and entropy for DPPS flip-flop did not increase linearly with the fraction of DPPS but did directly correlate with the molecular area. The DPPS component also exhibited enthalpy-entropy compensation which suggests that lipid hydration may play a significant role in membrane dynamics.

Vibrational spectroscopy of water in hydrated lipid multi-bilayers. III. Water clustering and vibrational energy transfer

Water clustering and connectivity around lipid bilayers strongly influences the properties of membranes and is important for functions such as proton and ion transport. Vibrational anisotropic pump-probe spectroscopy is a powerful tool for understanding such clustering, as the measured anisotropy depends upon the time-scale and degree of intra- and intermolecular vibrational energy transfer. In this article, we use molecular dynamics simulations and theoretical vibrational spectroscopy to help interpret recent experimental measurements of the anisotropy of water in lipid multi-bilayers as a function of both lipid hydration level and isotopic substitution. Our calculations are in satisfactory agreement with the experiments of Piatkowski, Heij, and Bakker, and from our simulations we can directly probe water clustering and connectivity. We find that at low hydration levels, many water molecules are in fact isolated, although up to 70% of hydration water forms small water clusters or chains. At intermediate hydration levels, water forms a wide range of cluster sizes, while at higher hydration levels, the majority of water molecules are part of a large, percolating water cluster. Therefore, the size, number, and nature of water clusters are strongly dependent on lipid hydration level, and the measured anisotropy reflects this through its dependence on intermolecular energy transfer.

Effect of Some Formulation Variables on the Entrapment Efficiency and in Vitro Release of Ketoprofen from Ketoprofen Niosomes

The objective of the present study was to formulate Ketoprofen Niosomes based on fixing the levels of different formulation variables which affect Niosomal preparation to study the effects of two formulation variables; changing the method of preparation by using either lipid hydration method or ether injection method, and changing the incorporated surfactant by using either Span 60 surfactant or mixed Span 60 surfactant with Tween grades surfactants (20, 40, 60, and 80 respectively) aiming of studying the effects of these variables on the entrapment efficiency and in vitro release of Ketoprofen from Ketoprofen Niosomes.

Potential therapeutic effect of nanobased formulation of rivastigmine on rat model of Alzheimer's disease

BackgroundTo sustain the effect of rivastigmine, a hydrophilic cholinesterase inhibitor, nanobased formulations were prepared. The efficacy of the prepared rivastigmine liposomes (RLs) in comparison to rivastigmine solution (RS) was assessed in an aluminium chloride (AlCl3)-induced Alzheimer’s model.MethodsLiposomes were prepared by lipid hydration (F1) and heating (F2) methods. Rats were treated with either RS or RLs (1 mg/kg/day) concomitantly with AlCl3 (50 mg/kg/day).ResultsThe study showed that the F1 method produced smaller liposomes (67.51 ± 14.2 nm) than F2 (528.7 ± 15.5 nm), but both entrapped the same amount of the drug (92.1% ± 1.4%). After 6 hours, 74.2% ± 1.5% and 60.8% ± 2.3% of rivastigmine were released from F1 and F2, respectively. Both RLs and RS improved the deterioration of spatial memory induced by AlCl3, with RLs having a superior effect. Further biochemical measurements proved that RS and RLs were able to lower plasma C-reactive protein, homocysteine and asymmetric dimethy-larginine levels. RS significantly attenuated acetylcholinesterase (AChE) activity, whereas Na+/K+-adenosine triphosphatase (ATPase) activity was enhanced compared to the AlCl3-treated animals; however, RLs succeeded in normalization of AChE and Na+/K+ ATPase activities. Gene-expression profile showed that cotreatment with RS to AlCl3-treated rats succeeded in exerting significant decreases in BACE1, AChE, and IL1B gene expression. Normalization of the expression of the aforementioned genes was achieved by coadministration of RLs to AlCl3-treated rats. The profound therapeutic effect of RLs over RS was evidenced by nearly preventing amyloid plaque formation, as shown in the histopathological examination of rat brain.ConclusionRLs could be a potential drug-delivery system for ameliorating Alzheimer’s disease.

Suppression of Cooperative Motions in Phospholipid Membranes by Osmotic Stress: Deuterium NMR Relaxation Study

Understanding membrane dynamics is crucial to explaining the function of membrane proteins. Phospholipids are commonly employed as model systems to investigate biological membranes. The complex dynamic organization of phospholipid membranes spans several frequency decades, starting from sub-picosecond local motions to millisecond collective dynamics [1]. Such motional frequencies can be accessed using various NMR relaxation methods. To address membrane dynamics mediated by osmotic stress, we measured 2H longitudinal (R1Z) and transverse quadrupolar echo (R2QE) relaxation rates for the liquid-crystalline phase of DMPC-d54 membrane bilayers. Osmotic stress was applied by both dehydration and osmolyte concentration [2]. The R1Z values of individual acyl segments were independent of osmotic stress while the segmental order parameters (SCD) and R1Z profiles followed a theoretical square-law functional dependence [3]. The R2QE rates were found to be sensitive to osmotic pressure as well as the acyl position, thus yielding two important observations: enhanced transverse relaxation rates with increased amount of water per lipid, and limiting lower R2QE values as we dehydrate the membrane. The R2QE rates of the acyl segments and respective SCD values tend to follow a square-law behavior [3] with increasing lipid dehydration. At higher hydration the square-law behavior is limited to those acyl segments deeper in the hydrophobic region, with a break as the head group is approached. These results clearly indicate that water enhances slow cooperative motions whereas they are suppressed by dehydration. Additional complementary Carr-Purcell-Meiboom-Gill (CPMG) dispersion measurements map the frequency dependence of relaxation rates. Such studies in presence of membrane proteins give insight into optimized lipid hydration for their biological functions.[1] A. Leftin et al. (2011) BBA 1808, 818-839.[2] K.J. Mallikarjunaiah et al. (2011) BJ100, 98-107.[3] M.F. Brown (1982) JCP77, 1576-1599.

A criterion to identify the equilibration time in lipid bilayer simulations

With the aim of establishing a criterion for identifying when a lipid bilayer has reached steady state using the molecular dynamics simulation technique, lipid bilayers of different composition in their liquid crystalline phase were simulated in aqueous solution in presence of CaCl$_2$ as electrolyte, at different concentration levels. In this regard, we used two different lipid bilayer systems: one composed by 288 DPPC (DiPalmitoylPhosphatidylCholine) and another constituted by 288 DPPS (DiPalmitoylPhosphatidylSerine). In this sense, for both type of lipid bilayers, we have studied the temporal evolution of some lipids properties, such as the surface area per lipid, the deuterium order parameter, the lipid hydration and the lipid-calcium coordination. From their analysis, it became evident how each property has a different time to achieve equilibrium. The following order was found, from faster property to slower property: coordination of ions $\approx$ deuterium order parameter $>$ area per lipid $\approx$ hydration. Consequently, when the hydration of lipids or the mean area per lipid are stable we can ensure that the lipid membrane has reached the steady state.Received: 3 September 2012, Accepted: 24 October 2012; Edited by: M. C. Barbosa; DOI:http://dx.doi.org/10.4279/PIP.040005Cite as: R. D. Porasso, J. J. López Cascales, Papers in Physics 4, 040005 (2012)

A Study of the Antioxidant Effect of Flavonic Compounds for Preventing Lipid Oxidation by Using Fluorescence Spectroscopy

In this paper a study of the effect of flavonic compounds in preventing and/or reducing the membrane lipid oxidation due to free radical attack was performed by using fluorescence spectroscopy techniques. Lipid peroxidation was investigated by using liposomes-artificial membrane models, which were prepared by lipid hydration method. Their oxidation was performed with a 15-W ultraviolet germicidal lamp having wavelength radiation 253.7 nm. In the first series of experiments, the protective effect of two synthetic antioxidants (quercetin and caffeic acid) against free radicals was monitored, while in the second series two 70% hydroalcoholic extracts from blueberry and blackcurrant leaves was used. It was determined that natural antioxidants have a much higher antioxidant power against free radicals than synthetic compounds but they degrade after two hours of oxidation. Liposomes are better protected when using natural antioxidants, but their degradation is completed more quickly, than in the case of synthetic antioxidants.

THE USE OF KETOPROFEN NIOSOMES FOR FORMULATION OF SUSTAINED RELEASE TABLET DOSAGE FORM

The objective of this study was to formulate sustained release tablet dosage form (100 mg) using Ketoprofen Niosomes. Ketoprofen Niosomes were prepared by lipid hydration method and then dried and compressed into sustained release tablets (100 mg). The tablet excipients employed include: Avicel PH 101, Avicel PH 102, and Mannitol as diluents, Cab-O-Sil (colloidal silica) as a glidant, Starch as a disintegrant and Magnesium Stearate as lubricating agent. The tablets were tested for weight variation, hardness, friability, thickness, disintegration, drug content and release ratio. Finally the release kinetics of Ketoprofen tablets were calculated.

Dermal delivery by niosomes of black tea extract as a sunscreen agent

The primary objective of this study was to investigate the feasibility of using niosomes as a delivery vehicle for the dermal administration in vitro of black tea extract (BTE) as a sunscreen. Multi-lamellar niosomes were obtained by means of a previously reported method of lipid hydration films. In vitro penetration experiments through nude mouse skin were carried out to evaluate the potential of niosomes as a dermal formulation. The nude mouse skin membrane allowed the effects of penetration with a niosome formulation to be evaluated. Penetration rates of caffeine- and gallic acid-loaded niosomes in a steady state were higher than dispersion in aqueous solutions. For skin permeation, higher transdermal absorption rates were seen with solutions of caffeine and gallic acid. In the near future, BTE as a sunscreen agent will be dermally delivered by niosomes.

Effect of temperature on the formation of liquid phase-separating giant unilamellar vesicles (GUV)

Giant unilamellar vesicles (GUVs) are widely used as model systems to study both, lipid and membrane protein behavior. During their preparation by the commonly applied electroformation method, a number of issues must be considered to avoid the production of artifacts due to a poor lipid hydration and protein degradation. Here we focus on the effect of preparation temperature on GUVs composed of the most commonly used domain-forming mixture dioleoylelphospatidylcholine/shingomyelin/cholesterol (DOPC/SM/chol) (2/2/1). Lower production temperatures are generally preferable when aiming at a functional reconstitution of proteins into the membrane. On the other hand, lower growth temperature is suspected to alter the lipid composition and the yield of phase-separating vesicles. By confocal imaging, we find that vesicles prepared significantly above and below the melting temperature Tm have the same overall morphology, similar size distributions of vesicles and a similar area coverage by liquid-ordered (Lo) domains. However, a large population analysis indeed reveals a different overall yield of phase-separating vesicles. Two-focus scanning fluorescence correlation spectroscopy measurements did not show any divergence of lipid analog mobility in (Lo) and (Ld) phases in vesicles prepared at different temperatures, indicating that the lowered growth temperature did not influence the lipid organization within the two phases. Moreover, the expected advantages of lower preparation temperature for proteo-GUVs could be exemplified by the reconstitution of voltage dependent anion channel (VDAC) into DOPC/SM/chol GUVs, which aggregates at high, but not at low preparation temperatures.

Impact of Embedded Endocannabinoids and Their Oxygenation by Lipoxygenase on Membrane Properties

N-Arachidonoylethanolamine (anandamide) and 2-arachidonoylglycerol are the best characterized endocannabinoids. Their biological activity is subjected to metabolic control whereby a dynamic equilibrium among biosynthetic, catabolic, and oxidative pathways drives their intracellular concentrations. In particular, lipoxygenases can generate hydroperoxy derivatives of endocannabinoids, endowed with distinct activities within cells. The in vivo interaction between lipoxygenases and endocannabinoids is likely to occur within cell membranes; thus, we sought to ascertain whether a prototypical enzyme like soybean (Glycine max) 15-lipoxygenase-1 is able to oxygenate endocannabinoids embedded in synthetic vesicles and how these substances could affect the binding ability of the enzyme to different lipid bilayers. We show that (i) embedded endocannabinoids increase membrane fluidity; (ii) 15-lipoxygenase-1 preferentially binds to endocannabinoid-containing bilayers; and that (iii) 15-lipoxygenase-1 oxidizes embedded endocannabinoids and thus reduces fluidity and local hydration of membrane lipids. Together, the present findings reveal further complexity in the regulation of endocannabinoid signaling within the central nervous system, disclosing novel control by oxidative pathways.

GAFFlipid: a General Amber Force Field for the accurate molecular dynamics simulation of phospholipid

Previous attempts to simulate phospholipid bilayers using the General Amber Force Field (GAFF) yielded many bilayer characteristics in agreement with experiment, however when using a tensionless NPT ensemble the bilayer is seen to compress to an undesirable extent resulting in low areas per lipid and high order parameters in comparison to experiment. In this work, the GAFF Lennard-Jones parameters for the simulation of acyl chains are corrected to allow the accurate and stable simulation of pure lipid bilayers. Lipid bilayers comprised of six phospholipid types were simulated for timescales approaching a quarter of a microsecond under tensionless constant pressure conditions using Graphics Processing Units. Structural properties including area per lipid, volume per lipid, bilayer thickness, order parameter and headgroup hydration show favourable agreement with available experimental values. Expanding the system size from 72 to 288 lipids and a more experimentally realistic 2 × 288 lipid bilayer stack induces little change in the observed properties. This preliminary work is intended for combination with the new AMBER Lipid11 modular force field as part of on-going attempts to create a modular phospholipid AMBER force field allowing tensionless NPT simulations of complex lipid bilayers.

Hydration-Mediated Slow Dynamics in Phospholipid Membranes

Biological activities of integral membrane proteins depend on the nature of the surrounding lipid bilayer [1]. The dynamic organization of lipid bilayer systems spans a wide frequency range encompassing individual fast acyl motions, molecular rotations, lipid protrusions, and collective bilayer fluctuations [2]. Time scales of these motional modes typically span a large range from sub-picoseconds up to milliseconds, and can be investigated using solid-state NMR spectroscopy. To correlate structural changes of lipid bilayers mediated by osmotic stress [3] with biological function and molecular dynamics, we measured 2H longitudinal (R1Z) and transverse (R2CP) relaxation rates in the liquid-crystalline phase of DMPC-d54 membrane bilayers at various hydration levels. The R1Z experiments used a conventional inversion-recovery pulse sequence, while R2CP rates were measured from quadrupolar-echo intensities using a Carr-Purcell-Meiboom-Gill pulse train. By Fourier transforming individual echoes with different pulse spacings we map the frequency dependence of relaxation rates and motional modes of individual acyl segments. Empirical correlations of acyl chain segmental order parameters (SCD) and R1Zprofiles followed a theoretical square-law functional dependence [4]. Our preliminary results show that R2CP is sensitive to hydration levels as well as the acyl position, thus indicating the presence of slow dynamic modes. The R2CPrates of the acyl segments near the polar head groups increase with lipid dehydration, while the acyl segments deeper in the hydrophobic region show lower R2CP values at high dehydration levels. Similar studies in presence of peptides and proteins can give insight into optimized lipid hydration for protein functionality and slow cooperative motions.[1] M.F. Brown (1994) Chem. Phys. Lipids 73, 159–180.[2] A. Leftin et al. (2011) Biochim. Biophys. Acta 1808, 818–839.[3] K.J. Mallikarjunaiah et al. (2011) Biophys. J. 100, 98–107.[4] M.F. Brown (1982) J. Chem. Phys. 77, 1576–1599.

Effect of pH on Physicochemical Properties and Encapsulation Efficiency of PEGylated Linolenic Acid Vesicles

The preparation of vesicle from a mixture of linolenic acid and 1,2-dipalmitoyl-sn-glycerol-3-phosphoethanolamine-N-[methoxy-(polyethylene glycol) -2000] (DPPE-PEG2000) has been successfully carried out by using dry lipid hydration method. The effect of pH on particle size, zeta potential, encapsulation efficiency and critical vesiculation concentration (CVC) of the prepared vesicle suspensions in aqueous were studied. Macroscopic stability of the vesicles was also evaluated through their particle size and zeta potential for a period of 30 days. We found that CVC vary according to the pH, with higher pH of the bulk solution, CVC is higher. Vesicles formed at pH 8.5 were the most stable suspension throughout a period of 30 days compared to those at pH 7.5 and pH 9.0. Addition of DPPE-PEG2000 into the preparation of vesicle at pH 8.5 caused a reduction of the vesicle size to the scale of nanometer which is an advantage to their application. On the other hand, encapsulation of calcein and vitamin E were carried out. Certain amount of these compounds could be successfully loaded into the resulting liposomes under this experimental condition.

Water Adsorption Isotherms of Lipids

Hydration of bilayer lipids is a fundamental property of biological membranes. The available database of lipid hydration isotherms is fitted over the entire range of water activities by using a statistical mechanical approach that is an extension of the common Brunauer-Emmett-Teller model, to include differential energies of association for water molecules beyond the first strongly bound layer. Three-parameter fits are obtained that can be used to represent the experimental isotherms to a good degree of accuracy over the complete range of water-binding activities. Fits are also made in terms of the hydration pressure and correlation length of water ordering, by using the polarization theory of lipid hydration. The relationship of the latter approach to measurements of hydration forces between lipid bilayers is discussed.

Exploring the correlation between lipid packaging in lipoplexes and their transfection efficacy.

Whilst there is a large body of evidence looking at the design of cationic liposomes as transfection agents, correlates of formulation to function remain elusive. In this research, we investigate if lipid packaging can give further insights into transfection efficacy. DNA lipoplexes composed of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) or 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) in combination with 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) or 1,2-stearoyl-3-trimethylammonium-propane (DSTAP) were prepared by the lipid hydration method. Each of the formulations was prepared by hydration in dH2O or phosphate buffer saline (PBS) to investigate the effect of buffer salts on lipoplex physicochemical characteristics and in vitro transfection. In addition, Langmuir monolayer studies were performed to investigate any possible correlation between lipid packaging and liposome attributes. Using PBS, rather than dH2O, to prepare the lipoplexes increased the size of vesicles in most of formulations and resulted in variation in transfection efficacies. However, one combination of lipids (DSPE:DOTAP) could not form liposomes in PBS, whilst the DSPE:DSTAP combination could not form liposomes in either aqueous media. Monolayer studies demonstrated saturated lipid combinations offered dramatically closer molecular packing compared to the other combinations which could suggest why this lipid combination could not form vesicles. Of the lipoplexes prepared, those formulated with DSTAP showed higher transfection efficacy, however, the effect of buffer on transfection efficiency was formulation dependent.