Formation Of Unilamellar Vesicles Research Articles

High-Speed Imaging of Giant Unilamellar Vesicle Formation in cDICE

High-Speed Imaging of Giant Unilamellar Vesicle Formation in cDICE

Synergistic antibacterial effect of the pistachio green hull extract-loaded porphysome decorated with 4-nitroimidazole against bacteria

‘Active targeting’ refers to modifying a nanocarrier’s surface with targeting ligands. This study introduced an efficient approach for immobilizing imidazole-based drugs onto the metallated-porphyrin complex within the porphysome nanocarrier. To enhance cellular and bacterial uptake, a Ni-porphyrin with a fatty acid tail was synthesized and placed in the bilayer center of DPPC, facilitating receptor-mediated endocytosis. The Ni-porphyrin in the head group of the Ni-porphyrin-tail was placed superficially in the polar region of the membrane. Spherical unilamellar vesicle formation (DPPC: Ni-porphyrin-tail 4:1 mole ratio), as metallo-porphysome, was achieved through supramolecular self-assembly in an aqueous buffer. These vesicles exhibited a diameter of 279 ± 7 nm and a zeta potential of −15.3 ± 2.5 mV, showcasing their unique cytocompatibility. Nitroimidazole was decorated on the surface of metallo-porphysomes and pistachio green hull extract (PGHE) was loaded into the carrier for synergistic activity against (E. coli) and (S. aureus) bacteria strains. The physicochemical properties of Nitroimidazole-porphysome-PGHE, including size, zeta potential, morphology, loading efficiency, and release profile under various pH and temperature conditions in simulated gastrointestinal fluids were characterized. This combination therapy prevented bacterial cell attachment and biofilm formation in Caco-2 cells, as colon epithelial cells. The remarkable benefit of this system is that it does not affect cell viability even at 0.5 mg/ml. This study demonstrates the potential of a new co-delivery system using biocompatible metallo-porphysomes to decrease bacterial infections.

Investigation of the vesicle-to-micelle transition of 11-amino undecanoic acid derived sulphonamide and a comprehensive study of its interaction with protein

In the present manuscript, an amphiphile sulphonamide based surfactant benzenesulphonyl-11-amino sodium undecanoate (BASU) is designed and synthesized. The surface activity of the amphiphile in the solutions is studied at neutral pH so that the resulting amphiphile self-organizes and transfers from large unilamellar vesicles to small micelles from dilute to concentrated solutions. During the aggregate transitions, the common surfactants tend to form the small aggregate at low concentrations; but BASU shows the large vesicle structure at low concentration of ~3 mM and converts into the small micelle at ~9 mM. Therefore, different techniques have been used, such as, tensiometry, conductometry, fluorimetry and DLS and some microscopic characterization, e.g., confocal fluorescence microscopy to reveal the aggregate assembly and transition mechanism. The isothermal titration calorimetry is used for quantitative measurement of thermodynamic properties of self-assembly formation and the process is found spontaneous and entropically favorable. The permeability of the vesicle membrane bilayer is explored by a kinetic study. Effects of salt and cholesterol on the aggregate of respective amphiphile are also investigated. The interaction of surfactant with both human and bovine serum albumin is analyzed through UV–visible and fluorescence techniques to draw a comparative study. Antibacterial activity is tested by both spectral and zone inhibition methods and its application for mixed amphiphiles (e.g., BASU/CTAB) is found. Therefore, according to the ability of formation of unilamellar vesicles (ULV) and its stability, permeability and antibacterial activity, the amphiphile can have potential applications in the medicinal field.

In vitro and Ex vivo Analysis of Pitavastatin-Loaded Liposomes for the Treatment of Hyperlipidemia

Pitavastatin is a BCS class II drug with low solubility and high permeability. So, the purpose of the study was to develop and characterize pitavastatin-loaded liposomes to achieve sustained release of the drug by increasing the solubility as well as bioavailability in treating hyperlipidemia. All formulations were prepared using the ether injection method (EIM) to determine the effect of three independent factors lecithin, cholesterol, and chitosan/PEG-6000 on two dependent variables of percent drug entrapment efficiency (% DEE) and percent cumulative drug release (% CDR) at low (-1) and high (+1) levels, respectively. The prepared liposomes were analyzed using various techniques, including percent DEE, percent CDR, FTIR, S0EM, and zeta potential. After 8 hours of dissolution, the lowest and highest drug releases were 78.01 and 88.70 percent, 68.12 and 80.19 percent, and 66.22 and 78.91 percent, for FCH-4 and FCH-2, FLE-1 and FLE-3, and FPEG-4 and FPEG-3, respectively. Additionally, permeability experiments were conducted by in vitro and ex vivo methods. The particle size of liposomes was between 3.51 μm to 4.19 μm that gave evidence of the formation of uni-lamellar vesicles. The release kinetics were investigated using a variety of mathematical models. SEM and FTIR analyses indicated that liposomes have a spherical and smooth surface with no interaction between medications and excipients. Comparative in vitro and ex vivo studies demonstrated that pitavastatin calcium-loaded liposomes may be viable for patients with hyperlipidemia due to their enhanced solubility and bioavailability. Dhaka Univ. J. Pharm. Sci. 22(1): 43-54, 2023 (June)

Microscopic diffusion in cationic vesicles across different phases

Understanding the phase behavior and microscopic diffusion mechanism of cationic vesicles is crucial for controlled-release kinetics in gene/DNA transfection. Here, we report our findings on the structure, phase behavior, and microscopic dynamics of a vesicle based on a cationic double-chained surfactant, dihexadecyldimethylammonium bromide (DHDAB), which is a promising candidate for a nonviral gene/DNA carrier. Small-angle neutron-scattering measurements reveal that DHDAB aggregates are in the form of unilamellar vesicles. Calorimetric studies show a strong thermal hysteresis in the heating and cooling cycles following different pathways. Two first-order transitions are observed at 303 and 318 K in the heating cycle. Fourier transform infrared spectroscopy reveals that the first transition (at 303 K) is associated with a polymorphic (solid-solid) transition from coagel to an intermediate crystalline (IC) phase, and subsequently the second transition (at 318 K) is an order-disorder transition from IC to the fluid phase. Interestingly, it is found that in the cooling cycle, the fluid phase directly transforms back into the coagel phase without passing through the IC phase. This is also confirmed by an elastic fixed window scan (EFWS) using incoherent neutron scattering. The phase behavior of DHDAB is in strong contrast to that of its longer-chain counterpart, dioctadecyldimethylammonium bromide (DODAB), where the intermediate gel phase was observed during the cooling cycle. Moreover, no polymorphic transition was observed in the heating cycle of DODAB, in which the coagel phase directly converts to the fluid phase. These sharply contrasting phase behaviors for DHDAB and DODAB suggest the strong dependence of membrane phase behavior to the alkyl chain length. EFWS measurements reveal that the phase transitions observed in the DHDAB membrane are associated with strong changes in the DHDAB dynamics. The changes in conformational entropy of DHDAB molecules estimated from EFWS confirm that the coagel-fluid and IC-fluid phase transitions are accompanied by strong changes in the conformations of the alkyl chains. The microscopic dynamics of DHDAB molecules in each of these phases is investigated by employing quasielastic neutron-scattering (QENS). In the ordered phases (coagel and IC), only localized internal dynamics of the DHDAB is observed in the QENS spectra. However, in the fluid phase, the presence of a long-range lateral diffusion is detected in addition to the localized internal dynamics of the alkyl chain. The lateral motion is found to follow Fickian diffusion. The internal dynamics of DHDAB depends on the phase state of the bilayer. In the ordered phase, the internal dynamics is described by a fractional uniaxial rotational diffusion model. However, in the fluid phase, where alkyl tails are disordered with significant gauche defects, the internal dynamics is described using localized translation within confined spheres. This study highlights the rich phase behavior of the DHDAB membrane and the surfactant dynamics across these phases.

The Elucidation of the Molecular Mechanism of the Extrusion Process.

Extrusion is a popular method for producing homogenous population of unilamellar liposomes. The technique relies on forcing a lipid suspension through cylindrical pores in a polycarbonate membrane. The quantification of the extrusion and/or recalibration processes make possible the acquisition of experimental data, which can be correlated with the mechanical properties of the lipid bilayer. In this work, the force needed for the extrusion process was correlated with the mechanical properties of a lipid bilayer derived from other experiments. Measurements were performed using a home-made dedicated device capable of maintaining a stable volumetric flux of a liposome suspension through well-defined pores and to continuously measure the extrusion force. Based on the obtained results, the correlation between the lipid bilayer bending rigidity and extrusion force was derived. Specifically, it was found that the bending rigidity of liposomes formed from well-defined lipid mixtures agrees with data obtained by others using flicker-noise spectroscopy or micromanipulation. The other issue addressed in the presented studies was the identification of molecular mechanisms leading to the formation of unilamellar vesicles in the extrusion process. Finally, it was demonstrated that during the extrusion, lipids are not exchanged between vesicles, i.e., vesicles can divide but no membrane fusion or lipid exchange between bilayers was detected.

Investigation of the adsorption of a mixture of two anionic surfactants, AOT and SDBS, on silica at ambient temperature

Chemical flooding, one of the Enhanced Oil Recovery (EOR) techniques used to increase oil production, consists in the injection in the well of an aqueous formulation containing various chemical additives such as surfactants. However, its performance can be significantly altered by the loss of surfactants in reservoir rocks. More precisely, surfactant loss due to adsorption on the reservoir rock may have a non-negligible impact on the efficiency of the injected formulation. In this article, we considered the adsorption of a mixture of two anionic surfactants having an important industrial relevance for EOR applications. Adsorption was studied on silica, representative of reservoir rocks such as sandstone, by combining Quartz Crystal Microbalance with Dissipation monitoring (QCM-D) and neutron reflectivity experiments. A preliminary characterization of the surfactant mixture solution demonstrated the formation of unilamellar vesicles in the bulk. Whereas an adsorbed layer was measured with the single AOT vesicles, but not with the SDBS micelles, we observed that mixing both anionic surfactants change the adsorption phenomenon. Indeed, non-negligible adsorption was measured for the mixture even at concentrations where only slight adsorption had been observed with the individual surfactants. This suggests that the structure of the aggregates formed in the bulk has a non-negligible impact on the adsorption. We note that the addition of salt tends to enhance the adsorption by screening the repulsive interactions between both negatively charged surfactants and silica at neutral pH. The present work provides new insights into the description of the adsorption of a mixture of surfactants of same nature in unfavorable conditions.

Stable Ethosome-like Catanionic Vesicles for Transdermal Hydrophilic Drug Delivery with Predictable Encapsulation Efficiency.

Lipid-like pseudo-double-chained catanionic surfactants have emerged as the attractive materials to prepare potential vesicular carriers in drug and gene delivery applications. In particular, the semi-spontaneous process has been developed to fabricate ethosome-like catanionic vesicles for the transdermal drug delivery. In this work, Arbutin (a water-soluble drug) encapsulation efficiency of ethosome-like catanionic vesicles fabricated from decyltrimethylammonium-tetradecylsulfate (DeTMA-TS, CH3(CH2)9 N(CH3)3-CH3(CH2)13SO4) and decyltrimethylammonium-dodecylsulfate (DeTMA-DS, CH3(CH2)9N(CH3)3-CH3(CH2)11SO4) with various amounts of ethanol and cholesterol in tris buffer solution was experimentally determined. A simple unilamellar vesicle (ULV) model, resulting in the theoretical encapsulation efficiency within ±10% error for most vesicle compositions, was also developed. Such agreement indirectly confirmed the formation of unilamellar vesicles by the preparation method. Stable ethosome-like catanionic vesicles by using catanionic surfactants with the aid of suitable amounts of ethanol and cholesterol, which led to polydispersity index (PDI) values of vesicle size distribution less than 0.3, were successfully prepared and their hydrophilic drug encapsulation efficiencies can be accurately predicted. Furthermore, the linear correlations of the trap volume ratio with both vesicle size and concentration of the extra added CHOL also provide important guidelines for controlling the drug loading of ethosome-like catanionic vesicles. The accomplishments reached for the novel vesicles are useful for developing their transdermal drug delivery applications.

Formation of Uni-Lamellar Vesicles in Mixtures of DPPC with PEO-b-PCL Amphiphilic Diblock Copolymers.

The ability of mixtures of 1.2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and the amphiphilic diblock copolymers poly (ethylene oxide)-block-poly(ε-caprolactone) (PEO-b-PCL) to stabilize uni-lamellar nano-vesicles is reported. Small angle neutron scattering (SANS) is used to define their size distribution and bilayer structure and resolve the copresence of aggregates and clusters in solution. The vesicles have a broad size distribution which is compatible with bilayer membranes of relatively low bending stiffness. Their mean diameter increases moderately with temperature and their number density and mass is higher in the case of the diblock copolymer with the larger hydrophobic block. Bayesian analysis is performed in order to justify the use of the particular SANS fitting model and confirm the reliability of the extracted parameters. This study shows that amphiphilic block copolymers can be effectively used to prepare mixed lipid-block copolymer vesicles with controlled lamellarity and a significant potential as nanocarriers for drug delivery.

Spontaneous formation of unilamellar vesicles based on the surfactant 1-methylimidazolium bis-(2-ethylhexyl) phosphate, evaluated as a function of pH and in saline solution

In this work, we report the formation of unilamellar vesicles in water based on the protic ionic liquid-surfactant 1-methylimidazolium bis-(2-ethylhexyl) phosphate (imim-DEHP) explored under different experimental conditions (surfactant concentration, pH and ionic strength). Surface tension measurements were made depending on the surfactant concentration at different pHs and in saline solution. The size of the vesicles was measured by dynamic light scattering (DLS) and images of the systems were obtained by atomic force microscopy (AFM).Our results showed that the surfactant self-assembled forming spontaneously vesicles at different pHs (from 2–8) and in saline solution. The vesicle formation, especially at pH ≥ 4, is independent of the imim-DEHP concentration and the ionic strength of the medium used, which demonstrates great stability in the systems formed. When the pH is lower than the pKa of the polar head group, the surfactant produces very large polydisperse aggregates, which is due to an agglomeration of smaller vesicles. Very interesting is that the dependence of the vesicle size with the pH value is reversible therefore, this system could be used as drug delivery under different conditions. In addition to this, imim-DEHP vesicles were coated with chitosan to improve biocompatibility and avoid possible effects of cytotoxicity of these systems.In summary, imim-DEHP is a promising surfactant to be used in biological applications, for example as a nanocarrier for the incorporation of bioactive compounds into living organisms.

CARMUSTINE LOADED NANOSIZE LIPID VESICLES SHOWED PREFERENTIAL CYTOTOXICITY AND INTERNALIZATION IN U87MG CELL LINE ALONG WITH IMPROVED PHARMACOKINETIC PROFILE IN MICE: A STRATEGY FOR TREATMENT OF GLIOMA

Objective: Successful treatment of glioma still remains a tough challenge. The present study aims at the development and evaluation of carmustine loaded nanosize phospholipid vesicles (CNLVs) for the treatment of glioma.
 Methods: The experimental NLVs were developed by conventional lipid layer hydration technique and were characterized by different in vitro tools such as diffraction light scattering (DLS), zeta potential, field emission scanning electron microscopy (FESEM), cryo-transmission electron microscopy (cryo-TEM), in vitro drug loading capacity, drug release study etc. In vitro cytotoxicity and cellular uptake of the optimized drug-loaded NLVs were carried out in U87MG human glioblastoma cell line. In vivo pharmacokinetic study was conducted in Swiss albino mice.
 Results: DLS data showed an average vesicle diameter of 92 nm with narrow size distribution. Optimized CNLVs were spherical in shape with a smooth surface as depicted from FESEM data. Cryo-TEM study confirmed formation of unilamellar vesicles with intact outer bilayer. A reasonable drug loading of 7.8 % was reported for the optimized CNLVs along with a sustained release of CS over a 48 h study period. In vitro cytotoxicity assay revealed a considerable higher toxicity of CNLVs than free drugs in the U87MG cells. Confocal microscopy showed a satisfactory internalization of the optimized drug-loaded NLVs in the tested cell line. Pharmacokinetic data demonstrated an enhanced mean residence time of optimized CNLVs in blood than free drug.
 Conclusion: Results depicted the potential of experimental CNLVs for the treatment of glioma after further in vivo tests.

On the Mechanism of Bilayer Separation by Extrusion; or, Why Your Large Unilamellar Vesicles are Not Really Unilamellar

ABSTRACT Extrusion through porous filters is a widely used method for preparing biomimetic model membranes. Of primary importance in this approach is the efficient production of single bilayer (unilamellar) vesicles that eliminate the influence of interlamellar interactions and strictly define the bilayer surface area available to external reagents such as proteins. Sub-microscopic vesicles produced using extrusion are widely assumed to be unilamellar, and large deviations from this assumption would dramatically impact interpretations from many model membrane experiments. Using three probe-free methods—small-angle X-ray and neutron scattering (SAXS and SANS) and cryogenic electron microscopy (cryoEM)—we report unambiguous evidence of extensive multilamellarity in extruded vesicles composed of neutral phosphatidylcholine lipids, including for the common case of neutral lipids dispersed in physiological buffer and extruded through 100 nm diameter pores. In such preparations, only ~35% of lipids are externally accessible, and this fraction is highly dependent on preparation conditions. Charged lipids promote unilamellarity, as does decreasing solvent ionic strength, indicating the importance of electrostatic interactions in determining the lamellarity of extruded vesicles. Smaller extrusion pore sizes also robustly increase the fraction of unilamellar vesicles, suggesting a role for membrane bending. Taken together, these observations suggest a mechanistic model for extrusion, wherein formation of unilamellar vesicles involves competition between bilayer bending and adhesion energies. The findings presented here have wide-ranging implications for the design and interpretation of model membrane studies, especially ensemble-averaged observations relying on the assumption of unilamellarity. STATEMENT OF SIGNIFICANCE Extruded vesicles are a ubiquitous tool in membrane research. It is widely presumed that extrusion produces unilamellar (i.e., single bilayer) vesicles, an assumption that is often crucial for data analysis and interpretation. Using X-ray and neutron scattering and cryogenic electron microscopy, we show that a substantial amount of lipid remains inaccessible after extrusion due to an abundance of multilamellar vesicles (MLVs). While this is a general phenomenon for neutral lipids, MLV contamination can be reduced by several complementary approaches such as including charged lipids in the mixture, reducing the ionic strength of the aqueous medium, and reducing the extrusion pore size. These observations together suggest a mechanism by which extrusion strips MLVs of their layers.

On the Mechanism of Bilayer Separation by Extrusion, or Why Your LUVs Are Not Really Unilamellar

Extrusion through porous filters is a widely used method for preparing biomimetic model membranes. Of primary importance in this approach is the efficient production of single bilayer (unilamellar) vesicles that eliminate the influence of interlamellar interactions and strictly define the bilayer surface area available to external reagents such as proteins. Submicroscopic vesicles produced using extrusion are widely assumed to be unilamellar, and large deviations from this assumption would impact interpretations from many model membrane experiments. Using three probe-free methods—small angle X-ray and neutron scattering and cryogenic electron microscopy—we report unambiguous evidence of extensive multilamellarity in extruded vesicles composed of neutral phosphatidylcholine lipids, including for the common case of neutral lipids dispersed in physiological buffer and extruded through 100-nm diameter pores. In such preparations, only ∼35% of lipids are externally accessible and this fraction is highly dependent on preparation conditions. Charged lipids promote unilamellarity as does decreasing solvent ionic strength, indicating the importance of electrostatic interactions in determining the lamellarity of extruded vesicles. Smaller extrusion pore sizes also robustly increase the fraction of unilamellar vesicles, suggesting a role for membrane bending. Taken together, these observations suggest a mechanistic model for extrusion, wherein the formation of unilamellar vesicles involves competition between bilayer bending and adhesion energies. The findings presented here have wide-ranging implications for the design and interpretation of model membrane studies, especially ensemble-averaged observations relying on the assumption of unilamellarity.

Interaction of the mononucleotide UMP with a fluid phospholipid bilayer.

Interaction between mononucleotides and lipid membranes is believed to have played an important role in the origin of life on Earth. Studies on mononucleotide-lipid systems hitherto have focused on the influence of the lipid environment on the organization of the mononucleotide molecules, and the effect of the latter on the confining medium has not been investigated in detail. We have probed the interaction of the mononucleotide, uridine 5'-monophosphate (UMP), and its disodium salt (UMPDSS) with fluid dimyristoylphosphatidylcholine (DMPC) membranes, using small-angle X-ray scattering (SAXS), cryogenic scanning electron microscopy (cryo-SEM) and computer simulations. UMP adsorbs and charges the lipid membrane, resulting in the formation of unilamellar vesicles in dilute solutions. Adsorption of UMP reduces the bilayer thickness of DMPC. UMPDSS has a much weaker effect on interbilayer interactions. These observations are in very good agreement with the results of an all-atom molecular dynamics simulation of these systems. In the presence of counterions, such as Na+, UMP forms small aggregates in water, which bind to the bilayer without significantly perturbing it. The phosphate moiety in the lipid headgroup is found to bind to the hydrogens from the sugar ring of UMP, while the choline group tends to bind to the two oxygens from the nucleotide base. These studies provide important insights into lipid-nucleotide interactions and the effect of the nucleotide on lipid membranes.

Effects of artepillin C on model membranes displaying liquid immiscibility.

It has been hypothesized that the therapeutic effects of artepillin C, a natural compound derived from Brazilian green propolis, are likely related to its partition in the lipid bilayer component of biological membranes. To test this hypothesis, we investigated the effects of the major compound of green propolis, artepillin C, on model membranes (small and giant unilamelar vesicles) composed of ternary lipid mixtures containing cholesterol, which display liquid-ordered (lo) and liquid-disordered (ld) phase coexistence. Specifically, we explored potential changes in relevant membrane parameters upon addition of artepillin C presenting both neutral and deprotonated states by means of small angle X-ray scattering (SAXS), differential scanning calorimetry (DSC), and confocal and multiphoton excitation fluorescence microscopy. Thermotropic analysis obtained from DSC experiments indicated a loss in the lipid cooperativity of lo phase at equilibrium conditions, while at similar conditions spontaneous formation of unilamellar vesicles from SAXS experiments showed that deprotonated artepillin C preferentially located at the surface of the membrane. Time-resolved experiments using fluorescence microscopy showed that at doses above 100 µM, artepillin C in its neutral state interacted with both liquid-ordered and liquid-disordered phases, inducing curvature stress and promoting dehydration at the membrane interface.

Characterization of self-assembled hybrid siloxane-phosphocholine bilayers

We have synthesized six new hybrid siloxane phosphocholines (SiPCs) and examined their self-assembly behaviour in aqueous dispersions. Employing small angle X-ray scattering we have characterized SiPC bilayers. SiPCs exhibit differential self-assembly behaviour that results from the interplay between the siloxane fatty acid in the sn-2 position and the differing chain length fatty acids in the sn-1 position. SiPCs that possess a fatty acid chain of a C8–C14 chain length in the sn-1 position form unilamellar vesicles. Extending the fatty acid chain length to C16 and C18 allows for the formation of both unilamellar and multilamellar vesicles. We propose that the preferential formation of unilamellar vesicles is the result of an enhanced hydrophobic effect imparted by siloxane chains at the termini of lipid tails.

Role of oligo(malic acid) on the formation of unilamellar vesicles

Stable unilamellar dipalmitoylphosphatidylcholine vesicles were produced by using oligo(malic acid) and cholesterol. Detailed physico-chemical characterization prove that by using oligo(malic acid) the substitution of PEGylated lipids for sterically stabilization comes possible. The polymer molecules cover the outer surface of spherical-shaped vesicles, and an asymmetrical composition occurs in the two leaflets of the phospholipid bilayer. The oligo(malic-acid) and cholesterol are enriched in the outer side assuring the stabilization of vesicles. Cholesterol plays an important role in the self-assembly of components as it makes the entering of oligomers possible deep into the polar head-region of lipids. The presence of oligo(malic acid) molecules does not induce degradation by hydrolysis of lipid molecules but the vesicle system turns into a sensitive form giving a possibility for pH sensitive targeting. Preliminary investigation on the investigated oligo(malic acid)-stabilized vesicles do not show any toxic effect promising their applicability in the field of liposomal drug delivery.

Effect of PEG, Lipid Composition and Formulation on Vesicle Lamellarity: A Small Angle Neutron Scattering Study

Liposomes are vesicles comprising a phospholipid bilayer and inner aqueous cavity, and are well-established systems for drug delivery applications. They are able to encapsulate both hydrophobic and hydrophilic compounds. Among the parameters to account for when designing a liposomal drug carrier lamellarity (number of consecutive lipid bilayers within one vesicle) is very important: it affects both encapsulation efficiency and efflux rate through the membrane of the encapsulated drug. Despite the relevance of liposomes as drug carriers, a comprehensive study on how lipid composition and formulation method affect vesicle lamellarity is still lacking. Here, we combined small-angle neutron scattering (SANS) and cryo-TEM to elucidate the membrane structure of vesicles prepared using three different formulation methods (film hydration followed by freeze-thawing (FT) or agitation on a shaker (AS), and reverse phase evaporation (REV)) and extruded through a 100 nm membrane. We examined two phospholipids: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), both widely used in the liposome field. Our results show that POPC vesicles prepared via the FT or AS methods retain a substantial amount of multilamellarity after extrusion through 100 nm membranes. This is also true for DPPC vesicles, although the extent of residual multilamellarity is much lower. The introduction of a poly(ethylene)glycol (PEG)-modified lipid in the vesicle composition results in the formation of unilamellar vesicles after extrusion when the PEGylated lipid content is at least 0.5 mol% for POPC vesicles and 0.1 mol% for DPPC vesicles. As for the REV method, unextruded DPPC vesicles are already unilamellar, while unextruded POPC vesicles still exhibit some bilamellarity. Extruded, REV-prepared POPC vesicles are unilamellar instead. In conclusion, our results provide important insights into the effect of formulation method and lipid composition on producing liposomes with a defined membrane structure.

Synergistic Interaction between Cholesterol and Functionalized Ionic Liquid Based Surfactant Leading to the Morphological Transition

AbstractStable vesicular aggregate is the primary requisite to transport and deliver the biological and pharmaceutical standards. The serendipitous discovery of nonlipid vesicles made with the nonlipid building blocks, such as ionic liquid based surfactants (ILBSs) proved to be advantageous over others. In a quest to achieve stable, more rigid and hydrophobic vesicles from ILBSs, here we have investigated the lipid (cholesterol) induced unilamellar vesicle formation in the aqueous functionalized and nonfunctionalized ILBSs. The vesicles show outstanding stability with time, temperature and dilution with water. Spectroscopic (turbidity, steady state absorbance and fluorescence), scattering (dynamic light scattering) and microscopic (transmission electron microscopy) techniques were used to characterize the vesicles. The hydration behavior and rigidity associated with the vesicular bilayer on transforming from micellar assembly was characterized through steady state absorbance and fluorescence techniques. The unilamellar vesicles derived are compared with the conventional surfactant, i. e. cetyltrimethyl ammonium bromide.

Analysis of Trisiloxane Phosphocholine Bilayers.

We have synthesized unique siloxane phosphocholines and characterized their aggregates in aqueous solution. The siloxane phosphocholines form nearly monodisperse vesicles in aqueous solution without the need for secondary extrusion processes. The area/lipid, lipid volume, and bilayer thickness were determined from small-angle X-ray scattering experiments. The impetus for the spontaneous formation of unilamellar vesicles by these compounds is discussed.