Formation Of Small Unilamellar Vesicles Research Articles

Influence of archaeal lipids isolated from Aeropyrum pernix K1 on physicochemical properties of sphingomyelin-cholesterol liposomes

We investigated the influence of archaeal lipids (C25,25) isolated from thermophilic archaeon Aeropyrum pernix K1 on physicochemical properties of liposomes comprised of egg sphingomyelin (SM) and cholesterol (CH) using fluorescence emission anisotropy, calcein release studies, dynamic light scattering, transmissive electron microscopy and phase analysis light scattering. The 2 mol% addition of archaeal lipids enabled formation of small unilamellar vesicles by sonication while also having significant effect on reducing mean size, polydispersity index and zeta potential of C25,25/SM/CH vesicles. Increasing the ratio of C25,25 lipids in mixture of C25,25/SM/CH decreased lipid ordering parameter in dose dependent manner at different temperatures. We also demonstrated that adding 15 mol% C25,25 to SM/CH mixture will cause it to notably interact with fetal bovine serum which could make them a viable alternative adjuvant to synthetic ether-linked lipids in development of advanced liposomal vaccine delivery systems. The prospect of combining the proven strengths of SM/CH mixtures with the unique properties of C25,25 opens exciting possibilities for advancing drug delivery technologies, promising to yield formulations that are both highly effective and adaptable to a range of therapeutic applications.

Rapid formation of Small Unilamellar Vesicles (SUV) through low-frequency sonication: An innovative approach

Liposomes are membrane models and excellent Drug Delivery Systems. However, their preparation is expensive, labor intensive, time consuming, and sometimes toxic. Recently, we published an innovative methodology for the production of homogeneous Small Unilamellar Vesicles (SUV) through a simple, fast, relatively low cost, and reproducible process that resulted in very stable vesicles. The methodology involves a small amount of F127 triblock Pluronic® copolymer (0.02% m/V) to a phospholipid (DPPC, DOPC, and DSPC), followed by the solid dispersion methodology. After that, during the thin-film hydration process (of lipids and F127), SUVs are quickly formed after 30 s of sonication using bath equipment at a low frequency of 42 kHz. The resultant colloidal solution was homogeneous with liposomes lower than ˜100 nm of hydrodynamic diameter. The SUV formation is highly temperature dependent. However, it functions independently from the lipid´s phase (gel or liquid-crystal phases). A preparation with Pluronic P123 did not lead to homogeneous SUV. We found that the conditions for SUV formation feature a mixture of F127 and lipids at above a critical temperature. This temperature is not the copolymer´s CMT (micelle is not required). Interestingly, the long PEO groups of F127 play an essential role in this SUV formation, which is proposed to be governed by the “Budding Off” model. The findings show a complex combination of factors: a sum of the sonoporation, the oscillation effects of the compressed/dilated regions, the frequency of oscillation, and the temperature-dependence on long PEO groups. Also, the outer lipid monolayer interaction might by responsible for generating "daughter" vesicles from "mother" vesicles in the mechanism.

Baicalin loaded in folate-PEG modified liposomes for enhanced stability and tumor targeting

Bioavailability of baicalin (BAI), an example of traditional Chinese medicine, has been modified by loading into liposome. Several liposome systems of different composition i.e., lipid/cholesterol (L), long-circulating stealth liposome (L-PEG) and folate receptor (FR)—targeted liposome (L-FA) have been used as the drug carrier for BAI. The obtained liposomes were around 80nm in diameter with proper zeta potentials about −25mV and sufficient physical stability in 3 months. The entrapment efficiency and loading efficiency of BAI in the liposomes were 41.0–46.4% and 8.8–10.0%, respectively. The morphology details of BAI lipsosome systems i.e., formation of small unilamellar vesicles, have been determined by cryogenic transmission electron microscopy (cryo-TEM) and small angle X-ray scattering (SAXS). In vitro cytotoxicity of BAI liposomes against HeLa cells was evaluated by MTT assay. BAI loaded FR-targeted liposomes showed higher cytotoxicity and cellular uptake compared with non-targeted liposomes. The results suggested that L-FA-BAI could enhance anti-tumor efficiency and should be an effective FR-targeted carrier system for BAI delivery.

How does the surface charge of ionic surfactant and cholesterol forming vesicles control rotational and translational motion of rhodamine 6G perchlorate (R6G ClO₄)?

The rotational dynamics and translational diffusion of a hydrophilic organic molecule, rhodamine 6G perchlorate (R6G ClO4) in small unilamellar vesicles formed by two different ionic surfactants, cetyltrimethylammonium bromide (CTAB) and sodium dodecyl sulfate (SDS), with cholesterol have been investigated using fluorescence spectroscopic methods. Moreover, in this article the formation of vesicle using anionic surfactant, SDS at different cholesterol-to-surfactant molar ratio (expressed by Q value (Q = [cholesterol]/[surfactant])) has also been reported. Visual observation, dynamic light scattering (DLS) study, turbidity measurement, steady state fluorescence anisotropy (r0) measurement, and eventually microscopic images reveal the formation of small unilamellar vesicles in aqueous solution. Also, in this study, an attempt has been made to observe whether the cationic probe molecule, rhodamine 6G (R6G) experiences similar or different microenvironment in cholesterol-SDS and cholesterol-CTAB assemblies with increase in cholesterol concentration. The influence of cholesterol on rotational and translational diffusion of R6G molecules has been investigated by monitoring UV-vis absorption, fluorescence, time-resolved fluorescence anisotropy, and finally fluorescence correlation spectroscopy (FCS) measurements. In cholesterol-SDS assemblies, due to the strong electrostatic attractive interaction between the negatively charged surface of vesicle and cationic R6G molecules, the rotational and diffusion motion of R6G becomes slower. However, in cholesterol-CTAB aggregates, the enhanced hydrophobicity and electrostatic repulsion induces the migration of R6G from vesicle bilayer to aqueous phase. The experimental observations suggest that the surface charge of vesicles has a stronger influence than the hydrophobicity of the vesicle bilayer on the rotational and diffusion motion of R6G molecules.

Studies of a Channel-Forming Peptide Inserted into Liposomes formed by POPC:POPS and POPC:POPE

Peptide-based channel replacement therapy attempts to use channel-forming peptides to establish new ion conductive pathways through the cell membrane. In the present study, a 22 residue amino acid peptide derived from the second transmembrane segment of the alpha-1-subunit of the glycine receptor (M2GlyR) was investigated because it has previously shown high conductance as a channel-forming peptide in MDCK cells monolayers in Ussing chamber experiments.As membrane models, liposomes formed by different phospholipids common to mammalian plasma membranes were made by extrusion at different ratios and then the ion channel peptide was added into the artificial bilayer in order to study the peptide-lipid interactions. These mixtures were analyzed using Trp fluorescence blue shifts, which is an indication of peptide insertion, Transmission Electron Microscopy (TEM) as well as Circular Dichroism (CD).The Trp fluorescence results showed the highest blue shift for POPC:POPS and POPC:POPE at 7:3 and 6:4 ratios respectively. These results indicated that the peptide was inserted into the artificial bilayer in a phospholipids’ ratio and composition dependent manner. TEM analysis showed the formation of Small Unilamellar Vesicles (SUVs) and Large Unilamellar Vesicles (LUVs) in a size range of 80-120 nm with both systems. The circular dichroism spectra showed an alpha helical content for the peptide without liposomes under 40% TFE and SDS. However, the CD for the peptide/liposomes mixtures showed predominantly random coiled structures.Taken together, these results suggest that an artificial membrane model containing different phospholipids can be a useful tool of studying this channel-forming peptide insertion and peptide/lipid interaction.

31P NMR and AFM studies on the destabilization of cell and model membranes by the major bovine seminal plasma protein, PDC‐109

The effect of PDC-109 binding to dimyristoylphosphatidylcholine (DMPC) and dipalmitoylphosphatidylglycerol (DPPG) multilamellar vesicles (MLVs) and supported membranes was investigated by (31)P NMR spectroscopy and atomic force microscopy. Additionally, the effect of cholesterol on the binding of PDC-109 to phosphatidylcholine (PC) membranes was studied. Binding of PDC-109 to MLVs of DMPC and DPPG induced the formation of an isotropic signal in their (31)P NMR spectra, which increased with increasing protein/lipid ratio and temperature, consistent with protein-induced disruption of the MLVs and the formation of small unilamellar vesicles or micelles but not inverse hexagonal or cubic phases. Incorporation of cholesterol in the DMPC MLVs afforded a partial stabilization of the lamellar structure, consistent with previous reports of membrane stabilization by cholesterol. AFM results are consistent with the above findings and show that addition of PDC-109 leads to a complete breakdown of PC membranes. The fraction of isotropic signal in (31)P NMR spectra of DPPG in the presence of PDC-109 was less than that of DMPC under similar conditions, suggesting a significantly higher affinity of the protein for PC. Confocal microscopic studies showed that addition of PDC-109 to human erythrocytes results in a disruption of the plasma membrane and release of hemoglobin into the solution, which was dependent on the protein concentration and incubation time.

Transition from Vesicles to Small Nanometer Scaled Vesicles, Arising from the Manipulation of Curvature in Dialkyl Chain Cationic/Nonionic Surfactant Mixed Aggregates by the Addition of Straight Chain Alkanols

The addition of straight chain alkanols to the dialkyl chain cationic/nonionic surfactant mixtures of dihexadecyl dimethyl ammonium bromide, DHDAB, and dodecaethylene monododecyl ether, C(12)E(12), has been used to manipulate the mean curvature of the self-assembled aggregates. This induces some significant structural changes and notably the formation of small unilamellar vesicles, nanometer scaled vesicles, L(sv). These structural changes have been measured and quantified using small angle neutron scattering, SANS. At a solution concentration of 25 mM, the DHDAB/C(12)E(12) mixtures have a structural evolution, from C(12)E(12) rich to DHDAB rich solution compositions, of small globular micelles, L(1), to micellar/vesicle coexistence, L(1)/L(v) or L(v)/L(1), to vesicle structures, L(v), bilamellar or multilamellar vesicles, blv or mlv. The impact of the addition of straight chain alkanols (in the range octanol to hexadecanol) depends upon the alkyl chain length and the amount of alcohol added. Furthermore, the effect of the addition of octanol and decanol appears to be distinctly different from that of the larger straight chain alkanols of dodecanol and hexadecanol. For the addition of octanol and decanol to C(12)E(12) rich DHDAB/C(12)E(12) mixtures, the alcohol is solubilized into the micellar core, and as the amount of alcohol added increases, significant micellar growth is ultimately observed. However, notably at intermediate DHDAB/C(12)E(12) solution compositions, in the region of L(1)/L(v) or L(v)/L(1) coexistance in the absence of alcohol, the addition of octanol or decanol promotes the formation of relatively small unilamellar vesicles, L(sv), nanometer sized vesicles, with a mean diameter in the range 70-140 A. For solutions that are rich in DHDAB, the addition of octanol or decanol results in a transition to L(v)/L(sv) coexistence and ultimately to L(v) formation. In contrast, the addition of the larger straight chain length alkanols, dodecanol or hexadecanol, to DHDAB/C(12)E(12) mixtures results in a somewhat different behavior. In this case, the addition of dodecanol or hexadecanol results in the transition from L(1) to L(1)/L(v) to L(v) occurring for solutions less rich in DHDAB than is observed in the absence of alcohol. That is, there is an enhanced tendency toward the formation of structures with a lower net curvature, either blv or mlv. Notably, for these mixtures, the small unilamellar nanometer scaled vesicle phase, L(sv), is absent.

Surfactin-Triggered Small Vesicle Formation of Negatively Charged Membranes: A Novel Membrane-Lysis Mechanism

The molecular mode of action of the lipopeptide SF with zwitterionic and negatively charged model membranes has been investigated with solid-state NMR, light scattering, and electron microscopy. It has been found that this acidic lipopeptide (negatively charged) induces a strong destabilization of negatively charged micrometer-scale liposomes, leading to the formation of small unilamellar vesicles of a few 10s of nanometers. This transformation is detected for very low doses of SF (Ri=200) and is complete for Ri=50. The phenomenon has been observed for several membrane mixtures containing phosphatidylglycerol or phosphatidylserine. The vesicularization is not observed when the lipid negative charges are neutralized and a cholesterol-like effect is then evidenced, i.e., increase of gel membrane dynamics and decrease of fluid membrane microfluidity. The mechanism for small vesicle formation thus appears to be linked to severe changes in membrane curvature and could be described by a two-step action: 1), peptide insertion into membranes because of favorable van der Waals forces between the rather rigid cyclic and lipophilic part of SF and lipid chains and 2), electrostatic repulsion between like charges borne by lipid headgroups and the negatively charged SF amino acids. This might provide the basis for a novel mode of action of negatively charged lipopeptides.

Spectroscopic studies of D-α-tocopherol concentration-induced transformation in egg phosphatidylcholne vesicles

The effects of embedding up to 60 mol% of α-tocopherol (α-Toc) on the morphology and structure of the egg phosphatidylcholine (PC) membrane were studied using spectroscopic techniques. The resulting vesicles were subjected to turbidometric and dynamic light scattering measurements to evaluate their size distribution. The α-Toc intrinsic fluorescence and its quenching was used to estimate the tocopherol position in the membrane. Optical microscopy was used to visualize morphological changes in the vesicles during the inclusion of tocopherol into the 2 mg/ml PC membrane. The incorporation of up to 15 mol% of tocopherol molecules into PC vesicles is accompanied by a linear increase in the fluorescence intensity and the simultaneous formation of larger, multilamellar vesicles. Increasing the tocopherol concentration above 20 mol% induced structural and morphological changes leading to the disappearance of micrometer-sized vesicles and the formation of small unilamellar vesicles of size ranging from 30 to 120 nm, mixed micelles and non-lamellar structures.

Dioctadecyldimethylammonium bromide vesicles prepared by the surfactant removal method

Dioctadecyldimethylammonium bromide (DODAB) is a double chain vesicle-forming cationic surfactant, whereas octa-ethyleneglycol mono- n-dodecyl ether (C 12E 8) is a single chain micelle-forming nonionic surfactant. At room temperature (ca. 22 °C) C 12E 8 molecules self-assemble in water as micelles while DODAB is insoluble. A mixture of DODAB and C 12E 8, however, can be soluble in water at room temperature depending on the relative amount of the compounds. We report the formation of small unilamellar vesicles (SUVs) by dialyzing at room temperature a mixture of 1.0 mM DODAB with 10 mM C 12E 8 in water. Extended bilayers are formed as well in equilibrium with vesicles. Such structures are viewed by a cryogenic transmission electron microscopy (cryo-TEM) image.

Structural and Macroscopic Characterization of a Gel Phase of Densely Packed Monodisperse, Unilamellar Vesicles

In the phase diagram of the surfactant system sodium oleate/octanol/water a very stiff gel phase has been found, which is usually not present in similar systems. The phases of this system have been characterized by electric conductivity and rheological measurements. The structures present have been investigated by means of freeze-fracture electron microscopy and small-angle neutron scattering (SANS). These experiments have shown that addition of octanol to the surfactant solution for concentrations higher than 130 mM Na oleate leads to the formation of small unilamellar vesicles. For sufficiently high octanol content these vesicles are so monodisperse that they are able to form a densely packed system with long-range order and with a shear modulus that is about 100 times higher than normally found for vesicle systems. Upon dilution with water the vesicles swell while keeping the packing fraction constant until a maximum diameter of about 450 A is reached, beyond which no further growth of the vesicles can...

Supramolecular Aggregates of Azobenzene Phospholipids and Related Compounds in Bilayer Assemblies and Other Microheterogeneous Media: Structure, Properties, and Photoreactivity1

A number of phosphatidyl choline derivatives containing trans-azobenzene units in the fatty ester backbone have been synthesized and studied in aqueous dispersions both pure and in the presence of saturated and unsaturated phospholipids. The structures of the assemblies formed have been investigated by microcalorimetry, dynamic light scattering, cryo-transmission electron microscopy, and reagent entrapment. While many of the mixed phospholipid dispersions give evidence for the formation of small unilamellar vesicles, the aqueous dispersions of pure azobenzene phospholipids (APL's) give evidence for several different structures, including relatively large plates in at least one case. The azobenzenes show strong evidence of “H” aggregate formation both in the pure and mixed dispersions. The aggregation number has been estimated for several of the APL's and found to be typically 3 or a multiple thereof. On the basis of simulations and studies with similar stilbene phospholipids as well as on the strong induced circular dichroism signals observed for the aggregate, we infer a chiral “pinwheel” unit aggregate structure similar to that found for several aromatics. The azobenzenes in the aqueous dispersions have been found to photoisomerize to give cis-rich photostationary states; the cis-azobenzenes show no evidence for aggregation and no induced circular dichroism. The cis-azobenzenes can be isomerized back to the trans either by irradiation or by thermal paths. Mixed aqueous dispersions of trans-APL's with saturated or unsaturated phospholipids can be prepared which entrap the fluorescent dye carboxyfluorescein (CF) under conditions where the CF fluorescence is very low due to self-quenching. By varying the APL/host phospholipid ratio the azobenzene can be aggregate, monomer, or dimer. In cases where the azobenzene is monomer or dimer, irradiation produces complete isomerization but little “leakage” of CF from the vesicle interior. In contrast, where the azobenzene is predominantly aggregate, irradiation results in both photoisomerization and reagent release. That photoisomerization in the latter case can result in “catastrophic” destruction of the vesicle can also be shown by cryo-transmission electron microscopy.

Micelle to Vesicle Transition Induced by Cosurfactant: Rheological Study and Direct Observations

We report a micelle−vesicle transition induced by the addition of a neutral cosurfactant, hexanol, into a wormlike micellar solution of gemini cationic surfactant. This transition was investigated with rheology measurements as well as direct imaging techniques. It was observed by cryo transmission electron microscopy that micelles first become elongated with an increase of hexanol, which increases the viscosity. As the hexanol concentration is increased, another more fluid phase appears, where micelles are highly branched and coexist with small vesicles. One can describe this transition as a decrease of the spontaneous curvature caused by the cosurfactant molecule, which induces the elongation, and then the branching of micelles as well as the formation of small unilamellar vesicles. With a further increase of hexanol, these vesicles grow in size, which allows their observation by optical microscopy with differential interferential contrast. Some of these vesicles show long tubule-like shape, typically a ...

Thermodynamics of Vesicle Formation from a Mixture of Anionic and Cationic Surfactants

Detailed model calculations on mixed sodium dodecyl sulfate/dodecylammonium chloride vesicles show that the work of bending a planar bilayer into a geometrically closed bilayer vesicle rises steeply as the molar ratio between aggregated anionic and cationic surfactant approaches unity. Likewise, the bending work increases as the mole fraction of either of the surfactants approaches unity, resulting in a bending free energy minimum on each side of the equimolar composition. In the vicinity of that composition the bending work is too large to permit vesicle formation to any appreciable extent, while at compositions where one of the surfactants is in excess, the bending free energy is much lower, thus enabling the formation of small unilamellar vesicles (R < 500 Å). These calculation results are in good qualitative agreement with recent experimental findings on mixed anionic/cationic vesicles. Moreover, the fluctuations in composition, chain packing density, shape, and size contribute to making the vesicle size distribution fairly polydisperse with a relative standard deviation of σR/Rmax equal to 0.283.

Spontaneous formation of small unilamellar vesicles by pH jump: a pH gradient across the bilayer membrane as the driving force

31P NMR and infrared spectroscopic methods have been used to study the formation of small unilamellar vesicles by the pH-jump method. It is shown that increasing the pH of different lamellar phospholipid dispersions (phosphatidic acids and phosphatidylserines) induces a pH gradient. This pH gradient is estimated to be 4 +/- 1 pH units, and its direction is such that the inner monolayer of the vesicles is at lower pH. There is spectroscopic evidence for tighter packing of the lipid hydrocarbon chains in the inner monolayer, probably due to the constraints imposed by the high curvature of the small vesicles formed. These results are discussed in terms of the driving force of the spontaneous vesiculation.

Mechanism of spontaneous vesiculation.

Both naturally occurring and synthetic phosphatidic acid (PtdOH) molecules show the phenomenon of spontaneous vesiculation on jump in pH value. This method involves a transient increase in pH of smectic PtdOH dispersions to values between 10 and 12. Such a pH increase induces spontaneous vesiculation with the formation of small unilamellar vesicles of diameter less than 50 nm as shown by 31P NMR. Both high-resolution and broad-line 31P NMR were used to study the mechanism of this process. When the pH of unsonicated PtdOH dispersions is raised to pH 10-12, lipid molecules on the outer monolayer of the bilayer become fully ionized. The second pK value of PtdOH in bilayers is 8.6 +/- 0.3, determined by 31P NMR. PtdOH molecules on the inner monolayer remain partially protonated. 31P NMR provides unambiguous evidence that the "pH-jump" treatment produces a pH gradient across the PtdOH bilayer. The orientation of the pH gradient is such that the pH in the external medium is 3-5 pH units higher than the internal pH. Associated with the pH gradient is a transverse packing asymmetry: partially protonated PtdOH molecules in the inner layer of the bilayer are more tightly packed than fully ionized molecules present in the outer layer. The pH gradient generated by the pH jump is proposed as the energy source that drives the spontaneous formation of highly curved vesicles.

Attachment of immunoglobulin to liposomal membrane via protein carbohydrate

A general method has been developed for the covalent attachment of immunoglobulin molecules to the outer layer of liposomal membranes. Aldehyde groups are generated by the mild oxidation with periodate or galactose oxidase of the carbohydrate groups on the constant region of the heavy chain. The oxidized protein is then reacted with a hydrazide group linked to a membrane component. Detailed studies were carried out with monomers of a monoclonal human IgM and two monoclonal murine IgM antibodies specific for the 1-dimethylaminonaphthalene-5-sulfonyl (Dns) group. Two hydrazide-containing hydrophobic reagents were used: N α -lauroyl, N ϵ -Dns-lysine hydrazide and lauric acid hydrazide. The number of protein aldehyde groups formed was assayed by reaction with N-(2,4-dinitrophenyl)- β-alanylglycylglycine hydrazide. Measurement of the intrinsic affinity for Dns-lysine of the processed anti-Dns IgMs demonstrated no substantial impairment of the specific reactivity of the antibody either from the oxidation step or the subsequent attachment to small unilamellar vesicles. The extent of attachment of antibody to small unilamellar vesicles was evaluated with respect to the mol% of hydrazide in the membrane, the duration of the incubation period for the aldehyde-hydrazide reaction and the ratio of protein to hydrazide. The yield of attached protein was significantly dependent on each of these experimental parameters over the ranges tested. Under the most favorable conditions the extent of covalent attachment of IgMs to small unilamellar vesicles was 535 μg of protein per μ mol of phospholipid, corresponding to 0.3 mol% of protein. Under these conditions, 61% of the total protein was associated with the small unilamellar vesicle fraction after fractionation by gel filtration. The attachment of the antibody to small unilamellar vesicles did not destroy the integrity of the vesicles, as demonstrated by the retention of carboxyfluorescein following initial encapsulation during the formation of small unilamellar vesicles.