Interaction of sodium N-lauroylsarcosinate with N-alkylpyridinium chloride surfactants: spontaneous formation of pH-responsive, stable vesicles in aqueous mixtures

The interaction of sodium N-lauroylsarcosinate (SLS) with N-cetylpyridinium chloride (CPC) and N-dodecylpyridinium chloride (DPC) was investigated in aqueous mixtures. A strong interaction between the anionic and cationic surfactants was observed. The interaction parameter, β was determined for a wide composition range and was found to be negative. The mixed systems were found to have much lower critical micelle concentration (cmc) and surface tension at cmc. The surfactant mixtures exhibit synergism in the range of molar fractions investigated. The self-assembly formation in the mixtures of different compositions and total concentrations were studied using a number of techniques, including surface tension, fluorescence spectroscopy, dynamic light scattering (DLS), transmission electron microscopy (TEM), confocal fluorescence microscopy (CFM). Thermodynamically stable unilamellar vesicles were observed to form upon mixing of the anionic and cationic surfactants in a wide range of composition and concentrations in buffered aqueous media. TEM as well as DLS measurements were performed to obtain shape and size of the vesicular structures, respectively. These unilamellar vesicles are stable for periods as long as 3 months and appear to be the equilibrium form of aggregation. Effect of pH, and temperature on the stability was investigated. The vesicular structures were observed to be stable at pH as low as 2.0 and at biological temperature (37°C). In presence of 10 mol% of cholesterol the mixed surfactant vesicles exhibited leakage of the encapsulated calcein dye, showing potential application in pH-triggered drug release.     

Hollow giant lipid vesicles prepared by coaxially electrospraying solutions of phospholipid and degradable polyelectrolyte

Hollow giant lipid vesicles were prepared in a single step by coaxially electrospraying separate solutions of phospholipid and a degradable polyelectrolyte. We synthesized a hydrolytically degradable cationic polyelectrolyte, poly(β-amino esters) (PBAE), and employed it as a degradable microgel template to form giant vesicles. Droplets of the phospholipid solution and the degradable polyelectrolyte solution were electrosprayed from coaxial double needles into a receiving solution. The PBAE formed a microgel by crosslinking with multivalent anions in the receiving solution, and the phospholipids formed bilayers on the microgel. Hollow giant lipid vesicles were successfully obtained and the mean diameters were 7.6 μm (C.V. 58 %). Substrates (calcein, dextran, and polymeric microparticles) were successfully encapsulated in the giant vesicles. Microscopic observations of microparticle mobility inside a giant vesicle indicated the fluidity of its aqueous interior. Investigations using fluorescently labeled PBAE also suggested the degradation of PBAE, and the release of fluorescent PBAE fragments from the encapsulated microgel, to form hollow giant lipid vesicles.     

Characterization of fluorinated catansomes: a promising vector in drug-delivery

Catansomes, which are vesicles prepared from mixtures of oppositely charged surfactants, have been suggested as effective alternatives to phospholipid vesicles, i.e., liposomes, in applications such as drug-delivery. This is mainly due to their enhanced chemical and physical stability as well as to their relatively easy preparation, which is an advantage for large-scale productions. In this study we have investigated catansomes prepared from a perfluorinated anionic surfactant (sodium perfluorooctanoate) premixed with a hydrogenated cationic surfactant (dodecyltrimethylammonium bromide or 1-dodecylpyridinium chloride). The aim was to gain insights into the physicochemical properties of these systems, such as size, stability, surface charge, and membrane morphology, which are essential for their use in drug-delivery applications. The catansomes were mostly unilamellar and 100-200 nm in size, and were stable for more than five months at room temperature. After loading the catansomes with the fluorescent marker calcein, they were found to exhibit an appreciable encapsulation efficiency and a low calcein leakage over time. The addition of fatty acids to calcein-loaded catansomes considerably promoted the release of calcein, and the rate and efficiency of calcein release were found to be proportional to the fatty acid concentration and chain length. Our results prove the feasibility of utilizing catansomes as drug-delivery vehicles as well as provide a means to efficiently release the encapsulated load.     

Rapid access to phospholipid analogs using thiol-yne chemistry

Phospholipids and glycolipids constitute an essential part of biological membranes, and are of tremendous fundamental and practical interest. Unfortunately, the preparation of functional phospholipids, or synthetic analogs, is often synthetically challenging. Here we utilize thiol-yne click chemistry methodology to gain access to phospho- and glycolipid analogs. Alkynyl hydrophilic head groups readily photoreact with numerous thiol modified lipid tails to yield the appropriate dithioether phospho- or glycolipids. The resulting structures closely resemble the structure and function of native diacylglycerolipids. Dithioether phosphatidylcholines (PCs) are suitable for forming giant unilamellar vesicles (GUV), which can be used as vessels for cell-free expression systems. The unnatural thioether linkages render the lipids resistant to phospholipase A2 hydrolysis. We utilize the improved stability of these lipids to control the shrinkage of GUVs composed of a mixture of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and dioleyl-dithioether PC, concentrating encapsulated nanoparticles. We imagine that these readily accessible lipids could find a number of applications as natural lipid substitutes.     

Making a tool of an artifact: the application of photoinduced Lo domains in giant unilamellar vesicles to the study of Lo/Ld phase spinodal decomposition and its modulation by the ganglioside GM1

Electroformed giant unilamellar vesicles containing liquid-ordered Lo domains are important tools for the modeling of the physicochemical properties and biological functions of lipid rafts. Lo domains are usually imaged using fluorescence microscopy of differentially phase-partionioning membrane-embedded probes. Recently, it has been shown that these probes also have a photosensitizing effect that leads to lipid chemical modification during the fluorescence microscopy experiments. Moreover, the lipid reaction products are able as such to promote Lo microdomain formation, leading to potential artifacts. We show here that this photoinduced effect can also purposely be used as a new approach to study Lo microdomain formation in giant unilamellar vesicles. Photosensitized lipid modification can promote Lo microdomain appearance and growth uniformly and on a faster time scale, thereby yielding new information on such processes. For instance, in egg phosphatidylcholine/egg sphingomyelin/cholesterol 50:30:20 (mol/mol) giant unilamellar vesicles, photoinduced Lo microdomain formation appears to occur by the rarely observed spinodal decomposition process rather than by the common nucleation process usually observed for Lo domain formation in bilayers. Moreover, temperature and the presence of the ganglioside GM1 have a profound effect on the morphological outcome of the photoinduced phase separation, eventually leading to features such as bicontinuous phases, phase percolation inversions, and patterns evoking double phase separations. GM1 also has the effect of destabilizing Lo microdomains. These properties may have consequences for Lo nanodomains stability and therefore for raft dynamics in biomembranes. Our data show that photoinduced Lo microdomains can be used to obtain new data on fast raft-mimicking processes in giant unilamellar vesicles.     

Visualization and Quantification of Transmembrane Ion Transport into Giant Unilamellar Vesicles

Transmembrane ion transporters (ionophores) are widely investigated as supramolecular agents with potential for biological activity. Tests are usually performed in synthetic membranes that are assembled into large unilamellar vesicles (LUVs). However transport must be followed through bulk properties of the vesicle suspension, because LUVs are too small for individual study. An alternative approach is described whereby ion transport can be revealed and quantified through direct observation. The method employs giant unilamellar vesicles (GUVs), which are 20–60 μm in diameter and readily imaged by light microscopy. This allows characterization of individual GUVs containing transporter molecules, followed by studies of transport through fluorescence emission from encapsulated indicators. The method provides new levels of certainty and relevance, given that the GUVs are similar in size to living cells. It has been demonstrated using a highly active anion carrier, and should aid the development of compounds for treating channelopathies such as cystic fibrosis.     

Interaction between water-soluble peptidic CdSe/ZnS nanocrystals and membranes: formation of hybrid vesicles and condensed lamellar phases

Due to their tunable optical properties and their well-defined nanometric size, core/shell nanocrystals (quantum dots, QDs) are extensively used for the design of biomarkers as well as for the preparation of nanostructured hybrid materials. It is thus of great interest to understand their interaction with soft lipidic membranes. Here we present the synthesis of water-soluble peptide CdSe/ZnS QDs and their interaction with the fluid lipidic membrane of vesicles. The use of short peptides results in the formation of small QDs presenting both high fluorescence quantum yield and high colloidal stability as well as a mean hydrodynamical diameter of 10 nm. Their interaction with oppositely charged vesicles of various surface charge and size results in the formation of hybrid giant or large unilamellar vesicles covered with a densely packed layer of QDs without any vesicle rupture, as demonstrated by fluorescence resonance energy transfer experiments, zetametry, and optical microscopy. The adhesion of nanocrystals onto the vesicle membrane appears to be sterically limited and induces the reversion of the surface charge of the vesicles. Therefore, their interaction with small unilamellar vesicles induces the formation of a well-defined lamellar hybrid condensed phase in which the QDs are densely packed in the plane of the layers, as shown by freeze-fracture electron microscopy and small-angle X-ray scattering. In this structure, strong undulations of the bilayer maximize the electrostatic interaction between the QDs and the bilayers, as previously observed in the case of DNA polyelectrolytes interacting with small vesicles.     

pH-responsive poly(methacrylic acid)-grafted hollow silica vesicles

Poly(methacrylic acid)-grafted hollow silica vesicles (PMAA-g-hollow silica vesicles) were obtained through a grafting-from approach. PMAA brushes were formed by performing atom-transfer radical polymerisation of sodium methacrylate with an initiator attached to the hollow silica spheres. PMAA-g-hollow silica vesicles were characterised by using TEM, thermogravimetric analysis (TGA) and FTIR spectroscopy. pH-dependent ξ potential and (1)H NMR spectra of PMAA-g-hollow silica vesicles were measured, and the results indicated that MAA brushes in PMAA-g-hollow silica vesicles had a lower ionisation degree and low solubility in acidic aqueous solution, for example, pH 3.4, but a higher ionisation degree and high solubility when the pH was higher than 7. Also it was demonstrated that calcein blue and fluorescein isothiocyanate (FITC) labelled dextran (M(n):10 kDa) could be encapsulated in the interiors of the PMAA-g-hollow silica vesicles with a negligible amount in PMAA brushes at pH 2, and pH-triggered release of calcein blue and FITC-labelled dextran from PMAA-g-hollow silica vesicles was observed at pH 7.4.     

New poly(phenyleneethynylene)s with cationic,facially amphiphilic structures

Polymers based on meta substituted phenylene ethylene are prepared with patterned polar and nonpolar groups to favor an extended conformation. These polymers were characterized at the air-water interface by Langmuir techniques and found to form stable monolayers with an extended conformation based on molecular models. In addition, these polymers show phospholipid membrane activity as measured by induced leakage of calcein from large unilamellar vesicles. These polymers represent new facially amphiphilic structures which are cationic in nature and surface active.     

DNA-Based Artificial Receptors as Transmembrane Signal Transduction Systems for Protocellular Communication

The ability to reproduce signal transduction and cellular communication in artificial cell systems is significant in synthetic protobiology. Here, we describe an artificial transmembrane signal transduction through low pH-mediated formation of the i-motif and dimerization of DNA-based artificial membrane receptors, which is coupled to the occurrence of fluorescence resonance energy transfer and the activation of G-quadruplex/hemin-mediated fluorescence amplification inside giant unilamellar vesicles. Moreover, an intercellular signal communication model is established when the extravesicular H⁺ input is replaced by coacervate microdroplets, which activate the dimerization of the artificial receptors, and subsequent fluorescence production or polymerization in giant unilamellar vesicles. This study represents a crucial step towards designing artificial signalling systems with environmental response, and provides an opportunity to establish signalling networks in protocell colonies.     

Hypelcin A,an α-aminoisobutyric acid containing antibiotic peptide,induced fusion of egg yolk-l-α-phosphatidylcholine small unilamellar vesicles

Hypelcin A, an α-aminoisobutyric acid-containing antibiotic peptide inducing fusion of egg yolk-l-α-phosphatidylcholine (egg PC) small unilamellar vesicles (SUVs), was investigated by lipid-mixing assay based on resonanceenergy transfer between fluorescent probes, electron microscopy, light scattering, and¹H-nuclear magnetic-resonance spectroscopy. At a high peptide-to-lipid ratio of approximately 1:5, the peptide fuses several SUVs of 20–30 nm in diameter into a 40–100 nm vesicle. Under mild conditions where the permeability enhancement (leakage of a trapped fluorescent dye, calcein) of lipid bilayers are observed (peptide to lipid ratios around 1/100), the fusion of the SUVs also occurs, although the fusion requires a somewhat larger amount of the peptide than the leakage does. Furthermore, at higher lipid concentrations, where the aggregation step is sufficiently rapid, the fusion rate is determined by the amount of the membrane bound peptide per lipid molecule, as is the leakage rate. In contrast, for egg PC large unilamellar vesicles (110 nm), hypelcin A induces the leakage, but not the fusion. We conclude that the leakage is not due to the fusion.     

Dynamics imaging of lipid phases and lipid-marker interactions in model biomembranes

Biomembranes are complex systems that regulate numerous biological processes. Lipid phases that constitute these membranes influence their properties and transport characteristics. Here, we demonstrate the potential of short-range dynamics imaging (excited-state lifetime, rotational diffusion, and order parameter) as a sensitive probe of lipid phases in giant unilamellar vesicles (GUVs). Liquid-disordered and gel phases were labeled with Bodipy-PC at room temperature. Two-photon fluorescence lifetime imaging microscopy of single-phase GUVs reveals more heterogeneity in fluorescence lifetimes of Bodipy in the gel phase (DPPC: 3.8+/-0.6 ns) as compared with the fluid phase (DOPC: 5.2+/-0.2 ns). The phase-specificity of excited-state lifetime of Bodipy-PC is attributed to the stacking of ordered lipid molecules that possibly enhances homo-FRET. Fluorescence polarization anisotropy imaging also reveals distinctive molecular order that is phase specific. The results are compared with DiI-C12-labeled fluid GUVs to investigate the sensitivity of our fluorescence dynamics assay to different lipid-marker interactions. Our results provide a molecular perspective of lipid phase dynamics and the nature of their microenvironments that will ultimately help our understanding of the structure-function relationship of biomembranes in vivo. Furthermore, these ultrafast excited-state dynamics will be used for molecular dynamics simulation of lipid-lipid, lipid-marker and lipid-protein interactions.     

Size, charge, and interactions with giant lipid vesicles of quantum dots coated with an amphiphilic macromolecule

Semiconductor colloidal quantum dots (QDs) are promising fluorescent probes for biology. Initially synthesized in organic solvents, they can be dispersed in aqueous solution by noncovalent coating with amphiphilic macromolecules, which renders the particles hydrophilic and modifies their interactions with other biological compounds. Here, after coating QDs with an alkyl-modified polyacrilic acid, we investigated their colloidal properties in aqueous buffers by electrophoresis, electron microscopy, light scattering, and rate zonal centrifugation. Despite polymer dispersity and variation of the size of the inorganic nanoparticles, the polymer-dot complexes appeared relatively well-defined in terms of hydrodynamic radius and surface charge. Our data show that these complexes contain isolated QD surrounded by a polymer layer with thickness 8-10 nm. We then analyzed their interaction with giant unilamellar vesicles, either neutral or cationic, by optical microscopy. At neutral pH, we found the absence of binding of the coated particles to lipid membrane, irrespective of their lipid composition. An abrupt surface aggregation of the nanoparticles on the lipid membrane was observed in a narrow pH range (6.0-6.2), indicative of critical binding triggered by polymer properties. The overall features of QDs coated with amphiphilic polymers open the route to using these nanoparticles in vivo as optically stable probes with switchable properties.     

Vesicles from docosahexaenoic acid

In dilute aqueous solution and at room temperature, cis-4,7,10,13,16,19-docosahexaenoic acid (DHA) self-assembles into vesicles (self-closed bilayers), if the molar ratio of the neutral form of DHA to anionic DHA is kept between 1:1 and 1:3 (corresponding to a bulk pH between 8.5 and 9.2 for a system with 10 mM DHA). By using polycarbonate membrane extrusion, stable unilamellar DHA vesicles with an average diameter of 80 nm can be prepared at pH 8.8. Cryo-transmission electron microscopy indicates that the width of the DHA bilayers in the vesicles is clearly below twice the length of an extended DHA molecule, indicating a high conformational flexibility of DHA within the vesicle bilayer. These DHA bilayers have a similar thickness like bilayers of vesicles prepared at pH 8.5 from oleic acid (cis-9-octadecenoic acid). Using calcein as fluorescent reference compound, it is shown that water-soluble molecules can be encapsulated inside DHA vesicles which may make them interesting for medical or food applications.     

Membrane fusion of giant unilamellar vesicles of neutral phospholipid membranes induced by La3+

Membrane fusions of vesicles of biomembranes play various important roles in cells, but their mechanisms are unclear and controversial. In the present study, we found that 30 microM to 1 mM La3+ induced membrane fusion of two giant unilamellar vesicles (GUVs) composed of a mixture of dioleoylphosphatidylcholine (DOPC) and dipalmitoleoylphosphatidylethanolamine (DPOPE). We succeeded in observing a process of this membrane fusion in detail. First, two GUVs became strongly associated, with a partition membrane between them composed of two bilayers, one from each GUV. Then, the partition membrane was suddenly broken at one site on its edge. The area of this breakage site gradually spread, until it was completely separated from the GUV to complete the membrane fusion. Here, we propose a new model (i.e., the partition breakage model) for the mechanism of La3+ -induced membrane fusion of GUVs.     

Multilamellar LipoCEST Agents Obtained from Osmotic Shrinkage of Paramagnetically Loaded Giant Unilamellar Vescicles (GUVs)

Moving from nano‐ to micro‐systems may not just be a matter of scale, but it might imply changes in the properties of the systems that can open new routes for the development of efficient MRI contrast agents. This is the case reported in the present paper, where giant liposomes (giant unilamellar vesicles, GUVs) loaded with Ln^III complexes have been studied as chemical exchange saturation transfer (CEST) MRI contrast agents. The comparison between nanosized liposomes (small unilamellar vesicles, SUVs) and GUVs sharing the same formulation led to differences that could not be accounted for only in terms of the increase in size (from 100–150 nm to 1–2 μm). Upon osmotic shrinkage, GUVs yielded a saturation‐transfer effect three order of magnitude higher than SUVs consistent with the increase in vesicles volume. Confocal microscopy showed that the shrinkage of GUVs resulted in multilamellar particles whereas SUVs are known to yield asymmetrical, discoidal shape.     

Tris(Znsalen) cryptand minimizes Znsalen aggregation arising from intermolecular Zn…O interaction

Exploring the factors to control Znsalen aggregation is of importance to design functional materials in catalysis, optical materials and biological imaging. In this work, we synthesized and characterized four cryptand type triZnsalen complexes and found that cryptand structure could efficiently minimize intermolecular Zn···O interaction. More importantly, encapsulated by PLGA nanoparticles, cryptand triZnsalen 1 displayed visible intracellular fluorescence whereas monomeric Znsalen 5 could not. These results provide a new access to design new luminescent materials with the potential application in optics and biological studies.     

Freezing or wrapping: the role of particle size in the mechanism of nanoparticle-biomembrane interaction

Understanding the interactions between nanoparticles (NPs) and biological matter is a high-priority research area because of the importance of elucidating the physical mechanisms underlying the interactions leading to NP potential toxicity as well as NP viability as therapeutic vectors in nanomedicine. Here, we use two model membrane systems, giant unilamellar vesicles (GUVs) and supported monolayers, to demonstrate the competition between adhesion and elastic energy at the nanobio interface, leading to different mechanisms of NP-membrane interaction relating to NP size. Small NPs (18 nm) cause a "freeze effect" of otherwise fluid phospholipids, significantly decreasing the phospholipid lateral mobility. The release of tension through stress-induced fracture mechanics results in a single microsize hole in the GUVs after interaction. Large particles (>78 nm) promote membrane wrapping, which leads to increased lipid lateral mobility and the eventual collapse of the vesicles. Electrochemical impedance spectroscopy on the supported monolayer model confirms that differently sized NPs interact differently with the phospholipids in close proximity to the electrode during the lipid desorption process. The time scale of these processes is in accordance with the proposed NP/GUV interaction mechanism.     

Biomembrane Interactions of Functionalized Cryptophane‐A: Combined Fluorescence and 129Xe NMR Studies of a Bimodal Contrast Agent

Fluorescent derivatives of the ¹²⁹Xe NMR contrast agent cryptophane‐A were obtained by functionalization with near infrared fluorescent dyes DY680 and DY682. The resulting conjugates were spectrally characterized, and their interaction with giant and large unilamellar vesicles of varying phospholipid composition was analyzed by fluorescence and NMR spectroscopy. In the latter, a chemical exchange saturation transfer with hyperpolarized ¹²⁹Xe (Hyper‐CEST) was used to obtain sufficient sensitivity. To determine the partitioning coefficients, we developed a method based on fluorescence resonance energy transfer from Nile Red to the membrane‐bound conjugates. This indicated that not only the hydrophobicity of the conjugates, but also the phospholipid composition, largely determines the membrane incorporation. Thereby, partitioning into the liquid‐crystalline phase of 1,2‐dipalmitoyl‐sn‐glycero‐3‐phosphocholine was most efficient. Fluorescence depth quenching and flip‐flop assays suggest a perpendicular orientation of the conjugates to the membrane surface with negligible transversal diffusion, and that the fluorescent dyes reside in the interfacial area. The results serve as a basis to differentiate biomembranes by analyzing the Hyper‐CEST signatures that are related to membrane fluidity, and pave the way for dissecting different contributions to the Hyper‐CEST signal.     

Wrapping of nanoparticles by membranes

How nanoparticles interact with biomembranes is central for understanding their bioactivity. Biomembranes wrap around nanoparticles if the adhesive interaction between the nanoparticles and membranes is sufficiently strong to compensate for the cost of membrane bending. In this article, we review recent results from theory and simulations that provide new insights on the interplay of bending and adhesion energies during the wrapping of nanoparticles by membranes. These results indicate that the interplay of bending and adhesion during wrapping is strongly affected by the interaction range of the particle–membrane adhesion potential, by the shape of the nanoparticles, and by shape changes of membrane vesicles during wrapping. The interaction range of the particle–membrane adhesion potential is crucial both for the wrapping process of single nanoparticles and the cooperative wrapping of nanoparticles by membrane tubules.