Rapid preparation of giant unilamellar vesicles (original) (raw)
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Chemistry & Biodiversity, 2012
Dedicated to Professor Dieter Seebach on the occasion of his 75th birthday The lipid-coated ice-droplet hydration method was applied for the preparation of milliliter volumes of a suspension of giant phospholipid vesicles containing in the inner aqueous vesicle pool in high yield either calcein, a-chymotrypsin, fluorescently labeled bovine serum albumin or dextran (FITC-BSA and FITC-dextran; FITC ΒΌ fluorescein isothiocyanate). The vesicles had an average diameter of ca. 7-11 mm and contained 20-50% of the desired molecules to be entrapped, the entrapment yield being dependent on the chemical structure of the entrapped molecules and on the details of the vesicle-formation procedure. The lipid-coated ice droplet hydration method is a multistep process, based on i) the initial formation of a monodisperse water-in-oil emulsion by microchannel emulsification, followed by ii) emulsion droplet freezing, and iii) surfactant and oil removal, and replacement with bilayer-forming lipids and an aqueous solution. If one aims at applying the method for the entrapment of enzymes, retention of catalytic activity is important to consider. With a-chymotrypsin as first model enzyme to be used with the method, it was shown that high retention of enzymatic activity is possible, and that the entrapped enzyme molecules were able to catalyze the hydrolysis of a membrane-permeable substrate which was added to the vesicles after their formation. Furthermore, one of the critical steps of the method that leads to significant release of the molecules from the water droplets was investigated and optimized by using calcein as fluorescent probe.
Biophysical Journal, 2007
In recent years, giant unilamellar vesicles (GUVs) have become objects of intense scrutiny by chemists, biologists, and physicists who are interested in the many aspects of biological membranes. In particular, this ''cell size'' model system allows direct visualization of particular membrane-related phenomena at the level of single vesicles using fluorescence microscopyrelated techniques. However, this model system lacks two relevant features with respect to biological membranes: 1), the conventional preparation of GUVs currently requires very low salt concentration, thus precluding experimentation under physiological conditions, and 2), the model system lacks membrane compositional asymmetry. Here we show for first time that GUVs can be prepared using a new protocol based on the electroformation method either from native membranes or organic lipid mixtures at physiological ionic strength. Additionally, for the GUVs composed of native membranes, we show that membrane proteins and glycosphingolipids preserve their natural orientation after electroformation. We anticipate our result to be important to revisit a vast variety of findings performed with GUVs under low-or no-salt conditions. These studies, which include results on artificial cell assembly, membrane mechanical properties, lipid domain formation, partition of membrane proteins into lipid domains, DNA-lipid interactions, and activity of interfacial enzymes, are likely to be affected by the amount of salt present in the solution.
Forming giant vesicles with controlled membrane composition, asymmetry, and contents
Proceedings of the National Academy of Sciences, 2011
Growing knowledge of the key molecular components involved in biological processes such as endocytosis, exocytosis, and motility has enabled direct testing of proposed mechanistic models by reconstitution. However, current techniques for building increasingly complex cellular structures and functions from purified components are limited in their ability to create conditions that emulate the physical and biochemical constraints of real cells. Here we present an integrated method for forming giant unilamellar vesicles with simultaneous control over (i) lipid composition and asymmetry, (ii) oriented membrane protein incorporation, and (iii) internal contents. As an application of this method, we constructed a synthetic system in which membrane proteins were delivered to the outside of giant vesicles, mimicking aspects of exocytosis. Using confocal fluorescence microscopy, we visualized small encapsulated vesicles docking and mixing membrane components with the giant vesicle membrane, resulting in exposure of previously encapsulated membrane proteins to the external environment. This method for creating giant vesicles can be used to test models of biological processes that depend on confined volume and complex membrane composition, and it may be useful in constructing functional systems for therapeutic and biomaterials applications.
Recent Biophysical Issues About the Preparation of Solute-Filled Lipid Vesicles
Mechanics of Advanced Materials and Structures, 2014
Here we report some recent biophysical issues on the preparation of solute-filled lipid vesicles and their relevance to the construction of "synthetic cells." First, we introduce the "semi-synthetic minimal cells" as the liposome-based cell-like systems, which contain a minimal number of biomolecules required to display simple and complex biological functions. Next, we focus on recent aspects related to the construction of synthetic cells. Emphasis is given to the interplay between the methods of synthetic cell preparation and the physics of solute encapsulation. We briefly introduce the notion of structural and compositional "diversity" in synthetic cell populations.
Characterization of giant vesicles formed by phase transfer processes
2009
Vesicles are of great interest as drug delivery system or models for cell membranes. For many applications, it is necessary to produce vesicles which are unilamellar, monodisperse, easy to adjust in size, and which can be filled with various types of active compounds. In a series of experiments, we produced giant vesicles with dimension of several millimeters by phase transfer processes. This new technique allowed synthesizing defined vesicles with lipid and surfactant membranes. The preparation of these aggregates occurred in two steps. First, we filled some amount of water into a cuvette and covered this liquid with an oil phase. Surfactants or lipids were solved either in the water or the oil phase. In the second step, a water droplet filled with methyl blue and saccharose was formed with a syringe in the oil phase. Due to density difference, the water droplet passed the plane oil/water interface and during this process it was transformed into a vesicle. The giant liposomes, thus formed, showed a high sensitivity against variations of the osmotic pressure, and their stability reached from seconds to hours. Due to the phase transfer process, the vesicle membranes often contained incorporated lenses of oil. If this hydrophobic liquid was released from the membrane, the vesicles decayed into smaller liposomes with a broad particle size distribution.
Interdigitation-fusion: a new method for producing lipid vesicles of high internal volume
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1994
Previously we demonstrated that fused phospholipid sheets can be formed from small unilamellar vesicles (SUVs) comprised of saturated symmetric chain lipids by exposing them to concentrations of ethanol sufficient to cause bilayer interdigitation Biochim. Biophys. Acta 1146, 247-257). Here we report that these sheets spontaneously form large, predominately unilamellar vesicles, when exposed to temperatures above their main phase transition temperature (Tm). These vesicles, termed interdigitation-fusion vesicles (IFVs), have mean diameters between 1 and 6/zm, and, once produced, are stable both above and below the T m of the lipid. The average captured volume of IFVs is dependent upon lipid chain length, the concentration of ethanol used to induce interdigitation-fusion, and size of the precursor liposomes. IFVs comprised of DPPC and DSPC had averaged captured volumes of 20-25 ~l//zmol lipid. IFVs produced from SUVs containing only DPPG or DPPC/DPPG mixtures had captured volumes equivalent to those made from pure DPPC SUVs indicating that charge can be introduced without consequence to the IFV process. Inclusion of cholesterol in precursor vesicles reduced IFV captured volume in a concentration dependent fashion by interfering with interdigitation. Cholesterol could be incorporated, however, into IFVs through admixture with the already formed phospholipid sheets producing far less comprise to captured volume. IFVs are useful as model systems or drug carriers, since their large internal volume allows for efficient encapsulation particularly with regard to compounds such as iodinated radiocontrast agents which otherwise interfere with vesicularization.