One Step Membrane Incorporation of Viral Antigens as a Vaccine Candidate Against HIV

Liposomes can been used as potential immunoadjuvants, because they have the ability to elicit both a cellular mediated immune response and a humoral immune response. Studies have shown liposomes to be effective immunopotentiators in hepatitis A and influenza vaccines. For all these purposes, liposomes can be prepared by different methods. After disperging suitable membrane lipids in an aqueous phase and spontaneous formation of multilamellar large vesicles (MLV), mechanical procedures such as ultrasonication, homogenization by a French press or by other high pressure devices and, or extrusion through polycarbonate membranes with defined pore sizes lead to a reduction in size and number of lamellae of the vesicles. A second group of preparation procedures uses suitable detergents, e.g., bile salts or alkylglycosides. A third group of procedures starts with dissolving the lipids in an organic solvent and mixing it with an aqueous phase. The concentration of the organic solvent is then reduced by suitable procedures.

Here we present a new technique for the preparation of liposomes with associated membrane proteins, where lipid vesicles are formed immediately after injection into a micellar protein solution. The model membrane protein used for these studies is a truncated recombinant gp41 produced in E. coli. This viral membrane antigen is a possible candidate protein for the establishment of HIV-vaccines.

The data presented here, show an efficient and reproducible one step membrane protein encapsulation procedure into liposomes in a closed and sterile containment. We examined encapsulation efficiency, membrane protein conformation and immunogenicity of this possible liposomal vaccine candidate, which can be produced in GMP-compliant quality with the described technique.     

Enhanced protein loading into liposomes by the multiple crossflow injection technique

Methods for encapsulation of a drug into liposomes should preferably result in a high encapsulation efficiency and a high encapsulation capacity. Our studies were focussed on the establishment of an efficient encapsulation procedure of the radical scavenging protein, rh-Cu/Zn-SOD, into liposomes with the cross flow injection method. Limitations to increase the encapsulation efficiency are caused by the enclosed aqueous volume, by the lipid concentration, the aspired vesicle size and the final ethanol concentration. Our research was performed to maximize the encapsulation following several strategies of injecting higher lipid concentrations into the aqueous phase. The one way triple technique, a sophisticated preparation procedure is presented, which enables three times higher encapsulation rates in comparison to standard procedures. Additionally, scalability studies demonstrate reproducibility independent of the preparation volume. Vesicle size distribution and encapsulation efficiency remain constant. Furthermore, special attention is paid on reproducibility of prepared liposomes, scale-up and on long term stability of the lipid vesicles.     

ENHANCED PROTEIN LOADING INTO LIPOSOMES BY THE MULTIPLE CROSSFLOW INJECTION TECHNIQUE

ABSTRACT

Methods for encapsulation of a drug into liposomes should preferably result in a high encapsulation efficiency and a high encapsulation capacity. Our studies were focussed on the establishment of an efficient encapsulation procedure of the radical scavenging protein, rh-Cu/Zn-SOD, into liposomes with the cross flow injection method. Limitations to increase the encapsulation efficiency are caused by the enclosed aqueous volume, by the lipid concentration, the aspired vesicle size and the final ethanol concentration. Our research was performed to maximize the encapsulation following several strategies of injecting higher lipid concentrations into the aqueous phase. The one way triple technique, a sophisticated preparation procedure is presented, which enables three times higher encapsulation rates in comparison to standard procedures. Additionally, scalability studies demonstrate reproducibility independent of the preparation volume. Vesicle size distribution and encapsulation efficiency remain constant. Furthermore, special attention is paid on reproducibility of prepared liposomes, scale-up and on long term stability of the lipid vesicles.     

Constant Pressure-controlled Extrusion Method for the Preparation of Nano-sized Lipid Vesicles

Liposomes are artificially prepared vesicles consisting of natural and synthetic phospholipids that are widely used as a cell membrane mimicking platform to study protein-protein and protein-lipid interactions³, monitor drug delivery^4,5, and encapsulation⁴. Phospholipids naturally create curved lipid bilayers, distinguishing itself from a micelle.⁶ Liposomes are traditionally classified by size and number of bilayers, i.e. large unilamellar vesicles (LUVs), small unilamellar vesicles (SUVs) and multilamellar vesicles (MLVs)⁷. In particular, the preparation of homogeneous liposomes of various sizes is important for studying membrane curvature that plays a vital role in cell signaling, endo- and exocytosis, membrane fusion, and protein trafficking⁸. Several groups analyze how proteins are used to modulate processes that involve membrane curvature and thus prepare liposomes of diameters <100 - 400 nm to study their behavior on cell functions³. Others focus on liposome-drug encapsulation, studying liposomes as vehicles to carry and deliver a drug of interest⁹. Drug encapsulation can be achieved as reported during liposome formation⁹. Our extrusion step should not affect the encapsulated drug for two reasons, i.e. (1) drug encapsulation should be achieved prior to this step and (2) liposomes should retain their natural biophysical stability, securely carrying the drug in the aqueous core. These research goals further suggest the need for an optimized method to design stable sub-micron lipid vesicles.Nonetheless, the current liposome preparation technologies (sonication¹⁰, freeze-and-thaw¹⁰, sedimentation) do not allow preparation of liposomes with highly curved surface (i.e. diameter <100 nm) with high consistency and efficiency^10,5, which limits the biophysical studies of an emerging field of membrane curvature sensing. Herein, we present a robust preparation method for a variety of biologically relevant liposomes.Manual extrusion using gas-tight syringes and polycarbonate membranes^10,5 is a common practice but heterogeneity is often observed when using pore sizes <100 nm due to due to variability of manual pressure applied. We employed a constant pressure-controlled extrusion apparatus to prepare synthetic liposomes whose diameters range between 30 and 400 nm. Dynamic light scattering (DLS)¹⁰, electron microscopy¹¹ and nanoparticle tracking analysis (NTA)¹² were used to quantify the liposome sizes as described in our protocol, with commercial polystyrene (PS) beads used as a calibration standard. A near linear correlation was observed between the employed pore sizes and the experimentally determined liposomes, indicating high fidelity of our pressure-controlled liposome preparation method. Further, we have shown that this lipid vesicle preparation method is generally applicable, independent of various liposome sizes. Lastly, we have also demonstrated in a time course study that these prepared liposomes were stable for up to 16 hours. A representative nano-sized liposome preparation protocol is demonstrated below.     

Carrier and adjuvant properties of liposome-borne tumor-specific antigens

Summary The purpose of this study was to examine (1) the association of tumor extract proteins with phospholipid vesicles of varying physiochemical properties, and (2) the adjuvant and carrier properties of liposome-borne tumor antigens in the in vivo induction of an antitumor immune response. Cell surface antigens of the 3-methylcholanthrene-induced fibrosarcoma of C3H/HeJ mice, MCA-F, were extracted using 2.5% 1-butanol. Crude and electrofocused antigen preparations capable of eliciting a protective antitumor immune response were used to prepare liposome vaccines. The incorporation of extract proteins into liposomes formed by butanol dialysis (BVD) was three- to five-fold greater than the encapsulation of protein into the aqueous compartment of multilamellar vesicles (MLV). The electrochemical properties of the BDV had a significant effect on the induction of an antitumor response: Antigens borne on negatively charged, but not uncharged, liposomes were effective in protecting hosts against supralethal tumor challenge, and displayed a specific activity 20- to 50-fold greater than soluble antigen. Antigens carried by MLV were not effective in generating an immunoprotective response. The lipophilic characteristics of butanol-extracted antigens allowed (1) the passive adsorption of immunoprotective tumor antigen onto the surface of preformed vesicles, and (2) adsorption of MCA-F antigen onto the surface of an antigenically distinct tumor MCA-D. In the latter experiment, adsorption of MCA-F-specific antigen onto MCA-D cells resulted in a change in the membrane antigen phenotype as measured by indirect immunofluorescence. Although butanol released a lipophilic moiety from cells which spontaneously reassociated with phospholipid bilayers, no evidence for a lipoidal antigen was obtained when tumor-derived lipids were used as immunogens. This study demonstrates that butanol-extracted tumor antigen is lipophilic without being a lipid, and that negatively charged liposomes can be effective as carriers and adjuvants for tumor antigens in the induction of an antitumor immune response. Abbreviations used: BDV, butanol dialysis vesicles; CBE-F, crude butanol extract from the MCA-F tumor; Chol, cholesterol; FI, fluorescense index; PBS, Dulbecco's calcium- and magnesium-free phosphate-buffered saline, pH 7.4; PC, phosphatidylcholine; PG, phosphatidylglycerol; pIEF, preparative isoelectric focusing; MLV, multilamellar vesicles; REV, reverse-evaporation vesicles     

Functional reconstitution of membrane proteins in monolayer liposomes from bipolar lipids of Sulfolobus acidocaldarius.

Membranes of Sulfolobus acidocaldarius, an extreme thermophilic archaebacterium, are composed of unusual bipolar lipids. They consist of macrocyclic tetraethers with two polar heads linked by two hydrophobic C40 phytanyl chains which are thought to be arranged as a monolayer in the cytoplasmic membrane. Fractionation of a total lipid-extract from S. acidocaldarius yielded a lipid fraction which forms closed and stable unilamellar liposomes in aqueous media. Beef heart cytochrome c-oxidase could be functionally reconstituted in these liposomes. In the presence of reduced cytochrome c, a protonmotive force (delta p) across the liposomal membrane was generated of up to -92 mV. Upon fusion of these proteoliposomes with membrane vesicles of Lactococcus lactis, the delta p generated by cytochrome c-oxidase activity was capable to drive uphill transport of leucine. Electron microscopic analysis indicated that the tetraether lipids form a single monolayer liposome. The results demonstrate that tetraether lipids of archaebacteria can form a suitable matrix for the function of exogenous membrane proteins originating from a regular lipid bilayer.     

Large-scale preparation of clove essential oil and eugenol-loaded liposomes using a membrane contactor and a pilot plant

Based on our previous study where optimal conditions were defined to encapsulate clove essential oil (CEO) into liposomes at laboratory scale, we scaled-up the preparation of CEO and eugenol (Eug)-loaded liposomes using a membrane contactor (600?mL) and a pilot plant (3?L) based on the principle of ethanol injection method, both equipped with a Shirasu Porous Glass membrane for injection of the organic phase into the aqueous phase. Homogenous, stable, nanometric-sized and multilamellar liposomes with high phospholipid, Eug loading rates and encapsulation efficiency of CEO components were obtained. Saturation of phospholipids and drug concentration in the organic phase may control the liposome stability. Liposomes loaded with other hydrophobic volatile compounds could be prepared at large scale using the ethanol injection method and a membrane for injection.     

Rapid and efficient method for the size separation of homogeneous fluorescein-encapsulating liposomes

Liposomes are colloidal structures formed by the self-assembly of lipid molecules in solution into spherical, self-closed structures through their amphiphilic properties. All liposome preparation protocols reported consist of several steps of preparation, homogenization, and purification, which are labor-intensive, arduous, and lengthy to execute. In this work, a new procedure has been developed to reduce the time of the postrehydration sizing of liposomes from multilamellar vesicles, while improving the uniformity of the resulting liposomes produced and achieving high encapsulation efficiencies. For the homogenization step, the typically used method of filter extrusion was substituted by centrifugation. Purification of liposomes to eliminate nonencapsulated molecules and lipids is routinely carried out via gel permeation chromatography, an extremely lengthy procedure, and in the method we report, this lengthy step was replaced by the use of molecular-weight cut-off filters. Using this novel method, large unilamellar vesicles were produced and the time required, postrehydration, was dramatically reduced from almost 48 to less than 2 hours, with a highly uniformly sized population of liposomes being produced—the homogeneity of the liposome population achieved using our method was 99%, as compared to 88% attained by using the traditional method of production. We have used this approach to encapsulate fluorescein isothiocyanate (FITC), and 160,000 FITC molecules were encapsulated and the liposomes were demonstrated to be stable for at least 10 weeks at 4°C.     

Essential oils encapsulated in liposomes: a review

Abstract

In the recent years there has been an increased interest toward the biological activities of essential oils. However, essential oils are unstable and susceptible to degradation in the presence of oxygen, light and temperature. So, attempts have been made to preserve them through encapsulation in various colloidal systems such as microcapsules, microspheres, nanoemulsions and liposomes. This review focuses specifically on encapsulation of essential oils into liposomes. First, we present the techniques used to prepare liposomes encapsulating essential oils. The effects of essential oils and other factors on liposome characteristics such as size, encapsulation efficiency and thermal behavior of lipid bilayers are then discussed. The composition of lipid vesicles membrane, especially the type of phospholipids, cholesterol content, the molar ratio of essential oils to lipids, the preparation method and the kind of essential oil may affect the liposome size and the encapsulation efficiency. Several essential oils can decrease the size of liposomes, homogenize the liposomal dispersions, increase the fluidity and reduce the oxidation of the lipid bilayer. Moreover, liposomes can protect the fluidity of essential oils and are stable at 4–5?°C for 6 months at least. The applications of liposomes incorporating essential oils are also summarized in this review. Liposomes encapsulating essential oils are promising agents that can be used to increase the anti-microbial activity of the essential oils, to study the effect of essential oils on cell membranes, and to provide alternative therapeutic agents to treat several diseases.     

Manufacture of liposomes by isopropanol injection: characterization of the method

Unilamellar liposomes are conventionally prepared by rapid injection of an ethanolic solution of lipids into an aqueous medium. The aim of the present study was to control, more efficiently, vesicle diameter by using an alternative solvent. The results show that isopropanol injection is a good alternative to ethanol injection for the manufacture of liposomes. Particle size can be controlled by the variation of process parameters, such as stirring speed of the aqueous phase and injection flow rate of lipid-isopropanol solution. Diameter of vesicles obtained by this method is less affected by the nature of phospholipid, as well as lipid concentration, than in the ethanol-injection process. In addition, the vesicles are generally smaller (approximately 40-210?nm). Accurate characterization of the particles, by fluorescence, (31)P-NMR, and cryo-transmission electron microscopy, showed that particles are formed of a single lipid bilayer around an aqueous cavity. We thus provide the scientific community with a fully characterized alternative method to produce unilamellar vesicles.     

Enveloped viruses as model membrane systems: microviscosity of vesicular stomatitis virus and host cell membranes.

The fluorescence probe 1,6-diphenyl-1,3,5-hexatriene was used to study and compare the dynamic properties of the hydrophobic region of vesicular stomatitis virus grown on L-929 cells, plasma membrane of L-929 cells prepared by two different methods, liposomes prepared from virus lipids and plasma membrane lipids, and intact L-929 cells. The rate of penetration of the probe into the hydrophobic region of the lipid bilayer was found to be much faster in the lipid vesicle bilayer as compared with the intact membrane, but in all cases the fluorescence anisotropy was constant with time. The L-cell plasma membranes, the vesicles prepared from the lipids derived from the plasma membranes, and intact cells are found to have much lower microviscosity values than the virus or virus lipid vesicles throughout a wide range of temperatures. The microviscosity of plasma membrane and plasma membrane lipid vesicles was found to depend on the procedure for plasma membrane preparation as the membranes prepared by different methods had different microviscosities. The intact virus and liposomes prepared from the virus lipids were found to have very similar microviscosity values. Plasma membrane and liposomes prepared from plasma membrane lipids also had similar microviscosity values. Factors affecting microviscosity in natural membranes and artificially mixed lipid membranes are discussed.     

Preparation of liposomes of defined size distribution by extrusion through polycarbonate membranes

Liposomes of defined size and homogeneity have been prepared by sequential extrusion of the usual multilamellar vesicles through polycarbonate membranes. The process is easy, reproducible, produces no detectable degradation of the phospholipids, and can double the encapsulation efficiency of the liposome preparation. Multilamellar vesicles extruded by this technique are shown by both negative stain and freeze-fracture electron microscopy to have mean diameters approaching the pore diameter of the polycarbonate membrane through which they were extruded. When sequentially extruded down through a 0.2 μm membrane, the resulting vesicles exhibit a very homogeneous size distribution with a mean diameter of 0.27 μm while maintaining an acceptable level of encapsulation of the aqueous phase.     

Preparation of submicron unilamellar liposomes by freeze-drying double emulsions

A novel method is described for the preparation of sterile submicron unilamellar liposomes. The method is based on the lyophilization of double emulsions containing disaccharides as lyoprotectants in both the inner and outer aqueous phase. Using various phospholipids or mixtures of lipids as emulsifiers, the double emulsions can be prepared by a two-step emulsification, including hydrophilic agents in the inner aqueous phase or lipophilic agents in the oil phase. Then, the double emulsions are lyophilized after sterilization by passing them through a 0.22-μm pore filter. Rehydration of the lyophilized products results in liposomes with a relatively high encapsulation efficiency (for calcein, 87%; 5-fluorouracil, 19%; flurbiprofen, 93%) and a size below 200 nm measured by the dynamic light scattering technique (DLS) and the atomic force microscopy (AFM). The liposomes were found to be unilamellar from freeze-fracture electron micrographs and X-ray diffraction patterns. In addition, the liposomes can be reconstituted just before use by rehydration of the lyophilized products which are relatively stable. Thus, this reproducible and simple technique can be used to prepare sterilized, submicron unilamellar liposomes with a relatively high encapsulation efficiency, and excellent stability during long-term storage.     

Interaction between corneocytes and stratum corneum lipid liposomes in vitro

Small unilamellar vesicles were made from a mixture of epidermal ceramides (45%), cholesterol (35%), free fatty acids (15%) and cholesteryl sulfate (5%). Isolated corneocytes prepared from pig epidermis were added to the liposomes and the interaction between corneocytes and liposomes was studied by (1) thin-section electron microscopy and (2) monitoring the release of aqueous contents of the vesicles by following the fluorescence intensity of carboxyfluorescein entrapped in the vesicles. The vesicles adsorbed readily onto the corneocytes and slowly transformed into lamellar sheets. Enhanced fluorescence intensity indicated a corneocyte-induced membrane fusion process that resulted in the release of aqueous contents of the vesicles. The results suggest a cohesive role for the corneocyte cell envelope, which consists of a monomolecular layer of lipids covalently bound to the outside of a cross-linked protein envelope. This may be one of the major factors in the reassembly of extruded membranous disks into lamellar sheets which occurs during the final stages of epidermal differentiation.     

Manufacture of liposomes by isopropanol injection: characterization of the method

Unilamellar liposomes are conventionally prepared by rapid injection of an ethanolic solution of lipids into an aqueous medium. The aim of the present study was to control, more efficiently, vesicle diameter by using an alternative solvent. The results show that isopropanol injection is a good alternative to ethanol injection for the manufacture of liposomes. Particle size can be controlled by the variation of process parameters, such as stirring speed of the aqueous phase and injection flow rate of lipid-isopropanol solution. Diameter of vesicles obtained by this method is less affected by the nature of phospholipid, as well as lipid concentration, than in the ethanol-injection process. In addition, the vesicles are generally smaller (approximately 40–210?nm). Accurate characterization of the particles, by fluorescence, ³¹P-NMR, and cryo–transmission electron microscopy, showed that particles are formed of a single lipid bilayer around an aqueous cavity. We thus provide the scientific community with a fully characterized alternative method to produce unilamellar vesicles.     

Preparation of submicron unilamellar liposomes by freeze-drying double emulsions

A novel method is described for the preparation of sterile submicron unilamellar liposomes. The method is based on the lyophilization of double emulsions containing disaccharides as lyoprotectants in both the inner and outer aqueous phase. Using various phospholipids or mixtures of lipids as emulsifiers, the double emulsions can be prepared by a two-step emulsification, including hydrophilic agents in the inner aqueous phase or lipophilic agents in the oil phase. Then, the double emulsions are lyophilized after sterilization by passing them through a 0.22-microm pore filter. Rehydration of the lyophilized products results in liposomes with a relatively high encapsulation efficiency (for calcein, 87%; 5-fluorouracil, 19%; flurbiprofen, 93%) and a size below 200 nm measured by the dynamic light scattering technique (DLS) and the atomic force microscopy (AFM). The liposomes were found to be unilamellar from freeze-fracture electron micrographs and X-ray diffraction patterns. In addition, the liposomes can be reconstituted just before use by rehydration of the lyophilized products which are relatively stable. Thus, this reproducible and simple technique can be used to prepare sterilized, submicron unilamellar liposomes with a relatively high encapsulation efficiency, and excellent stability during long-term storage.     

Liposomes containing colloidal gold are a useful probe of liposome-cell interactions

A method is described for the preparation of liposomes containing colloidal gold as an electron-dense marker to trace liposome-cell interactions. Since gold sols would precipitate at the high concentrations necessary for loading a large proportion of liposomes, gold sols were formed within preformed liposomes which had encapsulated gold chloride. The optimal conditions for encapsulating the marker were ascertained for liposomes prepared by the method of reverse-phase evaporation. Gold sols formed rapidly at ambient temperature and without organic solvent, and produced homogeneous populations of gold granules inside liposomes. Most vesicles contained the marker, allowing us to determine unambiguously the intracellular fate of liposomes and their contents. The in vitro experiments showed that gold-liposomes were internalized by African green monkey kidney cells in a manner similar to receptor-mediated endocytosis of well-characterized ligands. Preliminary in vivo studies also indicated that liposomes were endocytosed by Kupffer cells via the coated vesicle pathway.     

Generation of multilamellar and unilamellar phospholipid vesicles

Multilamellar and unilamellar vesicles can be generated by a variety of techniques which lead to systems with differing lamellarity, size, trapped volume and solute distribution. The straight-forward hydration of lipid to produce multilamellar vesicles (MLVs) results in systems which exhibit low trapped volumes and where solutes contained in the aqueous buffer are partially excluded from the MLV interior. Large trapped volumes and equilibrium solute distributions can be achieved by freeze-thawing or by ‘reverse phase’ procedures where the lipid is hydrated after being solubilized in organic solvent. Unilamellar vesicles can be produced directly from MLVs by extrusion or sonication or, alternatively, can be obtained by reverse phase or detergent removal procedures. The advantages and limitations of these techniques are discussed.     

A lipid based depot (DepoFoam technology) for sustained release drug delivery

Encapsulation of drugs into multivesicular liposomes (DepoFoam) offers a novel approach to sustained-release drug delivery. While encapsulation of drugs into unilamellar and multilamellar liposomes, and complexation of drugs with lipids, resulted in products with better performance over a period lasting several hours to a few days after intravascular administration, DepoFoam-encapsulation has been shown to result in sustained-release lasting over several days to weeks after non-vascular administration. The routes of administration most viable for delivery of drugs via DepoFoam formulations include intrathecal, epidural, subcutaneous, intramuscular, intra-atricular, and intraocular. DepoFoam particles are distinguished structurally from unilamellar vesicles, multilamellar vesicles, and neosomes in that each particle comprises a set of closely packed non-concentric vesicles. The particles are tens of microns in diameter and have large trapped volume, thereby affording delivery of large quantities of drugs in the encapsulated form in a small volume of injection. A number of methods based on a manipulation of the lipid and aqueous composition can be used to control the rate of sustained-release from a few days to several weeks.     

Pediocin PA-1, a bacteriocin from Pediococcus acidilactici PAC1.0, forms hydrophilic pores in the cytoplasmic membrane of target cells.

Pediocin PA-1 is a bacteriocin which is produced by Pediococcus acidilactici PAC1.0. We demonstrate that pediocin PA-1 kills sensitive Pediococcus cells and acts on the cytoplasmic membrane. In contrast to its lack of impact on immune cells, pediocin PA-1 dissipates the transmembrane electrical potential and inhibits amino acid transport in sensitive cells. Pediocin interferes with the uptake of amino acids by cytoplasmic membrane vesicles derived from sensitive cells, while it is less effective with membranes derived from immune cells. In liposomes fused with membrane vesicles derived from both sensitive and immune cells, pediocin PA-1 elicits an efflux of small ions and, at higher concentrations, an efflux of molecules having molecular weights of up to 9,400. Our data suggest that pediocin PA-1 functions in a voltage-independent manner but requires a specific protein in the target membrane.