Cationic liposomes produced via ethanol injection method for dendritic cell therapy

Cationic liposomes can be designed and developed in order to be an efficient gene delivery system for mammalian cells. Dendritic cell (DC) vaccines can be used to treat cancer, as cationic liposomes can deliver tumor antigens to cells while cells remain active. However, most methods used for liposome production are not able to reproduce in large scale the physicochemical and biological properties of liposomes produced in laboratory scale. In this context, ethanol injection method achieved promising results, although requiring post-treatment for size reduction and/or to remove residual ethanol. Thus, the purpose of this study was to generate cationic liposomes suitable for gene therapies via ethanol injection method in only one step (VEI) and compared to those submitted to a size reduction processes by microfluidization (MFV). For this, the method to produce cationic liposomes composed of egg phosphatidylcholine (EPC), 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) and 1,2-dioleoylphosphatidylethanolamine (DOPE) was optimized using a statistical design approach. As a result, the size of VEI decreased from 290?nm to 110?nm and the polydispersity from 0.54 to 0.17. In the case of MFV, size decreased from 128?nm to 107?nm and polydispersity from 0.40 to 0.18. ST and MFV before and after optimization were also characterized in terms of morphology by transmission electron microscopy (TEM) and structure by differential scanning calorimetry (DSC). Finally, to show their potential in gene/immune therapies applications, DCs were stimulated by such liposomes. Cells internalized liposomes, increasing expression of the costimulatory molecule CD86 and inducing T lymphocyte proliferation.     

Modified and derived ethanol injection toward liposomes: development of the process

A modified and derived ethanol injection (MDEI) process was developed to produce liposomes. The aim of the present study was to more efficiently control the vesicle diameter than with the conventional ethanol injection method. A hot ethanolic solution of lipids (60°C) was injected into a hot aqueous buffer (70°C). Then, ethanol was removed by rotary evaporation under reduced pressure. The size of the liposomes could be controlled by the ratio of ethanol to hydroalcoholic solution before evaporation. The concentration of lipids, the charge of lipids, and the type of aqueous phase had little effect on the vesicle diameter when the process involved a ratio of 33% (v/v) ethanol. In addition, it was possible to obtain lipid concentrations 10- to 30-fold higher that the conventional ethanol injection method. The encapsulation of a hydrophilic compound was feasible with this MDEI process. The observation by cryogenic transmission electron microscopy revealed that these liposomes were predominantly unilamellar at a ratio as high as 33 or 50% (v/v) ethanol. Thus, the results showed that MDEI is an appropriate alternative for the manufacture of liposomes with respect to the ethanol injection process.     

CT imaging with iopromide liposomes in a rabbit model

The aim of this study was to evaluate the computed tomography (CT)-imaging potential of iopromide-carrying liposomes (SPC/CH/SPG, 6:3:1) of approximately 200?nm in diameter in healthy rabbits and in rabbits with implanted liver tumors in an intraindividual comparison with iopromide. Normal rabbits and animals with VX2 tumors implanted into the liver received iopromide (600?mg of iodine/kg, bolus injection) and, 1 or 2 days later, iopromide liposomes (300?mg of iodine/kg, bolus injection or 10-minute infusion). CT imaging up to 1 hour after administration was performed, focusing on the aorta, vena cava, kidney, spleen, and liver. Pharmacokinetic parameters for CT enhancement were calculated. Detectability and delineation of liver lesions were assessed on a 4-grade scale, and differences were evaluated statistically. Using half the iodine dose, iopromide liposomes achieved similar blood-pool enhancement as iopromide. Detectability and delineation of liver lesions were easy/good in the arterial phase after iopromide injection, but poor in the venous and equilibration phases. Iopromide liposomes resulted in a long-lasting, good detectability and delineation of liver lesions similar or superior to that observed after iopromide in the arterial phase.     

Kinetic and equilibrium studies of bile salt–liposome interactions

Abstract

Research has suggested that exposure to sub-micellar concentrations of bile salts (BS) increases the permeability of lipid bilayers in a time-dependent manner. In this study, incubation of soy phosphatidylcholine small unilamellar vesicles (liposomes) with sub-micellar concentrations of cholate (C), deoxycholate (DC), 12-monoketocholate (MKC) or taurocholate (TC) in pH 7.2 buffer increased membrane fluidity and negative zeta potential in the order of increasing BS liposome-pH 7.2 buffer distribution coefficients (MKC?<?C?≈?TC?<?DC). In liposomes labeled with the dithionite-sensitive fluorescent lipid N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)phosphatidylethanolamine (NBD-PE) in both leaflets and equilibrated with sub-micellar concentrations of BS, fluorescence decline during continuous exposure to dithionite was biphasic involving a rapid initial phase followed by a slower second phase. Membrane permeability to dithionite as measured by the rate of the second phase increased in the order control?<?MKC?<?TC?～?C?<?DC. In liposomes labeled with NBD-PE in the inner leaflet only and incubated with the same concentrations of C, DC and MKC, membrane permeability to dithionite initially increased very rapidly in the order MKC?<?C?<?DC before impermeability to dithionite was restored after which fluorescence decline was consistent with NBD-PE flip-flop. For liposomes incubated with TC, membrane permeability to dithionite was only slightly increased and the decline in fluorescence was mainly the result of NBD-PE flip-flop. These results provide evidence that BS interact with lipid bilayers in a time-dependent manner that is different for conjugated and unconjugated BS. MKC appears to cause least disturbance to liposomal membranes but, when the actual MKC concentration in liposomes is taken into account, MKC is actually the most disruptive.     

Enhancement of apparent substrate selectivity of proteinase K encapsulated in liposomes through a cholate-induced alteration of the bilayer permeability

Proteinase K-containing liposomes with highly selective membrane permeability properties were prepared. The selectivity obtained was with respect to the two substrate molecules added to the external aqueous phase of the liposomes: acetyl-L-Ala-Ala-Ala-p-nitroanilide (Ac-AAA-pNA) and succinyl-L-Ala-Ala-Ala-p-nitroanilide (Suc-AAA-pNA). The liposome-forming lipid used was POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) and modulation of the membrane permeability was achieved using the detergent cholate. Proteinase K-containing mixed liposomes (PKCL) were prepared by adding cholate to preformed proteinase K-containing POPC liposomes (PKL) at a defined effective cholate/POPC molar ratio in the liposomal bilayer membrane R(e). Proteinase K was kept inside PKCL with a negligible amount of leakage into the bulk aqueous phase at R(e) < or = 0.30. At higher R(e), leakage of proteinase K was pronounced, even under conditions where POPC/cholate mixed liposomes seemed to be still intact (0.30 < R(e) < or = 0.39). At R(e) < or = 0.30, the reactivity of proteinase K in the PKCL measured with the externally added substrate Ac-AAA-pNA increased with increasing R(e), while the reactivity measured with Suc-AAA-pNA remained low, regardless of the R(e) value. This showed that externally added Ac-AAA-pNA molecules permeated the liposomal membrane more easily than Suc-AAA-pNA by modulating the membrane with cholate. Consequently, Ac-AAA-pNA was hydrolyzed in PKCL with considerably higher apparent substrate selectivity in comparison with the cases of proteinase K in PKL and free proteinase K (without liposomal encapsulation). The results obtained clearly demonstrate that the prepared PKCL can be utilized as a kind of nano-scaled bioreactor system which can take up a particular target substrate with high apparent substrate selectively from the external phase of the liposomes. Inside the liposomes, the target substrate is then converted into the corresponding products.     

A new method for liposome preparation using a membrane contactor

In this article, we present a novel, scalable liposomal preparation technique suitable for the entrapment of pharmaceutical agents into liposomes. This new method is based on the ethanol-injection technique and uses a membrane contactor module, specifically designed for colloidal system preparation. In order to investigate the process, the influence of key parameters on liposome characteristics was studied. It has been established that vesicle-size distribution decreased with a decrease of the organic-phase pressure, an increase of the aqueous-phase flow rate, and a decrease of the phospholipid concentration. Additionally, special attention was paid on reproducibility and long-term stability of lipid vesicles, confirming the robustness of the membrane contactor-based technique. On the other hand, drug-loaded liposomes were prepared and filled with two hydrophobic drug models. High entrapment-efficiency values were successfully achieved for indomethacin (63%) and beclomethasone dipropionate (98%). Transmission electron microscopy images revealed nanometric quasispherical-shaped multilamellar vesicles (size ranging from 50 to 160?nm).     

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.     

MODIFIED ETHANOL INJECTION METHOD FOR LIPOSOMES CONTAINING β-SITOSTEROL β-D-GLUCOSIDE

A modified ethanol injection method for liposomes containing soybean phosphatidylcholine (SPC), cholesterol (Ch), β-sitosterol β-D-glucoside (Sit-G) and oleic acid (OA) was developed, that can produce homogeneous unilamellar liposomes without the use of sonication and dialysis. In this method, water is poured into a concentrated lipid-ethanol solution and then ethanol is removed in an evaporator. Dilution with water causes spontaneous formation of small and homogenous unilamellar vesicles from micellar aggregate. The size of liposomes can be controlled by the ratio of ethanol to water. OA and Sit-G were distributed at the surface of liposomes and were recognized by Concanavalin A, respectively. This easy and quick method for preparation of liposomes may be applicable in many areas.     

Ethanol injection method for hydrophilic and lipophilic drug-loaded liposome preparation

In this article, a hydrophobic (beclomethasone dipropionate; BDP) and a hydrophilic (cytarabine; Ara-C) drugs have been encapsulated in liposomes in order to be administered via the pulmonary route. For this aim, a liposome preparation method, which is easy to scale up, the ethanol injection method, has been selected. The effects of critical process and formulation parameters have been investigated. The drug-loaded liposomes were prepared and characterized in terms of size, zeta potential, encapsulation efficiency, release study, cell uptake, and aerodynamic behavior. Small multilamellar vesicles, with sizes ranging from about 80 to 170?nm, were successfully obtained. Results indicated a significant influence of phospholipid and cholesterol amounts on liposome size and encapsulation efficiency. The higher encapsulation efficiencies were about 100% for the hydrophobic drug (BDP) and about 16% for the hydrophilic one (Ara-C). The in vitro release study showed a prolonged release profile for BDP, in contrast with Ara-C, which was released more rapidly. The cell-uptake test revealed that fluorescent liposomes have been well internalized into the cytoplasm of SW-1573 human lung carcinoma cells, confirming the possibility to use liposomes for lung cell targeting. Nebulized Ara-C and BDP liposomes presented aerodynamic diameters compatible with deep lung deposition. In conclusion, the elaborated liposomes seem to be promising carriers for both Ara-C and BDP pulmonary delivery.     

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.     

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.     

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.     

Expression of hepatitis B virus surface antigen in adult rat liver. Co-introduction of DNA and nuclear protein by a simplified liposome method.

We established a simple and efficient method for gene transfer in vitro (to cultured cells) and in vivo (to an adult organ) using liposomes. Plasmid DNA and proteins were efficiently co-encapsulated in liposomes by agitation and sonication, and were co-introduced into cells by hemagglutinating virus of Japan (HVJ)-mediated membrane fusion. Introduction of the Escherichia coli beta-galactosidase gene with non-histone chromosomal protein high mobility group 1 (HMG1) into LLCMK2 cells resulted in about 3 times higher beta-galactosidase activity than that on introduction of the gene alone. Two days after injection of HVJ-liposomes containing the beta-galactosidase gene and HMG1 under the perisplanchnic membrane of adult rat liver, hepatic cells near the injection site were found by 5-bromo-4-chloro-3-indolyl beta-D-galactoside staining to have beta-galactosidase activity. After similar injection of HVJ-liposomes containing the hepatitis B virus surface antigen (HBsAg) gene and HMG1, HBsAg was detected in the serum for 9 days with a maximum of 25-45 ng/ml on day 2 after the injection.     

Characterization of a liposome-based artificial skin membrane for in vitro permeation studies using Franz diffusion cell device

A prerequisite for successful transdermal or dermal drug therapy is the drug ability to penetration through the skin, especially stratum corneum (SC). The most acceptable technique for measuring skin permeation in vitro is the application of both the Franz diffusion cell device and the skin model. In the skin model, a liposome-based artificial skin membrane (LASM) consisting of tight layers of liposomes immobilized on a filter was prepared and characterized. Using porcine ear skin, rat skin and Strat-M? artificial membrane as control, the LASM was then evaluated in permeation studies with five active compounds: ferulic acid, paeoniflorin, albiflorin, tetrahydrocolumbamine, and tetrahydropalmatine. The scanning electron microscope images demonstrated complete filling of the membrane pores with lipids and the formation of a continuous liposomal coating. The contents of egg phosphatidylcholine (EPC) and cholesterol in LASM were measured to be 12.08?±?0.18 and 4.41?±?0.04?mg/cm², respectively. Moreover, revealed by the measurement of electrical resistance, the LASM remains intact for at least 12?h with the incubation of 20% ethanol. The results of permeation studies demonstrated a good correlation (r^2?=?0.9743, r?=?0.9871) of P_app values between the drugs’ permeation through LASM and porcine ear skin. In addition, by ATR-FTIR analysis, a slighter shift of CH₂ stretching frequency between LASM and porcine ear skin was observed compared with the shift between Strat-M? membrane and porcine ear skin. In summary, for the first time, the LASM has been proved to be a valuable alternative to porcine ear skin in permeation studies using Franz diffusion cell device.     

GMP Production of Liposomes—A New Industrial Approach

A new scalable liposome production system is presented, which is based on the ethanol injection technique. The system permits liposome manufacture regardless of production scale, as scale is determined only by free disposable vessel volumes. Once the parameters are defined, an easy scale up can be performed by just changing the process vessels. These vessels are fully sterilizeable and all raw materials are transferred into the sanitized and sterilized system via 0.2 μm filters to guarantee an aseptic production.

Liposome size can be controlled by the local lipid concentration at the injection point depending on process parameters like injection pressure, lipid concentration and injection rate. These defined process parameters are furthermore responsible for highly reproducible results with respect to vesicle diameters and encapsulation rates Compared to other technologies like the film method which is normally followed by size reduction through high pressure homogenization, ultrasonication or extrusion, no mechanical forces are needed to generate homogeneous and narrow distributed liposomes.

Another important advantage of this method is the suitability for the entrapment of many different drug substances such as large hydrophilic proteins by passive encapsulation, small amphiphilic drugs by a one step remote loading technique or membrane association of antigens for vaccination approaches     

Formation and maintenance of tubular membrane projections require mechanical force, but their elongation and shortening do not require additional force

Living cells develop their own characteristic shapes depending on their physiological functions, and their morphologies are based on the mechanical characteristics of the cytoskeleton and of membranes. To investigate the role of lipid membranes in morphogenesis, we constructed a simple system that can manipulate liposomes and measure the forces required to transform their shapes. Two polystyrene beads (1 microm in diameter) were encapsulated in giant liposomes and were manipulated using double-beam laser tweezers. Without any specific interaction between the lipid membrane and beads, mechanical forces could be applied to the liposome membrane from the inside. Spherical liposomes transformed into a lemon shape with increasing tension, and tubular membrane projections were subsequently generated in the tips at either end. This process is similar to the liposomal transformation caused by elongation of encapsulated cytoskeletons. In the elongation stage of lemon-shaped liposomes, the force required for the transformation became larger as the end-to-end length increased. Just before the tubular membrane was generated, the force reached the maximum strength (approximately 11 pN). However, immediately after the tubular membrane developed, the force suddenly decreased and was maintained at a constant strength (approximately 4 pN) that was independent of further tube elongation or shortening, even though there was no excess membrane reservoir as occurs in living cells. When the tube length was shortened to approximately 2 microm, the liposome reversed to a lemon shape and the force temporarily increased (to approximately 7 pN). These results indicate that the simple application of mechanical force is sufficient to form a protrusion in a membrane, that a critical force and length is needed to form and to maintain the protrusion, and suggest that the lipid bilayer itself has the ability to buffer the membrane tension.     

GMP production of liposomes--a new industrial approach

A new scalable liposome production system is presented, which is based on the ethanol injection technique. The system permits liposome manufacture regardless of production scale, as scale is determined only by free disposable vessel volumes. Once the parameters are defined, an easy scale up can be performed by just changing the process vessels. These vessels are fully sterilizeable and all raw materials are transferred into the sanitized and sterilized system via 0.2 microm filters to guarantee an aseptic production.Liposome size can be controlled by the local lipid concentration at the injection point depending on process parameters like injection pressure, lipid concentration and injection rate. These defined process parameters are furthermore responsible for highly reproducible results with respect to vesicle diameters and encapsulation rates Compared to other technologies like the film method which is normally followed by size reduction through high pressure homogenization, ultrasonication or extrusion, no mechanical forces are needed to generate homogeneous and narrow distributed liposomes.Another important advantage of this method is the suitability for the entrapment of many different drug substances such as large hydrophilic proteins by passive encapsulation, small amphiphilic drugs by a one step remote loading technique or membrane association of antigens for vaccination approaches.     

Preparation of connexin43‐integrated giant Liposomes by a baculovirus expression–liposome fusion method

Connexin‐43 (Cx43) containing giant liposomes (GL) were prepared by a baculovirus expression–liposome fusion method. Recombinant budded viruses expressing Cx43 were prepared and then fused with GLs containing DOPG/DOPC at pH 4.5. Connexon formation on the GL membrane was observed by transmission electron microscope. Hydrophilic fluorescent dye transfers were observed through a Cx43‐mediated pathway not only between Sf9 (Spodoptera frugiperda) cells with Cx43 but also from giant Cx43 liposomes to Cx43‐expressing U2OS cells (human osteosarcoma cell). The functional connexin‐containing liposome is expected to be useful for cellular cytosolic delivery systems. The original orientation and function of Cx43 was maintained after integration into the liposomes. The liposome fusion method will create new opportunities as a tool for analysis of channel membrane proteins. Biotechnol. Bioeng. 2010;107: 836–843. © 2010 Wiley Periodicals, Inc.     

Estimation of the membrane permeability of liposomes via use of eosin Y chemiluminescence catalysed by peroxidase encapsulated in liposomes.

The initial rate of horseradish peroxidase (HRP)-catalysed chemiluminescence (CL) reaction in an aqueous compartment of liposomes was applied to the estimation of membrane permeability of liposomes. HRP-encapsulated liposomes were prepared by an extrusion method, and a CL reagent and H(2)O(2) were added into the liposomes suspensions. Fluorescein, eosin Y and phloxin B, which are xanthene dyes with different chemical structures, were used as CL reagents. Xanthene dye and H(2)O(2) permeate into the inner phase of liposomes, resulting in initiation of the HRP-catalysed xanthene dye CL reaction with H(2)O(2). The initial rate of the CL reaction was independent of the xanthene dye used. The reproducibility of the initial rate with eosin Y was better than that with fluorescein and phloxin B. When the membrane permeability of the liposomes was changed by altering the concentration of cholesterol in them, the initial rate of the eosin Y CL reaction was dependent on the membrane permeability of the liposomes.     

The LEA-like protein HSP 12 in Saccharomyces cerevisiae has a plasma membrane location and protects membranes against desiccation and ethanol-induced stress

The LEA-like protein HSP 12 was identified as having a plasma membrane location in yeast. Gold particles, indicative of the presence of HSP 12, were observed on the external side of the plasma membrane when yeast grown to stationary phase were subjected to immunocytochemical analysis. Growth of yeast in the osmolyte mannitol resulted in an increased number of gold particles that were now observed to be present on both sides of the plasma membrane. No gold particles were observed using a mutant strain of the same yeast that did not express HSP 12. A model liposome system encapsulating the fluorescent dye calcein was used to investigate the protection by HSP 12 of membranes during desiccation. HSP 12 was found to act in an analogous manner to trehalose and protect liposomal membrane integrity against desiccation. The interaction between HSP 12 and the liposomal membrane was judged to be electrostatic as membrane protection was only observed with positively charged liposomes and not with either neutral or negatively charged liposomes. The ability of the wild-type and mutant yeast to grow in media containing ethanol was compared. It was found that yeast not expressing the HSP 12 protein were less able to grow in media containing ethanol. HSP 12 was shown to confer increased integrity on the liposomal membrane in the presence of ethanol. Ethanol, like mannitol, was found to induce HSP 12 protein synthesis. However, yeast grown in both ethanol and mannitol showed a decreased HSP 12 response compared with yeast grown in the presence of either osmolyte alone.