Differential binding of plasma proteins by liposomes loaded with lipophilic prodrugs of methotrexate and melphalan in the bilayer

Immediately upon contact with blood, nanosized drug delivery systems become coated with a so-called protein corona. The quantitative and qualitative composition of the corona defines not only the behavior of the nanocarrier in the circulation but, ultimately, the pharmacokinetics and biodistribution of the encapsulated drug as well. In turn, the composition of the protein corona depends on the surface properties of the nanoparticles, such as size and distribution of charge and functional groups on the particle surface. Liposomes belong to the most bio- and hemocompatible drug delivery systems feasible for intravenous route of administration required in chemotherapy of metastasizing tumors. However, knowledge on the interactions of liposomes of various compositions with blood plasma proteins remains fragmentary. Moreover, all nanosized drug delivery systems are potential targets for the innate immunity system, primarily the complement (C) system, which underlies frequent cases of hypersensitivity reactions. Recently, in a panel of in vitro hemocompatibility tests, we demonstrated that liposomes built of natural phospholipids — egg phosphatidylcholine and phosphatidylinositol from Saccharomyces cerevisiae — and loaded with diglyceride conjugates of anticancer drugs melphalan and methotrexate, did not affect the morphology and numbers of the main blood cell types. While preparations with melphalan prodrug were also inert in coagulation and C activation tests, methotrexate-loaded liposomes caused impaired coagulation and C activation. The aim of this work was to study the interactions of liposomes carrying prodrugs of melphalan and methotrexate with blood plasma proteins in vitro. Data on protein binding capacity of liposomes obtained with classical gel permeation chromatography techniques allowed for prediction of rather rapid elimination of the liposomes from circulation. A number of differences revealed through immunoblotting of the liposome-bound proteins agree with the previously obtained data on C activation. The possible mechanism of C activation by methotrexate-containing liposomes is discussed.     

Adsorption of non-membrane proteins on the surface of model phospholipid membranes

1. Two new methods are proposed for enhancement of the binding of hydrophilic proteins by liposomes. 2. An alkylating derivative of phosphatidic acid has been obtained by its reaction with N,N,N′-tris(2-chloroethyl)-N′- (p-formylphenyl)propylene-1,3-diamine. The alkylating activity of this derivative is very low due to the electron-acceptor effect of the formyl residue. Phosphatidylcholine liposomes which contain this alkylating derivative in the lipid bilayer may be obtained. The compound residing in the outer monolayer may be reduced by NaBH₄. Upon reduction, the formyl residue is transformed into a hydroxymethyl residue. Therefore, the alkylating group of the compound is activated, and proteins may be attached covalently to the outer monolayer by alkylation with such chemically reactive liposomes. 3. Reaction of alkylating liposomes with myoglobin results in covalent binding of this hydrophilic protein. Complement-mediated leakage of such myoglobin-carrying liposomes may be induced by antibodies against myoglobin. 4. Modification of hydrophilic proteins with dansyl chloride results, even at small extents of modification, in a dramatic increase of the affinity of such proteins to phosphatidylcholine liposomes.     

Characterization of liposomes coated with S-layer proteins from lactobacilli

The stability of liposomes coated with S-layer proteins from Lactobacillus brevis and Lactobacillus kefir was analyzed as a previous stage to the development of a vaccine vehicle for oral administration. The interactions of the different S-layer proteins with positively charged liposomes prepared with soybean lecithin or dipalmitoylphosphatidylcholine were studied by means of the variation of the Z potential at different protein-lipid ratios, showing that both proteins were able to attach in a greater extent to the surface of soybean lecithin liposomes. The capacity of these particles to retain carboxyfluorescein or calcein by exposure to bile salts, pancreatic extract, pH change and after a thermal shock showed that both S-layer proteins increased the stability of the liposomes in the same magnitude. The non-glycosylated protein from L. brevis protects more efficiently the liposomes at pH 7 than those from L. kefir even without treatment with glutaraldehyde.     

Adaptation of the Wine Bacterium Oenococcus oeni to Ethanol Stress: Role of the Small Heat Shock Protein Lo18 in Membrane Integrity

Malolactic fermentation in wine is often carried out by Oenococcus oeni. Wine is a stressful environment for bacteria because ethanol is a toxic compound that impairs the integrity of bacterial membranes. The small heat shock protein (sHsp) Lo18 is an essential actor of the stress response in O. oeni. Lo18 prevents the thermal aggregation of proteins and plays a crucial role in membrane quality control. Here, we investigated the interaction between Lo18 and four types of liposomes: one was prepared from O. oeni grown under optimal growth conditions (here, control liposomes), one was prepared from O. oeni grown in the presence of 8% ethanol (here, ethanol liposomes), one was prepared from synthetic phospholipids, and one was prepared from phospholipids from Bacillus subtilis or Lactococcus lactis. We observed the strongest interaction between Lo18 and control liposomes. The lipid binding activity of Lo18 required the dissociation of oligomeric structures into dimers. Protein protection experiments carried out in the presence of the liposomes from O. oeni suggested that Lo18 had a higher affinity for control liposomes than for a model protein. In anisotropy experiments, we mimicked ethanol action by temperature-dependent fluidization of the liposomes. Results suggest that the principal determinant of Lo18-membrane interaction is lipid bilayer phase behavior rather than phospholipid composition. We suggest a model to describe the ethanol adaptation of O. oeni. This model highlights the dual role of Lo18 in the protection of proteins from aggregation and membrane stabilization and suggests how modifications of phospholipid content may be a key factor determining the balance between these two functions.     

Examination of the specificity of tumor cell derived exosomes with tumor cells in vitro

Small endogenous vesicles called exosomes are beginning to be explored as drug delivery vehicles. The in vivo targets of exosomes are poorly understood; however, they are believed to be important in cell-to-cell communication and may play a prominent role in cancer metastasis. We aimed to elucidate whether cancer derived exosomes can be used as drug delivery vehicles that innately target tumors over normal tissue. Our in vitro results suggest that while there is some specificity towards cancer cells over “immortalized” cells, it is unclear if the difference is sufficient to achieve precise in vivo targeting. Additionally, we found that exosomes associate with their cellular targets to a significantly greater extent (> 10-fold) than liposomes of a similar size. Studies on the association of liposomes mimicking the unique lipid content of exosomes revealed that the lipid composition contributes significantly to cellular adherence/internalization. Cleavage of exosome surface proteins yielded exosomes exhibiting reduced association with their cellular targets, demonstrating the importance of proteins in binding/internalization. Furthermore, although acidic conditions are known to augment the metastatic potential of tumors, we found that cells cultured at low pH released exosomes with significantly less potential for cellular association than cells cultured at physiological pH.     

Binding of DNA to liposomes containing different derivatives of sphingosine

Binding of DNA to dimyristoylphosphatidylcholine (DMPC) liposomes containing different sphingosine derivatives was investigated. DNA labelled with adriamycin was used as a fluorescence quencher and its membrane association was observed by resonance energy transfer from liposomes incorporating a pyrene-derivatized lipid bisPDPC as a donor and containing 19 mol% of sphingosine, dihydro-, phyto- or dimethylsphingosine. As revealed by differential scanning calorimetry, the thermal phase behaviour of multilamellar liposomes containing these sphingolipids was also significantly altered by DNA. Attachment of DNA to liposomes containing sphingosylphosphorylcholine was much weaker, and no binding of DNA to membranes containing N-acetylsphingosine, N-stearoylsphingosine or sphingomyelin was observed. The membrane binding of DNA was dependent on pH and could be reversed by the inclusion of phosphatidic acid (eggPA) into the liposomes. Analogously, the association of cytochrome c with eggPA could be reversed by the DNA-binding sphingosines. These findings lend support to our previous proposal that the DNA-sphingosine interaction is electrostatic and requires the presence of a positive charge in the latter. Accordingly, sphingosines carrying a protonated amino group attach DNA to membranes, while blocking of the amino group by N-acylation abolishes this interaction.     

Quantitative Analysis of the Lamellarity of Giant Liposomes Prepared by the Inverted Emulsion Method

The inverted emulsion method is used to prepare giant liposomes by pushing water-in-oil droplets through the oil/water interface into an aqueous medium. Due to the high encapsulation efficiency of proteins under physiological conditions and the simplicity of the protocol, it has been widely used to prepare various cell models. However, the lamellarity of liposomes prepared by this method has not been evaluated quantitatively. Here, we prepared liposomes that were partially stained with a fluorescent dye, and analyzed their fluorescence intensity under an epifluorescence microscope. The fluorescence intensities of the membranes of individual liposomes were plotted against their diameter. The plots showed discrete distributions, which were classified into several groups. The group with the lowest fluorescence intensity was determined to be unilamellar by monitoring the exchangeability of the inner and the outer solutions of the liposomes in the presence of the pore-forming toxin α-hemolysin. Increasing the lipid concentration dissolved in oil increased the number of liposomes ∼100 times. However, almost all the liposomes were unilamellar even at saturating lipid concentrations. We also investigated the effects of lipid composition and liposome content, such as highly concentrated actin filaments and Xenopus egg extracts, on the lamellarity of the liposomes. Remarkably, over 90% of the liposomes were unilamellar under all conditions examined. We conclude that the inverted emulsion method can be used to efficiently prepare giant unilamellar liposomes and is useful for designing cell models.     

Binding of a pleurotolysin ortholog from Pleurotus eryngii to sphingomyelin and cholesterol-rich membrane domains

A mixture of sphingomyelin (SM) and cholesterol (Chol) exhibits a characteristic lipid raft domain of the cell membranes that provides a platform to which various signal molecules as well as virus and bacterial proteins are recruited. Several proteins capable of specifically binding either SM or Chol have been reported. However, proteins that selectively bind to SM/Chol mixtures are less well characterized. In our screening for proteins specifically binding to SM/Chol liposomes, we identified a novel ortholog of Pleurotus ostreatus, pleurotolysin (Ply)A, from the extract of edible mushroom Pleurotus eryngii, named PlyA2. Enhanced green fluorescent protein (EGFP)-conjugated PlyA2 bound to SM/Chol but not to phosphatidylcholine/Chol liposomes. Cell surface labeling of PlyA2-EGFP was abolished after sphingomyelinase as well as methyl-β-cyclodextrin treatment, removing SM and Chol, respectively, indicating that PlyA2-EGFP specifically binds cell surface SM/Chol rafts. Tryptophan to alanine point mutation of PlyA2 revealed the importance of C-terminal tryptophan residues for SM/Chol binding. Our results indicate that PlyA2-EGFP is a novel protein probe to label SM/Chol lipid domains both in cell and model membranes.     

A lipophilic complex with 186Re188Re incorporated in liposomes suitable for radiotherapy

Liposomes with a 70 nm diameter were made by the detergent removal technique on a gel filtration column. The complex oxodichloroethoxy-bis-(triphenylphosphine)rhenium(V) = (Rephos) was irradiated by neutrons in the reactor at PSI. 45 ± 5% of the radioactive complex was incorporated into the bilayer of the liposomes during the liposome formation. The stability of these radioactive liposomes was tested by dialysis: a loss of 40% of the radioactivity identified as perrhenate was observed after 8 days. Addition of the antioxidant ascorbic acid diminished the loss to 20%. Such liposomes carrying the lipophilic radioactive Re-complex can potentially be used in β-radiotherapy. The γ-lines of the two rhenium isotopes are helpful for localizing them in therapy controls by a γ-camera, a big advantage compared to other nuclides proposed for therapy (e.g. ⁹⁰Yttrium).     

Sequence and functional similarities between pro-apoptotic Bid and plant lipid transfer proteins

Pro-apoptotic proteins of the Bcl-2 family are known to act on mitochondria and facilitate the release of cytochrome c, but the biochemical mechanism of this action is unknown. Association with mitochondrial membranes is likely to be important in determining the capacity of releasing cytochrome c. The present work provides new evidence suggesting that some pro-apoptotic proteins like Bid have an intrinsic capacity of binding and exchanging membrane lipids. Detailed analysis indicates a significant sequence similarity between a subset of Bcl-2 family proteins including Bid and Nix and plant lipid transfer proteins. The similar structural signatures could be related to common interactions with membrane lipids. Indeed, isolated Bid shows a lipid transfer activity that is even higher than that of plant lipid transfer proteins. To investigate the possible relevance of these structure-function correlations to the apoptotic action of Bid, cell free assays were established with isolated mitochondria, recombinant Bid and a variety of exogenous lipids. Micromolar concentrations of lysolipids such as lysophosphatidylcholine were found to change the association of Bid with mitochondria and also stimulate the release of cytochrome c promoted by Bid. The changes in mitochondrial association and cytochrome c release were enhanced by the presence of liposomes of lipid composition similar to that of mitochondrial membranes. Thus, a mixture of liposomes, mitochondria and key lysolipids could reproduce the conditions enabling Bid to transfer lipids between donor and acceptor membranes, and also change its reversible association with mitochondria. Bid was also found to enhance the incorporation of a fluorescent lysolipid, but not of a related fatty acid, into mitochondria. On the basis of the results presented here, it is hypothesised that Bid action may depend upon its capacity of exchanging lipids and lysolipids with mitochondrial membranes. The hypothesis is discussed in relation to current models for the integrated action of pro-apoptotic proteins of the Bcl-2 family.     

New method for site-specific modification of liposomes with proteins using sortase A-mediated transpeptidation

A new method was developed for site-specific modifications of liposomes by proteins via sortase A (SrtA)-mediated transpeptidation reactions. In this regard, the enhanced green fluorescent protein (eGFP) was biologically engineered to carry at its polypeptide C-terminus the LPATG motif recognized by SrtA and used as the protein donor for linking to liposomes that were decorated with phospholipids carrying a diglycine motif as the other SrtA substrate and the eGFP acceptor. Under the influence of SrtA, eGFP was efficiently attached to liposomes, as proved by analyzing the enzymatic reaction products and the resultant fluorescent liposomes. It was observed that increasing the concentration and the distance of the diglycine motif on and from the liposome surface could significantly improve the efficiency of liposome modification by proteins. It is anticipated that this strategy can be widely useful for the modification of liposomes by other proteins.     

Effect of Multimerization on Membrane Association of Rous Sarcoma Virus and HIV-1 Matrix Domain Proteins

In most retroviruses, plasma membrane (PM) association of the Gag structural protein is a critical step in viral assembly, relying in part on interaction between the highly basic Gag MA domain and the negatively charged inner leaflet of the PM. Assembly is thought to begin with Gag dimerization followed by multimerization, resulting in a hexameric lattice. To directly address the role of multimerization in membrane binding, we fused the MA domains of Rous sarcoma virus (RSV) and HIV-1 to the chemically inducible dimerization domain FK506-binding protein (FKBP) or to the hexameric protein CcmK4 from cyanobacteria. The cellular localization of the resulting green fluorescent protein (GFP)-tagged chimeric proteins was examined by fluorescence imaging, and the association of the proteins with liposomes was quantified by flotation in sucrose gradients, following synthesis in a reticulocyte extract or as purified proteins. Four lipid compositions were tested, representative of liposomes commonly reported in flotation experiments. By themselves, GFP-tagged RSV and HIV-1 MA proteins were largely cytoplasmic, but both hexamerized proteins were highly concentrated at the PM. Dimerization led to partial PM localization for HIV-1 MA. These in vivo effects of multimerization were reproduced in vitro. In flotation analyses, the intact RSV and HIV-1 Gag proteins were more similar to multimerized MA than to monomeric MA. RNA is reported to compete with acidic liposomes for HIV-1 Gag binding, and thus we also examined the effects of RNase treatment or tRNA addition on flotation. tRNA competed with liposomes in the case of some but not all lipid compositions and ionic strengths. Taken together, our results further underpin the model that multimerization is critical for PM association of retroviral Gag proteins. In addition, they suggest that the modulation of membrane binding by RNA, as previously reported for HIV-1, may not hold for RSV.     

Stairway to Asymmetry: Five Steps to Lipid-Asymmetric Proteoliposomes

Membrane proteins are embedded in a complex lipid environment that influences their structure and function. One key feature of nearly all biological membranes is a distinct lipid asymmetry. However, the influence of membrane asymmetry on proteins is poorly understood, and novel asymmetric proteoliposome systems are beneficial. To our knowledge, we present the first study on a multispanning protein incorporated in large unilamellar liposomes showing a stable lipid asymmetry. These asymmetric proteoliposomes contain the Na⁺/H⁺ antiporter NhaA from Salmonella Typhimurium. Asymmetry was introduced by partial, outside-only exchange of anionic phosphatidylglycerol (PG), mimicking this key asymmetry of bacterial membranes. Outer-leaflet and total fractions of PG were determined via ζ-potential (ζ) measurements after lipid exchange and after scrambling of asymmetry. ζ-Values were in good agreement with exclusive outside localization of PG. The electrogenic Na⁺/H⁺ antiporter was active in asymmetric liposomes, and it can be concluded that reconstitution and generation of asymmetry were successful. Lipid asymmetry was stable for more than 7 days at 23°C and thus enabled characterization of the Na⁺/H⁺ antiporter in an asymmetric lipid environment. We present and validate a simple five-step protocol that addresses key steps to be taken and pitfalls to be avoided for the preparation of asymmetric proteoliposomes: 1) optimization of desired lipid composition, 2) detergent-mediated protein reconstitution with subsequent detergent removal, 3) generation of lipid asymmetry by partial exchange of outer-leaflet lipid, 4) verification of lipid asymmetry and stability, and 5) determination of protein activity in the asymmetric lipid environment. This work offers guidance in designing asymmetric proteoliposomes that will enable researchers to compare functional and structural properties of membrane proteins in symmetric and asymmetric lipid environments.     

Quantitative Analysis of the Lamellarity of Giant Liposomes Prepared by the Inverted Emulsion Method

The inverted emulsion method is used to prepare giant liposomes by pushing water-in-oil droplets through the oil/water interface into an aqueous medium. Due to the high encapsulation efficiency of proteins under physiological conditions and the simplicity of the protocol, it has been widely used to prepare various cell models. However, the lamellarity of liposomes prepared by this method has not been evaluated quantitatively. Here, we prepared liposomes that were partially stained with a fluorescent dye, and analyzed their fluorescence intensity under an epifluorescence microscope. The fluorescence intensities of the membranes of individual liposomes were plotted against their diameter. The plots showed discrete distributions, which were classified into several groups. The group with the lowest fluorescence intensity was determined to be unilamellar by monitoring the exchangeability of the inner and the outer solutions of the liposomes in the presence of the pore-forming toxin α-hemolysin. Increasing the lipid concentration dissolved in oil increased the number of liposomes ∼100 times. However, almost all the liposomes were unilamellar even at saturating lipid concentrations. We also investigated the effects of lipid composition and liposome content, such as highly concentrated actin filaments and Xenopus egg extracts, on the lamellarity of the liposomes. Remarkably, over 90% of the liposomes were unilamellar under all conditions examined. We conclude that the inverted emulsion method can be used to efficiently prepare giant unilamellar liposomes and is useful for designing cell models.     

Arabidopsis CURVATURE THYLAKOID1 Proteins Modify Thylakoid Architecture by Inducing Membrane Curvature

Chloroplasts of land plants characteristically contain grana, cylindrical stacks of thylakoid membranes. A granum consists of a core of appressed membranes, two stroma-exposed end membranes, and margins, which connect pairs of grana membranes at their lumenal sides. Multiple forces contribute to grana stacking, but it is not known how the extreme curvature at margins is generated and maintained. We report the identification of the CURVATURE THYLAKOID1 (CURT1) protein family, conserved in plants and cyanobacteria. The four Arabidopsis thaliana CURT1 proteins (CURT1A, B, C, and D) oligomerize and are highly enriched at grana margins. Grana architecture is correlated with the CURT1 protein level, ranging from flat lobe-like thylakoids with considerably fewer grana margins in plants without CURT1 proteins to an increased number of membrane layers (and margins) in grana at the expense of grana diameter in overexpressors of CURT1A. The endogenous CURT1 protein in the cyanobacterium Synechocystis sp PCC6803 can be partially replaced by its Arabidopsis counterpart, indicating that the function of CURT1 proteins is evolutionary conserved. In vitro, Arabidopsis CURT1A proteins oligomerize and induce tubulation of liposomes, implying that CURT1 proteins suffice to induce membrane curvature. We therefore propose that CURT1 proteins modify thylakoid architecture by inducing membrane curvature at grana margins.     

Purification and Biochemical Characterization of the Lambda Holin

Holins are small phage-encoded cytoplasmic membrane proteins, remarkable for their ability to make membranes permeable in a temporally regulated manner. The purification of S105, the λ holin, and one of the two products of gene S is described. Because the wild-type S105 holin could be only partially purified from membrane extracts by ion-exchange chromatography, an oligohistidine tag was added internally to the S105 sequence for use in immobilized metal affinity chromatography. An acceptable site for the tag was found between residues 94 and 95 in the highly charged C-terminal domain of S. This allele, designated S105H94, had normal lysis timing under physiological expression conditions. The S105H94 protein was overproduced, purified, and characterized by circular dichroism spectroscopy, which revealed approximately 40% alpha-helix conformation, consistent with the presence of two transmembrane helices. The purified protein was then used to achieve release of fluorescent dye loaded in liposomes in vitro, whereas protein from an isogenic construct carrying an S mutation known to abolish hole formation was inactive in this assay. These results suggest that S is a bitopic membrane protein capable of forming aqueous holes in bilayers.     

A simple method for producing a technetium-99m-labeled liposome which is stable In Vivo

A new method for labeling preformed liposomes with technetium-99m (^99mTc) has been developed which is simple to perform and stable in vivo. Previous ^99mTc-liposome labels have had variable labeling efficiencies and stability. This method consistently achieves high labeling efficiencies (> 90%) with excellent stability. A commercially available radiopharmaceutical kit—hexamethylpropyleneamine oxime (HM-PAO)—is reconstituted with ^99mTcO^−₄ and then incubated with preformed liposomes that encapsulate glutathione. The incubation takes only 30 min at room temperature. Liposomes that co-encapsulate other proteins such as hemoglobin or albumin, in addition to glutathione, also label with high efficiency. Both in vitro and in vivo studies indicate good stability of this label. Rabbit images show significant spleen and liver uptake at 2 and 20 h after liposome infusion without visualization of thyroid, stomach or bladder activity.This labeling method can be used to study the biodistribution of a wide variety of liposome preparations that are being tested as novel drug delivery systems. This method of labeling liposomes with ^99mTc may also have applications in diagnostic imaging.     

Evaluation of temperature and guanidine hydrochloride-induced protein–liposome interactions by using immobilized liposome chromatography

Hydrophobic interactions between nine model proteins and net-neutral lipid bilayer membranes (liposomes) under stress conditions were quantitatively examined by using immobilized liposome chromatography (ILC). Small or large unilamellar liposomes were composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and immobilized in a gel matrix by utilizing covalent coupling between amino-containing lipids and activated gel beads or avidin–biotin biospecific binding. Retardation of bovine carbonic anhydrase (CAB) in ILC was pronounced at particular temperatures (50 and 60 °C) where the local hydrophobicity of theses protein molecules becomes sufficiently large. Protein-induced leakage of a hydrophilic dye (calcein) from immobilized liposomes interior was also drastically enhanced at particular temperatures where large retardation was observed. For other proteins examined, similar results were also observed. The specific capacity factor of the proteins characteristic for the ILC and the amount of calcein released from immobilized liposomes were successfully expressed as a function of the product of the local hydrophobicities of proteins and liposomes, regardless of protein species and the type of the stress conditions applied (denaturant and heating). These findings indicate that lipid membranes have an ability to non-specifically recognize local hydrophobicities of proteins to form stress-mediated supramolecular assemblies with proteins, which may have potential applications in bioprocesses such as protein refolding and separation. ILC was thus found to be a very useful method for the quantitative detection of dynamic protein–liposome interactions triggered by stress conditions.     

Glutathione disulfide liposomes – A research tool for the study of glutathione disulfide associated functions and dysfunctions

Glutathione disulfide (GSSG) is the oxidized form of glutathione (GSH). GSH is a tripeptide present in the biological system in mM concentration and is the major antioxidant in the body. An increase in GSSG reflects an increase in intracellular oxidative stress and is associated with disease sates. The increase has also been demonstrated to lead to an increase in protein S-glutathionylation that can affect the structure and function of proteins. Protein S-glutathionylation serves as a regulatory mechanism during cellular oxidative stress. Though GSSG is commercially available, its roles in various GSSG-associated normal/abnormal physiological functions have not been fully delineated due to the reason that GSSG is not cell membrane permeable and a lack of method to specifically increase GSSG in cells. We have developed cationic liposomes that can effectively deliver GSSG into cells. Various concentrations of GSSG liposomes can be conveniently prepared. At 1 mg/mL, the GSSG liposomes effectively increased intracellular GSSG by 27.1±6.9 folds (n=3) in 4 h and led to a significant increase in protein S-glutathionylation confirming that the increased GSSG is functionally effective. The Trypan blue assay demonstrated that GSSG liposomes were not cytotoxic; the cell viability was greater than 95% after cells were treated with the GSSG liposomes for 4 h. A stability study showed that the dry form of the GSSG liposomes were stable for at least 70 days when stored at ?80 °C. Our data demonstrate that the GSSG liposomes can be a valuable tool in studying GSSG-associated physiological/pathological functions.     

Liposomes as Immunoadjuvants and Vaccine Carriers: Antigen Entrapment

Successful use of liposomes as immunological adjuvants in vaccines requires simple, easy to scale up technology capable of high-yield antigen entrapment. Recent work from this laboratory has led to the development of techniques that can generate liposomes of various sizes containing soluble antigens such as proteins or particulate antigens such as whole, live, or attenuated bacteria or viruses. Entrapment of proteins is carried out by the dehydration-rehydration procedure, which entails freeze-drying of a mixture of "empty" small unilamellar vesicles and free antigens. Upon rehydration, the large multilamellar vesicles that are formed incorporate up to 80% of the antigen used. When such liposomes are microfluidized in the presence of nonentrapped material, their size is reduced to about 100 nm in diameter, with much of the originally entrapped antigen still associated with the vesicles. A similar technique applied to the entrapment of particulate antigens (e.g., Bacillus subtilis spores) consists of freeze-drying giant vesicles (4-5 μm in diameter) in the presence of spores. On rehydration and sucrose gradient fractionation of the suspension, up to 27% of the spores used are associated with generated giant liposomes of similar mean size.